effects of halothane, isoflurane, sevoflurane and desflurane on contraction of ventricular myocytes...

5266062.xml Molecular and Cellular Biochemistry 5266062 February 20, 2004 16:7 TECHBOOKS

UNCORRECTEDPROOF

Molecular and Cellular Biochemistry xxx: 1–7, 2004.c© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

Effects of halothane, isoflurane, sevoflurane anddesflurane on contraction of ventricular myocytesfrom streptozotocin-induced diabetic rats

1

2

3

Mark Graham,1 Anwar Qureshi,2 Rabiah Noueihed,3 Simon Harrison1

and Frank Christopher Howarth24

51School of Biomedical Sciences, University of Leeds, UK; 2Department of Physiology, United Arab Emirates University, AlAin, U.A.E.; 3Department of Anaesthesia & Intensive Care, Tawam Hospital, Al Ain, U.A.E.

67

Abstract8

Various clinically used volatile general anaesthetics (e.g. sevoflurane, halothane, isoflurane and desflurane) have been shownto have significant negative inotropic effects on normal ventricular muscle. However, little is known about their effects inventricular tissue from diabetic animals. Streptozotocin (STZ)-induced diabetes is known to induce changes in the amplitudeand time course of shortening and one report suggests that the inotropic effects of anaesthetics are ameliorated in papillarymuscles from diabetic animals. The aim of these studies was to investigate this further in electrically stimulated (1 Hz) ventricularmyocytes. Cells were superfused with either normal Tyrode (NT) solution or NT containing anaesthetic (1 mM) for a period of2 min (at 30–32◦C). Myocytes from STZ rats were shown to have a significantly longer time to peak shortening (p > 0.001, n =50) and the amplitude of shortening tended to be greater but this was not significant (p = 0.13, n = 50). Halothane, isoflurane,desflurane and sevoflurane significantly (p < 0.05) reduced the magnitude of shortening of control cells by 72.5 ± 3.2%, 46.5± 9.7%, 28.9 ± 4.3% and 22.8 ± 5.6%, respectively (n > 11 per group) but their steady-state negative inotropic effect wasfound to be no different in cells from STZ-treated rats (73.0 ± 4.8%, 40.7 ± 4.7%, 25.0 ± 5.2% and 19.8 ± 5.2%, respectively,n > 10 per group). Therefore, we conclude that the inotropic effects of volatile anaesthetics were not altered by STZ treatment.(Mol Cell Biochem xxx: 1–7, 2004)

9101112131415161718192021

Key words: volatile anaesthetics, contraction, halothane, isoflurane, sevoflurane, desflurane, diabetes, heart, streptozotocin,ventricular myocytes

2223

Introduction24

Administration of streptozotocin (STZ) to young rats causes25selective necrosis of pancreatic insulin-producing β-cells.26Some of the metabolic defects seen in STZ-treated rats27including hypoinsulinaemia, hyperglycaemia, glucosuria,28polydipsia, polyphagia, hypercholesterolaemia and hyper-29triglyceridaemia can also be seen in Type 1 patients [1]. De-30fective contractile function of cardiac muscle is frequently31observed in STZ-induced diabetic rats [1]. In ventricular my-32ocytes these defects include prolonged time course and re-33

Address for offprints: Dr F. C. Howarth, Department of Physiology, Faculty of Medicine & Health Sciences, United Arab Emirates University, P.O. Box 17666,Al Ain, U.A.E. (E-mail: [email protected])

duced amplitude of shortening [2–5]. Altered mechanisms 34of Ca2+ transport including altered activity of sarcolemmal 35Na+/Ca2+ exchange, Ca2+ ATPase, Na+/K+ ATPase, sar- 36coplasmic reticulum (SR) Ca2+ content, uptake and release 37partly underlie these contractile defects [2, 4, 6]. 38

Previous data have shown that the negative inotropic effect 39of halothane and isoflurane was blunted in papillary muscles 40isolated from the left ventricle of streptozotocin-induced dia- 41betic rats [7]. We have carried out further experiments on iso- 42lated single ventricular myocytes to extend these observations 43and also to include two additional anaesthetics, sevoflurane 44


