Join Patrick Schönleitner, PhD and Francisco Altamirano, PhD as they share their work involving simultaneous measurements of intracellular calcium, membrane voltage, and contractility of cardiomyocytes. The heart’s electrical activity coordinates the contraction of the heart chambers to pump blood to other organs and sustain life. The movement of ions (charged atoms) through ion channels promotes voltage changes across the cardiomyocyte plasma membrane, known as action potentials. The arrival of an action potential depolarizes the plasma membrane and activates Ca2+ influx via L-type Ca2+ channels. Ca2+ then activates Ryanodine Receptors to release further Ca2+ into the cytosol and stimulate contraction. Both duration and shape of the cardiac action potential regulate Ca2+ fluxes and contractility in cardiomyocytes. Cardiac excitation-contraction coupling is the process used to describe the progression from membrane depolarization to calcium influx and intracellular release to myocyte contraction. In the heart, these processes are tightly intertwined, and crucial information can be missed when recording them individually. Recent technological advances enable the simultaneous multi-parametric measurement of membrane voltage, intracellular calcium, and contractility to reliably capture the complex excitation-contraction cascade in great detail. During the first half of this webinar, Patrick focuses on the theoretical background and address technical considerations and methodological limitations researchers need to consider to successfully run multi-parametric experiments and analyze the resulting data. Francisco then discusses how action potentials regulate Ca2+ fluxes to regulate cardiomyocyte contractile function. In addition, he discusses the advantages/limitations of mouse models and human inducible pluripotent stem cell (hiPSC)-derived cardiomyocytes to determine molecular mechanisms driving alterations in action potentials and Ca2+ handling.

1. Unraveling Excitation-Contraction
Coupling: Simultaneous Measures of
Intracellular Calcium and Action
Potentials
Francisco Altamirano, PhD
Assistant Professor
Cardiovascular Sciences
Houston Methodist Research Institute
Patrick Schönleitner, MD, PhD
Research Scientist
Research & Development
IonOptix

2. Experts share their work involving simultaneous
measurements of intracellular calcium, membrane
voltage, and contractility of cardiomyocytes.
Unraveling Excitation-Contraction
Coupling: Simultaneous Measures of
Intracellular Calcium and Action
Potentials

3. Unraveling Excitation-Contraction
Coupling: Simultaneous Measures
of Intracellular Calcium and
Action Potentials
Patrick Schönleitner, MD, PhD
Research Scientist
Research & Development
IonOptix
Copyright 2021 P. Schönleitner and InsideScientific. All Rights Reserved.

4. Welcome
• Introduction to EC coupling and voltage sensitive dyes
• Choosing the right dyes
• The Good, The Bad, The Ugly: From Limitations to Opportunities
• Analysing Action Potentials
• An Experiment in 30 minutes
• Pacing frequency matters
• Conclusion

5. From Excitation to Contraction …
Bers D. M., Nature (2002)
Action
Potential
Calcium
Transient
Contraction
Membrane
Potential
Calcium
handling
Contractility
Ca2+ buffering,
mechano chemo
transduction

6. The traditional approach
Voigt N. 2012 (Circulation)
Input
• Patch clamp Setup (Amplifier, AD converter,
micromanipulator, pipette puller, antivibration table…..)
• Calcium & Contractility Setup
• Someone with experience and patience
• 15-30 min for one myocyte
Output
• <10 cells/sample

7. A brief history of voltage sensitive dyes
Morad M, Salama G, 1978 (J.Physiol.)
• Early 1970s in neurons
• Late 1970s in myocardium with
Merocyanine-540
• 1985 New generation with ANEP dyes (Di-
4-ANEPPS)
• 2012 PeT dyes (VoltageFluor 2.4.Cl ) and
new high ΔF ANEP dyes

8. Choosing the right dye – Di-4-ANEPPS
• Excitation on “red shoulder” for Maximal ΔF →
Requires high light intensity
• Broad spectrum → Trickier to combine with
other dyes
• Often low ΔF → Averaging APs
• Photodamage APD prolongation → limited
recording time
Excitation Emission
YU T.Y. et al., 2015 (Comput Methods Biomech Biomed Eng Imaging Vis)

