Chronically elevated bilirubin protects from cardiac reperfusion injury in the male Gunn rat.

AIMS: Bilirubin is associated with reduced risk of cardiovascular disease, as evidenced in conditions of mild hyperbilirubinaemia (Gilbert's Syndrome). Little is known regarding myocardial stress resistance in hyperbilirubinaemic conditions or whether life-long exposure modifies cardiac function, which might contribute to protection from cardiovascular disease. METHODS: Hyperbilirubinaemic rats and littermate controls underwent echocardiography at 3, 6 and 12 months of age, with hearts subsequently assessed for resistance to 30 min of ischaemia. Heart tissue was then collected for assessment of bilirubin content. RESULTS: No difference in baseline cardiac function was evident until 6 months onwards, where Gunn rats demonstrated aortic dilatation and reduced peak ejection velocities. Additionally, duration of ventricular ejection increased progressively, indicating a negative inotropic effect of bilirubin in vivo. Ex vivo analysis of baseline function revealed reduced left ventricular pressure development (LVDP) and contractility in hyperbilirubinaemic rats. Furthermore, stress resistance was improved in Gunn hearts: post-ischaemic recoveries of LVDP (76 ± 22% vs. 29 ± 17% Control, P < 0.01) and coronary flow (96 ± 9% vs. 86 ± 16% Control, P < 0.01) were improved in Gunn hearts, accompanied by reduced infarct area (21 ± 5% vs. 47 ± 15% Control, P < 0.01), and ventricular malondialdehyde and protein carbonyl content. Expression of myocardial nitric oxide-regulating genes including Nos1 and Noa1 were not significantly different. CONCLUSIONS: These data reveal life-long hyperbilirubinaemia induces age-dependent hypocontractility in male Gunn rats, and improved stress resistance. In addition, bilirubin exerts sex-independent effects on vascular structure, myocardial function and ischaemic tolerance, the latter likely mediated via bilirubin's antioxidant properties.

The effects of visceral obesity and androgens on bone: trenbolone protects against loss of femoral bone mineral density and structural strength in viscerally obese and testosterone-deficient male rats.

SUMMARY: In males, visceral obesity and androgen deficiency often present together and result in harmful effects on bone. Our findings show that both factors are independently associated with adverse effects on femoral bone structure and strength, and trenbolone protects rats from diet-induced visceral obesity and consequently normalises femoral bone structural strength. INTRODUCTION: In light of the rapidly increasing incidence of obesity and osteoporosis globally, and recent conjecture regarding the effects of visceral adiposity and testosterone deficiency on bone health, we investigated the effects of increased visceral adipose tissue (VAT) mass on femoral bone mineral density (BMD), structure and strength in normal weight rats with testosterone deficiency. METHODS: Male Wistar rats (n = 50) were fed either standard rat chow (CTRL, n = 10) or a high-fat/high-sugar diet (HF/HS, n = 40). Following 8 weeks of feeding, rats underwent sham surgery (CTRL, n = 10; HF/HS, n = 10) or orchiectomy (HF/HS + ORX, n = 30). Following a 4-week recovery period, mini-osmotic pumps containing either vehicle (CTRL, n = 10; HF/HS, n = 10; HF/HS + ORX, n = 10), 2.0 mg kg day(-1), testosterone (HF/HS + ORX + TEST, n = 10) or 2.0 mg kg day(-1) trenbolone (HF/HS + ORX + TREN, n = 10) were implanted for 8 weeks of treatment. Dual-energy X-ray absorptiometry and three-point bending tests were used to assess bone mass, structure and strength of femora. RESULTS: Diet-induced visceral obesity resulted in decreased bone mineral area (BMA) and content (BMC) and impaired femoral stiffness and strength. Orchiectomy further impaired BMA, BMC and BMD and reduced energy to failure in viscerally obese animals. Both TEST and TREN treatment restored BMA, BMC, BMD and energy to failure. Only TREN reduced visceral adiposity and improved femoral stiffness and strength. CONCLUSIONS: Findings support a role for both visceral adiposity and testosterone deficiency as independent risk factors for femoral osteoporosis, adverse bone geometry and impaired bone strength in male rats. Trenbolone may be a more effective candidate for androgen replacement therapy than testosterone in viscerally obese testosterone-deficient males.

Selective pinning control of the average disease transmissibility in an HIV contact network.

