Eugene F. du Toit

Endothelin release during ischaemia and reperfusion of isolated perfused rat hearts

The hypothesis tested was that release of endogenous endothelin plays a role in events associated with or leading to myocardial ischaemia and/or post-ischaemic reperfusion damage. Release of endogenous endothelin into the coronary perfusate of isolated perfused rat hearts during ischaemia and reperfusion was measured with a sensitive radioimmunoassay using a polyclonal antibody with 100% cross-reactivity for all three endothelin isomers. Basal endothelin release was 0.69 +/- 0.02 pg/min/g wet heart weight (n = 35) and was constant up to 180 min. During low-flow hypoxic ischaemia for 180 min (PO2 approximately 250 mmHg) and in the presence of 1% foetal calf serum, the release rate was reduced to below 10% of controls (P < 0.01) and increased four-fold on reperfusion (P = 0.05). The influence of endothelin on vascular and myocardial reperfusion damage was studied with exogenous endothelin-2. After 1 h of low-flow ischaemia, endothelin-2 increased the coronary perfusion pressure to a similar extent as in non-ischaemic hearts, but with a 30-times higher potency. The threshold dose for the constrictive effect was approximately 100 to 300 pg per heart, about ten times more than was recovered in the coronary effluent upon reperfusion. The influence of endothelin on myocardial reperfusion mechanical function (stunning) was assessed with 100 ng endothelin-2, a dose some 3500-fold higher than the amount released during 30 min reperfusion. This dose, given at the onset of reperfusion, improved post-ischaemic aortic output recovery during the first 20 min of reperfusion, but worsened it thereafter (up to 40 min). These data indicate that, in the isolated perfused rat heart model, (1) endothelin is released in measurable amounts into the coronary circulation, (2) the release is much reduced during ischaemia and increased on early reperfusion following prolonged ischaemia, (3) based on the amounts released and the post-ischaemic sensitization of the coronary vasculature to endothelin, the peptide could contribute to reperfusion vascular damage, and (4) endothelin is unlikely to influence stunning owing to the extremely high dose necessary to alter reperfusion mechanical function.

Chronic melatonin consumption prevents obesity‐related metabolic abnormalities and protects the heart against myocardial ischemia and reperfusion injury in a prediabetic model of diet‐induced obesity

Abstract:  Obesity, a major risk factor for ischemic heart disease, is associated with increased oxidative stress and reduced antioxidant status. Melatonin, a potent free radical scavenger and antioxidant, has powerful cardioprotective effects in lean animals but its efficacy in obesity is unknown. We investigated the effects of chronic melatonin administration on the development of the metabolic syndrome as well as ischemia–reperfusion injury in a rat model of diet‐induced obesity (DIO). Male Wistar rats received a control diet, a control diet with melatonin, a high‐calorie diet, or a high‐calorie diet with melatonin (DM). Melatonin (4 mg/kg/day) was administered in the drinking water. After 16 wk, biometric and blood metabolic parameters were measured. Hearts were perfused ex vivo for the evaluation of myocardial function, infarct size (IFS) and biochemical changes [activation of PKB/Akt, ERK, p38 MAPK, AMPK, and glucose transporter (GLUT)‐4 expression). The high‐calorie diet caused increases in body weight (BW), visceral adiposity, serum insulin and triglycerides (TRIG), with no change in glucose levels. Melatonin treatment reduced the BW gain, visceral adiposity, blood TRIG, serum insulin, homeostatic model assessment index and thiobarbituric acid reactive substances in the DIO group. Melatonin reduced IFS in DIO and control groups and increased percentage recovery of functional performance of DIO hearts. During reperfusion, hearts from melatonin‐treated rats had increased activation of PKB/Akt, ERK42/44 and reduced p38 MAPK activation. Chronic melatonin treatment prevented the metabolic abnormalities induced by DIO and protected the heart against ischemia–reperfusion injury. These beneficial effects were associated with activation of the reperfusion injury salvage kinases pathway.

