Quantification of Sarcoplasmic Reticulum Ca²⁺ Release in Primary Ventricular Cardiomyocytes

Area of Science:

• Cardiovascular Physiology
• Molecular Cardiology
• Biophysics

Background:

• Cardiac function relies on precise electrical signaling and calcium (Ca2+) handling.
• The cardiac ryanodine receptor type 2 (RyR2) is crucial for releasing Ca2+ from the sarcoplasmic reticulum (SR), essential for muscle contraction.
• RyR2 dysfunction is implicated in severe cardiac arrhythmias.

Purpose of the Study:

• To provide standardized protocols for studying RyR2-mediated Ca2+ release in isolated cardiomyocytes.
• To enable detailed analysis of factors influencing RyR2 channel function, including small molecules and proteins.
• To leverage Mavacamten for characterizing RyR2 activity across varying Ca2+ concentrations.

Main Methods:

• Isolation of high-quality cardiomyocytes from mouse models.
• Preparation of cardiomyocytes for advanced Ca2+ imaging techniques.
• Confocal microscopy and quantitative analysis of Ca2+ sparks and waves using SparkMaster 2 software.

Main Results:

• Established protocols for reproducible cardiomyocyte isolation and sample preparation.
• Developed a framework for quantitative analysis of RyR2-mediated Ca2+ release events.
• Demonstrated the utility of Mavacamten in characterizing RyR2 function under specific experimental conditions.

Conclusions:

• The presented protocols offer a robust methodology for investigating RyR2 function and its regulation in cardiac cells.
• These methods facilitate the study of Ca2+ handling abnormalities contributing to heart disease.
• The approach allows for comprehensive characterization of RyR2 activity and its modulation by pharmacological agents.