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Histamine has a positive inotropic effect on ventricular myocardium and stimulation of histamine H2 receptors increases the intracellular cAMP level via Gs protein, as dose stimulation of β-adrenergic receptors, and worsens heart failure. To test whether a histamine H2 receptor blocker had a beneficial effect in addition to β-adrenergic receptor blockade, we investigated the cardioprotective effect of famotidine, a histamine H2 receptor blocker, in dogs receiving a β-blocker. We induced heart failure in dogs by rapid ventricular pacing (230 beats/min). Animals received no drugs (control group), famotidine (1 mg/kg daily), carvedilol (0.1 mg/kg daily), or carvedilol plus famotidine. Both cardiac catheterization and echocardiography were performed before and 4 weeks after the initiation of pacing. Immunohistochemical studies showed the appearance of mast cells and histamine in the myocardium after 4 weeks of pacing. In the control group, the left ventricular ejection fraction (LVEF) was decreased after 4 weeks compared with before pacing (71 ± 2 vs. 27 ± 2%, p

Cell-free hemoglobin (Hb) derivatives that have been developed as Hb-based artificial oxygen carrier cause both coronary vasoconstriction and platelet aggregation due to the scavenging actions of nitric oxide (NO). Recently, native Hb is found to undergo S-nitrosylation, which regulates blood flow, whereas artificial oxygen carriers are lacking of S-nitrosylation. Therefore, S-nitrosylated and pegylated hemoglobin (SNO-PEG-Hb) was prepared to overcome the above defects, where pegylation was included to avoid extravasation and to prolong the circulatory half-live. Since SNO-PEG-Hb possesses SNO property, we tested whether SNO-PEG-Hb increases coronary blood flow (CBF) and improves the severity of myocardial ischemia. In 19 open chest dogs, the left anterior descending coronary artery was perfused with blood from the carotid artery via the bypass tube, and then CBF and coronary perfusion pressure (CPP) were measured. After hemodynamic stabilization, CPP was reduced so that CBF decreased to 33% of the baseline and thereafter CPP was maintained constant. Ten minutes after the onset of coronary hypoperfusion, we infused 10% SNO-PEG-Hb into the coronary artery (2.5 ml/min). SNO-PEG-Hb increased CBF (28.1+/-3.3 to 43.3+/-3.9 ml/100 g/min, p

We have previously reported that the prolonged transient acidosis during early reperfusion mediates the cardioprotective effects in canine hearts. Recently, postconditioning has been shown to be one of the novel strategies to mediate cardioprotection. We tested the contribution of the prolonged transient acidosis to the cardioprotection of postconditioning. Open-chest anesthetized dogs subjected to 90-min occlusion of the left anterior descending coronary artery and 6-h reperfusion were divided into four groups: 1) control group; no intervention after reperfusion ( n = 6); 2) postconditioning (Postcon) group; four cycles of 1-min reperfusion and 1-min reocclusion ( n = 7); 3) Postcon + sodium bicarbonate (NaHCO3) group; four cycles of 1-min reperfusion and 1-min reocclusion with the administration of NaHCO3( n = 8); and 4) NaHCO3group; administration of NaHCO3without postconditioning ( n = 6). Infarct size, the area at risk (AAR), collateral blood flow during ischemia, and pH in coronary venous blood were measured. The phosphorylation of Akt and extracellular signal-regulated kinase (ERK) in ischemic myocardium was assessed by Western blot analysis. Systemic hemodynamic parameters, AAR, and collateral blood flow were not different among the four groups. Postconditioning induced prolonged transient acidosis during the early reperfusion phase. Administration of NaHCO3completely abolished the infarct size-limiting effects of postconditioning. Furthermore, the phosphorylation of Akt and ERK in ischemic myocardium induced by postconditioning was also blunted by the cotreatment of NaHCO3. In conclusion, postconditioning mediates its cardioprotective effects possibly via prolonged transient acidosis during the early reperfusion phase with the activation of Akt and ERK.

