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

Area of Science:

• Cardiovascular Research
• Molecular Cardiology
• Gene Therapy

Background:

• Increasing global prevalence of heart failure necessitates novel therapeutic strategies.
• Protein kinase C alpha (PKCα) is implicated in heart failure pathogenesis.
• Phosphorylation of PKCα at threonine 497 (T497) is crucial for its activation and stability.

Purpose of the Study:

• To investigate the therapeutic potential of ablating PKCα T497 phosphorylation in heart failure.
• To assess the efficacy of CRISPR-Cas9 adenine base editing for introducing the T497A mutation in vivo and in vitro.

Main Methods:

• Generation of germline phospho-resistant PKCα (T497A) mutant mice using CRISPR-Cas9.
• Postnatal genome editing in wild-type mice via adeno-associated virus 9-delivered base editors to introduce the T497A substitution.
• Modeling heart failure using transverse aortic constriction, followed by cardiac function, histological, and transcriptomic analyses.
• Editing of human induced pluripotent stem cells to introduce the PRKCA T497A mutation for in vitro studies on cardiomyocyte function and calcium homeostasis.

Main Results:

• The T497A mutation led to PKCα protein degradation and prevented its activation.
• PKCα phospho-resistant mice were protected against transverse aortic constriction-induced cardiac hypertrophy, fibrosis, and functional decline.
• In vivo base editing achieved similar cardioprotective effects, and in vitro studies demonstrated protection of edited cardiomyocytes from AngII-induced impairments.

Conclusions:

• Ablating PKCα T497 phosphorylation confers significant cardioprotection.
• CRISPR-Cas9 adenine base editing targeting PRKCA T497A is a viable strategy for treating heart failure.
• This gene editing approach holds potential as a therapeutic strategy for human cardiac diseases.