Ca(2+) homeostasis in sealed t-tubules of mouse ventricular myocytes.

We have recently shown that in mouse ventricular myocytes, t-tubules can be quickly and tightly sealed during the 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 the dynamics of Ca(2+) movements associated with sealing of t-tubules. The data show that under normal conditions sealed t-tubules contain Ca(2+) at concentrations below 100µM. However, blockade of voltage-dependent Ca(2+) channels with 10µM nicardipine, or increasing extracellular concentration of K(+) from 5.4mM to 20mM led to several fold increase in concentration of t-tubular Ca(2+). Alternatively, the release of Ca(2+) from sarcoplasmic reticulum using 10mM caffeine led to the restoration of t-tubular Ca(2+) towards extracellular levels within few seconds. Sealing of t-tubules in the presence of extracellular 1.5mM Ca(2+) and 5.4mM extracellular K(+) led to occasional and sporadic intracellular Ca(2+) transients. In contrast, sealing of t-tubules in the presence of 10mM caffeine was characterized by a significant long lasting increase in intracellular Ca(2+). The effect was completely abolished in the absence of extracellular Ca(2+) 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 Ca(2+) and present a major route for Ca(2+) influx into the cytosol during sealing process.

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

It has recently been shown that various stress-inducing manipulations in isolated ventricular myocytes may lead to significant remodelling of t-tubules. Osmotic stress is one of the most common complications in various experimental and clinical settings. This study was therefore designed to determine the effects of a physiologically relevant type of osmotic stress, hyposmotic challenge, to the integrity of the t-tubular system in mouse ventricular myocytes using the following 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) hyposmotic solution, but not its application to myocytes, led to a ∼27% reduction in membrane capacitance, a ∼2.5-fold reduction in the amplitude of the IK1 tail current and a ∼2-fold reduction in the so-called IK1 'inactivation' (due to depletion of t-tubular K(+)) at negative membrane potentials; all these data were consistent with significant detubulation. Confocal imaging experiments also demonstrated that extracellularly applied dextrans become trapped in sealed t-tubules only upon removal of hyposmotic solutions, i.e. during the shrinking phase, but not during the initial swelling period. In light of these data, relevant previous studies, including those on excitation-contraction coupling phenomena during hyposmotic stress, may need to be reinterpreted, and the experimental design of future experiments should take into account the novel findings.

MsrA protects cardiac myocytes against hypoxia/reoxygenation induced cell death.

Reactive oxygen species (ROS) are critical in tissue responses to ischemia-reperfusion. The enzyme methionine sulfoxide reductase-A (MsrA) is capable of protecting cells against oxidative damage by reversing damage to proteins caused by methionine oxidation or by decreasing ROS through a scavenger mechanism. The current study employed adenovirus mediated over-expression of MsrA in primary neonatal rat cardiac myocytes to determine the effect of this enzyme in protecting against hypoxia/reoxygenation in this tissue. Cells were transduced with MsrA encoding adenovirus and subjected to hypoxia/reoxygenation. Apoptotic cell death was decreased by greater than 45% in cells over-expressing MsrA relative to cells transduced with a control virus. Likewise total cell death as determined by levels of LDH release was dramatically decreased by MsrA over-expression. These observations indicate that MsrA is protective against hypoxia/reoxygenation stress in cardiac myocytes and point to MsrA as an important therapeutic target for ischemic heart disease.