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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE294246 | GEO | 2026/03/04

REPOSITORIES: GEO

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Project description: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.

Project description:Compelling evidence suggests that mitochondrial dysfunction contributes to the pathogenesis of heart failure, including defects in the substrate oxidation, and the electron transport chain (ETC) and oxidative phosphorylation (OXPHOS). However, whether such changes occur early in the development of heart failure, and are potentially involved in the pathologic events that lead to cardiac dysfunction is unknown. To address this question, we conducted transcriptomic/metabolomics profiling in hearts of mice with two progressive stages of pressure overload-induced cardiac hypetrophy: i) cardiac hypertrophy with preserved ventricular function achieved via transverse aortic constriction for 4 weeks (TAC) and ii) decompensated cardiac hypertrophy or heart failure (HF) caused by combining 4 wk TAC with a small apical myocardial infarction. Transcriptomic analyses revealed, as shown previously, downregulated expression of genes involved in mitochondrial fatty acid oxidation in both TAC and HF hearts compared to sham-operated control hearts. Surprisingly, however, there were very few changes in expression of genes involved in other mitochondrial energy transduction pathways, ETC, or OXPHOS. Metabolomic analyses demonstrated significant alterations in pathway metabolite levels in HF (but not in TAC), including elevations in acylcarnitines, a subset of amino acids, and the lactate/pyruvate ratio. In contrast, the majority of organic acids were lower than controls. This metabolite profile suggests “bottlenecks” in the carbon substrate input to the TCA cycle. This transcriptomic/metabolomic profile was markedly different from that of mice PGC-1a/b deficiency in which a global downregulation of genes involved in mitochondrial ETC and OXPHOS was noted. In addition, the transcriptomic/metabolomic signatures of HF differed markedly from that of the exercise-trained mouse heart. We conclude that in contrast to current dogma, alterations in mitochondrial metabolism that occur early in the development of heart failure reflect largely post-transcriptional mechanisms resulting in impedance to substrate flux into the TCA cycle, reflected by alterations in the metabolome. Microarray gene expression profiling was performed with bi-ventricle RNA isolated from (1) 3 month-old female C57Bl6/J mice with transverse aortic constriction (CH), vs sham-operated control (Sham-CH), (2) 3 month-old female C57Bl6/J mice with heart failure (HF), vs. sham-operated control (Sham-HF); (3) 2 month-old female C57Bl6/J mice with voluntary wheel training for 2 months (Run), vs. Sedentary control (Sed). Five biological replicates are included for each group.

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

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