measuring work in single isolated cardiomyocytes: replicating the cardiac cycle
TRANSCRIPT
Measuring Work in Single Isolated Cardiomyocytes:Replicating the Cardiac Cycle
Andy HentonInsideScientific
Sponsored by:
Michiel Helmes, PhDVUMC &IonOptix
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.
Measuring Work in Single Isolated Cardiomyocytes: Replicating the Cardiac Cycle
Michiel Helmes PhD
Department of PhysiologyVU University Medical Center
Amsterdam& IonOptix
Copyright 2015 IonOptix & InsideScientific. All Rights Reserved.
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
This webinar IS NOT about PV-loops!
• What we will be discussing is how to measure mechanical work in singleintact 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:
Where did the journey start?
• Le Guennec et al, ‘90
• Force measurements on isolated intact myocytes
• Carbon fibers, really low force levels
Where did the journey start?
• Le Guennec et al, ‘90
• Force measurements on isolated intact myocytes
• Carbon fibers, really low force levels
• Yasuda in ‘01, and Nishimura in ’04
• Bending of carbon fibers to measureforce
• 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
• 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
• 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
• 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…
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
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 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
cantilever
attachmentneedle
read out fiber
• Optical
• Fully submersible
• nN sensitivity
• High resonance frequency (8kHz)
• Stable baseline
IonOptix OptiForce, Revolutionary New Class of Force Transducer
Front view
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
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 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
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
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
LVV (or cell length)
LVP
(o
r fo
rce)
Isovolumic Contraction
100
10
100 ~
LVV (or cell length)
LVP
(o
r fo
rce)
End-systole
Ejection Phase
100
10
100~10
LVV (or cell length)
LVP
(o
r fo
rce)
Isovolumic Relaxation
100
10
~10
LVV (or cell length)
LVP
(o
r fo
rce)
Pressure-Volume Loop
Work (J) = Δ P*ΔV
Modulating Force Development By Changing Cell Length
length
forc
e
(I)
(II)
(III)
(IV)
(I)Start contraction, Pre-load > force < afterloadDo nothing
force > afterloadShorten the cell
End of active contractionPre-load > force < afterloadDo nothing
DiastoleForce < pre-loadStretch the cell
(IV)
(II)
(III)
First algorithm used to create work loops:
motor
force
After load
Pre load
d ba c d
Len
gth
ch
ange
(μ
m)
Forc
e (µ
N)
* Mouse myocyte, room temperature
d
b
ac
afterload
preload
Forc
e (u
N)
Length change
First work-loops with feed-back based force control
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
Afterload
Preload
Forc
e (
μN
) Isometric contraction
With force clamp
Time (s)
Len
gth
(μ
m)
length
forc
e
(I)
(II)
(III)
(IV)
Afterload
Preload
• Force clamps
• Improved end-systolic switch
• Pacing mark initiates new loop
• Improved speed of algorithm and motor
length
forc
e
(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’
Forc
e (
μN
)
Length (μm)
Force vs length
3 0 4 0 5 0 6 0
1
2
3
4
5
L e n g th ( m )
Fo
rc
e (
N)
2 .0 2 .5 3 .0 3 .5 4 .0
0
5
1 0
1 5
A fte r-L o a d ( N )
Wo
rk
(p
J)
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
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…
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 afterloadusing signal generators
-> Necessary tools to explore the parameter space of preload, afterload and pacing frequency or to do repeated measurements
Recording @ 2 Hz
Recording @, 4 Hz
Recording @ 8 Hz
SL = 1.98 µm 2.03 µm2.02 µm
Forc
e Length
Varying pre- and afterloadFo
rce
Len
gth
End Diastolic and End Systolic force length relation
• 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...
• Work-loop ≠ PV-loop; more sophisticated algorithms neededThe infrastructure is in place
• Force measurements need to be transformed into stressMeasuring cross sectional area reliably is difficult on a standard microscope
• Compliance in the attachment of the celllimits 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...
Improved attachment with the IonOptix cell holders
Slide courtesy of Ben Prosser, U. of Pennsylvania
images courtesy of Ben Prosser, U. of Pennsylvania
Images courtesy of Ben Prosser, U. of Pennsylvania
• Laser etched cell holder
• Cavity is formed to accomodatemyocyte
• 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
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 detailedscientific questions
3. work-loops are very useful in detecting changes in diastolic properties
Why do “work-loops” on single cells?
Post-rest potentiation, constant length
8 Hz
Post-rest potentiation has a diastolic and systolic component
• At constant length, the systoliccomponent (increased calcium release) dominates the change in signal
• The change in diastolic force (lower calcium level throughprolonged re-uptake) is relatively small
force
sarc len
length
4 Hz
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 andsystole can now be observed
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 calcium affect the diastolic and systolic phaseequally
• 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
Forc
e (
μN
)
after-load
pre-load
No BDM 5 mM BDM
The effect on length when force is constant
Switch to 5 mM BDM
Forc
e (μ
N)
Sarc
Len
(μm
)Le
ngt
h c
han
ge (
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
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
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
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?
Determining the work maximum for each preload
0
2
4
6
8
0 2 4 60
10
20
30
40
50
60
0 5 10
Wo
rk (
pJ)
Pow
er (
pW
)
After-load (μN) Pacing frequency (Hz)
Physiological heart ratesa) b) c)
Isometric (w = 0)
Isotonic (w=0)
Forc
e
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 )
Fo
rc
e (
N)
2 .0 2 .5 3 .0 3 .5 4 .0
0
5
1 0
1 5
A fte r-L o a d ( N )
Wo
rk
(p
J)
• 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
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
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
Michiel Helmes, [email protected]
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
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.