UNCORRECTEDPROOF

2

and desflurane. Our aim was to assess whether anaesthetic-45induced negative inotropy was ameliorated in STZ-treated46animals and whether this occurred at the level of the ventric-47ular myocyte.48

Materials and methods49

Induction of diabetes50

Diabetes was induced by a single i.p. injection of STZ (6051mg · kg−1 body weight; Sigma) administered to young male52Wistar rats (200–250 g; bred in-house). STZ was dissolved in53a citrate buffer solution (0.1 mM citric acid, 0.1 mM sodium54citrate; pH 4.5). Age-matched controls received an equiva-55lent volume of the citrate buffer solution alone. Both groups56of animals were maintained on the same diet and water ad57libitum until they were used 8–12 weeks later. Principles of58laboratory animal care were followed throughout. Approval59of this project was obtained from the Faculty of Medicine60& Health Sciences, United Arab Emirates University, Ethics61Committee.62

Ventricular myocyte isolation63

Single ventricular myocytes were isolated according to pre-64viously described techniques with minor modifications [3,658]. In brief, rats were killed humanely by decapitation with66a guillotine. Hearts were then removed rapidly, mounted on67a Langendorff perfusion apparatus and perfused retrogradely68at a constant flow of 8 ml · g−1 (heart · min)−1 with a HEPES-69based salt solution (isolation solution—see below) contain-70ing 0.75 mM Ca2+. Perfusion flow rate was adjusted to allow71for differences in heart weight between STZ-treated and con-72trol animals. When the coronary circulation was cleared of73blood, perfusion was continued for 4 min with Ca2+-free iso-74lation solution containing 0.1 mM EGTA, and then for 6 min75with the solution containing 0.05 mM Ca2+, 0.75 mg · ml−1

76collagenase (type 1; Worthington, NJ) and 0.075 mg · ml−1

77protease (type X1V; Sigma). After this time, the ventricles78were excised from the heart, minced and gently shaken in79collagenase-containing isolation solution supplemented with80

Table 1. General characteristics of STZ-treated and control animals

Control STZ

Body weight (g) 276 ± 10 (11) 238 ± 9 (6)∗Heart weight (g) 1.15 ± 0.03 (11) 1.07 ± 0.02 (6)Blood glucose (mg · dl−1) 86.4 ± 3.0 (11) 345.2 ± 41.7 (6)∗∗

Data are mean ± SEM, numbers in parenthesis indicate number ofanimals. Independent samples t-test, ∗p < 0.05, ∗∗p < 0.001.

1% BSA. Cells were filtered from this solution at 4-min 81intervals and resuspended in isolation solution containing 820.75 mM Ca2+. 83

Measurement of shortening 84

Ventricular myocytes were allowed to settle on the glass bot- 85tom of a Perspex chamber mounted on the stage of an inverted 86

Fig. 1. (A) Fast time base records of myocyte shortening from control andSTZ-treated rats. Mean data (± S.E.M.) describing myocyte shortening (ex-pressed as a % of resting cell length, RCL); (B) time to half peak (THALFPK)shortening; (C) time to peak (TPK) shortening; (D) time to half (THALF)relaxation; and (E) for myocytes isolated from control (open bars) and STZ-treated (hashed bars) rats. ∗∗p < 0.01, independent samples t-test betweencontrol and STZ. n = 50 myocytes for both control (from 4 rats) and STZ(from 3 rats) groups.


UNCORRECTEDPROOF

3

microscope (Axiovert 35, Zeiss, Germany). Electrically87stimulated (1 Hz) myocytes maintained at 30–32◦C were88superfused (3–5 ml · min−1) with HEPES-based NT solution89pH adjusted to 7.4. Once contractility had reached steady90state, the superfusate was changed to NT solution containing91either halothane, isoflurane, sevoflurane or desflurane at a92concentration of 1 mM. This concentration of each anaes-93thetic was used to induce robust negative inotropic effects94for all four anaesthetics for comparative purposes; however,95it should be noted that this concentration would exceed96that usually achieved during clinical practice. After 2-min97exposure to anaesthetic solution the superfusing solution was98returned to control HEPES-based NT. Unloaded shortening99was used as an index of contractility [9]. The shortening of100myocytes was followed using a video-edge detection system101(VED-114, Crystal Biotech, Northborough, MA). Amplitude102of shortening (expressed as a percentage of resting cell103length; RCL), time to peak (TPK) shortening and time from104peak to half (THALF) relaxation were measured using Signal105