Medication is applied to the HIV-infected nodes of high-risk contact networks with the aim of controlling the spread of disease to a predetermined maximum level. This intervention, known as pinning control, is performed both selectively and randomly in the network. These strategies are applied to 300 independent realizations per reference level of incidence on connected undirectional networks without isolated components and varying in size from 100 to 10,000 nodes per network. It is shown that a selective on-off pinning control strategy can control the networks studied with limited steady-state error and, comparing the medians of the doses from both strategies, uses 51.3% less medication than random pinning of all infected nodes. Selective pinning could possibly be used by public health specialists to identify the maximum level of HIV incidence in a population that can be achieved in a constrained funding environment.

Myocardial structure, function and ischaemic tolerance in a rodent model of obesity with insulin resistance.

Obesity and its comorbidities (dyslipidaemia, insulin resistance and hypertension) that together constitute the metabolic syndrome are all risk factors for ischaemic heart disease. Although obesity has been reported to be an independent risk factor for congestive heart failure, whether obesity-induced heart failure develops in the absence of increased afterload (induced by hypertension) is not clear. We have previously shown that obesity with insulin resistance decreases myocardial tolerance to ischaemia-reperfusion, but the mechanism for this decreased tolerance remains unclear. We hypothesize that obesity with insulin resistance induces adverse cardiac remodelling and pump dysfunction, as well as adverse changes in myocardial prosurvival reperfusion injury salvage kinase (RISK) pathway signalling to reduce myocardial tolerance to ischaemia-reperfusion. Wistar rats were fed an obesogenic (obese group) or a standard rat chow diet (control group) for 32 weeks. Echocardiography was performed over the 32 weeks before isolated Langendorff-perfused hearts were subjected to 40 min coronary artery ligation followed by reperfusion, and functional recovery (rate-pressure product), infarct size and RISK pathway function were assessed (Western blot analysis). Obesity with insulin resistance increased myocardial lipid accumulation but had no effect on in vivo or ex vivo left ventricular structure/function. Hearts from obese rats had lower reperfusion rate-pressure products (13115 ± 562 beats min(-1) mmHg for obese rats versus 17781 ± 1109 beats min(-1) mmHg for control rats, P < 0.05) and larger infarcts (36.3 ± 5.6% of area at risk in obese rats versus 14.1 ± 2.8% of area at risk in control rats, P < 0.01) compared with control hearts. These changes were associated with reductions in RISK pathway function, with 30-50 and 40-60% reductions in Akt and glycogen synthase kinase 3 beta (GSK-3ß) expression and phosphorylation, respectively, in obese rat hearts compared with control hearts. Total endothelial nitric oxide synthase expression was reduced by 25% in obese rats. We conclude that obesity with insulin resistance had no effect on basal cardiac structure or function but decreased myocardial tolerance to ischaemia-reperfusion. This reduction in ischaemic tolerance was likely to be due to compromised RISK pathway function in obese, insulin-resistant animals.

The effect of creatine supplementation on myocardial function, mitochondrial respiration and susceptibility to ischaemia/reperfusion injury in sedentary and exercised rats.

AIM: To investigate the effects of dietary creatine supplementation alone and in combination with exercise on basal cardiac function, susceptibility to ischaemia/reperfusion injury and mitochondrial oxidative function. There has been an increase in the use of creatine supplementation among sports enthusiasts, and by clinicians as a therapeutic agent in muscular and neurological diseases. The effects of creatine have been studied extensively in skeletal muscle, but not in the myocardium. METHODS: Male Wistar rats were swim-trained for 8 weeks, 5 days per week. Hearts were excised and either freeze-clamped for biochemical analysis or perfused on the isolated heart perfusion system to assess function and ischaemia/reperfusion tolerance. Mechanical function was documented in working heart and retrograde mode. The left coronary artery was ligated and infarct size determined. Mitochondrial oxidative capacity was quantified. RESULTS: Aortic output recovery of hearts from the sedentary controls (CSed) was significantly higher than those from creatine-supplemented sedentary (CrSed), creatine-supplemented exercised (CrEx) as well as control exercised (CEx) groups. Ischaemic contracture of hearts from CrEx was significantly higher than that of CSed. There were no differences in infarct size and mitochondrial oxygen consumption. CONCLUSION: This study suggests that creatine supplementation has no effects on basal cardiac function but reduces myocardial tolerance to ischaemia in hearts from exercise-trained animals, by increasing the ischaemic contracture and decreasing reperfusion aortic output. Exercise training alone also significantly decreased aortic output recovery. However, the exact mechanisms for these adverse myocardial effects are unknown and need further investigation.

