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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
•Press down gently until slight deformation of cell membrane 
•Attachment occurs immediately 
Attaching a Cell with MyoTak 
Prosser and Khairallah 
Click Here to View Video
•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 
Click Here to View Video
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 
Click Here to View Video
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 
•Work in progress 
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 
Click Here to View Video
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 
•Work in progress 
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 
Click Here to View Video
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) 
Force 
Length 
1 μN
Force (μN) 
SarcLen (μm) 
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 
200 nN 
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 (or force) 
End-diastole 
(LVP is ‘left ventricular pressure’, LVV is ‘left ventricular volume’)
100 
10 
10~100 
LVV (or cell length) 
LVP (or force) 
Isovolumic Contraction
100 
10 
100 ~ 
LVV (or cell length) 
LVP (or force) 
End-systole 
Ejection Phase
100 
10 
100~10 
LVV (or cell length) 
LVP (or force) 
Isovolumic Relaxation
100 
10 
~10 
LVV (or cell length) 
LVP (or force) 
Complete Pressure-Volume Loop 
Work (J) = Δ P*ΔV 
Diastolic Filling Phase
Modulating Force Development By Changing Cell Length 
length 
force 
(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) 
Click Here to View Video
After-load 
Pre-load 
Force (μN) 
Isometric contraction 
With force clamp 
Time (s) 
Length (μm) 
length 
force 
(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 
force 
(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’ 
Force (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
Rat cardiac myocyte at room temperature 
•Stepping up the pre-load 
•ESFL is unchanged 
Increasing pre-load 
End Systolic Force Length Relation 
Rat cardiac myocyte at room temperature
Rat cardiac myocyte at room temperature 
•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 Systolic Force Length Relation 
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 
Force (μN) 
after-load 
pre-load 
No BDM 
5 mM BDM
Switch to 5 mM BDM 
Force (μN) 
Sarc Len (μm) 
Length change (μm) 
Time (s) 
(data at room temperature) 
Length change 
Force (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
Force 
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 
Work (pJ) 
(after) load (μN) 
Isometric (w = 0) 
Isotonic (w=0) 
Force 
Length 
w=ΔF.Δl
Force-Length Loops & Mechanical Work 
4 Hz / 240bpm 
8 Hz / 480bpm 
6 Hz / 360bpm 
Work (pJ) 
(after) load (μN) 
Force (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 
Power (pJ.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
ACCESS THE RECORDING AND SUPPLIMENTARY MATERIALS FOR THIS EVENT AND OTHERS AT 
http://goo.gl/SbcfX5 
JOIN OUR GROUP ON LINKEDIN FOR INFORMATION ON UPCOMING EVENTS, ON-DEMAND WEBINARS, AND ADDITIONAL LAB RESOURCES
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.

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Measuring Force in Single Heart Cells

  • 1.
  • 2. 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.
  • 3. 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
  • 4. 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
  • 5. 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
  • 6. 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
  • 7. How Does Stretch Regulate Subcellular Ca2+ Signaling? Iribe et al., Circ Res. 2009 Fluo-4 Loaded Rat Ventricular Myocyte Calcium Spark Rate
  • 8. 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
  • 9. 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
  • 10. 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
  • 11. 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
  • 12. 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
  • 13. 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
  • 14. •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
  • 15. •Press down gently until slight deformation of cell membrane •Attachment occurs immediately Attaching a Cell with MyoTak Prosser and Khairallah Click Here to View Video
  • 16. •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 Click Here to View Video
  • 17. 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 Click Here to View Video
  • 18. 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
  • 19. • 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
  • 20. 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
  • 21. •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
  • 22. •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 20 hz 1 hz Force Sarcomere length Calcium 2 hz
  • 23. 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 Click Here to View Video
  • 24. 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 •Work in progress Improved assay for cardiac force vs. length relationships
  • 25. 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!
  • 26. 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
  • 27. 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.
