Sirtuin1-p53, forkhead box O3a, p38 and post-infarct cardiac remodeling in the spontaneously diabetic Goto-Kakizaki rat.

BACKGROUND: Diabetes is associated with changes in myocardial stress-response pathways and is recognized as an independent risk factor for cardiac remodeling. Using spontaneously diabetic Goto Kakizaki rats as a model of type 2 DM we investigated whether post-translational modifications in the Akt - FOXO3a pathway, Sirt1 - p53 pathway and the mitogen activated protein kinase p38 regulator are involved in post-infarct cardiac remodeling METHODS: Experimental myocardial infarction (MI) was induced by left anterior descending coronary artery ligation in spontaneously diabetic Goto-Kakizaki rats and non-diabetic Wistar controls. Cardiac function was studied by echocardiography. Myocardial hypertrophy, cardiomyocyte apoptosis and cardiac fibrosis were determined histologically 12 weeks post MI or Sham operation. Western blotting was used to study Caspase-3, Bax, Sirt1, acetylation of p53 and phosphorylation of p38, Akt and FOXO3a. Electrophoretic mobility shift assay was used to assess FOXO3a activity and its nuclear localization. RESULTS: Post-infarct heart failure in diabetic GK rats was associated with pronounced cardiomyocyte hypertrophy, increased interstitial fibrosis and sustained cardiomyocyte apoptosis as compared with their non-diabetic Wistar controls. In the GK rat myocardium, Akt- and FOXO3a-phosphorylation was decreased and nuclear localization of FOXO3a was increased concomitantly with increased PTEN protein expression. Furthermore, increased Sirt1 protein expression was associated with decreased p53 acetylation, and phosphorylation of p38 was increased in diabetic rats with MI. CONCLUSIONS: Post-infarct heart failure in diabetic GK rats was associated with more pronounced cardiac hypertrophy, interstitial fibrosis and sustained cardiomyocyte apoptosis as compared to their non-diabetic controls. The present study suggests important roles for Akt-FOXO3a, Sirt1 - p53 and p38 MAPK in the regulation of post-infarct cardiac remodeling in type 2 diabetes.

Carbon monoxide releasing molecule improves structural and functional cardiac recovery after myocardial injury.

Carbon monoxide (CO), produced by heme oxygenase-1 (HO-1), is an endogenous paracrine factor involved in the regulation of cardiovascular structure and function. We studied the effects of a synthetic CO releasing molecule (CORM-3) on cardiac recovery and myocardial microRNA expression after myocardial infarction (MI). Male Wistar rats with MI (n = 75) or sham-operated controls (n = 75) were treated from day 4 to day 14 after MI either with synthetic CORM-3 or with inactive iCORM and killed 2, 4 or 8 weeks post-MI. Infarct size, vascular and capillary densities, the amount of cardiomyocytes in the infarct area, and cardiomyocyte proliferation and apoptosis were determined. PCR was used for microRNA and mRNA quantification, western blotting to evaluate protein expression and echocardiography to assess cardiac structure and function. CORM-3 treatment increased vascular density (P< 0.05 vs. iCORM) and the proportion of cardiomyocytes (P< 0.05 vs. iCORM) in the infarct area. Ejection fraction improved (P< 0.05) and left ventricular volumes decreased (P< 0.05) in CORM-3 treated MI groups compared to iCORM treatment. CORM-3 treatment decreased the amount of proliferating Ki67 positive cardiomyocytes in the infarct/border area at week 2 after MI compared to iCORM treatment, whereas the amount of apoptotic cardiomyocytes did not differ between CORM-3 and iCORM groups. Compared to iCORM treatment, CORM-3 decreased expression on miR-206 in the remote area at week 2 after MI. The CO releasing molecule CORM-3 improved structural and functional cardiac recovery after MI. Modulation of HO-1-CO axis may prove novel drug targets to facilitate cardiac recovery after myocardial injury.

