measuring force in single heart cells

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Measuring Force and Mechano-Signaling in Single Muscle Cells

Benjamin L. Prosser, PhD

Department of Physiology

Pennsylvania Muscle Institute

Perelman School of Medicine

University of Pennsylvania

Copyright InsideScientific & IonOptix. All Rights Reserved.

Image courtesy of Science Signaling, AAAS

Why measure force in muscle cells?

• Assay intrinsic cellular mechanisms that drive cardiac performance

– Frank-starling, Anrep (Slow Force Response), Force-frequency relationships

• Evaluate the interplay between mechanical, electrical, and biochemical signaling

• Cardiomyocyte physiology and pathology changes under mechanical load!

a b c d f

0.1µm

1µN

[Ca2+i]

Force

Sarcomere

Length

1s

0.5 µN

1 min

8% ΔL

a

b

c d

f

FSM

SFR

FSM SFR

Single myocyte force

Mechanotransduction

Mechanisms of cellular mechano-transduction?

• The process of converting mechanical stimuli into cellular responses

• The heart experiences acute and chronic mechanical stimuli

• Strain (preload), stress (afterload), compression, torsion, shear

Strength/duration of mechanical stimulus, genetic predisposition

physiological hypertrophy, increased [Ca2+]i and contractility

pathological remodeling, [Ca2+]i instability, oxidative stress, arrhythmia, heart failure

Physiological/Adaptive Pathological/Maladaptive

Techniques for stretching heart cells…

1. Apply hydrostatic or osmotic pressure

2. Stick cells to flexible membranes (Pimentel et al., 2001) (Petroff et al., 2001)

3. Poke cells with a glass stylus (Dyachenko, Isenberg et al., 2009)

4. Suck cells into pipettes (Zeng, Bett & Sachs 2000, Palmer and Frindt 1996)

5. Attach them to micromanipulators Carbon fibers (Le Guennec et al., 1990, Yasuda et al., 2001, Iribe et al., 2006)

carbon fiber

Limitations • Non-physiological

• Lack of dynamic control

• Unable to measure force

• Unreliable attachment, low throughput

Our goals • Improve strength and reliability

of attachment

• Directly measure force under physiological conditions

How Does Stretch Regulate Subcellular Ca2+ Signaling?

Iribe et al., Circ Res. 2009

Fluo-4 Loaded Rat Ventricular Myocyte Calcium Spark Rate

Before Coating

MyoTak™

After Coating

Glass Micro-Rods

20 µm

Patch

pipette

Length

Controller

MyoTak Force

Transducer

4 s

• Stiff optical-fiber glass rods (25 μm diameter)

• Coated with biocompatible adhesive

• Simultaneously control length, record force, and monitor cellular signaling

MyoTak™ to Assay Mechano-Transduction

Prosser, Ward, Lederer

Before Coating

MyoTak™

After Coating

Glass Micro-Rods

20 µm

Δ Length

Fluo-4 Calcium

Force 4 s

10 µm

2 ΔF/F0

1 µN

• Stiff optical-fiber glass rods (25 μm diameter)

• Coated with biocompatible adhesive

• Simultaneously control length, record force, and monitor cellular signaling

MyoTak™ to Assay Mechano-Transduction

Prosser, Ward, Lederer

MyoTak™ Biological Adhesive

• Mimics physiological cell attachment to extracellular matrix (bio-compatible)

• Two primary components:

1. Mix of extracellular matrix proteins optimized for viscosity and stickiness in solution and at temperature

2. Rough 1 μm proteinaceous “pre-coat” that increases surface area of contact between cell membrane and MyoTak coated rod

to force transducer

to length controller

cardiomyocyte

MyoTak coated micro-rods

A’ B’

25 µm 25 µm

Coating micro-rods with MyoTak™

• Proper coating is everything!

• 2 step process: 1) coating with pre-coat, 2) coating with glue.

• Should be done under the microscope

• Dip rods in 1-2μl drop of pre-coat

• 30s – 2 minute dip in pre-coat

• Air dry: > 30 minutes is ideal, but not necessary

Step One – Pre-coat A - uncoated

B – pre-coated

100 µm

100 µm

Step Two – Glue-coat

A

B

Coating micro-rods with MyoTak™

• Monitor viscosity of glue

• 1-5 minutes depending on temperature, age of glue

• Visible bubble of glue on rod tip immediately after withdrawing from glue

• 1-2 minute air dry

• Once hydrated, keep hydrated!

