Simultaneous Recording of Subcellular Ca²⁺ Signals from the Cytosol and Sarco/Endoplasmic Reticulum: Compartmentalized Dye Loading, Imaging, and Analysis

An increase in the cytosolic Ca²⁺ concentration triggers the contraction in cardiomyocytes. In these cells sarcoplasmic reticulum (SR) is the major source of Ca²⁺, and the release from this store is mediated by the ryanodine receptors (RyRs). These receptors are regulated by cytosolic and intra-SR [Ca²⁺]. The cytosolic Ca²⁺ regulation is well established, but there are some limitations to determine indirectly the intra-SR Ca²⁺ concentration and understand its role in the RyRs regulation. Therefore, the interest to directly measure the free intra-SR Ca²⁺ concentration ([Ca²⁺]_SR) has led to the application of a low-affinity Ca²⁺ indicator (Fluo-5N AM) to follow changes of [Ca²⁺]_SR in cardiomyocytes. However the loading of this AM-ester dye into the SR has remained a challenge in freshly isolated mouse cardiomyocytes. Here, we describe an optimized protocol to measure changes of [Ca²⁺]_SR in mouse cardiomyocytes using fluorescent Ca²⁺ indicators and confocal microscopy. The application of this protocol allows to evaluate directly intra-SR Ca²⁺ in real time in various mouse models of cardiac disease, including transgenic animals harboring mutants of RyRs or other Ca²⁺ signaling proteins.