Low-flow Ischemia Research Articles

Adenosine enhances cytosolic phosphorylation potential and ventricular contractility in stunned guinea pig heart: receptor-mediated and metabolic protection

Mechanisms of adenosine (ADO) protection of reperfused myocardium are not fully understood. We tested the hypothesis that ADO (0.1 mM) alleviates ventricular stunning by ADO A(1)-receptor stimulation combined with purine metabolic enhancements. Langendorff guinea pig hearts were stunned at constant left ventricular end-diastolic pressure by low-flow ischemia. Myocardial phosphate metabolites were measured by (31)P-NMR, with phosphorylation potential {[ATP]/([ADP].[P(i)]), where brackets indicate concentration} estimated from creatine kinase equilibrium. Creatine and IMP, glycolytic intermediates, were measured enzymatically and glycolytic flux and extracellular spaces were measured by radiotracers. All treatment interventions started after a 10-min normoxic stabilization period. At 30 min reperfusion, ventricular contractility (dP/dt, left ventricular pressure) was reduced 17-26%, ventricular power (rate-pressure product) by 37%, and [ATP]/([ADP].[P(i)]) by 53%. The selective A(1) agonist 2-chloro-N(6)-cyclo-pentyladenosine marginally preserved [ATP]/([ADP].[P(i)]) and ventricular contractility but not rate-pressure product. Purine salvage precursor inosine (0.1 mM) substantially raised [ATP]/([ADP].[P(i)]) but weakly affected contractility. The ATP-sensitive potassium channel blocker glibenclamide (50 microM) abolished ADO protection of [ATP]/([ADP].[P(i)]) and contractility. ADO raised myocardial IMP and glucose-6-phosphate, demonstrating increased purine salvage and pentose phosphate pathway flux potential. Coronary hyperemia alone (papaverine) was not cardioprotective. We found that ADO protected energy metabolism and contractility in stunned myocardium more effectively than both the A(1)-receptor agonist 2-chloro-N(6)-cyclo-pentyladenosine and the purine salvage precursor inosine. Because ADO failed to stimulate glycolytic flux, the enhancement of reperfusion, [ATP]/([ADP].[P(i)]), indicates protection of mitochondrial function. Reduced ventricular dysfunction at enhanced [ATP]/([ADP].[P(i)]) argues against opening of mitochondrial ATP-sensitive potassium channel. The results establish a multifactorial mechanism of ADO antistunning, which appears to combine ADO A(1)-receptor signaling with metabolic adenylate and antioxidant enhancements.

Abnormal function and glucose metabolism in the type-2 diabetic db/db mouse heartThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute.

This study examined cardiac function and glucose metabolism in the 6-month-old db/db mouse, a model of type-2 diabetes. Cine magnetic resonance spectroscopy (MRI) was used to measure cardiac function in vivo. The db/db mice had decreased heart rates (17%, p<0.01) and stroke volumes (21%, p<0.05) that resulted in lower cardiac output (35%, p<0.01) than controls. Although there was no difference in ejection fraction between the 2 groups, db/db mouse hearts had a 35% lower maximum rate of ejection (p<0.01) than controls. In a protocol designed to assess maximal insulin-independent glucose uptake, hearts were isolated and perfused in Langendorff mode and subjected to 0.75 mL.min(-1).(g wet mass)(-1) low flow ischemia for 32 min. Glucose uptake during ischemia was 21% lower than in controls, and post-ischemic recovery of cardiac function was decreased by 30% in db/db mouse hearts (p<0.05). Total cardiac GLUT 4 protein was 56% lower (p<0.01) in db/db mice than in controls. In summary, the db/db mouse has abnormal left ventricular function in vivo, with impaired glucose uptake during ischemia, leading to increased myocardial damage.

