Real-time intra-store confocal Ca2+ imaging in isolated mouse cardiomyocytes.

To initiate the contraction of cardiomyocytes, Ca2+ is released from the SR to the cytosol via ryanodine receptors (RyRs), which are activated by the Ca2+-induced Ca2+ release mechanism (CICR). The activity of RyRs is regulated by both, cytosolic and SR luminal Ca2+. Deregulation of the CICR, by dysfunctional SR Ca2+ release or uptake, is frequently associated with cardiac pathologies (e.g. arrhythmias, CPVT, heart failure). Recently, the interest to directly measure changes of the free Ca2+ concentration within the SR ([Ca2+]SR) has led to the application of low affinity Ca2+ indicators (mag-fluo-4, Fluo-5N) to follow changes of [Ca2+]SR in cardiomyocytes from some species. However, direct measurement of Ca2+ signals from the SR have not been possible in freshly isolated mouse cardiomyocytes. Here, we show a new protocol optimized to measure changes of [Ca2+]SR in mouse cardiomyocytes using fluorescent Ca2+ indicators and confocal microscopy. The application of this protocol permits the design of experimental studies with direct evaluation of SR Ca2+ in real time in various mouse models of cardiac disease, including transgenic animals harboring mutants of RyRs or other Ca2+ signaling proteins. The technique, in combination with these models, will help to understand how these diseases and mutations affect Ca2+ signals within the SR and the Ca2+ sensitivity of the RyRs for cytosolic and SR luminal Ca2+, thereby contributing to arrhythmias or weak heart beat.