Cholesterol Protects Against Acute Stress-Induced T-Tubule Remodeling in Mouse Ventricular Myocytes

Efficient excitation-contraction coupling in ventricular myocytes depends critically on the presence of the t-tubular network. It has been recently demonstrated that cholesterol, a major component of the lipid bilayer, plays an important role in long-term maintenance of the integrity of t-tubular system although mechanistic understanding of underlying processes is essentially lacking. Accordingly, in this study we investigated the contribution of membrane cholesterol to t-tubule remodeling in response to acute hyposmotic stress. Experiments were performed using isolated left ventricular cardiomyocytes from adult mice. Depletion and restoration of membrane cholesterol was achieved by applying methyl-β-cyclodextrin (MβCD) and water soluble cholesterol (WSC), respectively, and t-tubule remodeling in response to acute hyposmotic stress was assessed using fluorescent dextran trapping assay and by measuring t-tubule dependent IK1 tail current (IK1,tail). The amount of dextran trapped in t-tubules sealed in response to stress was significantly increased when compared to control cells, and reintroduction of cholesterol to cells treated with MβCD restored the amount of trapped dextran to control values. Alternatively, application of WSC to normal cells significantly reduced the amount of trapped dextran further suggesting the protective effect of cholesterol. Importantly, modulation of membrane cholesterol (without osmotic stress) led to significant changes in various parameters of IK1, tail strongly suggesting significant but essentially hidden remodeling of t-tubules prior to osmotic stress. Results of this study demonstrate that modulation of the level of membrane cholesterol has significant effects on the susceptibility of cardiac t-tubules to acute hyposmotic stress

Functional consequences of Kir2.1/Kir2.2 subunit heteromerization

Kir2 subunits form channels that underlie classical strongly inwardly rectifying potassium currents. While homomeric Kir2 channels display a number of distinct and physiologically important properties, the functional properties of heteromeric Kir2 assemblies, as well as the stoichiometries and the arrangements of Kir2 subunits in native channels, remain largely unknown. Therefore, we have implemented a concatemeric approach, whereby all four cloned Kir2 subunits were linked in tandem, in order to study the effects of Kir2.1 and Kir2.2 heteromerization on properties of the resulting channels. Kir2.2 subunits contributed stronger to single-channel conductance than Kir2.1 subunits, and channels containing two or more Kir2.2 subunits displayed conductances indistinguishable from that of a Kir2.2 homomeric channel. In contrast, single-channel kinetics was a more discriminating property. The open times were significantly shorter in Kir2.2 channels compared with Kir2.1 channels and decreased nearly proportionally to the number of Kir2.2 subunits in the heteromeric channel. Similarly, the sensitivity to block by barium also depended on the proportions of Kir2.1 to Kir2.2 subunits. Overall, the results showed that Kir2.1 and Kir2.2 subunits exert neither a dominant nor an anomalous effect on any of the properties of heteromeric channels. The data highlight opportunities and challenges of using differential properties of Kir2 channels in deciphering the subunit composition of native inwardly rectifying potassium currents