β1-Adrenoreceptor activation contributes to ischemia-reperfusion damage as well as playing a role in ischemic preconditioning

Protein kinase A (PKA) activation has been implicated in early-phase ischemic preconditioning. We recently found that during ischemia PKA activation causes inactivation of cytochrome-c oxidase (CcO) and contributes to myocardial damage due to ischemia-reperfusion. It may be that beta-adrenergic stimulation during ischemia via endogenous catecholamine release activates PKA. Thus beta-adrenergic stimulation may mediate both myocardial protection and damage during ischemia. The present studies were designed to determine the role of the beta(1)-adrenergic receptor (beta(1)-AR) in myocardial ischemic damage and ischemic preconditioning. Langendorff-perfused rabbit hearts underwent 30-min ischemia by anterior coronary artery ligation followed by 2-h reperfusion. Occlusion-reperfusion damage was evaluated by delineating the nonperfused volume of myocardium at risk and volume of myocardial necrosis after 2-h reperfusion. In some hearts ischemic preconditioning was accomplished by two 5-min episodes of global low-flow ischemia separated by 10 min before coronary occlusion-reperfusion. Orthogonal electrocardiograms were recorded, and coronary flow was monitored by a drip count. Three hearts from each experimental group were used to determine mitochondrial CcO and aconitase activities. Two-hour reperfusion after occlusion caused an additional decrease in CcO activity vs. that after 30-min occlusion alone. Blocking the beta(1)-AR during occlusion-reperfusion reversed CcO activity depression and preserved myocardium at risk for necrosis. Similarly, mitochondrial aconitase activity exhibited a parallel response after occlusion-reperfusion as well as for the other interventions. Furthermore, classic ischemic preconditioning had no effect on CcO depression. However, blocking the beta(1)-AR during preconditioning eliminated the cardioprotection. If the beta(1)-AR was blocked after preconditioning, the myocardium was preserved. Interestingly, in both of the latter cases the depression in CcO activity was reversed. Thus the beta(1)-AR plays a dual role in myocardial ischemic damage. Our findings may lead to therapeutic strategies for preserving myocardium at risk for infarction, especially in coronary reperfusion intervention.

Glucose and insulin improve cardiac efficiency and postischemic functional recovery in perfused hearts from type 2 diabetic (db/db) mice

Hearts from type 2 diabetic (db/db) mice demonstrate altered substrate utilization with high rates of fatty acid oxidation, decreased functional recovery following ischemia, and reduced cardiac efficiency. Although db/db mice show overall insulin resistance in vivo, we recently reported that insulin induces a marked shift toward glucose oxidation in isolated perfused db/db hearts. We hypothesize that such a shift in metabolism should improve cardiac efficiency and consequently increase functional recovery following low-flow ischemia. Hearts from db/db and nondiabetic (db/+) mice were perfused with 0.7 mM palmitate plus either 5 mM glucose (G), 5 mM glucose and 300 microU/ml insulin (GI), or 33 mM glucose and 900 microU/ml insulin (HGHI). Substrate oxidation and postischemic recovery were only moderately affected by GI and HGHI in db/+ hearts. In contrast, GI and particularly HGHI markedly increased glucose oxidation and improved postischemic functional recovery in db/db hearts. Cardiac efficiency was significantly improved in db/db, but not in db/+ hearts, in the presence of HGHI. In conclusion, insulin and glucose normalize cardiac metabolism, restore efficiency, and improve postischemic recovery in type 2 diabetic mouse hearts. These findings may in part explain the beneficial effect of glucose-insulin-potassium therapy in diabetic patients with cardiac complications.

Glucosamine cardioprotection in perfused rat hearts associated with increasedO-linkedN-acetylglucosamine protein modification and altered p38 activation

We have shown that, in the perfused heart, glucosamine improved functional recovery following ischemia and that this appeared to be mediated via an increase in O-linked N-acetylglucosamine (O-GlcNAc) levels on nucleocytoplasmic proteins. Several kinase pathways, specifically Akt and the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2, which have been implicated in ischemic cardioprotection, have also been reported to be modified in response to increased O-GlcNAc levels. Therefore, the goals of this study were to determine the effect of ischemia on O-GlcNAc levels and to evaluate whether the cardioprotection resulting from glucosamine treatment could be attributed to changes in ERK1/2, Akt, and p38 phosphorylation. Isolated rat hearts were perfused with or without 5 mM glucosamine and were subjected to 5, 10, or 30 min of low-flow ischemia or 30 min of low-flow ischemia and 60 min of reperfusion. Glucosamine treatment attenuated ischemic contracture and improved functional recovery at the end of reperfusion. Glucosamine treatment increased flux through the hexosamine biosynthesis pathway and increased O-GlcNAc levels but had no effect on ATP levels. Glucosamine did not alter the response of either ERK1/2 or Akt to ischemia-reperfusion; however, it significantly attenuated the ischemia-induced increase in p38 phosphorylation and paradoxically increased p38 phosphorylation at the end of reperfusion. These data support the notion that O-GlcNAc may play an important role as an internal stress response and that glucosamine-induced cardioprotection may be mediated via the p38 MAPK pathway.

