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.

TRPV4 in porcine lens epithelium regulates hemichannel-mediated ATP release and Na-K-ATPase activity

In several tissues, transient receptor potential vanilloid 4 (TRPV4) channels are involved in the response to hyposmotic challenge. Here we report TRPV4 protein in porcine lens epithelium and show that TRPV4 activation is an important step in the response of the lens to hyposmotic stress. Hyposmotic solution (200 mosM) elicited ATP release from intact lenses and TRPV4 antagonists HC 067047 and RN 1734 prevented the release. In isosmotic solution, the TRPV4 agonist GSK1016790A (GSK) elicited ATP release. When propidium iodide (PI) (MW 668) was present in the bathing medium, GSK and hyposmotic solution both increased PI entry into the epithelium of intact lenses. Increased PI uptake and ATP release in response to GSK and hyposmotic solution were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18α-glycyrrhetinic acid and probenecid. Increased Na-K-ATPase activity occurred in the epithelium of lenses exposed to GSK and 18α-glycyrrhetinic acid + probenecid prevented the response. Hyposmotic solution caused activation of Src family kinase and increased Na-K-ATPase activity in the lens epithelium and TRPV4 antagonists prevented the response. Ionomycin, which is known to increase cytoplasmic calcium, elicited ATP release, the magnitude of which was no greater when lenses were exposed simultaneously to ionomycin and hyposmotic solution. Ionomycin-induced ATP release was significantly reduced in calcium-free medium. TRPV4-mediated calcium entry was examined in Fura-2-loaded cultured lens epithelium. Hyposmotic solution and GSK both increased cytoplasmic calcium that was prevented by TRPV4 antagonists. The cytoplasmic calcium rise in response to hyposmotic solution or GSK was abolished when calcium was removed from the bathing solution. The findings are consistent with hyposmotic shock-induced TRPV4 channel activation which triggers hemichannel-mediated ATP release. The results point to TRPV4-mediated calcium entry that causes a cytoplasmic calcium increase which is an essential early step in the mechanism used by the lens to sense and respond to hyposmotic stress.