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Measuring Work in Single Isolated Cardiomyocytes:
Replicating the Cardiac Cycle
Andy Henton
InsideScientific
Sponsored by:...
InsideScientific is an online educational environment
designed for life science researchers. Our goal is to aid in
the sha...
Measuring Work in Single Isolated
Cardiomyocytes: Replicating the
Cardiac Cycle
Michiel Helmes PhD
Department of Physiolog...
IonOptix MyoStretcher
 Attach, Stretch, and Record Force in Isolated Cardiac Myocytes
 Create “Work-Loops” and measure p...
This webinar IS NOT about PV-loops!
• What we will be discussing is how to measure mechanical work in single
intact cardio...
Where did the journey start?
• Le Guennec et al, ‘90
• Force measurements
on isolated intact
myocytes
• Carbon fibers, rea...
Where did the journey start?
• Le Guennec et al, ‘90
• Force measurements
on isolated intact
myocytes
• Carbon fibers, rea...
• Yasuda in ‘01, and
Nishimura in ’04
• Bending of carbon
fibers to measure
force
• This is a first attempt
at force contr...
• In 2006, Iribe et. al
use carbon fibers
with feed forward
control
• It works, but is slow
• Equally important,
forces ar...
• Work-loops of a single myocyte, constructed using feed-forward control of force
• Feed-forward vs feed-back
The journey ...
• Feed-back instead of feed-forward would have been ideal, but
couldn’t be done
• The end of the road for carbon fibers an...
Reinvigorated interest
with MyoTak
• MyoTak Glue is introduced as a cell
adhesive (Prosser et al., Science 2011)
• Mimics ...
Basic Layout of The MyoStretcher
3D micromanipulator
optical rail, microscope mount
arms to reach
experimental chamber
• on pressure lead
We wanted force
control/force clamps, but…
• Force measurements
using fiber bending are
not suitable fo...
cantilever
attachment
needle
read out fiber
• Optical
• Fully submersible
• nN sensitivity
• High resonance
frequency (8kH...
Raw data from a rat myocyte undergoing a stretch and subsequent release while being paced at 2 Hz
This force transcuer is ...
Cell Chamber View
Force ProbePiezo Motor
System on a Microscope
1. MyoTak -- to attach the cells
2. Mechanics -- to pick up and stretch the myocyte
3. Force transducer -- to get an accur...
The cardiac
cycle
Aorta
Left Atrium
Mitral Valve
Aortic Valve
Left Ventricle
(cardiac cycle animations courtesy of Dr. Gen...
100
10
10
LVV (or cell length)
LVP(orforce)
End-diastole
(LVP is ‘left ventricular pressure’, LVV
is ‘left ventricular vol...
100
10
10~100
LVV (or cell length)
LVP(orforce)
Isovolumic Contraction
100
10
100 ~
LVV (or cell length)
LVP(orforce)
End-systole
Ejection Phase
100
10
100~10
LVV (or cell length)
LVP(orforce)
Isovolumic Relaxation
100
10
~10
LVV (or cell length)
LVP(orforce)
Pressure-Volume Loop
Work (J) = Δ P*ΔV
Modulating Force Development By Changing Cell Length
length
force
(I)
(II)
(III)
(IV)
(I)
Start contraction,
Pre-load > fo...
d ba c d
Lengthchange(μm)Force(µN)
* Mouse myocyte, room temperature
d
b
ac
afterload
preload
Force(uN)
Length change
Firs...
1. MyoTak -- to attach the cells
2. Lighter mechanics & faster piezo –to pick up and stretch the myocyte with precision an...
Afterload
Preload
Force(μN)
Isometric contraction
With force clamp
Time (s)
Length(μm)
length
force
(I)
(II)
(III)
(IV)
Af...
length
force
(I)
(II)
(III)
(IV)
Afterload
Preload
• Control is good at RT
• Square loops
• No correction for
arterial res...
3 0 4 0 5 0 6 0
1
2
3
4
5
L e n g th ( m )
Force(N)
2 .0 2 .5 3 .0 3 .5 4 .0
0
5
1 0
1 5
A fter-L o ad ( N )
Work(pJ)
V...
1. MyoTak -- to attach the cells
2. Lighter mechanics & faster piezo –to pick up and stretch the myocyte with precision an...
Improving the experiment…
Force
Length
Typical protocol:
Pre-load
After-load
(rat cardiac myocytes, 37°C, paced at 2 Hz)
•...