B’ A’

Glue in air Before glue

Before glue

Glue in solution

A B

100 µm 100 µm

25 µm

Coating micro-rods with MyoTak

• Single coat should last 2-4 hours

• Glue can be washed off in 10% acetic acid

• Rods can be re-used

Finished Product

Glue

Pre-coat

• 2-10μm layer of glue

• Rods oriented parallel to cell membrane

3D reconstruction of fluorescent MyoTak coated rods attached to cardiomyoctye

Prosser et. al., Science 2011

Click Here to View Video

http://youtu.be/knCT_69Ps-s

• Press down gently until slight deformation of cell membrane

• Attachment occurs immediately

Attaching a Cell with MyoTak

Prosser and Khairallah



• Passive stretch experiments simple and straighforward

• Active contraction measurements require more skill

• Introduce slack, stimulate cell, allow it come to steady state, then stretch to desired diastolic length

Stretching a Cell with MyoTak

Prosser and Khairallah



Prosser et. al., Science 2011

• Monitoring sarcomere length provides confidence in robust attachment

1 vs. 4 Hz rhythmic stretch

Prosser et al., Cardiovascular Research 2013



1. Mechanical stretch rapidly enhances calcium release mechanisms

2. Microtubule cytoskeleton transduces the mechanical signal

3. Stretch rapidly increases the production of reactive oxygen species (ROS) by Nox2

4. ROS act on ryanodine receptor calcium channels to enhance calcium release

Stretch Relax

ΔF/F0

5 s

Stretch-dependent ROS and calcium signaling Prosser et al., Science 2011; Prosser et al. Cardiovascular Research 2013; Khairallah et al., Science Signaling 2013; Iribe et al., Circ Res 2009

X vs. T surface plot - Calcium sparks

• Stretch triggers arrhythmogenic calcium waves in Duchenne Muscular Dystrophy model

• Conserved stretch-dependent mechanism that also regulates calcium homeostasis in skeletal muscle

Cardiomyopathy (DCM)

ΔF/F0

5 s

Stretch Relax

Rate of ROS

(dDCF/dt)

1 A.U.

DCM

wt

ROS

5s

10 A.U.

DCM

wt

8% stretch

DCM

rat cardiomyocytes

mouse myofiber

flexor digitorum brevis CD-1 - 8 weeks age

• Glass rods insufficient to maintain attachment of contracting skeletal muscle

• Different attachment modality required to accommodate much larger forces

Stretching Skeletal Muscle

• MyoTak coated laser-etched cell holder

• Allows precise control of skeletal muscle length, assay of larger forces

• Work in progress

Controlling length and measuring force in skeletal muscle fibers

Chris Ward, Jackie Kerr

Myofiber

channel

Myotak coated cell holder

• MyoTak coated laser-etched cell holder

• Allows precise control of skeletal muscle length, assay of larger forces


Controlling length and measuring force in skeletal muscle fibers

Chris Ward, Jackie Kerr

20 hz 1 hz

Force

Sarcomere length

Calcium

2 hz

8μm

30μm

Optical force transducer and laser-etched cell holder

Force

transducer Piezo Myocyte holder coated with MyoTak-647

Prosser and Helmes (Ionoptix)

0.2μN

0.2 s

Raw force recording



Glass Rod Cell Holder

cell

z

y

x

cell

myotak

Glass Rod

Cell holder

• Etched concavity cups over cell

• Greatly increases surface area of attachment


Improved assay for cardiac force vs. length relationships

Summary…

• The isolated, intact cardiac myocyte is an ideal model to study

physiologically relevant mechanics and mechano-signaling

• New tools provide a robust, high-throughput assay of

mechano-signaling in heart cells

• Proper coating and practice are key!

Acknowledgements

Prosser Lab • Patrick Robison

• Alexey Bogush

• Michael Neinast

University of Maryland • Jon Lederer – BioMET

• Skeletal Muscle crew:

– Chris Ward

– Jackie Kerr

– Ramzi Khairallah – (Now Loyola University Chicago)

Funding • National Heart, Lung, and Blood Institute, National

Institutes of Health (NHLBI, NIH)

• National Institute of Arthritis and Musculo-Skeletal Disease (NIAMS), NIH

Technical Development • Michiel Helmes, Ionoptix

• Konrad Gueth, Harm Knot

• Siskiyou

Developing a Force Transducer for Single Myocyte Experimentation Measuring The Power Curve of a Heart Cell

Michiel Helmes PhD

Department of Physiology VU University Medical Center

Amsterdam & IonOptix

Copyright InsideScientific & IonOptix. All Rights Reserved.

