energetics of mouse papillary muscle - equella · specific changes in work and enthalpy output; ......

EEnneerrggeettiiccss ooff

mmoouussee ppaappiillllaarryy mmuussccllee

Cecilia Widén

MSc. Biomedicine

A thesis submitted in fulfillment of the requirements of the degree of

Doctor of Philosophy

Heart Foundation Research Centre

School of Physiotherapy & Exercise Science

Faculty of Health

Griffith University

Queensland

AUSTRALIA April 2006

i

SSuummmmaarryy

The overall aim of this Thesis was to characterise the energetic properties of the mouse

papillary muscle as this preparation could become a useful model to study alterations of

energetic aspects of cardiac pathologies and heart-focussed genetic changes.

Measurements of resting and active metabolism of the papillary muscles were made in

vitro using the myothermic technique.

In the first study the mechanism underlying impaired contractility of post-ischaemic rat

papillary muscle was investigated. The rat preparation is well established and was used

to develop protocols and approaches that could later be used as the basis for studies with

mouse papillary muscle. The muscles were exposed to simulated ischaemia for 60 min

and change in energetics was studied 30 min into the reperfusion phase. The work

output was reduced to 66 ± 3% of the pre-ischaemia value and the enthalpy output

decreased to 71 ± 3% of pre-ischaemia value. However, there was no change in either

initial, 19 ± 3%, or net mechanical efficiency, 9.0 ± 0.9%. These data, in combination

with studies of Ca2+

handling, suggests that the reduced work output was caused by

attachment of fewer cross-bridges in each twitch, but with no change in work generated

by each cross-bridge.

The following two studies involved characterisation of the energetics of the mouse

papillary muscle and included measurements of resting and active metabolism. The

resting metabolic rate varied with muscle size but the mean initial value was ∼25 mW

ii

g-1

and the estimated steady value ∼5 mW g-1

. The resting metabolic rate declined

exponentially with time towards a steady value, with a time constant of 18 ± 2 min.

There was no alteration in isometric force output during this time. The magnitude of

resting metabolism depended inversely on muscle mass, more than doubled following a

change in substrate from glucose to pyruvate and was increased 2.5-fold when the

osmolarity of the bathing solution was increased by addition of 300 mM sucrose.

Addition of 30 mM BDM affected neither the time course of the decline in metabolic

rate nor the eventual steady value.

The energy requirements associated with contractile activity were ∼7 mJ g-1

twitch-1

at a

contraction frequency of 1 Hz. The enthalpy output was not affected by changing

substrate from glucose to pyruvate but did decrease with an increase in temperature. The

enthalpy output was partitioned into force-dependent and force-independent

components using BDM to selectively inhibit cross-bridge cycling. The force-

independent enthalpy output was 18.6 ± 1.9% of the initial enthalpy output. Muscle

initial efficiency was ∼32% and net efficiency ∼17% when shortening at a realistic

velocity. The enthalpy output decreased with increased contraction frequency but was

independent of shortening velocity. On the basis of these values, it was calculated that

the twitch energetics were consistent with ATP splitting by half the cross-bridges and

the pumping of one Ca2+

into the SR for every three cross-bridge cycles. The lack of

influence of shortening velocity on energy cost supports the idea that the amount of

energy to be used is determined early in a twitch and is not greatly influenced by events

that occur during the contraction.

The suitability of the mouse papillary muscle as a model to study ischaemia and

reperfusion damage was also assessed. This preparation is excellent for studying muscle

specific changes in work and enthalpy output; however, due to the long-term instability

and variability amongst preparations, the suitability of this preparation in prolonged

experiments remains uncertain.

iii

DDeeccllaarraattiioonn

This work has previously not been submitted for a degree or diploma in any university.

To all the best of my knowledge and belief, the thesis contains no material previously

published or written by another person except where due reference is made in the thesis

itself.

Cecilia Widén

v

TTaabbllee ooff ccoonntteennttss

LIST OF FIGURES...........................................................................................IX

LIST OF TABLES ............................................................................................XI

LIST OF PUBLICATIONS.............................................................................. XIII

ACKNOWLEDGMENTS..................................................................................XV

CHAPTER 1: INTRODUCTION......................................................................... 1

CHAPTER 2: BRIEF BACKGROUND ................................................................ 3

2.1 Cardiac energetics..................................................................................................3 2.1.1 Initial biochemical reactions.................................................................................4

2.1.1.1 ATP hydrolysis ................................................................................................................4 2.1.1.2 Creatine kinase reaction...................................................................................................8

2.1.2 Recovery biochemical reactions ...........................................................................8

2.2 Papillary muscles as a model of ventricular muscle energetics .......................10

2.3 Isolated mouse papillary model ..........................................................................11

CHAPTER 3: METHODS ............................................................................... 13

3.1 Papillary muscle preparation and dissection.....................................................13

vi

3.2 Overview of experimental apparatus .................................................................14 3.2.1 Measurement of muscle force production ..........................................................15

3.2.2 Measurement of muscle length changes .............................................................16

3.2.3 Principles of measuring muscle heat output using a thermopile ........................16 3.2.3.1 Calibration of thermopile...............................................................................................16 3.2.3.2 Conversion of thermopile signals into heat production .................................................19

3.2.4 Determination of stimulus heat...........................................................................20

3.3 Data recording......................................................................................................21

3.4 Partitioning of initial and recovery metabolism................................................22 3.4.1 Partitioning initial metabolism into force-dependent and force-independent

components .........................................................................................................24

3.5 Calculations of efficiency.....................................................................................24 3.5.1 Mechanical efficiency.........................................................................................24

3.5.1.1 Initial and net mechanical efficiency .............................................................................24 3.5.2 Mitochondrial efficiency.....................................................................................25

3.6 Ratio of recovery heat output to initial heat output .........................................25

3.7 Oxygenation of papillary muscles.......................................................................26

3.8 Data normalisation...............................................................................................27

CHAPTER 4: MECHANISM OF DEPRESSED WORK OUTPUT IN POST-ISCHAEMIC CARDIAC MUSCLE................................................ 29

4.1 Introduction..........................................................................................................29

4.2 Methods.................................................................................................................30 4.2.1 Initial and recovery metabolism .........................................................................31

4.2.2 Experimental protocols .......................................................................................32

4.2.3 Statistical analysis...............................................................................................33

4.2.4 Analysis of diffusive O2 supply..........................................................................33

4.3 Results .................................................................................................................34 4.3.1 Control experiments............................................................................................34

4.3.2 Effects of simulated ischaemia on force output..................................................36

4.3.3 Effects of simulated ischaemia and reperfusion on mechanical and energetic

parameters...........................................................................................................36

4.3.4 Partitioning energy cost between force-dependent and force-independent

components .........................................................................................................37

4.3.5 Time course of oxidative recovery metabolism..................................................39

4.4 Discussion..............................................................................................................40 4.4.1 Impaired work output due to fewer attached cross-bridges ................................41

4.4.2 Mitochondrial efficiency.....................................................................................44

4.4.3 Recovery time course..........................................................................................44

4.5 Conclusion.............................................................................................................45

vii

CHAPTER 5: RESTING METABOLISM OF MOUSE PAPILLARY MUSCLE ....... 47

5.1 Introduction..........................................................................................................47

5.2 Methods.................................................................................................................49 5.2.1 Heat measurements .............................................................................................49

5.2.2 Calculation of rate of resting heat production ....................................................50

5.2.3 Measurement of myocyte sarcomere length by diffraction of laser light ...........50




5.3 Results .................................................................................................................53 5.3.1 Resting metabolic rate depended on time and muscle mass...............................53

5.3.2 The effect of BDM on resting metabolic rate .....................................................56

5.3.3 The effect of hyperosmolarity on resting metabolic rate ....................................56

5.3.4 The effect of metabolic substrate on resting metabolism and force output ........57

5.4 Discussion..............................................................................................................58 5.4.1 Comparison with other studies ...........................................................................58

5.4.2 Adequacy of diffusive oxygen supply ................................................................61

5.5 Recommendations for performing experiments with isolated papillary

muscles .................................................................................................................63

CHAPTER 6: CHARACTERISATION OF ACTIVE METABOLISM..................... 65

6.1 Introduction..........................................................................................................65

6.2 Methods.................................................................................................................66 6.2.1 Mechanical output...............................................................................................67

6.2.2 Contribution of recovery heat to initial heat measurements ...............................67


6.2.4 Calculation of number of cross-bridge cycles ....................................................69



6.3 Results .................................................................................................................71 6.3.1 Force output of mouse papillary muscle.............................................................71

6.3.2 Energy cost of a twitch and effects of contraction frequency, substrate and

temperature .........................................................................................................72

6.3.3 Effects of shortening on twitch energy cost........................................................74

6.3.4 Mechanical efficiency.........................................................................................76

6.3.5 Ratio of recovery to initial enthalpy output ........................................................76

6.3.6 Partitioning energy cost between force-dependent and force-independent

components .........................................................................................................77

6.4 Discussion..............................................................................................................77 6.4.1 Number of cross-bridge cycles per twitch ..........................................................78

6.4.2 Amount of Ca2+

released from the SR in each twitch.........................................80

6.4.3 Partitioning of energy between force-dependent and force-independent

components .........................................................................................................82

viii

6.4.4 Mitochondrial efficiency.....................................................................................83

6.5 Conclusion.............................................................................................................83

CHAPTER 7: CAN MOUSE PAPILLARY MUSCLES BE USED IN PROLONGED

EXPERIMENTS? ....................................................................... 85

7.1 The observations ..................................................................................................86

7.2 Solution .................................................................................................................87

7.3 Experimental set-up .............................................................................................88

7.4 Adequate oxygenation..........................................................................................89

7.5 Method of euthanasia...........................................................................................89

7.6 Assessment of the suitability of isolated mouse papillary muscles for

investigating cardiac muscle physiology ............................................................89

CHAPTER 8: CONCLUDING COMMENTS ...................................................... 91

8.1 Resting metabolism ..............................................................................................91

8.2 Active metabolism ................................................................................................92

8.3 Ischaemia and reperfusion ..................................................................................93

8.4 Conclusion.............................................................................................................94

APPENDIX I ................................................................................................ 95

APPENDIX II ................................................................................................ 97

REFERENCES................................................................................................ 99

ix

LLiisstt ooff ffiigguurreess

Fig. 2.1. Initial and recovery reactions........................................................................................ 10

Fig. 3.1. Calibration of force transducer. .................................................................................... 15

Fig. 3.2. Time-course of cooling of silver blocks. ...................................................................... 18

Fig. 3.3. Calibration curve for the Seebeck coefficient............................................................... 18

Fig. 3.4. Heat loss correction....................................................................................................... 20

Fig. 3.5. Stimulus heat using a mouse papillary muscle. ............................................................ 21

Fig. 3.6. Example of the time course of force production and enthalpy output. ......................... 23

Fig. 4.1. Comparison of twitches recorded using isometric and realistic contraction protocols. 33

Fig. 4.2. Simulations of time courses of muscle oxygenation at the onset and end of a period of

simulated ischaemia. ................................................................................................. 35

Fig. 4.3. Changes in force output during ischaemia and reperfusion of rat papillary muscle. .... 38

Fig. 4.4. Effects of simulated ischaemia on relative work and relative enthalpy output............. 39

Fig. 4.5. Effect of simulated ischaemia on time course of rate of recovery heat output. ............ 40

Fig. 4.6. Effect of simulated ischaemia on cross-bridge-dependent and -independent energy cost.

................................................................................................................................... 43

Fig. 5.1. Example of myocyte sarcomere.................................................................................... 51

Fig. 5.2. Example of decline in resting heat rate with time during an experiment...................... 54

Fig. 5.3. Exponential decline in resting metabolism. .................................................................. 55

Fig. 5.4. Effect of muscle mass on resting metabolic rate........................................................... 55

Fig. 5.5. Example of the effect of hyperosmolarity on resting heat output and force production.

................................................................................................................................... 57

Fig. 5.6. The effect of glucose and pyruvate on the resting metabolic rate of mouse papillary

muscle. ...................................................................................................................... 58

x

Fig. 5.7. Prediction of in vivo resting metabolism....................................................................... 60

Fig. 5.8. Adequacy of diffusive O2 supply to resting mouse papillary muscle. .......................... 62

Fig. 6.1. Simulations of time course of PO2 at muscle centre during contraction series............. 71

Fig. 6.2. Contraction frequency dependence of normalised isometric force output.................... 72

Fig. 6.3. Enthalpy output per twitch in isometric contractions. .................................................. 73

Fig. 6.4. Effect of shortening velocity on work-loop. ................................................................. 75

Fig. 6.5. Enthalpy output per twitch in shortening contractions. ................................................ 75

Fig. 6.6. Initial and net mechanical efficiency. ........................................................................... 76

Fig. 6.7. Example of determining force-independent enthalpy output........................................ 77

Fig. 6.8. Dependence of Ca2+

released and ATP used on magnitude of force-independent

enthalpy output.......................................................................................................... 82

Fig. 7.1. Changes in mechanical performance with time. ........................................................... 86

xi

LLiisstt ooff ttaabblleess

Table 2.1. Reported values of free energy of ATP hydrolysis. ..................................................... 6

Table 3.1. Thermopile characteristics. ........................................................................................ 17

Table 3.2. Estimates of quantization noise and noise from other sources................................... 22

Table 4.1. Left ventricular rat papillary muscle characteristics. ................................................. 31

Table 4.2. Energetic variables before and after 60 min of simulated ischaemia......................... 37

Table 4.3. Recovery time constant before and after 60 min of simulated ischaemia.................. 40

Table 4.4. Theoretical changes in εI for different mechanisms underlying depressed work output.

................................................................................................................................... 42

Table 5.1. Characteristics of mouse papillary muscles. .............................................................. 55

Table 6.1. Characteristics of mouse papillary muscles. .............................................................. 67

Table 6.2 Effect of glucose and pyruvate on enthalpy output (n = 11). ..................................... 73

Table 6.3. Characteristics of mechanical and energetic properties of mouse papillary muscles

performing isometric contractions (2 Hz) at 22, 27 and 37°C using glucose as a

substrate..................................................................................................................... 74

xiii

LLiisstt ooff ppuubblliiccaattiioonnss

The following publications are listed in support of this Thesis:

Papers Widén C. and Barclay, C.J. (2006). ATP-splitting by half the cross-bridges can explain

the twitch energetics of mouse papillary muscle. Journal of Physiology 573: 5-15.

Widén C. and Barclay, C.J. (2005). Resting metabolism of mouse papillary muscle.

Pflugers Archiv 450: 209-216.

Abstract Widén C. and Barclay, C.J. (2005). Active metabolism of mouse papillary muscle.

Proceedings of the Australian Physiological Society 36: 114P.

xv

AAcckknnoowwlleeddggmmeennttss

I would sincerely like to thank my supervisor, mentor and friend Dr Chris Barclay for

his guidance, patience and encouragement throughout this candidature. “I think you’re

brilliant”, said Cissi! Also, you can stop looking in your mailbox now because I am

awarding you this year’s Nobel Prize in physiology and medicine!

Many thanks also to the Heart Foundation Research Centre and Griffith University for

supporting my PhD candidature.

I would also like to express my gratitude to the many wonderful people I have met

during my stay here in Australia. A very special thank you to my office buddies Tracey

Norling and Andrew Petersen for keeping me from going insane and Luke de Beus for

his invaluable help, especially with presentations and computers. I would also like to

thank my fellow students and colleagues at the School of Physiotherapy & Exercise

Science and the School of Medical Sciences.

Thank you also to the people I have met outside of the university through beach

volleyball and the gym. I have truly enjoyed our Sunday morning boot camp sessions

and I will be forever grateful for the friendship I have formed with the Lancettes!

Thanks also to Carole Rushton for helping me with secret women’s business!

Finally, I couldn’t have said it better than A.V. Hill, but the “slavery of writing” is

finally over!

1

CChhaapptteerr 11:: IInnttrroodduuccttiioonn

Muscles are biological machines that convert the chemical energy obtained from

breakdown of metabolic substrates into mechanical work. Muscle energetics is the study

of the processes involved in this energy conversion. A muscle pathology that potentially

lends itself to investigation based on energetics is the damage to cardiac muscle that

results from prolonged ischaemia (insufficient blood supply) and subsequent

reperfusion. It seems likely that at least part of the cause of post-ischaemic dysfunction

relates to cellular elements involved in energy conversion, especially the mitochondria

(transfer energy from metabolic substrates to ATP) and myosin cross-bridges (convert

energy from ATP into work). Much current research on the causes of ischaemia and

reperfusion damage is carried out using hearts and cardiac muscle from genetically

modified mice. However, relatively little is known about mouse heart energetics and

nothing is known about specific mouse cardiac muscle energetics. The overall aim of

this study was to characterise the energetics of the mouse papillary muscle and to

develop a protocol to study alterations of energetic aspects of cardiac muscle during

ischaemia and reperfusion.

To lay the theoretical foundations for the work described in the Thesis, a brief

background section has been provided (Chapter 2). This describes the function of the

papillary muscle, the biochemical reactions that underlie muscle contraction and also

provides a review of published values of the change in free energy of ATP hydrolysis.

2

This section is followed by a description of the methods that are common to most of the

experimental chapters (Chapter 3). Details specific to particular investigations are given

in the appropriate chapters.

The first experimental chapter (Chapter 4) describes an initial set of experiments

designed to investigate the mechanism of impaired work output arising from ischaemia

and reperfusion. This work was done using rat papillary muscle. This preparation is well

established and was used to develop protocols and approaches that could later be used

as the basis for studies with mouse papillary muscle.

The following chapters describe the characterisation of the energetics of mouse

papillary muscle. In Chapter 5 experiments to measure the resting metabolism (energy

required to maintain cell structure and integrity) are described. An important aspect of

this study was that of diffusive O2 supply to ensure that the muscles had an adequate O2

supply to meet the metabolic demand. This is important because, at least when first

dissected, the resting metabolic rate of papillary muscles is very high. Active

metabolism (energy required for contractile activity) was measured using both isometric

and realistic contraction protocols (Chapter 6) and experiments were performed to

determine the effects of contraction frequency, substrate, temperature and shortening on

twitch energy use. From these measurements it was concluded that about half of the

cross-bridges cycle during one twitch and that one Ca2+

is pumped into the sarcoplasmic

reticulum for every three cross-bridge cycles. In the final experimental chapter, the

suitability of the mouse papillary muscle as a model to study prolonged ischaemia is

discussed (Chapter 7).

3

CChhaapptteerr 22:: BBrriieeff bbaacckkggrroouunndd

This chapter provides a brief background on cardiac energetics and introduces the

biochemical reactions that underlie muscle contraction. The importance of ATP as the

energy source for muscle activity is highlighted and a review of reported literature

values of the change in free energy of ATP hydrolysis is presented. The difficulty of

interpreting the performance of the heart in terms of muscle specific energy output is

explained and the advantages of using an isolated cardiac muscle preparation as a model

of ventricular muscle function are discussed.

2.1 Cardiac energetics

Muscles convert the chemical free energy obtained from ATP hydrolysis into heat, and

if allowed to shorten, work. The First Law of Thermodynamics states that (in a closed

system∗) energy can be converted from one form to another but cannot be created or

destroyed. In the context of muscle contraction all the energy, or more properly,

enthalpy produced by the biochemical reactions underlying muscle contraction appears

as either heat or work. The chemical reactions are made up of two sets of processes: (1)

the breakdown of high-energy phosphates, which occurs simultaneously with

contraction, and (2) the regeneration of high-energy phosphates. The first set of

∗ By definition, a system in which the total energy of the system remains constant.

4

processes is called initial processes and the second, recovery processes. When there is

an adequate supply of O2 and metabolic substrate, recovery reactions are primarily those

of oxidative phosphorylation (Crow & Kushmerick, 1982; Paul, 1983; Smith et al.,

2005).

2.1.1 Initial biochemical reactions

Two biochemical reactions, the breakdown of adenosine triphosphate (ATP) and the

creatine kinase (CK) reaction, are commonly referred to as the initial reactions since

they take place within the time course of a contraction.

2.1.1.1 ATP hydrolysis

Muscles convert the chemical free energy obtained from ATP breakdown into heat and

work. During hydrolysis, bonds of the molecule are broken and energy is released.

iATP ADP P→ + (1)

where ADP is adenosine diphospate and Pi is inorganic phosphate. The energy produced

is known as enthalpy (∆H) and a negative value is indicative of liberation of energy (or

a decrease in the energy content of the molecules) from the process. For convenience, in

the remainder of this Thesis, enthalpy and free energy values are expressed as absolute

values. The enthalpy change of this process is affected by factors such as pH,

temperature (T), ionic conditions (I) and the free magnesium (Mg2+

) concentration.

Alberty (2003) reported a molar enthalpy value of approximately 26 kJ mol-1

at pH 7,

[Mg2+] = 0.1 mM, T = 40°C and I = 0.25 M.

The enthalpy term consists of free energy (∆G) and entropy (T∆S; where T is the

absolute temperature and ∆S is the change of entropy).

H G T S∆ = ∆ + ∆ (2)

Only the free energy can potentially be converted to work. The entropy and any free

energy not converted to work is converted to heat. The free energy change associated

with ATP hydrolysis, under specified intracellular conditions, can be described as

follows.

[ ][ ]ln

[ ]

iATP ATP

ADP PG G RT

ATP

° ∆ = ∆ +

(3)

5

where R is the Universal gas constant (8.314 J mol-1

K-1

). °

ATP∆G is the standard energy

of ATP hydrolysis, which is the free energy change that occurs under specified

conditions of pH, free [Mg2+], temperature and ionic strength and with the ratio [ADP]

[Pi]/[ATP] = 1. Although there are several published values for °

ATP∆G , they are mostly

around 30 kJ mol-1

and the most commonly used value is 30.5 kJ mol-1

, which was

calculated using the equilibrium constant for CK under conditions approximating those

in muscle cells (Lawson & Veech, 1979). The second term on the right-hand side of

Equation (3) quantifies the difference in chemical potential between the products and

the reactants.

An estimate of ∆GATP can be obtained by substituting typical values into Equation (3).

For example, if [ADP] = 50 µM, [Pi] = 1 mM and [ATP] = 8 mM (Kammermeier et al.,

1982), then the change in free energy is:

∆GATP = 30.5 + (0.0083 × 310) × ln ((50 × 10-6

× 1 × 10-3

) / (8 × 10-3

)) = 61.4 kJ mol-1

In muscle ∆GATP is important because it is the fraction of energy from ATP hydrolysis

that can potentially be converted into work. A summary of published values for cardiac

muscle is shown in Table 2.1. Most studies used NMR to measure [ATP] and [Pi] in

isolated, perfused hearts. Determining [ADP] is difficult because its levels are usually

below detection threshold of NMR and it must be estimated from the CK equilibrium

constant. There is considerable variation in published values of the latter and its value is

particularly sensitive to pH (Golding et al., 1995). Even with chemical analysis of

muscle extracts, [ADP] is difficult to estimate as the relative amounts of bound and free

ADP (both of which are measured but only the free affects ∆GATP) are uncertain.