Objectives We investigated the effects of erythropoietin (EPO) on neovascularization and cardiac function after myocardial infarction (MI). Background Erythropoietin exerts antiapoptotic effects and mobilizes endothelial progenitor cells (EPCs). Methods We intravenously administered EPO (1,000 IU/kg) immediately [EPO(0) group], 6 h [EPO(6h) group], or 1 week [EPO(1wk) group] after the permanent ligation of the coronary artery in dogs. Control animals received saline immediately after the ligation. Results The infarct size 6 h after MI was significantly smaller in the EPO(0) group than in the control group (61.5 ± 6.0% vs. 22.9 ± 2.2%). One week after MI, the circulating CD34-positive mononuclear cell numbers in both the EPO(0) and the EPO(6h) groups were significantly higher than in the control group. In the ischemic region, the capillary density and myocardial blood flow 4 weeks after MI was significantly higher in both the EPO(0) and the EPO(6h) groups than in the control group. Four weeks after MI, left ventricular (LV) ejection fraction in the EPO(6h) (48.6 ± 1.9%) group was significantly higher than that in either the control (41.9 ± 0.9%) or the EPO(1wk) (42.6 ± 1.2%) group but significantly lower than that in the EPO(0) group (56.1 ± 2.3%). The LV end-diastolic pressure 4 weeks after MI in both the EPO(0) and the EPO(6h) groups was significantly lower than either the control or the EPO(1wk) group. Hematologic parameters did not differ among the groups. Conclusions In addition to its acute infarct size-limiting effect, EPO enhances neovascularization, likely via EPC mobilization, and improves cardiac dysfunction in the chronic phase, although it has time-window limitations.

The prevalence of atrial fibrillation (AF) increases in patients with hypertension. Angiotensin II is involved in structural atrial remodeling, which contributes to the onset and maintenance of AF in paced animal models. We investigated the role of angiotensin II in atrial structural remodeling in rats with hypertension. Ten-week-old male Wistar-Kyoto rats were randomly divided into 4 groups: a control group (no treatment), an Nomega-nitro-L-arginine methyl ester (L-NAME) group (administered L-NAME, an inhibitor of nitric oxide synthase, 1 g/l in drinking water), an L-NAME+candesartan group (L-NAME plus candesartan-an angiotensin II receptor blocker (ARB)-at 0.1 mg/kg/day), and an L-NAME + hydralazine group (L-NAME plus hydralazine at 120 mg/l in drinking water). Eight weeks after treatment, the L-NAME group showed significantly higher systolic blood pressure than the control group (197 +/- 12 vs.138 +/- 5 mmHg, p < 0.05). Candesartan or hydralazine with L-NAME reduced systolic blood pressure to baseline. Chronic inhibition of NO synthesis increased the extent of fibrosis and transforming growth factor-beta expression in atrial tissue, and both of these effects were prevented by candesartan, but not by hydralazine. Cardiac hypertrophy and dysfunction were induced in the L-NAME group, and these effects were also prevented by candesartan, but not by hydralazine. In contrast, the decrease in thrombomodulin expression in the atrial endocardium in hypertensive rats was restored by candesartan and hydralazine. The ARB prevented atrial structural remodeling, a possible contributing factor for the development of AF, in the hearts of rats with hypertension induced by long-term inhibition of NO synthesis.

Background— Some studies have shown that metformin activates AMP-activated protein kinase (AMPK) and has a potent cardioprotective effect against ischemia/reperfusion injury. Because AMPK also is activated in animal models of heart failure, we investigated whether metformin decreases cardiomyocyte apoptosis and attenuates the progression of heart failure in dogs. Methods and Results— Treatment with metformin (10 μmol/L) protected cultured cardiomyocytes from cell death during exposure to H 2 O 2 (50 μmol/L) via AMPK activation, as shown by the MTT assay, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling staining, and flow cytometry. Continuous rapid ventricular pacing (230 bpm for 4 weeks) caused typical heart failure in dogs. Both left ventricular fractional shortening and left ventricular end-diastolic pressure were significantly improved in dogs treated with oral metformin at 100 mg · kg −1 · d −1 (n=8) (18.6±1.8% and 11.8±1.1 mm Hg, respectively) compared with dogs receiving vehicle (n=8) (9.6±0.7% and 22±0.9 mm Hg, respectively). Metformin also promoted phosphorylation of both AMPK and endothelial nitric oxide synthase, increased plasma nitric oxide levels, and improved insulin resistance. As a result of these effects, metformin decreased apoptosis and improved cardiac function in failing canine hearts. Interestingly, another AMPK activator (AICAR) had effects equivalent to those of metformin, suggesting the primary role of AMPK activation in reducing apoptosis and preventing heart failure. Conclusions— Metformin attenuated oxidative stress–induced cardiomyocyte apoptosis and prevented the progression of heart failure in dogs, along with activation of AMPK. Therefore, metformin may be a potential new therapy for heart failure.

Marked sarcomere disorganization is a well-documented characteristic of cardiomyocytes in the failing human myocardium. Myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v), which is involved in the development of human cardiomyopathy, is an important structural protein that affects physiologic cardiac sarcomere formation and heart development. Integrated cDNA expression analysis of failing human myocardia uncovered a novel protein kinase, cardiac-specific myosin light chain kinase (cardiac-MLCK), which acts on MLC2v. Expression levels of cardiac-MLCK were well correlated with the pulmonary arterial pressure of patients with heart failure. In cultured cardiomyocytes, knockdown of cardiac-MLCK by specific siRNAs decreased MLC2v phosphorylation and impaired epinephrine-induced activation of sarcomere reassembly. To further clarify the physiologic roles of cardiac-MLCK in vivo, we cloned the zebrafish ortholog z–cardiac-MLCK. Knockdown of z–cardiac-MLCK expression using morpholino antisense oligonucleotides resulted in dilated cardiac ventricles and immature sarcomere structures. These results suggest a significant role for cardiac-MLCK in cardiogenesis.