Diet-induced obesity alters signalling pathways and induces atrophy and apoptosis in skeletal muscle in a prediabetic rat model.

Pro‐inflammatory and stress‐activated signalling pathways are important role players in the pathogenesis of obesity and insulin resistance. Obesity and type II diabetes are associated with chronic, low‐grade inflammation and elevated tumour necrosis factor‐α (TNF‐α) levels. There is increasing evidence that TNF‐α may play a critical role in skeletal muscle atrophy. However, the effects of obesity‐induced insulin resistance on these signalling pathways are poorly understood in skeletal muscle. Therefore, the present study addressed the effects of obesity‐induced insulin resistance on the activity of the ubiquitin ligases, nuclear factor‐κB, p38 MAPK and phosphoinositide 3‐kinase signalling pathways in the gastrocnemius muscle and compared these with muscle of standard chow‐fed control rats. Male Wistar rats were randomly allocated to a control diet group (standard commercial chow; 60% carbohydrates, 30% protein and 10% fat) or a cafeteria diet group (65% carbohydrates, 19% protein and 16% fat) for 16 weeks. Blood analysis was conducted to determine the impact of the model of obesity on circulating insulin, glucose, free fatty acids, TNF‐α and angiotensin II concentrations. The experimental animals were 18% heavier and had 68% greater visceral fat mass than their control counterparts and were dyslipidaemic. Significant increases in the ubiquitin ligase and MuRF‐1, as well as in caspase‐3 and poly‐ADP‐ribose polymerase cleavage were observed in the muscle of obese animals compared with the control rats. We propose that dyslipidaemia may be a mechanism for the activation of inflammatory/stress‐activated signalling pathways in obesity and type II diabetes, which will lead to apoptosis and atrophy in skeletal muscle.

A potential role for angiotensin II in obesity induced cardiac hypertrophy and ischaemic/reperfusion injury.

BackgroundThe mechanisms for obesity induced myocardial remodelling and subsequent mechanical dysfunction are poorly understood. There is good evidence that angiotensin II and TNFα have strong growth promoting properties and are elevated with obesity. In addition, these two peptides may interact to exacerbate myocardial ischaemic/reperfusion injury.HypothesisObesity increases systemic and myocardial renin–angiotensin system (RAS) activity and TNFα levels and contributes to obesity induced cardiac remodelling and ischaemic/reperfusion injury.MethodsMale Wistar rats were placed on a standard rat chow diet or cafeteria diet for 16 weeks. Two additional groups of rats received the respective diets and losartan (30 mg/ kg/d) in their drinking water. Hearts were perfused on the isolated working rat heart perfusion system and mechanical function was documented before and after 15 min normothermic total global ischaemia. Blood and myocardial samples were collected for angiotensin II, TNFα and NADPH oxidase activity determinations.ResultsThe rats on the cafeteria diet became obese compared to rats on the standard rat chow (438 ± 5.9 g vs 393 ± 7.3 g for control, p < 0.05). Obesity was associated with elevated serum angiotensin II (0.050 ± 0.015 pmol/ml vs. 0.035 ± 0.003 pmol/ml, p < 0.05) and TNFα levels (42.8 ± 5.93 pg/ml vs. 13.18 ± 2.50 pg/ml, p < 0.05), and increased heart to body weight ratios (3.1 ± 0.04 mg/g vs. 2.8 ± 0.03 mg/g, p < 0.05). Losartan had no effect on body weight but decreased basal myocardial angiotensin II and TNFΑ levels as well as heart to body weight ratio in the obese and lean controls (2.5 ± 0.05 mg/g and 2.6 ± 0.04 mg/g relative to their controls, p < 0.05). Hearts from obese rats had lower reperfusion aortic outputs (AO) than their concurrent controls (18.42 ± 1.17 ml/min vs. 27.8 ± 0.83 ml/min, p < 0.05). Losartan improved aortic output recoveries in obese rats (23.0 ± 1.71 ml/min, p < 0.05).ConclusionsObesity increased serum angiotensin II and TNFα levels, blood pressure, and heart weight to body weight ratios. These changes were associated with decreased basal and post–ischaemic myocardial mechanical function. Chronic AT1 receptor antagonism prevented the adverse changes in heart weight, mechanical function and susceptibility to ischaemic/reperfusion injury. Although current data do not exclude additional mechanisms for obesity induced cardiac remodelling, they suggest that angiotensin II may contribute to obesity induced cardiac remodelling and ischaemic/reperfusion injury.

