Caveolae Act As Membrane Reserves Which May Limit ICl,swell Activation During Cardiac Myocyte Swelling

Abstract

The channel responsible for swelling-activated chloride current (ICl,swell) is a mechanosensor which responds to changes in membrane tension during cell swelling, and regulates cell volume. It has been proposed that the ICl,swell channel (or elements that regulate this) are dependent on caveolae, and we have previously shown that disrupting caveolae increases the rate of hyposmotic cardiac myocyte swelling. Here we test the hypothesis that the role of caveolae as a membrane reserve limits activation of ICl,swell. Rat ventricular myocytes were treated with methyl- β-cyclodextrin (MBCD) to disrupt caveolae and exposed to 0.02T solution (until cell lysis) or 0.64T solution for 10-15 min (swelling). Maximum cell volume achieved prior to lysis was calculated from a video image. Swollen cells (0.64T) were fixed for electron microscopy, and the negative inotropic response to swelling used as an index of ICl,swell activation. Following disruption of caveolae, the time to lysis in 0.02T solution was significantly reduced compared with control cells. In cells fixed for EM, caveolae were defined as invaginations or closed subsarcolemmal vesicles with a diameter of ≈50-100 nm. MBCD and 0.064T hyposmotic swelling significantly reduced the total number of caveolae by 75 and 50% respectively. Both ‘open’ and ‘closed’ caveolae were reduced by MBCD, but swelling only affected the ‘closed’ population. The negative inotropic response observed 6 and 10 min after exposure to 0.064T solution was blocked by the ICl,swell inhibitor DIDS but enhanced by disruption of caveolae. Our data suggest that swelling causes flattening of ‘open’ caveolae, in tandem with sarcolemmal incorporation of ‘closed’ caveolae. We propose that disrupting caveolae removes essential membrane reserves, thereby speeding cell swelling in hyposmotic conditions and promoting activation of mechanosensitive ICl,swell channels. Supported by CRISTAL and the British Heart Foundation.