Fig. 2. (A) Slow time base recordings of cell shortening before during and following a 2-min exposure to 1 mM halothane (Hal) in control and STZ myocytes.Mean data (± S.E.M.) of (B) cell shortening and (C) TPK shortening for myocytes isolated from control (open bars) and STZ-treated (hashed bars) ratssuperfused with normal tyrode (NT) solution or in the presence of 1 mM halothane. +p < 0.05, ++p < 0.01, paired t-test, NT versus halothane; ∗p < 0.05, ∗∗p< 0.01 independent samples t-test, control versus STZ (n = 14 control and 11 STZ myocytes, taken from 4 control and 3 STZ-treated animals).

Averager v 6.37 software (Cambridge Electronic Design 106Ltd., UK) [3, 8]. 107

Solutions 108

The cell isolation solution contained (in mM) 130.0 NaCl, 1095.4 KCl, 1.4 MgCl2, 0.4 NaH2PO4, 5 HEPES, 10 glucose, 11020 taurine and 10 creatine set to pH 7.3 with NaOH. The 111NT solution contained (in mM): NaCl 140; KCl 5; MgCl2 1; 112glucose 10; HEPES 5; CaCl2 1 set to pH 7.4 with NaOH. 113

Statistical analysis 114

Results are expressed as the mean ± S.E.M of n observations. 115Statistical comparisons of the effects of STZ-induced dia- 116betes and effects of exposure to anaesthetics were performed 117using either independent samples t-test or paired samples t- 118test (SPSS, v 11.0). P values less than 0.05 were considered 119significant. 120


UNCORRECTEDPROOF

4

Results121

General characteristics of STZ-treated rats122

The general characteristics of STZ-treated rats compared123with their age-matched controls are shown in Table 1. Dia-124betes was confirmed in STZ-treated rats by a significant, ∼4-125fold, elevation of blood glucose similar to that observed pre-126viously. STZ-treated rats had significantly lower body weight127compared with controls; heart weight tended to be reduced128in STZ-treated animals but this did not reach significance129(p = 0.096).130

General characteristics of ventricular myocytes131from control and STZ-treated rats132

There were no clear visual differences between rod-shaped133myocytes from control and STZ-treated animals. Resting cell134length (RCL) of control myocytes was 127 ± 3 µm (n = 50)135

Fig. 3. (A) Slow time base recordings of cell shortening before during and following a 2-min exposure to 1 mM sevoflurane (Sevo) in control and STZmyocytes. Mean data (± S.E.M.) of (B) cell shortening and (C) TPK shortening for myocytes isolated from control (open bars) and STZ-treated (hashed bars)rats superfused with NT solution or in the presence of 1 mM sevoflurane. +p < 0.05, ++p < 0.01, paired t-test, NT versus sevoflurane; ∗∗p < 0.01 independentsamples t-test, control versus STZ (n = 12 control and 14 STZ myocytes taken from 3 control and 3 STZ-treated animals).

and was significantly (p < 0.05) reduced by STZ-treatment 136(RCL of STZ myocytes, 118 ± 3 µm, n = 50). Figure 1A 137illustrates fast time base records of shortening from a repre- 138sentative ventricular myocyte isolated from a control and an 139STZ-treated rat. Although the amplitude of shortening tended 140to be greater in STZ cells (Fig. 1B) this did not reach statisti- 141cal significance. Figures 1C–1E show mean data describing 142the time course of contractile shortening in cells from control 143and STZ-treated rats. Figures 1C and 1D show that there was 144significant (p < 0.001) prolongation in both time to half peak 145(THALFPK) and TPK shortening but no significant effect of 146STZ treatment on THALF relaxation (Fig. 1E). 147

Effects of anaesthetics on amplitude and time course 148of shortening 149

Figure 2A shows slow time base records of ventricular 150myocyte shortening before during and following a 2-min 151exposure to 1 mM halothane in a representative control and 152STZ cell. Application of halothane led to an initial increase 153


