CXCR4 attenuates cardiomyocytes mitochondrial dysfunction to resist ischaemia-reperfusion injury.

The chemokine (C-X-C motif) receptor 4 (CXCR4) is expressed on native cardiomyocytes and can modulate isolated cardiomyocyte contractility. This study examines the role of CXCR4 in cardiomyocyte response to ischaemia-reperfusion (I/R) injury. Isolated adult rat ventricular cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) to simulate I/R injury. In response to H/R injury, the decrease in CXCR4 expression was associated with dysfunctional energy metabolism indicated by an increased adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratio. CXCR4-overexpressing cardiomyocytes were used to determine whether such overexpression (OE) can prevent bio-energetic disruption-associated cell death. CXCR4 OE was performed with adenoviral infection with CXCR4 encoding-gene or non-translated nucleotide sequence (Control). The increased CXCR4 expression was observed in cardiomyocytes post CXCR4-adenovirus transduction and this OE significantly reduced the cardiomyocyte contractility under basal conditions. Although the same extent of H/R-provoked cytosolic calcium overload was measured, the hydrogen peroxide-induced decay of mitochondrial membrane potential was suppressed in CXCR4 OE group compared with control group, and the mitochondrial swelling was significantly attenuated in CXCR4 group, implicating that CXCR4 OE prevents permeability transition pore opening exposure to overload calcium. Interestingly, this CXCR4-induced mitochondrial protective effect is associated with the enhanced signal transducer and activator of transcription 3 (expression in mitochondria. Consequently, in the presence of H/R, mitochondrial dysfunction was mitigated and cardiomyocyte death was decreased to 65% in the CXCR4 OE group as compared with the control group. I/R injury leads to the reduction in CXCR4 in cardiomyocytes associated with the dysfunctional energy metabolism, and CXCR4 OE can alleviate mitochondrial dysfunction to improve cardiomyocyte survival.

High levels of glucose induce "metabolic memory" in cardiomyocyte via epigenetic histone H3 lysine 9 methylation.

Diabetic patients continue to develop inflammation and cardiovascular complication even after achieving glycemic control, suggesting a "metabolic memory". Metabolic memory is a major challenge in the treatment of diabetic complication, and the mechanisms underlying metabolic memory are not clear. Recent studies suggest a link between chromatin histone methylation and metabolic memory. In this study, we tested whether histone 3 lysine-9 tri-methylation (H3K9me3), a key epigenetic chromatin marker, was involved in high glucose (HG)-induced inflammation and metabolic memory. Incubating cardiomyocyte cells in HG resulted in increased levels of inflammatory cytokine IL-6 mRNA when compared with myocytes incubated in normal culture media, whereas mannitol (osmotic control) has no effect. Chromatin immunoprecipitation (ChIP) assays showed that H3K9me3 levels were significantly decreased at the promoters of IL-6. Immunoblotting demonstrated that protein levels of the H3K9me3 methyltransferase, Suv39h1, were also reduced after HG treatment. HG-induced apoptosis, mitochondrial dysfunction and cytochrome-c release were reversible. However, the effects of HG on the expression of IL-6 and the levels of H3K9me3 were irreversible after the removal of HG from the culture. These results suggest that HG-induced sustained inflammatory phenotype and epigenetic histone modification, rather than HG-induced mitochondrial dysfunction and apoptosis, are main mechanisms responsible for metabolic memory. In conclusion, our data demonstrate that HG increases expression of inflammatory cytokine and decreases the levels of histone-3 methylation at the cytokine promoter, and suggest that modulating histone 3 methylation and inflammatory cytokine expression may be a useful strategy to prevent metabolic memory and cardiomyopathy in diabetic patients.

Effect of impaired glucose tolerance on cardiac dysfunction in a rat model of prediabetes.

BACKGROUND: The effect of impaired glucose tolerance (IGT) on cardiac function during the chronic prediabetes state is complicated and plays an important role in clinical outcome. However, the molecular mechanisms are not fully understood. This study was designed to observe cardiac dysfunction in prediabetic rats with IGT and to determine whether glucose metabolic abnormalities, inflammation and apoptosis are linked to it. METHODS: The IGT rat models were induced by streptozocin, and the heart functions were assessed by echocardiography. Myocardial glucose metabolism was analyzed by glycogen periodic acid-Schiff staining, and the pro-apoptotic effect of IGT was evaluated by TUNEL staining. Additionally, caspase-3 activation, macrophage migration inhibitory factor (MIF) and G-protein coupled receptor kinase 2 (GRK2) were detected by Western blotting in cardiac tissue lysates. RESULTS: Area-under-the-curve of blood glucose in rats injected with streptozotocin was higher than that in controls, increased by 16.28%, 38.60% and 38.61% at 2, 4 and 6 weeks respectively (F = 15.370, P = 0.003). Abnormal cardiac functions and apoptotic cardiomyocytes were observed in the IGT rats, the ejection fraction (EF) being (68.59 ± 6.62)% in IGT rats vs. (81.07 ± 4.59)% in controls (t = 4.020, P = 0.002). There was more glucose which was converted to glycogen in the myocardial tissues of IGT rats, especially in cardiac perivascular tissues. Compared to controls, the cleaved caspase-3, MIF and GRK2 were expressed at higher levels in the myocardial tissues of IGT rats. CONCLUSIONS: IGT in the prediabetes period resulted in cardiac dysfunction linked to abnormal glycogen storage and apoptosis. Additionally, MIF and GRK2 may be involved in the pathogenesis of cardiac dysfunction in prediabetes and their regulation may contribute to the design of novel diagnostic and therapeutic strategies for those who have potential risks for diabetic cardiovascular complications.

