Quantification of Sarcoplasmic Reticulum Ca Release in Primary Ventricular Cardiomyocytes.

In the heart, ion channels generate electrical currents that signal muscle contraction through changes in intracellular calcium concentration, i.e., [Ca]. The cardiac ryanodine receptor type 2 (RyR2) is the predominant ion channel responsible for increasing intracellular [Ca] by releasing Ca from the sarcoplasmic reticulum (SR). Timely Ca release is necessary for appropriate cardiac function, and dysfunction can cause or contribute to life-threatening diseases such as arrhythmia. Quantification of SR-Ca release in the form of sparks and waves can provide valuable insight into RyR2 opening, and factors that influence or regulate channel function. Here, we provide a series of protocols that outline processes for (1) obtaining high-quality isolated cardiomyocytes, (2) preparing samples for experimentally investigating factors that influence RyR2 function, and (3) data acquisition and analysis. Notably, our protocols leverage the potency of the recently developed myosin ATPase inhibitor, Mavacamten. This affords the opportunity to characterize the effects of small molecules or reconstituted proteins/enzymes on RyR2-Ca release events across a range of [Ca]. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Cardiomyocyte isolation from mouse Basic Protocol 2: Preparation of cardiomyocytes for Ca imaging Basic Protocol 3: Confocal microscopy and quantitative Ca analysis using SparkMaster 2.