A Brief History…

(LeGuennec et al. JMCC 1990, Iribe et al, Am J Phys 2006, King et al J Gen Phys 2010, Chuan et al. Bioph J 2012)

Challenges…

• No commercially available supply of carbon fibers

• Equipment was complicated

• Low forces

We have been able to measure force for a while

Reinvigorated with Myotak

• Myotak Glue (Prosser et al., Science 2011)

• Measuring force development in mice and rats

• Triple the force

• Development MyoStretcher



Basic Layout of The Myostretcher

3D micromanipulator

optical rail, microscope mount

arms to reach experimental chamber

Cell Chamber View

Force Probe Piezo Motor

System on a Microscope

How to measure force? Fiber bending or force

transducer?

• on pressure lead

• Force measurements using fiber bending are cheap, cheerful and reliable

• cannot control length very well

• Calibration is difficult

• Classic force transducer are not very suitable for this force range

• Air-water interface creates drift problems

• Relatively low resonance frequency, susceptible to noise, slow response times

Fiber bending

Force transducer

Turning an Interferometer into a Force Transducer

• Measures distance between optical fiber and cantilever with nm accuracy

• Displacement x spring constant = force

• Optical, submersible

mouse myocyte, room temperature

0.5

μN

‘Classic’ force transducer (ASI 403)

Early prototype of OptiForce

0.5

μN

cantilever

attachment needle

read out fiber

• Optical

• Fully submersible

• nN sensitivity ( <1 nN possible)

• High resonance frequency (8kHz)

• Stable baseline

IonOptix OptiForce, Revolutionary New Class of Force Transducer

Front view

• Force response to moderate stretches

• Used to establish EDFL and ESFL (end-diastolic and end-systolic force length relation)

Length Dependent Activation in a Rat Myocyte (@ 37°C)

Forc

e

Len

gth

1 μ

N

Forc

e (μ

N)

Sarc

Len

(μ

m)

Detecting very subtle changes in force development

• Excellent signal-to-noise

• Unfiltered data

• Notable drop in diastolic force when switching from 2 to 1 Hz pacing

(mouse myocyte, room temperature, switch from 2 Hz to 1 Hz pacing frequency)

How low can we go? Myofibrils

20

0 n

N

Sarcomere length

Force

Motor Displacement

(single myofibril, skeletal muscle, RT)

• Myofibrils

• Cardiac iPS (induced Pluripotent Stem-Cells)?

How Low Can We Go?...

How High Can We Go?... The read-out is independent from the probe, we can make probes for any force level! • Trabeculea

• Skeletal muscle

• Whatever you can think of!

What can you do with a fast, stable and sensitive force transducer?

Force

Cell Length

Modulate the force generation within a contractile cycle by stretching or

shortening the myocytes

Could we mimick the cardiac cycle by controlling pre- and after-load using feed-back?

Pressure-Volume Loops Single Cell Work Loops

• Pressure curve

• Volume (ejection) curve

• Combine to create PV loop

• Just a reminder…

(from: ‘Cardiovascular physiology concepts’ by R. Klabunde)

The cardiac cycle

Aorta

Left Atrium

Mitral Valve

Aortic Valve

Left Ventricle

(cardiac cycle animations courtesy of Dr. Gentaro Iribe)

• With a simple model of the ventricle

100

10

10

LVV (or cell length)

LVP

(o

r fo

rce)

End-diastole

(LVP is ‘left ventricular pressure’, LVV is ‘left ventricular volume’)

100

10

10~100


LVP

(o

r fo

rce)

Isovolumic Contraction

100

10

100 ~


LVP

(o

r fo

rce)

End-systole

Ejection Phase

100

10

100~10


LVP

(o

r fo

rce)

Isovolumic Relaxation

100

10

~10


LVP

(o

r fo

rce)

Complete Pressure-Volume Loop Work (J) = Δ P*ΔV

Diastolic Filling Phase

Modulating Force Development By Changing Cell Length

length

forc

e

(I)

(II)

(III)

(IV)

(I) Start contraction, Pre-load > force < afterload Do nothing

force > afterload Shorten the cell

End of active contraction Pre-load > force < afterload Do nothing

Diastole Force < pre-load Stretch the cell

(IV)

(II)

(III)

Algorithm used to create work loops:

motor

force

:

