The SGLT2 Inhibitor Empagliflozin Directly Binds Cardiac JNK3, Uncouples c-Jun/c-Fos Signaling, and Confers Cardioprotection in Diabetic Cardiomyopathy

Background: Sodium-glucose cotransporter 2 (SGLT2) inhibitors, including empagliflozin (EMPA), confer significant cardioprotection in type 2 diabetes in addition to lowering glucose levels. However, their direct targets in cardiac tissue and molecular mechanisms underlying their effects in diabetic cardiomyopathy (DCM) remain unclear. The aim of the present study was to identify a direct cardiac target of EMPA and define the downstream signaling mechanisms mediating its cardioprotective effects in DCM. Methods: A kinase activity screening of 107 kinases was used to identify candidate targets for EMPA, followed by protein-ligand docking and surface plasmon resonance to validate direct binding to c-Jun N-terminal kinase 3 (JNK3). The cardioprotective effects of EMPA (10 mg/kg/day, 10 weeks) were evaluated in male db/db mice. Mechanistic studies were performed using AC16 cardiomyocytes under high glucose and palmitic acid (HG/PA) conditions. JNK3 function was examined using pharmacological inhibition with SR-3576, overexpression, and small interfering RNA knockdown. Results: EMPA improved systolic and diastolic function, reduced cardiac fibrosis and mitochondrial injury in db/db mice, accompanied by suppression of c-Jun/c-Fos signaling. Docking and binding analyses demonstrated selective interaction between EMPA and JNK3 over JNK1/2. EMPA inhibited JNK3-dependent c-Jun/c-Fos signaling without altering pan-JNK phosphorylation, indicating selective disruption of JNK3-c-Jun coupling. In AC16 cells, EMPA or JNK3 inhibition reduced c-Jun phosphorylation and restored mitochondrial function. JNK3 overexpression impaired mitochondrial function. EMPA reversed these effects, whereas JNK3 knockdown improved mitochondrial function independently of EMPA treatment. Conclusion: EMPA directly targets JNK3, selectively uncoupling it from c-Jun/c-Fos signaling and thus preserving mitochondrial function and limiting fibrosis in DCM. These findings identify JNK3 as a novel direct target of EMPA in the heart and provide a mechanistic basis to explain the cardioprotective effect of SGLT2 inhibitors.