α-Ketoglutarate improves cardiac insufficiency through NAD+-SIRT1 signaling-mediated mitophagy and ferroptosis in pressure overload-induced mice

Abstract

Background: In heart failure (HF), mitochondrial dysfunction and metabolic remodeling lead to a reduction in energy productivity and aggravate cardiomyocyte injury. Supplementation with α-ketoglutarate (AKG) alleviated myocardial hypertrophy and fibrosis in mice with HF and improved cardiac insufficiency. However, the myocardial protective mechanism of AKG remains unclear. We verified the hypothesis that AKG improves mitochondrial function by upregulating NAD⁺ levels and activating silent information regulator 2 homolog 1 (SIRT1) in cardiomyocytes.

Methods: In vivo, 2% AKG was added to the drinking water of mice undergoing transverse aortic constriction (TAC) surgery. Echocardiography and biopsy were performed to evaluate cardiac function and pathological changes. Myocardial metabolomics was analyzed by liquid chromatography‒mass spectrometry (LC‒MS/MS) at 8 weeks after surgery. In vitro, the expression of SIRT1 or PINK1 proteins was inhibited by selective inhibitors and siRNA in cardiomyocytes stimulated with angiotensin II (AngII) and AKG. NAD⁺ levels were detected using an NAD test kit. Mitophagy and ferroptosis levels were evaluated by Western blotting, qPCR, JC-1 staining and lipid peroxidation analysis.

Results: AKG supplementation after TAC surgery could alleviate myocardial hypertrophy and fibrosis and improve cardiac function in mice. Metabolites of the malate-aspartate shuttle (MAS) were increased, but the TCA cycle and fatty acid metabolism pathway could be inhibited in the myocardium of TAC mice after AKG supplementation. Decreased NAD⁺ levels and SIRT1 protein expression were observed in heart of mice and AngII-treated cardiomyocytes. After AKG treatment, these changes were reversed, and increased mitophagy, inhibited ferroptosis, and alleviated damage in cardiomyocytes were observed. When the expression of SIRT1 was inhibited by a selective inhibitor and siRNA, the protective effect of AKG was suppressed.

Conclusion: Supplementation with AKG can improve myocardial hypertrophy, fibrosis and chronic cardiac insufficiency caused by pressure overload. By increasing the level of NAD⁺, the SIRT-PINK1 and SIRT1-GPX4 signaling pathways are activated to promote mitophagy and inhibit ferroptosis in cardiomyocytes, which ultimately alleviates cardiomyocyte damage.

Keywords: Angiotensin II; Cardiac insufficiency; Ferroptosis; Mitophagy; NAD; SIRT1; Transverse aortic constriction; α-Ketoglutarate.