Myocardial ischemia/reperfusion injury in the inducible nitric oxide synthase knockout mice.

Inducible nitric oxide synthase (iNOS) plays an important role in the inflammatory process of certain major cardiac disorders including myocardial infarction and allograft rejection. However, the role of iNOS in acute myocardial ischemia has not been well defined. We determined the effects of genetically disruption of the intact iNOS system on cardiac tolerance to ischemia/reperfusion injury. Adult male wild-type (WT) and iNOS knockout (KO) B6,129 mice were subjected to 20 min global ischemia and 30 min reperfusion in a Langendorff isolated perfused heart model (37 degrees C, n = 10/each group). Ventricular contractile function, heart rate, coronary flow, and leakage of intracellular enzymes (CK and LDH) were not significantly different between the groups during pre-ischemia as well as reperfusion period (P > 0.05). Myocardial infarct size was also not significantly different between WT (20.2+/-2.0% of risk area) and KO mice (23.5+/-3.8%; Mean+/-SEM, P > 0.05). However, the post-ischemic heart rate was significantly preserved in KO as compared to WT (P < 0.05). We conclude that disruption of iNOS gene does not exacerbate ischemia/ reperfusion injury in the heart.     

Altered cardiac metabolic phenotype after prolonged inhibition of NO synthesis in chronically instrumented dogs

Acute inhibition of nitric oxide (NO) synthase causes a reversible alteration in myocardial substrate metabolism. We tested the hypothesis that prolonged NO synthase inhibition alters cardiac metabolic phenotype. Seven chronically instrumented dogs were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1) po) for 10 days to inhibit NO synthesis, and seven were used as controls. Cardiac free fatty acid, glucose, and lactate oxidation were measured by infusion of [(3)H]oleate, [(14)C]glucose, and [(13)C]lactate, respectively. After 10 days of L-NAME administration, despite no differences in left ventricular afterload, cardiac O(2) consumption was significantly increased by 30%, consistent with a marked enhancement in baseline oxidation of glucose (6.9 +/- 2.0 vs. 1.7 +/- 0.5 micromol.min(-1).100 g(-1), P < 0.05 vs. control) and lactate (21.6 +/- 5.6 vs. 11.8 +/- 2.6 micromol.min(-1).100 g(-1), P < 0.05 vs. control). When left ventricular afterload was increased by ANG II infusion to stimulate myocardial metabolism, glucose oxidation was augmented further in the L-NAME than in the control group, whereas free fatty acid oxidation decreased. Exogenous NO (diethylamine nonoate, 0.01 micromol.kg(-1).min(-1) iv) could not reverse this metabolic alteration. Consistent with the accelerated rate of carbohydrate oxidation, total myocardial pyruvate dehydrogenase activity and protein expression were higher (38 and 34%, respectively) in the L-NAME than in the control group. Also, protein expression of the constitutively active glucose transporter GLUT-1 was significantly elevated (46%) vs. control. We conclude that prolonged NO deficiency causes a profound alteration in cardiac metabolic phenotype, characterized by selective potentiation of carbohydrate oxidation, that cannot be reversed by a short-term infusion of exogenous NO. This phenomenon may constitute an adaptive mechanism to counterbalance cardiac mechanical inefficiency.     

Inhibition of mitochondrial permeability transition improves functional recovery and reduces mortality following acute myocardial infarction in mice

Inhibition of mitochondrial permeability transition pore (mPTP) opening by cyclosporin A or ischemic postconditioning attenuates lethal reperfusion injury. Its impact on major post-myocardial infarction events, including worsening of left ventricular (LV) function and death, remains unknown. We sought to determine whether pharmacological or postconditioning-induced inhibition of mPTP opening might improve functional recovery and survival following myocardial infarction in mice. Anesthetized mice underwent 25 min of ischemia and 24 h (protocol 1) or 30 days (protocol 2) of reperfusion. At reperfusion, they received no intervention (control), postconditioning (3 cycles of 1 min ischemia-1 min reperfusion), or intravenous injection of the mPTP inhibitor Debio-025 (10 mg/kg). At 24 h of reperfusion, mitochondria were isolated from the region at risk for assessment of the Ca(2+) retention capacity (CRC). Infarct size was measured by triphenyltetrazolium chloride staining. At 30 days of reperfusion, mortality and LV contractile function (echocardiography) were evaluated. Postconditioning and Debio-025 significantly improved Ca(2+) retention capacity (132 +/- 13 and 153 +/- 31 vs. 53 +/- 16 nmol Ca(2+)/mg protein in control) and reduced infarct size to 35 +/- 4 and 32 +/- 7% of area at risk vs. 61 +/- 6% in control (P < 0.05). At 30 days, ejection fraction averaged 74 +/- 6 and 77 +/- 6% in postconditioned and Debio-025 groups, respectively, vs. 62 +/- 12% in the control group (P < 0.05). At 30 days, survival was improved from 58% in the control group to 92 and 89% in postconditioned and Debio-025 groups, respectively. Inhibition of mitochondrial permeability transition at reperfusion improves functional recovery and mortality in mice.     

