It has been recently shown that various stress-inducing manipulations in isolated ventricular myocytes may lead to significant remodeling of t-tubules. Osmotic stress is one of the most common complications in various experimental and clinical settings. Therefore, this study was designed to determine the effects of a physiologically relevant type of osmotic stress, hypo-osmotic challenge, to the integrity of t-tubular system in mouse ventricular myocytes using two approaches: (1) electrophysiological measurements of membrane capacitance and inward rectifier (IK1) tail currents originating from K+ accumulation in t-tubules, and (2) confocal microscopy of fluorescent dextrans trapped in sealed t-tubules. Importantly, we found that removal of 0.6 Na (60% NaCl) hypo-osmotic solution, but not its application to myocytes, led to ~27% reduction in membrane capacitance, ~2.5-fold reduction in the amplitude of IK1 tail current and ~2-fold reduction in so-called IK1 ‘inactivation’ (due to depletion of t-tubular K+) at negative membrane potentials – all data being consistent with significant detubulation. Confocal imaging experiments also demonstrated that extracellularly applied dextrans become trapped in sealed t-tubules only upon removal of hypo-osmotic solutions, i.e. during shrinking phase, but not during initial swelling period. In light of these data, relevant previous studies, including those on EC coupling phenomena during hypo-osmotic stress, may need to be reinterpreted and the experimental design of future experiments should take into account the novel findings.

Cardiac ventricular myocytes possess an extensive t-tubular system that facilitates the propagation of membrane potential across the cell body. It is well established that ionic currents at the restricted t-tubular space may lead to significant changes in ion concentrations, which, in turn, may affect t-tubular membrane potential. In this study, we used the whole cell patch-clamp technique to study accumulation and depletion of t-tubular potassium by measuring inward rectifier potassium tail currents ( IK1,tail), and inward rectifier potassium current ( IK1) “inactivation”. At room temperatures and in the absence of Mg2+ ions in pipette solution, the amplitude of IK1,tail measured ∼10 min after the establishment of whole cell configuration was reduced by ∼18%, but declined nearly twofold in the presence of 1 mM cyanide. At ∼35°C IK1,tail was essentially preserved in intact cells, but its amplitude declined by ∼85% within 5 min of cell dialysis, even in the absence of cyanide. Intracellular Mg2+ ions played protective role at all temperatures. Decline of IK1,tail was accompanied by characteristic changes in its kinetics, as well as by changes in the kinetics of IK1 inactivation, a marker of depletion of t-tubular K+. The data point to remodeling of t tubules as the primary reason for the observed effects. Consistent with this, detubulation of myocytes using formamide-induced osmotic stress significantly reduced IK1,tail, as well as the inactivation of inward IK1. Overall, the data provide strong evidence that changes in t tubule volume/structure may occur on a short time scale in response to various types of stress.