Circular RNA circCHPC is a regulator of hypertrophy and calcium handling in cardiomyocytes

Heart failure is the leading cause of death with a high socioeconomic burden and patient numbers are predicted to increase in the upcoming years. Market approval of novel drugs to treat those patients were rare in the last years. Therefore, novel preventive and therapeutic approaches are urgently needed. The class of non-coding RNAs and more specifically circular RNAs (circRNAs) have an unused potential as targets for pharmaceutical pipelines. CircRNAs are formed by back-splicing of mRNA precursors and exhibit a covalent loop. They are stable, highly conserved and often specifically expressed in organs and disease conditions. The identification of circRNAs and the understanding of their molecular mechanism will provide new targets for the development novel therapeutic strategies for the treatment of cardiovascular diseases.

Here, circRNAs deregulated in a murine model of pressure overload-induced cardiac hypertrophy were identified by global circRNA profiling. One of the significantly deregulated and conserved circRNA was circCHPC (cardiac hypertrophy protecting circRNA). In line with the murine regulation, circCHPC was downregulated in failing cardiac tissue from patients, while the host gene remained unaltered. CircCHPC is highly expressed in the heart and there mostly abundant in cardiomyocytes. To understand the molecular role of circCHPC in cardiomyocytes, we performed loss of function experiments with siRNAs targeting the specific backsplice site of the circRNA. The loss of circCHPC resulted in a hypertrophic response by increased expression of Nppa, Nppb and Rcan1 in neonatal mouse cardiomyocytes (NMCMs) comparable to the hypertrophic stimulation with LIF. In line, the cardiomyocytes showed increased cell size after the knockdown, which we also observed in human iPSC-derived cardiomyocytes. The AAV-mediated overexpression resulted in the successful increase of circCHPC expression, which was able to rescue the increased cell size in hypertrophic cardiomyocytes. Also the expression of Rcan1 was reduced by circCHPC overexpression in hypertrophic cardiomyocytes. By global transcriptome analysis of cardiomyocytes after circCHPC silencing and overexpression, calcium-related processes were among the top deregulated processes. The expression of proteins essential for the calcium handling of murine and human cardiomyocytes like SERCA2a and RYR2 were quantified by Western blotting after circCHPC modulation. The silencing of circCHPC decreased SERCA2a expression, which was not visible after circCHPC overexpression, while RYR2 remained constant upon circCHPC modulation. Therefore, we analyzed calcium transients with the calcium sensor Fura-2 in human iPSC-derived cardiomyocytes. The knockdown of circCHPC resulted in a significant reduction of the calcium transient amplitude, time to peak and relaxation velocity, while those parameters were significantly increased after circCHPC overexpression. This points to a similar calcium dysfunction after circCHPC silencing as known from hypertrophic cardiomyocytes, while the overexpression showed reciprocal effects.

Overall, we identified circCHPC as a promising candidate for therapeutic strategies in cardiac hypertrophy and calcium handling. CircCHPC overexpression mitigated both features of characteristic HF in vitro, which strongly warrants further investigation.