4805GLP-1(28–36) prevents ischemic cardiac injury by modulating metabolism and activating soluble adenylyl cyclase in coronary vascular cells

Abstract

Background: Cardiovascular disease (CVD) is increasingly attributable to type- 2 diabetes (T2D) and obesity, with the incidence of T2D predicted to double by 2030. However, mechanisms underlying the pathophysiology of CVD in T2D are poorly understood. Pre-clinical studies in animals and recent human clinical trials have demonstrated robust cardioprotective actions of incretin glucagon-like peptide-1 (GLP-1), related peptides and drugs. How GLP-1 prevents acute myocardial ischemia-reperfusion injury (IRI) as well as chronic myocardial ischemic injury has not been established. We previously described that GLP-1(28-36), a small metabolite of GLP-1, is as protective as the parent peptide in ex vivo isolated heart IRI and in vivo experimental myocardial infarction (MI) models in mice, and that these effects require soluble adenylyl cyclase (sAC), but not the known GLP- 1 receptor. Here we define the molecular mechanisms and cell types in which GLP-1(28-36) activates sAC. Methods and results: Immunoblots reveal that mouse and human coronary artery smooth muscle cells (caSMC) and human coronary artery endothelial cells (caEC) express sAC, while mouse neonatal ventricular cardiomyocytes (CM), a mouse atrial CM cell line (HL-1), and human embryonic stem cell (ESC)-derived CM do not. Using pharmacological inhibitors of sAC, sAC-null mice and siRNA against sAC, we demonstrate that GLP-1(28-36) causes sAC-dependent increases in cAMP, activation of PKA, and cytoprotection from oxidative stress injury in both mouse and human caSMC. Moreover, GLP-1(28-36) increases cAMP responses in human caEC and probenecid, which blocks the anion (HCO3) entry required for sAC activity, abrogates these effects. By contrast, human ESCderived CM shows no cAMP or cytoprotection responses to GLP-1(28-36). Next, we show in caSMC that GLP-1(28-36) causes dose-dependent increases in intracellular ATP, a known substrate of sAC. Affinity pull-down experiments using biotinylated-GLP-1(28-36), proteomic analysis with mass spectrometry, and confirmatory immunoblots suggest that GLP-1(28-36) interacts with mitochondrial trifunctional protein alpha (MTPα), an enzyme involved in fatty acid metabolism, and does not directly interact with sAC. Using extracellular flux analysis of caSMC and caEC, and siRNA against MTPα, we demonstrate that the ability of GLP-1(28-36) to shift substrate utilization towards the energy efficient glucose utilization and away from the high energy consuming fatty acid oxidation is dependent on MTPα. Conclusion: GLP-1(28-36) is a small peptide that targets a novel molecular (MTPα-ATP-sAC) and cellular (caSMC and caEC) mechanisms for the treatment of myocardial ischemic injury.