Reported values of ∆GATP range between 54 and 70 kJ mol-1

and the average of all

studies is ∼59 kJ mol-1

(see Table 2.1). It is notable that in contrast to the suggestion of

Dobson (Dobson & Headrick, 1995; Dobson & Himmelreich, 2002; Dobson, 2003)

there does not appear to be any systematic variation with animal size. Furthermore,

there is no clear difference between values from chemical analysis and NMR.

6

Species Technique ∆GATP References

(kJ mol-1

)

Rat Biochemical analysis ∼57a Hassinen & Hiltunen (1975)

Rat Biochemical analysis ∼53b Nishiki et al. (1978)

Rat HPLC 60.5 Kammermeier et al. (1982)

Hamster Biochemical analysis 61.2 Sievers et al. (1983)

Rat Biochemical analysis 55.2 Fiolet et al. (1984)

Ferret NMR ∼60c Allen et al. (1985)

Guinea pig Biochemical analysis ∼62 Zweier & Jacobus (1987)

Rat HPLC 56.6 Griese et al. (1988)

Rat Biochemical analysis ∼55 Masuda et al. (1990)

Rat Biochemical analysis 57.3d Siegmund et al. (1991)

Rat Biochemical analysis 58.2d Fiolet et al. (1991)

Rat NMR ∼58e Barbour et al. (1991)

Rat MRS 60.9 Figueredo et al. (1992)

Rat Biochemical analysis 58.8d Koop & Piper (1992)

Rat NMR 63.9f

Headrick et al. (1994)

Rat HPLC 59.9 Headrick et al. (1994)

Rat NMR 64.7g Dobson & Headrick (1995)

Rabbit NMR 63.2g Dobson & Headrick (1995)

Dog Estimated 61.9g Dobson & Headrick (1995)

Human Estimated 60.2g Dobson & Headrick (1995)

Rat Biochemical analysis ∼62 Headrick (1996)

Guinea pig NMR 61.8 Kelm et al. (1997)

Rat NMR ∼55h Balschi et al. (1997)

Mouse NMR ∼54 Saupe et al. (1998)

Mouse NMR 59.7 Spindler et al. (1998)

Rat NMR ∼59 Saupe et al. (1999)

Mouse NMR & Biochemical 54.4 Saupe et al. (2000)

Mouse MRI/MRS ∼60i Chacko et al. (2000)

Human MRI/MRS ∼59i Chacko et al. (2000)

Pig Biochemical analysis 57.8j Heusch et al. (2000)

Pig Biochemical analysis 58.6j Schulz et al. (2001)

Dog MR 56.3k Bottomley & Weiss (2001)

Mouse NMR 69.9l Dobson & Himmelreich (2002)

Rat NMR 67.5l Dobson & Himmelreich (2002)

Table 2.1. Reported values of free energy of ATP hydrolysis.

7

Guinea pig NMR 66.5l Dobson & Himmelreich (2002)

Mouse NMR 58.5 Spindler et al. (2002)

Mouse NMR & Biochemical 60.0 Weiss et al. (2002)

Mouse NMR & Biochemical 54.3 Javadpour et al. (2003)

Human MRS 59.7m Weiss et al. (2005)

Mouse NMR & Biochemical ∼59n Day et al. (2006)

a Average from reported ∆GATP for beating (55.6 kJ mol

-1) and arrested (57.7 kJ mol

-1)

heart.

b Average from five experimental conditions (different afterloads, ionotropic

stimulation and arrested heart).

c Average from reported values from measurements allowing glycolysis (61.5 kJmol

-1)

and where glycolysis had been prevented (58.9 kJ mol-1

).

d Ventricular myocytes.

e Average value from measurements made at two different Mg

2+ concentrations (56.7 kJ

mol-1

at [Mg2+

]o = 1.2 mM and 59.3 kJ mol-1

at [Mg2+

]o = 4.8 mM).

f Measurements made in situ. [Pi] was below NMR detection but estimated to be 0.83

mM.

g The phosphorylation ratio was determined in hearts under resting conditions for the

rat, rabbit and dog under anesthesia and in resting non-anesthetized healthy human

subjects. Cytosolic [ATP] for dog myocardium and [total creatine] was used from

other studies. Myocardial pH and [Pi] was not measured in the human heart because of

the low signal/noise ratio of Pi in the NMR spectra; resting human skeletal muscle

values of pH 7.2 and free [Mg2+

] and [Pi] of dog heart were used.

h Average ∆GATP from three different work states: (1) heart rate, 300 beats per minute

(bpm) (55.7 kJ mol-1

), (2) 450 bpm (54.5 kJ mol-1

), (3) 450 bpm and 80 µg L-1

dobutamine (53.2 kJ mol-1

).

i In mice: based on literature values of [total creatine] = 30 mM, [ATP] = 9.6 mM, pHi

= 7.2. [Pi] estimated at ≤2 mM, based on the inorganic phosphate peak relative to that

of ATP, when Pi was unambiguously identified. In humans: based on literature values

of [total creatine] = 43.3 mM, [ATP] = 12.08 mM, pHi = 7.2. [Pi] estimated at ≤2 mM.

The PCr/ATP ratio in the mouse heart measured in vivo, the human ratio determined

from previous measurements by the authors and other sources.

j Chemical analysis using biopsy samples.

k Unable to reliably quantify [Pi], referred to other sources.

l Concentrations of ATP, PCr and Pi, pH and [Mg

2+] determined from in situ

31P-NMR

spectra. Total creatine (PCr + Cr) measured enzymatically on freeze-clamped tissue

and intracellular creatine concentration calculated from subtracting the NMR derived

PCr from the total creatine concentration.

m Unable to reliably quantify [Pi], assumed Pi of ∼1 µmol g

-1.

n Offered no explanation on how the changes in free energy were calculated.

8

2.1.1.2 Creatine kinase reaction

The ADP formed in Equation (1) is rapidly regenerated into ATP at the expense of

phosphocreatine (PCr), a reaction catalysed by CK.

ADP PCr ATP Cr+ → + ∆H = 12 kJ mol-1

(Teague & Dobson, 1992) (4)

where Cr is creatine. When pH = 7.3, [Mg2+] = 0.4 mM, T = 38°C and I = 0.25 M

(conditions approximating those in muscle cells) the equilibrium constant for this

reaction is

' [ ] [ ]62

[ ] [ ]CK

ATP CrK

PCr ADP= = (Golding et al., 1995) (5)

The large equilibrium constant indicates that this reaction maintains ATP at a relatively

constant level in the cell. The net chemical reaction of Equations (1) and (4) is the

breakdown of PCr.

iPCr Cr P→ + (6)

where ∆H ≈ 34 kJ mol-1

when pH = 7, [Mg2+

] = 0.1 mM (see Figure 2, Woledge &

Reilly, 1988).

2.1.2 Recovery biochemical reactions

The initial biochemical processes occurring in muscle contraction are reversed by

oxidative phosphorylation. Creatine and Pi diffuse or are shuttled to the mitochondria

where ATP rephosphorylates free Cr to PCr, a reaction catalysed by the mitochondrial

isoenzyme of CK (Fig. 2.1).

Cr ATP PCr ADP+ → + (7)

The PCr can then diffuse back to the sites of ATP consumption and can be used again

by the ATPases that fuel ion pumps and cross-bridge cycling. ATP is formed during

oxidative phosphorylation by the breakdown of metabolic substrates such as

carbohydrates and fats.

2 2 2iSubstrate ADP P ATP CO H O+ + + Ο → + + (8)

The net recovery chemical reaction is PCr resynthesis and substrate utilization.

2 2 2iSubstrate P Cr CO H O PCr+ Ο + + → + + (9)

9

By combining net initial and net recovery reactions, Equations (6) and (9), the net

overall reaction of muscle contraction is thus the oxidation of substrates.

2 2 2Substrate CO H O+ Ο → + (10)

The heart normally utilises both carbohydrates and fats as metabolic substrates. The

molar enthalpy change for substrate oxidation can be used to convert enthalpy output to

equivalent O2 consumption. The molar enthalpy change for glucose oxidation is 2820 kJ

mol-1

(Crabtree & Nicholson, 1988). Breakdown of 1mole of glucose requires six moles

of O2, so the energetic equivalent of the O2 consumed is 2820/6 = 470 kJ (mol O2)-1

.

This can be converted into units of mJ µL-1

with the Ideal Gas Law.

PV nRT= (11)

where P is pressure (atm), V is volume (L), n is quantity of gas (mol), R is gas constant

(0.0821 L atm mol-1

K-1

) and T is temperature (K). By rearranging the formula, the

molar gas volume at a given temperature, for instance 27°C as used in most of the

experiments in the current project, can be calculated:

-1nRT 1 mol × 0.0821 L atm mol × 300 KV = = = 24.6 L

P 1 atm

Thus, the energetic equivalent of the O2 consumed when temperature is 27°C, and

glucose is the substrate, is 470 kJ mol-1

/24.6 L mol-1

= 19.1 mJ µL-1

.

Interestingly, the breakdown of lipids gives a similar energy yield per mol of O2

consumed. The oxidation of palmitate (∆Hpalmitate = 9790 kJ mol-1

), the most abundant

fatty acid in the body, requires 23 moles of O2, so that the energy yield is 9790/23 = 426

kJ (mol O2)-1

. This corresponds to 426 kJ mol-1

/24.6 mol L-1

= 17.3 mJ µL-1

.

10

2.2 Papillary muscles as a model of ventricular muscle energetics

The structure of the musculature of the heart is complex (for a review, see Stevens &

Hunter, 2003). Ventricular pressure development arises from the actions of layers of

myocytes (i.e. cardiac muscle cells) with different fibre alignments relative to the long

axis of the heart. The loading experienced by myocytes is likely to differ among regions

of the heart with the consequence that it is difficult to precisely measure the work and

the energy used to perform that work by any specific section of the cardiac muscle. This

Fig. 2.1. Initial and recovery reactions.

Two groups of biochemical reactions take place in muscle contraction, initial

and recovery reactions. The initial reactions shown in the upper half of the

diagram involve consumption of high-energy phosphates whereas recovery

reactions shown in the lower half of the diagram involve the regeneration of

high-energy phosphates. In the presence of adequate supplies of metabolic

substrate and O2, recovery reactions are primarily those of mitochondrial

oxidative phosphorylation.

11

can be overcome by using a small section of ventricular tissue where the myocytes are

aligned from end-to-end, such as the papillary muscle.

The papillary muscle is of cylindrical shape with its fibres aligned relatively parallel to

the long axis of the muscle and has tissue at either end that can be tied or clipped for

attaching to experimental apparatus. Papillary muscles are discrete bundles of fibres

projecting from the wall of the ventricle to the mitral or bicuspid valve in the left

ventricle and the tricuspid valve in the right ventricle. Their function is to prevent the

valves protruding into the atrium during ventricular contraction. Papillary muscles

(from cat, rabbit, ferret, guinea pig and rat), have been used in a number of studies

(Chapman, 1972; Loiselle & Gibbs, 1979; Allen et al., 1989) and are a good model of

ventricular muscle function (Rayhill et al., 1994; Gibbs & Barclay, 1998). In fact, in

1998 Professor Gibbs declared that that there is little evidence suggesting that the

mechanical or energetic results obtained with papillary muscles are different from the

results obtained from the in vivo, whole heart situation (Gibbs & Barclay, 1998).

Examples of indices of cardiac function that have direct correlates with muscle function

include the pressure−volume index and isovolumic pressure development. The

pressure−volume diagram of the heart has a two-dimensional analogue in the

force−length diagram for isolated papillary muscle (Hisano & Cooper, 1987; Mast &

Elzinga, 1990; Baxi et al., 2000; Mellors & Barclay, 2001). The area enclosed by a

pressure−volume loop for a heart and a force−length loop for a papillary muscle is

closely related to the energy (or O2) consumption (Hisano & Cooper, 1987; Mast &

Elzinga, 1990; Suga, 1990, 2003a, b). The papillary muscle equivalent of isovolumic

pressure development is isometric force development. In addition, isolated papillary

muscle preparations can provide information about myocyte force generation, the

relationship between energy supply and demand and kinetics of mitochondrial

metabolism (Barclay et al., 2003).

2.3 Isolated mouse papillary model

With the development of genetically modified mice, there is a need for a cardiac muscle

model for determining the physiological and functional consequences of the various

genetic manipulations. A few studies have used the mouse papillary muscle to

investigate consequences of heart-focussed genetic changes (He et al., 1997; Meyer et

al., 1999; Bluhm et al., 2000; Pyle et al., 2002) but all of them have used an isometric

12

contraction protocol and none has attempted a more realistic contraction protocol or

performed energetic measurements. The suitability of isometric contractions as a model

of cardiac function has been questioned (Sonnenblick, 1962; Mellors & Barclay, 2001;

Redel et al., 2002) as no mechanical work is done and the protocol thus bears little

resemblance to the in situ situation of papillary or ventricular muscles. Therefore, a

protocol designed to closely simulate the reported changes in muscle shortening

(Semafuko & Bowie, 1975) will be used in most sections of this study (Mellors &

Barclay, 2001).

13

CChhaapptteerr 33:: MMeetthhooddss

The general experimental approach used in the experiments described in this Thesis is

measurement of the changes in muscle enthalpy content that accompany contraction.

The enthalpy output comes from the biochemical reactions that underlie contraction (i.e.

the hydrolysis and regeneration of ATP). Enthalpy changes appear as both heat and

mechanical work. The thermal changes can be measured using the myothermic

technique, an elegant and ingenious system (Warshaw, 2005) that allows partitioning of

energy used for processes that require and supply ATP and that has excellent temporal

and chemical resolution (Woledge, 1998). In this chapter, methods common to most of

the experiments are described. Where particular methods were used for a section of the

study, these are described in the relevant section.

3.1 Papillary muscle preparation and dissection

Papillary muscles were dissected from the left ventricle of hearts from six to twelve

week old male Swiss mice or Wistar rats. The animals were rendered unconscious by

inhalation of 80% CO2–20% O2 gas mixture and killed by cervical dislocation.

Euthanasia by short exposure to this gas mixture has previously been shown not to stop

the heart beating (Kohler et al., 1999). All animal-handling procedures were approved

by the Griffith University Animal Ethics Committee. The chest was opened and the

heart rapidly excised and transferred to oxygenated (95% O2–5% CO2) Krebs-Henseleit

14

solution of the following composition (mM): 118 NaCl, 4.75 KCl, 1.18 KH2PO4, 1.18

MgSO4, 24.8 NaHCO3, 2.5/1.5 (mice and rats, respectively) CaCl2, 10 glucose. The

value for ionized [Ca2+

] measured in the blood of mice is ∼1.4 mM (Sutherland et al.,

2003), a concentration that was used in the Krebs buffer in preliminary experiments

using the mouse papillary muscle. However, in those experiments it was found difficult

to reliably obtain preparations that contracted consistently and that continued to contract

for the duration of an experiment. When 2.5 mM Ca2+

was used muscles contracted

vigorously and continued to do so for at least 90 min after dissection.

The heart was gently massaged in the oxygenated saline buffer, with the apex removed

to facilitate removal of blood, and then placed in Krebs solution containing 30 mM 2,3–

butanedione monoxime (BDM) (Sigma, St. Louis, MO, USA) to prevent the myocytes

from contracting. BDM was included in the solution only during dissection to avoid

contracture upon freeing the muscle from its in situ length constraints; it does not alter

energetic properties once washed out (Kiriazis & Gibbs, 1995). The heart remained

immersed in oxygenated BDM-Krebs-Henseleit solution throughout the dissection

period. The right ventricular free wall was removed and the inter-ventricular septum

was bisected and pinned open, exposing the papillary muscles in the left ventricle.

Papillary muscles were dissected free from the wall of the heart and T-shaped platinum

clips (Ford et al., 1977; Donald et al., 1980) were attached to the tendon at one end of

the muscle and to a piece of ventricular wall at the other.

Muscles were stimulated using rectangular electric pulses (amplitude, 4–6 V; duration,

1–2 ms) that were passed along thin platinum wires, which were wound around the rods

up to the hook so that the platinum clips were in direct contact with the platinum wire.

The thin diameter (15 or 25 µm) prevented the wires affecting movement of the rod

connected to the motor or recording of force output.

3.2 Overview of experimental apparatus

In the experimental chamber used in most experiments (for an exception, see Chapter

5), muscles were mounted between a semi-conductor force transducer (AE801,

SensorOne, CA, USA) and a servo-controlled motor (322B, Aurora Scientific Inc.,

Ontario, Canada) via fine stainless steel wires that provided a low compliance linkage

between the preparation and the recording equipment. The muscle lay along the active

15

thermocouples of a thin-film, antimony–bismuth thermopile (Mulieri et al., 1977;

Barclay et al., 1995).

3.2.1 Measurement of muscle force production

The force produced was measured using a silicon strain gauge force transducer. A

stainless steel pin was glued on to the transducer using a fast-setting adhesive (Prism

406, Loctite, Welwyn Garden City, UK) and the stainless steel wire connecting the

muscle to the transducer was attached to the pin by a drop of wax.

A set of weights, the masses of which were determined using an analytical balance

(Scout II, Ohaus Corporation, NJ, USA), was used to calibrate the force transducer. The

masses of the weights were chosen to cover the range of expected muscle force outputs.

The force transducer was positioned vertically and the weights were hung from the pin,

thus exerting force in the same direction as the force applied by a muscle preparation in

an experiment.

The relationship between force output and applied load was linear and the data were

fitted with a straight line (Fig. 3.1). The calibration factor, which corresponds to the

inverse slope of the fitted line, was 782.9 mN V-1

for the force transducer used in most

of the experiments described in this Thesis. The force transducer output was amplified

by a factor of 50, 100 or 250.

0 10 20 30 40Force (mN)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Fo

rce

tra

nsd

uce

r o

utp

ut

(V)

Fig. 3.1. Calibration of force transducer. The relationship between transducer output and applied force for the force

transducer used in this project. The output has been adjusted to account for the

amplifier gain so the values shown are the true transducer output. The straight

line was fitted using linear regression; its slope is 1.28 mV mN-1

.

16

3.2.2 Measurement of muscle length changes

The remaining end of the muscle was attached to an aluminium lever via a stainless

steel rod. The lever was attached to the servo-controlled motor. The motor system

allowed muscle length to be controlled and measured simultaneously. Lever position,

and thus muscle length, was controlled using the output of a 12-bit digital-to-analogue

converter (DAS-1802AO, Keithley Instruments, Cleveland, OH, USA). The patterns of

muscle length changes were generated using software.

3.2.3 Principles of measuring muscle heat output using a thermopile

Muscle heat production was determined by measuring the change in muscle temperature

using a thermopile. A thermopile consists of a number of thermocouples connected in

series. The thermocouples are arranged so that every second thermocouple lies along the

centre of the thermopile (the “active” thermocouples) and the alternate thermocouples

(the reference thermocouples) are positioned on the edges of the thermopile. The edges

were clamped between the jaws of the aluminium frame that supported the thermopile

so that the reference thermocouples were maintained at close to the temperature of the

frame. Frame temperature was kept constant by circulating water through channels in

the frame. The muscle preparation was positioned along the active thermocouples, thus

the muscle regulated the temperature of the active region. Upon stimulation, the muscle

contracted and released heat, increasing the temperature of the active thermocouples

relative to the reference thermocouples. The temperature difference between the active

and reference junctions generated a signal with an amplitude proportional to the change

in muscle temperature.

3.2.3.1 Calibration of thermopile

To convert the thermopile output into temperature units, the Seebeck coefficient of the

thermopile must be known. This can be determined using the Peltier effect: when a

current is passed through a thermopile, heat is absorbed at one set of junctions and

evolved at the other. The Seebeck coefficient can be calculated from the relationship

between the heat capacity of the thermopile and the rate at which its output changes

upon starting or stopping the current (Kretzschmar & Wilkie, 1972; Woledge et al.,

1985).

In practice, a series of small silver blocks (12−60 mg; heat capacity 0.234 mJ K-1

mg-1

),

with a drop of glycerol to aid thermal transfer with the thermopile (89% glycerol, 11%

17

water; heat capacity 2.59 mJ K-1

mg-1

), were placed over the active junctions of the

thermopile. A current was passed through the thermopile to heat the active junctions and

the silver block. Once a steady temperature had been reached, the current was turned off

and the time-course of the cool-off measured (Equation (12) Fig. 3.2). The relationship

between the initial rate of cooling and the added heat capacity is described as follows.

2 2

0

( )thermopile Ag glycerol

dV ITn

dt C C

α

+

=+

(p.188, Woledge et al., 1985) (12)

where dV0/dt is the initial rate of change of the thermopile output (V s-1

) , I is the

heating current (A), T is the absolute temperature (K), n is the number of active

thermocouples, α is the Seebeck coefficient (V K-1

couple-1

) and CAg+glycerol (J K-1

) is the

sum of the heat capacities of the silver block and the glycerol and Cthermopile the heat

capacity of the region of the thermopile beneath the silver block.

If the current passed through the thermopile is small, heating by the Peltier effect

(proportional to the current) is much greater than that by the Joule effect (proportional

to the square of the current). For example, the heating current used was 123 µA, the

temperature 300 K, and typically the resistance of 16 thermocouples was ∼180 Ω, which

gives a rate of heating due to the Peltier effect (= ITnα) of 5.0 × 10-5

W and due to the

Joule effect (= I2R) of 2.8 × 10

-6 W. That is, heating due to the Peltier effect was ∼18

times greater than that due to the Joule effect. This would result in the Seebeck

coefficient being overestimated by ∼2%. No correction was made for this effect.

Knowing the cool-off rate constant, the initial thermopile output and the added heat

capacity of the silver block and the glycerol, the Seebeck coefficient was determined by

fitting Equation (12) using a non-linear least squares regression (Fig. 3.3). This was

performed using Mathcad’s (version 11.0, Mathsoft Inc., USA) “genfit” function.

Values for the Seebeck coefficient for each thermopile used in this Thesis are presented

in Table 3.1.

Thermopile 1 2 3

Active region (mm) 4 5 2

n (couple) 16 20 8

α (µV K-1

couple-1

) 65.0 75.6 78.3

Output (mV °C-1

) 1.30 1.21 0.63

Table 3.1. Thermopile characteristics.

18

Fig. 3.2. Time-course of cooling of

silver blocks. An example of the time-course with

which the silver blocks cooled

following a period of Peltier heating.

Records from two silver blocks

weighing 12 and 60 mg are shown. The

bigger silver block took longer to cool

than the smaller block. The time-course

of cooling could be described by a

single exponential curve. The black

lines show the measured thermopile

output and the white lines show fitted

exponential curves. Data from the first

1 s were excluded from the fitting

because that section was largely due to

the change in temperature at the

reference junctions. This was rapidly

reversed due to the proximity of the

frame.

0 10 20 30 40 50 60

Time (s)

0.0

0.2

0.4

0.6

0.8

1.0

Th

erm

op

ile o

utp

ut

(V)

12 mg 60 mg

Fig. 3.3. Calibration curve for the

Seebeck coefficient. Data from one thermopile showing the

relationship between the initial rate of

cooling of the thermopile and the added

heat capacity. Initial rate of cooling

was calculated as the product of the

initial value of the thermopile output

and the rate constant for the heat loss.

