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.

1. Measuring Work in Single Isolated Cardiomyocytes:
Replicating the Cardiac Cycle
Andy Henton
InsideScientific
Sponsored by:
Michiel Helmes, PhD
VUMC &
IonOptix

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 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. 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. 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. Where did the journey start?
• Le Guennec et al, ‘90
• Force measurements
on isolated intact
myocytes
• Carbon fibers, really
low force levels

7. Where did the journey start?
• Le Guennec et al, ‘90
• Force measurements
on isolated intact
myocytes
• Carbon fibers, really
low force levels

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. • 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. • 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. • 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. 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. Basic Layout of The MyoStretcher
3D micromanipulator
optical rail, microscope mount
arms to reach
experimental chamber

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. 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. 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. Cell Chamber View
Force ProbePiezo Motor
System on a Microscope

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. 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. 100
10
10
LVV (or cell length)
LVP(orforce)
End-diastole
(LVP is ‘left ventricular pressure’, LVV
is ‘left ventricular volume’)

21. 100
10
10~100
LVV (or cell length)
LVP(orforce)
Isovolumic Contraction

22. 100
10
100 ~
LVV (or cell length)
LVP(orforce)
End-systole
Ejection Phase

23. 100
10
100~10
LVV (or cell length)
LVP(orforce)
Isovolumic Relaxation

24. 100
10
~10
LVV (or cell length)
LVP(orforce)
Pressure-Volume Loop
Work (J) = Δ P*ΔV

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. 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. 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. 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. 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. 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. 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. 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. Recording @ 2 Hz

34. Recording @, 4 Hz

35. Recording @ 8 Hz

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. • 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. • 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. Improved attachment with the IonOptix cell holders
Slide courtesy of Ben Prosser, U. of Pennsylvania
images courtesy of Ben Prosser, U. of Pennsylvania

40. Images courtesy of Ben Prosser, U. of Pennsylvania

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. 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. 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. 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. Change in work-loops when switching from 8Hz to 1 Hz
rat myocyte, 37˚C

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. • 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. 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. 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. 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. 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. 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. • 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. 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. 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. 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

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protocols, research tools and
laboratory services.