The Anti-Fibrotic Activity of Sodium Glucose Co-Transporters (Sglts) Inhibition: Therapeutic Potential in Diabetes Mellitus Cardiomyopathy

Background: Cardiovascular (CV) complications are major causes of mortality in diabetes mellitus (DM) patients. Myocardial fibrosis is one of the major pathological consequences of long-standing DM. Interstitial and perivascular fibrosis are pathological hallmarks of DM cardiomyopathy. However, the mechanisms underlying CV dysfunction in DM cardiomyopathy are not clear, and treatment is not well established. Recent trials demonstrated that sodium glucose co-transporter (SGLT)2 inhibitor has significant improvement on CV mortality. However, mechanism by which SGLTs inhibition improves CV outcomes are not fully understood. Although significant reduction in cardiac interstitial fibrosis and perivascular fibrosis was found on empagliflozin, the role of SGLT1 and or SGLT2 inhibition on the myocardial fibroblastic activities and their underlying signaling mechanisms need further investigation. Accordingly, SGLTs inhibitor may rescue DM cardiomyopathy by improving cardiac fibrosis. Therefore, our purpose is to investigate the anti-fibrotic activity of SGLTs inhibitor in DM cardiomyopathy, and whether inhibition of SGLTs will improve myocardial dysfunction through modulation on cardiac structural remodeling and electrical activity. In the first year, we will investigate on anti-fibrotic activity of SGLTs inhibitor in the cardiac fibroblast with or without hyperglycemic condition. In the second year, we will evaluate whether SGLTs inhibitors will modulate cardiac fibrosis, apoptosis, autophagy and related downstream signaling pathway in type 2 DM cardiomyopathy. We will further investigate whether SGTLs inhibitor will regulate calcium (Ca2+) and sodium homeostasis and reactive oxygen species to rescue DM-related cardiomyopathy. In the third year experiment, we will perform small RNA deep sequencing to analyze the differentially miRNAs expressions in the hearts of DM rats on SGLTs inhibitor and explore the potential underlying signaling sequences. Methods: First year: Migration, proliferation, collagen production, nitric oxide production assay, Ca2+ fluorescence imaging, picrosirius red staining and Western blots will be evaluated in human cardiac fibroblasts without or with glucose (25mM), with and without KGA-2727(selective SGLT1inhibitor,100M), empagliflozin(selective SGLT2 inhibitor, 100M), LX4211(dual SGLT1 and SGLT2 inhibitor, 100M) for 48 hours and co-administration of L-NAME (NOS inhibitor, 1μmol/L), EGTA (free Ca2+ chelator, 1nmol/L), or SCH79797 (PAR1 inhibitor, 1μmol/L).Second Year: Type 2 DM rats will be induced and structural and functional modulation in DM cardiomyopathy will be evaluated by echocardiography, electrocardiography, blood sampling, histopathological and immunochemistry examination. Cardiac ventricular fibroblasts will be isolated from 16 week old male Wistar rats group as control, type 2 DM treated with or without KGA-2727(0.3mg/kg), empagliflozin(10mg/kg) or LX4211(10mg/kg) orally for 4 weeks. Migration and proliferation assay, oxidative stress assay, collagen measurement, cytokine array, Western blot, whole patch clamp studies for action potential duration and ionic currents will be evaluated in the ventricular fibroblasts of control, type 2 DM treated with or without KGA-2727, empagliflozin, LX4211. Third year: small RNA deep sequencing will be performed in the cardiac ventricular fibroblast of rodents to analyze the differentially miRNA expressions and identify the regulatory pathways between control and type 2 DM rats with or without SGLTs inhibitors. Expected Results: SGLTs inhibitor modulate cardiac fibrosis, Ca2+ /Na+ regulation in DM cardiomyopathy.