Chronically administered acetaminophen and the ischemia/reperfused myocardium

Male and female Hartley strain guinea pigs weighing 280 +/- 10 g were given acetaminophen-treated water ad libitum for 10 days. Sham-treated control animals were given similar quantities of untreated tap water (vehicle-treated control group). On Day 10, hearts were extracted, instrumented, and exposed to an ischemia (low-flow, 20 min)/reperfusion protocol. Our objective was to compare and contrast ventricular function, coronary circulation, and selected biochemical and histological indices in the two treatment groups. Left ventricular developed pressure in the early minutes of reperfusion was significantly greater in the presence of acetaminophen, e.g., at 1 min, 40 +/- 4 vs 21 +/- 3 mmHg (P < 0.05). Coronary perfusion pressure was significantly less from 3 to 40 min of reperfusion in the presence of acetaminophen. Creatine kinase release in vehicle-treated hearts rose from 42 +/- 14 (baseline) to 78 +/- 25 units/liter by the end of ischemia. Corresponding values in acetaminophen-treated hearts were 36 +/- 8 and 44 +/- 14 units/liter. Acetaminophen significantly (P < 0.05) attenuated release of creatine kinase. Chemiluminescence, an indicator of the in vitro production of peroxynitrite via the in vivo release of superoxide and nitric oxide, was also significantly attenuated by acetaminophen. Electron microscopy indicated a well-preserved myofibrillar ultrastructure in the postischemic myocardium of acetaminophen-treated hearts relative to vehicle-treated hearts (e.g., few signs of contraction bands, little or no evidence of swollen mitochondria, and well-defined light and dark bands in sarcomeres with acetaminophen; opposite with vehicle). We conclude that chronic administration of acetaminophen provides cardioprotection to the postischemic, reperfused rodent myocardium.     

Coronary nitric oxide production controls cardiac substrate metabolism during pregnancy in the dog

The aim of this study was to examine the role of nitric oxide (NO) in the control of cardiac metabolism at 60 days of pregnancy (P60) in the dog. There was a basal increase in diastolic coronary blood flow during pregnancy and a statistically significant increase in cardiac output (55 +/- 4%) and in cardiac NOx production (44 +/- 4 to 59 +/- 3 nmol/min, P < 0.05). Immunohistochemistry of the left ventricle showed an increase in endothelial nitric oxide synthase staining in the endothelial cells at P60. NO-dependent coronary vasodilation (Bezold-Jarisch reflex) was increased by 20% and blocked by N(G)-nitro-l-arginine methyl ester (l-NAME). Isotopically labeled substrates were infused to measure oleate, glucose uptake, and oxidation. Glucose oxidation was not significantly different in P60 hearts (5.4 +/- 0.5 vs. 6.2 +/- 0.4 micromol/min) but greatly increased in response to l-NAME injection (to 19.9 +/- 0.9 micromol/min, P < 0.05). Free fatty acid (FFA) oxidation was increased in P60 (from 5.3 +/- 0.6 to 10.4 +/- 0.5 micromol/min, P < 0.05) and decreased in response to l-NAME (to 4.5 +/- 0.5 micromol/min, P < 0.05). There was an increased oxidation of FFA for ATP production but no change in the respiratory quotient during pregnancy. Genes associated with glucose and glycogen metabolism were downregulated, whereas genes involved in FFA oxidation were elevated. The acute inhibition of NO shifts the heart away from FFA and toward glucose metabolism despite the downregulation of the carbohydrate oxidative pathway. The increase in endothelium-derived NO during pregnancy results in a tonic inhibition of glucose oxidation and reliance on FFA uptake and oxidation to support ATP synthesis in conjunction with upregulation of FFA metabolic enzymes.     