Effects of angiotensin II blockade on cardiomyocyte regeneration after myocardial infarction in rats.

INTRODUCTION: We studied the effects of angiotensin type 1 receptor blockade (ARB) on formation of new cardiomyocytes, neovascularization and ventricular remodelling after myocardial infarction (MI). METHODS: Male Wistar rats with MI or sham-operated controls were treated with either losartan or vehicle. Bromodeoxyuridine (BrdU) was given to identify newly formed cardiac cells. Immunohistochemical analysis was used to quantify proliferative and apoptotic cardiomyocytes, vascular structures and c-Kit+ stem/progenitor cells, western blotting to evaluate gene expression, and planimetry and echocardiography to assess cardiac structure and function. RESULTS: The number of BrdU+ cardiomyocytes increased similarly in the vehicle and losartan treated MI groups. The number of apoptotic or proliferating cardiomyocytes did not differ between losartan and vehicle treated rats. Losartan induced an increase in capillary and BrdU+ vascular densities in the infarct border zone. Losartan treatment completely prevented post-MI cardiac hypertrophy. In the non-infarcted myocardium the amount of all BrdU+ cells (including non-cardiomyocyte cells) was highest in the vehicle treated MI rats at week 4. CONCLUSIONS: The number of newly formed cardiomyocytes increased after MI. Angiotensin II blockade neither stimulated nor prevented cardiomyocyte regeneration. ARB treatment increased vascular densities in the infarct border zone and modulated remodelling of the non-infarcted myocardium preventing effectively post-MI cardiac hypertrophy.

Heme oxygenase-1 induction protects the heart and modulates cellular and extracellular remodelling after myocardial infarction in rats.

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme, which regulates cell proliferation and has potential antifibrogenic properties. In the present study, we investigated the effects of pre-emptive HO-1 induction by cobalt protoporphyrin IX on the healing of myocardial infarction in rats. The proliferation and repair of cardiac cells was assessed by immunostaining of Ki67 and proliferating cell nuclear antigen, and apoptosis of cardiomyocytes by terminal deoxynucleotidyl transferase dUTP nick end labelling. Compared with control hearts, HO-1 induction reduced apoptosis and increased proliferation and repair of cardiomyocytes in the infarct border area during the first few days after infarction. Concomitantly, HO-1 decreased accumulation and proliferation of fibroblasts, and down-regulated procollagen type I expression in the infarct area. Furthermore, HO-1 increased expression of the anti-inflammatory cytokine, transforming growth factor-ß1, suggesting that the cardioprotective effect of HO-1 in the early phase of infarct healing may result partly from the suppression of the inflammatory response. In the remote myocardium, HO-1 inhibited both proliferation and apoptosis of cardiomyocytes, attenuated heart failure-induced increase in the repair of cardiomyocytes and decreased perivascular fibrosis, thereby potentially alleviating adverse ventricular remodelling. The cardioprotective effects of HO-1 in the late phase of infarct healing may be mediated partly by down-regulation of the profibrotic connective tissue growth factor (CTGF), as HO-1 decreased CTGF expression at week 4. In conclusion, our findings suggest an important role for HO-1 in maintaining cellular homeostasis in the postinfarction heart. Modulation of the HO-1 pathway may provide a new therapeutic approach to enhance the recovery of myocardial infarction and protect against pathological myocardial changes.

Heme oxygenase-1 and carbon monoxide promote neovascularization after myocardial infarction by modulating the expression of HIF-1alpha, SDF-1alpha and VEGF-B.