Melatonin and the metabolic syndrome: a tool for effective therapy in obesity-associated abnormalities?

The metabolic syndrome (MetS) is a cluster of metabolic abnormalities associated with increased risk for cardiovascular diseases. Apart from its powerful antioxidant properties, the pineal gland hormone melatonin has recently attracted the interest of various investigators as a multifunctional molecule. Melatonin has been shown to have beneficial effects in cardiovascular disorders including ischaemic heart disease and hypertension. However, its role in cardiovascular risk factors including obesity and other related metabolic abnormalities is not yet established, particularly in humans. New emerging data show that melatonin may play an important role in body weight regulation and energy metabolism. This review will address the role of melatonin in the MetS focusing on its effects in obesity, insulin resistance and leptin resistance. The overall findings suggest that melatonin should be exploited as a therapeutic tool to prevent or reverse the harmful effects of obesity and its related metabolic disorders.

The effects of gender and obesity on myocardial tolerance to ischemia.

Obesity is increasing at an alarming rate globally. Several studies have shown that premenopausal women have a reduced risk of CV disease and a reduced myocardial susceptibility to ischemia/reperfusion injury. The effect of obesity on myocardial tolerance to ischemia in women has not been established. To determine how obesity affects myocardial susceptibility to ischemia/reperfusion injury in both males and females, we fed male and female Wistar rats a high caloric diet (HCD) or a control rat chow diet (CD) for 18 weeks. Rats were subsequently fasted overnight, anesthetized and blood was collected. In separate experiments, 18-week-fed (HCD and CD) rats underwent 45 min in vivo coronary artery ligation (CAL) followed by 2 hours reperfusion. Hearts were stained with TTC and infarct size determined. Both male and female HCD fed rats had increased body and visceral fat weights. Homeostasis model assessment (HOMA) index values were 13.95+/-3.04 for CD and 33.58+/-9.39 for HCD male rats (p<0.01) and 2.98+/-0.64 for CD and 2.99+/-0.72 for HCD fed female rats. Male HCD fed rats had larger infarct sizes than CD fed littermates (43.2+/-9.3 % vs. 24.4+/-7.6 %, p<0.05). Female HCD and CD diet fed rats had comparable infarct sizes (31.8+/-4.3 % vs. 23.9+/-3.3 %). We conclude that male rats on the HCD became viscerally obese, dyslipidemic and insulin-resistant, while female HCD fed rats became viscerally obese without developing dyslipidemia or insulin resistance. Obesity increased myocardial infarct size in males but not the females.

Impaired contractile function and mitochondrial respiratory capacity in response to oxygen deprivation in a rat model of pre-diabetes.

AIM: Obesity is a major contributor to the global burden of disease and is closely associated with the development of type 2 diabetes and cardiovascular diseases. This study tested the hypothesis that mitochondrial respiratory capacity of the pre-diabetic heart is decreased leading to impaired contractile function and tolerance to ischaemia/reperfusion. METHODS: Eight-week-old male Wistar rats were fed a high caloric diet for 16 weeks after which anthropometric, metabolic, cardiac and mitochondrial parameters were evaluated vs. age-matched lean controls. Cardiac function (working heart perfusions) and mitochondrial respiratory capacity were assessed at baseline and in response to acute oxygen deprivation. RESULTS: Rats fed the high caloric diet exhibited increased body weight and visceral fat vs. the control group. Heart weights of obese rats were also increased. Triglyceride, fasting plasma insulin and free fatty acid levels were elevated, while high-density lipoprotein cholesterol levels were reduced in the obese group. Contractile function was attenuated at baseline and further decreased after subjecting hearts to ischaemia-reperfusion. Myocardial infarct sizes were increased while ADP phosphorylation rates were diminished in obese rats. However, no differences were found for mtDNA levels and the degree of oxidative stress-induced damage. CONCLUSIONS: These data show that decreased mitochondrial bioenergetic capacity in pre-diabetic rat hearts may impair respiratory capacity and reduce basal contractile function and tolerance to acute oxygen deprivation.

Protection of the ischaemic heart: investigations into the phenomenon of ischaemic preconditioning.