Acute administration of vitamin E triggers preconditioning via K(ATP) channels and cyclic-GMP without inhibiting lipid peroxidation

Vitamin E (VitE) is considered an antioxidant agent. One or more brief periods of ischemia (isc), followed by short reperfusion (rep), increase the tolerance of the heart to a subsequent prolonged ischemia, a phenomenon known as ischemic preconditioning (PC). Mitochondrial KATP channels (mitoKATP), cyclic-GMP (cGMP), and free radicals are involved in the mechanism of PC, whereas some antioxidants abolish this benefit. The purpose of this study was to evaluate the effect of VitE on infarct size, PC, and the oxidative status in vivo. Male rabbits were divided into seven groups and were subjected to myocardial ischemia (isc) and reperfusion (rep) with the following interventions: (1) control (no intervention); (2) E150 (iv VitE at a dose of 150 mg/kg for 75 min, starting 40 min before index isc and lasting through 5 min of rep); (3) E300 (iv VitE 300 mg/kg as previously described); (4) PC (two cycles of 5 min isc and 10 min rep), (5) combined E150-PC; and (6) combined E300-PC. In the last two groups VitE was given 40 min before index ischemia. Blood samples were taken for malondialdehyde (MDA) and conjugated dienes (CDs) measurement. In a second series of experiments heart tissue samples were taken at the time of long ischemia for MDA and CD determination and for cGMP assay. In order to test whether combined treatment with VitE (as the E150 group) and the mitoKATP blocker 5-hydroxydecanoic acid (5-HD) changes the infarct size, an additional group was assessed in the first series of experiments. Tissue VitE concentration was evaluated in myocardium. VitE at both doses reduced the infarct size (19.7 +/- 2.8% for E150 and 18.8 +/- 4.9% for E300 vs 47.4 +/- 2.6% in control, P < 0.05) without attenuating the effect of PC (10.2 +/- 3.1% for E150-PC, 12.4 +/- 2.2% for E300-PC, vs 13.5 +/- 3.3% for PC). Combined VitE and 5-HD treatment abrogates this benefit (37.4 +/- 6.5%, P < 0.05 vs E150 and NS vs control). VitE increases intracellular cGMP and CDs levels (P < 0.05 vs control) to the same extent as PC (P < 0.05 vs control), with no effect on MDA (P = NS between all the groups). Peripheral markers of oxidative stress are increased during reperfusion in all groups (P < 0.05 vs baseline). Overall, VitE limits infarct size via mitoKATP and cGMP, while preserving the benefit of ischemic PC.     

Hypoxic conditioning suppresses nitric oxide production upon myocardial reperfusion

Physiologically modulated concentrations of nitric oxide (NO) are generally beneficial, but excessive NO can injure myocardium by producing cytotoxic peroxynitrite. Recently we reported that intermittent, normobaric hypoxia conditioning (IHC) produced robust cardioprotection against infarction and lethal arrhythmias in a canine model of coronary occlusion-reperfusion. This study tested the hypothesis that IHC suppresses myocardial nitric oxide synthase (NOS) activity and thereby dampens explosive, excessive NO formation upon reperfusion of occluded coronary arteries. Mongrel dogs were conditioned by a 20 d program of IHC (FIO(2) 9.5-10%; 5-10 min hypoxia/cycle, 5-8 cycles/d with intervening 4 min normoxia). One day later, ventricular myocardium was sampled for NOS activity assays, and immunoblot detection of the endothelial NOS isoform (eNOS). In separate experiments, myocardial nitrite (NO(2)(-)) release, an index of NO formation, was measured at baseline and during reperfusion following 1 h occlusion of the left anterior descending coronary artery (LAD). Values in IHC dogs were compared with respective values in non-conditioned, control dogs. IHC lowered left and right ventricular NOS activities by 60%, from 100-115 to 40-45 mU/g protein (P < 0.01), and decreased eNOS content by 30% (P < 0.05). IHC dampened cumulative NO(2)(-) release during the first 5 min reperfusion from 32 +/- 7 to 14 +/- 2 mumol/g (P < 0.05), but did not alter hyperemic LAD flow (15 +/- 2 vs. 13 +/- 2 ml/g). Thus, IHC suppressed myocardial NOS activity, eNOS content, and excessive NO formation upon reperfusion without compromising reactive hyperemia. Attenuation of the NOS/NO system may contribute to IHC-induced protection of myocardium from ischemia-reperfusion injury.     

Protective effect of a low K+ cardioplegic solution on myocardial Na,K-ATPase activity.

