Myocardial susceptibility to ischaemia/reperfusion in obesity: a re-evaluation of the effects of age.

BACKGROUND: Reports on the effect of age and obesity on myocardial ischaemia/reperfusion (I/R) injury and ischaemic preconditioning are contradictory. The aim of this study was to re-evaluate the effects of age and diet-induced obesity (DIO) on myocardial I/R injury and preconditioning potential. METHODS: Four groups of Wistar male rats were used: age-matched controls (AMC) receiving standard rat chow for (i) 16 weeks and (ii) 16 months respectively; DIO rats receiving a sucrose-supplemented diet for (iii) 16 weeks and (iv) 16 months respectively. The ages of groups (i) and (iii) were 22 weeks ("young") and groups (ii) and (iv) 17 months ("middle-aged") at time of experimentation. Isolated perfused working hearts were subjected to 35 min regional ischaemia/1 h reperfusion. Endpoints were infarct size (tetrazolium staining) and functional recovery. Hearts were preconditioned by 3 × 5 min ischaemia/5 min reperfusion. Results were processed using GraphPad Prism statistical software. RESULTS: Age did not affect baseline heart function before induction of ischaemia and I/R damage as indicated by infarct size and similar values were obtained in hearts from both age groups. Age also had no effect on functional recovery of hearts during reperfusion after regional ischaemia in AMC rats, but cardiac output during reperfusion was better in hearts from middle-aged than young DIO rats. The diet reduced infarct size in hearts from young rats (% of area at risk: AMC: 32.4 ± 3.6; DIO: 20.7 ± 2.9, p < 0.05), with no differences in hearts from middle-aged rats (AMC: 24.6 ± 4.6; DIO: 28.3 ± 13.5, p = NS). Compared to their respective AMC, diet-induced obesity had no significant effect on functional recovery of hearts from both age groups after exposure to regional ischaemia. When exposed to the more severe stress of global ischaemia, the functional recovery potential of middle-aged DIO rats appeared to be impeded compared to hearts of young DIO rats, while age had no effect on the functional recovery of AMC hearts. Preconditioning reduced infarct size in hearts from young control rats and both middle-aged groups, but not from young DIO rats. Age had a significant effect on functional recovery in preconditioning: it was improved in hearts from young control and DIO rats, but depressed in both middle-aged groups. CONCLUSIONS: The data showed that middle-age and obesity had no effect on baseline myocardial function and did not increase susceptibility to I/R damage upon exposure to regional ischaemia. On the contrary, obesity reduced I/R damage in young rats. Preconditioned aging hearts showed a decreased infarct size, but a reduction in functional recovery.

ANG II type I receptor antagonism improved nitric oxide production and enhanced eNOS and PKB/Akt expression in hearts from a rat model of insulin resistance.

Exogenous insulin therapy improves endothelial function in insulin resistant patients, indirectly indicating that nitric oxide synthase activity and NO production may be impaired. Insulin stimulates production of NO by activating a signaling pathway including insulin receptor substrate-1, phosphatidylinositol-3-kinase and protein kinase B (PKB/Akt). Angiotensin II type I (AT1) receptor-evoked oxidative stress is implicated in the inactivation of NO, impairing endothelium-dependent vasodilatation. Blocking the actions of Angiotensin II with an AT1 receptor antagonist (Losartan), has beneficial effects in patients with insulin resistance or type 2 diabetes mellitus. This study investigated whether elevated Angiotensin II influences myocardial insulin resistance, insulin signaling and NO production in a rat model of diet-induced obesity (DIO) by antagonizing the actions of the AT1 receptor with Losartan. Isolated, perfused hearts, Western blotting and flow-cytometric methods were utilized to determine myocardial function, expression and phosphorylation of key proteins and NO production, respectively. Results showed that hearts from DIO rats are insulin resistant (higher serine phosphorylation of IRS-1, lower insulin-stimulated phosphorylation of PKB/Akt and eNOS, lower NO production) and had poorer functional recovery and larger infarct development after ischaemia/reperfusion. Losartan improved the impaired functional recovery, and NO production and enhanced eNOS expression and phosphorylation and reduced infarct size in hearts from the DIO animals. Data obtained from Losartan treatment also revealed that Angiotensin II signaling modulates myocardial PKB/Akt expression. We conclude that Angiotensin II signaling exacerbates inhibition of NO production in insulin resistance and that this can be improved by AT1 antagonism.

Melatonin has profound effects on mitochondrial dynamics in myocardial ischaemia/reperfusion.