A Comparison between Ranolazine and CVT-4325, a Novel Inhibitor of Fatty Acid Oxidation, on Cardiac Metabolism and Left Ventricular Function in Rat Isolated Perfused Heart during Ischemia and Reperfusion

Inhibition of fatty acid oxidation has been reported to be cardioprotective against myocardial ischemic injury; however, recent studies have questioned whether the cardioprotection associated with putative fatty acid oxidation inhibitors, such as ranolazine and trimetazidine, are due to changes in substrate oxidation. Therefore, the goals of this study were to compare the effects of ranolazine with a new fatty acid oxidation inhibitor, CVT-4325 [(R)-1-(2-methylbenzo[d]thiazol-5-yloxy)-3-(4-((5-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)methyl)-piperazin-1-yl)propan-2-ol], on carbohydrate and fatty acid oxidation and on left ventricular (LV) function in the response to ischemia/reperfusion in rat isolated perfused hearts. Metabolic fluxes were determined in hearts perfused in an isovolumic Langendorff mode using 13C nuclear magnetic resonance isotopomer analysis or in isolated working hearts using [14C]glucose and [3H]palmitate, with and without 10 microM ranolazine or 3 microM CVT-4325. Isovolumic perfused hearts were also subjected to 30 min of low-flow ischemia (0.3 ml/min) and 60 min of reperfusion, and working hearts were subjected to 15 min of zero-flow ischemia and 60 min of reperfusion. Regardless of the experimental protocol, ranolazine had no effect on carbohydrate or fatty acid oxidation, whereas CVT-4325 significantly reduced fatty acid oxidation up to approximately 7-fold with a concomitant increase in carbohydrate oxidation. At these same concentrations, although ranolazine significantly improved LV functional recovery following ischemia/reperfusion, CVT-4325 had no significant protective effect. These results demonstrate that at pharmacologically relevant concentrations, ischemic protection by ranolazine was not mediated by inhibition of fatty acid oxidation and conversely that inhibition of fatty acid oxidation with CVT-4325 was not associated with improved LV functional recovery.

Rosiglitazone treatment improves cardiac efficiency in hearts from diabetic mice

Isolated perfused hearts from type 2 diabetic (db/db) mice show impaired ventricular function, as well as altered cardiac metabolism. Assessment of the relationship between myocardial oxygen consumption (MVO2) and ventricular pressure-volume area (PVA) has also demonstrated reduced cardiac efficiency in db/db hearts. We hypothesized that lowering the plasma fatty acid supply and subsequent normalization of altered cardiac metabolism by chronic treatment with a peroxisome proliferator-activated receptor-γ (PPARγ) agonist will improve cardiac efficiency in db/db hearts. Rosiglitazone (23 mg/kg body weight/day) was administered as a food admixture to db/db mice for five weeks. Ventricular function and PVA were assessed using a miniaturized (1.4 Fr) pressure-volume catheter; MVO2 was measured using a fibre-optic oxygen sensor. Chronic rosiglitazone treatment of db/db mice normalized plasma glucose and lipid concentrations, restored rates of cardiac glucose and fatty acid oxidation, and improved cardiac efficiency. The improved cardiac efficiency was due to a significant decrease in unloaded MVO2, while contractile efficiency was unchanged. Rosiglitazone treatment also improved functional recovery after low-flow ischemia. In conclusion, the present study demonstrates that in vivo PPARγ-treatment restores cardiac efficiency and improves ventricular function in perfused hearts from type 2 diabetic mice.