Long duration ischemia in hypothermic conditions followed by reperfusion alters membrane transport function and in particular Na,K-ATPase. We compared the protective effect of two well-described cardioplegic solutions on cardiac Na,K-ATPase activity during reperfusion after hypothermic ischemia. Isolated perfused rat hearts (n = 10) were arrested with CRMBM or UW cardioplegic solutions and submitted to 12 hr of ischemia at 4 degrees C in the same solution followed by 60 min of reperfusion. Functional recovery and Na,K-ATPase activity were measured at the end of reperfusion and compared with control hearts and hearts submitted to severe ischemia (30 min at 37 degrees C) followed by reflow. Na,K-ATPase activity was not altered after 12 hr of ischemia and 1 hr reflow when the CRMBM solution was used for preservation (55 +/- 2 micromolPi/mg prot/hr) compared to control (53 +/- 2 micromol Pi/mg prot/hr) while it was significantly altered with UW solution (44 +/- 2 micromol Pi/mg prot/hr, p < 0.05 vs control and CRMBM). Better preservation of Na,K-ATPase activity with the CRMBM solution was associated with higher functional recovery compared to UW as represented by the recovery of RPP, 52 +/- 12% vs 8 +/- 5%, p < 0.05 and coronary flow (70 +/- 2% vs 50 +/- 8%, p < 0.05). The enhanced protection provided by CRMBM compared to UW may be related to its lower K+ content.     

Limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation prevents excess mitochondrial Ca2+ accumulation and attenuates myocardial injury.

BACKGROUND: intracellular Na+ accumulation during ischemia and reperfusion leads to cytosolic Ca2+ overload through reverse-mode operation of the sarcolemmal Na+ -Ca2+ exchanger. Cytosolic Ca2+ accumulation promotes mitochondrial Ca2+ (Ca2+ m) overload, leading to mitochondrial injury. We investigated whether limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation (VF) attenuates Ca2+ m overload and lessens myocardial dysfunction in a rat model of VF and closed-chest resuscitation. METHODS: hearts were harvested from 10 groups of 6 rats each representing baseline, 15 min of untreated VF, 15 min of VF with chest compression given for the last 5 min (VF/CC), and 60 min postresuscitation (PR). VF/CC and PR included four groups each randomized to receive before starting chest compression the new NHE-1 inhibitor AVE4454B (1.0 mg/kg), the Na+ channel blocker lidocaine (5.0 mg/kg), their combination, or vehicle control. The left ventricle was processed for intracellular Na+ and Ca2+ m measurements. RESULTS: limiting sarcolemmal Na+ entry attenuated cytosolic Na+ increase during VF/CC and the PR phase and prevented Ca2+ m overload yielding levels that corresponded to 77% and 71% of control hearts at VF/CC and PR, without differences among specific Na+ -limiting interventions. Limiting sarcolemmal Na+ entry attenuated reductions in left ventricular compliance during VF and prompted higher mean aortic pressure (110 +/- 7 vs. 95 +/- 11 mmHg, P < 0.001) and higher cardiac work index (159 +/- 34 vs. 126 +/- 29 g x m x min(-1) x kg(-1), P < 0.05) with lesser increases in circulating cardiac troponin I at 60 min PR. CONCLUSIONS: Na+ -limiting interventions prevented excess Ca2+ m accumulation induced by ischemia and reperfusion and ameliorated myocardial injury and dysfunction.     

Effects of avertin versus xylazine-ketamine anesthesia on cardiac function in normal mice

Anesthetic regimens commonly administered during studies that assess cardiac structure and function in mice are xylazine-ketamine (XK) and avertin (AV). While it is known that XK anesthesia produces more bradycardia in the mouse, the effects of XK and AV on cardiac function have not been compared. We anesthetized normal adult male Swiss Webster mice with XK or AV. Transthoracic echocardiography and closed-chest cardiac catheterization were performed to assess heart rate (HR), left ventricular (LV) dimensions at end diastole and end systole (LVDd and LVDs, respectively), fractional shortening (FS), LV end-diastolic pressure (LVEDP), the time constant of isovolumic relaxation (tau), and the first derivatives of LV pressure rise and fall (dP/dt(max) and dP/dt(min), respectively). During echocardiography, HR was lower in XK than AV mice (250 +/- 14 beats/min in XK vs. 453 +/- 24 beats/min in AV, P < 0.05). Preload was increased in XK mice (LVDd: 4.1 +/- 0.08 mm in XK vs. 3.8 +/- 0.09 mm in AV, P < 0.05). FS, a load-dependent index of systolic function, was increased in XK mice (45 +/- 1.2% in XK vs. 40 +/- 0.8% in AV, P < 0.05). At LV catheterization, the difference in HR with AV (453 +/- 24 beats/min) and XK (342 +/- 30 beats/min, P < 0.05) anesthesia was more variable, and no significant differences in systolic or diastolic function were seen in the group as a whole. However, in XK mice with HR <300 beats/min, LVEDP was increased (28 +/- 5 vs. 6.2 +/- 2 mmHg in mice with HR >300 beats/min, P < 0.05), whereas systolic (LV dP/dt(max): 4,402 +/- 798 vs. 8,250 +/- 415 mmHg/s in mice with HR >300 beats/min, P < 0.05) and diastolic (tau: 23 +/- 2 vs. 14 +/- 1 ms in mice with HR >300 beats/min, P < 0.05) function were impaired. Compared with AV, XK produces profound bradycardia with effects on loading conditions and ventricular function. The disparate findings at echocardiography and LV catheterization underscore the importance of comprehensive assessment of LV function in the mouse.     