Exposure of the heart to one or more short episodes of ischaemia/reperfusion protects the heart against a subsequent prolonged period of ischaemia, as evidenced by a reduction in infarct size and an improvement in functional recovery during reperfusion. Elucidation of the mechanism of this endogenous protection could lead to the development of pharmacological mimetics to be used in the clinical setting. The aim of our studies was therefore to gain more information regarding the mechanism of ischaemic preconditioning, using the isolated perfused working rat heart as model. A preconditioning protocol of 1 x 5 or 3 x 5 min of ischaemia, interspersed with 5 min of reperfusion was found to protect hearts exposed to 25 min of global ischaemia or 35-45 min of regional ischaemia. These models were used throughout our studies. In view of the release of catecholamines by ischaemic tissue, our first aim was to evaluate the role of the alphaadrenergic receptor in ischaemic preconditioning. However, using a multi-cycle ischaemic preconditioning protocol, we could not find any evidence for alpha-1 adrenergic or PKC activation in the mechanism of preconditioning. Cyclic increases in the tissue cyclic nucleotides, cAMP and cGMP were found, however, to occur during a multi-cycle preconditioning protocol, suggesting roles for the beta-adrenergic signalling pathway and nitric oxide (NO) as triggers of cardioprotection. This was substantiated by the findings that (1) administration of the beta-adrenergic agonist, isoproterenol, or the NO donors SNAP or SNP before sustained ischaemia also elicited cardioprotection similar to ischaemic preconditioning; (2) beta-adrenergic blockade or nitric oxide synthase inhibition during an ischaemic preconditioning protocol abolished protection. Effectors downstream of cAMP, such as p38MAPK and CREB, were also demonstrated to be involved in the triggering process. Our next step was to evaluate intracellular signalling during sustained ischaemia and reperfusion. Our results showed that ischaemic preconditioned-induced cardioprotection was associated with a significant reduction in tissue cAMP, attenuation of p38MAPK activation and increased tissue cGMP levels and HSP27 activation, compared to non-preconditioned hearts. The role of the stress kinase p38MAPK was further investigated by using the inhibitor SB203580. Our results suggested that injury by necrosis and apoptosis share activation of p38MAPK as a common signal transduction pathway and that pharmacological targeting of this kinase offers a tenable option to manipulate both these processes during ischaemia/reperfusion injury.

A role for the RISK pathway and K(ATP) channels in pre- and post-conditioning induced by levosimendan in the isolated guinea pig heart.

BACKGROUND AND PURPOSE: Myocardial reperfusion injury prevents optimal salvage of the ischaemic myocardium, and adjunct therapy that would significantly reduce reperfusion injury is still lacking. We investigated whether (1) the heart could be pre- and/or post-conditioned using levosimendan (levosimendan pre-conditioning (LPC) and levosimendan post-conditioning (LPostC)) and (2) the prosurvival kinases and/or the sarcolemmal or mitochondrial K(ATP) channels are involved. EXPERIMENTAL APPROACH: Isolated guinea pig hearts were treated with two 5 min cycles of levosimendan (0.1 microM) interspersed with vehicle perfusion, or two 5 min cycles of ischaemia/reperfusion, before coronary artery ligation (CAL) for 40 min at 36.5 degrees C. Hearts were treated with mitochondrial or sarcolemmal K(ATP) channel blockers before LPC or LPostC. For post-conditioning, hearts received three 30 s cycles of ischaemia/reperfusion or levosimendan/vehicle. Hearts were pretreated with levosimendan immediately before CAL (without washout). Cardiac function, infarct size and reperfusion injury salvage kinase activity was assessed. KEY RESULTS: LPC and LPostC halved the infarct size compared with controls (P<0.05). Treatment with K(ATP) channel blockers before LPC or LPostC reversed this decrease. Pretreating hearts with levosimendan increased activity of extracellular signal-regulated kinase (ERK) 42/44 on reperfusion and had the most marked infarct-lowering effect (P<0.05). CONCLUSIONS AND IMPLICATIONS: (1) Hearts could be pharmacologically pre- and post-conditioned with levosimendan; (2) levosimendan pretreatment is the most effective way to reduce infarct size, possibly by increasing ERK 42/44 activity; (3) benefits of LPC and LPostC were abolished by both K(ATP) channel blockers and (4) LPC may be useful before elective cardiac surgery, whereas LPostC may be used after acute coronary artery events.