Both these values were determined

from the exponential curves fitted to

the cool-off data (Fig. 3.2). The dashed

line is the line of best fit described by

Equation (12) and fitted through the

data using non-linear regression (pp

683-688, Press, 1992). The Seebeck

coefficient for this example was 78.3

µV K-1

couple-1

.

0.000 0.005 0.010 0.015 0.020

Added heat capacity (J K-1)

0.0

0.5

1.0

1.5

2.0

Initia

l co

olin

g r

ate

(V

s-1

)

10-5

19

3.2.3.2 Conversion of thermopile signals into heat production

To calculate heat production from the measured change in muscle temperature it is

necessary to (1) convert the thermopile output into temperature units, (2) correct the

records for heat lost from the active thermocouples to the frame while recording and (3)

multiply the corrected temperature change by the heat capacity with which the heat

produced by the muscle is shared.

The output of the thermopile (∆V) can be converted into units of temperature change

(∆T) by dividing the output by the product of the number of thermocouples and the

Seebeck coefficient:

VT

nα

∆∆ = (13)

Due to the temperature difference between the muscle and the frame, heat is constantly

lost from a preparation. The corrected temperature at time t (Tc(t)) is given by the

integral of the measured temperature (Tm) with respect to time multiplied by the rate of

heat loss (k) (Equation (14), Fig. 3.4).

0( ) t

c mT t k T dt= ∫ (14)

The cooling after Peltier heating was used not only to measure the rate of heat loss but

also to calculate muscle heat capacity (pp 187-188, Woledge et al., 1985).

0 /muscle

ITnC

dT dt

α= (15)

where dT0/dt is the initial rate of cooling (in temperature units) after Peltier heating.

Note that from Equation (13) dT0/dt = (dV0/dt)/nα. Finally, by rearranging Equations

(13) and (15) an expression for calculating the heat (∆Q) produced by the muscle is

derived.

0 0/ /muscle

V ITn ITnQ TC V

n dT dt dV dt

α α

α

∆∆ = ∆ = ⋅ = ∆ ⋅ (16)

20

3.2.4 Determination of stimulus heat

When a muscle is stimulated, the current running through the muscle and any adhering

solution produces heat by the Joule effect. The amount of heat produced by the stimulus

current was measured by stimulating an artificial muscle created from agar gel made

with Krebs solution and with the same dimensions as a muscle. The stimulus heat was

∼0.1 µJ pulse-1

using the artificial muscle (amplitude 6 V, duration 2 ms). This typically

corresponded to less than ~2% of the net heat produced by a muscle in response to a

stimulus pulse. Another way of illustrating the magnitude of the stimulus heat relative to

muscle heat production is shown in Fig. 3.5. This muscle failed to respond to the first

four stimulus pulses (for unknown reasons) but then contracted upon delivery of the

fifth pulse. The first four pulses produced very small increments in the cumulative heat

record. When the muscle contracted there was a large, rapid increase in heat output. In

that example the heat per 1 ms stimulus pulse was ∼0.08 mJ g-1

twitch-1

which was

∼1.5% of the initial heat produced in the twitch. Given that the net heat is approximately

twice the initial heat (see Section 3.6), this estimate of stimulus heat corresponds to

0 5 10 15 20

Time (s)

0

50

100

150

Mu

scle

te

mp

era

ture

(m

°C)

Measured temperature

Corrected temperature

0 40 80

Time (s)

0

50

100

150

Te

mp

era

ture

(m

°C)

Fig. 3.4. Heat loss correction. Temperature signals were corrected for heat lost from the preparation due to the

temperature difference between the muscle and the frame. The recording shows

the measured (dotted line) and corrected (solid line) temperature from a

contracting mouse papillary muscle stimulated at a contraction frequency of 2

Hz lasting 20 s. The inset shows the complete time course of change in muscle

temperature. The vertical dashed line indicates the time at which contractions

ended and the recorded muscle temperature was then ∼10 m°C. Heat was

produced at a rate greater than the resting rate for ∼60 s after the end of the

contraction series, indicating the ongoing recovery metabolism. Muscle mass:

1.57 mg; length: 3.8 mm.

21

∼0.8% of the likely net heat. If this value were doubled to allow comparison with 2 ms

pulses, as used for the artificial muscle, the result is consistent with the estimated

stimulus heat measured using the agar “muscles”.

3.3 Data recording

The thermopile output was low pass filtered (cut-off frequency, 100 Hz) and amplified

using a series arrangement of two low-noise amplifiers (15C-3A, Ancom Instruments,

Cheltenham, UK; SR560, Stanford Research, CA, USA). The output of the thermopile

was sampled at 220 Hz. Software developed using TestPoint (Capital Equipment

Corporation, Middleborough, MA, USA) was used to control data recording and to

analyse the data.

All signals were recorded using a 12-bit A/D converter with an input range of ±10 V

(DAS-1802AO, Keithley Instruments, Cleveland, OH, USA). The resolution of the A/D

converter (i.e. the voltage change giving a change of 1 digit) is 20/212

= 20/4096 = 4.88

mV. The force, length and temperature signals were amplified prior to recording to

reduce the quantization noise (4.88 mV/signal amplitude) to an acceptable level; that is,

to <1% of the signal amplitude or to less than the amplitude of noise from other sources,

whichever was larger. Estimates of the relative amplitude of quantization noise and

noise from other sources are provided in Table 3.2.

Fig. 3.5. Stimulus heat using a mouse

papillary muscle. Records from a mouse papillary muscle

that was stimulated five times at a

contraction frequency of 2 Hz (amplitude

6 V, duration 1 ms). The muscle did not

respond to the first four stimuli but did

respond to the fifth, producing an active

force of ∼18 mN mm-2

. The amount of

stimulus heat was estimated by measuring

the cumulative heat produced from the

first four stimulus pulses (dashed line).

Muscle mass; 1.50 mg, muscle length; 3.7

mm, temperature; 37°C. 0.0 0.5 1.0 1.5 2.0 2.5

Time (s)

0

5

10

15

20

25

Fo

rce

ou

tpu

t (m

N m

m-2

)0

2

4

6

8

10

12

He

at o

utp

ut (m

J g

-1)

Stimulus

Heat

Force

22

Force Length Temperature

mN 5 mm 0.3 m°C 5

V 0.6 V 0.9 V∗ 0.5

Nq (%) 0.8 Nq (%) 0.5 Nq (%) 1

N0 (%) <1 N0 (%) <0.5 N0 (%) 2

3.4 Partitioning of initial and recovery metabolism

The heat and the work liberated from a contracting muscle arise from enthalpy changes

associated with the biochemical reactions that underlie contraction. Enthalpy output

associated with contractile activity can be separated into initial enthalpy output and

recovery enthalpy output because of the difference in the time courses with which they

are produced during the transition from rest to steady activity. Initial enthalpy is

produced simultaneously with contractile activity and thus its production commences in

synchrony with the activity (see Fig. 3.6A & Fig. 3.6B). In contrast, recovery enthalpy

output increases exponentially from its resting value towards its steady state value with

a time constant, in the rat and mouse papillary muscle at 27°C, of about 10 and 12 s,

respectively. In this study, the net enthalpy output produced during and after a short set

of contractions (lasting 20 s) was measured (Fig. 3.6C & Fig. 3.6D) and the initial

enthalpy output was determined from the enthalpy produced during the first three

contraction cycles, before the rate of recovery enthalpy production became significant

(Fig. 3.6B).

The relative contributions of initial and recovery processes to enthalpy output in the first

three cycles of a contraction protocol were estimated as follows. An exponential

increase in the rate of recovery heat production ( RQg

) can be described by:

-

,( ) 1-t

R R SSQ t Q eτ

=

g g

(17)

where R,SSQg

is the steady-state recovery heat rate.

Table 3.2. Estimates of quantization noise and noise from other sources.

Nq = relative amplitude of quantization error.

N0 = typical relative amplitude of electrical noise. ∗ Typical gain 100 000.

23

0

5

10

15

20

25

30

Fo

rce

ou

tpu

t (m

N m

m-2

)

1 s

A

0

5

10

15

En

tha

lpy o

utp

ut (m

J g

-1)

IH1

IH2

IH3

1 s

B

0 20 40 60 80

Time (s)

0

5

10

15

20

25

30

Fo

rce

ou

tpu

t (m

N m

m-2

)

C

0 20 40 60 80

Time (s)

Net

enthalpy

0

50

100

150

200

En

tha

lpy o

utp

ut (m

J g

-1)

D

Fig. 3.6. Example of the time course of force production and enthalpy

output. An example of force production and enthalpy output of a mouse papillary

muscle. The muscle performed 20 isometric contractions at a frequency of 1 Hz.

A. Force output for the first three twitches. B. Cumulative enthalpy production

during the first three twitches. Because the muscle was not shortening, there was

no work production so enthalpy output was equal to the heat output. The solid

line indicates the measured heat output and the dashed line indicates the

estimated time course of recovery heat production (from Equation (18)). The

quantities indicated by the arrows labelled IH1, IH2 and IH3 represent the

cumulative initial heat production up to the end of each of the first three

contraction cycles. C. The time course of force output for the whole contraction

series. D. The cumulative enthalpy output during and after the 20 contractions.

The vertical dashed line indicates the time at which contractions ended. The rate

of heat production remained above the resting heat rate for ~60 s, indicating the

ongoing recovery metabolism. Muscle mass 1.72 mg; length 3.4 mm.

24

The cumulative recovery enthalpy production associated with contractile activity (QR) is

given by the integral of Equation (17) and assuming that RQ (0)g

= 0:

( )-

, ,( ) -t

R R SS R SSQ t Q t e Qττ τ

= + ⋅ ⋅

g g

(18)

Recovery heat production starts quite quickly in cardiac muscle so a detailed assessment

of the likely contribution of recovery heat to initial heat measurements was made. This

is described in Section 4.2.1 for rat papillary muscles and 6.2.2 for mouse papillary

muscle.

3.4.1 Partitioning initial metabolism into force-dependent and force-independent components

Initial enthalpy output was partitioned using an isometric contraction protocol (2 Hz)

into a force-dependent component (i.e. enthalpy output associated with cross-bridge

activity) and a force-independent, or activation, component (i.e. enthalpy output

associated primarily with ion pumping) by selectively inhibiting cross-bridge cycling

with BDM and/or exposure to hyperosmotic solution (Alpert et al., 1989). The

relationship between initial enthalpy output and force-time integral (FTI) was

determined by making incremental reductions in FTI by step-wise increases in BDM

concentration from 2 to 10 mM and using 5 mM BDM in combination with 150 mM

sucrose. The magnitude of force-independent enthalpy output was determined by

extrapolating the enthalpy output−FTI relationship to zero FTI.

3.5 Calculations of efficiency

3.5.1 Mechanical efficiency

In general terms, efficiency is the ratio of the work performed to the energy expended to

do that work. Mechanical efficiency is defined as the ratio of the work output to the

enthalpy change accompanying the work performance. The precise definition depends

on whether just the enthalpy from initial reactions is used or whether that from recovery

reactions is also included.

3.5.1.1 Initial and net mechanical efficiency

The initial mechanical efficiency (εI) is the ratio of the work output to the initial

enthalpy output (∆HI). In this project ∆HI was defined to be the enthalpy produced

25

between the start of a series of contractions and the end of the third contraction cycle

(for details see Fig. 3.6).

I

I

W

Hε =

∆ (19)

In isometric contractions, ∆HI is equivalent to the heat produced in the three cycles and

in shortening contractions it is the sum of the heat and work produced. Contraction

frequency was typically 2 Hz, so ∆HI was measured over the first 1.5 s of activity.

Net mechanical efficiency (εNet) was defined as the ratio of the work output produced in

a series of contractions to the total enthalpy produced, in excess of that due to resting

metabolism, during and after the contractions series .

Net

Net

W

Hε =

∆ (20)

3.5.2 Mitochondrial efficiency

An index of mitochondrial function is the efficiency (ηR) with which the mitochondria

transfer free energy from metabolic substrate to ATP (Barclay et al., 2003; Smith et al.,

2005). This can be calculated from the ratio of εNet and εI.

Net ATPR

I PCr

G

H

εη

ε

∆= ⋅

∆ (21)

3.6 Ratio of recovery heat output to initial heat output

The ratio of the amount of recovery heat relative to the amount of initial enthalpy output

(R/I) quantifies the coupling between energy supply and energy demand and its value

reflects the cost of oxidative recovery metabolism relative to the amount of ATP used

during contraction. R/I can be derived from the initial and net mechanical efficiency (for

details, see Barclay & Weber, 2004).

1I

Net

R

I

ε

ε= − (22)

The initial and net values are not strictly comparable as they come from different sets of

contractions. However, Mast & Elzinga (1988) showed that initial and recovery heat

ratios are the same for whole series of contractions as for sub-sets. Moreover, the values

obtained for rat and mouse papillary muscles are similar to those reported previously in

26

isometric contractions for rabbit papillary muscles (1.14 and 1.18; Mast & Elzinga,

1988; 1.10; Mast et al., 1990) as well as for rat papillary muscles (1.16, Barclay et al.,

2003) using a shortening protocol. In the studies by Mast et al. (1990) and Barclay et al.

(2003) a mathematical approach was used to partition the initial and recovery

components.

3.7 Oxygenation of papillary muscles

The isolated muscle preparation lacks normal circulation and thus the sole source of O2

is diffusion from the outer surface of the muscle. In a thick piece of muscle or a muscle

with a high metabolic rate, O2 diffusion may be insufficient to match the muscle’s

metabolic needs, resulting in formation of an anoxic core. In this Thesis, the adequacy

of oxygenation of the papillary muscles was assessed using a mathematical model of

diffusion of O2 into muscles as described by A.V. Hill (1928; 1965). In this analysis it

was assumed that the papillary muscle was cylindrical, of uniform radius and that

negligible O2 diffusion occurs through the ends of the cylinder.

The adequacy of diffusive O2 supply to papillary muscles during protocols in which

muscles performed a short series of contractions was assessed by numerical solution of

Equation (23) describing diffusion of O2 into muscles of cylindrical geometry with a

rate of O2 consumption that varied with time (p. 229, Hill, 1965) as described in detail

by Barclay (2005).

2 2 2

2

2

1( )

O O OP P PK A t

t r r r

δ δ δ

δ δ δ

= + ⋅ −

(Barclay, 2005) (23)

where t is time, PO2 is the partial pressure of O2, r is the radial distance from the

muscle’s centre and A(t) is the time-dependent rate of O2 consumption. The equation

was solved using a programme written using Mathcad. The contribution of myoglobin-

facilitated O2 diffusion to O2 supply was not included in the model because myoglobin

contributes little to total O2 flux within isolated papillary muscles when the external PO2

is >0.2 atm (Loiselle, 1987). It was assumed that the rate of mitochondrial O2

consumption was PO2-dependent such that it varied sigmoidally with PO2 at values

below ∼10 mm Hg, as observed experimentally (Wittenberg & Wittenberg, 1985).

27

3.8 Data normalisation

At the end of experiments, the platinum clips were cut off the preparation, the muscle

was lightly blotted and its mass determined using an electronic balance (Cahn 25, Cahn

Instruments, Cerritos, CA, USA). Some of the mouse papillary muscles were kept after

the end of the experiment and later placed to dry in an oven with the temperature set at

80°C. The preparations were re-weighed and the wet-to-dry mass ratio determined. The

mean ratio of wet mass to dry mass was 4.9 ± 0.2 (n = 22). The average cross-sectional

area was calculated by dividing mass by length, and assuming muscle density was 1.06

g cm-3

. Active force was normalised by cross-sectional area. Work and enthalpy output

were normalised by muscle wet mass. All data are presented as the mean ± S.E.M.

29

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oouuttppuutt iinn ppoosstt--iisscchhaaeemmiicc ccaarrddiiaacc mmuussccllee

4.1 Introduction

Exposure of cardiac muscle to ischaemia lasting more than a few minutes results in a

prolonged depression of cardiac contractile function even after blood flow is restored

(for a review, see Bolli & Marban, 1999). Hearts in this state are referred to as

“stunned” and are characterised by reduced cardiac output (Heyndrickx et al., 1975;

Braunwald & Kloner, 1982). The mechanism underlying the impaired contractility is

unclear but must ultimately reflect changes in work generation by myosin cross-bridges

during their cyclic interactions with actin filament. Decreased cardiac muscle work

output can potentially arise from either of two mechanisms (or a combination of both): a

decrease in the number of cross-bridges contributing to muscle work output or a

decrease in the work generated by each cross-bridge. In the first case, the amount of

work produced in each cross-bridge cycle would be the same as that before ischaemia

but a smaller proportion of cross-bridges are attached and generating force at any instant

during contraction so that the total work output is decreased. In the second case, the

number of attached cross-bridges does not alter but the work produced in each cross-

bridge cycle is decreased.

30

The likely effects of these two mechanisms on muscle efficiency potentially provide a

means of distinguishing between the mechanisms. Efficiency is the ratio of the work

produced to the energy used to produce that work. If, as seems likely in most conditions,

each cross-bridge cycle is associated with splitting of one ATP molecule, then the

efficiency of a population of cross-bridges is the ratio of the average cross-bridge work

output to the number of cross-bridge cycles that produced that work. In the first

mechanism described above, the work produced in each cross-bridge cycle would be

unaffected by ischaemia, so efficiency would also be unaltered. For the second

mechanism, however, less work would be performed in each ATP splitting cross-bridge

cycle, so cross-bridge efficiency would be decreased.

The aim of the current study was to identify the mechanism underlying the impaired

work output of post-ischaemic cardiac muscle by comparing the efficiency of cardiac

muscle measured before exposure to ischaemia with that measured following

reperfusion. The experiments were performed using an isolated rat papillary muscle

preparation so that precise measurements could be made of both work output and

energy use. Work output of a papillary muscle can be accurately measured because,

unlike the ventricular wall, the myocytes are aligned parallel to the long axis of the

muscle. Energy use was determined by measuring the energy liberated during

contraction as heat and work. This enthalpy output arises from the enthalpy changes

accompanying the biochemical reactions that underlie contraction and the amount of

enthalpy produced is proportional to the amount of ATP consumed. Another advantage

of an isolated muscle preparation is that it is possible to closely specify the “ischaemic”

conditions. In this study, ischaemia was simulated by removing superfusate from the

muscles and by replacing the O2 supply with N2.

4.2 Methods

The materials and methods used for the experiments reported in this chapter have been

described in Chapter 3. Only those methods specific to these experiments will be

described here. Characteristics of the rat papillary muscles are given in Table 4.1.

31

Number of preparations 15

Wet muscle mass (mg) 3.7 ± 0.2

Muscle length (mm) 6.2 ± 0.3

Cross-sectional area (mm2) 0.57 ± 0.02

Muscle radius (mm) 0.43 ± 0.01

4.2.1 Initial and recovery metabolism

The enthalpy output produced by the contracting muscle was measured in the form of

net enthalpy output (∆HNet) and initial enthalpy output (∆HI). The former was the total,

suprabasal enthalpy output produced in response to a 20 s series of contractions and the

latter was the cumulative enthalpy produced during the first three contraction cycles.

The initial and recovery enthalpy output were separated as described in Section 3.4 and

used to measure force-independent enthalpy output (∆HP). The relative contribution of

recovery heat to the measured initial heat was calculated using Equation (18). The

steady state of recovery heat output ( R,SSQg

in Equation (18)) was estimated on the basis

that the amount of recovery heat required to completely reverse the initial heat was 1.1

× the amount of initial heat (i.e. R/I ratio, Table 4.2) and that in an energetic steady state

this amount of recovery heat would be produced within each contraction cycle (i.e. in

0.5 s when contracting at 2 Hz). The value of the R/I ratio was calculated as described

previously (see Section 3.6, Equation (22)) using data obtained from rat papillary

muscles in the current study.

The initial enthalpy output was ~3.2 mJ g-1

twitch-1

, at a contraction frequency of 2 Hz,

so in one cycle R,SSQg

= 1.1 × 3.2 × 2 cycles s-1

= 7 mW g-1

. Substituting this value and

the time-constant for recovery metabolism (~10 s; see Table 4.3) into Equation (18), the

amounts of recovery heat produced between the start of recording and the ends of the

first, second and third contraction cycles were 0.05, 0.20, and 0.44 mJ g-1

, respectively,

which was 1.6, 3.1 and 4.6% of the total heat produced by the end of those cycles (Fig.

3.6B). The post-ischaemic recovery time constant was ∼6 s but the initial enthalpy

output was smaller and the R/I unaltered so the contribution of recovery heat remained

the same.

The initial enthalpy output was used to partition energy use into force-dependent and

force-independent components (see Section 3.4.1) and to calculate initial efficiency.

Table 4.1. Left ventricular rat papillary muscle characteristics.

32

Recordings of net enthalpy output were used to calculate the overall energetic cost of

contractile activity, the net efficiency and the time course of recovery heat output.

4.2.2 Experimental protocols

After mounting in the experimental chamber, the length of the muscles was adjusted to

that at which twitch force was maximal (Lmax) and they were then allowed to equilibrate

for 60 min. The protocol consisted of pre-ischaemia measurements, followed by either

30 or 60 min of simulated ischaemia and 30 min of simulated reperfusion. Ischaemia

was simulated by withdrawing the bathing solution from around the muscle and by

replacing the 95% O2–5% CO2 gas flow with 95% N2–5% CO2. Gas was supplied to the

muscle chamber via a humidifier that saturated the gas with water vapour before it

reached the muscles. This prevented muscles from dehydrating when the solution was

removed from the chamber (e.g. during simulated ischaemia). At the end of either 30 or

60 min, solution aerated with 95% O2 was returned to the muscle.

Two control experiments were performed. In the first, muscles remained in solution

throughout the protocol and were continuously aerated with 95% O2 whereas in the

second the solution was removed from the muscle but aeration with 95% O2 was

maintained. This enabled the effect of removing the saline from the muscle to be

distinguished from effects due to the withdrawal of O2.

A total of five measurements were made: three to investigate changes in mechanical and

energetic output and two to determine the contribution of force-independent heat to total

enthalpy output. Muscle enthalpy output was measured before ischaemia and after 5 and

30 min of reperfusion using separate series of isometric and more realistic contractions.

The realistic contraction protocol (Mellors & Barclay, 2001), illustrated in Fig. 4.1, was

designed to mimic the in situ pattern of length changes reported for papillary muscles

(Semafuko & Bowie, 1975). The isometric contraction protocol was used for

measurements of the force-independent enthalpy output. These measurements were

made at the end of the equilibration period and 40 min after the end of the ischaemic

phase. Force-independent enthalpy output was calculated by extrapolation of the two-

point, enthalpy output versus force-time integral plot constructed from measurements

made with the muscle in normal Krebs solution and in an hyperosmotic solution of

Krebs with 5 mM BDM and 150 mM sucrose added (Alpert et al., 1989). For all

measurements using both isometric and realistic protocols, muscles performed 40

contractions at a contraction frequency of 2 Hz. During simulated ischaemia muscles

33

remained isometric, were stimulated at 0.5 Hz and both total and passive force outputs

were recorded continuously. Passive force was defined as the force measured in the

absence of contractile activity and total force was the sum of the passive force and the

additional force produced in response to stimulation.

