Ablation of PKCα phosphorylation cascades by CRISPR-Cas9 Base Editing Rescues Heart Failure

BACKGROUND: The global prevalence of heart failure is increasing significantly, and the prognosis for patients with heart failure remains poor, highlighting the urgent need for novel therapeutic strategies. Protein kinase C alpha (PKCα) plays a central role in heart failure progression. Phosphorylation of PRKCA at T497 triggers a series of phosphorylation cascades, leading to its activation. Ablation of T497 phosphorylation leads to reduced protein stability and activity. However, there is no therapeutic strategy for heart failure targeting PKCα T497. METHODS: We employed CRISPR-Cas9 adenine base editing to ablate the phosphorylation site T497 of PKCα. We generated mice harboring a PKCα phosphoresistant T497A mutation in the germline and administered adeno-associated virus 9 (AAV9) harboring base editor of PrkcaT497A to postnatal wild-type (WT) mice to explore its clinical feasibility. To model for heart failure, mice were subjected to transverse aortic constriction (TAC). Cardiac function, hypertrophy, fibrosis, and transcriptional changes were evaluated by echocardiography, wheat germ agglutinin staining, Masson’s trichrome staining, and RNA sequencing, respectively. The editing efficiency of PrkcaT497A was assessed using Sanger sequencing and deep amplicon sequencing. Cellular Ca2+ homeostasis was analyzed using epifluorescence microscopy in Fura-2–loaded human induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) carrying the PRKCAT497A mutation under chronic angiotensin II (AngII) stimulation. RESULTS: The T497A mutation in PKCα prevented subsequent phosphorylation and led to PKCα degradation. Four weeks after TAC surgery, WT mice showed dramatically impaired cardiac function, cardiac remodeling, and increased lung weight, whereas PKCα phosphoresistant mice were protected from these effects. PKCα phosphoresistant mice also showed protection against heart failure-related aberrant changes in cardiac gene expression, cardiac hypertrophy, and fibrosis. Likewise, mice administrated with AAV9-base editors exhibited similar cardioprotective effects against TAC-induced heart failure. In vitro, PKCα-edited iPSC-CMs were protected from AngII-induced impairments in contractility and Ca2+ transients observed in WT iPSC-CMs. CONCLUSIONS: The editing of PRKCAT497A through adenine base editing may potentially provide a promising therapeutic approach for human cardiac diseases.