Resolution of hypo-osmotic stress in isolated mouse ventricular myocytes causes sealing of t-tubules

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 (I K1 ) 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 I K1 tail current and ~2-fold reduction in so-called I K1 ‘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.