9. Rhod2
Choosing the right dye – Fluovolt
• Excitation at peak absorption
• High ΔF → Fast acquisition
• Narrower Emission spectrum → Simultaneous Ca2+ recordings e.g. RHOD2
Myocam
Rhod2
Emission
Rhod2
Excitation
Fluovolt
Excitation
Fluovolt
Emission
Rhod2
light source
Fluovolt
Detector
Rhod2
Detector
Fluovolt
light source
Fluovolt
Myocyte

10. Excellent spectral separation
20 beat average
0.0 0.2 0.4 0.6
500
550
600
650
700
Time (s)
Fluovolt
(counts)
0.0 0.1 0.2 0.3 0.4 0.5
100
150
200
250
300
Time (s)
Fluovolt
Leakage
(counts)
Minimal Fluovolt Bleed into
Ca2+ Channel
Fluovolt
Detector
Rhod2
Detector
Fluovolt
light source
Myocyte loaded with Fluovolt
0.0 0.1 0.2 0.3 0.4 0.5
0
50
100
150
200
Time (s)
Rhod2
Leakage
(counts)
0.0 0.1 0.2 0.3 0.4 0.5
300
350
400
450
500
Time (s)
Rhod2
(counts)
No RHOD2 bleed into
AP channel
Rhod2
light source
Fluovolt
Detector
Rhod2
Detector
Myocyte loaded with RHOD2

11. Our first multiparametric experiment
0.9
1.0
1.1
1.2
1.3
V
m
(F/F
0
)
0.6
1.0
1.4
1.8
2.2
CaT
(F/F
0
)
0.0 0.1 0.2 0.3 0.4 0.5
1.70
1.75
1.80
1.85
Time (s)
SL
(m)
0.0 0.5 1.0 1.5 2.0 2.5
1.70
1.75
1.80
1.85
Time (s)
SL
(m)
3000
3500
4000
4500
V
m
(Counts)
1000
1500
2000
2500
CaT
(Counts)
• Rat ventricular myocytes
• Loading at 37 C
• 1:1000 Fluovolt
• 1 um Rhod2-AM
• Acquisition at 2000hz for single wavelength
dyes
• 250hz for sarcomere length
Myocam-SL
RHOD2-CaT
Fluovolt
-Vm

12. The Ugly – Methodological limitations
• Non ratiometric Vm recording → no easy way to account for motion artefacts (not applicable to single
cells)
• Difficult to calibrate Calcium signal → only relative diastolic/systolic Calcium
• Repeated measurements sensitive to dye leakage
• No direct control over Vm
Bito V. et. al. 2012 (Exp. Physiol.)

13. The bad – Obstacles to overcome
• Preparation dependent phototoxicity
(not in monolayers)
• Signal to noise ratio vs. acquisition speed vs. light intensity
• Loading protocol will depend on species and preparations
Mouse Ventricular Myocyte
Fluovolt
–
Raw

14. The Good – Comprehensive EC assessment
Action potential Calcium Transient Contractility
EAD F/F0 Contraction Amplitude
APD 10-90% Time to Peak (10-90%) Time to 10-90% Contraction
Diastolic interval Time to Baseline (10-90%) Time to 10-90% Relaxation
Beat rate CaT decay (Tau) Max Contraction Velocity
Cycle length
ΔCaT-APD
Phase plots (CaT vs Vm, CaT vs Shortening)
0.0 0.1 0.2 0.3 0.4 0.5
1.70
1.75
1.80
1.85
Time (s)
SL
(m)
0.0 0.1 0.2 0.3 0.4 0.5
0.6
1.0
1.4
1.8
2.2
Time (s)
CaT
(F/F
0
)
0.0 0.1 0.2 0.3 0.4 0.5
0.9
1.0
1.1
1.2
1.3
Time (s)
V
m
(F/F
0
)
Amplitude
RT 10-90%
Max Vel.
Tau
F/F0
DI
APD70

15. Can we analyse action potentials?
Calcium Transient Action Potential
Return Fit Return Fit
1. Simulated human ventricular action potentials O’Hara et al. 2011 (PLoS Comput. Biol.)
2. Varying APD by modifying potassium currents
3. Add noise to signal at different SNRs
4. Analyse with IW and compare with noiseless APs.

