Hans Strijdom

Myocardial susceptibility to ischemic-reperfusion injury in a prediabetic model of dietary-induced obesity

We assessed the myocardial susceptibility to ischemic-reperfusion injury in obese rat hearts in the absence and the presence of predicted circulating concentrations of insulin and fatty acids. Feeding rats a high-calorie diet resulted in increases in body weight, visceral fat content, cardiac hypertrophy, plasma insulin, nonesterified free fatty acid, and triglyceride concentrations. In the absence of both insulin and fatty acids in the coronary perfusate, the hearts of obese rats developed an increased infarct size (41.9 +/- 1.9% for obese vs. 22.9 +/- 2.3% for control, P < 0.05) and a reduced percent recovery of aortic output (4.2 +/- 4.2% for obese vs. 27.7 +/- 3.4% for controls, P < 0.05) after coronary artery occlusion and reperfusion. In the presence of insulin in the coronary perfusate, a cardioprotective effect was noted in both groups, an action that was greater in hearts from obese compared with control rats and which abolished the obesity-induced changes in infarct size (13.8 +/- 1.2% for controls vs. 21.0 +/- 1.6% for obese), and percent recovery of aortic output (60.2 +/- 4.7% for controls vs. 45.7 +/- 9.4% for obese). Fatty acids (0.7 mM, control; and 1.5 mM, obese) added to the coronary perfusate with in vivo concentrations of insulin dramatically increased infarct size (48.2 +/- 3.1% for obese, and 37.5 +/- 2.7% for control; P < 0.05 vs. without fatty acids) and decreased percent aortic output recovery (control, 10.4 +/- 5.2%, and obese 7.8 +/- 3.5%; P < 0.05 vs. without fatty acids) in both groups to similar values. In conclusion, in obesity, the impact of an increased susceptibility of the myocardium to ischemic-reperfusion injury on myocardial injury is likely to be overshadowed by the comparatively greater roles played by predicted increases in circulating insulin and fatty acids found in vivo. These data support the notion that adiposity per se is unlikely to be a valuable predictor of outcomes in ischemic-reperfusion injury.

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.

Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway.

The roles of endothelial nitric oxide synthase (eNOS), and its putative association with protein kinase B (PKB), and inducible nitric oxide synthase (iNOS) are not well characterized in hypoxic cardiac cells and there is a lack of studies that measure nitric oxide (NO) directly. Objective To measure NO production in cardiomyocytes and cardiac microvascular endothelial cells (CMECs) under baseline and hypoxic conditions and to evaluate the expression, regulation and activation of eNOS, iNOS and PKB. The effect of PI3-K/PKB inhibition on NO production and eNOS expression/activation was also investigated. Methods Adult rat cardiomyocytes and rat CMECs were made hypoxic by cell pelleting and low PO2 incubation. Intracellular NO was measured by FACS analysis of DAF-2/DA fluorescence, and eNOS, iNOS and PKB were evaluated by Western blotting or flow cytometry. Upstream PKB inhibition was achieved with wortmannin. Results (1) NO levels increased in both cell types after exposure to hypoxia. (2) In hypoxic CMECs, eNOS was upregulated and activated, no iNOS expression was observed and PKB was activated. (3) In myocytes, hypoxia did not affect eNOS expression, but increased its activation. Activated PKB also increased during hypoxia. FACS analysis showed increased iNOS in hypoxic myocytes. (4) Wortmannin resulted in decreased hypoxia-induced NO production and reduced activated eNOS levels. Conclusions Cardiomyocytes and CMECs show increased NO production during hypoxia. eNOS seems to be the main NOS isoform involved as source of the increased NO generation, although there may be a role for iNOS and other non-eNOS sources of NO in the hypoxic myocytes. Hypoxia-induced PKB and eNOS activation occurred simultaneously in both cell types, and the PI3-K/PKB pathway was associated with hypoxia-induced NO production via eNOS activation.

Nitric oxide production is higher in rat cardiac microvessel endothelial cells than ventricular cardiomyocytes in baseline and hypoxic conditions: a comparative study

The relative importance of endothelium‐ and cardiomyocyte‐derived nitric oxide (NO) is unknown, with a lack of direct studies on cardiac microvessel endothelial cells (CMEC) and cardiomyocytes regarding relative cellular NO production. Aims: To assess and compare baseline and hypoxia‐induced NO and ONOO– production in cardiomyocytes and CMEC. Methods: Rat cardiomyocytes were isolated, and cultured rat CMEC were purchased commercially. Hypoxia (± NOS inhibitors) was induced by mineral oil layering or hypoxic culture. NO and ONOO– were detected by FACS analysis of DAF‐2/DA and DHR123, respectively. Total eNOS was determined by Western blot analysis. Results: 1) Baseline NO production in CMEC was sevenfold (cultured cells) and 26‐fold (isolated cells) higher than in cardiomyocytes, 2) eNOS expression was 22‐fold higher in CMEC, 3) hypoxia increased NO production in both cell types, albeit to a larger extent in CMEC, 4) in hypoxic cardiomyocytes, nonselective NOS and iNOS‐specific inhibition attenuated NO production, whereas in CMEC, iNOS‐specific inhibition was ineffective, and 5) baseline ONOO– production was 2.2 times greater in CMEC than in cardiomyocytes. Conclusion: Using a novel approach, this study demonstrated that CMEC produce more baseline NO than cardiomyocytes, and that hypoxia activates NOS to increase NO production in both cell types. Baseline eNOS content was higher in CMEC than in cardiomyocytes, suggesting that differences in baseline NO production were eNOS‐associated.

