Endothelin-1-induced remodelling of murine adult ventricular myocytes

Abstract

The precise role of hormones binding to Gαq protein-coupled receptors (H-GαqPCRs) in chronic heart diseases remains poorly understood. To address this, we used a model of cultured adult rat ventricular myocytes stimulated with endothelin-1 (ET-1) or phenylephrine (PE) over a period of 8 days in vitro (DIV). Chronically treated cells showed an increased number of arrhythmogenic Ca2+ transients when electrically paced at 0.5Hz. While their post-rest behaviour was preserved, from DIV6 onwards the amplitude of caffeine-evoked Ca2+ transients was increased in hormone-treated cells, suggesting an elevated sarcoplasmic reticulum Ca2+ load. The duration of electrically evoked global Ca2+ transients gradually increased over the culturing time indicating decreased activity of processes removing cytosolic Ca2+. In treated cells, spontaneous Ca2+ sparks displayed smaller amplitudes from DIV6 onwards, and a slower decay period for PE (from DIV3) and for ET-1 (from DIV6). This cellular functional remodelling was associated with changes in gene expression: chronic ET-1 treatment decreased PKCγ transcripts, whereas PE increased PKCγ and SERCA2a transcripts as probed by qPCR. Western blot analysis confirmed the upregulation of PKCγ with PE. To study ET-1 receptor desensitization in vivo, osmotic minipumps containing either NaCl or ET-1 were implanted in mice and Ca2+ signalling was studied in acutely isolated ventricular myocytes after 2 weeks of chronic treatment. Interestingly, while cellular responses to isoproterenol stimulation were preserved in ET-1 treated animals, the inotropic response of myocytes to ET-1 stimulation was abrogated. We therefore conclude that chronic stimulation of cardiac myocytes by H-GαqPCRs induces cellular remodelling of Ca2+ cycling with altered PKCγ expression and promotion of arrhythmogenic cellular responses.