Circadian regulation of Ca_V1.2 expression by RORα in the mouse heart

Abstract

Background. In addition to show autonomous beating rhythmicity, the physiological functions of the heart present daily periodic oscillations. Notably the ventricular repolarization itself varies throughout the circadian cycle which was mainly related to the periodic expression of K+ channels. However, the involvement of the L-type Ca2+ channel (CaV) 1.2 encoded by Cacna1c gene) in these circadian variations remains elusive. Methods. We used a transgenic mouse model (PCa-luc) that expresses the luciferase reporter under the control of the cardiac Cacna1c promoter and analyzed promoter activity by bioluminescent imaging, qPCR, immunoblot, Chromatin immunoprecipitation assay (ChIP) and CaV1.2 activity. Results. Under normal 12:12h light-dark cycle, we observed in vivo a biphasic diurnal variation of promoter activities peaking at 9 and 19.5 Zeitgeber time (ZT). This was associated with a periodicity of Cacna1c mRNA levels preceding 24-h oscillations of CaV1.2 protein levels in ventricle (with a 1.5 h phase shift) but not in atrial heart tissues. The periodicity of promoter activities and CaV1.2 proteins, which correlated with biphasic oscillations of L-type Ca2+ current conductance, persisted in isolated ventricular cardiomyocytes from PCa-Luc mice over the course of the 24-h cycle, suggesting an endogenous cardiac circadian regulation. Comparison of 24-h temporal patterns of clock gene expressions in ventricles and atrial tissues of the same mice revealed conserved circadian oscillations of the core clock genes except for the retinoid-related orphan receptor alpha gene (RORalpha), which remained constant throughout the course of a day in atrial tissues. In vitro we found that RORalpha is recruited to two specific regions on the Cacna1c promoter and that incubation with specific RORalpha inhibitor disrupted 24-h oscillations of ventricular promoter activities and CaV1.2 protein levels. Similar results were observed for pore forming subunits of the K+ transient outward currents, KV4.2 and KV4.3. Conclusions. These findings raise the possibility that the RORalpha-dependent rhythmic regulation of cardiac CaV1.2 and KV4.2/4.3 throughout the daily cycle may play an important role in physiopathology of heart function.