Roles of FGF21 signaling in mediating the protective effects of exercise on diabetic cardiomyopathy

Diabetic patients are at high risk of developing diabetic cardiomyopathy (DCM). Although exercise is an effective non-pharmacological strategy for the prevention and treatment of DCM, the underlying molecular mechanisms remain poorly understood. Fibroblast growth factor 21 (FGF21), a peptide hormone with multiple salutary effects on cardiometabolic complications, has been identified as an exercise-responsive factor. Nevertheless, the roles of FGF21 in cardiac function under exercise intervention have not yet been explored. This study aims to investigate: 1) whether FGF21 signaling is involved in exercise-induced improvement in type-2 DCM, 2) whether FGF21 mediates the cardiac protection of exercise via its direct actions in cardiomyocytes, and 3) the mechanism whereby FGF21 signaling contributes to the beneficial effects of exercise in diabetic hearts. Our results show that global-FGF21 knockout (FGF21 KO) and wild-type (WT) mice exhibit similar symptoms of cardiac dysfunction induced by the combination of high-fat diet feeding and streptozotocin injection (HFD/STZ). Predictably, 6-week of treadmill exercise significantly improves HFD/STZ-induced cardiomyopathy in WT mice. However, such cardioprotective effects of exercise are abrogated in FGF21 KO mice, Furthermore, we identified that muscle-specific β-klotho knockout (KLB mKO) mice are also refractory to the beneficial effects of exercise in diabetic hearts, suggesting FGF21 signaling is essential for exercise-induced improvement in DCM. Histological and biochemical analyses additionally revealed that exercise alleviates HFD/STZ-induced mitochondrial dysfunctions, including accumulation of mitochondrial reactive oxygen species (ROS) and lipid droplets, as well as impairments in fatty acid oxidation and ATP production in the hearts of WT mice, whereas FGF21 KO mice are resistant to such beneficial effects of exercise on mitochondrial functions. Further biochemical screening demonstrated that exercise reverses diabetes-induced impairments of complex II&V, long-chain acyl-CoA dehydrogenase (LCAD), and superoxide dismutase-2 (SOD2) activities in WT mice, but not in FGF21 KO mice. Given the important roles of protein deacetylation in the regulation of mitochondrial metabolism and functions, we hypothesized that FGF21 signaling may mediate the acetylation levels of mitochondrial enzymes by modulating the expression of deacetylases sirtuins. Western blot analyses revealed that exercise-induced sirtuin-3 (Sirt3) expression is severely compromised in the hearts of FGF21 KO mice. Conversely, restoration of cardiac Sirt3 levels, using adeno-associated virus serotype-9 (AAV9), alleviates mitochondrial dysfunctions and DCM in FGF21 KO mice. In vitro experiments demonstrated that recombinant FGF21 activates AMP-activated protein kinase and forkhead box O3a (AMPK-FOXO3a) pathways, and induces Sirt3 expression in H9C2 cardiomyocytes. However, the activation of FOXO3a and induction of Sirt3 are abolished after treatment with an AMPK inhibitor. Moreover, chromatin immunoprecipitation (ChIP) assays revealed that FOXO3a binds to the proximal Sirt3 gene promoter regions and enhances transcriptional activity of the Sirt3 promoter, further suggesting that AMPK-FOXO3a signaling contributes to FGF21-induced upregulation of Sirt3 in H9C2 cells. Collectively, these findings suggest that FGF21 signaling plays an essential role in exercise-induced improvement in DCM. Mechanistically, FGF21 stimulates Sirt3 expression via AMPK-FOXO3a signaling in cardiomyocytes. Cardiac Sirt3 reduces hyperacetylation of mitochondrial enzymes, further enhancing mitochondrial activities, thereby improving mitochondrial functions under exercise intervention.