Localization and Role of Transient Receptor Potential Cation Channels in Rabbit Ventricular Myocytes

Abstract

Several members of the transient receptor potential cation (TRPC) channel family are stretch activated. They are thought to play a critical role in mechano-electrical coupling in cardiac myocytes and in cardiac hypertrophic remodeling. However, studies on their subcellular localization in cardiomyocytes are conflicting, and their functions in cardiac myocytes are barely understood. In this study, we investigated the spatial distribution of TRPC channels in isolated ventricular myocytes from adult rabbit using immunolabeling and three-dimensional confocal microscopy. Colocalization of TRPC with (i) sarcolemma labeled with wheat germ agglutinin (WGA), (ii) sarcoplasmic reticulum (SR) labeled for SR calcium ATPase (SERCA2), and (iii) cytoskeletal proteins including sarcomeric α-actinin, desmin, vinculin and β-tubulin, was assessed by a quantitative approach. Furthermore, we measured stretch-activated current with whole-cell voltage clamping. Our results from confocal imaging revealed a transverse striated distribution of TRPC1 and TRPC6 channels. We found colocalization of TRPC1 and TRPC6 with sarcomeric α-actinin, SERCA2A and desmin, but not with WGA, vinculin and β-tubulin. Additionally, no stretch-activated current was detected when stretching isolated cardiomyocytes to approximately 10% of their resting length. Taken together, our data indicate that TRPC1 and TRPC6 are not located in the sarcolemma, but in the membrane of the SR adjacent to the Z-disks in rabbit ventricular myocytes. We suggest that a potential role of TRPC1 and TRPC6 channels is in stretch-dependent calcium leak from the SR and/or counter current balancing calcium release from the SR through ryanodine receptors.