Dr. Joe Soughayer and Dr. Adam Veteto provide an in-depth overview of excitation-contraction (E-C) coupling in cardiomyocytes and the tools to characterize this. Cardiac excitation-contraction coupling is the process by which electrical excitation drives cardiomyocyte mechanical shortening. Following membrane depolarization, calcium influx through voltage-gated calcium channels triggers calcium release from intracellular stores. Increased cytosolic calcium binds to troponin, allowing sliding of actin thin filaments against myosin thick filaments. This results in myocyte contraction. Cytosolic calcium extrusion and sequestration induces myofilament relaxation, readying the cardiomyocyte to begin again. Understanding this tightly regulated process, as well as how perturbations disrupt it, is critical to understanding heart function and dysfunction. In this webinar, Dr. Soughayer and Dr. Veteto present tools to characterize E-C coupling in isolated adult cardiomyocytes and iPSC-/ESC-derived cardiomyocytes. They describe how these tools work and how they quantify important functional outputs describing the speed and amplitude of calcium release and reuptake, as well as downstream myocyte shortening and relengthening. Real world examples of data collection, analysis, and interpretation are also shown. Key Topics Include: - How do measurements in isolated cardiomyocytes and iPSC-CM monolayers differ from whole heart studies? - What does characterization of excitation-contraction coupling reveal about cardiac physiology/pathophysiology? - What quantitative approaches can you take to understand excitation-contraction coupling?

1. Understanding the Heart’s
Intracellular Orchestra: Functional
Characterization of Excitation-
Contraction Coupling in
Cardiomyocytes
Adam Veteto, PhD
Medical Pharmacology and Physiology
IonOptix/University of Missouri
Applications Scientist/Visiting Scientist
Joe Soughayer, PhD
Business Development
Manager
IonOptix

2. Copyright 2022. All Rights Reserved. Contact Presenter for Permission
Understanding the Heart’s
Intracellular Orchestra:
Functional Characterization of
Excitation-Contraction
Coupling in Cardiomyocytes
Joe Soughayer, PhD
Business Development
Manager
IonOptix

3. Introduction

4. A brief history

5. MYOFILAMENTS
Na+
Ca2+
Na+
/Ca2+
Exchanger
L-Type
Ca2+ Channel
SR Ca2+
ATPase
Na+ /K+
ATPase
Ryanodine
Receptor
RyR
LTCC NCX
[Ca
2+
]
i
Length
Domeier, Unpublished
Excitation-Contraction Coupling

6. Excitation-Contraction Coupling
Lu & Pu, Biophysical Reviews, 2020

7. Excitation-Contraction Coupling
Streng et al, JMCC, 2013

8. MYOFILAMENTS
Na+
Ca2+
Na+
/Ca2+
Exchanger
L-Type
Ca2+ Channel
SR Ca2+
ATPase
Na+ /K+
ATPase
Ryanodine
Receptor
RyR
LTCC NCX
[Ca
2+
]
i
Length
Domeier, Unpublished
Excitation-Contraction Coupling

9. Contractility

10. Contractility:
CytoMotion
• Simple, easy-to-use
contrast-based algorithm
• Fast motion detection in
amorphous myocytes
• Quantitative contractility
kinetics
• Beat frequency w/
variance

11. Contractility:
Cell length
• Contrast-based edge
detection and tracking
• Whole myocyte length
• Quantitative amplitude and
kinetic data

12. Contractility:
Sarcomere length
• Robust, easy-to-use
algorithm
• Quantitative amplitude
and kinetic data
• Meaningful absolute
measurement

13. Calcium

14. Calcium
• Can be combined with
Contracility data
• Available with quantita-
tive, ratiometric
indicators, like Fura-2,
and faster, brighter
intensiometric indicators
like Fluo-4
• LEDs provide clean,
monochromatic excitation
• PMTs provide fast,
sensitive emission
collection

15. Action Potentials
(and other dyes)

16. Action Potentials
(and other dyes)
• Membrane potential,
e.g., FluoVolt
• Other calcium indicators,
e.g., Rhod-2
• Genetically encoded
indicators, e.g., GCaMP
• pH, e.g., SNARF
• Endogenous
fluorophores, e.g., NADH
Schönleitner, www.ionoptix.com

17. Systems

18. CytoMotion Lite
• Designed to be used with
spontaneously beating
myocytes
• Low-cost simple system
with only a camera, laptop,
and software
• Measure beat frequency
and contractility kinetics in
hiPSC-CMs

19. Contractility Only
• Measure cell and
sarcomere length in both
spontaneously beating and
stimulated myocytes
• Includes A/D interface as
well as field stimulator and
stimulation/perfusion cell
chamber