  • 28. 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
  • 29. Reinvigorated with Myotak •Myotak Glue (Prosser et al., Science 2011) •Measuring force development in mice and rats •Triple the force •Development MyoStretcher Click Here to View Video
  • 30. Basic Layout of The Myostretcher 3D micromanipulator optical rail, microscope mount arms to reach experimental chamber
  • 31. Cell Chamber View Force Probe Piezo Motor System on a Microscope
  • 32. How to measure force? Fiber bending or force transducer?
  • 33. •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
  • 34. 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
  • 35. 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
  • 36. •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) Force Length 1 μN
  • 37. Force (μN) SarcLen (μm) 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)
  • 38. How low can we go? Myofibrils 200 nN 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!
  • 39. 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?
  • 40. 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)
  • 41. 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
  • 42. 100 10 10 LVV (or cell length) LVP (or force) End-diastole (LVP is ‘left ventricular pressure’, LVV is ‘left ventricular volume’)
  • 43. 100 10 10~100 LVV (or cell length) LVP (or force) Isovolumic Contraction
  • 44. 100 10 100 ~ LVV (or cell length) LVP (or force) End-systole Ejection Phase
  • 45. 100 10 100~10 LVV (or cell length) LVP (or force) Isovolumic Relaxation
  • 46. 100 10 ~10 LVV (or cell length) LVP (or force) Complete Pressure-Volume Loop Work (J) = Δ P*ΔV Diastolic Filling Phase
  • 47. Modulating Force Development By Changing Cell Length length force (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
  • 48. •Initially isometric, no movement piezo •Enabling force control; piezo starts to correct Controlling force (top) with length changes (bottom) Click Here to View Video
  • 49. After-load Pre-load Force (μN) Isometric contraction With force clamp Time (s) Length (μm) length force (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
  • 50. length force (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’ Force (μN) Length (μm) Force vs length
  • 51. 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
  • 52. Rat cardiac myocyte at room temperature •Stepping up the pre-load •ESFL is unchanged Increasing pre-load End Systolic Force Length Relation Rat cardiac myocyte at room temperature
  • 53. Rat cardiac myocyte at room temperature •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 Systolic Force Length Relation End Diastolic Force Length Relation
  • 54. •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 Force (μN) after-load pre-load No BDM 5 mM BDM
  • 55. Switch to 5 mM BDM Force (μN) Sarc Len (μm) Length change (μm) Time (s) (data at room temperature) Length change Force (μN) pre-load No BDM 5 mM BDM
  • 56. 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
  • 57. Force Length Real-Time Force vs. Length Loops •Instantaneous feedback on the loop quality Force Length
  • 58. Same cell, Same Protocol, 4 Hz / 240 BPM…
  • 59. From Force-Length Loops to Power Curves 4 Hz / 240bpm Work (pJ) (after) load (μN) Isometric (w = 0) Isotonic (w=0) Force Length w=ΔF.Δl
  • 60. Force-Length Loops & Mechanical Work 4 Hz / 240bpm 8 Hz / 480bpm 6 Hz / 360bpm Work (pJ) (after) load (μN) Force (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®
  • 61. Constructing The Power Curve Of A Cardiac Myocyte Power (pJ.s-1) Physiological heart rates Freq (Hz)
  • 62. 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
  • 63. (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!
  • 64. Thank You Vumc Amsterdam: Prof. J van der Velden A. Najafi VU Physics Department: Prof. D. Iannuzzi E. Breel IonOptix: T. Udale
  • 65. 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
  • 66. ACCESS THE RECORDING AND SUPPLIMENTARY MATERIALS FOR THIS EVENT AND OTHERS AT http://goo.gl/SbcfX5 JOIN OUR GROUP ON LINKEDIN FOR INFORMATION ON UPCOMING EVENTS, ON-DEMAND WEBINARS, AND ADDITIONAL LAB RESOURCES
  • 67. 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.