Heme oxygenase-1 (HO-1), a known cytoprotective enzyme implicated also in the cell cycle regulation and angiogenesis, exerts many of its beneficial effects through carbon monoxide (CO). We studied the roles of HO-1 and CO in cardiac regeneration after myocardial infarction. Prior to coronary artery ligation, male Wistar rats were given either cobolt protoporphyrin IX to induce HO-1 or CO-donor methylene chloride. Cardiac regeneration was assessed by immunohistochemistry and confocal microscopy. CO significantly increased the accumulation of c-kit+ stem/progenitor cells into the infarct area and induced formation of new coronary arteries by promoting a substantial differentiation of c-kit+ cells into vascular smooth muscle cells (c-kit+/GATA6+ cells). Furthermore, CO increased proliferation of cardiomyocytes in the infarct border area at 4weeks post-infarction. This suggests proliferation of newly formed cardiomyocytes derived from c-kit+ cells as 10% of c-kit+ cells expressed early cardiac marker Nkx2.5. Increased expression of hypoxia-inducible factor-1alpha (HIF-1alpha), stromal cell derived factor-1alpha (SDF-1alpha) and vascular endothelial growth factor-B (VEGF-B) were found in the infarct areas of CO-donor pretreated hearts suggesting that these factors potentially promoted the migration of c-kit+ cells into the infarct area and subsequent vasculogenesis and myocardial regeneration by CO. HO-1 increased both capillary and vascular densities, while only a small increase of c-kit+ cells was found. HO-1 upregulated SDF-1alpha, but did not have effect on HIF-1alpha and VEGF-B. In conclusion, HO-1 and CO have differential roles and mechanisms of action in cardiac regeneration. Modulation of the HO-1/CO axis may provide a novel tool for the repair of cardiac injury.

Oral levosimendan prevents postinfarct heart failure and cardiac remodeling in diabetic Goto-Kakizaki rats.

BACKGROUND: Diabetes increases the risk for fatal myocardial infarction and development of heart failure. Levosimendan, an inodilator acting both via calcium sensitization and opening of ATP-dependent potassium channels, is used intravenously for acute decompensated heart failure. The long-term effects of oral levosimendan on postinfarct heart failure are largely unknown. OBJECTIVE: To examine whether oral treatment with levosimendan could improve cardiac functions and prevent cardiac remodeling after myocardial infarction in a rodent model of type 2 diabetes, the Goto-Kakizaki rat. METHODS: Myocardial infarction (MI) was induced to diabetic Goto-Kakizaki and nondiabetic Wistar rats by coronary ligation. Twenty-four hours after surgery, Goto-Kakizaki and Wistar rats were randomized into four groups: MI group without treatment, MI group with levosimendan for 12 weeks (1 mg/kg per day), sham-operated group, sham-operated group with levosimendan. Blood pressure, cardiac functions as wells as markers of cardiac remodeling were determined. RESULTS: In Goto-Kakizaki rats, MI induced systolic heart failure, pronounced cardiac hypertrophy in the remote area, and sustained cardiomyocyte apoptosis. Postinfarct cardiac remodeling was associated with increased atrial natriuretic peptide, interleukin-6 and connective tissue growth factor mRNA expressions, as well as three-fold increased cardiomyocyte senescence, measured as cardiac p16 mRNA expression. Levosimendan improved cardiac function and prevented postinfarct cardiomyocyte hypertrophy, cardiomyocyte apoptosis, and cellular senescence. Levosimendan also ameliorated MI-induced atrial natriuretic peptide, IL-6, and connective tissue growth factor overexpression as well as MI-induced disturbances in calcium-handling proteins (SERCA2, Na-Ca exchanger) without changes in diabetic status or systemic blood pressure. In nondiabetic Wistar rats, MI induced systolic heart failure; however, the postinfarct cardiac remodeling was associated with less pronounced cardiac hypertrophy, cardiomyocyte apoptosis, inflammatory reaction, and induction of cellular senescence. Levosimendan only partially prevented postinfarct heart failure and cardiac remodeling in Wistar rats. CONCLUSION: Our findings suggest a therapeutic role for oral levosimendan in prevention of postinfarct heart failure and cardiac remodeling in type 2 diabetes and underscore the importance of sustained cardiomyocyte apoptosis and induction of cellular senescence in the pathogenesis.