Research focus recently shifted to mitochondrial dynamics and the role of fusion and fission in cardioprotection. The aim of this study was to evaluate (i) the function and dynamics of mitochondria isolated from hearts exposed to ischaemia/reperfusion (I/R) (ii) the effects of melatonin, a powerful cardioprotectant, on mitochondrial dynamics in I/R. Isolated perfused rat hearts were stabilized for 30 min, subjected to 20 min global ischaemia, followed by 30 min reperfusion. Tissue was collected, mitochondria isolated for measurement of mitochondrial oxidative function and lysates from mitochondrial and cytosolic fractions prepared for western blotting. Melatonin (0.3 or 50 µM) was administered for 10 min immediately before the onset of ischaemia and for 10 min at the onset of reperfusion. Infarct size was assessed after 35 min regional ischaemia/60 min reperfusion using triphenyltetrazolium staining. The results show that reperfusion significantly reduced mitochondrial QO2 (states 3 and 4), with minor effects by melatonin. Cytosolic Beclin 1 and the LC3 II/I ratio were reduced by ischaemia and increased by reperfusion. Both ischaemia and reperfusion reduced mitochondrial PINK1 and Parkin levels, while reperfusion increased p62. An alternative mitophagy pathway mediated by Rab9 is activated during myocardial ischaemia/reperfusion. Ischaemia reduced and reperfusion increased cytosolic ULK1 expression, associated with redistribution of Rab9 and Drp1 between the cytosol and mitochondria. Melatonin significantly reduced mitochondrial p62 expression upon reperfusion. Throughout the protocol, melatonin significantly (i) increased cytosolic total (t) and phospho (p) ULK1, and Rab9 levels (ii) increased the cytosolic and reduced the mitochondrial pDrp1 levels and p/t Drp1 ratio, suggesting inhibition of mitochondrial fission. Fusion was affected to a lesser extent. Cardioprotection by melatonin is associated with substantial effects on mitophagy, the significance thereof remains to be established.

Effect of captopril on changes in rats' hearts induced by long-term irradiation.

The aim of this study was to test the efficacy of captopril, an angiotensin-converting enzyme inhibitor and a known suppressor of fibrosis, in preventing late radiation-induced cardiac pathology. Myocardial functional, histochemical and ultrastructural-morphometric studies were done on perfused hearts of rats isolated 3 and 6 months after 60Co gamma irradiation with 20 Gy and age-matched controls. At each time the animals were divided into the following groups: nonirradiated controls; irradiated once with 20 Gy; irradiated as above and given daily doses of captopril; daily doses of captopril without irradiation. The results showed that captopril, while ameliorating the decrease in the indices of capillary function, increase in mast cells, fibrosis, number of atrial granules, and changes in nerve terminals, failed to prevent the progressive functional deterioration of the hearts after irradiation. These findings suggest that an intramyofiber derangement may be involved in the long-term myocardial complications of irradiation.

The cardioprotective effect of an aqueous extract of fermented rooibos (Aspalathus linearis) on cultured cardiomyocytes derived from diabetic rats.

Diabetic cardiomyopathy (DCM) is a disorder of the heart muscle that contributes to cardiovascular deaths in the diabetic population. Excessive generation of free radicals has been directly implicated in the pathogenesis of DCM. The use of antioxidants, through dietary supplementation, to combat increased cellular oxidative stress has gained popularity worldwide. Aspalathus linearis (rooibos) is a popular herbal tea that contains a novel antioxidant, aspalathin. Literature has reported on the antidiabetic, anti-inflammatory and free radical scavenging effects of rooibos. However, its protective effect against DCM has not been established. Therefore, this study investigated whether chronic exposure to an aqueous extract of fermented rooibos (FRE) has an ex vivo cardioprotective effect on hearts obtained from streptozotocin (STZ) induced diabetic rats. Adult Wistar rats were injected with 40 mg/kg of STZ. Two weeks after STZ injection, cardiomyocytes were isolated and cultured. Cultured cardiomyocytes were treated with FRE (1 and 10 µg/ml), vitamin E (50 µg/ml), and n-acetyl cysteine (1mM) for 6h, before exposure to either hydrogen peroxide (H2O2) or an ischemic solution. Cardiomyocytes exposed to H2O2 or an ischemic solution showed a decrease in metabolic activity and glutathione content with a concomitant increase in apoptosis and intracellular reactive oxygen species. Pretreatment with FRE was able to combat these effects and the observed amelioration was better than the known antioxidant vitamin E. This study provides evidence that an aqueous extract of fermented rooibos protects cardiomyocytes, derived from diabetic rats, against experimentally induced oxidative stress and ischemia.

The role of the MAP-kinase superfamily in beta-amyloid toxicity.

