Mechanisms of Empaglifozin on Cardiac Metabolism with Electrical and Structural Remodelings: Implications in Cardio-Protection of Sodium Glucose Transporter 2 Inhibitor

Background: Cardiovascular (CV) complications are considered to be the major causes of mortality in diabetes mellitus (DM) patients. Although advances in medical management have reduced CV mortality in DM patients by about 40%, the actual number of deaths is predicted to rise as a result of DM and aging population. However, the mechanisms underlying CV dysfunction in DM are not clear, and the treatment of DM cardiomyopathy was not well established. Recently, clinical studies found that sodium glucose cotransporter (SGLT)2 inhibitor, empagliflozin, has significant CV benefits with improvement of hypertension and heart failure. However, the mechanisms on which SGLT2 inhibitor improves CV outcomes are not fully understood. Metabolic disorder plays a vital role in DM cardiomyopathy. Our previous studies have shown that DM impaired cardiac metabolism with electrical and structural remodeling from dysregulated 5'AMPactivated protein kinase (AMPK) signaling and peroxisome proliferator activated receptors (PPARs). Accordingly, SGLT2 inhibitor may rescue DM cardiomyopathy by improving cardiac metabolism. Therefore, the purpose of this study is to investigate the myocardial effect of SGLT2 inhibitor-empagliflozin on DM cardiomyopathy, we will study whether empagliflozin may improve the metabolisms disorder in DM through modulation on cardiac electrical and structural remodeling. In addition, we will evaluate whether SGLT2 inhibition will be novel strategy for treating heart failure. In the first year experiments, we will evaluate the effect of empagliflozin on cardiac function and study the myocardial fatty acid and glucose metabolism, and PPARs expression in DM rats. We will also study whether empagliflozin modulate cardiac electrical activity, calcium (Ca2+) homeostasis and related molecular mechanisms. In the second year experiment, we will investigate whether empagliflozin modulates cardiac fibroblasts activity to rescue DM cardiomyopathy. In the third year experiment, we will study whether empagliflozin can improve heart failure through its distinctive SGLT2 inhibition effects. We will evaluate the effects of empagliflozin on heart failure-induced electrical and structural remodeling and explore the related molecular mechanisms. Methods: First year: Fasting plasma glucose, metabolic and inflammatory parameters, electrocardiogram, echocardiogram, and blood pressure will be obtained before inducing type 2 DM (fed with 60% high fat diet and streptozotocin, 35 mg/kg intraperitoneally on the 3rd week of high fat diet), and before and after treatment with empagliflozin (10 mg/kg, oral gavage, for 4 weeks). Non-invasive micro PET scan will be used to evaluate the in vivo efficacy of empagliflozin on myocardial energy metabolism in the control and type 2 DM rats with or without empagliflozin. mRNA or protein expressions of PPAR isoforms, acetyl-CoA carboxylase (ACC), sterol regulatory element-binding protein 1c (SREBP1c), carnitine palmitoyltransferase 1A (CPT1A), and microsomal triglyceride transfer protein (MTTP), fatty acid substrates (AMPK-2, pAMPK-2, pACC, CPT1, PGC1-, CD 36, DGAT 2, DGAT1), down-steam signaling pathways involving calcineurin NFAT, P38 MAPK, ERK1/2, CaMK II kinase, and glucose metabolism (glucose transporter (GLUT-2, GLUT-4, IRS, iRS, Akt, pAKT), and Ca2+ regulatory protein will be performed using real time PCR or Western blot. Intracellular Ca2+ and electrophysiological characteristics will be recorded using fluorimetric ratio technique and whole cell patch clamped in isolated single ventricular cardiomyocytes. Second Year: Cardiac fibroblasts will be isolated from 16 week old control, type 2 DM male rats with or without empagliflozin (10 mg/kg, oral gavage). Ventricular primary isolated fibroblasts control, type 2 DM male rats with or without empagliflozin will be subjected to proliferation assay, oxidative stress assay, cell adhesion and migration analysis, collagen measurement, cytokine array. Tissue will be fixed in 2% paraformaldehyde, embedded in paraffin and sectioned at 5 μm will be stained with Masson’s trichrome (MT) or Sirius red (SR), and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay for histological analysis for myocardial fibrosis and apoptosis. mRNA expressions of alphasmooth muscle actin, type I Collagen, type III collagen, acidic ribosomal binding protein, tumor necrosis factor (TNF)-, and interleukin (IL)-6 will be performed using real time PCR. Protein expressions of TNF- , IL-6, angiotensin II receptors, type 1 (ATR1), receptor advanced glycated end-product (RAGE) will be evaluated by Western blot. Third year: Male Wistar rats at 12 weeks age will be grouped into control, isoproterenol-induced (100 mg/kg, intraperitoneal for 1week) with or without empagliflozin, 10 mg/kg, oral gavage) for 4 weeks. Fasting plasma glucose, metabolic and inflammatory parameters, electrocardiogram, echocardiogram, blood pressure will be measured before and after treatment with empagliflozin in each group. Histology, mRNA expressions or western blot of PPAR isoforms, ACC, SREBP1c, CPT1A, and MTTP, fatty acid substrates (AMPK-2, pAMPK-2, pACC, CPT1, PGC1-, CD 36, DGAT 2, DGAT1), down-steam signaling pathways involving calcineurin NFAT, P38 MAPK, ERK1/2, CaMK II kinase, and glucose metabolism (glucose transporter (GLUT-2, GLUT-4, IRS, iRS, Akt, pAKT), TNF-, IL-6, ATR1, RAGEs, Ca2+regulation proteins will be evaluated by Western blot or real time PCR. Intracellular Ca2+ and electrophysiological characteristics will be recorded using fluorimetric ratio technique and whole cell patch clamped in isolated single ventricular cardiomyocytes. Preliminary results: SGLT2 inhibitor-empagliflozin reverses the prolonged cQT interval, action potential duration, and Ca2+ transient and decreases the incidence of Ca2+ sparks in type 2 DM. Additionally, empagliflozin reverses dilated left ventricular chamber sizes associated with improved cardiac functions in type 2 DM rats. Expected Results: SGLT2 inhibitor-empagliflozin modulate Ca2+ regulation, fatty acid and glucose metabolism, inflammation and PPARs in DM and heart failure cardiomyopathy.