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

BACKGROUND: The mechanisms for obesity induced myocardial remodelling and subsequent mechanical dysfunction are poorly understood. There is good evidence that angiotensin II and TNFalpha have strong growth promoting properties and are elevated with obesity. In addition, these two peptides may interact to exacerbate myocardial ischaemic/reperfusion injury. HYPOTHESIS: Obesity increases systemic and myocardial renin-angiotensin system (RAS) activity and TNFalpha levels and contributes to obesity induced cardiac remodelling and ischaemic/reperfusion injury. METHODS: Male 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, TNFalpha and NADPH oxidase activity determinations. RESULTS: The 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 TNFalpha 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 TNFAlpha 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). CONCLUSIONS: Obesity increased serum angiotensin II and TNFalpha levels, blood pressure, and heart weight to body weight ratios. These changes were associated with decreased basal and post-ischaemic myocardial mechanical function. Chronic AT(1) 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.

Proposed mechanisms for the anabolic steroid-induced increase in myocardial susceptibility to ischaemia/reperfusion injury.

UNLABELLED: Androgenic anabolic steroids (AAS) are often used by athletes to enhance athletic performance but are strongly associated with detrimental cardiovascular effects including sudden cardiac death. HYPOTHESIS: AAS use increases myocardial susceptibility to ischaemia/reperfusion injury. METHODS: Rats were trained (swimming) with or without intramuscular injection of nandrolone laurate (0.375 mg/kg). Untrained rats with or without nandrolone served as controls. Hearts were mounted on the Langendorff perfusion apparatus and mechanical function was measured before and after 20-min normothermic global ischaemia. Myocardial tissue samples were collected for determination of tissue cyclic nucleotide and TNFalpha concentrations. RESULTS: Anabolic steroids decreased the rate pressure product (RPP) of the exercise-trained rat heart [34 582 +/- 1 778 mmHg/min vs 28 868 +/- 2 446 mmHg/min for exercise-trained steroid-treated hearts (p < 0.05)]. Reperfusion RPP was lower in both the sedentary, and the exercise-trained, steroid-treated hearts than in their concurrent vehicle-treated controls (18 276 +/- 2 026 mmHg/min vs 12 018 +/- 1 725 mmHg/min for sedentary steroid-treated hearts and, 21 892 +/- 2 912 mmHg vs 12 838 +/- 1 536 mmHg/min for exercise-trained steroid-treated hearts). Myocardial TNFalpha [267.75 +/- 44.25 pg/g vs 190.00 +/- 15.75 pg/g (p < 0.05)] and cAMP concentrations [406.04 +/- 18.41 pmol/g vs 235.6 +/- 43.26 pmol/g (p < 0.05)] were elevated in the steroid-treated hearts when compared with their untreated counterparts. CONCLUSIONS: Supraphysiological doses of anabolic steroids, whether taken during exercise training or under sedentary conditions increase myocardial susceptibility to ischaemia/reperfusion injury in our model. This increased susceptibility may be related to steroid-induced increases in the pre-ischaemic myocardial cAMP concentrations and/or increases in both pre-ischaemic and reperfusion TNFalpha concentrations.

Dietary red palm oil improves reperfusion cardiac function in the isolated perfused rat heart of animals fed a high cholesterol diet.

It has been shown that dietary red palm oil (RPO) supplementation improved reperfusion function. However, no exact protective cellular mechanisms have been established. Our aim was to search for a possible cellular mechanism and a role for fatty acids. Rats were fed a standard rat chow, plus cholesterol and/or RPO-supplementation for 6 weeks. Functional recovery, myocardial phospholipid and cAMP/cGMP levels were determined in isolated rat hearts subjected to 25 min of normothermic total global ischaemia. Dietary RPO in the presence of cholesterol improved aortic output (AO) recovery (63.2+/-3.06%, P<0.05) vs. cholesterol only (36.5+/-6.2%). The improved functional recovery in hearts supplemented with RPO vs. control was preceded by an elevation in the cGMP levels early in ischaemia (RPO 132.9+/-36.3% vs. control 42.7+/-24.4%, P<0.05). Concurrently, cAMP levels decreased (RPO -8.3+/-6.9% vs. control 19.9+/-7.7%, P<0.05). Our data suggest that dietary RPO-supplementation improved reperfusion AO through mechanisms that may include activation of the NO-cGMP and inhibition of the cAMP pathway.

Relation of cyclic nucleotide ratios to ischemic and reperfusion injury in nitric oxide-donor treated rat hearts.