Anti-shock effects of human superoxide dismutase in splanchnic artery occlusion (SAO) shock

We studied the effects of human superoxide dismutase (h-SOD) in splanchnic artery occlusion (SAO) shock. Pentobarbital anesthetized rats subjected to total occlusion of the superior mesenteric and the celiac arteries for 40 min developed a severe shock state usually resulting in a fatal outcome within 20 min after the release of the occlusion. h-SOD (10 mg/kg) was infused intravenously starting at reperfusion and lasting for 10 min. SAO shock rats treated with h-SOD maintained postreperfusion MABP at significantly higher values compared to rats receiving the vehicle (final MABP 84 +/- 6 vs 46 +/- 1 mm Hg, P less than 0.01, respectively). Treatment with h-SOD attenuated the plasma accumulation of free amino-nitrogen compounds (P less than 0.01 from vehicle) as well as the activity of the lysosomal protease cathepsin D (P less than 0.05 from vehicle). Furthermore, the plasma activity of a myocardial depressant factor was significantly lower in h-SOD-treated rats than in SAO rats receiving only the vehicle (27 +/- 1 vs 64 +/- 3 U/ml, P less than 0.01). SAO shock rats treated with h-SOD also exhibited a significantly higher survival rate than the SAO shock +/- vehicle group (88% vs 11%, P less than 0.01, respectively). These results support the role of oxygen-derived radicals in the pathophysiology of SAO shock, and indicate that h-SOD effectively ameliorates the deleterious effects of oxygen radicals in this severe model of ischemia and reperfusion.     

Increased mitochondrial calcium coexists with decreased reperfusion injury in postconditioned (but not preconditioned) hearts

Ca(2+) is the main trigger for mitochondrial permeability transition pore opening, which plays a key role in cardiomyocyte death after ischemia-reperfusion. We investigated whether a reduced accumulation of mitochondrial Ca(2+) might explain the attenuation of lethal reperfusion injury by postconditioning. Anesthetized New Zealand White rabbits underwent 30 min of ischemia, followed by either 240 (infarct size protocol) or 60 (mitochondria protocol) min of reperfusion. They received either no intervention (control), preconditioning by 5-min ischemia and 5-min reperfusion, postconditioning by four cycles of 1-min reperfusion and 1-min ischemia at the onset of reflow, or pharmacological inhibition of the transition pore opening by N-methyl-4-isoleucine-cyclosporin (NIM811; 5 mg/kg iv) given at reperfusion. Area at risk and infarct size were assessed by blue dye injection and triphenyltetrazolium chloride staining. Mitochondria were isolated from the risk region for measurement of 1) Ca(2+) retention capacity (CRC), and 2) mitochondrial content of total (atomic absorption spectrometry) and ionized (potentiometric technique) calcium concentration. CRC averaged 0.73 +/- 0.16 in control vs. 4.23 +/- 0.17 mug Ca(2+)/mg proteins in shams (P < 0.05). Postconditioning, preconditioning, or NIM811 significantly increased CRC (P < 0.05 vs. control). In the control group, total and free mitochondrial calcium significantly increased to 2.39 +/- 0.43 and 0.61 +/- 0.10, respectively, vs. 1.42 +/- 0.09 and 0.16 +/- 0.01 mug Ca(2+)/mg in sham (P < 0.05). Surprisingly, whereas total and ionized mitochondrial Ca(2+) decreased in preconditioning, it significantly increased in postconditioning and NIM811 groups. These data suggest that retention of calcium within mitochondria may explain the decreased reperfusion injury in postconditioned (but not preconditioned) hearts.     

