Sarcolemmal distribution of ICa and INCX and Ca autoregulation in mouse ventricular myocytes

The balance of Ca influx and efflux regulates the Ca load of cardiac myocytes, a process known as autoregulation. Previous work has shown that Ca influx, via L-type Ca current (ICa), and efflux, via Na- Ca exchange (NCX), occur redominantly at t-tubules; however the role of t-tubules in autoregulation is unknown. We therefore investigated the sarcolemmal distribution of ICa and INCX, and autoregulation, in mouse ventricular myocytes using whole cell voltage-clamp and simultaneous Ca measurement in intact and detubulated (DT) cells. In contrast to rat, INCX was located predominantly at the surface membrane, and the hysteresis between INCX and Ca observed in intact myocytes was preserved following detubulation. Immunostaining showed both NCX and ryanodine
48 receptors (RyRs) at the t-tubules and surface membrane, consistent with co-localization of NCX and RyR at both sites. Unlike INCX, ICa was found predominantly in the t-tubules. Recovery of Ca transient amplitude to steady-state (autoregulation) following application of 200 µM or 10 mM caffeine was slower in DT cells than in intact cells. However during application of 200 µM caffeine, to increase SR Ca release, DT and intact cells recovered at the same rate. It appears likely that this asymmetric response to changes in SR Ca release is a consequence of the distributions of ICa, which is reduced in DT cells and is required to re-fill the SR after depletion, and NCX, which is little affected by DT, remaining available to remove Ca when SR Ca release is increased.