Pyridostigmine Protects Against Diabetic Cardiomyopathy by Regulating Gut Microbiota and Branched-Chain Amino Acid Catabolism to Attenuate Mitochondria Dysfunction

Background: Recent studies have reported that disruption of gut microbes and their metabolites is associated with diabetic cardiomyopathy, but the mechanism by which gut microbes improve diabetic cardiomyopathy remains unclear. Method: Male C57BL/6J mice with high-fat diet and streptozotocin-induced diabetic cardiomyopathy were studied in comparison with control littermates. Diabetic mice were either untreated or subjected to daily intragastric of pyridostigmine. After 10 weeks of hyperglycaemia, vagus activity, cardiac function and cardiac structure were measured by heart rate variability assessment, echocardiography, and immunohistochemistry. The intestinal barrier and gut microbiota were evaluated by fluorescence in situ hybridization and high-throughput sequencing. Additionally, plasma and cardiac branched-chain amino acid (BCAA) distribution and cardiac BCAA catabolism were determined. The structure and respiratory function of mitochondria were measured to assess cardiac mitochondria performance. Results: Intestinal permeability and tight junctions were impaired, bacterial translocation was increased, vagal activity was decreased in mice with diabetic cardiomyopathy mice. Additionally, gut microbes in mice with diabetic cardiomyopathy were disrupted, especially key microbes related to diabetes and BCAA production. Pyridostigmine, which reversibly inhibits cholinesterase to improve autonomic imbalance, enhanced vagus nerve activity, improved insulin resistance and cardiac damage, and alleviated intestinal barrier injury and gut microbiota disruption. Specifically, pyridostigmine decreased the abundance of diabetes-non-protective microbes and increased that of diabetes-protective microbes and BCAA-producing microbes. Pyridostigmine decreased cardiac BCAA concentrations by impairing gut microbe-mediated BCAA production. Furthermore, pyridostigmine upregulated BCAT2 and PP2Cm expression and decreased P-BCKDHA/BCKDHA and BCKDK expression, thus improving cardiac BCAA catabolism. Interestingly, the mitochondrial structural and functional disruption in mice with diabetic cardiomyopathy was attenuated after pyridostigmine administration, which may indicate one of the mechanisms by which BCAAs reduce cardiac damage. Conclusions: In conclusion, intestinal barrier, gut microbiota and vagal activity were impaired in mice with diabetic cardiomyopathy. Pyridostigmine ameliorated insulin resistance and cardiomyopathy, with an effect related to regulated gut microbes and its metabolite BCAA catabolism to attenuate mitochondria dysfunction of heart. These results provide novel insights for the development of a therapeutic strategy for diabetes-induced cardiac damage that targets gut microbes and BCAA catabolism.