BACKGROUND: 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, 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. PRKCA
METHODS: 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 thegene () 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 ofwas assessed using Sanger sequencing and deep amplicon sequencing. To further explore its clinical potential, we introduced themutation into human induced pluripotent stem cells by nucleofection-mediated adenine base editing. Cahomeostasis was analyzed in Fura-2-loaded human induced pluripotent stem cell-derived cardiomyocytes withunder chronic AngII (angiotensin II) stimulation. Prkca Prkca Prkca PRKCA PRKCA T497A T497A T497A2+ T497A
RESULTS: 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 cardiomyocyte were protected from AngII-induced impairments in contractility and Catransients. 2+
CONCLUSIONS: The editing ofthrough adenine base editing represents a potential therapeutic approach for human cardiac diseases. PRKCA T497A
CRISPR Gene Editing