Muscle metaboreflex-induced increases in cardiac sympathetic activity vasoconstrict the coronary vasculature.

Ischemia of active skeletal muscle evokes a powerful blood pressure-raising reflex termed the muscle metaboreflex (MMR). MMR activation increases cardiac sympathetic nerve activity, which increases heart rate, ventricular contractility, and cardiac output (CO). However, despite the marked increase in ventricular work, no coronary vasodilation occurs. Using conscious, chronically instrumented dogs, we observed MMR-induced changes in arterial pressure, CO, left circumflex coronary blood flow (CBF), and coronary vascular conductance (CVC) before and after alpha1-receptor blockade (prazosin, 100 microg/kg iv). MMR was activated during mild treadmill exercise by partially reducing hindlimb blood flow. In control experiments, MMR activation caused a substantial pressor response-mediated via increases in CO. Although CBF increased (+28.1 +/- 3.7 ml/min; P < 0.05), CVC did not change (0.45 +/- 0.05 vs. 0.47 +/- 0.06 ml x min(-1) x mmHg(-1), exercise vs. exercise with MMR activation, respectively; P > 0.05). Thus all of the increase in CBF was due to the increase in arterial pressure. In contrast, after prazosin, MMR activation caused a greater increase in CBF (+55.9 +/- 17.1 ml/min; P < 0.05 vs. control) and CVC rose significantly (0.59 +/- 0.08 vs. 0.81 +/- 0.17 ml x min(-1) x mmHg(-1), exercise vs. exercise with MMR activation, respectively; P < 0.05). A greater increase in CO also occurred (+2.01 +/- 0.1 vs. +3.27 +/- 1.1 l/min, control vs. prazosin, respectively; P < 0.05). We conclude that the MMR-induced increases in sympathetic activity to the heart functionally restrain coronary vasodilation, which may limit increases in ventricular function.     

Hepatic portal venous delivery of a nitric oxide synthase inhibitor enhances net hepatic glucose uptake

Hepatic portal venous infusion of nitric oxide synthase (NOS) inhibitors causes muscle insulin resistance, but the effects on hepatic glucose disposition are unknown. Conscious dogs underwent a hyperinsulinemic (4-fold basal) hyperglycemic (hepatic glucose load 2-fold basal) clamp, with assessment of liver metabolism by arteriovenous difference methods. After 90 min (P1), dogs were divided into two groups: control (receiving intraportal saline infusion; n = 8) and LN [receiving N(G)-nitro-L-arginine methyl ester (L-NAME), a nonspecific NOS inhibitor; n = 11] intraportally at 0.3 mg x kg(-1) x min(-1) for 90 min (P2). During the final 60 min of study (P3), L-NAME was discontinued, and five LN dogs received the NO donor SIN-1 intraportally at 6 mug x kg(-1) x min(-1) while six received saline (LN/SIN-1 and LN/SAL, respectively). Net hepatic fractional glucose extraction (NHFE) in control dogs was 0.034 +/- 0.016, 0.039 +/- 0.015, and 0.056 +/- 0.019 during P1, P2, and P3, respectively. NHFE in LN was 0.045 +/- 0.009 and 0.111 +/- 0.007 during P1 and P2, respectively (P < 0.05 vs. control during P2), and 0.087 +/- 0.009 and 0.122 +/- 0.016 (P < 0.05) during P3 in LN/SIN-1 and LN/SAL, respectively. During P2, arterial glucose was 204 +/- 5 vs. 138 +/- 11 mg/dl (P < 0.05) in LN vs. control to compensate for L-NAME's effect on blood flow. Therefore, another group (LNlow; n = 4) was studied in the same manner as LN/SAL, except that arterial glucose was clamped at the same concentrations as in control. NHFE in LNlow was 0.052 +/- 0.008, 0.093 +/- 0.023, and 0.122 +/- 0.021 during P1, P2, and P3, respectively (P < 0.05 vs. control during P2 and P3), with no significant difference in glucose infusion rates. Thus, NOS inhibition enhanced NHFE, an effect partially reversed by SIN-1.     