CARDIAC ISCHEMIA‐REPERFUSION DAMAGE IN PARTIAL HEXOKINASE (HK) II KNOCKOUT MICE

Overexpression of HKI and HKII in cultured cells protects against oxidant-induced cell death. It is unknown whether cardiac alterations of HKII affect ischemia-reperfusion injury in the whole heart. In the present study we examined ischemia-reperfusion injury in isolated hearts of partial HKII (+/−) knockout (HKKO) and wild-type mice (WT). Langendorff-perfused hearts (HKKO: n=7; WT: n=8) were subjected to 40 min low-flow (5% of baseline flow) ischemia and 120 min reperfusion. Necrosis was determined by lactate dehydrogenase (LDH) enzyme release during the reperfusion period. At baseline, no significant differences were observed between groups: end-diastolic pressure (EDP) is 7.5 ± 1.0 mm Hg, developed left ventricular pressure (DLVP) is 98.7 ± 4.6 mm Hg, heart rate is 369 ± 12 beats/min and flow is 15.1 ± 1.4 ml/min/gww. Following 120 min reperfusion, recovery of DLVP was 68.3 ± 8.3 % (WT) versus 55.6 ± 9.1% (HKKO) and EDP was 11.6 ± 3.7 mm Hg (WT) versus 29.0 ± 6.8 mm Hg (HKKO). Between groups contracture development during ischemia reached EDP >10 mm Hg at different time points: 34 min for WT and 12 min for HKKO. LDH release was 1.8 ± 0.35 versus 3.3 ± 0.75 μmol/gww/120 min reperfusion for WT versus HKKO, respectively. The data suggest for the first time that HKII may be an important mediator of ischemia-reperfusion damage in the intact heart.

Low carbohydrate diet reduces myocardial glycogen stores and increases myocardial injury following ischemia‐reperfusion

Low carbohydrate diets (LCDs) are popular for weight loss, but the safety of such diets is unknown. Myocardial glycogen may be an important energy source during ischemia, when circulating energy substrate and O2 supplies are reduced. We hypothesized that LCD lowers cardiac glycogen and increases cardiac injury during ischemia and reperfusion. Rats were fed LCD (60% kcal from fat/30% protein/10% carbohydrate) or a low fat diet (LFD; 16%/19%/65%) for 2 weeks (N=18 each diet). 6 from each group were sacrificed for biochemical analysis, 6 were subjected to 30 min of low flow ischemia (LFI; 0.3ml/min), and 6 to a 60 min reperfusion protocol following 30 min of LFI after hearts were isolated and perfused with a physiologic perfusion mixture. LCD led to significant increase of circulating free fatty acids, 3-hydroxybutyrate and decrease in insulin, and a decrease in myocardial and liver glycogen (Fig B). LCD resulted in impaired left ventricular performance during LFI, with worsened ischemic contracture and increased total lactate dehydrogenase (LDH) release (Fig. A). Cardiac glycogen was lower after LFI (0.42±0.04 and 0.44±0.02 mg/g tissue; P < 0.05 vs. pre-LFI). These results demonstrate that LCD lowers cardiac glycogen and increases myocardial injury following ischemia-reperfusion, and suggest that glycogen stores play a critical role in prevention of ischemic injury. Figure A and BOpen in figure viewerPowerPoint A: Total LDH release during LFI and reperfusion. B: Glycogen content in myocardium and liver (mg glucose/g wet tissue weight). ∗ P<0.05 vs. LFD.

Adenosine provides contractile support in the low‐flow ischemic heart

Low-flow ischemia decreases heart contractile performance. Adenosine A2A receptor (A2AR) activation elicits an increased contractile performance in normoxic hearts. The myocardial level of adenosine increases when the supply of oxygen is restricted. The effect of adenosine to increase contractile performance (+dP/dtmax) was investigated in the low-flow ischemic (LFI) heart and compared to the adenosine effect in the non-ischemic fully oxygenated (NFO) heart. Perfused rat hearts were rendered LFI by reducing perfusion from 18 to 6 ml/min and paced to contract at 320 beats/min. The maximum rate of left ventricular pressure development (+dP/dtmax) was used as an index of contractile performance. Adenosine (10 μM) increased +dP/dtmax with LFI and NFO hearts by 3.2 ± 1.6% and 9.6 ± 2.1%, respectively. However, adenosine in the presence of the adenosine A1 receptor (A1R) antagonist DPCPX (0.1 μM; dipropylcyclopentylxanthine) caused a greater increase in +dP/dtmax in LFI (26.6 ± 4.1%) compared to NFO (15.8 ± 3.3%) hearts. This is a significant improvement in contractile function in the LFI heart. The A2AR antagonist ZM-241385 (0.1 μM) prevented the increase in +dP/dtmax caused by adenosine in the presence of DPCPX in both LFI and NFO hearts. The results suggest that A2AR stimulation in the low-flow ischemic heart provides important inotropic support in the absence of A1R activation. (PHS Grants: HL-66045 & AG-11481)

