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

Abstract

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca²⁺ reservoir for contraction, which reuptakes intracellular Ca²⁺ during relaxation. The SR Ca²⁺ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca²⁺ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca²⁺ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa²⁺ reserve and diastolic Ca²⁺ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca²⁺ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca²⁺ 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 Ca²⁺-ATPase (SERCA) and Na⁺-Ca²⁺ exchanger (NCX) to diastolic calcium removal was evaluated.