Background— Adenosine inhibits proliferation of cardiac fibroblasts and hypertrophy of cardiomyocytes, both of which may play crucial roles in cardiac remodeling. In the present study, we investigated whether chronic stimulation of adenosine receptors begun after myocardial infarction (MI) prevents cardiac remodeling. Methods and Results— MI was produced in Wistar rats by permanent ligation of the left anterior descending coronary artery. One week after the onset of MI, animals were randomized into 8 groups: vehicle, dipyridamole (DIP; the adenosine uptake inhibitor, 50 mg/kg), 2-chroloadenosine (CADO; the stable analogue of adenosine, 2 mg/kg), and CADO in the presence of the nonselective adenosine receptor antagonist 8-sulfophenyltheophylline (8-SPT) or the selective antagonist for adenosine A1, A2a, A2b, or A3 receptor. Three weeks after treatment, hemodynamic and echocardiographic parameters in the DIP and CADO groups were significantly improved compared with the vehicle group. These hemodynamic and echocardiographic improvements were blunted by either 8-SPT or the selective adenosine A2b antagonist MRS1754 but not by the selective antagonists for other subtypes of adenosine receptors. The collagen volume fraction was smaller, and gene expression of the molecules associated with cardiac remodeling such as matrix metalloproteinase in noninfarcted areas was reduced in the DIP and CADO groups compared with the vehicle group, both of which were attenuated by either 8-SPT or MRS1754. Conclusions— Long-term stimulation of adenosine A2b receptors begun after MI attenuates cardiac fibrosis in the noninfarcted myocardium and improves cardiac function. Drugs that stimulate adenosine A2b receptors or increase adenosine levels are new candidates for preventing cardiac remodeling after MI.

Recent studies suggest that G-CSF prevents cardiac remodeling following myocardial infarction (MI) likely through regeneration of the myocardium and coronary vessels. However, it remains unclear whether G-CSF administered at the onset of reperfusion prevents ischemia/reperfusion injury in the acute phase. We investigated acute effects of G-CSF on myocardial infarct size and the incidence of lethal arrhythmia and evaluated the involvement of the phosphatidylinositol-3 kinase (PI3K) in the in vivo canine models.In open-chest dogs, left anterior descending coronary artery (LAD) was occluded for 90 minutes followed by 6 hours of reperfusion. We intravenously administered G-CSF (0.33 micro/kg/min) for 30 minutes from the onset of reperfusion. Wortmannin, a PI3K inhibitor, was selectively administered into the LAD after the onset of reperfusion.G-CSF significantly (p0.05) reduced myocardial infarct size (38.7+/-4.3% to 15.7+/-5.3%) and the incidence of ventricular fibrillation during reperfusion periods (50% to 0%) compared with the control. G-CSF enhanced Akt phospholylation in ischemic canine myocardium. Wortmannin blunted both the infarct size-limiting and anti-arrhythmic effects of G-CSF. G-CSF did not change myeloperoxidase activity, a marker of neutrophil accumulation, in the infarcted myocardium.An intravenous administration of G-CSF at the onset of reperfusion attenuates ischemia/reperfusion injury through PI3K/Akt pathway in the in vivo model. G-CSF administration can be a promising candidate for the adjunctive therapy for patients with acute myocardial infarction.

We have previously reported that histamine H(2) blockers may be cardioprotective in patients with chronic heart failure. Since both endogenous histamine and histamine H(2) receptors are present in heart tissue, we tested the hypothesis that the blockade of histamine H(2) receptors mediates protection against reversible or irreversible ischemia and reperfusion injury. In open-chest dogs, the left anterior descending coronary artery was occluded for 90 minutes, followed by reperfusion for 6 hours. Administration of famotidine and cimetidine from 10 minutes before occlusion until after 1 hour of reperfusion reduced infarct size (17.0 +/- 4.1% and 17.8 +/- 2.9% vs. 36.9 +/- 5.9% of the solvent group, respectively) Famotidine administration only during the reperfusion period for 1 hour also attenuated infarct size (22.5 +/- 3.5%). There were no differences in either area at risk or collateral flow among the groups. In another set of experiments, we decreased coronary perfusion pressure in dogs so that the coronary blood flow decreased to 50% of the non-ischemic level. In such conditions, we observed the increases in histamine release compared with non-ischemic conditions (0.04 +/- 0.03 to 0.28 +/- 0.13 ng/ml, p < 0.05). Famotidine improved anaerobic myocardial metabolism gauged by both lactate extraction ratio and myocardial oxygen consumption. We conclude that the blockade of histamine H(2) receptors mediates improvements in the anaerobic myocardial metabolism, and thus protects against ischemia and reperfusion injury.