Bradykinin is a Mediator, but Unlikely a Trigger, of Antiarrhythmic Effects of Ischemic Preconditioning

Brief reversible ischemic episodes (ischemic preconditioning, IPC) protect the heart against arrhythmias during a subsequent prolonged low-flow ischemia. We have recently shown that this protection involves release of bradykinin, activation of bradykinin B2 receptors followed by opening of sarcolemmal, but not mitochondrial ATP-sensitive K+ channels. The goal of this study was to clarify a trigger and/or mediator role of bradykinin in the antiarrhythmic effects of IPC during low-flow ischemia. Isolated perfused rat hearts underwent 60 minutes of low-flow ischemia induced by reducing perfusion pressure followed by 60 minutes of reperfusion. Preconditioning was induced by 2 x 5 minutes episodes of zero-flow ischemia. In yet other groups, preconditioned or non-preconditioned hearts were treated either with bradykinin (10 nmol/L) or with HOE 140 (bradykinin B2 receptor antagonist, 100 nmol/L). IPC reduced the number of ventricular premature beats, as well as the incidence of ventricular tachycardia and of ventricular fibrillation during low-flow ischemia. In addition, this protection was abolished by HOE 140 given during low-flow ischemia. Pharmacological preconditioning using short bradykinin perfusion instead of IPC did not show antiarrhythmic effects. However, bradykinin administered during low-flow ischemia and reperfusion reduced the number of ventricular premature beats and the incidence of ventricular tachycardia and of ventricular fibrillation during low-flow ischemia. Bradykinin is a mediator, but unlikely a trigger, of antiarrhythmic effects of IPC during low-flow ischemia.

Inhibition of MMP-2 activation and release as a novel mechanism for HDL-induced cardioprotection

High density lipoproteins (HDL) protect the heart against ischemia/reperfusion (I/R) injury, and matrix metalloproteinase-2 (MMP-2) directly contributes to cardiac contractile dysfunction after I/R. To investigate the possible involvement of MMP-2 inhibition in HDL-mediated cardioprotection, isolated rat hearts underwent 20 min of low-flow ischemia and 30 min of reperfusion. Plasma-derived and synthetic HDL attenuated the I/R-induced cardiac MMP-2 activation and release in a dose-dependent way. The attenuation of I/R-induced MMP-2 activation by HDL correlated with the reduction of post-ischemic contractile dysfunction and cardiomyocyte necrosis. These results indicate prevention of MMP-2 activation as a novel mechanism for HDL-mediated cardioprotection.

Prevention of the ischemia-induced decrease in mitochondrial Tom20 content by ischemic preconditioning

Preserved mitochondrial function (respiration, calcium handling) and integrity (cytochrome c release) is central for cell survival following ischemia/reperfusion. Mitochondrial function also requires import of proteins from the cytosol via the translocase of the outer and inner membrane (TOM and TIM complexes). Since mitochondrial function following ischemia/reperfusion is better preserved by ischemic preconditioning (IP), we now investigated whether expression of parts of the import machinery is affected by ischemia/reperfusion without or with IP in vivo. We analyzed the mitochondrial content of the presequence receptor Tom20, the pore forming unit Tom40 and Tim23. Goettinger minipigs were subjected to 90 min of low-flow ischemia without or with preconditioning by 10 min ischemia and 15 min reperfusion. Mitochondria were isolated from the ischemic or preconditioned anterior wall of the left ventricle and from the control posterior wall. Infarct size was significantly reduced by IP (20.1 ± 1.6% of area at risk (non-preconditioned) vs. 6.5 ± 2.5% of area at risk (IP)). Using Western blot analysis, the ratio of Tom20 (normalized to Ponceau S) between mitochondria isolated from the anterior ischemic and posterior control wall was reduced (0.72 ± 0.11, a.u., n = 8), whereas the mitochondrial Tom20 content was preserved by IP (1.17 ± 0.16 a.u., n = 7, P < 0.05). The mitochondrial Tom40, Tim23 and adenine nucleotide transporter (ANT) contents were not significantly different between non-preconditioned and preconditioned myocardium. The preservation of the mitochondrial Tom20 protein level may contribute to the improved mitochondrial function after IP.