20. Calcium and
Contractility
• Combine contractility
measurements with
calcium and/or other
fluorometry
• Adds LED(s), optics, beam
splitters, and PMT(s)

21. MultiCell Lite
• Repeated measurements
(pairwise data collection)
• Adds motorized stage and
mark and find software
• Includes support for multiwell
plates

22. MultiCell
• Fast cell to cell movement-
motorized objective
• Temperature controlled
• Capable of >1,000 myocyte
measurement per hour
(depending on acquisition
parameters)

23. Summary

24. Copyright 2022. All Rights Reserved. Contact Presenter for Permission
Understanding the Heart’s
Intracellular Orchestra:
Functional Characterization of
Excitation-Contraction
Coupling in Cardiomyocytes
Adam Veteto, PhD
Medical Pharmacology and Physiology
Applications Scientist/Visiting Scientist
IonOptix/University of Missouri

25. Applications in
Cardiac Research

26. Investigating Novel
Animal Models

27. Calcium and
Contractility
• Female Ossabaw swine
were subjected to
Western Diet and Aortic
Banding (WD-AB) to
mimic HFpEF with
metabolic involvement
• Left ventricular
cardiomyocytes were
enzymatically isolated
Olver et al., 2019

28. MYOFILAMENTS
Ca2+
L-Type
Ca2+
Channel
SR Ca2+
ATPase
Ryanodine
Receptor
RyR
LTCC
[Ca
2+
]
i
Length
Dobutamine
PKA
1
2
3
3
ß1 Receptor
Dobutamine and the Sympathetic Response
Veteto, Unpublished

29. Calcium and
Contractility
Olver et al., 2019

30. Olver et al., 2019
Calcium and
Contractility

31. Olver et al., 2019
Calcium and
Contractility

32. Olver et al., 2019
Calcium and
Contractility

33. Olver et al., 2019
Calcium and
Contractility

34. Olver et al., 2019
Calcium and
Contractility

35. Characterizing
Novel Drugs

36. Novel Myosin II ATP-ase Inhibitor: Mechanism
Streng, 2013
Novel “CK” Myosin
II ATP-ase Inhibitor

37. Novel Myosin II ATP-ase Inhibitor: Dose Response
Veteto, Unpublished

38. Novel Myosin II ATP-ase Inhibitor: Dose Response
Veteto, Unpublished

39. Novel Myosin II ATP-ase Inhibitor: Dose Response
Veteto, Unpublished

40. Novel Myosin II ATP-ase Inhibitor: Dose Response
Veteto, Unpublished

41. Assaying Cultured
Cell Maturation
and Viability

42. CytoMotion:
Beat Frequency
and iPSC
Maturation
Time (sec)
Time (sec)
C
Beat
Frequency
(Hz)
Baseline Baseline
ISO ISO
Pixel
Correlation
(AU)
Pixel
Correlation
(AU)
Manders, Unpublished

43. CytoMotion:
Beat Frequency
and iPSC
Maturation
Time (sec)
Time (sec)
C
Beat
Frequency
(Hz)
Baseline Baseline
ISO ISO
Pixel
Correlation
(AU)
Pixel
Correlation
(AU)
Manders, Unpublished

44. MYOFILAMENTS
Ca2+
L-Type
Ca2+
Channel
SR Ca2+
ATPase
Ryanodine
Receptor
RyR
LTCC
[Ca
2+
]
i
Length
Isoproterenol
PKA
1
2
3
3
ß1 Receptor
Isoproterenol and the Sympathetic Response
Veteto, Unpublished

45. CytoMotion:
Beat Frequency
and iPSC
Maturation
Time (sec)
Time (sec)
C
Beat
Frequency
(Hz)
Baseline Baseline
ISO ISO
Pixel
Correlation
(AU)
Pixel
Correlation
(AU)
Manders, Unpublished

46. CytoMotion: Drug-Dependent Changes in iPSC Contractility
A
B C
Time
to
Relaxation
50%
(sec)
Schonleitner, Unpublished

47. CytoMotion: Drug-Dependent Changes in iPSC Contractility
A
B C
Time
to
Relaxation
50%
(sec)
Schonleitner, Unpublished

48. MYOFILAMENTS
Ca2+
L-Type
Ca2+
Channel
RyR
LTCC NCX
[Ca
2+
]
i
Length
Verapamil
Verapamil: Mechanism of Action
Veteto, Unpublished

49. CytoMotion: Drug-Dependent Changes in iPSC Contractility
A
B C
Time
to
Relaxation
50%
(sec)
Schonleitner, Unpublished

50. CytoMotion: Drug-Dependent Changes in iPSC Contractility
A
B C
Time
to
Relaxation
50%
(sec)
Schonleitner, Unpublished

51. Conclusion

52. Thank you for participating!
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