Heat pretreatment differentially affects cardiac fatty acid accumulation during ischemia and postischemic reperfusion

We investigated whether the cardioprotection induced by heat stress (HS) pretreatment is associated with mitigation of phospholipid degradation during the ischemic and/or postischemic period. The hearts, isolated from control rats and from heat-pretreated rats (42 degrees C for 15 min) either 30 min (HS0.5-h) or 24 h (HS24-h) earlier, were subjected to 45 min of no-flow ischemia, followed by 45 min of reperfusion. Unesterified arachidonic acid (AA) accumulation was taken as a measure for phospholipid degradation. Significantly improved postischemic ventricular functional recovery was only found in the HS24-h group. During ischemia, AA accumulated comparably in control and both HS groups. During reperfusion in control and HS0.5-h hearts, AA further accumulated (control hearts from 82 +/- 33 to 109 +/- 51 nmol/g dry wt, not significant; HS-0.5h hearts from 52 +/- 22 to 120 +/- 53 nmol/g dry wt; P < 0.05). In contrast, AA was lower at the end of the reperfusion phase in HS24-h hearts than at the end of the preceding ischemic period (74 +/- 18 vs. 46 +/- 23 nmol/g dry wt; P < 0.05). Thus accelerated reperfusion-induced degradation of phospholipids in control hearts is completely absent in HS24-h hearts. Furthermore, the lack of functional improvement in HS0.5-h hearts is also associated with a lack of beneficial effect on lipid homeostasis. Therefore, it is proposed that enhanced membrane stability during reperfusion is a key mediator in the heat-induced cardioprotection.     

Ventricular-arterial coupling in a rat model of reduced arterial compliance provoked by hypervitaminosis D and nicotine

The vitamin D(3) and nicotine (VDN) model is one of isolated systolic hypertension (ISH) in which arterial calcification raises arterial stiffness and vascular impedance. The effects of VDN treatment on arterial and cardiac hemodynamics have been investigated; however, a complete analysis of ventricular-arterial interaction is lacking. Wistar rats were treated with VDN (VDN group, n = 9), and a control group (n = 10) was included without the VDN. At week 8, invasive indexes of cardiac function were obtained using a conductance catheter. Simultaneously, aortic pressure and flow were measured to derive vascular impedance and characterize ventricular-vascular interaction. VDN caused significant increases in systolic (138 +/- 6 vs. 116 +/- 13 mmHg, P < 0.01) and pulse (42 +/- 10 vs. 26 +/- 4 mmHg, P < 0.01) pressures with respect to control. Total arterial compliance decreased (0.12 +/- 0.08 vs. 0.21 +/- 0.04 ml/mmHg in control, P < 0.05), and pulse wave velocity increased significantly (8.8 +/- 2.5 vs. 5.1 +/- 2.0 m/s in control, P < 0.05). The arterial elastance and end-systolic elastance rose significantly in the VDN group (P < 0.05). Wave reflection was augmented in the VDN group, as reflected by the increase in the wave reflection coefficient (0.63 +/- 0.06 vs. 0.52 +/- 0.05 in control, P < 0.05) and the amplitude of the reflected pressure wave (13.3 +/- 3.1 vs. 8.4 +/- 1.0 mmHg in control, P < 0.05). We studied ventricular-arterial coupling in a VDN-induced rat model of reduced arterial compliance. The VDN treatment led to development of ISH and provoked alterations in cardiac function, arterial impedance, arterial function, and ventricular-arterial interaction, which in many aspects are similar to effects of an aged and stiffened arterial tree.     

Urocortin 1 administration from onset of rapid left ventricular pacing represses progression to overt heart failure

Urocortin 1 (Ucn1) may be involved in the pathophysiology of heart failure (HF), but the impact of Ucn1 administration on progression of the disease is unknown. The aim of this study was to investigate the effects of Ucn1 in sheep from the onset of cardiac overload and during the subsequent development of HF. Eight sheep underwent two 4-day periods of HF induction by rapid left ventricular pacing (225 beats/min) in conjunction with continuous infusions of Ucn1 (0.1 microg.kg(-1).h(-1) iv) and a vehicle control (0.9% saline). Compared with control, Ucn1 attenuated the pacing-induced decline in cardiac output (2.43 +/- 0.46 vs. 3.70 +/- 0.89 l/min on day 4, P < 0.01) and increases in left atrial pressure (24.9 +/- 1.0 vs. 11.9 +/- 1.1 mmHg, P < 0.001) and peripheral resistance (38.7 +/- 9.4 vs. 25.2 +/- 6.1 mmHg.l(-1).min, P < 0.001). Ucn1 wholly prevented increases in plasma renin activity (4.02 +/- 1.17 vs. 0.87 +/- 0.1 nmol.l(-1).h(-1), P < 0.001), aldosterone (1,313 +/- 324 vs. 413 +/- 174 pmol/l, P < 0.001), endothelin-1 (3.8 +/- 0.5 vs. 2.0 +/- 0.1 pmol/l, P < 0.001), and vasopressin (10.8 +/- 4.1 vs. 1.8 +/- 0.2 pmol/l, P < 0.05) during pacing alone and blunted the progressive increases in plasma epinephrine (2,132 +/- 697 vs. 1,250 +/- 264 pmol/l, P < 0.05), norepinephrine (3.61 +/- 0.73 vs. 2.07 +/- 0.52 nmol/l, P < 0.05), and atrial (P < 0.05) and brain (P < 0.01) natriuretic peptide levels. Ucn1 administration also maintained urine sodium excretion (0.75 +/- 0.34 vs. 1.59 +/- 0.50 mmol/h on day 4, P < 0.05) and suppressed pacing-induced declines in creatinine clearance (P < 0.05). These findings indicate that Ucn1 treatment from the onset of cardiac overload has the ability to repress the ensuing hemodynamic and renal deterioration and concomitant adverse neurohumoral activation, thereby delaying the development of overt HF. These data strongly support a use for Ucn1 as a therapeutic option early in the course of the disease.     