Nitric oxide triggers classic ischemic preconditioning.

Abstract: The role of NO in the classic ischemic preconditioning phenomenon of the myocardium is not well defined, and was investigated by using the isolated perfused rat heart as a model. Hearts were preconditioned with 3 × 5 minute ischemia in the presence and absence of the NOS inhibitors l‐NAME (50 μM) and l‐NNA (50 μM), and the guanylyl cyclase inhibitor ODQ (20 μM). These inhibitors significantly attenuated the protective effect of preconditioning against 25‐min global ischemia (as measured by functional recovery), specifically if administered during the triggering phase. Cyclic infusions (3 × 5 min) of the NO‐donors SNAP (50 μM) and SNP (100 μM) elicited protection against both 25‐min global or low‐flow ischemia. Hearts preconditioned with NO donors displayed significantly superior functional reserve, if stimulated with adrenaline, compared to hearts preconditioned with ischemia. Although the NO donors SNAP and SNP both activated p38 MAPK during the preconditioning protocol, protection was accompanied by significantly decreased p38 MAPK activity during sustained ischemia, as was the case in ischemic preconditioning. We conclude that (1) NO is a trigger for classic preconditioning, (2) cGMP generation plays an important role in its protection, (3) attenuation of p38 MAPK during sustained ischemia accompanies NO preconditioning and may mediate cardiac protection, and (4) preconditioning with NO may be more advantageous than using ischemia.

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.

Effect of Sildenafil on Reperfusion Function, Infarct Size, and Cyclic Nucleotide Levels in the Isolated Rat Heart Model

We have previously shown that NO-donor induced elevation in myocardial cGMP levels is associated with improved reperfusion function of the isolated rat heart. The phosphodiesterase 5 (PDE 5) inhibitor, sildenafil could potentially increase myocardial cGMP levels and thus protect the heart against ischaemic/reperfusion injury.Methods: To test our hypothesis we treated the isolated working rat heart with vehicle, OR sildenafil (10, 20, 50, 100, 200 nM), OR sildenafil (50 nM) plus a sarcolemmal (HMR 1098) or a mitochondrial (5-Hydroxydecanoate (5-HD)) KATP channel blocker. Hearts were then subjected to 20 min global, or 35 min regional ischaemia at 37∘C before reperfusion function (aortic output, coronary flow and aortic pressure) and infarct size were documented. Pre-ischaemic, ischaemic and reperfusion myocardial cAMP and cGMP concentrations were determined.Results: Low concentrations of sildenafil (10, 20 and 50 nM) improved reperfusion aortic output (AO) recovery (61.4± 4.5%, 64.8 ± 5.2% and 62.3 ± 5.0% vs. 45.4 ± 3.8% for controls (p < 0.05)) and infarct size, while high concentrations (200 nM) worsened AO recovery (24.9 ± 4.9.0%, p < 0.05). Myocardial cGMP levels of ischaemic tissue were elevated (34.7 ± 2.4 vs. 27.3 ± 2.2 pmol/g ww) and cAMP levels were suppressed (0.59 ± 0.03 vs. 0.87 ± 0.06 nmol/g ww) in the sildenafil (50 nM) treated hearts. Co-perfusion with sildenafil plus HMR 1098 decreased AO recovery (21.7 ± 7.6% vs. 62.3 ± 5.0% for sildenafil alone, p < 0.05) and increased infarct size (29.7 ± 2.04% vs. 8.6 ± 2.39% for sildenafil alone, p < 0.05).Similarly, sildenafil plus 5-HD decreased reperfusion AO recovery (44.4 ± 6.0% vs. 62.3 ± 5.0% for sildenafil alone, p < 0.05) and increased infarct size (33.8 ± 1.62% vs. 8.6 ± 2.39% for sildenafil alone, p < 0.05).Conclusions: (1) Pretreatment with low concentrations of sildenafil (20–50 nM) improves, while higher concentrations (200 nM) worsen reperfusion function in this model. (2) Low concentrations of sildenafil (20–50 nM) decrease infarct size while the higher concentrations had no effect. (3) These protective properties of low concentrations of sildenafil may be related to its cGMP elevating and cAMP suppressing effects in the ischaemic heart. (4) Possible end-effectors for sildenafil in the ischaemic heart include the mitochondrial and sarcolemmal KATP channel.