UNCORRECTEDPROOF

5

Fig. 4. (A) Slow time base recordings of cell shortening before during and following a 2-min exposure to 1 mM isoflurane (Iso) in control and STZ myocytes.Mean data (± S.E.M.) of (B) cell shortening and (C) TPK shortening for myocytes isolated from control (open bars) and STZ-treated (hashed bars) ratssuperfused with NT solution or in the presence of 1 mM isoflurane. ++p < 0.01, paired t-test, NT versus isoflurane; ∗∗p < 0.01, independent samples t-testbetween control and STZ (n = 12 control and 12 STZ myocytes taken from 3 control and 3 STZ-treated animals).

in contractile shortening which then declined to a steady state154for the duration of the exposure as has been observed previ-155ously [10, 11]. Contractions returned to pre-anaesthetic levels156on removal of halothane. Figure 2B illustrates the extent of157shortening (expressed as a percentage of resting cell length)158in a group of control and STZ cells. As described above, there159was no significant difference in the amplitude of contraction160between control and STZ myocytes. Following equilibration161with halothane, the amplitude of shortening was significantly162(p < 0.01) reduced by 72.5 ± 3.2% (n = 14) and 73.0 ± 4.8%163(n = 11) in control and STZ myocytes, respectively. Figure1642C shows that halothane significantly (p < 0.05) reduced the165TPK shortening in both control [10, 11] and STZ myocytes166consistent with halothane increasing the off-rate constant for167Ca2+ from troponin-C [10, 11], however, the STZ-induced168prolongation of TPK observed in anaesthetic-free solution169was maintained in the presence of halothane.170

Figure 3A shows slow time base records of cell shorten-171ing during an exposure to 1 mM sevoflurane. Application of172sevoflurane led to a decrease in the magnitude of shortening173

(Fig. 3B) but to a lesser extent than observed with halothane. 174Shortening was significantly reduced by 22.8 ± 5.6% (p < 1750.05, n = 12) in control cells and 19.8 ± 5.2% (p < 0.01, n 176= 14) in STZ cells. On removal of sevoflurane, contractions 177increased above the pre-anaesthetic level before returning to 178steady state. The prolongation of TPK induced by sevoflu- 179rane was still evident in the presence of sevoflurane (Fig. 3C); 180however, sevoflurane induced a shortening of TPK in control 181cells only. 182

Figures 4 and 5 illustrate the effects of 1 mM isoflurane 183and desflurane, respectively on contraction in control and 184STZ cells. Both anaesthetics led to a significant reduction in 185contractility which was readily reversed upon removal of the 186anaesthetic. Isoflurane reduced contractions by 46.5 ± 9.7% 187(p < 0.01, n = 12) in control cells and by 40.7 ± 4.7 (p < 1880.01, n = 12) in STZ cells (Fig. 4B), whereas desflurane had a 189lesser inhibitory effect reducing contractions by 28.9 ± 4.3% 190(p < 0.01, n = 12) in control compared to 25.0 ± 5.2% (p < 1910.01, n = 13) in STZ cells (Fig. 5B). As for halothane, isoflu- 192rane and desflurane significantly (p < 0.01) reduced the TPK 193


UNCORRECTEDPROOF

6

Fig. 5. (A) Slow time base recordings of cell shortening before during and following a 2-min exposure to 1 mM desflurane (Des) in control and STZ myocytes.Mean data (± S.E.M.) of (B) cell shortening, (C) TPK shortening for myocytes isolated from control (open bars) and STZ-treated (hashed bars) rats superfusedwith NT solution or in the presence of 1 mM desflurane. ++p < 0.01, paired t-test, NT versus desflurane; ∗∗p < 0.01, independent samples t-test betweencontrol and STZ (n = 12 control and 13 STZ myocytes taken from 3 control and 3 STZ-treated animals).

shortening in both control and STZ myocytes however the194STZ-induced prolongation of TPK observed in anaesthetic-195free solution was maintained.196

