measuring force in single heart cells
DESCRIPTION
Traditional shortening experiments utilizing isolated myocytes have provided scientists a valuable functional assay. These studies, however, have been limited to techniques that could not replicate the mechanical environment of the heart. New technology now provides investigators the ability to reliably attach myocytes, mechanically stretch them, make direct force measurements and control cell length intelligently. This webinar reviews best-practices and techniques for attaching, stretching, and studying isolated cells. During this exclusive webinar sponsored by IonOptix, presenters Ben Prosser and Michiel Helmes discuss methodology, best-practices, and show attendees how to attach isolated myocytes to ensure accurate force measurements. In addition, Ben Prosser reviews an application of myocyte stretching and loading. Michiel Helmes discusses the importance of both mechanical loading and measuring force, and how controlling myocyte length to regulate force development enables generation of work loops and a host of mechanical studies. Key Topics: Why it is important to mechanically load single myocytes and how to do master the technique The value of measuring force in single cell applications of cardiac function research Why it is important to control myocyte length The value of combining force measurements with other indices of contraction and cellular function Presenters: Benjamin L. Prosser, PhD Department of Physiology Perelman School of Medicine University of Pennsylvania Michiel Helmes, PhD Department of Physiology VU University Medical Center Amsterdam & IonOptixTRANSCRIPT
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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)
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
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)
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)
Click Here to View Video
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
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
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
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FOR THIS EVENT AND OTHERS AT
http://goo.gl/SbcfX5
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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.