Mechanism of decreased adenosine protection in reperfusion injury of aging rats

The purpose of this study was to determine whether the protective effects of adenosine on myocardial ischemia-reperfusion injury are altered with age, and if so, to clarify the mechanisms that underlie this change related to nitric oxide (NO) derived from the vascular endothelium. Isolated perfused rat hearts were exposed to 30 min of ischemia and 60 min of reperfusion. In the adult hearts, administration of adenosine (5 micromol/l) stimulated NO release (1. 06 +/- 0.19 nmol. min(-1). g(-1), P < 0.01 vs. vehicle), increased coronary flow, improved cardiac functional recovery (left ventricular developed pressure 79 +/- 3.8 vs. 57 +/- 3.1 mmHg in vehicle, P < 0.001; maximal rate of left ventricular pressure development 2,385 +/- 103 vs. 1,780 +/- 96 in vehicle, P < 0.001), and reduced myocardial creatine kinase loss (95 +/- 3.9 vs. 159 +/- 4.6 U/100 mg protein, P < 0.01). In aged hearts, adenosine-stimulated NO release was markedly reduced (+0.42 +/- 0.12 nmol. min(-1). g(-1) vs. vehicle), and the cardioprotective effects of adenosine were also attenuated. Inhibition of NO production in the adult hearts significantly decreased the cardioprotective effects of adenosine, whereas supplementation of NO in the aged hearts significantly enhanced the cardioprotective effects of adenosine. The results show that the protective effects of adenosine on myocardial ischemia-reperfusion injury are markedly diminished in aged animals, and that the loss in NO release in response to adenosine may be at least partially responsible for this age-related alteration.     

Low-pressure reperfusion alters mitochondrial permeability transition

We hypothesized that low-pressure reperfusion may limit myocardial necrosis and attenuate postischemic contractile dysfunction by inhibiting mitochondrial permeability transition pore (mPTP) opening. Male Wistar rat hearts (n = 36) were perfused according to the Langendorff technique, exposed to 40 min of ischemia, and assigned to one of the following groups: 1) reperfusion with normal pressure (NP = 100 cmH(2)O) or 2) reperfusion with low pressure (LP = 70 cmH(2)O). Creatine kinase release and tetraphenyltetrazolium chloride staining were used to evaluate infarct size. Modifications of cardiac function were assessed by changes in coronary flow, heart rate (HR), left ventricular developed pressure (LVDP), the first derivate of the pressure curve (dP/dt), and the rate-pressure product (RPP = LVDP x HR). Mitochondria were isolated from the reperfused myocardium, and the Ca(2+)-induced mPTP opening was measured using a potentiometric approach. Lipid peroxidation was assessed by measuring malondialdehyde production. Infarct size was significantly reduced in the LP group, averaging 17 +/- 3 vs. 33 +/- 3% of the left ventricular weight in NP hearts. At the end of reperfusion, functional recovery was significantly improved in LP hearts, with RPP averaging 10,392 +/- 876 vs. 3,969 +/- 534 mmHg/min in NP hearts (P < 0.001). The Ca(2+) load required to induce mPTP opening averaged 232 +/- 10 and 128 +/- 16 microM in LP and NP hearts, respectively (P < 0.001). Myocardial malondialdehyde was significantly lower in LP than in NP hearts (P < 0.05). These results suggest that the protection afforded by low-pressure reperfusion involves an inhibition of the opening of the mPTP, possibly via reduction of reactive oxygen species production.