Rapid nongenomic actions of aldosterone independent of mineralocorticoid receptors (MRs) on vascular tone are divergent. Until now, the rapid nongenomic actions of aldosterone on vascular tone of coronary artery and cardiac function in the in vivo ischemic hearts were not still fully estimated. Furthermore, although aldosterone can modulate protein kinase C (PKC) activity, there is no clear consensus whether PKC is involved in the nongenomic actions of aldosterone on the ischemic hearts. In open chest dogs, the selective infusion of aldosterone into the left anterior descending coronary artery (LAD) reduced coronary blood flow (CBF) in the nonischemic hearts in a dose-dependent manner. Also, in the ischemic state that CBF was decreased to 33% of the baseline, the intracoronary administration of aldosterone (0.1 nmol/L) rapidly decreased CBF (37.4±3.8 to 19.3±5.2 mL/100 g/min; P P P

Although recent studies suggest that erythropoietin (EPO) may reduce multiple features of the myocardial ischemia/reperfusion injury, the cellular mechanisms and the clinical implications of EPO-induced cardioprotection are still unclear. Thus, in this study, we clarified dose-dependent effects of EPO administered just before reperfusion on infarct size and the incidence of ventricular fibrillation and evaluated the involvement of the phosphatidylinositol-3 (PI3) kinase in the in vivo canine model. The canine left anterior descending coronary artery was occluded for 90 min followed by 6 h of reperfusion. A single intravenous administration of EPO just before reperfusion significantly reduced infarct size (high dose (1,000 IU/kg): 7.7 +/- 1.6%, low dose (100 IU/kg): 22.1 +/- 2.4%, control: 40.0 +/- 3.6%) in a dose-dependent manner. Furthermore, the high, but not low, dose of EPO administered as a single injection significantly reduced the incidence of ventricular fibrillation during reperfusion (high dose: 0%, low dose: 40.0%, control: 50.0%). An intracoronary administration of a PI3 kinase inhibitor, wortmannin, blunted the infarct size-limiting and anti-arrhythmic effects of EPO. Low and high doses of EPO equally induced Akt phosphorylation and decreased the equivalent number of TUNEL-positive cells in the ischemic myocardium of dogs. These effects of EPO were abolished by the treatment with wortmannin. In conclusion, EPO administered just before reperfusion reduced infarct size and the incidence of ventricular fibrillation via the PI3 kinase-dependent pathway in canine hearts. EPO administration can be a realistic strategy for the treatment of acute myocardial infarction.

ObjectivesThe purpose of this study was to investigate whether liposomal adenosine has stronger cardioprotective effects and fewer side effects than free adenosine.BackgroundLiposomes are nanoparticles that can deliver various agents to target tissues and delay degradation of these agents. Liposomes coated with polyethylene glycol (PEG) prolong the residence time of drugs in the blood. Although adenosine reduces the myocardial infarct (MI) size in clinical trials, it also causes hypotension and bradycardia.MethodsWe prepared PEGylated liposomal adenosine (mean diameter 134 ± 21 nm) by the hydration method. In rats, we evaluated the myocardial accumulation of liposomes and MI size at 3 h after 30 min of ischemia followed by reperfusion.ResultsThe electron microscopy and ex vivo bioluminescence imaging showed the specific accumulation of liposomes in ischemic/reperfused myocardium. Investigation of radioisotope-labeled adenosine encapsulated in PEGylated liposomes revealed a prolonged blood residence time. An intravenous infusion of PEGylated liposomal adenosine (450 μg/kg/min) had a weaker effect on blood pressure and heart rate than the corresponding dose of free adenosine. An intravenous infusion of PEGylated liposomal adenosine (450 μg/kg/min) for 10 min from 5 min before the onset of reperfusion significantly reduced MI size (29.5 ± 6.5%) compared with an infusion of saline (53.2 ± 3.5%, p < 0.05). The antagonist of adenosine A1, A2a, A2b, or A3subtype receptor blocked cardioprotection observed in the PEGylated liposomal adenosine-treated group.ConclusionsAn infusion as PEGylated liposomes augmented the cardioprotective effects of adenosine against ischemia/reperfusion injury and reduced its unfavorable hemodynamic effects. Liposomes are promising for developing new treatments for acute MI.