Hyperglycemic myocardial damage is mediated by proinflammatory cytokine: macrophage migration inhibitory factor.

BACKGROUND: Diabetes has been regarded as an inflammatory condition which is associated with left ventricular diastolic dysfunction (LVDD). The purpose of this study was to examine the expression levels of macrophage migration inhibitory factor (MIF) and G protein-coupled receptor kinase 2 (GRK2) in patients with early diabetic cardiomyopathy, and to investigate the mechanisms involved in MIF expression and GRK2 activation. METHODS: 83 patients in the age range of 30-64 years with type 2 diabetes and 30 matched healthy men were recruited. Left ventricular diastolic function was evaluated by cardiac Doppler echocardiography. Plasma MIF levels were determined by ELISA. To confirm the clinical observation, we also studied MIF expression in prediabetic rats with impaired glucose tolerance (IGT) and relationship between MIF and GRK2 expression in H9C2 cardiomyoblasts exposed to high glucose. RESULTS: Compared with healthy subjects, patients with diabetes have significantly increased levels of plasma MIF which was further increased in diabetic patients with Left ventricular diastolic dysfunction (LVDD). The increased plasma MIF levels in diabetic patients correlated with plasma glucose, glycosylated hemoglobin and urine albumin levels. We observed a significant number of TUNEL-positive cells in the myocardium of IGT-rats but not in the control rats. Moreover, we found higher MIF expression in the heart of IGT with cardiac dysfunction compared to that of the controls. In H9C2 cardiomyoblast cells, MIF and GRK2 expression was significantly increased in a glucose concentration-dependant manner. Furthermore, GRK2 expression was abolished by siRNA knockdown of MIF and by the inhibition of CXCR4 in H9C2 cells. CONCLUSIONS: Our findings indicate that hyperglycemia is a causal factor for increased levels of pro-inflammatory cytokine MIF which plays a role in the development of cardiomyopathy occurring in patients with type 2 diabetes. The elevated levels of MIF are associated with cardiac dysfunction in diabetic patients, and the MIF effects are mediated by GRK2.

High levels of glucose induce apoptosis in cardiomyocyte via epigenetic regulation of the insulin-like growth factor receptor.

Diabetic hyperglycemia result in cardiovascular complications, but the mechanisms by which high levels of glucose (HG) cause diabetic cardiomyopathy are not known. We investigate whether HG-induced repression of insulin-like growth factor 1 receptor (IGF-1R) mediated by epigenetic modifications is one potential mechanism. We found that HG resulted in decreased IGF-1 receptor (IGF-1R) mRNA levels, and IGF-1R protein when compared with H9C2 rat cardiomyocyte cells incubated in normal glucose. HG also induced apoptosis of H9C2 cells. The effects of HG on reduced expression of IGF-1R and increased apoptosis were blocked by silencing p53 with small interference RNA but not by non-targeting scrambled siRNA. Moreover, HG negatively regulated IGF-1R promoter activity as determined by ChIP analysis, which was dependent on p53 since siRNA-p53 attenuated the effects of HG on IGF-1R promoter activity. HG also increased the association of p53 with histone deacetylase 1 (HDAC1), and decreased the association of acetylated histone-4 with the IGF-1R promoter. Furthermore, HDAC inhibitor relieved the repression of IGF-1R following HG state. These results suggest that HG-induced repression of IGF-1R is mediated by the association of p53 with the IGF-1R promoter, and by the subsequent enhanced recruitment of chromatin-modifying proteins, such as HDAC1, to the IGF-1R promoter-p53 complex. In conclusion, our data demonstrate that HG decreases expression of IGF-1R and decreases the association of acetylated histone-4 with the IGF-1R promoter. These studies may help delineate the complex pathways regulating diabetic cardiomyopathy, and have implications for the development of novel therapeutic strategies to prevent diabetic cardiomyopathy by epigenetic regulation of IGF-1R.

High glucose induces apoptosis in AC16 human cardiomyocytes via macrophage migration inhibitory factor and c-Jun N-terminal kinase.

1. It is known that high glucose can induce cardiomyocyte apoptosis and that macrophage migration inhibitory factor (MIF) may be involved in the development of diabetes. However, the relationship between high glucose and MIF in diabetic cardiomyopathy remains unclear. 2. In the present study, AC16 human cardiomyocytes were cultured in the presence of 25 mmol/L glucose for 20, 30 and 60 min before being subjected to western blot analyses to determine MIF expression and c-Jun N-terminal kinase (JNK) activation. In addition, AC16 cells were pretreated with 2.5 µmol/L SP600125 (a JNK inhibitor), 40 µmol/L (s,r)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1; an MIF antagonist) or 0.1% dimethylsulphoxide (DMSO; vehicle) for 1 h prior to exposure to 25 mmol/L glucose and culture for 72 h, followed by annexin V-fluorescein isothiocyanate/propidium iodide staining and flow cytometry analysis. Caspase 3 activity and phosphorylation of JNK were also analysed by western blotting. 3. The high concentration of glucose increased expression of endogenous MIF and JNK phosphorylation in AC16 cardiomyocytes. Pretreatment of cells with SP600125 and ISO-1 reduced glucose-induced apoptosis and caspase 3 activity. Furthermore, JNK phosphorylation was attenuated by inhibition of endogenous MIF. 4. In conclusion, myocardial cell apoptosis induced by high glucose involves the overexpression of MIF and activation of the JNK signalling pathway. The identification of a high glucose-MIF-JNK pathway will help determine potential new targets in the treatment of diabetic cardiomyopathy.