The mitogen-activated protein kinase (MAP kinase) pathway participates in a number of reactions of the cell when responding to various external stimuli. These stimuli include growth factor binding to its receptor as well as stressful situations such as hypoxia and oxidative stress. It has been postulated that one of the mechanisms by which beta-amyloid exerts its toxic effects is to produce oxidative stress. This study therefore investigated whether the MAP-kinase pathway was activated in cells following exposure to beta-amyloid. Neuroblastoma (N2alpha) cells were used in all experiments. The cells were exposed to 50, 100, and 500 microM glutamate, and 10, 30, and 50 microM beta-amyloid, for 24 h. The methylthiazolyl tetrazolium salt (MTT) assay was performed to determine the degree of toxicity. The generation of hydrogen peroxide was detected by fluorescence microscopy using the dye dihydrochlorofluorescein diacetate (DCDHF). Extracellular-signal-regulated kinase (ERK) and p38 MAP-kinase phosphorylation, as representatives of the MAP-kinase pathway, was determined. Treating N2alpha cells with beta-amyloid resulted in a greater than 50% reduction in cell viability. These cells also showed a significantly higher presence of hydrogen peroxide. Western Blot analysis revealed that the phosphorylation of p38 MAP kinase was dose-dependently increased in cells exposed to glutamate and beta-amyloid. On the other hand, the phosphorylation of ERK was significantly reduced in these cells. These data therefore suggest that the toxic effects of beta-amyloid involve the generation of hydrogen peroxide, leading to the activation of p38 and the down-regulation of ERK.

Melatonin protects against ischaemic-reperfusion myocardial damage.

OBJECTIVES: Melatonin, a hormonal product of the pineal gland, is now known to be a multi-faceted free radical scavenger and anti-oxidant. Since little information is available regarding the action of melatonin on the heart, we studied the effects of melatonin on adult ventricular myocytes subjected to chemical hypoxia and reoxygenation. METHODS: Adult rat ventricular myocytes were preloaded with tetramethylrhodamine (TMRM) in combination with one of the following fluorophores: dichlorodihydrofluorescein diacetate (DCDHF), dihydrorhodamine 123 (DHR) or fluo 3 (Fluo) and then investigated with confocal laser scanning microscopy. Chemical hypoxia was induced by addition of 1.5 mM KCN and 20 mM deoxyglucose to the superfusion buffer. Melatonin (50-100 microM) was added at intervals during the protocol. RESULTS: Cells subjected to 12.5 min chemical hypoxia showed marked morphological changes, increased fluorescence intensity of DCDHF, DHR and Fluo, suggesting Ca2+ accumulation and generation of H2O2 and reactive oxygen species. The number of cells showing increased fluorescence also increased significantly. Melatonin (50 and 100 microM) caused a significant reduction in morphological changes, number of cells with increased fluorescence and fluorescence intensity of DHR and Fluo, (but not DCDHF). CONCLUSION: Melatonin effectively reduced damage induced by chemical hypoxia in adult cardiomyocytes, probably by virtue of its effects on reactive oxygen species generation and intracellular Ca2+ accumulation.

Halothane protects the isolated rat myocardium against excessive total intracellular calcium and structural damage during ischemia and reperfusion.

A recent study from our laboratory demonstrated halothane to be a powerful protectant of the isolated rat heart during reperfusion after normothermic cardioplegic arrest. It was speculated that this protective effect might be due to prevention of excessive intracellular calcium. The aim of the present study was to evaluate the effect of halothane on the total intracellular calcium (Ca2+) content and on myocardial structure both at the end of normothermic cardioplegic arrest and at the end of reperfusion. Isolated perfused rat hearts were perfused for a control period of 30 min, followed by 40 min of normothermic cardioplegic arrest with or without reperfusion for 30 min. Halothane (1.5%) was administered continuously before and after arrest. Halothane caused a significant decrease of intracellular Ca2+ at the end of normothermic cardioplegic arrest and after reperfusion. Myocardial morphology was assessed by extensive light microscopy and ultrastructure was evaluated by electron microscopy. Grading of ischemic damage showed that exposure to normothermic cardioplegia resulted in marked ischemic injury, regardless of whether the hearts were treated with halothane. Reperfusion in the presence of halothane caused a significant reversal of ischemic damage and almost complete ultrastructural repair, whereas untreated hearts still exhibited severe edema, contracture, and contracture bands. Our results indicate that the beneficial effects of halothane on myocardial structural recovery during reperfusion is associated with a reduction in excessive intracellular Ca2+. The exact mechanism of this protective action is under investigation.