T-tubule recovery after detubulation in isolated mouse cardiomyocytes.

Remodeling of cardiac t-tubules in normal and pathophysiological conditions is an important process contributing to the functional performance of the heart. While it is well documented that deterioration of t-tubule network associated with various pathological conditions can be reversed under certain conditions, the mechanistic understanding of the recovery process is essentially lacking. Accordingly, in this study we investigated some aspects of the recovery of t-tubules after experimentally-induced detubulation. T-tubules of isolated mouse ventricular myocytes were first sealed using osmotic shock approach, and their recovery under various experimental conditions was then characterized using electrophysiologic and imaging techniques. The data show that t-tubule recovery is a strongly temperature-dependent process involving reopening of previously collapsed t-tubular segments. T-tubule recovery is slowed by (1) metabolic inhibition of cells, (2) reducing influx of extracellular Ca2+ as well as by (3) both stabilization and disruption of microtubules. Overall, the data show that t-tubule recovery is a highly dynamic process involving several central intracellular structures and processes and lay the basis for more detailed investigations in this area.

The mechanism of osmotically induced sealing of cardiac t tubules.

Cardiac t tubules undergo significant remodeling in various pathological and experimental conditions, which can be associated with mechanical or osmotic stress. In particular, it has been shown that removal of hyposmotic stress can lead to sealing of t tubules. However, the mechanisms underlying the sealing process remain essentially unknown. In this study we used dextran trapping assay to demonstrate that in adult mouse cardiomyocytes, t-tubular sealing can also be induced by hyperosmotic challenge and that both hypo- and hyperosmotic sealing display a clear threshold behavior requiring ≈100 mosmol/L minimal stress. Importantly, during both hypo- and hyperosmotic challenges, the sealing of t tubules occurs only during the shrinking phase. Analysis of the time course of t-tubular remodeling following removal of hyposmotic stress shows that t tubules become sealed essentially instantly, well before any significant reduction in cell size can be observed. Overall, the data support the hypothesis that the critical event in the process of t-tubular sealing during osmotic challenges is detachment (peeling) of the membrane from the underlying cytoskeleton due to suprathreshold stress.NEW & NOTEWORTHY This study provides new insights into how t-tubular membranes respond to osmotic forces. In particular, the data show that osmotically induced sealing of cardiac t tubules is a threshold phenomenon initiated by detachment of t-tubular membrane from the underlying cytoskeleton. The findings are consistent with the hypothesis that final sealing of t tubules is driven by negative hydrostatic intracellular pressure coincident with cell shrinking.

Small membrane permeable molecules protect against osmotically induced sealing of t-tubules in mouse ventricular myocytes.

Cardiac t-tubules are critical for efficient excitation-contraction coupling but become significantly remodeled during various stress conditions. However, the mechanisms by which t-tubule remodeling occur are poorly understood. Recently, we demonstrated that recovery of mouse ventricular myocytes after hyposmotic shock is associated with t-tubule sealing. In this study, we found that the application of Small Membrane Permeable Molecules (SMPM) such as DMSO, formamide and acetamide upon washout of hyposmotic solution significantly reduced the amount of extracellular dextran trapped within sealed t-tubules. The SMPM protection displayed sharp biphasic concentration dependence that peaks at ∼140 mM leading to >3- to 4-fold reduction in dextran trapping. Consistent with these data, detailed analysis of the effects of DMSO showed that the magnitude of normalized inward rectifier tail current (IK1,tail), an electrophysiological marker of t-tubular integrity, was increased ∼2-fold when hyposmotic stress was removed in the presence of 1% DMSO (∼140 mM). Analysis of dynamics of cardiomyocytes shrinking during resolution of hyposmotic stress revealed only minor increase in shrinking rate in the presence of 1% DMSO, and cell dimensions returned fully to prestress values in both control and DMSO groups. Application and withdrawal of 10% DMSO in the absence of preceding hyposmotic shock induced classical t-tubule sealing. This suggests that the biphasic concentration dependence originated from an increase in secondary t-tubule sealing when high SMPM concentrations are removed. Overall, the data suggest that SMPM protect against sealing of t-tubules following hyposmotic stress, likely through membrane modification and essentially independent of their osmotic effects.