4.2.3 Statistical analysis

Statistical significance of variations in force output and energetic variables with time

were assessed using repeated measures, one-way analysis of variance. A paired t-test

was used to compare measurements of force-independent enthalpy output made before

ischaemia with those made after 40 min of reperfusion. Decisions concerning statistical

significance were made at the 95% level of confidence.

4.2.4 Analysis of diffusive O2 supply

A mathematical model of diffusion of O2 into cylindrical muscles (see Section 3.7) was

used to assess the diffusive O2 supply to the isolated preparations, both to ensure

adequacy of supply during control and reperfusion phases and also to estimate the

degree of anoxia during the ischaemic phase and the time course of changes in muscle

oxygenation at the start and end of that phase. The O2 partial pressure (PO2) at the

muscle surface was measured under conditions matching those used during

Fig. 4.1. Comparison of twitches

recorded using isometric and realistic

contraction protocols. An example of changes in muscle length (A)

and force output (B) during an isometric

(dashed line) and realistic (solid line)

contraction cycle. In the realistic contraction

protocol, muscle length was held constant for

110 ms, then shortened by 10% Lmax in 185 ms;

the velocity was 0.54 (Lmax s-1

). The vertical

dotted lines indicate the time for the start and

end of the shortening phase. Once the muscle

was relaxed, it was returned to its original

length. Muscle mass; 3.64 mg; muscle length

6.7 mm.

-0.8

-0.6

-0.4

-0.2

0.0L

en

gth

(m

m)

0.0 0.1 0.2 0.3 0.4 0.5

Time (s)

0

5

10

15

20

25

Fo

rce

(m

N)

A.

B.

34

experiments. An O2-sensitive microelectrode (OX500, Unisense, Aarhus, Denmark) was

placed above the thermopile, in the location normally occupied by a muscle and the bath

was sealed using microscope slides. When the system was supplied with 95% O2, PO2 in

the muscle chamber was 0.84 atm. When the gas mixture changed to 95% N2, PO2

decreased to ∼0.05 atm.

The analysis took account of resting metabolic rate, variations in mitochondrial activity

at low PO2 and the time course of changes in O2 consumption. The results of the

analysis for the transitions from high PO2 to low PO2, and back again, are shown in Fig.

4.2. The figure shows both the fraction of the muscle cross-section that is oxygenated

(Fig. 4.2A) and also the PO2 values at the surface and centre of the muscles (Fig. 4.2B).

When aerated with 95% O2, diffusive O2 supply would have been adequate to keep the

muscles fully oxygenated. Upon switching to 95% N2, PO2 within the muscle would

have fallen rapidly so that within 30 s almost the entire muscle would have been anoxic.

Similarly, when 95% O2 was returned to the muscle surface, O2 would have diffused

quickly into the muscle so that the preparation was likely to have been fully oxygenated

in less than 25 s (i.e. the time at which the calculated PO2 at the muscle centre became

>0).

4.3 Results

4.3.1 Control experiments

The total and passive forces recorded from twitches delivered at 0.5 Hz throughout the

60 min ischaemia protocol are illustrated in Fig. 4.3. One of the sets of data shown in

Fig. 4.3 is for “control” measurements (open symbols; n = 4) in which the solution was

drained from around the muscle for 60 min but aeration with 95% O2 was maintained.

In that case, there were no significant changes in either passive or total force during the

protocol. In addition, neither enthalpy output, work output nor efficiency, measured in

series of twitches, was altered significantly. In the other set of control experiments both

the solu tion surrounding the muscle and 95% O2 supply were maintained throughout. In

this protocol, too, there were no significant changes in any of the measured variables.

These results show that the measured variables were stable with time in the absence of

anoxia and were unaffected by just removal of the solution surrounding the muscle.

35

0

20

40

60

80

100F

ractio

n o

f m

uscle

with

PO

2>

0

A.

0.0

0.2

0.4

0.6

0.8

1.0

PO

2 (

atm

)

N2 O2

10 s

Muscle surface

Muscle centre

B.

Fig. 4.2. Simulations of time courses of muscle oxygenation at the

onset and end of a period of simulated ischaemia. A. Time-course of muscle oxygenation, quantified as fraction of muscle cross-

sectional area with PO2>0, after switching from 95% O2 to 95% N2 (left panel)

and upon returning to 95% O2. It was assumed that the muscle was contracting at

0.5 Hz throughout the protocol. When O2 was returned to the anoxic muscle

(arrow, right panel), the initial increase in area oxygenated was very rapid, due

to the very high PO2 gradient across the muscle surface and because small

increases in the radius of the oxygenated region produce large increases in

oxygenated area. B. Time course of changes in PO2 at the muscle surface (solid

lines) and at the centre of the muscle (dashed lines). Steady-state surface PO2

was set to be that measured in the chamber (0.84 atm with 95% O2 and 0.05 atm

with 95% N2). When aerated with 95% O2, PO2 at the muscle centre was

calculated to be 0.4 atm. This decreased to 0 within 25 s of switching to 95% N2.

The arrows indicate the time at which 95% O2 was reintroduced into the muscle

chamber.

36

4.3.2 Effects of simulated ischaemia on force output

The second set of data in Fig. 4.3 (closed symbols; n = 6) are for muscles that had both

solution removed and were exposed to 95% N2 for 60 min. In that case, active twitch

force (i.e. the difference between total force and passive force) declined in the first 20

min of N2 exposure until it was the same as the passive force; that is, active force

production became completely inhibited. Upon the return of O2, active force production

rapidly recovered so that total force had returned to pre-ischaemia values in 10 min.

Note, however, that the recovery of mechanical function was much slower than the

estimated time-course of return of O2 to the muscle (Fig. 4.2). After recovering, force

output then declined again and was significantly lower than its pre-ischaemia value after

30 min of re-oxygenation. Passive force also altered significantly with time but only

after O2 was returned to the experimental chamber, when it was transiently higher than

the pre-ischaemia value.

The contrast between the absence of changes in the measured variables in the two

control groups and the clear changes in force output when O2 was removed indicate

that, for this muscle preparation, the absence of solution surrounding the muscle per se

did not affect contractile function but that anoxia resulted in both an immediate and,

after re-oxygenation, delayed reduction in the ability of the papillary muscles to produce

force.

4.3.3 Effects of simulated ischaemia and reperfusion on mechanical and energetic parameters

In Fig. 4.4, the effects of both 30 and 60 min simulated ischaemia on the work and

enthalpy outputs measured after reperfusion are illustrated. In control muscles, there

were no significant changes in work or enthalpy output measured 5 min and 30 min

after re-oxygenation compared to that before the simulated ischaemia. Thirty min of

ischaemia had no significant effect on either work output or enthalpy output, regardless

of whether it was measured after 5 or 30 min of re-oxygenation. In contrast, after 60

min ischaemia both work output and enthalpy output were significantly decreased at

both the post-ischaemia recording times (Table 4.2). For instance, after 30 min re-

oxygenation, work output was 66 ± 3% of the pre-ischaemia value and enthalpy output

was 71 ± 3% of its pre-ischaemia value.

Prior to exposure to N2, εnet was ∼9%; that is, work accounted for 9% of the enthalpy

produced. This value did not alter significantly after either 30 or 60 min of simulated

37

ischaemia. Similarly, εI was ∼19% before the ischaemic period and there was no

significant variation over the time course of the experiment. Thus, neither initial nor net

mechanical efficiency was affected by a period of simulated ischaemia.

4.3.4 Partitioning energy cost between force-dependent and force-independent components

Initial enthalpy output, measured over the first three contraction cycles, was partitioned

into force-dependent and force-independent components by selectively inhibiting force

output using 5 mM BDM and 150 mM sucrose. Force-independent enthalpy output

accounted for 19 ± 5% (n = 6) of the initial enthalpy output measured before ischaemia.

Using a paired comparison, there was no significant difference between the values

before ischaemia and those measured after 40 min of reperfusion.

Table 4.2. Energetic variables before and after 60 min of simulated

ischaemia.

Pre-ischaemia

Post-ischaemia

5 min

Post-ischaemia

30 min

∆HNet (mJ g-1

twitch-1

) 10.7 ± 0.3 6.1 ± 1.0 7.6 ± 0.3

∆HI (mJ g-1

twitch-1

) 3.2 ± 0.5 2.3 ± 0.5 2.4 ± 0.3

∆HP (mJ g-1

twitch-1

) 0.6 ± 0.1 - 0.62 ± 0.08

W (mJ g-1

twitch-1

) 1.0 ± 0.1 0.6 ± 0.1 0.66 ± 0.03

εNet (%) 9.0 ± 0.9 9 ± 1 8.2 ± 0.5

εI (%) 19 ± 3 19 ± 2 16 ± 3

R/I 1.1 ± 0.2 1.1 ± 0.2 0.9 ± 0.2

38

0 20 40 60 80 100

Time (min)

0

50

100

Re

lative

fo

rce

ou

tpu

t (%

)

O2 N2 O2

Ischaemia

Control

Fig. 4.3. Changes in normalised force output during ischaemia and

reperfusion of rat papillary muscle. Force output is expressed relative to isometric twitch force measured at the start

of the experiment. Symbols represent the mean values and error bars the S.E.M.

The open symbols (, passive force; , total force) are data from control

experiments in which muscles (n = 4) were exposed to 95% O2 throughout the

experiment although solution was withdrawn for 60 min starting 15 min after the

beginning of the recordings. The solid symbols (, passive force; , total force)

are for data from muscles (n = 6) which were exposed to 95% N2 for 60 min. In

muscles exposed to N2, active force output was abolished after 20 min (i.e. total

force equalled passive force) while passive force output increased gradually and

remained elevated upon return of 95% O2. Active force recovered to control

levels within 10 min of the return of O2 but then decreased significantly over the

next 20 min.

39

4.3.5 Time course of oxidative recovery metabolism

The time course of oxidative recovery metabolism was determined from the time course

of the decline in rate of heat output after a series of contractions. The time course was

well described by a single exponential and could thus be quantified by the time constant

of an exponential function fitted through the post-contraction heat rate data (Fig. 4.5).

Prior to exposure to ischaemic conditions, the mean time constant was 9.5 ± 0.7 s (Table

4.3). Following 30 and 60 min ischaemia the time constant was significantly shorter

after both 5 min (5.6 ± 0.2 s) and 30 min of reperfusion (6.5 ± 0.3). The mean

differences between the pre-ischaemia values were 3.8 ± 0.7 s after 5 min reperfusion

and 3.0 ± 0.7 s after 30 min reperfusion. In other words, after simulated ischaemia

recovery metabolism was complete in about two-thirds of the time it took before

ischaemia. This was not simply an effect of time in vitro because there was no change in

the time constant for control muscles (Table 4.3). The amount of recovery metabolism

relative to the amount of initial metabolism was not significantly different (R/I ratio,

Table 4.2) so, although the time course was altered, the coupling between initial and

recovery metabolism was preserved.

0

50

100

150R

ela

tive

wo

rk o

utp

ut

(%)

Control N2 30 N2 60

A. Work

0

50

100

150

Re

lative

en

tha

lpy o

utp

ut

(%)

Control N2 30 N2 60

Equilibration

Reperfusion 30 min

Reperfusion 5 min

B. Enthalpy

Fig. 4.4. Effects of simulated ischaemia on relative work and relative

enthalpy output. Measurements of work (A) and enthalpy (B) output were made at three different

times: during the equilibration phase (black) and 5 min (dark grey) and 30 min

(light grey) min after re-oxygenation. There was no statistically significant

difference after 30 min simulated ischaemia but after 60 min of simulated

ischaemia both work and enthalpy output were significantly reduced at both

measurement times. N230 and N260 min represents 30 min and 60 min of

simulated ischaemia, respectively.

40

Fig. 4.5. Effect of simulated ischaemia on

time course of rate of recovery heat

output. The time course of rate of recovery enthalpy

output was calculated by differentiation of the

cumulative enthalpy record from rat papillary

muscles before and after exposure to N2. The

records were normalised by the value measured

1 s after the end of the last contraction cycle.

The time constant of the decline in rate of

recovery heat output was determined by fitting

an exponential through the records using non-

linear, least squares regression. In this example

the time constant decreased from 12.2 s to 6.6 s

after exposure to N2. Muscle mass, 3.66 mg;

muscle length, 5.5 mm.

0.0

0.2

0.4

0.6

0.8

1.0

Re

lative

en

tha

lpy r

ate

20 s

Pre-ischaemia

Post-

ischaemia

4.4 Discussion

The main finding of this study was that ischaemia resulted in a substantial decrease in

the work output of rat papillary muscles with no alteration in net or initial mechanical

efficiency. Thus, the transduction of energy from metabolic substrates, via the

mitochondria and myosin cross-bridges, to mechanical work was unaffected by a

preceding period of ischaemia. Consequently, the reduced work output of the muscles

cannot be attributed to a deficit in energy supply pathways, but rather must be

associated with the processes involved in either initiation or generation of force; that is,

with either excitation-contraction coupling or with the cycling of myosin cross-bridges.

Impaired excitation-contraction coupling would decrease the influx of Ca2+

into

myocytes at the start of contraction thereby decreasing the number of cross-bridges that

could attach to actin. Alternatively, the impaired work output could result from a deficit

in cross-bridge work output. This could come about in two ways: fewer attached cross-

Table 4.3. Recovery time constant before and after 60 min of simulated

ischaemia.

Recovery time constant (s) Pre-ischaemia

Post-ischaemia

5 min

Post-ischaemia

30 min

Control∗

9.5 ± 0.7 10.2 ± 0.7 9.8 ± 0.6

Ischaemia 60 min† 9.4 ± 0.6 5.6 ± 0.2 6.5 ± 0.3

* 16 observations, 4 muscles.

† 22 observations, 11 muscles.

41

bridges or less work per cross-bridge cycle. The overall aim of this study was to

distinguish among these possibilities.

4.4.1 Impaired work output due to fewer attached cross-bridges

This distinction can be made using the initial mechanical efficiency values and

partitioning of energy use between force-dependent and force-independent processes as

follows. εI is the ratio of the work output to the initial enthalpy output and the latter is

the sum of the initial heat (QI) and work produced. Cross-bridge cycling accounts for all

the work performed and for part of the heat produced (QCB). The remainder of the heat

arises from ion pumping (QP), in particular Ca2+

pumping, so Equation (19) can be re-

expressed as follows.

I

I CB P

W W

W Q W Q Qε = =

+ + + (24)

Equation (24) can be used to calculate the expected difference in εI in post-ischaemic

muscles for the mechanisms proposed to account for decreased work output. The

calculation was performed assuming that εI was 0.19 (Table 4.2), ion pumping

accounted for ~19% of ∆HI and that W declined to 0.66 of the pre-ischaemic value (Fig.

4.4). The results of the calculations are presented in Table 4.4, in which the predicted

post-ischaemic values of εI and the factors in Equation (24) expressed as percentages of

the pre-ischaemic values, are shown for the three following mechanisms. (1) The

decrease in work output was due to release of less Ca2+

. This would reduce QP, QCB and

W by the same proportion (i.e. to 0.66 of their control values). (2) Work output

decreased due to fewer cross-bridge cycles occurring but with no change in Ca2+

release. In this case, both W and QCB would be decreased to 0.66 of their control values

but QP would be unaltered. (3) The decline in work output was entirely due to a

decrease in the work performed in each cross-bridge cycle but with no change in either

the number of cross-bridge cycles or the amount of Ca2+

released (i.e. only W

decreased). A comparison between the data obtained in the current study and the

expected values for each mechanism can be made with reference to Table 4.2 and Fig.

4.6.

42

For Mechanisms 1 and 2, εI would be either unaltered or only slightly decreased

whereas for Mechanism 3, εI would be reduced to about two-thirds of its control value

(Table 4.4). However, there was no significant change in εI (Table 4.2, Fig. 4.6A). This

suggests that Mechanism 3 was the least likely mechanism to have accounted for the

change in work output.

Mechanisms 2 and 3 can be distinguished from Mechanism 1 on the basis of changes in

QP (i.e. force-independent heat output). For Mechanisms 2 and 3 QP would be unaltered

by ischaemia whereas for Mechanism 1 QP would be reduced to two-thirds of its pre-

ischaemic value. There was no significant change in QP (Fig. 4.6B), suggesting

Mechanism 1 was unlikely to have contributed. It should be noted that there was

considerable variation in post-ischaemic QP values. However, the notion that the amount

of Ca2+

released would not be altered after re-oxygenation is supported by previous

work in which the free Ca2+

transients in ferret papillary muscle were shown to rapidly

regain their pre-ischaemic characteristics upon re-oxygenation (Allen et al., 1989).

Note, however, that there are almost certainly changes in Ca2+

cycling at any time when

∆GATP (which determines the amount of Ca2+

that can be accumulated in the

sarcoplasmic reticulum) is decreased, such as during and immediately after ischaemia.

The final evidence to clarify the most likely of the three mechanisms to have accounted

for the impaired contractility is that Mechanism 3 would be expected to be associated

with an unaltered QCB (i.e. heat output associated with cross-bridge cycling) whereas

Mechanisms 1 and 2 would be expected to lead to a reduction in QCB. The calculated

QCB did decrease significantly and by the amount expected for Mechanisms 1 and 2

(Fig. 4.6C).

Table 4.4. Theoretical (expected) values of εI for different mechanisms

underlying depressed work output.

Relative values†

Mechanism∗ W QP QCB εI

1 66 66 66 100

2 66 100 66 90

3 66 100 100 66

* 1: decreased number of cross-bridges due to decreased Ca2+

release; 2: decreased

number of cross-bridges but no alteration in Ca2+

release; 3: decreased work per

cross-bridge cycle but unaltered number of cross-bridge cycles.

† Symbols refer to Equation (24).

43

To summarise, the effects of ischaemia on QCB and εI are not consistent with

Mechanism 3 and the changes in QP are probably inconsistent with predictions based on

Mechanism 1. Thus, the mechanism most likely to account for the depressed post-

ischaemic work output is that fewer cross-bridge cycles occur in each contraction,

despite little alteration in Ca2+

release. In other words, the sensitivity of the myofibrils

to Ca2+

is reduced. Several changes that occur in the intracellular environment after

ischaemia (e.g. decreased pH and accumulation of inorganic phosphate and reactive

oxygen species) can potentially reduce the sensitivity of the muscle to Ca2+

(Fabiato &

Fabiato, 1978; Allen & Orchard, 1983; Camara et al., 2004).

0

20

40

60

80

100

ε Ι (%

)

Mech 3

Pre Post

Mech 1 or 2

A.

0

20

40

60

80

100

QP (

% in

itia

l e

nth

alp

y)

Mech 1

Pre Post

Mech 2 or 3

B.

0

20

40

60

80

100

QC

B (

% in

itia

l e

nth

alp

y)

Mech 3

Pre Post

Mech 1 or 2

C.

Fig. 4.6. Effect of simulated ischaemia on cross-bridge-dependent and

-independent energy cost. The arrows indicate the values of the post-ischaemic data for the mechanisms

described in the text and Table 4.4. A. The initial efficiency expressed as a

percentage. The difference in initial efficiency between Mechanism 1 and 2 is

only 10%, which would be too small to be detected from the current data.

Mechanism 3 corresponds to an efficiency two-thirds of the pre-ischaemic value.

There was no significant change in efficiency, indicating Mechanism 1 or 2 were

likely to have caused the decreased in work. B. The measured force-independent

heat (QP), expressed as a percentage of the initial enthalpy. Sixty min of

simulated ischaemia had no significant effect on QP. It is most likely that

Mechanisms 2 or 3 are consistent with this result. C. Cross-bridge-dependent

heat output (QCB), expressed as a percentage of the initial enthalpy, calculated

for each muscle by subtracting from the measured initial enthalpy output the sum

of the work output and force-independent heat output. The cross-bridge-

dependent heat output was significantly lower after 60 min of simulated

ischaemia. The post-ischaemic data are consistent with the expected values for

Mechanisms 1 and 2. Data indicating pre-ischaemic and post-ischaemic values

are labelled Pre and Post, respectively.

44


It has been proposed that an important consequence of ischaemia and reperfusion

damage is impaired mitochondrial function. One index of mitochondrial function is the

efficiency (ηR) with which the mitochondria transfer free energy from metabolic

substrate to ATP (Barclay et al., 2003). This can be calculated from the ratio of εNet and

εI (Equation (21), Section 3.5.2).

Assuming that ∆GATP in rat cardiac muscle in the absence of ischaemia was ∼59 kJ

mol-1

(Table 2.1) and ∆HPCr was ∼35 kJ mol-1

(Woledge & Reilly, 1988), then the pre-

ischaemic value of ηR would have been (9/19) × (59/35) = 0.82, which indicates that

82% of the free energy produced by oxidation of the substrate was transferred to free

energy in ATP. Neither εNet nor εI changed significantly during the experiments, so if

the ratio of ∆GATP to ∆HPCr was also unaltered, then ηR would have been unaffected by

ischaemia. There is considerable evidence that, although ∆GATP is reduced during

ischaemia, its value returns to pre-ischaemic values quickly during reperfusion so that

after 30 min of reperfusion ∆GATP differs from pre-ischaemic value by <10% (Griese et

al., 1988; Headrick et al., 1990; Headrick, 1996; Schulz et al., 2001; Day et al., 2006).

Given that the R/I ratio was also unaltered after ischaemia, indicating that the coupling

between ATP use and ATP regeneration was preserved, then the only alteration in

mitochondrial function following the simulated ischaemia was the more rapid kinetics

(Fig. 4.5).

4.4.3 Recovery time course

The time course with which mitochondria respond to changes in energy demand due, for

example, to changes in heart rate or progressing from rest to steady activity is greatly

influenced by the function of creatine kinase (CK). When CK, either mitochondrial or

cytosolic, in rabbit and mouse hearts was inhibited pharmacologically or by genetic

modification, then the mitochondria responded more rapidly to changes in ATP demand

produced by the changes in heart rate (Harrison et al., 1999; Gustafson & Van Beek,

2002), supporting the idea that CK acts as an energetic buffer. The results in the current

study are, therefore, consistent with impaired CK activity following simulated

ischaemia. It has been shown before that ischaemia of even quite short duration (10−60

min), in rabbit hearts can cause loss of CK activity that persists for at least 30 min after

the end of ischaemia (Bittl et al., 1985). Therefore, our results are consistent with the

45

notion that the CK reaction in the rat papillary muscles became inhibited during

exposure to N2.

It should be noted that published reports of kinetics of mitochondrial oxidative

phosphorylation in response to changes in energy demand are somewhat contradictory.

The time course of changes in mitochondrial O2 consumption has been reported to be

prolonged in acidotic muscles (Mast & Elzinga, 1989) and to be unaltered in stunned

cardiac muscle following ischaemia (Zuurbier et al., 1997). However, interpretation of

the latter result was confounded by a substantial slowing of mitochondrial response over

the time course of an experiment in “control” muscles. In the current study, the time

constant of recovery heat production was stable in control muscles (Table 4.3), making

it easier to establish the nature of the response to simulated ischaemia.