A New Insight Into the Pathogenesis of Coronary Vasospasm

See related article, pages 682–689 To elucidate the control mechanisms of coronary vascular tone is one of the central interests in cardiac pathophysiology, because ischemic heart disease is one of the major causes of death in many countries. Although the coronary vascular tone is finally determined by the contractile state of smooth muscle, exo-smooth muscle mechanisms including autonomic nerves, endothelial cells, and blood cells have been shown indispensable for the control. Particular types of receptors, ion channels, and intracellular signal-cascades exist in coronary smooth muscle cells and mediate the controls by exo-smooth muscle elements (Figure). For example, stimulation of α1-adrenergic receptor, which is physiologically achieved by noradrenaline (NA) released from sympathetic presynapses, increases the intracellular Ca2+ (Ca2+i) via phosphatidylinositol (PI)-turnover and also probably via opening of receptor-activated Ca2+-permeable TRP channel. Membrane depolarization does so via opening voltage-dependent Ca2+ channels. The augmentation of Ca2+i results in contraction of the smooth muscle cells and thus constriction of the coronary artery.1 Activation of β2-adrenergic receptor, in turn, inhibits the coronary smooth muscle contraction through protein-kinase A pathway.2 The contraction is also suppressed by protein-kinase G activated by a dilating factor, nitric oxide (NO), which is produced in the endothelial cells. The mechanism for control of vascular tone . Activation of voltage-gated Ca2+ channel at the presynapse of sympathetic nerve increases intracellular Ca2+, which triggers a release of norepinephrine (NE). NE stimulates adrenergic α1 receptor (α1R) in the smooth muscle cells and drives PI-turnover as described in this scheme. The intracellular Ca2+, which flows into the cells via smooth-muscle’s Ca2+ channel or moves from endoplasmic reticulum (ER), contracts the cells. The constriction of smooth muscle cells is prevented by activation …

Decrease in fatty acid oxidation increases tolerance of the ageing heart to ischaemic injury

Ageing is associated with reduced tolerance to myocardial infarction. Alterations in mitochondrial oxidative metabolism are important determinants in the contractile recovery of the adult heart after ischaemia. However, the profile of substrate metabolism during reperfusion in the ageing heart is unknown. The aim of this study was to investigate energy substrate preference and functional recovery following low flow ischaemia in the ageing heart. Hearts from 6 and 24 mo male Wistar rats were perfused in an isovolumic mode with buffer containing 3% albumin and a physiological mixture of substrates including 0.3mM [U-13C] palmitate, 1mM [3-13C] lactate and 5mM glucose. Cardiac work was increased by dobutamine infusion for 20 min (2.8±0.1 μg/min/g heart weight) followed by 30 min low flow ischaemia and 30 min reperfusion. Ischaemia caused similar reductions in left ventricular developed pressure and cardiac work in adult and senescent hearts. On reperfusion, 24 mo hearts demonstrated improved functional recovery relative to 6-mo hearts. This was associated with a significant decline in palmitate oxidation and increase in lactate oxidation. Thus, the ageing heart has increased tolerance to severe ischaemia, which may be attributed to the reduction in fatty acid oxidation. JS is funded by a PhD scholarship from the University of Hull and Academic Dept of Cardiology.