Recording @ 2 Hz
Recording @, 4 Hz
Recording @ 8 Hz
SL = 1.98 µm 2.03 µm2.02 µm
Force
Length
Varying pre- and afterloadForceLength
End Diastolic and End Systolic force length...
• Measurements on intact loaded myocytes have come a long way
• The development of a revolutionary new force transducer al...
• Work-loop ≠ PV-loop; more sophisticated algorithms needed
The infrastructure is in place
• Force measurements need to be...
Improved attachment with the IonOptix cell holders
Slide courtesy of Ben Prosser, U. of Pennsylvania
images courtesy of Be...
Images courtesy of Ben Prosser, U. of Pennsylvania
• Laser etched cell holder
• Cavity is formed to accomodate
myocyte
• Currently 30 micron opening, 10
micron depth, can be...
1. Because it was a cool thing to do?
2. Myocytes are more accessible than muscle strips
• Ease of use
• No extra-cellular...
Post-rest potentiation, constant length
8 Hz
Post-rest potentiation has a diastolic and systolic component
• At constant l...
Post-rest potentiation, work loops
Post-rest potentiation has a diastolic and systolic component
force
sarc len
length
1 H...
Change in work-loops when switching from 8Hz to 1 Hz
rat myocyte, 37˚C
How do work-loops amplify changes in diastole?
• Linear end systolic and end
diastolic force length relation
• Changes in ...
• BDM inhibits cross
bridge formation, ESFL
goes down
• But also improves
relaxation, so EDFL will
go down as well
• The d...
The effect on length
when force is constant
Switch to 5 mM BDM
Force(μN)SarcLen(μm)Lengthchange(μm) Time (s)
• Myocoyte wi...
Length control:
Decreased performance
Force control:
Improved performance
A different perspective
• BDM depresses both the...
Why do “work-loops”? continued…
• The external work done by a myocyte encompasses
changes in both systolic and diastolic f...
How to maximize the measurable effect of a drug treatment
Effect of 100nM Isoproterenol
• Work-loop measurements can
show ...
Determining the work maximum for each preload
0
2
4
6
8
0 2 4 6
0
10
20
30
40
50
60
0 5 10
Work(pJ)
Power(pW)
After-load (...
• 100nM Iso increases work/loop 2-4
fold (n = 10)
• Compared to a 50-75% increase in
isometric force (trabeculae at 37˚C)
...
Work-loop measurements lend themselves well...
• To establish the maximum amount of work a cell can produce
• Detect chang...
I’d like to thank:
• Aref Najafi – who did most of the actual experiments
• Prof. Jolanda van der Velden – in whose group ...
Michiel Helmes, PhD
michiel@ionoptix.com
Thank You!
For additional information on solutions for high speed
quantitative fl...
Follow us on
Join our group
InsideScientific is an online
educational environment designed
for life science researchers.
O...
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Measuring Work in Single Isolated Cardiomyocytes: Replicating the Cardiac Cycle

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A special webinar for basic cardiovascular researchers interested in a novel technique for measuring work output and replicating the four phases of the cardiac cycle at the single cell level.

The study of isolated cardiac myocytes provide a wealth of basic cellular and molecular information without the complications often associated with heterogeneous multicellular preparations. The overwhelming majority of data presented in myocyte studies, however, are reported in mechanically unloaded conditions. Join us for a practical demonstration of an exciting new technique where mechanical control of the cell reveals the myocyte's force-length relationship by varying pre- and afterload to achieve isometric, isotonic, and, ultimately, work-loop style contractions analogous to the pressure-volume relationship in whole heart studies.

In this exclusive webinar sponsored by IonOptix, Michiel Helmes presents methodology and best-practices that scientists should follow in order to replicate the cardiac cycle in an isolated cardiomyocyte. He discusses how this research method can be used to better address contractile function in cardiovascular disease studies and highlight critical features of the IonOptix MyoStretcher system that are important for this emerging and novel technique.

Published in: Science
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Measuring Work in Single Isolated Cardiomyocytes: Replicating the Cardiac Cycle

  1. 1. Measuring Work in Single Isolated Cardiomyocytes: Replicating the Cardiac Cycle Andy Henton InsideScientific Sponsored by: Michiel Helmes, PhD VUMC & IonOptix
  2. 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. 3. Measuring Work in Single Isolated Cardiomyocytes: Replicating the Cardiac Cycle Michiel Helmes PhD Department of Physiology VU University Medical Center Amsterdam & IonOptix Copyright 2015 IonOptix & InsideScientific. All Rights Reserved.
