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 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 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 100 μM. However, blockade of voltage-dependent Ca2+ channels with 10 μM nicardipine, or increasing extracellular concentration of K+ from 5.4 mM to 20 mM led to several fold increase in concentration of t-tubular Ca2+. Alternatively, release of Ca2+ from sarcoplasmic reticulum using 10 mM caffeine led to restoration of t-tubular Ca2+ towards extracellular levels within few seconds. Sealing of t-tubules in the presence of extracellular 1.5 mM Ca2+ and 5.4 mM extracellular K+ led to occasional and sporadic intracellular Ca2+ transients. In contrast, sealing of t-tubules in the presence of 10 mM caffeine was characterized by 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 cytosol during sealing process.

Sarcolemmal distribution of ICa and INCX and Ca2+ autoregulation in mouse ventricular myocytes

“…Although t-tubules uncoupled from the surface membrane by DT may reseal within the cell and, in principle, sequester and release Ca 2+ ( 5 , 24 ), it seems unlikely that they can explain the current data, since they are electrically uncoupled from the surface membrane. Therefore, Ca 2+ channels in such resealed tubules will not undergo activation (and thus inactivation), an idea supported by the lack of Ca 2+ release in the center of DT myocytes ( 5 ).…”

Caspofungin Modulates Ryanodine Receptor-Mediated Calcium Release in Human Cardiac Myocytes

“…Caffeine (CAF) is well known to release Ca 2ϩ most probably from sarcoplasmic reticulum (SR) by activation of RYN receptors. Therefore, depleting caffeine-sensitive SR Ca 2ϩ stores was found to inhibit CAS (140 g/ml)-induced Ca 2ϩ release (21,22). Also, RYN used in doses that inhibit ryanodine receptors was able to inhibit CAS-induced increase in [Ca 2ϩ ] i .…”

Cholesterol Protects Against Acute Stress-Induced T-Tubule Remodeling in Mouse Ventricular Myocytes

“…Myocytes were isolated from the hearts essentially as described in the study by Moench and Lopatin (2014) and used for experiments within 1–8 h post-isolation.…”