16. Accurate and precises analysis APD
B
a
s
e
l
i
n
e
5
0
%
I
K
R
1
5
0
%
I
K
R
0.0
0.1
0.2
0.3
0.4
Time
(s)
B
a
s
e
l
i
n
e
5
0
%
I
K
R
1
5
0
%
I
K
R
B
a
s
e
l
i
n
e
5
0
%
I
K
R
1
5
0
%
I
K
R
B
a
s
e
l
i
n
e
5
0
%
I
K
R
1
5
0
%
I
K
R
B
a
s
e
l
i
n
e
5
0
%
I
K
R
1
5
0
%
I
K
R
0.00
0.25
0.50
0.75
1.00
R
B
a
s
e
l
i
n
e
5
0
%
I
K
R
1
5
0
%
I
K
R
B
a
s
e
l
i
n
e
5
0
%
I
K
R
1
5
0
%
I
K
R
B
a
s
e
l
i
n
e
5
0
%
I
K
R
1
5
0
%
I
K
R
PSNR 36 8 2
∞
PSNR 36 8 2
∞
Baseline
50%
IKR
150%
IKR
Action Potential Duration 70%
Goodness of Return fit

17. An experiment in 30 minutes – Introduction
Hoffmann, P., & Warner, B. (2006)

18. An experiment in 30 minutes – Methods

19. An experiment in 30 minutes – Methods
Baseline
repeat baseline measurement
Find 40 centres of contraction and
measure each for 5s
30nM Verapamil 100nM Verapamil
repeat baseline measurement
Ca2+
Vm
Motion
Incubation Incubation
1200
CaT
1200
AP
1200
Contractions

20. An experiment in 30 minutes – Methods

21. An experiment in 30 minutes – Results
B
a
s
e
l
i
n
e
3
0
n
M
V
e
r
a
p
a
m
i
l
1
0
0
n
M
V
e
r
a
p
a
m
i
l
✱✱✱✱
✱✱✱✱
✱✱✱✱
B
a
s
e
l
i
n
e
3
0
n
M
V
e
r
a
p
a
m
i
l
1
0
0
n
M
V
e
r
a
p
a
m
i
l
0.00
0.05
0.10
0.15
0.20
Time
(s)
✱✱✱✱
✱✱✱✱
✱✱✱✱
APD 70
APD 30
50ms
0.1
F/F
0
Baseline
30 nM Verapamil
100 nM Verapamil
Fluovolt
n/N=40/1

22. B
a
s
e
l
i
n
e
3
0
n
M
V
e
r
a
p
a
m
i
l
1
0
0
n
M
V
e
r
a
p
a
m
i
l
1.0
1.2
1.4
1.6
CaT
amplitude
(F/F
0
)
✱✱✱✱
✱✱✱✱
✱✱✱✱
An experiment in 30 minutes – Results
50ms
0.1
F/F
0
Baseline
30 nM Verapamil
100 nM Verapamil
CaT
Rhod2
B
a
s
e
l
i
n
e
3
0
n
M
V
e
r
a
p
a
m
i
l
1
0
0
n
M
V
e
r
a
p
a
m
i
l
0.00
0.05
0.10
0.15
0.20
0.25
Time
(s)
ns
✱✱✱✱
✱✱✱✱
Return time 50%
n/N=40/1