Effect of nitrovasodilators and inhibitors of nitric oxide synthase on ischaemic and reperfusion function of rat isolated hearts

1 The functional role of the nitric oxide (NO)/guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) pathway in experimental myocardial ischaemia and reperfusion was studied in rat isolated hearts. 2 Rat isolated hearts were perfused at constant pressure with Krebs‐Henseleit buffer for 25 min (baseline), then made ischaemic by reducing coronary flow to 0.2 ml min−1 for 25 or 40 min, and reperfused at constant pressure for 25 min. Drugs inhibiting or stimulating the NO/cyclic GMP pathway were infused during the ischaemic phase only. Ischaemic contracture, myocardial cyclic GMP and cyclic AMP levels during ischaemia, and recovery of reperfusion mechanical function were monitored. 3 At baseline, heart rate was 287±12 beats min−1, coronary flow was 12.8±0.6 ml min−1, left ventricular developed pressure (LVDevP) was 105±4 mmHg and left ventricular end‐diastolic pressure 4.6±0.2 mmHg in vehicle‐treated hearts (control; n=12). Baseline values were similar in all treatment groups (P>0.05). 4 In normoxic perfused hearts, 1 μM NG‐nitro‐L‐arginine (L‐NOARG) significantly reduced coronary flow from 13.5±0.2 to 12.1±0.1 ml min−1 (10%) and LVDevP from 97±1 to 92±1 mmHg (5%; P<0.05, n=5). 5 Ischaemic contracture was 46±2 mmHg, i.e. 44% of LVDevP in control hearts (n=12), unaffected by low concentrations of nitroprusside (1 and 10 μM) but reduced to ∼30 mmHg (∼25%) at higher concentrations (100 or 1000 μM; P<0.05 vs control, n=6). Conversely, the NO synthase inhibitor L‐NOARG reduced contracture at 1 μM to 26±3 mmHg (23%), but increased it to 63±4 mmHg (59%) at 1000 μM (n=6). Dobutamine (10 μM) exacerbated ischaemic contracture (81±3 mmHg; n=7) and the cyclic GMP analogue Sp‐8‐(4‐p‐chlorophenylthio)‐3′,5′‐monophosphorothioate (Sp‐8‐pCPT‐cGMPS; 10 μM) blocked this effect (63±1 mmHg; P<0.05 vs dobutamine alone, n=5). 6 At the end of reperfusion, LVDevP was 58±5 mmHg, i.e. 55% of pre‐ischaemic value in control hearts, significantly increased to ∼80% by high concentrations of nitroprusside (100 or 1000 μM) or L‐NOARG at 1 μM, while a high concentration of L‐NOARG (1000 μM) reduced LVDevP to ∼35% (P<0.05 vs control; n=6). 7 Ischaemia increased tissue cyclic GMP levels 1.8 fold in control hearts (P<0.05; n=12); nitroprusside at 1 μM had no sustained effect, but increased cyclic GMP ∼6 fold at 1000 μM; L‐NOARG (1 or 1000 μM) was without effect (n=6). Nitroprusside (1 or 1000 μM) marginally increased cyclic AMP levels whereas NO synthase inhibitors had no effect (n=6). 8 In conclusion, the cardioprotective effect of NO donors, but not of low concentrations of NO synthase inhibitors may be due to their ability to elevate cyclic GMP levels. Because myocardial cyclic GMP levels were not affected by low concentrations of NO synthase inhibitors, their beneficial effect on ischaemic and reperfusion function is probably not accompanied by reduced formation of NO and peroxynitrite in this model.

