SPAK regulates myocardial chloride homeostasis and drives dilated cardiomyopathy through mitochondrial dysfunction and energy metabolic disruption

Background Dilated cardiomyopathy (DCM) is a major cause of heart failure and sudden cardiac death, with myocardial ion homeostasis imbalance and mitochondrial dysfunction as core pathological mechanisms. However, the regulatory role of chloride (Cl⁻) homeostasis and its key mediators in DCM remains unclear. Ste20-related proline-alanine-rich kinase (SPAK) regulates cellular ion transport, but its specific function in cardiomyocytes and regulatory mechanisms on mitochondrial function/energy metabolism in DCM are not fully elucidated. Purpose In the present study, we aimed to elucidate the role of SPAK on cardiac function and the regulatory mechanisms of SPAK on the heart and mitochondrial function and energy metabolism. Methods Cardiomyocyte-specific SPAK knockout (SPAK-/-) and control (SPAKf/f) mice were generated. Cardiac function was assessed by transthoracic echocardiography; survival was analyzed by Kaplan-Meier curves. Histopathology (HE, Sirius Red, WGA, TUNEL) and TEM were used to evaluate cardiac morphology, fibrosis, apoptosis, and mitochondrial ultrastructure. To identify the mechanism of SPAK-regulated cardiac function, we applied proteomics and targeted metabolomics, Western blot, ion detection, and qRT-PCR. In vitro, NRCM experiments (shRNA-mediated SPAK knockdown) were performed to verify molecular mechanisms. Results SPAKCKO induced progressive DCM and reduced the survival rate of the mice. Cardiac function (LVEF, LVFS) decreased and ventricular dilation (EDV, ESV) increased from 3 months; typical DCM features (cardiac enlargement, fibrosis) appeared after 4 months. SPAK deletion also promoted cardiomyocyte apoptosis and pyroptosis, with upregulated Myh7, Col3a1, NLRP3, and GSDMD. Severe mitochondrial dysfunction and abnormal mitophagy were observed in vivo and in vitro, by disorganized mitochondrial cristae, reduced ATP and membrane potential, dysregulated ULK1/Parkin/P62 LC3BⅡ/LC3BⅠ expression, and abnormal activation of the mTOR/AKT pathway. SPAK specifically maintained myocardial Cl⁻ homeostasis (serum Cl⁻ abnormal; K⁺/Na⁺ unchanged). Proteomics (4293 proteins) showed differential proteins enriched in mitochondrial function, and mitochondrial complex genes (ATP5mj, COX6a2) were downregulated. Metabolomics showed an abnormal TCA cycle, with citric acid accumulated, while other key metabolites (α-ketoglutaric acid, pyruvate, ATP, NADH) decreased. Conclusion SPAK is essential for myocardial Cl⁻ homeostasis. Its deletion disrupts Cl⁻ balance, induces mitochondrial dysfunction (cristae disorder, ATP reduction), abnormal mitophagy (ULK1/Parkin/P62/LC3B dysregulation), and mTOR/AKT activation, causing energy metabolic disorder (TCA/glycolysis block). These trigger cardiomyocyte death and remodeling, driving DCM. We revealed a novel SPAK-Cl⁻ homeostasis-mitochondrial-energy metabolism axis and identified SPAK as a potential therapeutic target.EchocardiographyHistopathology