After load

Pre load

• Initially isometric, no movement piezo

• Enabling force control; piezo starts to correct

Controlling force (top) with length changes (bottom)



After-load

Pre-load

Forc

e (

μN

) Isometric contraction

With force clamp

Time (s)

Len

gth

(μ

m)

length

forc

e

(I)

(II)

(III)

(IV)

After load

Pre load

• Dissecting the force trace in 4 phases

• Force max and min user defined

• Length changes modulate force

length

forc

e

(I)

(II)

(III)

(IV)

After load

Pre load

• Blue: isomertric contraction, no work

• Red: force control creates the work loop

motor

After-load

Pre-load

Mechanical work = Force x length = area in loop, ‘work loop’

Forc

e (

μN

)

Length (μm)

Force vs length

Isometric isotonic Varying the after-load

force

length

• Shallow loops: almost isotonic

• Continuously increasing afterload

• Establishes the ESFL

• At very low lenghts not linear

Rat cardiac myocyte at room temperature

End Systolic Force Length Relation


• Stepping up the pre-load

• ESFL is unchanged

Increasing pre-load




• Third pre-load level

• Establishes the EDFL

• ESFL is unchanged

• Frank-Starling in a single cell

The Frank Starling Law of the Heart at the Myocyte Level


End Diastolic Force Length Relation

• BDM inhibits active force development

• BDM infusion improves relaxation

• Length increase leads to force increase

• Doubles effective work

Effect of low levels of BDM on diastolic dysfuntion

(data at room temperature) Length change

Forc

e (

μN

)

after-load

pre-load

No BDM 5 mM BDM

Switch to 5 mM BDM

Forc

e (

μN

) Sa

rc L

en

(μ

m)

Len

gth

ch

ange

(μ

m)

Time (s)

(data at room temperature) Length change

Forc

e (

μN

)

pre-load

No BDM 5 mM BDM

Improving the experiment…

Force

Length Protocol:

Pre-load

After-load

(rat cardiac myocytes, 37°C, paced at 2 Hz)

1. temperature control

2. automated force level changes using built in signal generators

Forc

e

Length

Real-Time Force vs. Length Loops

• Instantaneous feedback on the loop quality

Force

Length

Same cell, Same Protocol, 4 Hz / 240 BPM…

From Force-Length Loops to Power Curves

4 Hz / 240bpm

Wo

rk (

pJ)

(after) load (μN)

Isometric (w = 0)

Isotonic (w=0)

Forc

e

Length

w=ΔF.Δl

Force-Length Loops & Mechanical Work 4 Hz / 240bpm 8 Hz / 480bpm 6 Hz / 360bpm

Wo

rk (

pJ)

(after) load (μN)

Forc

e (

μN

)

Length (μm)

Top: Force-length loops Bottom: mechanical work plotted for each contraction

• Repeated for 1, 2, 4, 6 and 8 Hz

• Preparation is stable

• Analyzing the work from each FL-loops

• Using LabChart®

Constructing The Power Curve Of A Cardiac Myocyte

Po

we

r (p

J.s-1

)

Physiological heart rates

Freq (Hz)

Summary…

• We have developed a force transducer that bridges the gap between AFM (pN) and classic force transducers (uN and up)

• It has been designed for compatibility with physiology experiments

• Here we use the transducer we can do force control at the myocyte level

• We can now mimic the cardiac cycle at the single myocyte level and measure the power a myocyte can generate

(from J. Spudich, Bioph J, 2014) HCM is recognized as hyper contractile, suggesting that the power output is higher than that of the normal heart. Conversely, the clinical features of DCM patients are characterized by reduced systolic function, … , leading to lower output than that of the normal heart. … therapies could be directed toward either reducing the power output or increasing it… Life, however, is not that simple

But at least we now have another good tool to study it!

Thank You

Vumc Amsterdam:

Prof. J van der Velden A. Najafi

VU Physics Department:

Prof. D. Iannuzzi E. Breel

IonOptix: T. Udale

Thank You!

For additional information on Force Measurements, Calcium & Contractility Experiments, Cell Pacing, Myocyte Harvesting, and Tissue Bath Fluorometry please visit:

http://goo.gl/C7oADl

http://www.ionoptix.com/

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InsideScientific is an online educational environment designed

for life science researchers. Our goal is to aid in the sharing and

distribution of scientific information regarding innovative

technologies, protocols, research tools and laboratory services.

http://www.insidescientific.com/