23. B
a
s
e
l
i
n
e
3
0
n
M
V
e
r
a
p
a
m
i
l
1
0
0
n
M
V
e
r
a
p
a
m
i
l
0
10
20
30
40
50
Contraction
(Δ%)
✱✱✱✱
✱✱✱✱
✱✱✱✱
An experiment in 30 minutes – Results
200ms
10%
Baseline
30 nM Verapamil
100 nM Verapamil
Contraction
Contraction
Relaxation time 50%
B
a
s
e
l
i
n
e
3
0
n
M
V
e
r
a
p
a
m
i
l
1
0
0
n
M
V
e
r
a
p
a
m
i
l
0.00
0.05
0.10
0.15
0.20
0.25
Time
(s)
ns
✱✱✱✱
✱✱✱✱
n/N=40/1

24. An experiment in 30 minutes – Conclusion
• Automated recordings of iPSC derived myocytes increase throughput
• Reliable repeated measurements increase power of experiment
• Assessment of the whole EC cascade by combining Voltage, Calcium and Contractility
• 40 samples are enough to resolve 5ms APD differences within 30 minutes
(at α < 0.001 and 1-β = 0.999)

25. Pacing frequency Matters – Introduction
Verkerk A, 1999 (Am. J. Physiol. )
Na+


26. Pacing frequency Matters – Introduction
Fluovolt
Fluovolt
Rhod2
Rhod2
2H
z
4H
z
2
H
z
4
H
z
0.10
0.15
0.20
0.25
0.30
Time
(s)
✱✱✱✱
CaT
2 Hz
4 Hz
50ms
0.1
F/F
0
2
H
z
4
H
z
1.0
1.1
1.2
1.3
1.4
1.5
CaT
amplitude
(F/F
0
)
✱✱✱✱
CaT Amplitude RT 70
Action Potential
2 Hz
4 Hz
50ms
0.1
F/F
0
2 Hz 4 Hz
0.00
0.05
0.10
0.15
0.20
Time
(s)
✱✱✱✱
APD 70
APD 30
n/N=36/1
2
H
z
4
H
z
✱✱✱✱

27. Conclusion
• Simultaneous Multiparametric recording of Vm, Ca2+ and Contraction is feasible and easy
to implement
• Cell preparation, dye loading and protocols need to be adapted for each model
• Enables comprehensive evaluation of EC coupling
• Cytosolver is sufficiently precise and accurate for basic AP parameters
• Multiparametric recordings in combination with our HTS can be an alternative for
traditional safety testing approaches

28. Simultaneous Voltage, Calcium and
Contractility Measurements to Study
the Role of Polycystin-1 in
Cardiomyocytes
Francisco Altamirano, PhD
Assistant Professor
Cardiovascular Sciences
Houston Methodist Research Institute
Copyright 2021 F. Altamirano and InsideScientific. All Rights Reserved.

29. Autosomal Dominant Polycystic Kidney Disease
(ADPKD)
Nat Rev Dis Primers. 2018;4:50

30. Cardiovascular alterations in ADPKD
• Hypertension
• Left ventricular hypertrophy
• Systolic/Diastolic dysfunction
• Cardiac valvular disease
• Aortic root dilatation
• Arterial aneurysms/dissections
• Pericardial effusion
• Higher incidence of atrial fibrillation
• Etc.
Kidney Int Rep. 2020; 5(4): 396–406

31. Altamirano, et al. Circulation. 2019 Sep 10;140(11):921-936.
Cardiomyocyte-specific Polycystin-1 deletion
promotes cardiac dysfunction

32. Impaired Ca2+ cycling and contractility in
Polycystin-1-deficient cardiomyocytes
Altamirano, et al. Circulation. 2019 Sep 10;140(11):921-936.

33. Altamirano, et al. Circulation. 2019 Sep 10;140(11):921-936
Polycystin-1 deletion shortens action potential
duration in cardiomyocytes
J Physiol. 2004 Sep 1; 559(Pt 2): 593–609
J Physiol. 2003 Jan 1;546(Pt 1):5-18
Action potential duration
Activation of L-type
Ca2+ channels
SR Ca2+ stores

34. Altamirano, et al. Circulation. 2019 Sep 10;140(11):921-936.
Polycystin-1 deletion shortens action potential
duration and impairs Ca2+ cycling
Whole-cell voltage clamp
+ Fluorescence detection (Fluo-4)
Ca2+
AP-clamp (same WT cell) Square pulses in WT and KO cells

35. HEK-293T
Ito
Similar results with Kv1.5 (IKslow1) and Kv2.1 (IKslow2)
Polycystin-1 inhibits Kv channels (Ito, IKslow1, IKslow2)
Altamirano, et al. Circulation. 2019 Sep 10;140(11):921-936.