4.5 Conclusion

In conclusion, the results of the current study, taken in concert with those of a previous

study of Ca2+

handling, indicate that the most likely cause of the depressed work output

of the post-ischaemic papillary muscles in this study was attachment of fewer cross-

bridges in each twitch but with no change in Ca2+

release or in work generated by each

attached cross-bridge. Furthermore, there were no signs of impairment of mitochondrial

function per se because neither the R/I ratio nor the estimated mitochondrial efficiency

were altered by ischaemia. However, the mitochondria responded more rapidly to

changes in energy demands, which may reflect impaired creatine kinase activity after

ischaemia and reperfusion.

47

CChhaapptteerr 55:: RReessttiinngg mmeettaabboolliissmm ooff mmoouussee

ppaappiillllaarryy mmuussccllee

5.1 Introduction

The isolated mouse papillary muscle is useful for determining the physiological

consequences of heart-focussed genetic manipulations. Papillary muscles are composed

of ventricular myocytes aligned along the long axis of the muscle, making them ideal

for measurement of ventricular muscle force generation, work output and energy

expenditure (Gibbs et al., 1967; Hisano & Cooper, 1987; Dietrich & Elzinga, 1993;

Baxi et al., 2000; Barclay et al., 2003). An important consideration for any isolated

muscle preparation is the adequacy of O2 supply. The only source of O2 for an isolated

muscle preparation is diffusive supply from the muscle surface. If the rate of O2

consumption is sufficiently high, diffusive O2 supply may be inadequate to oxygenate

the entire muscle cross-section, leaving an anoxic region in the centre of the muscle. An

anoxic region of muscle will eventually be unable to generate force or use energy, thus

reducing mass specific values for these variables, and, as observed in ischaemic cardiac

muscle, contracture is likely to develop.

The adequacy of diffusional O2 supply is governed by the balance between the rate at

which O2 diffuses into the muscle, quantified by the diffusivity, and the rate at which

the tissue consumes O2. The probability of an anoxic core forming is enhanced by

conditions that increase metabolic rate relative to O2 diffusivity. Muscles from small

48

animals, such as mice, have inherently high metabolic rates compared to muscles from

larger animals. Furthermore, it is common for investigations using mouse papillary

muscles to be performed at 37°C (He et al., 1997; Meyer et al., 1999; Bluhm et al.,

2000). Metabolic rate is more temperature sensitive than O2 diffusivity (Mahler et al.,

1985) so higher experimental temperatures increase the probability of anoxic core

formation.

In most studies that have used mouse papillary muscles there appears to have been a

poor appreciation of the potential limitations of diffusive O2 supply (He et al., 1997;

Meyer et al., 1999; Bluhm et al., 2000; Stull et al., 2002; Wang et al., 2002). In one

study in which the size of muscle required to avoid anoxia was assessed (Redel et al.,

2002), the assessment was based on published data for rat papillary muscles but the

metabolic rate of mouse cardiac muscle is likely to be greater than that of rat cardiac

muscle (Gibbs & Loiselle, 2001). Another study used metabolic data from isolated

beating mouse hearts to analyse O2 diffusion into isolated mouse trabeculae (Stuyvers et

al., 2002). However, the rate of O2 consumption used in those authors’ calculations is in

error through not appreciating that the original value (Gustafson & Van Beek, 2000)

was expressed per gram dry weight. Consequently, those authors (2002) overestimated

the rate of O2 consumption by a factor of ~5 and concluded, incorrectly, that even thin

mouse trabeculae (half-thickness ~0.11 mm) would become anoxic at metabolic rates

occurring during contractile activity. These two examples highlight the fundamental

problem for analysis of diffusional O2 supply to isolated mouse cardiac muscles: the

metabolic rate of these muscles has not been measured.

Metabolism can be divided into a resting component, corresponding to the metabolism

required to support cellular processes other than those related to contractile activity, and

a contractile or active component, representing the metabolism associated with

excitation, activation and contraction. In cardiac muscle, resting metabolism accounts

for 20 to 35% of the metabolism of a beating heart (Gibbs & Loiselle, 2001), a much

higher fraction than for other striated muscles. The first factor to be considered in

assessing the viability of in vitro preparations is the ability of diffusive O2 supply to

meet the metabolic demands of the resting muscle. The aims of the current study were

to measure the resting metabolic rate of mouse papillary muscles and to assess the

ability of O2 diffusion to meet the resting energy demands.

The resting metabolism of rat, cat and rabbit papillary muscles has been measured

previously. It was observed that resting metabolic rate declined exponentially with time

49

during an experiment (Loiselle & Gibbs, 1979, 1983; Loiselle, 1985c). For instance, the

resting metabolic rate of rat papillary muscles was ∼12 mW g-1

1 h after the heart was

removed from the animal but 2 h later it had decreased to ∼7 mW g-1

and subsequently

remained at that value. This characteristic could potentially result in a transient, central

anoxia in the muscles early in an experiment, when metabolic rate is high, with the

possibility that ischaemia and reperfusion damage then occurs as the resting metabolic

rate declines and O2 supply becomes adequate. Therefore, in the current study, the time

course of changes in resting metabolism was characterised.

5.2 Methods

The details of dissection of the preparations were given in Chapter 3. The following

sections describe methods specific to the experiments on resting metabolism.

In the experimental chamber, the clip at one end of the muscle was connected to a semi-

conductor force transducer (AE801, SensorOne, CA, USA) and the other end was held

in a clamp. Preparations were placed on the thermopile so that the section of ventricular

wall and the clip holding that end of the preparation were not on the recording region.

That end of the thermopile had thermocouples that were not connected to the recording

region to ensure that heat loss from the preparation was uniform along its length and

thus prevented the development of thermal gradients along the muscles length due to

varied heat loss through the thermocouples. Muscles were stimulated via two platinum

wire electrodes placed on either side of the preparation.

5.2.1 Heat measurements

The thermopile was enclosed in a glass chamber containing Krebs-Henseleit solution.

The chamber was submerged in a 40 L temperature-controlled water bath (F-10-HC,

Julabo Labortechnik, Germany) maintained at 27°C. The thermopile output was

proportional to the difference in temperature between the muscle and the frame of the

thermopile. The frame was kept at a constant temperature by contact with the

temperature-controlled reservoir. The rate of muscle heat output was calculated from the

difference in muscle temperature between that measured when the chamber was full of

solution (heat produced by the muscle dissipates into the solution so muscle temperature

equals the chamber temperature) and that measured when the solution was drained from

the chamber (air is a poor conductor of heat so muscle temperature increases until rate

of heat loss through the thermocouples to the frame equals the rate of heat production).

50

The chamber was continuously aerated with 95% O2–5% CO2 that had been thermally

equilibrated and saturated with water vapour.

5.2.2 Calculation of rate of resting heat production

Resting metabolic rate was defined as the rate measured when the muscle was

mechanically quiescent and had been so long enough for metabolic activity associated

with any preceding contractile activity to have ceased. In preliminary experiments, it

was found that after a series of contractions active metabolic rate decreased with a time

constant of ∼15 s at 27°C. Therefore, contractile activity must have ended at least 5 × 15

= 75 s prior to measurement of resting metabolism. In the current study, muscles

performed a brief series of twitches, to measure contractile force, after each

measurement of resting metabolism and the muscle was then quiescent for 10 min

before the next measurement.

The rate of heat production of the resting muscles (AR) was calculated from the change

in thermopile output (∆V, corrected for amplifier gain) that occurred upon draining the

solution from the muscle chamber using the following formula (Hill, 1928).

R

VkCA

nα

∆= (25)

where n is the number of thermocouples, α the Seebeck coefficient (µV °C-1

couple-1

), k

the rate of heat loss from the muscle (s-1

) and C the heat capacity of the muscle and any

adhering saline (mJ °C-1

). C and k were determined from the time course of cooling

following heating of the muscle using the Peltier effect (Kretzschmar & Wilkie, 1972).

To check the resolution of the method, a series of measurements was made using a dead

papillary muscle (stored in ethanol for 24 h prior to measurements and then rehydrated

in saline). The “resting heat rate” was -0.1 ± 0.4 mW g-1

(mean ± standard deviation; 21

measurements). For comparison, the minimum measured resting heat rate for live

preparations was 8.4 ± 1.6 mW g-1

.

5.2.3 Measurement of myocyte sarcomere length by diffraction of laser light

The performance of the heart depends on the length of the sarcomeres (the basis of

Starling’s Law of the heart). The sarcomere is the functional contractile unit of the

muscle cell and consists of two opposing sets of thin filaments (composed of actin)

extending towards the middle of the sarcomere where they overlap with the thick

51

filament (myosin) (see Fig. 5.1). The varying optical density of regions of sarcomeres

allows muscles to act as a diffraction grating when monochromatic light is shone

through the fibres. The sarcomere length sets the width of the diffracting slits.

The equation for determining sarcomere length, SL, is:

2 LSL

X

λ= (26)

where λ is the wavelength of light (in this case 632.8 nm), L the distance from the

muscle fibre to the screen for measuring the diffraction pattern and X the distance

between the two first order diffraction bands.

In a series of preliminary experiments, muscle length was set to that giving a passive

force of 5 mN mm-2

. It has been shown that with this pre-load sarcomere length is ~2.1

µm (see Fig. 4 in ref. Stuyvers et al., 2002). The muscles were then fixed in ethanol

(Josephson & Stokes, 1994). After 24 h, the muscles were transferred to glycerol, small

bundles of myocytes were teased free and their sarcomere length measured using the

diffraction pattern formed when laser light was shone through the myocytes. The mean

sarcomere length was 2.12 ± 0.03 µm (mean ± SEM; n = 5 preparations). Subsequently,

at the start of each experiment, muscles were stretched until the passive force was ~5

mN mm-2

.

Fig. 5.1. Example of myocyte sarcomere. The thin filaments are composed of actin and the associated proteins troponin

and tropomyosin. Actin is attached to the Z-lines at either end of the sarcomere

and the thin filaments extend in towards the middle of the sarcomere where they

overlap with thick filaments. The latter are mainly composed of the contractile

protein myosin. The area consisting of thin filaments is called the I-band and the

region where the thin filaments overlap with the thick filaments is referred to as

the A-band. Finally, the remaining area with only thick filaments is named the

H-band. Illustration from Huxley (1957).

thick

filaments

thin

filaments

52


The thermopile system required 15 min for thermal stabilisation before the first

measurement of resting metabolism could be made. Measurements were then made at

10 min intervals for 40 min. In experiments in which the effect of metabolic substrate

(glucose or pyruvate) on resting metabolism was determined, the protocol was

performed as described above using one substrate and, 40 min after the first

measurement, the solution was changed to one containing the other substrate and the

measurement protocol was repeated. The order of presentation of the substrates was

alternated in successive experiments.

Sucrose was used to study the effect of hyperosmolarity on resting metabolism. For

these experiments, three measurements of resting heat rate were made in the standard

Krebs solution (∼150 mOsM) and then that solution was replaced with Krebs containing

300 mOsM sucrose in addition to the normal Krebs for the following three

measurements. Thus, the solution osmolarity went from 150 mOsM to 450 mOsM. The

solution was then changed back to the standard Krebs for three more measurements (see

Fig. 5.5).


The radius to which O2 can diffuse (the critical radius, RC) was calculated using the

equation derived by Hill (1928; 1965). For consideration of steady-state O2

consumption, Eq (23) simplifies to the following:

04C

R

KYR

A= (27)

where K is the diffusivity of O2 in muscle (cm2 atm

-1 min

-1), Yo is the partial pressure

of O2 (PO2) at the muscle surface (atm) and AR is the rate of O2 consumption of the

resting muscle (cm3 min

-1 g

-1). In this model it is assumed that the rate of O2

consumption is constant. K is 2.37 × 10-5

cm2 atm

-1 min

-1 at 22.8°C (Mahler et al.,

1985) and was adjusted to 27°C using a Q10 of 1.06 (Mahler et al., 1985), giving a value

of 2.43 × 10-5

cm2 atm

-1 min

-1. The PO2 in the chamber solution was measured using an

O2-sensitive microelectrode (OX500, Unisense, Aarhus, Denmark) and was 0.95 atm.

Rates of resting metabolism were converted to rates of O2 consumption using an

energetic equivalent of 19 mJ µL-1

(see Section 2.1.2).

53


The statistical significance of variations in metabolic rate with muscle mass was

analysed using a one-way analysis of variance. Comparisons of substrates and the effect

of hyperosmolarity were made using a paired Student’s t-test. Decisions concerning

statistical significance were made at the 95% level of confidence.

5.3 Results

5.3.1 Resting metabolic rate depended on time and muscle mass

The rate of heat output from resting papillary muscles decreased with time during an

experiment (Fig. 5.2). The records in Fig. 5.2 show thermopile records from one muscle

made at 10 min intervals. The amplitude of the record is proportional to resting heat

rate. The rate of heat production declined with time and the difference between

successive 10 min intervals decreased as the experiment progressed.

The time course of the decline could be well described by a single exponential function

with an asymptote approximating the eventual steady value (AR(∞), Equation (28)).

-( ) [ (0) - ( )] ( )tR R R RA t A A e Aτ= ∞ + ∞ (28)

where AR(0) was the first value measured in an experiment (i.e. 15 min after placing the

muscle in the chamber) and τ was the time constant. The mean time constant was 18 ± 2

min (n = 13) so that ~5 × 18 = 90 min were required before AR attained a steady value.

Fig. 5.3 illustrates that AR did eventually stop declining and become fairly steady.

Despite the changes in AR, there was no significant effect of time on isometric force

production during the time course of the experiments.

54

The absolute values of AR(t), for a given value of t, depended on muscle mass, being

greater for small muscles than large muscles. To illustrate this, muscles were sorted

according to mass and mean values calculated for each group (Table 5.1 and Fig. 5.4).

The mean resting metabolic rate of muscles with mass <1 mg (mean mass, 0.74 ± 0.03

mg; n = 3) was 47 ± 11 mW g-1

compared to a rate of 14 ± 2 mW g-1

for muscles >2 mg

(mean mass, 2.6 ± 0.2 mg; n = 4). There was no significant difference in the time

constant for decline in rate among the groups and the slope of a line through a plot of

the individual data as a function of muscle radius did not differ significantly from zero

(r2 = 0.04, n = 13).

On eight occasions, two papillary muscles from the same heart were tested. In those

cases, the second muscle was left attached to the ventricular wall of the isolated heart

for ~2 h, exposed to oxygenated Krebs, before it was transferred to the experimental

chamber. The initial values of AR for the second muscles were appropriate for their

mass, as judged by data from muscles that were tested shortly after excision of the heart,

and AR declined with the same time course as for the other muscles.

0

10

20

30

40R

ate

of

he

at

ou

tpu

t (µ

J s

-1)

25

15

55

7565

45

35

1 s

A.

0 20 40 60 80

Time (min)

0

6

12

18 Re

stin

g m

eta

bo

lism

(mW

g-1)

B.

Fig. 5.2. Example of decline in resting heat rate with time during an

experiment. A. Thermopile records from one papillary muscle (mass, 2.06 mg; length, 4.6

mm) at different times during an experiment. The time (in min) at which each

recording was made is given beside the record. The sections of records on the

left are the baseline measurements made while the muscle was immersed in

solution. The sections on the right were made after the solution had been

drained. B. Resting metabolic rate, normalised by muscle mass () taken from

the records in A. An exponential curve (solid line) was fitted through the points

by least squares regression. Time constant for this example was 28 min and the

estimated steady value was 3.4 mW g-1

.

55

Table 5.1. Characteristics of mouse papillary muscles.

<1 mg 1–2 mg >2 mg

Number of muscles 3 6 4

Wet mass (mg) 0.74 ± 0.03 1.5 ± 0.1 2.6 ± 0.2

Length (mm) 2.8 ± 0.3 3.8 ± 0.2 4.2 ± 0.4

Cross-sectional area (mm2) 0.26 ± 0.02 0.39 ± 0.04 0.61 ± 0.09

Radius (mm) 0.28 ± 0.01 0.35 ± 0.02 0.44 ± 0.03

Fig. 5.3. Exponential decline in resting

metabolism. Resting metabolic rate of a papillary muscle

(mass, 1.78 mg; length, 3.9 mm) measured at

10 min intervals. The fitted exponential curve

is shown by the solid line. The time constant

was 20 min and the steady value was 3.6 mW

g-1

.

0 50 100 150 200

Time (min)

0

5

10

15

20

Re

stin

g m

eta

bolis

m (

mW

g-1

)

Fig. 5.4. Effect of muscle mass on resting

metabolic rate. Muscles were divided according to wet mass

into three groups (<1mg, 1–2 mg, >2 mg) and

for each group, the mean resting metabolic rate

is plotted as a function of mean mass. The

number of muscles in each group is shown in

parentheses. Data are shown for the first

measurement made in an experiment (; solid

line) and for the estimated steady values (;

dashed line). The steady values were estimated

from the exponential curves fitted to the data

for each muscle (Fig. 5.2 and Fig. 5.3). The

symbols represent the mean values and the

error bars represent the SEM. The effect of

muscle mass on resting metabolic rate was

statistically significant.

0 1 2 3

Mass (mg)

0

10

20

30

40

50

60

Re

stin

g m

eta

bo

lism

(m

W g

-1)

AR(0)

AR(∞)

(3)

(6)

(4)

56

5.3.2 The effect of BDM on resting metabolic rate

To determine whether changes in either cross-bridge activity or Ca2+

cycling may have

contributed to the decline in rate with time, the resting metabolism of four muscles was

measured with 30 mM BDM in the Krebs solution. BDM at this concentration inhibits

both cross-bridge cycling and Ca2+

cycling (Backx et al., 1994). AR still declined

exponentially with a time constant of 17 ± 4 min (n = 4). Although the experiment was

not specifically designed to test whether the magnitude of AR at the start of an

experiment was affected by BDM, the mean value in the first measurement (9 ± 2 mW

g-1

; mean mass, 2.2 ± 0.6 mg) was comparable to that for muscles of similar mass

measured without BDM. After measuring AR in the presence of BDM for ~90 min, the

solution was changed to Krebs without BDM and, after a 15 min interval (sufficient for

mechanical activity to recover), AR was measured again. At that time, AR measured

without BDM present did not differ significantly from that measured previously with

BDM.

5.3.3 The effect of hyperosmolarity on resting metabolic rate

A characteristic of resting metabolism in striated muscles from other species is that it

increases with increasing osmolarity of the bathing solution (e.g. Loiselle et al., 1996).

To see whether this also applied to mouse papillary muscles, resting metabolism

measured in normal Krebs solution was compared to that measured in the presence of

the impermeant solute sucrose (300 mM ≡ 300 mOsmol L-1

, a 3-fold increase in

osmolarity). In the hyperosmotic solution, AR increased greatly (Fig. 5.5A) and active

force output (Fig. 5.5B) was almost abolished. The mean resting heat rate with sucrose

was 247 ± 20% (n = 4) of that in normal Krebs solution. Upon returning the muscle to

normal solution, both AR and force output returned to previous values within 15 min.

57

5.3.4 The effect of metabolic substrate on resting metabolism and force output

Two exogenous substrates are commonly used in experiments with isolated papillary

muscles: glucose and pyruvate. In all preparations studied (n = 10), resting metabolic

rate was higher with pyruvate than with glucose (Fig. 5.6). The mean difference (AR

with pyruvate–AR with glucose) was significant and was 2.8 ± 0.8 mW g-1

, which

corresponded to the value in pyruvate being 210 ± 50% of that in glucose. There was no

significant difference in the maximum isometric force output between the two substrates

(mean values: pyruvate, 20 ± 3 mN mm-2

; glucose, 19 ± 2 mN mm-2

(n = 9)).

Fig. 5.5. Example of the effect of

hyperosmolarity on resting heat

output and force production. A. The protocol used to determine the

effect of hyperosmolarity on resting

metabolism. The first three measurements

of resting metabolism () were made in

the presence of isosmotic saline (I). The

decline in rate was fitted with an

exponential curve (dashed line). Three

measurements were then made in the

hyperosmotic solution (time of exposure is

indicated by H and solid horizontal line;

resting heat rate, ), followed by another 4

in isosmotic solution. The magnitude of

the hyperosmotic stimulation of resting

metabolism (indicated by the arrow) was

determined by comparing the resting

metabolism to that predicted by

extrapolation of the exponential fitted

through the first three points (dashed line).

Hyperosmolarity caused a three-fold

increase in resting heat rate in this

example. B. Records of the time course of

isometric force production made before

(left), during (middle) and after (right)

exposure to the hyperosmolar solution.

Hyperosmolarity reversibly decreased

isometric force production.

0 20 40 60 80 100 120 140 160

Time (min)

0

2

4

6

8

Re

stin

g m

eta

bo

lism

(m

W g

-1)

A

H II

0

6

12

18

Fo

rce

ou

tpu

t (m

N m

m-2

)

500 ms

B

58

5.4 Discussion

The data described in this chapter are the first measurements of the metabolism of

mouse papillary muscles. The primary purpose of this study was to obtain data that

could be used to assess the adequacy of diffusive O2 supply. In addition, the

characterisation of resting metabolism of mouse papillary muscles provides some

further insight into the metabolism of in vitro preparations of cardiac muscle and these

will be discussed first.

5.4.1 Comparison with other studies

A prominent characteristic of the resting metabolism of papillary muscles is that it

declines with time (Figs. 5.2 and 5.3). This has been observed in papillary muscles from

cats and rats (Loiselle & Gibbs, 1979) but not those from guinea-pigs (Loiselle &

Gibbs, 1979; Daut & Elzinga, 1988). For papillary muscles from rats, the time constant

for the decline in resting heat rate is greater (~60 min at 27°C; Loiselle & Gibbs, 1979;

Loiselle, 1985b) than that for mouse papillary muscles (~20 min at 27°C). The cause of

the decline in resting metabolism with time remains obscure. It is not a reflection of a

dying muscle, because the mechanical performance does not alter with time, nor does it

represent an irreversible or only slowly reversible depression of metabolic capacity,

because the resting metabolism can be stimulated several-fold by changing substrate

from glucose to pyruvate and increasing the osmolarity of the bathing solution (Fig.

5.5). The decline is related to isolation of the muscles, since it probably does not occur

Fig. 5.6. The effect of glucose and

pyruvate on the resting metabolic

rate of mouse papillary muscle. Resting metabolic rate measured in the

presence of either 10 mM glucose or 10

mM pyruvate. Values in glucose are

indicated by symbols on the left and those

in pyruvate by symbols on the right. The

lines join values from the same muscle.

The measurements were made after 1 hour

in vitro and the order of presentation of

substrates was alternated to negate time-

dependent effects.

