Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca 2+ reservoir for contraction, which reuptakes intracellular Ca 2+ during relaxation. The SR Ca 2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca 2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca 2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa 2+ reserve and diastolic Ca 2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca 2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca 2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca 2+ -ATPase (SERCA) and Na + -Ca 2+ exchanger (NCX) to diastolic calcium removal was evaluated.