36. Altamirano, et al. Circulation. 2019 Sep 10;140(11):921-936.
Role of Polycystin-1 in cardiomyocytes

37. Differences in action potentials and currents between
human and mouse ventricular cardiomyocytes
Circulation Research. 2001;89:944–956

38. Hypothesis
PC1 controls action potential duration, Ca2+ handling and contractility
in human cardiomyocytes

39. Human-induced pluripotent stem cell derived
cardiomyocytes (hiPSC-CM)
(460 KDa)
(37 KDa)
α-actinin DAPI MLC-2v DAPI Troponin T DAPI
iPSC
CHIR Wnt
C59
d2 d4 d14 d24
Metabolic selection
( - glucose
+ lactate)
d60-90
maturation

40. Action potentials - FluoVolt (potentiometric dye)
Calcium - Calbryte 590 AM: cytosolic distribution (alternative
to Rhod-2/mitochondria-cytosol; higher Kd)
Contraction - Contrast algorithm/IonOptix
Simultaneous voltage, calcium and contractility
measurements in iPSC-CMs

41. Simultaneous voltage, calcium and contractility
measurements in iPSC-CMs

42. Simultaneous voltage, calcium and contractility
measurements in iPSC-CMs
N=14-16 cells

43. Immature vs mature cardiomyocytes
Nature Reviews Cardiology. 17, 341–359 (2020)

44. Physiol Rev 97: 227–252, 2017
α-actinin DAPI
Immature vs mature cardiomyocytes

45. Thyroid and Glucocorticoid hormones promote
functional T-tubule development in hiPSC-CM
Circulation Research. 2017;121:1323–1330 (Bjorn C. Knollman Lab)

46. Our lab
- iPSC-CM (day 80; ~95% MLC-2v)
- Plated in matrigel mattress with RPMI B27
supplemented with dexamethasone and thyroid
hormone. 5 days (preliminary data)
Simultaneous voltage, calcium and contractility in
mature hiPSC-CM
5 µm
Fluovolt
Calb-590

47. Edge detection / IonOptix
Our lab
- iPSC-CM (day 80; ~95% MLC-2v)
- Plated in matrigel mattress with RPMI B27
supplemented with dexamethasone and thyroid
hormone. 5 days (preliminary data)
Simultaneous voltage, calcium and contractility in
mature hiPSC-CM
5 µm
Fluovolt
Calb-590
Adult cardiomyocyte

48. Fluovolt
Calbryte-590
Simultaneous voltage, calcium and contractility in
mature hiPSC-CM
Edge detection
Pacing at 0.5 Hz
N=19-20 cells

49. Summary
• PC1 controls action potential duration, Ca2+ handling and contractility in both
human and mouse cardiomyocytes
• Simultaneous recordings of action potential, calcium and contractility are a
good model to study polycystin-1 function in cardiomyocytes.

50. Acknowledgements
Ravi
Susmitha
Dr. Cooke
Troy Hendrickson
William Perez
Sufen Wang
Liliana Tavares
Dr Valderrábano
UT Southwestern
Joseph A. Hill
Mayo Clinic
Peter Harris
Baltimore PKD Core
Terry Watnick
Patricia Outeda
Small Research Grant
FOCUS/PRIDE/NHLBI
Career Development
Award
Startup funds
Our Lab
Administrative team

51. Francisco Altamirano, PhD
Assistant Professor
Cardiovascular Sciences
Houston Methodist Research Institute
Patrick Schönleitner, MD, PhD
Research Scientist
Research & Development
IonOptix
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