0

5

10

15

Re

stin

g m

eta

bo

lism

(m

W g

-1)

Glucose Pyruvate

59

in isolated hearts (for a detailed discussion, see Gibbs & Loiselle, 2001). Gibbs &

Loiselle (2001) have suggested that the decline in resting metabolism may arise from

accumulation of an inhibitory endothelial factor that accumulates in the absence of

microvascular circulation. Our data suggest that the rate does not decline from the time

the heart is excised, as previously suggested (Loiselle & Gibbs, 1979; Loiselle, 1985b;

Gibbs & Loiselle, 2001) because in our experiments the resting metabolism was the

same whether muscles were tested within 20 min of excision of the heart or had

remained in the open, superfused ventricle for two hours before dissection. This

suggests that the decline in metabolic rate is initiated by dissection of the papillary

muscle from the ventricle. This raises the possibility that the resting metabolic heat rate

is high initially due to dissection damage, as has been described for skeletal muscle

(Biron et al., 1979). The only part of the papillary muscles that necessarily incurred

damage during dissection was the end that attached to the ventricular wall (one of the

clips holding the preparation was crimped onto a piece of ventricular wall muscle) but

heat output from that section of the preparation was not included in our recordings. It

has also been suggested that the first values measured (i.e. AR(0)) may be high due to

waves of contractile activity (Denis Loiselle, personal communication). However, there

was no visual evidence of writhing in these experiments and treatment with BDM did

not affect metabolic rate. Thus, it is unlikely that the high metabolic rate recorded at the

start of an experiment was related to dissection damage. However, the cause of the

change in resting metabolic rate with time in isolated papillary muscles remains unclear.

It is of interest to know how resting metabolic rates measured in papillary muscles

compare to those of intact mouse hearts. The resting metabolism of mouse hearts can be

estimated from published data for energy cost as a function of pressure−volume area

(PVA). The y-intercept of these relationships (i.e. the energy cost corresponding to 0

PVA is the sum of resting heat rate and metabolism associated with excitation-

contraction coupling (or activation metabolism). There are two reports that provide the

data required to estimate resting metabolic rate in mouse hearts.

60

It was assumed that ∼19% (see Section 6.3.6) of the force-dependent metabolism

corresponds to activation metabolism and that the remainder is associated with resting

metabolism. Using data from How et al. (2005), the resting metabolism was predicted to

be ∼20 mW g-1

when supplied with medium containing a low fatty acid concentration

(Fig. 5.7A). Kameyama et al. (1998), on the other hand, showed with their perfused

mouse hearts a very high y-intercept on the PVA graphs, corresponding to a resting

metabolism of ∼60 mW g-1

(Fig. 5.7B). These hearts were perfused with a buffer

containing pyruvate. In the current study, it was shown that pyruvate doubled the resting

metabolic rate of papillary muscles. If this is also the case with mouse hearts, this may

account for some of the difference in resting heart rate between these two studies. In

summary, these mouse heart studies suggest that resting metabolic rate of hearts is

higher than the steady values for papillary muscles but lower than the highest values

recorded for papillary muscles. However, it should be noted that neither of the heart

studies appeared to be aware of the potential for trans-epicardial O2 exchange in isolated

hearts (Loiselle, 1989; Gibbs & Loiselle, 2001). This has potential to cause substantial

0.000 0.005 0.010 0.015 0.020

Pressure-volume area (J beat-1 g dry wt-1)

0.0

0.5

1.0

1.5

2.0O

2 c

on

su

mp

tio

n (

ml O

2 b

ea

t-1 g

-1)

10-3A.

0 5 10 15 20

Pressure-volume area (mmHg ml g-1)

0.0

0.5

1.0

1.5

2.0

Activation metabolism

Resting metabolism

Pressure-dependent

Pressure-independent

10-3B.

Fig. 5.7. Prediction of in vivo resting metabolism. Schematic diagrams of published relationships between O2 consumption and

pressure−volume area in the mouse heart. The O2 consumption is equivalent to

the total energy cost. The energy cost of the heart is made up of two components,

pressure-dependent and pressure-independent, or activation, metabolism. The y-

intercept indicates the in vivo energetic requirements of pressure-independent

metabolism. This is in turn made up of two components, resting and activation

metabolism. From the relationships shown the contribution of resting

metabolism was predicted to be ∼20 mW g-1

(A) by How et al. (2005) and ∼60

mW g-1

(B) by Kameyama et al. (1998).

61

errors in calculated rates of O2 consumption. Therefore, it is difficult to be sure exactly

how mouse papillary muscle data compares with that for isolated mouse hearts.

The inverse relationship between muscle size and resting metabolism has been

described previously for rabbit papillary muscles (Loiselle & Gibbs, 1983), although in

the current study the magnitude (both absolute and relative) of this effect was greater

than that for rabbit muscles. An obvious explanation for such an effect is that larger

muscles are unable to get enough O2 by diffusion to support their metabolism, so an

anoxic core develops. However, this explanation has been shown to be incorrect, by

both modelling and experiment (Loiselle & Gibbs, 1983; Loiselle, 1985a; Loiselle,

1987). In fact, as will be demonstrated in the following section, it was the small

preparations used in the current study that were most likely to be O2 diffusion limited.

5.4.2 Adequacy of diffusive oxygen supply

The main purpose of these experiments was to obtain data to enable an assessment of

the adequacy of diffusive O2 supply to meet the metabolic requirements of resting

mouse papillary muscles. If the metabolic rate of an isolated muscle is high compared to

the rate at which O2 diffuses into the muscle, an anoxic region may develop in the core

of the muscle. Furthermore, if an anoxic region subsequently becomes oxygenated, for

example as metabolic rate declines with time, then the cellular characteristics associated

with ischaemia-reperfusion damage (for a review, see Bolli & Marban, 1999) may

become apparent.

The results of the analysis of diffusive O2 supply are illustrated in Fig. 5.8 in which the

resting metabolic rates of the muscles used in this study are plotted as a function of

muscle radius. Data are shown both for the first values measured (Fig. 5.8A) and for the

estimated steady values (Fig. 5.8B). The curved lines indicate the calculated critical

radius (RC; Equation (27)) for each combination of radius and metabolic rate. For

muscles lying to the right of these lines, O2 diffusion would have been inadequate to

meet the O2 demand. For the metabolic rates measured early in an experiment, the

calculations indicate that six muscles may have had inadequate O2 supply (Fig. 5.8A).

Three of these were muscles with masses below 0.8 mg. Once resting metabolic rate had

declined to its steady value, all the muscles used were below the critical radius for their

metabolic rate.

62

This analysis illustrates that despite the small size of the mouse papillary muscles, at

early times in an experiment their metabolic rate can be sufficiently high that formation

of an anoxic core is likely and, in an apparent paradox, this is particularly likely to occur

for the smallest muscles because they have high metabolic rates. Furthermore, because

the resting metabolic rate is doubled if pyruvate is used as a substrate, achieving

adequate oxygenation would be more difficult using that substrate; doubling metabolic

rate reduces RC by a factor of ≈ 0.7.

Experimental temperature also has an important effect on diffusive O2 supply with

lower temperatures favouring O2 supply because resting metabolism is more

temperature sensitive (Q10 ~1.3; Loiselle & Gibbs, 1983; Loiselle, 1985c) than O2

diffusivity (Q10 = 1.06; Mahler et al., 1985). Some mouse papillary muscle experiments

have been performed at 37°C (He et al., 1997; Meyer et al., 1999; Bluhm et al., 2000).

At that temperature, metabolic rate would be 1.3-times greater than the values in Fig.

5.8 and the critical radius would be decreased by a factor of 1.06 1.3 = 0.9. The

metabolic rate of contracting mouse papillary muscles has not yet been measured but

contractile activity at physiological frequencies might be expected to double metabolic

0.0 0.2 0.4 0.6

Radius (mm)

0

20

40

60

80

100R

estin

g m

eta

bo

lism

(m

W g

-1)

A.

0.0 0.2 0.4 0.6

Radius (mm)

0

20

40

60

80

100

B.

Fig. 5.8. Adequacy of diffusive O2 supply to resting mouse papillary

muscle. The resting metabolic rates measured 15 min after placing the muscles in the

chamber (A) and the estimated steady value (B) plotted as a function of muscle

radius. Muscle radii were calculated from the average cross-sectional area for

each muscle. Data are shown for muscles with glucose () and pyruvate () as

substrate. The curved lines are the critical radii for diffusive O2 supply at 27°C;

to the left of the curves, muscles are adequately oxygenated and to the right, O2

supply would have been inadequate to maintain PO2>0 throughout the muscle

cross-section. Critical radii were calculated using Equation (27).

63

rate (Gibbs & Loiselle, 2001), decreasing the critical radius by an additional factor of

0.7. If we consider a mid-range papillary muscle (mass ~1.5 mg; radius 0.35 mm), at

27°C it has an eventual steady AR of ~4.5 mW g-1

and RC would be 0.8 mm (RC>actual

radius implies adequate O2 supply). Two hours after dissection, at 37°C, contracting and

with pyruvate as substrate, total metabolic rate could be ~18 mW g-1

and RC would be

reduced to 0.4 mm but still greater than the critical radius. However, under those

conditions RC would be <0.3 mm, favouring anoxic core formation, throughout the first

30 min in vitro. These calculations emphasise that the adequacy of diffusive O2 supply

cannot be taken for granted simply because the preparation is small.

5.5 Recommendations for performing experiments with isolated papillary muscles

In summary, even for quiescent mouse papillary muscles it is possible that diffusive O2

supply is inadequate, especially shortly after removal of the muscle from the heart. On

the basis of the data obtained in this study we would recommend the following

procedures to minimise the chances of O2 supply becoming inadequate: (1) avoid using

very small papillary muscles (<1 mg); (2) use glucose rather than pyruvate as substrate;

(3) use a temperature lower than 37°C; (4) avoid or minimise contractile activity during

the equilibration period after dissecting the muscle. Implementing recommendations

(2)–(4) just during the time that AR is high (the first 40–60 min after dissection) would

also minimise the chances of developing anoxia early in an experiment.

65

CChhaapptteerr 66:: CChhaarraacctteerriissaattiioonn ooff aaccttiivvee

mmeettaabboolliissmm

This Chapter characterises the active metabolism of the mouse papillary muscle. The

energy requirements of a contracting muscle were examined using different frequencies

and in combination with various metabolic substrates and at different temperatures.

Muscle efficiency was studied using a realistic range of shortening velocities. Finally,

the number of cross-bridges that are activated in a cardiac twitch was calculated.

6.1 Introduction

The basic contractile event in striated muscle is the twitch, which is the response of a

muscle or fibre to a single neural or electrical stimulus. Given its fundamental nature, it

is of interest to characterise the molecular events that underlie the twitch. Under both

physiological and typical laboratory conditions peak twitch force is less than the

maximum force that a muscle or fibre can produce. Support for this idea comes from the

observations that skeletal muscle twitch force is less than tetanic force and that cardiac

twitch force can be increased substantially by inotropic agents and changes in

experimental conditions (e.g. cooling) that increase intracellular [Ca2+

] (for a review,

see Endoh, 2004). Because twitch force is sub-maximal, it is unlikely that the force

output reflects the simultaneous action of all the available myosin cross-bridges.

66

One purpose of this study was to quantify the fraction of cross-bridges that cycle during

a cardiac twitch. This has not been determined before. Under most conditions, each

force-generating interaction between a myosin cross-bridge and an adjacent actin

filament is associated with the hydrolysis of one ATP molecule. Therefore, one way of

counting the number of cross-bridge cycles that occur during a contraction is to measure

the number of ATP molecules used; that is, to measure the energy cost of the

contraction. In the current study, the fraction of cycling cross-bridges was estimated

from measurements of the energy cost of contraction using isolated papillary muscles

taken from the left ventricle of the mouse heart. Energy cost was determined using the

myothermic technique, which has the chemical and temporal resolution to accurately

monitor the biochemical changes occurring within a brief twitch (Woledge, 1998). In

addition, with this technique it is possible to separate energy used by the cross-bridges

from energy used by other cellular ATPases (i.e. those associated with pumping ions

across membranes) (Gibbs et al., 1988; Alpert et al., 1989). Mouse cardiac muscle was

used because the advent of heart-focussed genetic manipulations (e.g. Headrick et al.,

1998; Bluhm et al., 2000) has made it important to develop experimental techniques and

protocols that can be used to probe the basic physiology of cardiac muscle in this

species. For example, although in a number of studies the intracellular free Ca2+

and

Ca2+

content of the sarcoplasmic reticulum (SR) of mouse cardiac muscle have been

measured (Gao et al., 1998; Georgakopoulos & Kass, 2001; Stull et al., 2002), in no

cases has the amount of Ca2+

released been quantified.

This study is the first in which the energetics of contracting, isolated mouse cardiac

muscle have been measured. It is important to ensure that diffusive O2 supply is

adequate to meet the needs of isolated muscles so the metabolic data obtained in the

current study was combined with a theoretical model of diffusive O2 supply (Barclay,

2005) to confirm that the muscles used were small enough to maintain a favourable

balance between O2 supply and consumption.

6.2 Methods

The materials and methods used for the experiments reported in this chapter have been

described in Chapter 3. Only those methods specific to these experiments will be

described here. Muscle characteristics are listed in Table 6.1.

67

6.2.1 Mechanical output

The time course of a twitch was followed and the time taken to reach peak force

calculated. The time to peak force output reflects how quickly cross-bridges attach and

generate force, which allows comparison between muscles that produce different peak

forces under different conditions.

6.2.2 Contribution of recovery heat to initial heat measurements

As described in the Methods (Section 3.4), initial enthalpy output was calculated by

measuring the enthalpy produced over the first three contraction cycles. As for the rat

papillary muscle (Section 4.2.1), an estimate was made of how much recovery heat

might have been included in the measured enthalpy over the first three cycles. The R/I

for mouse papillary muscles contracting at 2 Hz was 1.2 (see Section 6.3.5) and was

independent of muscle mass. The steady state recovery heat output was estimated using

that R/I and assuming that in an energetic steady state this amount of recovery heat

would be produced within each contraction cycle (i.e. in 0.5 s when contracting at 2

Hz).

At a contraction frequency of 2 Hz, the initial enthalpy output was ~2.3 mJ g-1

twitch-1

so R,SSQg

= (1.2 × 2.3) / 0.5 = 5.5 mW g-1

. Substituting this value into Equation (18), the

amounts of recovery heat produced between the start of recording and the ends of the

first, second and third contraction cycles were 0.028, 0.11, and 0.25 mJ g-1

, respectively,

which was 1.2, 2.4 and 3.6% of the total heat produced by the end of those cycles (Fig.

3.6B).


Once in the experimental chamber, muscle length was adjusted to give a passive force

of 5 mN mm-2

; this length was designated L0. It has previously been shown that this

passive force corresponds to a sarcomere length of ~2.1 µm in these muscles (see

Table 6.1. Characteristics of mouse papillary muscles.

Number of muscles 24

Wet mass (mg) 1.7 ± 0.1

Length (mm) 3.4 ± 0.1


Radius (mm) 0.38 ± 0.01

68

Section 5.2.3). Muscles were then allowed to equilibrate for 60 min. When first

dissected, the resting metabolism of isolated papillary muscles is high and central

anoxia can develop (see Fig. 5.8). To minimise the possibility of this occurring, muscles

were not stimulated during the equilibration period. After 60 min, the muscles were

stimulated at 0.2 Hz for 10 min. For 5 min prior to making heat measurements, muscles

were not stimulated to allow metabolic rate to decrease to its resting value. This

provided the thermal baseline for heat measurements.

To investigate the effects of contraction frequency on number of cross-bridge cycles in

a twitch, an isometric contraction protocol was used; the protocol consisted of 20 s of

contractions at frequencies of 1, 2, 3 and 4 contractions s-1

.

The effect of metabolic substrate (glucose or pyruvate) on isometric force production

and enthalpy output was first measured using one substrate. After the last measurement

in that substrate the solution was changed to one containing the other substrate and

thirty min were allowed for stabilisation in the substrate before the first measurement

took place and the measurement protocol repeated. Muscles were allowed to equilibrate

in each substrate and then force and enthalpy output were measured using the

abovementioned frequencies. The order of presentation of the substrates was alternated

in successive experiments.

The effect of temperature (22, 27 and 37°C) was also investigated using 40 twitches at a

contraction frequency of 2 Hz.

To investigate the effects of shortening on energetics, a protocol that incorporated a

cyclic strain pattern in each twitch was used (Mellors & Barclay, 2001). The strain

pattern was designed to mimic strains experienced by papillary muscles in vivo

(Semafuko & Bowie, 1975). The protocol consisted of a 60 ms isometric phase after

delivery of the stimulus pulse, isovelocity shortening with an amplitude of 0.15L0 s-1

,

followed by isovelocity lengthening back to L0 (see Fig. 4.1). The velocity of the

shortening phase ranged between 1 and 5 mm s-1

(~0.4 to 1.7 L0 s-1

). These velocities

were calculated by dividing the strain amplitude by the reported duration of the ejection

phase in isolated, working mouse hearts (Larsen et al., 1999). To accommodate the sub-

physiological temperature used in this study, it was assumed that the Q10 for shortening

velocity (i.e. change in velocity for a 10°C temperature change) was 2. This particular

strain pattern was used because it was found in preliminary experiments to maximise

work output at 2 Hz.

69

6.2.4 Calculation of number of cross-bridge cycles

The number of cross-bridge cycles that occurred in a twitch was estimated from the

amount of ATP used and assuming that each cross-bridge cycle was associated with

hydrolysis of one ATP molecule. It was further assumed that under the conditions used

in this study ATP splitting was fully buffered by the CK reaction so that the enthalpy

output arises from the net breakdown of PCr. The number of ATP molecules hydrolysed

(NATP; ATP g-1

twitch-1

) was calculated as follows.

(1 )I CB A

ATP

PCr

H f NN

H

∆ −=

∆ (29)

∆HI is the initial heat per twitch (mJ g-1

), (1−fCB) is the force-independent enthalpy

output expressed as a fraction of the initial enthalpy output, ∆HPCr is the molar enthalpy

change for PCr hydrolysis and NA is Avogadro’s constant. ∆HPCr was calculated from

the R/I ratio (Section 6.3.5), as described by Woledge and colleagues (p.219, Woledge

et al., 1985; Woledge & Reilly, 1988), and was 34 kJ mol-1

. To compare this value to

the number of cross-bridges present in mouse cardiac muscle, we scaled NATP to a

volume of muscle containing a precisely known number of cross-bridges, the unit

sarcomere rhombus. This volume contains a total of 600 cross-bridges (for calculation

see Appendix I). Then the number of ATP used can be related to the number of cross-

bridge cycles that occurred in this volume per twitch (NCB) as follows.

sCB ATP

M

VN N

V

ρ= (30)

where Vs is the volume of a sarcomere rhombus, ρ is the density of muscle (1.06 g cm-

3) and VM is the fraction of muscle volume occupied by myofibrils. The volume of a

sarcomere unit cell was calculated assuming that sarcomere length was 2.1 µm (see

Section 5.2.3) and that the spacing between the thick filaments was 41 nm (Yagi et al.,

2004), giving a sarcomere cell volume of 3.5 × 10-15

cm3 (see Appendix I). VM takes

account of the volume density of myofibrils in myocytes (0.52 in mouse myocytes;

Barth et al., 1992) and the fraction of muscle volume that is occupied by myocytes (0.79

in rat myocardium; Dobson & Cieslar, 1997); therefore, VM is 0.52 × 0.79 = 0.41 ×

muscle volume.

70


The adequacy of diffusive O2 supply to papillary muscles was assessed as described in

Section 3.7. The value of PO2 at the muscle centre was calculated for 20 s of contractile

activity (as used in this study) and for contraction frequencies of 1, 2, 3 and 4 Hz (Fig.

6.1). PO2 at the muscle surface, measured using an O2-sensitive microelectrode (OX500,

Unisense, Aarhus, Denmark), was 0.84 atm (85 kPa). At rest, PO2 at the muscle centre

was calculated to be ~0.7 atm. Central PO2 decreased upon stimulation (Fig. 6.1).

However, even in the worst case (contraction frequency 4 Hz), at the end of the

contraction protocol central PO2 (minimum value ~4 kPa) would have remained greater

than the values at which mitochondrial respiration becomes compromised (~1.3 kPa;

Schenkman, 2001).


The statistical significance of variations in initial and net enthalpy use with contraction

frequency and shortening velocity was assessed using one-way analysis of variance

(ANOVA); for the contraction frequency data, a repeated measures one-way ANOVA

was used. The significance of variations in force production, measured at different times

during the contraction series, with contraction frequency were assessed using a 2-way

ANOVA. Where appropriate, post-hoc analyses were performed using Dunnett’s test for

comparisons with a control group (Hancock & Klockars, 1996). Decisions concerning

statistical significance were made at the 95% level of confidence.

71

6.3 Results

6.3.1 Force output of mouse papillary muscle

Metabolic substrate, pyruvate or glucose, had no effect on force or enthalpy output in

isometric contractions. Isometric force output decreased significantly with contraction

frequency. This analysis was performed using the average forces in each of the four 5 s

intervals in the contraction protocol. The effect of contraction frequency was

independent of the time interval over which force was averaged (Fig. 6.2). There was no

difference in peak force output between muscles in a bathing solution containing

glucose; 16 ± 2 mN mm2, and pyruvate; 15 ± 2 mN mm

2 (n = 11).

0 5 10 15 20

Time after start of contraction series (s)

0.0

0.2

0.4

0.6

0.8

PO

2 a

t m

uscle

ce

ntr

e (

atm

)

1 Hz

2 Hz

3 Hz

4 Hz

Fig. 6.1. Simulations of time course of PO2 at muscle centre during

contraction series. Simulations of the PO2 profile through muscle were made using Equation (23).

The diffusivity of O2 through muscle was taken to be 2.43 × 10-5

ml cm-1

min-1

atm-1

at 27°C (adjusted from value at 22.8°C using a Q10 of 1.06; Mahler et al.,

1985). Prior to the start of the contraction series, metabolic rate was assumed to

be constant and equal to the resting metabolic rate (∼5 mW g-1

). It was further

assumed that during the series of contractions the rate of O2 consumption

increased exponentially towards a steady value of 7 mJ g-1

twitch-1

with a time

constant equal to that for the decline in rate of heat output when contractile

activity ended (i.e. 12.2 ± 0.8 s, n = 11). Metabolic rates were converted to rates

of O2 consumption using an energetic equivalent of ∼19 mJ µL-1

, which was

calculated on the basis that the primary substrate for mitochondrial oxidation

was glucose for which the molar enthalpy is 2820 kJ mol-1

. Note, however, that

the calculated PO2 values are only ~2.5% smaller if it were assumed that fat

oxidation, yielding 17.3 mJ µL-1

, fuelled the contractions. The PO2 in the

solution surrounding the muscle was 0.84 atm. Muscle radius was taken to be

0.38 mm. Simulations were made for 20 s of contractile activity (as used in this

study) and for contraction frequencies of 1, 2, 3, and 4 Hz.

72

Fig. 6.2. Contraction frequency

dependence of normalised

isometric force output. Isometric force output is shown as a

function of contraction frequency. For

each muscle, the average force output

was calculated over 5 s intervals and

normalised by the average force over the

corresponding interval at 1 Hz. At all

frequencies the full protocol was of 20 s

duration. The symbols represent the

mean values of data from 11 muscles.