Discussion197

The main defect observed in myocytes from diabetic rats was198a prolonged time-course of contraction (Fig. 1A) a finding199that is consistent with several previous studies [2–5]. Altered200mechanisms of Ca2+ transport including altered activity of201sarcolemma membrane Na+/Ca2+ exchange, Ca2+ ATPase,202Na+/K+ ATPase, SR Ca2+ content, uptake and release partly203underlie these contractile defects [2, 4, 6]. However, the main204aim of these studies was to assess the effects of volatile anaes-205thetics on contractility given that previous data have sug-206gested that halothane and isoflurane had a reduced inhibitory207effect in papillary muscle from STZ-treated compared to con-208trol rats [7].209

The mechanisms underlying the steady-state negative in-210otropic effects of these anaesthetics in healthy ventricular211

muscle have yet to be fully elucidated; however, for halothane 212and isoflurane, this is thought to result from a combination of 213reduced myofilament Ca2+ sensitivity [11–13] and reduced 214cytosolic Ca2+ transient [11, 12, 14, 15], secondary to a re- 215duction in the inward L-type Ca2+ current, ICa [16–18]. For 216sevoflurane and desflurane, less is known but sevoflurane ap- 217pears to have little sustained effect on myofilament Ca2+ sen- 218sitivity [13] and induces only a modest reduction of ICa [19] 219which paradoxically appears to have little impact on the Ca2+ 220content of the sarcoplasmic reticulum [13] suggesting that 221Ca2+ efflux pathways may also be inhibited. Detailed experi- 222ments concerning the mechanisms of action of desflurane on 223myofilament Ca2+ sensitivity and cytosolic Ca2+ handling 224are still outstanding. 225

Data in Figs. 2 to 5 illustrate that there was no difference in 226the extent to which halothane, isoflurane, sevoflurane or des- 227flurane reduced contraction magnitude at steady-state in my- 228ocytes from control and STZ-treated rats. The reason for this 229discrepancy is unclear as many similarities exist between the 230earlier [7] and current study such as the dose of STZ adminis- 231tered to induce diabetes, resultant plasma glucose levels, the 232


UNCORRECTEDPROOF

7

age of the animals at induction of diabetes, the period of time233following induction before the animals were used and the234temperature at which the two studies were carried out (30–23532◦C). Furthermore, qualitatively similar effects of diabetes236on the magnitude and time course of contraction were ob-237served in this compared to other reports. Therefore, the reason238for this disparity is not evident though could reflect different239experimental preparations, i.e. papillary muscles versus iso-240lated myocytes. For example, papillary muscles have intact241endothelium whereas isolated ventricular myocytes do not. It242has been shown that STZ-induced diabetes leads to increases243in plasma endothelin-1 levels and that the positive inotropic244effect of endothelin-1 is greater in atrial muscle from diabetic245rats compared to control animals [20]. Therefore, it is pos-246sible that following STZ treatment the amelioration of the247negative inotropic effect of anaesthetics in papillary muscle248[7] may be mediated via anaesthetic effects on modified en-249docardial epithelium rather than directly modulating mech-250anisms of excitation–contraction coupling at the level of the251myocyte.252

In summary, exposure to volatile anaesthetics reduces con-253traction of ventricular myocytes; halothane was the most po-254tent negative inotropic agent, followed by isoflurane, desflu-255rane and sevoflurane. However, we found that contraction256was diminished to a similar extent in ventricular myocytes257isolated from control and STZ-treated rats and therefore con-258clude that the direct negative inotropic effects of these agents259at the level of the ventricular myocyte are not altered by STZ260treatment.261

Acknowledgements262

This research was supported by grants from the Faculty of263Medicine & Health Sciences, United Arab Emirates Uni-264versity, the Medical Research Council, UK and the British265Council.266

References267

1. Dhalla NS, Pierce GN, Innes IR, Beamish RE: Pathogenesis of cardiac268dysfunction in diabetes mellitus. Can J Cardiol 1: 263–281, 1985269

2. Choi KM, Zhong Y, Hoit BD, Grupp IL, Hahn H, Dilly KW,270Guatimosim S, Lederer WJ, Matlib MA: Defective intracellular Ca2+271signaling contributes to cardiomyopathy in Type 1 diabetic rats. Am J272Physiol 283: H1398–H1408, 2002273

3. Howarth FC, Qureshi MA, Bracken NK, Winlow W, Singh J: Time- 274dependent effects of streptozotocin-induced diabetes on contraction of 275ventricular myocytes from rat heart. Emirates Med J 19: 35–41, 2001 276

