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

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