Nsun2 controls cardiac homeostasis and hypertrophic response by regulating PRKACA expression.

Rationale: Internal modifications of mammalian RNA have been suggested to be essential for the maintenance of cardiac homeostasis. However, the role of RNA cytosine methylation (m5C) in the heart remains largely unknown. Methods: Bulk and single cell RNA sequencing data and tissues from the human hearts were exploited for analyzing the expression of RNA m5C modifying proteins. Neonatal rat and adult mouse cardiomyocytes were isolated to assess the impact of Nsun2 on cellular hypertrophic response. Cre/LoxP-mediated gene knockout and recombinant adeno-associated virus serotype 9 (rAAV9) were employed respectively to achieve cardiac-specific interference of the expression of related genes in mice that were subjected to heart stresses from aging, aortic constriction, and angiotensin II stimulation. RNA m5C immunoprecipitation sequencing (m5C-RIP-seq), RNA pull-down, polysome profiling, reporter gene analysis, and IonOptix measurement were conducted to elucidate the involved regulatory mechanisms. Results: Nsun2 expression was significantly elevated in human, rat, and mouse hypertrophic myocardial cells. Knockout of Nsun2 (αMHC-Cre^ERT2, Nsun2 flox^+/+) abolished the hypertrophic response of mice to diverse stresses, while accelerating the progression of heart failure. Mechanistically, Nsun2 specifically methylates PKA catalytic subunit alpha (PRKACA) mRNA, which substantially promotes PRKACA translation in a YBX1-dependent manner. Nsun2 ablation markedly attenuated the activation of PKA signaling, as evidenced by the reduced PKA activity and protein phosphorylation levels of PKA substrates, impaired myocyte contraction and relaxation, and disturbed calcium transients. Overexpressing Nsun2 and PRKACA-3'UTR transcripts in the myocardia sensitized and desensitized heart hypertrophic responses, respectively, whereas co-administration of the PKA inhibitor H-89 or overexpressing PRKACA-3'UTR transcript lacking Nsun2 methylating regions failed to produce corresponding responses, reiterating the significance of Nsun2-PRKACA regulation in the cardiac hypertrophic program. Conclusion: These observations reveal the importance of Nsun2-PRKACA regulation in cardiac homeostasis, which provides novel insights into heart function modulation and sheds light on future treatments for hypertrophic remodeling associated heart diseases.