Short-term melatonin consumption protects the heart of obese rats independent of body weight change and visceral adiposity.

Chronic melatonin treatment has been shown to prevent the harmful effects of diet‐induced obesity and reduce myocardial susceptibility to ischaemia‐reperfusion injury (IRI). However, the exact mechanism whereby it exerts its beneficial actions on the heart in obesity/insulin resistance remains unknown. Herein, we investigated the effects of relatively short‐term melatonin treatment on the heart in a rat model of diet‐induced obesity. Control and diet‐induced obese Wistar rats (fed a high calorie diet for 20 wk) were each subdivided into three groups receiving drinking water with or without melatonin (4 mg/kg/day) for the last 6 or 3 wk of experimentation. A number of isolated hearts were perfused in the working mode, subjected to regional or global ischaemia‐reperfusion; others were nonperfused. Metabolic parameters, myocardial infarct sizes (IFS), baseline and postischaemic activation of PKB/Akt, ERK42/44, GSK‐3β and STAT‐3 were determined. Diet‐induced obesity caused increases in body weight gain, visceral adiposity, fasting blood glucose, serum insulin and triglyceride (TG) levels with a concomitant cardiac hypertrophy, large postischaemic myocardial IFSs and a reduced cardiac output. Melatonin treatment (3 and 6 wk) decreased serum insulin levels and the HOMA index (P < 0.05) with no effect on weight gain (after 3 wk), visceral adiposity, serum TG and glucose levels. It increased serum adiponectin levels, reduced myocardial IFSs in both groups and activated baseline myocardial STAT‐3 and PKB/Akt, ERK42/44 and GSK‐3β during reperfusion. Overall, short‐term melatonin administration to obese/insulin resistant rats reduced insulin resistance and protected the heart against ex vivo myocardial IRI independently of body weight change and visceral adiposity.

Nitric Oxide Synthase (NOS) Does Not Contribute to Simulated Ischaemic Preconditioning in an Isolated Rat Cardiomyocyte Model

It is widely accepted that nitric oxide (NO) is a trigger and mediator of late ischaemic preconditioning (IP), however its role in classic (protection observed within 2–4 hours after the IP stimulus) IP is less certain. In addition, the contribution of cardiomyocyte nitric oxide synthase (NOS) activation to NO production in ischaemia is unknown. The aim of this study was therefore to investigate the role of NOS, NO, reactive oxygen species (ROS) and cGMP in IP in an isolated cardiomyocyte model.Methods: Adult rat cardiomyocytes were isolated by collagenase perfusion. Hypoxia was induced by covering pelleted cardiomyocytes with mineral oil. The IP protocol was one 10 min hypoxia/20 min reoxygenation cycle, followed by 2 hr sustained hypoxia. Non-IP cells were subjected to 2 hr sustained hypoxia only. The contribution of NO was investigated by NOS inhibition (L-NAME 50 μM) or by pre-treatment of cells with a NO donor (SNP 100 μM), and that of ROS by inclusion of ROS scavengers (MPG and N-acetyl-cysteine) or pre-treatment with H2O2. End-points were cellular cGMP content and cell viability as assessed by trypan blue exclusion (TBE) and cell morphology.Results: IP significantly improved myocyte viability (54% increase in TBE) at the end of sustained hypoxia. Treatment of cells with L-NAME and ROS scavengers during either the IP protocol or during sustained hypoxia had no effect on cell viability after 2 hr hypoxia, whereas viability of non-IP cells treated with L-NAME during sustained hypoxia improved significantly. cGMP levels were reduced in IP cells. Pre-treatment with SNP and H2O2 did not mimic IP.Conclusions: IP conferred cardioprotection in isolated cardiomyocytes. Protection in this model was not due to activation of cardiomyocyte NOS or ROS production. However, NOS activation induced by sustained hypoxia, appeared to be harmful to non-IP cells.

Aqueous extract of unfermented honeybush (Cyclopia maculata) attenuates STZ-induced diabetes and β-cell cytotoxicity.