  4. 4. IonOptix MyoStretcher  Attach, Stretch, and Record Force in Isolated Cardiac Myocytes  Create “Work-Loops” and measure power output  Use the MyoStretcher to Investigate: – Accurate diastolic calcium – Auxotonic and isometric contractions – Length-dependent activation – Force-velocity relationship Thank you to our event sponsor
  5. 5. This webinar IS NOT about PV-loops! • What we will be discussing is how to measure mechanical work in single intact cardiomyocytes, and how a simple model of the cardiac cycle can be created • The resulting “work-loops” are analogous to PV-loops in that they provide information about the contractile properties of the myocyte, and by extension, heart function What we will cover today: • History, recent developments, and a review of experimental results for isolated cardiomyocyte “work-loops” to date • The Technique: what we CAN do and CANNOT do at the bench-top • Why “work-loops” are valuable and why we should do them Before we get started:
  6. 6. Where did the journey start? • Le Guennec et al, ‘90 • Force measurements on isolated intact myocytes • Carbon fibers, really low force levels
  7. 7. Where did the journey start? • Le Guennec et al, ‘90 • Force measurements on isolated intact myocytes • Carbon fibers, really low force levels
  8. 8. • Yasuda in ‘01, and Nishimura in ’04 • Bending of carbon fibers to measure force • This is a first attempt at force control The journey continues... Nishimura, S. et al. AJP - Heart and Circulatory Physiology 2004 Vol. 287 no. 1, H196-H202
  9. 9. • In 2006, Iribe et. al use carbon fibers with feed forward control • It works, but is slow • Equally important, forces are still too low The journey continues... Le Guennec → Ed White → Peter Kohl
  10. 10. • Work-loops of a single myocyte, constructed using feed-forward control of force • Feed-forward vs feed-back The journey continues... Le Guennec → Ed White → Peter Kohl A B
  11. 11. • Feed-back instead of feed-forward would have been ideal, but couldn’t be done • The end of the road for carbon fibers and feed-forward force control? • It did set up a collaboration with the Lederer lab in Baltimore though The first set of challenges…
  12. 12. Reinvigorated interest with MyoTak • MyoTak Glue is introduced as a cell adhesive (Prosser et al., Science 2011) • Mimics physiological cell attachment to extracellular matrix and is bio-compatible • In parallel, IonOptix upgrades the MyoStretcher system to force transducer to length controller cardiomyocyte MyoTak coated micro-rods JY Le Guennec → Ed White → Peter Kohl → Gentaro Iribe → Jon Lederer, Chris Ward and Ben Prosser
  13. 13. Basic Layout of The MyoStretcher 3D micromanipulator optical rail, microscope mount arms to reach experimental chamber
  14. 14. • on pressure lead We wanted force control/force clamps, but… • Force measurements using fiber bending are not suitable for feed- back; data rate is too slow • Classic muscle physiology force transducers? Problems with sensitivity and stability in this force range • We had to come up with something better -> develop our own force transducer Fiber bending Force transducer
  15. 15. cantilever attachment needle read out fiber • Optical • Fully submersible • nN sensitivity • High resonance frequency (8kHz) • Stable baseline IonOptix OptiForce, Revolutionary New Class of Force Transducer Front view
  16. 16. Raw data from a rat myocyte undergoing a stretch and subsequent release while being paced at 2 Hz This force transcuer is suitable for developing a force control system at the nN level Optical force transducer that bridges the gap between AFM & regular force transducers
  17. 17. Cell Chamber View Force ProbePiezo Motor System on a Microscope
  18. 18. 1. MyoTak -- to attach the cells 2. Mechanics -- to pick up and stretch the myocyte 3. Force transducer -- to get an accurate, stable and reliable force signal 4. Hardware and software -- so the force transducer and piezo can interact (you can only control force by modulating myocyte length) – ex. LabView 5. Algorithm – sequence that more or less mimics the cardiac cycle that can be executed via #4 What you need to do force control and generate work loops
  19. 19. The cardiac cycle Aorta Left Atrium Mitral Valve Aortic Valve Left Ventricle (cardiac cycle animations courtesy of Dr. Gentaro Iribe) • Schematic of cardiac cycle and construction of PV-loop
  20. 20. 100 10 10 LVV (or cell length) LVP(orforce) End-diastole (LVP is ‘left ventricular pressure’, LVV is ‘left ventricular volume’)
  21. 21. 100 10 10~100 LVV (or cell length) LVP(orforce) Isovolumic Contraction
  22. 22. 100 10 100 ~ LVV (or cell length) LVP(orforce) End-systole Ejection Phase
  23. 23. 100 10 100~10 LVV (or cell length) LVP(orforce) Isovolumic Relaxation
  24. 24. 100 10 ~10 LVV (or cell length) LVP(orforce) Pressure-Volume Loop Work (J) = Δ P*ΔV