The data for different 5 s intervals are

distinguished as indicated by the key in

the Figure. Only at 4 Hz did the mean

values for all groups differ significantly

from those at 1 Hz (indicated by ∗).

0 1 2 3 4

Frequency (Hz)

0

20

40

60

80

100

Active

fo

rce

ou

tpu

t (%

)

0-5 s5-10 s10-15 s

15-20 s

6.3.2 Energy cost of a twitch and effects of contraction frequency, substrate and temperature

The energy cost of papillary muscle contraction was first assessed using a series of

isometric contractions at frequencies between 1 and 4 Hz. At a frequency of 1 Hz, the

initial enthalpy output, averaged over the first three contractions (Fig. 3.6B), was 3.3 ±

0.6 mJ g-1

twitch-1

and the net enthalpy output, from all the twitches, was 6.8 ± 1.1 mJ

g-1

twitch-1

(n = 11). Both the initial and net enthalpy outputs declined significantly with

increasing contraction frequency (Fig. 6.3). At a frequency of 4 Hz the mean initial

enthalpy output was 1.2 ± 0.2 mJ g-1

twitch-1

and the mean net enthalpy output was 3.9

± 0.6 mJ g-1

twitch-1

(n = 11). There was no significant difference in heat output

between the two substrates as the mean absolute heat output was 6.8 ± 1.1 mJ g-1

twitch-1

in glucose and 5.9 ± 1.2 mJ g-1

twitch-1

in pyruvate when stimulated at 1 Hz

(Table 6.2).

73

Increasing temperature from 22 to 27°C caused a reduction in peak twitch force and

enthalpy output and shortened the time taken for force to develop (Table 6.3). Two

muscles were also studied at 37°C and data from these preparations support the

conclusions from muscles studied at 22 and 27°C. At 37°C, twitch enthalpy output was

further reduced from 6.5 mJ g-1

twitch-1

(22°C) to 3.7 mJ g-1

twitch-1

. However, there

were no significant effects of temperature on rate of heat output or the time for recovery

heat rate to return to baseline (Table 6.3). The resulting Q10 values (where Q10 is the

magnitude of change in rate for 10°C change in temperature) of active rate of enthalpy

output were 0.78 between 22 and 27°C and 0.64 between 27 and 37°C (Table 6.3).

Fig. 6.3. Enthalpy output per twitch

in isometric contractions. The initial (grey bar) and net (black bar)

enthalpy output per twitch was measured

in isometric contractions. The heat output

of the papillary muscles was measured at

four different frequencies (1−4 Hz) using

glucose as substrate. The enthalpy output

per twitch decreased as twitch rate

increased. Asterisks (*) indicate statistical

significant difference between enthalpy

outputs of higher frequencies compared to

1 Hz.

0 1 2 3 4

Frequency (Hz)

0

2

4

6

8

En

tha

lpy o

utp

ut

(mJ g

-1 t

witch

-1)

Net enthalpy

Initial enthalpy

Table 6.2 Effect of glucose and pyruvate on enthalpy output (n = 11).

Contraction frequency

(Hz)

Glucose

(mJ g-1

twitch-1

)

Pyruvate

(mJ g-1

twitch-1

)

1 6.8 ± 1.1 5.9 ± 1.2

2 5.8 ± 0.8 6.3 ± 1.3

3 4.8 ± 0.7 4.6 ± 0.9

4 4.0 ± 0.6 5.1 ± 1.1

74

6.3.3 Effects of shortening on twitch energy cost

The effects of shortening velocity on energy cost were investigated using a contraction

frequency of 2 Hz with a period of shortening in each contraction at one of a range of

velocities. The shortening velocities used ranged from 0 (isometric) to 1.7 L0 s-1

and

enthalpy data were sorted into velocity bins, with bin width determined using Sturge’s

rule (no. bins = 1 + 3.322 × log(no. data points)). By plotting force output as a function

of change in muscle length a work-loop is formed. The work-loop has its three-

dimensional analogue in the pressure−volume diagram for the heart. Although the shape

of the work-loop changed with shortening velocity, there was only a marginal effect on

the work output (Fig. 6.4). Shortening velocity, in the range tested, had no significant

effect on either initial or net enthalpy output (Fig. 6.5). The net enthalpy output,

averaged across all velocities, was 5.6 ± 0.4 mJ g-1

twitch-1

and the corresponding initial

enthalpy output was 2.1 ± 0.2 mJ g-1

twitch-1

(n = 9).

Table 6.3. Characteristics of mechanical and energetic properties of

mouse papillary muscles performing isometric contractions (2 Hz) at

22, 27 and 37°C using glucose as a substrate.

22°C 27°C 37°C

Number of preparations 5 11 2

Active force (mN mm-2

) 20 ± 2 15 ± 2 14 ± 4

Twitch time (ms) 474 ± 5 381 ± 9 195 ± 27

Time to peak tension (ms) 253 ± 10 163 ± 2 95 ± 9

Enthalpy output (mJ g-1

twitch-1

) 6.5 ± 0.9 5.8 ± 0.8 3.70 ± 0.04

Time constant for recovery heat rate (s) 13 ± 2 13 ± 1 11 ± 2

75

Length (mm)

Fo

rce

(m

N)

0.53 L0 s-1

1.14 mJ g-1

0.79 L0 s-1

1.38 mJ g-1

1.05 L0 s-1

1.41 mJ g-1

1.32 L0 s-1

1.28 mJ g-1

1.58 L0 s-1

1.18 mJ g-1

Fig. 6.4. Effect of shortening velocity on work-loop. Work-loops produced by a mouse papillary muscle contracting at 2 Hz. In each

set of records the first stimulus pulse resulted in a large twitch force and the

second in much reduced force output. In the subsequent contractions there was a

gradual build-up of the force back towards the initial level. In this example the

shortening velocity varied from 0.5 to 1.6 L0 s-1

. Muscle mass: 1.57 mg; muscle

length: 3.8 mm.

0.0 0.5 1.0 1.5 2.0

Shortening velocity (L0 s-1)

0

2

4

6

8

En

tha

lpy o

utp

ut

(mJ g

-1 t

witch

-1)

Net enthalpy

Initial enthalpy

Fig. 6.5. Enthalpy output per twitch in shortening contractions. The initial (grey bar) and net (black bar) enthalpy output per twitch was

measured in contractions using a realistic contraction protocol. Enthalpy output

of a muscle shortening using a realistic contraction protocol. Heat and work were

expressed as a function of shortening velocity normalised to muscle length at a

realistic range of velocities.

76

6.3.4 Mechanical efficiency

There was no significant effect of shortening velocity on initial mechanical efficiency

(Fig. 6.6A). Peak initial efficiency was 31.1 ± 1.3%. Net mechanical efficiency includes

both the initial and recovery processes and there was significant effect of shortening

velocity on net mechanical efficiency (Fig. 6.6A). Peak net mechanical efficiency was

16.9 ± 1.5%. There was no significant effect of shortening velocity on the ratio between

net efficiency and initial efficiency (Fig. 6.6B). The mean ratio was 2.20 ± 0.09.

6.3.5 Ratio of recovery to initial enthalpy output

The ratio of recovery to initial enthalpy output (R/I) quantifies the coupling between

energy supply and energy demand and its value depends on the cost of recovery

metabolism relative to the cost associated with contractile activity. R/I was derived from

the initial and net mechanical efficiency (Equation (22)) and was 1.20 ± 0.09. This ratio

indicates the relative enthalpy change of values associated with PCr hydrolysis and its

0.0 0.5 1.0 1.5 2.0


0

5

10

15

20

25

30

35

Me

ch

an

ica

l e

ffic

ien

cy (

%)

ε initial

ε net

A.

0.0 0.5 1.0 1.5 2.0


0

1

2

3

4ε in

itia

l/εn

et

B.

Fig. 6.6. Initial and net mechanical efficiency. (A) Initial () and net () mechanical efficiency were expressed as functions of

shortening velocity normalised to muscle length. Peak efficiency was ∼31% and

net mechanical efficiency was ∼17%. There was a small but statistically

significant effect (∗) of shortening velocity on net mechanical efficiency. (B)

Variation in the ratio of initial to net mechanical efficiency with shortening

velocity. The mean ratio across all velocities was 2.2. There was no statistically

significant effect of shortening velocity on the ratio of initial and net mechanical

efficiency.

77

oxidative resynthesis. If ∆H for PCr hydrolysis is 34 kJ mol-1

(Woledge & Reilly,

1988), then ∆H for PCr resynthesis must be 1.2 × 34 = 41 kJ mol-1

.

6.3.6 Partitioning energy cost between force-dependent and force-independent components

Initial enthalpy output, measured over the first three contraction cycles, was partitioned

into force-dependent and force-independent components by selectively inhibiting force

output using BDM (Fig. 6.7). Force-independent enthalpy output accounted for 18.6 ±

1.9% (n = 7) of the initial enthalpy output.

0 20 40 60 80 100

FTI (%)

0

20

40

60

80

100

Initia

l h

ea

t (%

)

Fig. 6.7. Example of determining force-independent enthalpy output. Force-independent enthalpy output was determined from the relationship between initial

enthalpy output and force-time integral (FTI). FTI was varied by bathing muscles in Krebs

solution containing increasing concentrations of BDM. Initial enthalpy output is expressed

as a percentage of that measured in the absence of BDM. The lowest force-time integral was

obtained using a combination of 5 mM BDM and 150 mM sucrose. A line was fitted

through the data using the method of least squares; its equation is Relative initial heat = 0.82

× FTI + 13.6 (r2 = 0.97). The value of the y-intercept was used to quantify the force-

independent fraction of initial enthalpy output.

6.4 Discussion

The overall purpose of this part of the study was to characterise the active metabolism

of the mouse papillary muscle. The first point to note is that it was possible to make

reliable measurements of these preparations for the 90 min required to do these

experiments (i.e. 60 min equilibration and 30 min for measurements).

The peak isometric forces produced compared well with other reports of experiments

using left ventricular mouse papillary muscles for mechanical experiments (see Fig 5,

ref. Bluhm et al., 2000; Redel et al., 2002) but appears to be slightly lower than that

78

from papillary muscles from other species. For example, the peak isometric force for rat

muscles is typically between 30 and 50 mN mm-2

(Loiselle & Gibbs, 1979; Baxi et al.,

2000; Peterson et al., 2001). It is interesting to note, however, that there are several

published accounts of experiments with mouse papillary muscles which report very low

forces (eg ∼2−5 mN mm-2

) (He et al., 1997; Meyer et al., 1999; Bluhm et al., 2000;

Golenhofen et al., 2006) under similar conditions to those used in the current study. It

seems likely that the preparations in those studies were either not in good condition or

were incompletely stimulated.

6.4.1 Number of cross-bridge cycles per twitch

Using Equations (29) and (30), the number of ATP molecules split was calculated and

compared to the number of cross-bridges in the same volume of muscle. At 2 Hz and

contracting isometrically, it was calculated that 290 ATP molecules were used in the

unit sarcomere. There are 600 cross-bridges in that volume and if one cross-bridge cycle

involves splitting of one ATP, then the amount of ATP used is consistent with 48% of

the cross-bridges completing one ATP-splitting cycle in each twitch. These values can

also be expressed in µmol per g of muscle mass (i.e. taking into account non-

myofibrillar intracellular volume and extracellular volume). In that case, the amount of

ATP split is:

-1

23 -15

290 1ATP split = × 0.41 = 53 nmol g

6.023 × 10 3.5 × 10 × 1.06×

The cross-bridge concentration, worked out similarly, is 110 nmol g-1

.

The initial enthalpy measurements encompassed just the first three twitches so it is

possible that these calculations are not representative of energetics during more

prolonged activity. To see whether energy use averaged over 20 s can be accounted by a

similar number of ATP splitting cycles per twitch, the calculations were repeated using

the net enthalpy output produced in response to the complete 20 s protocol (i.e. 40

twitches when contraction frequency was 2 Hz). In that case, the denominator was the

molar enthalpy output associated with substrate oxidation, expressed per mol of ATP

formed. The enthalpy of glucose (the exogenous substrate provided in this study)

oxidation is 2820 kJ mol-1

(Crabtree & Nicholson, 1988). If this generates 38 ATP (i.e.

P/O2 ratio of 6.3) then glucose oxidation produces 2820/38 = 74 kJ per mole of ATP.

Substituting 5.6 mJ g-1

twitch-1

for ∆HI and 74 kJ mol-1

for ∆HPCr in Equation (29) gives

the number of ATP used per twitch per unit sarcomere cell of 336, which is equal to

79

56% of the number of cross-bridges. If the energy came from oxidation of endogenous

fat (which gives 76 kJ per mole of ATP) rather than glucose, then the number of ATP

used per twitch per unit sarcomere cell would be 328, which is equal to 55% of the

number of cross-bridges.

The simplest interpretation of this observation is that half the cross-bridges completed

one ATP-splitting cycle in each twitch. That only a fraction of cross-bridges complete

one ATP-splitting cycle has been suggested previously for rabbit papillary muscle (Mast

& Elzinga, 1990; see Discussion following Gibbs & Barclay, 1995) and could be

inferred from estimates of cross-bridge cycling rate for rat papillary muscle (e.g. Hoh et

al., 1988) but the current study is the first to attempt to quantify the fraction. It is

possible that fewer than half the cross-bridges completed more than one cycle, in which

case it might be expected that the number of cross-bridge cycles could be modulated by

events, such as shortening, occurring during the twitch. However, the lack of influence

of shortening velocity on energy cost is consistent with the idea that the amount of

energy to be used is determined early in a twitch (Gibbs & Barclay, 1995) and is not

greatly influenced by events that occur during the contraction. Two factors that strongly

influence twitch force and that are established at the start of a contraction are pre-load

and the amount of Ca2+

entering the cell, each of which, via different mechanisms (e.g.

Yagi et al., 2004), influences the number of cross-bridges that can bind. Contraction

frequency also influences the status of muscles at the start of the twitch by, for instance,

determining how much Ca2+

is available for release from the SR (e.g. Stull et al., 2002).

The enthalpy output observed at 4 Hz was ~60% of the value for 2 Hz, so the number of

cross-bridge ATP splitting cycles would have been reduced similarly. Consistent with

this the mean force output at 4 Hz was reduced by the same fraction as the enthalpy

output relative to that at 2 Hz.

The observation that, across a realistic range of shortening velocities, energy cost was

independent of shortening velocity is equivalent to stating that in a beating heart at

constant pre-load, varying stroke work does not alter energy cost. In terms of Suga’s

time-varying elastance model (for reviews, see Suga, 1990; Gibbs, 1995; Suga, 2003a),

the current papillary muscle protocol was equivalent to varying work output by altering

stroke volume (i.e. shortening) while maintaining a constant pressure−volume area

(PVA); rate of O2 consumption depends on PVA rather than upon stroke work (e.g.

Kameyama et al., 1998; Suga, 2003a; How et al., 2005). Equating energy cost to

80

number of cross-bridge cycles provides a mechanistic insight into this observation: the

number of cross-bridges cycles is not much altered by what the muscle does during the

twitch. Thus, sliding of the contractile filaments, at least when the sliding starts once

force has begun to develop (as in the current study), does not promote additional or

accelerated cross-bridge cycling, as occurs in skeletal muscle. Rather, the number of

cross-bridge cycles that will occur is set early in a twitch, presumably by the pre-load

and by the amount of Ca2+

released.

The twitch energy cost of mouse left ventricular papillary muscles was lower than that

for rat left papillary muscles (∼6 mJ g-1

twitch-1

in mouse (Table 6.2) versus ∼11 mJ g-1

twitch-1

in rat (Table 4.2)) stimulated at a contraction frequency of 2 Hz. In terms of the

analysis presented in this chapter this suggests that a greater fraction of cross-bridges

cycle in each twitch of rat papillary muscle compared to mouse. The number of cross-

bridge cycles in the rat preparations was about 70% of the total number of cross-bridges

in the muscle. ∆HPCr was calculated from the R/I ratio (1.1) and was 35 kJ mol-1

(Woledge & Reilly, 1988) and VM 0.61 in rat myocytes (Barth et al., 1992). It is

possible that the greater number of cross-bridge cycles estimated to occur in twitches of

rat muscles underlies the higher forces reported for these muscles as mentioned earlier

(Section 6.4).

6.4.2 Amount of Ca2+ released from the SR in each twitch

The approach taken to calculating NCB can also be applied to calculate the amount of

Ca2+

cycled through the SR of a mouse cardiac cell in each twitch. This was done by

modifying Equation (29) to take account of Ca2+

pump energetics and combining the

result with Equation (30).

2

2 sI A

CaPCr M

Vkf H NN

H V

ρ+

∆= ⋅

∆ (31)

where k is the fraction of the force-independent enthalpy output associated with Ca2+

pumping (assumed to be the same as in rabbit myocytes, 0.77; Delbridge et al., 1996)

and the factor of 2 reflects the stoichiometry of the SR Ca2+

pump (2 Ca2+

pumped for

each ATP hydrolysed). It should be noted that in mouse cardiac cells >90% of Ca2+

cycling is via the SR Ca2+

pump (Georgakopoulos & Kass, 2001). Substituting

appropriate values, again for the case for a muscle contracting at 2 Hz, gives a value of

98 Ca2+

released per twitch in the sarcomere cylinder volume, which, taking account of

the non-myofibrillar volume of muscle, is equivalent to 18 nmol g-1

twitch-1

. The SR

81

Ca2+

content in mouse myocytes contracting steadily at 0.5 Hz at 22°C has been

estimated to be 43 nmol (g muscle mass)-1

(Terracciano et al., 1998). Thus, the amount

estimated to be released in the current study is probably about half the SR Ca2+

content,

consistent with other estimates (e.g. Delbridge et al., 1996).

If there are 290 ATP-splitting cross-bridge cycles in the sarcomere unit volume in each

twitch and 98 Ca2+

are released into the same volume, then about three cross-bridge

cycles were completed for each Ca2+

released. Most Ca2+

released into muscle cells is

bound: peak free Ca2+

concentration (~0.5−1 nmol g-1

; Gao et al., 1998; Stull et al.,

2002) is <5% of the total Ca2+

released into the cell in each twitch (e.g. 18 nmol g-1

).

The concentration of troponin-C-tropomyosin regulatory units (each of which binds 1

Ca2+

in cardiac muscle) is 25% of the cross-bridge concentration (for a review, see

Gordon et al., 2000), which would be 0.25 × 110 = 28 nmol g-1

. If it were assumed that

all the 18 nmol g-1

Ca2+

released in a twitch were bound to troponin-C, then for mouse

muscle contracting at 2 Hz and 27°C about two-thirds of the regulatory units would

have been occupied by Ca2+

.

The dependence of the number of Ca2+

released per twitch on the assumed magnitude of

the force-independent enthalpy output is shown in Fig. 6.8. The greater the fraction of

enthalpy output that is independent of force generation, the greater the number of Ca2+

ions released. If it were assumed that under physiological conditions, the maximum

amount of Ca2+

that was released into the cell was just sufficient to saturate the

troponin-C Ca2+

binding sites, then this would correspond to 28 nmol Ca2+

g-1

twitch-1

or 150 Ca2+

per sarcomere cylinder. From Fig. 6.8, it can be seen that this would be

consistent with a relative force-independent enthalpy output of ~25%. This would then

be the maximum relative force-independent enthalpy output, which supports the idea

that estimates that were substantially higher than this value may have been in error.

Also shown in Fig. 6.8 is the variation in number of ATP molecules split with

magnitude of force-independent enthalpy output. This number decreases as force-

independent enthalpy output increases but, over the likely range of force-independent

enthalpy output, it is always greater than the number of Ca2+

ions released.

82

Fig. 6.8. Dependence of Ca2+

released and ATP used on

magnitude of force-independent

enthalpy output. The number of Ca

2+ released into a

sarcomere cylinder or the number of ATP

molecules hydrolysed in the sarcomere

cylinder volume per twitch is shown as a

function of the magnitude of the force-

independent enthalpy output (expressed

relative to the total initial enthalpy

output). The horizontal dashed line

corresponds to the number of troponin-C

Ca2+

binding sites in a sarcomere cylinder

in cardiac muscle (i.e. 150 or one-quarter

of the number of cross-bridges; Gordon et

al., 2000). The vertical dotted line is the

relative force-independent enthalpy output

measured in the current study.

0 10 20 30 40 50

Force-independent enthalpy output (%)

0

100

200

300

400

Nu

mb

er

of

mo

lecu

les

ATP

Ca2+

6.4.3 Partitioning of energy between force-dependent and force-independent components

The calculation of the number of ATP molecules split depends on the partitioning of

energy between cross-bridge and non-cross-bridge processes. In this study, it was

assumed that BDM inhibited cross-bridge cycling without affecting Ca2+

cycling (Alpert

et al., 1989; Higashiyama et al., 1994). Alpert et al. (1989) described a comprehensive

set of experiments, using rabbit papillary muscles, that were designed to establish

whether BDM selectively inhibited cross-bridge cycling and those experiments

supported the notion that this was correct. In contrast, the effects of BDM on the

relationship between rate of O2 consumption and pressure−volume area (PVA) for

blood-perfused dog hearts have been interpreted as indicating that BDM acted primarily

by reducing Ca2+

release (Takasago et al., 1997). If that also applied to the papillary

muscles used in this study, then force-time integral and enthalpy output would decline

in proportion so the enthalpy-FTI relation would pass through the origin; this was not so

(Fig. 6.7). It remains possible that the linear relationship between enthalpy output and

FTI reflects a partial inhibitory effect on Ca2+

cycling as well as a direct effect on cross-

bridge cycling; in that case, our estimate of force-independent enthalpy output

represents a lower limit.

Schramm et al. (1994) also used BDM (1−10 mM) to measure force-independent

enthalpy output of guinea pig trabeculae at 37°C. They found that force-independent

83

enthalpy output was 23% of the total energy cost. Gibbs and colleagues (Gibbs et al.,

1988; Kiriazis & Gibbs, 2001) used a different technique for measuring force-

independent enthalpy output: force output was reduced by rapidly shortening muscles

during the latent period between the delivery of the stimulus and the start of the

mechanical response. Using this technique, force-independent enthalpy output was

calculated to account for 25 to 30% of energy cost in papillary muscles from the rabbit

(Gibbs et al., 1988) and rat (Kiriazis & Gibbs, 2001). The cause of the discrepancy in

values from the latency release method and the BDM method is unclear, although it has

been suggested that in the former residual cross-bridge cycling may occur, increasing

estimates of force-independent enthalpy output (Alpert et al., 1989). Further insight into

the force-independent enthalpy output can be gained from the estimated amount of Ca2+

released from the SR in each twitch.


