Pharmacologic Inhibition of FOXO1 Improves Cardiac Function by Enhancing Glucose Metabolism and Attenuating Apoptosis in Type-1 Diabetic Rats

Abstract

IntroductionDiabetic cardiomyopathy, which comprises structural and functional abnormalities of the myocardium, is a major etiological factor for death in diabetic patients. Altered cardiac energy metabolism and increased cardiac apoptosis may underlie the pathology of diabetic cardiomyopathy. Forkhead transcriptional factor (FOXO1) is preferably activated in the myocardium of Type2 diabetic mice and mediates excessive lipid metabolism, resulting in diabetic cardiomyopahty. However, the effect of FOXO1 in cardiac glucose oxidation and apoptosis and its contribution to cardiac function in Type 1 diabetes-induced cardiomyopathy are largely unknown. Thus, the present study aimed to investigate whether or not pharmacological inhibition of FOXO1 by its selective inhibitor AS1842856 can attenuate diabetic cardiomyopathy in Type 1 diabetic rats and the underlying mechanisms.MethodsControl or streptozotocin (STZ, 65 mg/kg)-induced diabetic Sprague Dawley rats (n=7 per group) received vehicle or AS1842856 (50 mg/kg) by gavage twice daily for one week starting four weeks after STZ injection. On the day of harvest, the left ventricular (LV) functions of the rats were assessed by Pressure-volume (PV) loop analysis and the heart tissue were collected for immunofluorescence, qPCR, Western blotting, etc. The translocation of FOXO1 from cytoplasm to nucleus was performed by immunofluorescence. The mRNA expression of pyruvate dehydrogenase kinase 4 (PDK4) was measured by qPCR. The protein levels of FOXO1, phosphorylated-FOXO1(S256), PDK4, phosphorylated pyruvate dehydrogenase (pSer293-PDH), B-cell lymphoma 2 (BCl2), Bcl-2-like protein 4 (Bax), total and cleaved-caspase3 were detected by Western blotting analysis. TUNEL assay was performed for detection of apoptosis in the cardiac tissue.ResultsPV loop analysis indicated that heart rate (HR, bpm) was significantly reduced, and time constant of LV pressure decay (TAU, ms), slope of the end-diastolic pressure-volume relationship (EdPVR, mmHg/μl) were all significantly increased in Type-1 diabeticrats as compared to non-diabetic control rats (all P <0.05), suggesting that Type-1 diabetic rats indicated cardiac diastolic dysfunction. These changes were associated with remarkably increased FOXO1 activity (decreased P-FOXO1/FOXO1 ratio, increased FOXO1 nuclear translocation), reduced cardiac glucose oxidation (increased mRNA and protein expression of PDK4 and reduced expression of P-PDH) and enhanced cardiac apoptosis (reduced Bcl2/Bax ratio, increased cleaved-caspase3/total caspase 3 ratio and TUNEL positive cells) in Type-1 diabetic rats (all P <0.05, vs. Control rats). However, administration of FOXO1 inhibitor AS1842856 significantly inhibited FOXO1 activity (increased P-FOXO1/FOXO1 ratio, decreased FOXO1 nuclear translocation), increased HR, decreased TAU and EDSPVR, and reverted all the above-mentioned diabetes-induced biochemical changes, suggesting that pharmacological inhibition of FOXO1 may attenuate cardiomyopathy in Type-1 diabetic rats via enhanced glucose oxidation and reduced cardiac apoptosis.ConclusionFoxO1 in mediating PDK4 expression and subsequent PDH inactivation in the heart and also activation of cardiac apoptosis may represent major mechanisms whereby inhibition of FOXO1 improves cardiac function in early stage of diabetic cardiomyopathy in Type 1 diabetic rats and may provide a promising therapeutic target to treat the disease.