Ca2 + homeostasis in sealed t-tubules of mouse ventricular myocytes

Abstract

We have recently shown that in mouse ventricular myocytes, t-tubules can be quickly and tightly sealed during the resolution of hyposmotic shock of physiologically relevant magnitude. Sealing of t-tubules is associated with trapping extracellular solution inside the myocytes but the ionic homeostasis of sealed t-tubules and the consequences of potential transtubular ion fluxes remain unknown. In this study we investigated the dynamics of Ca2+ movements associated with sealing of t-tubules. The data show that under normal conditions sealed t-tubules contain Ca2+ at concentrations below 100μM. However, blockade of voltage-dependent Ca2+ channels with 10μM nicardipine, or increasing extracellular concentration of K+ from 5.4mM to 20mM led to several fold increase in concentration of t-tubular Ca2+. Alternatively, the release of Ca2+ from sarcoplasmic reticulum using 10mM caffeine led to the restoration of t-tubular Ca2+ towards extracellular levels within few seconds. Sealing of t-tubules in the presence of extracellular 1.5mM Ca2+ and 5.4mM extracellular K+ led to occasional and sporadic intracellular Ca2+ transients. In contrast, sealing of t-tubules in the presence of 10mM caffeine was characterized by a significant long lasting increase in intracellular Ca2+. The effect was completely abolished in the absence of extracellular Ca2+ and significantly reduced in pre-detubulated myocytes but was essentially preserved in the presence of mitochondrial decoupler dinitrophenol. This study shows that sealed t-tubules are capable of highly regulated transport of Ca2+ and present a major route for Ca2+ influx into the cytosol during sealing process.