Ca²⁺ removal mechanisms in mouse embryonic stem cell-derived cardiomyocytes

In mammalian adult cardiomyocytes, sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) plays a major role in controlling the decline of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in comparison with sarcolemmal Na(+)/Ca(2+) exchanger (NCX). However, the functional importance of SERCA and NCX in cytosolic Ca(2+) removal during early cardiomyogenesis is still debated. In this study, the functional contributions of Ca(2+) transporters to [Ca(2+)](i) decline in mouse embryonic stem cell-derived cardiomyocytes (mESCMs), a suitable model for investigation of early cardiogenesis, at various differentiation stages were investigated. We estimated that even at early differentiation stages of mESCMs, SERCA was responsible for approximately 76% of total Ca(2+) removal, while NCX was responsible for approximately 21%. The contributions of SERCA and NCX to cytosolic Ca(2+) clearance were increased to approximately 88% and decreased to approximately 10%, respectively, at the late differentiation stage. Dynamical analysis of the transient decay phases in normal and Na(+)-free solutions suggests that the contribution of NCX to [Ca(2+)](i) decline is more apparent in the terminal slow decay phase than that in the initial fast phase. When SR function was suppressed in type 2 ryanodine receptor-null mESCMs or with ryanodine receptor and SERCA inhibitors (ryanodine and thapsigargin), NCX acted as the main pathway for [Ca(2+)](i) decline. We conclude that the rapid [Ca(2+)](i) decline is mainly achieved by the SR uptake even at the early differentiation stage of mESCMs, while NCX acts as the main Ca(2+) remover when SR function is suppressed. These findings suggest a critical role of SR in the regulation of [Ca(2+)](i) homeostasis even in differentiating cardiomyocytes.