Direct Effects of Glucagon-Like Peptide-1 on Myocardial Contractility and Glucose Uptake in Normal and Postischemic Isolated Rat Hearts

Recent evidence suggests that glucagon-like peptide-1 (GLP-1) enhances recovery of left ventricular (LV) function after transient coronary artery occlusion. However, it is uncertain whether GLP-1 has direct effects on normal or ischemic myocardium and whether the mechanism involves increased myocardial glucose uptake. LV function and myocardial glucose uptake and lactate production were measured under basal conditions and after 30 min of low-flow ischemia and 30 min of reperfusion in the presence and absence of GLP-1-(7-36) amide. The response was compared with standard buffer alone or buffer containing insulin (100 microU/ml). GLP-1 decreased the left ventricular developed pressure (baseline: 100 +/- 2 mm Hg; GLP-1: 75 +/- 3 mm Hg, p < 0.05) and LV dP/dt (baseline: 4876 +/- 65 mm Hg/s; GLP-1: 4353 +/- 76 mm Hg/s, p < 0.05) in normal hearts. GLP-1 increased myocardial glucose uptake (baseline: 33 +/- 3 micromol/min/g; GLP-1: 81 +/- 7 micromol/min/g, p < 0.05) by increasing nitric oxide production and glucose transporter (GLUT)-1 translocation. GLP-1 enhanced recovery after 30 min of low-flow ischemia with significant improvements in LV end-diastolic pressure (control: 13 +/- 4 mm Hg; GLP-1: 3 +/- 2 mm Hg, p < 0.05) and LV developed pressure (control: 66 +/- 6 mm Hg; GLP-1: 98 +/- 5 mm Hg, p < 0.05). GLP-1 increased LV function, myocardial glucose uptake, and GLUT-1 and GLUT-4 translocation during reperfusion to an extent similar to that with insulin. GLP-1 has direct effects on the normal heart, reducing contractility, but increasing myocardial glucose uptake through a non-Akt-1-dependent mechanism, distinct from the actions of insulin. However, GLP-1 increased myocardial glucose uptake and enhanced recovery of cardiac function after low-flow ischemia in a fashion similar to that of insulin.

Inhibiting Effect of Amiodarone to Protective Action against Ischemia/Reperfusion Injury Induced by Ischemic Preconditioning

Background: Ischemic preconditioning (IPC) has protective effects on myocardial ischemia. These effects may be caused by ATP (adenosine triphosphate) sensitive potassium () channel activation. Amiodarone also has pharmacologic preconditioning effects, especially suppression of reperfusion arrhythmia. The mechanisms of amiodarone effects are different from that of IPC. The aim of this study using the isolated rat working heart was 1) to confirm whether IPC and amiodarone protected against reperfusion injury; and, if so, 2) to example whether combination of IPC and amiodarone demonstrated additive effect or not. Methods: Isolated rat hearts were stabilized for 30 minutes and were subdivided into four groups. Control group was subjected low-flow ischemia of 5% dextrose water for 30 minutes. IPC group was pretreated with IPC. In Amiodarone group, whole process was same as control group except amiodarone administration during low-flow ischemia. Amiodarone-IPC group, IPC was applied and also administrated amiodarone. Isovolumetric left ventricular end systolic pressure (LVESP), left ventricular end diastolic pressure (LVEDP), dP/ and heart rate were measured. Results: Group II showed more recovered LVESP, less developed ventricular stiffness, more preserved coronary effluent flow, earlier termination of arrhythmia and smaller infarct size than Group I. Group III showed preserved LVESP and dP/, less developed ventricular stiffness, shortest arrhythmia sustaining time, smallest infarct size among all groups. These myocardial protective effects were abolished in Group IV. Conclusions: We observed that IPC and amiodarone administration, each has myocardial protective effects against myocardial ischemia, but when they are dealt at the same time, their beneficial effects diminished.

A Newly Synthesised Molybdenum/Ascorbic Acid Complex Alleviates Some Effects of Cardiomyopathy in Streptozocin-Induced Diabetic Rats

