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

Abstract

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-5NAM) 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.