4. Yu Z, Tibbits GF, McNeill JH: Cellular functions of diabetic cardiomy- 277ocytes: Contractility, rapid-cooling contracture, and ryanodine binding. 278Am J Physiol 266: H2082–H2089, 1994 279

5. Ren J, Davidoff AJ: Diabetes rapidly induces contractile dysfunctions 280in isolated ventricular myocytes. Am J Physiol 272: H148–H158, 1997 281

6. Hattori Y, Matsuda N, Kimura J, Ishitani T, Tamada A, Gando S, 282Kemmotsu O, Kanno M: Diminished function and expression of the 283cardiac Na+-Ca2+ exchanger in diabetic rats: Implication in Ca2+ over- 284load. J Physiol 527: 85–94, 2000 285

7. Hattori Y, Azuma M, Gotoh Y, Kanno M: Negative inotropic effects of 286halothane, enflurane, and isoflurane in papillary muscles from diabetic 287rats. Anesth Analg 66: 23–28, 1987 288

8. Howarth FC, Qureshi MA, White E: Effects of hyperosmotic shrink- 289ing on ventricular myocyte shortening and intracellular Ca2+ in 290streptozotocin-induced diabetic rats. Pflugers Arch 444: 446–451, 2002 291

9. White E, Boyett MR, Orchard CH: The effects of mechanical loading 292and changes of length on single guinea-pig ventricular myocytes. J 293Physiol 482: 93–107, 1995 294

10. Wheeler DM, Rice RT, du Bell WH, Spurgeon HA: Initial contractile 295response of isolated rat heart cells to halothane, enflurane and isoflurane. 296Anesthesiology 86: 137–146, 1997 297

11. Harrison SM, Robinson MR, Davies LA, Hopkins PM, Boyett MR: 298Mechanisms underlying the inotropic action of the general anaesthetic 299halothane on intact rat ventricular myocytes. Br J Anaesth 82: 609–621, 3001999 301

12. Hanley PJ, Loiselle DS: Mechanisms of force inhibition by halothane 302and isoflurane in intact rat cardiac muscle. J Physiol 506: 231–244, 3031998 304

13. Davies LA, Gibson CN, Boyett MR, Hopkins PM, Harrison SM: Effects 305of isoflurane, sevoflurane, and halothane on myofilament Ca2+ sensitiv- 306ity and sarcoplasmic reticulum Ca2+ release in rat ventricular myocytes. 307Anesthesiology 93: 1034–1044, 2000 308

14. Bosnjak ZJ, Kampine JM: Effects of halothane on transmembrane po- 309tentials, calcium transients and papillary tension in the cat. Am J Physiol 310251: H374–H381, 1986 311

15. Wheeler DM, Rice RT, Lakatta EG: The action of halothane on sponta- 312neous contractile waves and stimulated contractions on isolated rat and 313dog heart cells. Anesthesiology 72: 911–920, 1990 314

16. Bosnjak ZJ, Supan FD, Rusch NJ: The effects of halothane, enflurane, 315and isoflurane on calcium current in isolated canine ventricular cells. 316Anesthesiology 74: 340–345, 1991 317

17. Eskinder H, Rusch NJ, Supan FD, Kampine JP, Bosnjak ZJ: The effects 318of volatile anesthetics on L- and T-type calcium channel currents in 319canine Purkinje cells. Anesthesiology 74: 919–926, 1991 320

18. Pancrazio JJ: Halothane and isoflurane preferentially depress a slow in- 321activating component of Ca2+ channel current in guinea-pig myocytes. 322J Physiol 494: 94–103, 1996 323

19. Park WK, Pancrazio JJ, Suh CK, Lynch C III: Myocardial depressant 324effects of sevoflurane. Anesthesiology 84: 1166–1176, 1996 325

20. Tada H, Muramatsu I, Nakai T, Kigoshi S, Miyabo S: Effects of chronic 326diabetes on the responsiveness to endothelin-1 and other agents of rat 327atria and thoracic aorta. Gen Pharmacol 25: 1221–1228, 1994 328

effects of halothane, isoflurane, sevoflurane and desflurane on contraction of ventricular myocytes...

Documents