Resolution of hyposmotic stress in isolated mouse ventricular myocytes causes sealing of t‐tubules

Abstract

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.

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

“…Measurements of calcium in sealed t-tubules, [Ca 2+ ] st , were performed using low affinity calcium indicator Rhod-5N trapped in t-tubules during resolution of hyposmotic shock [27]. A small aliquot (up to few hundred μl) of enzymatically digested myocytes was diluted in a large volume (usually 10 ml) of 0.6 Na solution in order to induce swelling of myocytes and the timer started.…”

“…This powerful experimental approach remains extremely useful for studies of cardiac t-tubules but, surprisingly, osmotic shock was not considered as one of the potential causes of t-tubular remodeling in vivo . In this regard, we have recently shown that in isolated mouse ventricular myocytes quick and significant loss of t-tubules can occur in response to resolution of hyposmotic shock of physiologically relevant magnitude [27]. In both types of osmotic shock (hyperosmotic or hyposmotic) the loss of t-tubules is due to their tight sealing [27, 28] rather than some kind of membrane disintegration.…”

“…In this regard, we have recently shown that in isolated mouse ventricular myocytes quick and significant loss of t-tubules can occur in response to resolution of hyposmotic shock of physiologically relevant magnitude [27]. In both types of osmotic shock (hyperosmotic or hyposmotic) the loss of t-tubules is due to their tight sealing [27, 28] rather than some kind of membrane disintegration. Similar “pinching off” of t-tubules has also been observed during culture of rat ventricular myocytes [29].…”

“…An important consequence of osmotically induced remodeling is a tight sealing of t-tubular lumen such that small molecules present in extracellular solution can be ‘efficiently’ trapped inside the myocytes [27, 28]. However, the fate of various components of trapped extracellular solution (e.g.…”

Label-free microscopy and stress responses reveal the functional organization of Pseudodiaptomus marinus copepod myofibrils

“…Muscle fibers swell when the external osmolarity is reduced and shrink when it is increased (Millman, 1998). Hypoosmotic shock in cardiac myocytes leads to significant changes in ion channels, transporters, intracellular organelles and key parameters characterizing their activity, such as membrane potential and currents and excitation contraction coupling (Moench et al, 2013;Vandenberg et al, 1996). After the combined temperature and haline stress, we hypothesize that energy in copepods muscles is rapidly released resulting in extension forces along the myofibrils.…”