CVB-D attenuates experimental diabetic cardiomyopathy by alleviating mitochondrial dysfunction via the JAK1-STAT1 signaling axis in vivo and in vitro.

BACKGROUND: Diabetic cardiomyopathy (DCM), as a prevalent cardiovascular complication in diabetes, involves cardiomyocyte dysfunction as a central pathological feature. Cyclovirobuxine D (CVB-D) is a naturally occurring bioactive alkaloid derived from Buxus microphylla. Emerging evidence suggests that CVB-D may ameliorate diabetes-associated cardiomyocyte failure. However, the protective effects of CVB-D against cardiomyocyte failure have not been extensively investigated, and the underlying molecular mechanisms remain unclear.

METHODS: A mouse model of diabetic cardiomyopathy was established by combining a high-fat diet (HFD) with streptozotocin (STZ). To examine the In vivo contribution of JAK1, AAV9 vectors targeting JAK1 were administered via tail-vein injection, with the corresponding negative-control vectors used in parallel. In vitro, a cardiomyocyte injury model was generated by exposing neonatal mouse ventricular myocytes (NMVMs) to palmitate and high-glucose (PA/HG) conditions. The cardioprotective effects of CVB-D were evaluated using Western blotting, flow cytometry, immunofluorescence microscopy, mitochondrial respiration assays, and ELISA-based measurements. Mechanistic investigations further integrated molecular docking, immunoprecipitation (IP), microscale thermophoresis (MST), surface plasmon resonance (SPR), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to define the molecular targets and JAK1/STAT1 signaling pathways underlying CVB-D activity.

RESULTS: CVB-D treatment robustly improved mitochondrial dysfunction in Diabetic cardiomyopathy and attenuated heart failure-like phenotypes in cardiomyocytes both in vivo and in vitro. Mechanistically, CVB-D reduced JAK1 expression and concomitantly diminished STAT1 phosphorylation, thereby alleviating cardiomyocyte injury. Moreover, convergent evidence from IP, MST, and SPR assays supported a central role for the JAK1-STAT1 axis in mediating the functional effects of CVB-D. LC-MS/MS analysis further identified STAT1 residues T598 and T699 as putative JAK1-dependent phosphorylation regulatory sites in NMVMs. Consistently, genetic knockdown or pharmacological inhibition of JAK1 improved DCM-related phenotypes, whereas enforced JAK1 expression or pharmacological activation blunted the protective effects of CVB-D, indicating that CVB-D-mediated cardioprotection requires suppression of JAK1-STAT1 signaling.

CONCLUSION: Our findings indicate that CVB-D enhances mitochondrial function by suppressing the JAK1-STAT1 signaling axis, thereby ameliorating heart failure associated with DCM. These results suggest that CVB-D may represent a promising therapeutic candidate for the treatment of DCM-related heart failure.