Nitric oxide (NO) donors given during ischemia possibly protect the myocardium by increasing tissue cyclic guanosine monophosphate (cGMP) and decreasing cytosolic Ca2+ levels. However, NO donors also elevate ischemic cyclic adenosine monophosphate (cAMP) levels, which exacerbates ischemic-reperfusion injury. The authors propose that suppression of this NO donor-induced increase in cAMP would improve the cardioprotective properties of these compounds. Langendorff perfused rat hearts were treated with sodium nitroprusside (SNP, 0.1 mM ) or glyceryl trinitrate (GTN, 1.0 microM ) and/or adenylyl cyclase (SQ, 50 microM ) or guanylyl cyclase (ODQ, 30-300 microM ) inhibitors during 40-min low-flow (0.2 ml/min) ischemia. Control reperfusion rate-pressure product (RPP) recoveries were 47 +/- 3% (n = 9) and improved to 59 +/- 1% (n = 11) (p < 0.05) with SNP treatment. Ischemic ODQ treatment decreased RPP recovery to 33 +/- 3% (n = 10) (p < 0.05). ODQ eliminated the cardioprotective effects of SNP (RPP recovery: 40 +/- 5% [n = 7] vs. 59 +/- 1% [p < 0.05]). Adenylyl cyclase inhibition improved RPP recovery from 59 +/- 1% (SNP) to 72 +/- 4% (SNP + SQ) (n = 11) (p < 0.05). The authors conclude that (a) suppression of the NO donor-induced elevations in ischemic cGMP levels (ODQ) worsened reperfusion RPP, (b) suppression of the NO donor-induced elevation in ischemic cAMP levels (SQ) further improved reperfusion RPP in NO donor-treated hearts, and (c) the severity of ischemic-reperfusion injury in the NO donor-treated heart was inversely related to ischemic-tissue cGMP levels and often directly related to the ischemic-tissue cAMP-to-cGMP ratio.

Levosimendan: effects of a calcium sensitizer on function and arrhythmias and cyclic nucleotide levels during ischemia/reperfusion in the Langendorff-perfused guinea pig heart.

The majority of clinically used inotropes act by increasing cytosolic calcium levels, which may hypothetically worsen reperfusion stunning and provoke arrhythmias. We tested the hypothesis that the calcium sensitizer levosimendan (levo) given during ischemia alone or ischemia and reperfusion would improve reperfusion function without promoting arrhythmias. The Langendorff-perfused guinea pig heart, subjected to 40-min low-flow ischemia (0.4 ml/min) with or without levo (10-300 nM) given during ischemia or ischemia/reperfusion was used. Left ventricular developed pressure (LVDP) was used as an index of mechanical function. The effect of levo (300 nM) or dobutamine (0.1 microM) on the incidence of ischemia/reperfusion arrhythmias was also investigated. Control hearts (vehicle-perfused) had LVDPs of 69.4 +/- 2.1 mm Hg whereas hearts treated with levo during ischemia and reperfusion (300 nM) had LVDPs of 104.5 +/- 2.7 mm Hg (p <.05). Hearts treated with levo during ischemia alone (10 nM) had reperfusion LVDPs of 95.8 +/- 4.2 mm Hg (p <.05) after 30-min reperfusion. Hearts treated with both levo and 10 microM glibenclamide (K(ATP) channel blocker) during ischemia had reperfusion LVDPs of 73.4 +/- 4.3 mm Hg after 30-min reperfusion. Of control hearts, 25% developed reperfusion ventricular tachycardia but not ventricular fibrillation. Levo-treated hearts had no ischemia/reperfusion arrhythmias whereas 83% (p <.05 versus control) of dobutamine-treated hearts developed ventricular tachycardia and 33% (p <.05 versus levo) developed reperfusion ventricular fibrillation. Levo improved reperfusion function without promoting arrhythmias in this model. This was possibly achieved by opening the K(ATP) channels during ischemia and sensitizing myocardial contractile apparatus instead of elevating cytosolic calcium levels in reperfused hearts.

