Ablation of PKCα Phosphorylation by CRISPR-Cas9 Base Editing Rescues Heart Failure.

Abstract

The prevalence of heart failure is increasing globally, with poor prognosis, highlighting the need for novel therapeutic strategies. PKCα (protein kinase C alpha), encoded by <i>PRKCA</i>, plays a central role in heart failure pathogenesis. Phosphorylation of PKCα at threonine 497 (T497) triggers a series of intramolecular phosphorylation events, leading to its activation. Ablation of T497 phosphorylation leads to reduced stability and activity of PKCα. We generated mice harboring a phospho-resistant PKCα (T497A) mutation in the germline using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated homology-directed repair. To assess the clinical feasibility of postnatal genome editing, we used CRISPR-Cas9 adenine base editing delivered by adeno-associated virus 9 to introduce the T497A substitution into the <i>Prkca</i> gene (<i>Prkca</i>^T497A) in wild-type mice. Mice underwent transverse aortic constriction to model heart failure. Cardiac function, hypertrophy, fibrosis, and transcriptional changes were evaluated by echocardiography, wheat germ agglutinin staining, Masson's trichrome staining, and RNA-sequencing. The editing efficiency of <i>Prkca</i>^T497A was assessed using Sanger sequencing and deep amplicon sequencing. To further explore its clinical potential, we introduced the <i>PRKCA</i>^T497A mutation into human induced pluripotent stem cells by nucleofection-mediated adenine base editing. Ca²⁺ homeostasis was analyzed in Fura-2-loaded human induced pluripotent stem cell-derived cardiomyocytes with <i>PRKCA</i>^T497A under chronic AngII (angiotensin II) stimulation. The T497A mutation in PKCα prevented its subsequent phosphorylation and led to PKCα protein degradation. Four weeks after transverse aortic constriction surgery, wild-type mice showed impaired cardiac function, cardiac remodeling, and increased lung weight. In contrast, PKCα phospho-resistant mice showed protection against heart failure-related aberrant changes in cardiac hypertrophy, fibrosis, and cardiac gene expression. Mice administered with adeno-associated virus 9 base editors to prevent T497 phosphorylation exhibited similar cardioprotective effects. In vitro, PKCα-edited induced pluripotent stem cell-derived cardiomyocytes were protected from AngII-induced impairments in contractility and Ca²⁺ transients. The editing of <i>PRKCA</i>^T497A through adenine base editing represents a potential therapeutic approach for human cardiac diseases.