  25. 25. 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) First algorithm used to create work loops: motor force After load Pre load
  26. 26. d ba c d Lengthchange(μm)Force(µN) * Mouse myocyte, room temperature d b ac afterload preload Force(uN) Length change First work-loops with feed-back based force control
  27. 27. 1. MyoTak -- to attach the cells 2. Lighter mechanics & faster piezo –to pick up and stretch the myocyte with precision and speed 3. Force transducer -- to get an accurate, stable and reliable force signal 4. Hardware and software – upgraded to an FPGA (a programmable, embedded chip designed for real time control) to increase the frequency with which we can run the control algorithm 5. Algorithm – sequence that BETTER mimics the cardiac cycle that can be executed via #4 What you need to do force control and generate work-loops well
  28. 28. Afterload Preload Force(μN) Isometric contraction With force clamp Time (s) Length(μm) length force (I) (II) (III) (IV) Afterload Preload • Force clamps • Improved end- systolic switch • Pacing mark initiates new loop • Improved speed of algorithm and motor
  29. 29. length force (I) (II) (III) (IV) Afterload Preload • Control is good at RT • Square loops • No correction for arterial resistance motor Afterload Preload Mechanical work = Force x length = area in loop, ‘work-loop’ Force(μN) Length (μm) Force vs length
  30. 30. 3 0 4 0 5 0 6 0 1 2 3 4 5 L e n g th ( m ) Force(N) 2 .0 2 .5 3 .0 3 .5 4 .0 0 5 1 0 1 5 A fter-L o ad ( N ) Work(pJ) Varying afterload at a fixed preload Mechanical work as a function of afterload (rat myocyte, RT) It worked, but better controls were needed for repeatable experiments
  31. 31. 1. MyoTak -- to attach the cells 2. Lighter mechanics & faster piezo –to pick up and stretch the myocyte with precision and speed 3. Force transducer -- to get an accurate, stable and reliable force signal 4. Hardware and software – upgraded to an FPGA (a programmable, embedded chip designed for real time control) to increase the frequency with which we can run the control algorithm 5. Algorithm – sequence that BETTER mimics the cardiac cycle that can be executed via #4 6. Control -- the ability to automatically set pre- and afterload levels based on actual force transient 7. Programming -- Implementation of signal generators in software so changes in pre- and afterload can be programmed 8. Temperature control! The final (?) additions to a complete solution…
  32. 32. Improving the experiment… Force Length Typical protocol: Pre-load After-load (rat cardiac myocytes, 37°C, paced at 2 Hz) • Automated selection of pre- and afterload based on force trace • Pre-defined changes in pre- and afterload using signal generators -> Necessary tools to explore the parameter space of preload, afterload and pacing frequency or to do repeated measurements
  33. 33. Recording @ 2 Hz
  34. 34. Recording @, 4 Hz
  35. 35. Recording @ 8 Hz
  36. 36. SL = 1.98 µm 2.03 µm2.02 µm Force Length Varying pre- and afterloadForceLength End Diastolic and End Systolic force length relation
  37. 37. • Measurements on intact loaded myocytes have come a long way • The development of a revolutionary new force transducer allows feed-back based force control on the myocyte level • We have used it to develop a system that can now reproducibly measure work- loops in myocytes • The work-loop algorithm mimics the the cardiac cycle (in a simplistic way) • We can vary the preload, afterload at will Summary so far...
  38. 38. • Work-loop ≠ PV-loop; more sophisticated algorithms needed The infrastructure is in place • Force measurements need to be transformed into stress Measuring cross sectional area reliably is difficult on a standard microscope • Compliance in the attachment of the cell limits the usefulness of the End Diastolic and End Systolic Force Length relation • Do we cover the physiological sarcomere length range? With the current attachment strength we can measure work-loops up to 2.1 µm SL Remaining Challenges...
  39. 39. Improved attachment with the IonOptix cell holders Slide courtesy of Ben Prosser, U. of Pennsylvania images courtesy of Ben Prosser, U. of Pennsylvania
  40. 40. Images courtesy of Ben Prosser, U. of Pennsylvania
  41. 41. • Laser etched cell holder • Cavity is formed to accomodate myocyte • Currently 30 micron opening, 10 micron depth, can be adjusted • Increases the attachment surface for the myocyte • Much stronger connection, less compliance Improved attachment with the IonOptix cell holders
  42. 42. 1. Because it was a cool thing to do? 2. Myocytes are more accessible than muscle strips • Ease of use • No extra-cellular matrix. Pro or con? • Ease of access for imaging and perfusion; you can ask very detailed scientific questions 3. work-loops are very useful in detecting changes in diastolic properties Why do “work-loops” on single cells?