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 microM N(G)-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 microM) but reduced to approximately 30 mmHg (approximately 25%) at higher concentrations (100 or 1000 microM; P<0.05 vs control, n=6). Conversely, the NO synthase inhibitor L-NOARG reduced contracture at 1 microM to 26+/-3 mmHg (23%), but increased it to 63+/-4 mmHg (59%) at 1000 microM (n=6). Dobutamine (10 microM) 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 microM) blocked this effect (63+/-11 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 approximately 80% by high concentrations of nitroprusside (100 or 1000 microM) or L-NOARG at 1 microM, while a high concentration of L-NOARG (1000 microM) reduced LVDevP to approximately 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 microM had no sustained effect, but increased cyclic GMP approximately 6 fold at 1000 microM; L-NOARG (1 or 1000 microM) was without effect (n=6). Nitroprusside (1 or 1000 microM) 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.

Inhibitors of Ca2+ ATPase pump of sarcoplasmic reticulum attenuate reperfusion stunning in isolated rat heart.

We tested for the first time the hypothesis that lessened uptake of Ca2+ into sarcoplasmic reticulum by inhibitors of the Ca(2+)-ATPase pump can decrease the severity of reperfusion stunning (postischemic mechanical dysfunction). We used two novel inhibitors of the Ca(2+)-ATPase pump: (a) cyclopiazonic acid (CPA, 10(-6)-10(-8)M), and (b) thapsigargin (10(-6) or 2.5 x 10(-8)M). The isolated working rat heart was subjected to 20-min global ischemia before 20-min reperfusion. The inhibitor was added either before onset of ischemia or at time of reperfusion. Reperfusion mechanical function (aortic output, AO) was measured and compared with the preischemia values. Pretreatment with CPA improved recovery of AO after 20-min reperfusion from 78.2 +/- 3.0 (n = 12) to 93.3 +/- 1.6% (n = 7) (p < 0.002) while CPA added during reperfusion only, improved AO recovery from 78.2 +/- 3.0 (n = 12) to 90.2 +/- 2.7% (n = 6) (p < 0.05). Pretreatment with thapsigargin (2.5 x 10(-8) M) improved reperfusion AO recovery from 63.5 +/- 1.1 (n = 6) to 96.8 +/- 4.2% (n = 6) (p < 0.002), but when thapsigargin was added only during reperfusion AO recovery did not change. We conclude that inhibition of the Ca2+ uptake pump represents a new principle of control of cell calcium fluxes and that CPA is more effective than thapsigargin. The proposed mechanism of protection against stunning may include inhibition of oscillations of intracellular calcium, and/or depletion of calcium in sarcoplasmic reticulum (SR).

Antiarrhythmic properties of specific inhibitors of sarcoplasmic reticulum calcium ATPase in the isolated perfused rat heart after coronary artery ligation.

OBJECTIVES: The hypothesis tested was that sequestration of calcium by the sarcoplasmic reticulum and internal calcium oscillations may play a role in the genesis of ischemic and reperfusion ventricular arrhythmias. BACKGROUND: Previous data suggest that inhibition of the release of intracellular calcium from the sarcoplasmic reticulum by ryanodine may prevent ventricular fibrillation. METHODS: The isolated Langendorff perfused rat heart was treated with two specific inhibitors of the calcium ATPase pump of the sarcoplasmic reticulum (thapsigargin [10(-6) mol/liter] or cyclopiazonic acid [10(-7) mol/liter]) for 5 min before left anterior descending coronary artery ligation was performed. One group of hearts was subject to 30 min of coronary artery ligation, and ischemic arrhythmias were monitored. In a second group, the incidence of reperfusion arrhythmias was measured after 10, 15, 20, 25 and 30 min of coronary artery ligation. RESULTS: Thapsigargin treatment during ischemia and reperfusion decreased the incidence of reperfusion ventricular fibrillation after 10 min of coronary artery ligation from 67% (n = 6) to 0% (n = 6) (p < 0.05), after 15 min from 81% (n = 16) to 25% (n = 20) (p < 0.002) and after 20 min of ischemia from 90% (n = 10) to 46% (n = 13) (p < 0.05). Thapsigargin treatment also decreased the incidence of ischemic ventricular fibrillation from 83% (n = 12) to 0% (n = 12) (p < 0.002). Cyclopiazonic acid treatment during ischemia and reperfusion likewise decreased the incidence of ischemic and reperfusion arrhythmias. CONCLUSIONS: The highly specific inhibitors of the calcium uptake pump of the sarcoplasmic reticulum--thapsigargin and cyclopiazonic acid--have antifibrillatory properties in the isolated perfused rat heart. They appear to act by restriction of calcium oscillations between the sarcoplasmic reticulum and the cytosol.