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.