  43. 43. Post-rest potentiation, constant length 8 Hz Post-rest potentiation has a diastolic and systolic component • At constant length, the systolic component (increased calcium release) dominates the change in signal • The change in diastolic force (lower calcium level through prolonged re-uptake) is relatively small force sarc len length 4 Hz
  44. 44. Post-rest potentiation, work loops Post-rest potentiation has a diastolic and systolic component force sarc len length 1 Hz8 Hz • With force clamps diastolic, systolic, and force are kept constant (except for an increased force overshoot due to imperfect control) • Length, instead of force, is the dependent variable and big changes in both diastole and systole can now be observed
  45. 45. Change in work-loops when switching from 8Hz to 1 Hz rat myocyte, 37˚C
  46. 46. How do work-loops amplify changes in diastole? • Linear end systolic and end diastolic force length relation • Changes in calcium affect the diastolic and systolic phase equally
  47. 47. • BDM inhibits cross bridge formation, ESFL goes down • But also improves relaxation, so EDFL will go down as well • The diastolic effect outweighs the systolic effect Effect of low levels of BDM on diastolic dysfunction (mouse, data at room temperature)Length change Force(μN) after-load pre-load No BDM 5 mM BDM
  48. 48. The effect on length when force is constant Switch to 5 mM BDM Force(μN)SarcLen(μm)Lengthchange(μm) Time (s) • Myocoyte with Ca++ overload • BDM reduces the stiffness of the cell in diastole • The myocyte is pulled at with the same force • The cell will stretch further
  49. 49. Length control: Decreased performance Force control: Improved performance A different perspective • BDM depresses both the ESFL and EDFL • Length dependent activation beats cross bridge inhibition control + 5mM BDM
  50. 50. Why do “work-loops”? continued… • The external work done by a myocyte encompasses changes in both systolic and diastolic forces but also takes length dependent activation into account • Therefore, this also makes it a particularly sensitive assay for drug testing
  51. 51. How to maximize the measurable effect of a drug treatment Effect of 100nM Isoproterenol • Work-loop measurements can show both the systolic and diastolic effects of beta-adrenergic stimulation • The effect of 100nM Iso is 2-4 fold increase in work per loop • How did we construct this figure?
  52. 52. Determining the work maximum for each preload 0 2 4 6 8 0 2 4 6 0 10 20 30 40 50 60 0 5 10 Work(pJ) Power(pW) After-load (μN) Pacing frequency (Hz) Physiological heart rates a) b) c) Isometric (w = 0) Isotonic (w=0) Force Length W=ΔF.Δl Finding the afterload that delivers maximum external work… 3 0 4 0 5 0 6 0 1 2 3 4 5 L e n g th ( m) Force(N) 2 .0 2 .5 3 .0 3 .5 4 .0 0 5 1 0 1 5 A fter-L o ad ( N ) Work(pJ)
  53. 53. • 100nM Iso increases work/loop 2-4 fold (n = 10) • Compared to a 50-75% increase in isometric force (trabeculae at 37˚C) • Improved signal/noise, increased statistical power Maximizing the effect of a drug
  54. 54. Work-loop measurements lend themselves well... • To establish the maximum amount of work a cell can produce • Detect changes in the work produced with changes in inotropy • Highlight changes in diastolic function or dysfunction • Finding drug effects by encompassing both systolic and diastolic effects What is next... • Further methodological improvements, mostly reducing end-compliance – Cell holders seem to be the solution • Further research: Calcium sensitizers and de-sensitizers in disease models? The Anrep effect? Summary and conclusion
  55. 55. I’d like to thank: • Aref Najafi – who did most of the actual experiments • Prof. Jolanda van der Velden – in whose group at the VUmc (Amsterdam) this work took place • Tom Udale at IonOptix – software and system design, cell holder design • Alex Nijmeijer – a world class FPGA programmer --- And the many others who contributed Acknowledgements
  56. 56. Michiel Helmes, PhD michiel@ionoptix.com Thank You! For additional information on solutions for high speed quantitative fluorescence, muscle mechanics, and tissue engineering -- in particular the MyoStretcher System for generating “work-loops” in isolated intact myocytes – please visit: www.ionoptix.com
  57. 57. Follow us on Join our group 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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