The RGK protein rrad regulates cardiac calcium levels in zebrafish

Abstract

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science, Innovation and Universities, Spain Background/Introduction High voltage-activated Ca2+ (CaV) channels are essential for excitation-contraction coupling. RGK proteins (Gem, Rad, Rem, and Rem2) are small Ras-like G proteins that bind CaV β-subunits and profoundly inhibit CaV1/CaV2 channels. A recent study showed that Rad inhibits CaV1.2 channels in cardiomyocytes, thus mediating the enhanced inotropy and chronotropy by β-adrenergic agonists during the fight-or-flight response. The zebrafish has emerged as a useful in vivo animal model in cardiovascular physiology and pathophysiology. The role of zebrafish rrad, the orthologe of the human RAD, in the regulation of cardiac Ca2+ homeostasis in zebrafish has not been investigated. Purpose The aim of this study was to investigate the role of rrad in cardiac Ca2+ regulation in zebrafish. Methods We evaluated the cardiac Ca2+ dynamics, heart rhythm, and hemodynamics in transgenic zebrafish larvae that overexpress rrad in the heart, as well as in heterozygous rrad knockout larvae. Measurements were done in the beating heart of 3 dpf transgenic zebrafish larvae expressing the fluorescent Ca2+ biosensor Twitch-4. Results In some larvae, overexpression of rrad induced a severe enlargement of the atrium with pericardial edema and a high mortality rate. In addition, the ventricle displayed low Ca2+ levels and reduced contractility. The remaining larvae had no altered morphology, but the ventricular Ca2+ levels were reduced. On the other hand, preliminary results showed that down regulation of rrad in zebrafish larvae increased atrial and ventricular Ca2+ levels, like what was found in Rad knockout mice. Conclusion Taken together, these findings suggest that rrad in the zebrafish heart, as in mammals, is an inhibitor of the Ca2+ entry, presumably through CaV1/CaV2 channels, and demonstrate the zebrafish's utility as a model to study the involvement of rrad in cardiac function.