The efficiency of mitochondrial oxidative phosphorylation was estimated as described

previously (Equation (21)). This equation uses the ratio εNet/εI which had a mean value

of 0.45 ± 0.02 (the inverse of the ratio of εI/εNet shown in Fig. 6.6B). Assuming ∆GATP is

59 kJ mol-1

(Table 2.1), ∆HPCr is 34 kJ mol-1

(Woledge & Reilly, 1988) and that the sole

source of uncertainty is the ratio of the efficiencies (a relative error of 0.02/0.45 = 0.04),

then ηR for mouse papillary muscle would be 0.81 ± 0.03. That is, the free energy in

ATP produced in the mitochondria was 81% of the free energy available in the

metabolic substrate. Thus, if exogenous glucose were the sole substrate, with a molar

free energy change of 2878 kJ mol-1

, then the amount of free energy in ATP from one

mole of glucose is (81/100) × 2878 = 2331 kJ mol-1

. If ∆GATP = 59 kJ mol-1

, then each

mole of substrate oxidised produced 2331/59 = 39 moles of ATP. If the O2:substrate

ratio for glucose oxidation is 6, then the P:O2 ratio is 39/6 = 6.5 (± 0.3; i.e. a 4%

uncertainty). This compares favourably with the expected stoichiometry of 6.3 that

arises from production of 38 ATP from each mole of substrate oxidised.

6.5 Conclusion

In conclusion, this study has demonstrated that the energetics of isolated preparations of

mouse cardiac muscle can be satisfactorily determined using the myothermic technique.

By combining a number of approaches used in previous studies, we have calculated that

the energy required for a twitch under the conditions used in this study can be accounted

84

for by ATP-splitting cycles by half the available cross-bridges and cycling of about one

Ca2+

for every three cross-bridge cycles.

85

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uusseedd iinn pprroolloonnggeedd eexxppeerriimmeennttss??

In Chapter 4 alterations in the cellular energy balance in isolated rat papillary muscles

following a period of simulated ischaemia were studied. Before the start of the

ischaemia protocol, the muscles were allowed to equilibrate for 60 min to allow resting

metabolism to decrease to a level that would not compromise the balance between O2

use and supply. The ischaemia protocol consisted of an equilibration period lasting 15

min, followed by 60 min exposure to conditions mimicking ischaemia, before the

preparation was allowed to recover for 30 min. Thus, the minimum duration of an

experiment was 2 hours 45 min. Therefore, the first step for replicating a protocol of

this type with mouse papillary muscles was to demonstrate that muscles would remain

viable (i.e. keep producing force) and, preferably, maintain a relatively stable

mechanical and energetic performance for almost 3 h in vitro. This proved extremely

difficult to achieve. This chapter describes some of the attempts made to improve the

stability of the performance of mouse papillary muscles and concludes with an

assessment of the suitability of mouse papillary muscles studying various aspects of

cardiac function.

86

7.1 The observations

In many trials, using a protocol like that used for the control studies in the study of

ischaemia in rat papillary muscles, it was found to be difficult to maintain a consistent

mechanical performance for 2 or 3 h. Instead, muscle performance often abruptly

decreased at some time after 90 min in vitro. An example of this behaviour is shown in

Fig. 7.1A, in which records made using the same stimulus pattern after 90 min and 155

min in vitro are compared. In the later recording active force output was much reduced

and passive force was elevated. However, this was not always the case, as shown by the

example in Fig. 7.1B. In that example, there was a small increase in passive force output

but little change in active force output over the same time interval. This inconsistent and

unpredictable behaviour would make it very difficult to identify changes due to an

experimental intervention such as ischaemia. A series of experiments was performed to

try to overcome the time-dependent changes in function and these are briefly described

in the following sections.

0

2

4

6

8

Fo

rce

ou

tpu

t (m

N)

10 s

155 min90 min

A.

0

5

10

15

20

10 s

105 min 175 min

B.

Fig. 7.1. Changes in mechanical performance with time. The stability of the mechanical performance of mouse papillary muscle is

illustrated above. A. The muscle was stimulated at a contraction frequency of 2

Hz and first measurement was made 90 min after the muscle was mounted in the

set-up and the force output remained >5 mN throughout the series of

contractions. The next measurement was made 65 min later and at that stage the

force output was reduced by about 80%, or was <1 mN. B. Another preparation

was stimulated at a contraction frequency of 1 Hz. The passive force increased

slightly over time but there was little change in active force output.

87

7.2 Solution

To ensure that the bathing solution was optimal, Krebs-Henseleit solution was made up

fresh on the day of the experiment. Solutions were usually made using deionised water

and a comparison was made with solutions made using more pure, osmotically filtered

water (18.2 Ω, Milli-RO 3 plus and Milli-Q plus 185, Millipore, Australia). However,

this had no discernable effect.

In initial experiments, the Krebs solution contained 1.5 mM Ca2+

. However, with this

solution muscle performance, from the start of experiments, was variable and many

muscles produced very little force. By increasing the concentration to 2.5 mM muscles

consistently produced more force and exhibited prolonged viability (see Section 3.1).

To further enhance the contractility of the muscle much effort was spent comparing

muscle performance using either glucose or pyruvate as the exogenous metabolic

substrate. It is well-accepted that the provision of pyruvate as a substrate has a positive

inotropic effect on cardiac muscle of many species. For instance it has been reported

that pyruvate, when substituted for glucose, increased the isometric force output in

isolated rabbit papillary muscles (Chapman, 1972; Chapman & Gibbs, 1974; Chapman

et al., 1976) and guinea pig trabeculae (Daut & Elzinga, 1989). The enhanced

contractile performance with pyruvate as a metabolic substrate has also been shown in

isolated perfused hearts from guinea pigs (Zweier & Jacobus, 1987; Mallet & Sun,

1999) and rats (Dos Santos et al., 2000). In contrast, the energetics of mouse papillary

muscles in this study was unaltered by substitution of pyruvate for glucose (both at 10

mM) (Table 6.2). This may be a chacteristic of mouse cardiac muscle because a similar

result has been observed in isolated, perfused mouse hearts (Flood et al., 2003). In fact,

in that study isovolumic pressure development was slightly lower in the presence of

pyruvate than glucose.

In a final modification to the bathing solution with the aim of enhancing the success rate

of prolonged experiments, the perfusion medium was supplemented with fetal bovine

serum. The inspiration for this came from a study by Lännergren & Westerblad (1987)

in which skeletal muscle fibre survival and force production improved when serum was

included in the solution. Unfortunately there were no beneficial effects of this on the

mouse papillary muscle.

88

7.3 Experimental set-up

Great care was taken to clean the apparatus thoroughly after each experiment and the

muscle bath was rinsed with both water and ethanol. The apparatus was also taken apart

for extra cleaning on a regular basis. All the tubes supplying the chamber with solution

and gas were also regularly replaced to prevent bacterial growth.

A thin layer of silicone heat transfer compound (Unick, Unick Chemical Corp.) was

always applied between the jaws of the thermopile frame and the thermopile and a

check was made to see whether leaching of this substance into the bathing solution may

have affected muscles. The silicone compound (detailed composition unknown) was

replaced with silicone vacuum grease, which has been used previously on the

thermopile used in this study. However, this change did not alter muscle performance.

The stimulating system was the part of the experimental set-up that was thought to be

the most likely to cause problems with consistent muscle performance. The stimulating

electrode arrangement was modified and replaced multiple times! The original

arrangement consisted of relatively thick platinum electrodes that were positioned to

touch each sides of the preparation. It was thought that this may damage the myocytes.

The first modification was to carefully tie very thin platinum wires (25 µm) in a knot

around each end of the muscle (Yin, 1990). This proved difficult and may also have

exposed the muscles to damage. Finally, the arrangement was altered so that muscles

were stimulated via the platinum clips that were used to connect the muscle to the rods

attached to the force transducer and motor. The stimulating wire was wound in fine

coils around the connecting rod and the hook connecting the muscle to the recording

unit. This proved the most reliable technique although it still proved necessary to

change the wires at regular intervals.

The treatment of the muscles during the stabilisation phase was also investigated. Two

approaches were taken: (1) stimulate muscles at 0.2 Hz for 60 min (as typically used for

rat papillary muscles) or (2) leave preparations unstimulated for the same amount of

time. However, there was no beneficial effect of either of these approaches on the

prolonged function of muscles.

89

7.4 Adequate oxygenation

The depressed active force output and raised passive force observed in some muscles

(Fig. 7.1A) is reminiscent of the effects of ischaemia and reperfusion (e.g. Fig. 4.3)

which lead to the idea that maybe, despite the model predictions, diffusive O2 supply to

the muscles was sometimes inadequate. The PO2 of the solution in the thermopile

chamber used for the resting heat experiments (Chapter 5) was higher than that in the

horizontal thermopile used for the other experiments (0.95 atm and 0.84 atm,

respectively), because the former could be bubbled more vigorously. So a trial was

performed in which some muscles were placed in the vertical chamber bubbling with

95% O2. However, muscles subjected to this protocol performed no better than those

placed in the horizontal thermopile chamber.

7.5 Method of euthanasia

The animals were rendered unconscious by inhalation of 80% CO2–20% O2 gas mixture

(Kohler et al., 1999) and killed by cervical dislocation. The inhalation of CO2 can

potentially lead to myocardial acidosis. However, the animals were only briefly exposed

to the gas before being killed and the hearts were still beating when removed from the

animal and were bright red in colour. There were no signs of consistent increase in force

output during the equilibration period, as might be expected if accumulated H+ were

being gradually removed from the cells (cardiac muscle force output is quite sensitive to

intracellular pH; Vaughan-Jones et al., 1987).

Furthermore, when mice were killed by cervical dislocation alone, in vitro performance

was not improved, supporting the idea that the method of euthanasia was not

compromising the mouse papillary muscles’ performance.

7.6 Assessment of the suitability of isolated mouse papillary muscles for investigating cardiac muscle physiology

The experiments described in this Thesis demonstrate that the isolated mouse papillary

muscle can be used to study cardiac muscle physiology. The preparations produce good

forces and the measured rates of energy use compare well with data from other species.

Furthermore, as long as experiments are of less than ∼2 h duration the inter-preparation

variability was acceptable. It has been demonstrated that the preparation can be used to

explore the integrated functioning of the activation and contraction processes. However,

90

the proviso is that the duration of the in vitro investigation should be at most 2 h. For

longer protocols, such as typically required for studies of ischaemia and reperfusion the

inability to obtain consistently stabile performance limits the usefulness of the

preparation.

It is interesting to note that in a recent study (Golenhofen et al., 2006) papillary muscles

from wild-type and transgenic mice were used to study changes in mechanical

performance during simulated ischaemia. Studying the paper in detail reveals a number

of limitations. (1) The peak active force output of these preparations was 2.5 ± 0.5 and

2.1 ± 0.5 mN mm-2

in wild-type and transgenic mice, respectively. This can be

compared to typical values of 20 mN mm-2

in the current study (see Section 5.3.4 and

6.3.1). A small fraction of this difference can be accounted for by the use of a higher

[Ca2+

] in the current study (2.5 mM versus 1.5 mM) as the force output doubles at the

higher concentration (Redel et al., 2002; Stuyvers et al., 2002) but the comparison

suggests that the muscles in the study by Golenhofen et al. (2006) were either in poor

condition or were inadequately stimulated. (2) Ischaemia was simulated by withdrawal

of glucose and by replacing O2 with 95% N2−5% CO2, but no data has been supplied

explaining the partial pressure of O2 in the chamber. In the current study, it was found

that considerable ingenuity and care was required to get chamber PO2 to very low

values. (3) The study lacked a suitable control group from which the effects of time in

vitro could be distinguished from those due to ischaemia. Instead muscles from wild-

type mice, also subjected to ischaemia, were used as a control for muscles from

transgenic mice. Ideally, these data would have been complemented with measurements

of the mechanical stability of muscles from the wild-type mice under non-ischaemic

conditions.

91

CChhaapptteerr 88:: CCoonncclluuddiinngg ccoommmmeennttss

In this Thesis the first measurements of the metabolism of mouse papillary muscles

have been described. These measurements encompassed both resting and active

metabolism and the partitioning of active metabolism between initial and recovery

reactions and between force-dependent and force-independent processes. In addition to

the work on mouse papillary muscles, there is also a section describing alterations in the

energetics of rat papillary muscles during and after a period of simulated ischaemia. In

this concluding chapter, the highlights of the study are briefly recounted and

experiments that may further address some of the issues raised in each chapter are

suggested.

8.1 Resting metabolism

The most striking outcome of experiments on resting metabolism was the exceptionally

high initial resting metabolic rate, particularly in small muscles (<1 mg). However, in

most respects, the characteristics of the resting metabolic rate of mouse papillary

muscles were similar to those of papillary muscles of other species. For example, it

declined exponentially with time (although with a much shorter time constant than that

for papillary muscles from larger species), was increased by changing substrate from

glucose to pyruvate and by exposure of the muscles to hyperosmotic solutions. The

difference in time course of the decline in resting metabolism between muscles from rat

92

and mouse may provide a clue to the mechanism underlying the decline: is it related, in

some way, to the size of the preparation? Is it diffusion related (in which case the

temperature dependence of the time constant would be quite small)?

Another interesting question from the resting metabolism study was how the resting

metabolism of the heart compares to that of the isolated papillary muscle. The available

data (see Section 5.4.1), obtained indirectly from the relationship between myocardial

O2 consumption and PVA, suggests that resting metabolism of isolated mouse hearts is

higher than the eventual steady values in the isolated papillary muscles. An interesting

project would be to measure resting metabolism of both the isolated heart and the

papillary muscle. Resting metabolism could first be measured in the isolated heart,

taking the transepicardial O2 exchange into account (Loiselle, 1989; Gibbs & Loiselle,

2001), followed by measurement of the resting metabolism of a papillary muscle

dissected from the same heart.

8.2 Active metabolism

Active metabolism of mouse papillary muscles was studied by using both isometric and

realistic contraction protocols. The active metabolic cost per twitch was lower in the

mouse papillary muscle compared to that of the rat papillary muscle but total

metabolic rate at realistic contraction frequencies would be higher in the mouse muscle

due to the higher heart rate of the mouse. Interestingly, there was no effect of pyruvate

on either force output or energy cost and, through reference to published data for

isolated mouse hearts, it was suggested that this may be a characteristic peculiar to

mouse cardiac muscle. The mechanical efficiency of mouse cardiac muscle was found

to be similar to that of cardiac muscle from other species, indicating that the need for

more rapid force dynamics and more rapid energy use and supply in the smaller muscles

does not compromise efficiency.

The enthalpy output of the mouse papillary muscle was used to calculate the number of

cross-bridge cycles that occur in a single twitch. It was concluded that the energy used

in a twitch of mouse papillary muscle can be accounted by cycling of about half the

cross-bridges and the uptake into the SR of about one Ca2+

for every three cross-bridge

cycles. Furthermore, the estimated number of Ca2+

released in a twitch was fewer than

the number of Ca2+

binding sites on troponin-C. Thus, it can be seen that twitch force

can be modulated (both increased and decreased) by varying the amount of Ca2+

93

released or, even in the absence of changes in Ca2+

release, by altering the kinetics of

cross-bridge attachment or detachment.

Overall, the experiments described in Chapter 6 demonstrated that energetic

measurements could be made using mouse papillary muscles and that it is possible to

use these to quantify the molecular and ionic changes that underlie contraction.

Most of the characterisation of mouse papillary muscles was carried out at a sub-

physiological temperature (27°C). This was regarded as a good balance between

preserving physiological properties evident at 37°C while ensuring that diffusive O2

supply is able to match metabolic O2 demands. However, recent modelling of diffusive

O2 supply to isolated papillary muscles (Barclay, 2005) suggested that these

experiments actually are favoured by the physiological temperature. This arises from the

negative temperature dependence of energy cost per twitch (Table 6.3), which

overcomes the small positive temperature dependence of resting metabolism. Therefore,

in future studies, it may be worth attempting to use physiological temperatures.

8.3 Ischaemia and reperfusion

Isolated rat papillary muscles were exposed to 60 min of simulated ischaemia, which

resulted in depressed work and enthalpy output but with no change in efficiency or

activation metabolism. These results suggested that the likely mechanism underlying the

depressed work output in post-ischaemic rat papillary muscle was that fewer cross-

bridges cycled in each contraction. This may come about because of reduced sensitivity

of the myofibrils to the Ca2+

release. Although this argument is supported by other

studies that investigated Ca2+

handling, these conclusions could be supported by further

work. For example, by measurements of Ca2+

transients or by comparing maximum

Ca2+

-activated force output pre- and post-ischaemia. It would also be of interest to

perform a test of the idea that reactive oxygen species might damage the myofibrils,

reducing their Ca2+

sensitivity (Bolli & Marban, 1999) by introducing antioxidants into

the superfusate.

One aim of this study was to investigate the effect of simulated ischaemia using the

mouse papillary muscle. This cannot be recommended under the conditions described

using the rat papillary muscle, as the prolonged stability of the mouse papillary muscle

is in doubt. However, one possibility could be to work out a compromise between the

94

long equilibration period (due to the high resting metabolic rate) and the start of the

ischaemic protocol.

8.4 Conclusion

It has been demonstrated that the mouse papillary muscle is a viable model for studying

energetic aspects of cardiac muscle contraction. This is important because, as a result of

the use of mice for studies involving genetic modifications, the mouse has become the

favoured animal model for work in the field of cardiovascular physiology. This

preparation would be ideal to study the physiological and functional consequences of

heart-focussed genetic manipulations. However, improvements in maintaining

preparation viability in vitro for periods greater than 2 h are required before extending

the scope of this model to ischaemia and reperfusion experiments.

95

AAppppeennddiixx II

The unit sarcomere cylinder is a region bounded transversely by four neighbouring thick

filaments and longitudinally by successive Z-lines and encloses four thin filaments (Fig.

AI. 1). The volume of a sarcomere unit cell was calculated assuming that sarcomere

length was 2.1 µm (see Section 5.2.3) and that the spacing between the thick filaments

was 41 nm (Yagi et al., 2004).

-6 ° -9 2 -15 3Volume sarcomere unit = 2.1 × 10 × sin 60 × (41 × 10 ) = 3.1 × 10 cm

This volume can be used to calculate the concentration of cross-bridges in mouse

papillary muscle. The length of the myosin filament containing cross-bridges is ∼700

nm. There are three myosin molecules (i.e. six cross-bridges) every 14.3 nm along the

filament (Cooke, 1986), giving a total of (700/14.3) × 6 = 294 cross-bridges per myosin

filament. From the geometrical arrangement of the filaments, each myosin filament can

interact with six neighbouring actin filaments so, 294/6 = 49 cross-bridges. As each thin

filament has three adjacent thick filaments, there are 3 × 49 = 147 cross-bridges that can

interact with each thin filament, so this volume contains a total of 588 cross-bridges. In

this case, the cross-bridge concentration in the sarcomere cell is 588/3.1×10-15

= 1.9 ×

1017

cm-3

= 0.32 mM.

Correcting for the volume density of myofibrils in myocytes (0.52 in mouse myocytes;

Barth et al., 1992) and the fraction of muscle volume that is occupied by myocytes (0.79

96

in rat myocardium; Dobson & Cieslar, 1997) the muscle cross-bridge concentration is

0.32 × 0.52 × 0.79 = 0.13 mM.

AI. 1. Unit sarcomere rhombus. Arrangement of thick () and thin (•) filaments in striated muscles (reproduced

from Squire et al., 1990, courtesy L. de Beus). Each myosin filament in a half

sarcomere is surrounded by six equally-spaced thin filaments. The unit

sarcomere rhombus is bounded transversely by four neighbouring thick filaments

and lengthwise by successive Z-lines and encloses four thin filaments, two in

each half of the sarcomere unit. The area enclosed by the four thick filaments is

given by the formula for the area of a rhombus: Area = sin(θ) × l2 where θ is the

internal angle of the sides and l the length of the sides. For mammalian striated

muscle, θ = 60° or 120° and l = 41 nm.

97

AAppppeennddiixx IIII

The energetic and mechanical properties of four right ventricular rat papillary muscles

were measured (Table AII. 1 and AII. 3 and Fig. AII. 2). The muscles were dissected

∼7−11 h after cardiectomy, mounted in the set-up and stabilised for 15 min before the

first measurement. During the stabilisation period, muscle length was adjusted to give

maximal force output (Lmax). The experiments were performed at 27°C and the muscles

stimulated to contract at a frequency of 2 Hz. The contraction protocol lasted for 20 s

and both isometric and realistic contractions were used. The realistic protocol was

similar to that used for measurements with the left ventricular muscle of the rat, that is,

shortening velocity was normalised to muscle length and corresponded to 0.54 Lmax s-1

.

AII. 1. Right rat ventricular papillary muscle characteristics.

Number of preparations 4

Wet muscle mass (mg) 1.2 ± 0.1

Muscle length (mm) 3.1 ± 0.2


Muscle radius (mm) 0.35 ± 0.02

Peak active force (mN mm-2

) 32 ± 2

Twitch time 409 ± 11

Time to peak tension (ms) 184 ± 8

98

Isometric Realistic

Net heat output (mJ g-1

twitch-1

) 7.6 ± 0.6 5.8 ± 0.6

Net work output (mJ g-1

twitch-1

) - 0.8 ± 0.1

Net enthalpy output (mJ g-1

twitch-1

) 7.6 ± 0.6 6.6 ± 0.7

Initial heat output (mJ g-1

twitch-1

) 4.2 ± 0.6 3.0 ± 0.4

Initial work output (mJ g-1

twitch-1

) - 1.00 ± 0.06

Net efficiency (%) - 12.7 ± 0.4

Initial efficiency (%) - 26.3 ± 4.1

R/I - 1.1 ± 0.3

Time constant for recovery heat rate, τ (s) 11 ± 2 11 ± 2

AII. 2. Examples of changes in

recordings of right rat ventricular

papillary muscle with time. Examples of recordings of the change in

muscle length, force output, temperature and

cumulative heat production in rat right

ventricular papillary muscles. This muscle

was mounted in the set-up ∼11 h after

cardiectomy and the first measurement was

made after an equilibration period of 15 min.

The first stimulus pulse resulted in a large

twitch force and the second a much reduced

force output. In the subsequent contractions

there was a gradual build-up of the force back

towards the initial level. A realistic

contraction protocol was used, indicated by

the change in muscle length. The protocol

consisted of a 110 ms long phase where the

muscle ends were held fixed, followed by a

shortening phase lasting 185 ms (amplitude

10% of muscle length), before being

relengthened at constant velocity. The

enthalpy output was recorded both in the form

of heat produced by the muscle and the work

generated. The peak force output was ∼26 mN

mm-2

and force output reached a steady-state

producing ∼24 mN mm-2

. The total amount of

heat and work produced was ∼300 mJ g-1

.

Muscle mass: 1.02 mg; muscle length: 2.9

mm.

-0.3

-0.2

-0.1

0.0

∆ L

en

gth

(m

m)

0

2

4

6

8

10

Fo

rce

(m

N)

0

2

4

6

8

10

∆ T

em

pe

ratu

re (

m°C

)

0 20 40 60 80Time (s)

0

100

200

300

400

En

tha

lpy o

utp

ut

(mJ g

-1)

Heat

Enthalpy

Work

AII. 3. Contractile properties of right papillary muscle.

99

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