Role for the Na+/h+ Exchanger in Reperfusion Stunning in Isolated Perfused Rat Heart

Summary: We tested the hypothesis that Na + /H+ exchange contributes to reperfusion stunning and arrhythmias. The effects of amiloride, an established inhibitor of Na +/H+ exchange, were compared with those of a new inhibitor (HOE 694). Working hearts, subjected to 20-min global ischemia and reperfused for 30 min, were pretreated (for 5 min before ischemia) or reperfused (initial 2 min) with HOE 694 or amiloride. Pretreatment with 10-7 M HOE increased recovery of aortic output (AO) after 30-min reperfusion: As a percentage of the preischemic controls, values were 38.5 ± 3.6% (n=7) for controls versus 50.6 ± 3.9% (n=5) for the treated group (p<0.05). Pretreatment with HOE (10-6 M) increased AO recoveries from 38.2 ± 2.0% (n=5) to 52.9 ± 2.7% (n=5) (p<0.05). Amiloride (10-5 M) pretreatment improved AO recoveries from 41.6 ± 2.7% (n=6) to 55.8 ± 4.0% (n=6) (p<0.05). Thus, pretreatment by both HOE 694 and amiloride decreased reperfusion stunning. Adding HOE (10-7 M) only in the reperfusion period increased AO recoveries from 36.4 ± 1.2% (n=6) to 62.4 ± 3.2% (n=11) (p<0.002). Amiloride (10-5 or 10-3 M) added only during reperfusion did not improve recovery of AO. HOE 694, active in much lower concentrations than amiloride, was the only compound active against stunning when added only during reperfusion. In addition, both compounds inhibited reperfusion arrhythmias when added at reperfusion. Hence, our data suggest that (a) HOE 694 is a more potent inhibitor of Na + /H + exchange than amiloride; (b) inhibition of Na +/H+ exchange by HOE 694 even during reperfusion only limits reperfusion stunning; and (c) Na+ /H+ exchange and, by implication, Na +/Ca2+ exchange during the early reperfusion period contributes to reperfusion stunning and arrhythmias in this model

Opioid receptors and cardioprotection – ‘opioidergic conditioning’ of the heart

Ischaemic heart disease (IHD) remains a major cause of morbidity/mortality globally, firmly established in Westernized or ‘developed’ countries and rising in prevalence in developing nations. Thus, cardioprotective therapies to limit myocardial damage with associated ischaemia–reperfusion (I–R), during infarction or surgical ischaemia, is a very important, although still elusive, clinical goal. The opioid receptor system, encompassing the δ (vas deferens), κ (ketocyclazocine) and μ (morphine) opioid receptors and their endogenous opioid ligands (endorphins, dynorphins, enkephalins), appears as a logical candidate for such exploitation. This regulatory system may orchestrate organism and organ responses to stress, induces mammalian hibernation and associated metabolic protection, triggers powerful adaptive stress resistance in response to ischaemia/hypoxia (preconditioning), and mediates cardiac benefit stemming from physical activity. In addition to direct myocardial actions, central opioid receptor signalling may also enhance the ability of the heart to withstand I–R injury. The δ‐ and κ‐opioid receptors are strongly implicated in cardioprotection across models and species (including anti‐infarct and anti‐arrhythmic actions), with mixed evidence for μ opioid receptor‐dependent protection in animal and human tissues. A small number of clinical trials have provided evidence of cardiac benefit from morphine or remifentanil in cardiopulmonary bypass or coronary angioplasty patients, although further trials of subtype‐specific opioid receptor agonists are needed. The precise roles and utility of this GPCR family in healthy and diseased human myocardium, and in mediating central and peripheral survival responses, warrant further investigation, as do the putative negative influences of ageing, IHD co‐morbidities, and relevant drugs on opioid receptor signalling and protective responses.