N6-methyladenosine regulator Rbm15b protects against adverse cardiac remodeling under pressure overload through modulating RNA methylation

Abstract Background and Purpose RNA modification controls protein synthesis. Although dysregulation of RNA modification has been implicated in the pathogenesis of heart failure, it remains largely unknown how RNA modification could be modulated for the treatment of heart failure. This study was performed to identify a protective RNA modification regulator in cardiomyocytes against pathological stress that mitigates adverse cardiac remodeling during pressure overload and to elucidate the underlying mechanism. Methods A whole-genome CRISPR activation screen was performed in HL-1 cardiomyocyte cell line transduced with dCAS9-VPR. Phenylephrine, cobalt chloride and hydrogen peroxide were used as pathological stressors. Neonatal rat ventricular myocytes (NRVMs) were used for further in vitro experiments. Doxorubicin was used as a pathological stressor. DNA damage was quantified via phospho-H2AX detection, and mitochondrial function was assessed by measuring mitochondrial membrane potential. RNA immunoprecipitation sequencing (RIP-seq) and immunoprecipitation coupled with mass spectrometry were performed in NRVMs overexpressing FLAG-tagged Rbm15b. For in vivo experiments, wild-type (WT) mice and transgenic mice with cardiomyocyte-specific Rbm15b overexpression (Rbm15b-TG), aged 8–12 weeks, underwent transverse aortic constriction (TAC) surgery to induce pressure overload. Echocardiographic and histological analyses were performed for the assessment of cardiac phenotype. Myocardial RNA methylation patterns were characterized using methylated RNA immunoprecipitation sequencing (MeRIP-seq). Results A whole-genome CRISPR activation screen identified Rbm15b as a cardioprotective N6-methyladenosine regulator under pathological stress conditions in HL-1 cardiomyocytes. Overexpression of Rbm15b in NRVMs enhanced cell viability and mitochondrial integrity under doxorubicin-induced stress. Transcriptomic analyses revealed that Rbm15b overexpression modulates genes involved in sarcomere structure and mitochondrial function. RIP-seq analyses demonstrated specific binding of Rbm15b to mRNAs associated with inflammation, mitochondrial function, and heart development. Additionally, mass spectrometry indicated Rbm15b participation in protein-protein interactions governing mitochondrial biogenesis and RNA metabolism. Compared with WT controls, Rbm15b-TG mice exhibited reduced pathological cardiac remodeling under pressure overload. RNA sequencing combined with MeRIP-seq and gene set enrichment analysis highlighted the central role of Rbm15b in regulating metabolic pathways as well as muscle and heart development pathways by modulating RNA methylation of genes involved in these pathways. Conclusions Rbm15b protects against adverse cardiac remodeling induced by pressure overload. This protection might be mediated, at least partially, by modulating inflammation, myofibril organization, and mitochondrial function via RNA methylation-dependent mechanisms.