New strategies, which include β-cell protection, are required in the treatment of T2D, as current drugs demonstrate little or no capacity to directly protect the vulnerable β-cell against diabetes-induced cytotoxicity. In this study we investigated the ameliorative effect of pre-treatment with an aqueous extract of unfermented Cyclopia maculata (honeybush) on STZ-induced diabetes and pancreatic β-cell cytotoxicity in Wistar rats after demonstrating a protective effect in vitro in RIN-5F cells. The amelioration of STZ-induced diabetes was seen in the reduction of the area under the curve, determined by the oral glucose tolerance test, as well as fasting glucose levels in extract-treated rats. Pre-treatment with extract also improved serum triglyceride levels and the glucose-to-insulin ratio. Pre-treatment with the extract or the drug, metformin, increased the β-cell area in islets, with a concomitant increase in β-cell proliferation at the higher extract dose (300 mg/kg/d), but not the lower dose (30 mg/kg/d). Subsequently, the in vitro tritiated thymidine incorporation assay showed that the extract was not mitogenic in RIN-5F cells. STZ-induced elevation of plasma nitrite levels was reduced in extract-treated rats, but no changes were observed in their serum catalase, serum glutathione, liver lipid peroxidation and liver nitrotyrosine levels. Pre-treating the rats with extract ameliorated the diabetic effect of STZ in Wistar rats, with evidence of pancreatic β-cells protection, attributed to the presence of high levels of antioxidants such as the xanthones, mangiferin and isomangiferin.

Impact of Aldosterone Receptor Blockade on the Deleterious Cardiac Effects of Adrenergic Activation in Hypertensive Rats

Although in hypertension β-adrenoreceptor activation promotes the transition from cardiac hypertrophy to pump dysfunction, the use of β-blockers is controversial. As adrenergic activation may mediate adverse effects on the heart through the renin-angiotensin-aldosterone system, we evaluated the effects of the aldosterone receptor blocker, spironolactone (SPIRO), on isoproterenol (ISO)-induced changes in left ventricular cavity size and pump function and the determinants thereof in spontaneously hypertensive rats (SHR). ISO administered for 4.5 months resulted in increases in left ventricular dimensions and a decrease in pump function in SHR but not in normotensive rats, changes that, without affecting blood pressure, were abolished by SPIRO. In SHR, 4-5 days of ISO increased myocardial matrix metalloproteinase-2 activity, which was associated with matrix metalloproteinase-2 but not tissue inhibitor of MMP expression; persisted at 4.5 months; and was prevented by SPIRO. Moreover, after 4.5 months, ISO increased non-cross-linked myocardial collagen concentrations in SHR, which was abolished by SPIRO. Although after 4.5 months, ISO was not associated with increased cardiomyocyte apoptosis, an early (4-5 days) ISO-induced apoptotic effect was noted, which was prevented by SPIRO. Hence, aldosterone receptor blockade may be sufficient to prevent those adverse effects of β-adrenoreceptor activation responsible for the transition from concentric cardiac hypertrophy to pump dysfunction in hypertension.

ATM Protein Kinase Signaling, Type 2 Diabetes and Cardiovascular Disease

The ataxia-telangiectasia mutated (ATM) protein kinase is well known to play a significant role in the response to double stranded DNA breaks in the nucleus. Recently, it has become apparent that ATM is also involved in a large number of cytoplasmic processes and responses, some of which may contribute to metabolic and cardiovascular complications when disrupted. Due to its involvement in these processes, therapeutic activation of ATM could potentially be a novel approach for the prevention or treatment of cardiovascular disease. However, relatively little is currently known about the cardiovascular role of ATM. In this review, we highlight studies that have shed some light on the role of ATM in the cardiovascular context, namely in oxidative stress, atherosclerosis and metabolism, insulin resistance and cardiac remodeling.

Effects of Sperm Processing Techniques Involving Centrifugation on Nitric Oxide, Reactive Oxygen Species Generation and Sperm Function~!2009-11-11~!2010-01-07~!2010-03-30~!

This study was aimed at investigating the effects of sperm centrifugation on nitric oxide (NO) and reactive oxygen species (ROS) generation as well as sperm motility and viability. Human spermatozoa were centrifuged for 10 and 30 minutes (400 x g) in the presence or absence of the NOS inhibitor, N G -nitro-L-arginine methyl ester (L-NAME); ROS scavenger, N-(2-mercaptopropionyl)Glycine (MPG) or the combination of L-NAME + MPG. Total sperm motility was significantly decreased with 30 minutes of centrifugation whereas progressive motility and cell viability were significantly decreased with 10 and 30 minutes of centrifugation. These effects were reversed with the administration of MPG or L- NAME + MPG. Ten minutes centrifugation significantly elevated ROS and NO production (P < 0.05). Thirty minutes centrifugation elevated ROS generation (P < 0.01) whereas NO was attenuated. This study has demonstrated that 10 and 30 minutes of sperm centrifugation were detrimental to both sperm motility and viability, but generally 30 minutes centrifugation was more detrimental to sperm than 10 minutes. It has also demonstrated that 10 minutes centrifugation led to both NO and ROS elevation whereas 30 minutes centrifugation led to ROS elevation and NO attenuation. We therefore recommend that sperm separation techniques should avoid using centrifugation or prolonged centrifugation in assisted reproductive technologies.