Exogenous insulin does not prevent cardiac failure in patients with type 1 diabetes mellitus and a cardioprotective insulin mimic is greatly needed. Certain transition metals are known to act as insulin mimics and may be cardio- protective. In this study, the ability of a newly synthesised molybdenum/ascorbic acid complex to strengthen cardiac function was investigated. Male CD rats were assigned to one of five groups: non-diabetic control, non-diabetic control treated with molybdenum/ascorbic acid complex, diabetic treated with sodium ascorbate, diabetic treated with molybdenum/ascorbic acid complex and untreated diabetics. Type 1 diabetes was induced by streptozocin injection. Once diabetes was confirmed, treatment was initiated by adding either the molybdenum/ascorbic acid complex or sodium ascorbate to the drinking water and continued for 6 weeks. Following the treatment period, the animals were terminated, and their hearts were excised and mounted in a working heart perfusion apparatus. Blood samples were taken for plasma glucose and plasma lipid level determination. Cardiac function was evaluated using 1 hour of low-flow ischaemic stress followed by 30 minutes of reperfusion. Hearts from the animals treated with the molybdenum/ascorbic acid complex displayed the best aerobic performance of all the diabetic animals. Blood glucose levels and blood lipid levels were significantly lower in animals treated with the complex than in other diabetic animals. The group treated with the complex also had a lower drinking rate than the other diabetic groups. Furthermore, hearts from animals treated with the molybdenum/ascorbic acid complex showed a greater degree of recovery from low-flow ischaemia than any other group. The molybdenum/ascorbic acid complex showed some significant insulin-mimic and cardioprotective effects. Further development of this complex could provide a drug useful for alleviating some of the cardiovascular problems associated with diabetes mellitus.

Changes in First-Pass Interstitial Kinetics of DTPA in Myocardium Submitted to Low-Flow Ischemia

This study aimed to determine the changes during ischemia in the myocardial first-pass kinetics of DTPA, an extracellular tracer that is currently used for assessing myocardial perfusion with magnetic resonance imaging (Magnevist). Using an indicator-dilution technique, first-pass kinetics of DTPA were compared between normoxia (n=11) and low-flow ischemia (n=11) in isolated rabbit hearts perfused with red blood cell-enhanced perfusate. There was no difference between ischemia and normoxia in the interstitial extraction and clearance rates of DTPA. Interstitial distribution volume of DTPA was, however, lower in ischemia than in normoxia (in percent of myocardial volume: 15+/-11% vs 25+/-11%, P=0.02) as a result of a relationship with coronary flow (P<0.001). During low-flow myocardial ischemia, DTPA kinetics are unchanged, except for the interstitial distribution volume that is decreased, presumably because of the shrinkage of extracellular fluid. These kinetic properties are favorable for detecting myocardial ischemia at rest with magnetic resonance imaging.

Role of Hydrogen Sulfide in the Cardioprotection Caused by Ischemic Preconditioning in the Rat Heart and Cardiac Myocytes

Endogenous H(2)S is synthesized mainly by cystathionine gamma-lyase in the heart. The present study investigated the role of H(2)S in cardioprotection induced by ischemic preconditioning. We have examined the effect of endogenous H(2)S and exogenous application of NaHS (H(2)S donor) on cardiac rhythm in the isolated rat heart subjected to low-flow ischemia insults as well as cell viability and function in isolated myocytes exposed to simulated ischemia solution. Preconditioning with NaHS (SP) or ischemia (IP) for three cycles (3 min each cycle separated by 5 min of recovery) significantly decreased the duration and severity of ischemia/reperfusion-induced arrhythmias in the isolated heart while increasing cell viability and the amplitude of electrically induced calcium transients after ischemia/reperfusion in cardiac myocytes. Both IP and SP also significantly attenuated the decreased H(2)S production during ischemia. Moreover, decreasing endogenous H(2)S production significantly attenuated the protective effect of IP in both the isolated heart and isolated cardiac myocytes. Blockade of protein kinase C with chelerythrine or bisindolylmaleimide I as well as ATP-sensitive K(+) (K(ATP)) channel with glibenclamide (a nonselective K(ATP) blocker) and HMR-1098 (1-[[5-[2-(5-Chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea) (a sarcolemmal K(ATP) channel blocker) reversed the cardioprotection induced by SP or IP. However, blockade of mitochondrial K(ATP) channels with 5-hydroxydecanoic acid had no effect on the cardioprotection of SP, suggesting that, unlike the mechanism involved in IP, mitochondrial K(ATP) channels most probably do not play a major role in the cardioprotection of SP. Our findings suggest that endogenous H(2)S contributes to cardioprotection induced by IP, which effect may involve protein kinase C and sarcolemmal K(ATP) channels.