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Background: Ischemic preconditioning (IPC) elicits two distinct windows of cardioprotection, an early phase that lasts for 1-2 h and a delayed phase that lasts for 24-72 h. However, there is conflicting data as to how long the heart is resistant to IPC-induced cardioprotection after the initial protection wanes, leading to the demonstration of IPC-resistance. This resistance to IPC appears to be dependent on the timing of the next IPC stimulus, the species of animals used and the model studied. Furthermore, the mechanisms responsible IPC-resistance are unknown. It is also important to demonstrate therapeutic interventions that will produce cardioprotection during this period of IPC-resistance., Methods and Results: To examine potential mechanisms responsible for acute IPC-induced resistance, the NHE-1 inhibitor EMD 85131 (2-methyl-5-methylsulfonyl-1-(1-pyrrollyl)-benzoylguanidine), which exerts its effects via mechanisms distinct from IPC, and the K(ATP) channel opener bimakalim, which bypasses the signaling mechanisms of IPC to directly open K(ATP) channels, were examined in a canine model of IPC-resistance. One 10 min. IPC stimulus followed by 10 min. of reperfusion produced a significant reduction in IS/AAR compared to Control (7.1 +/- 2.6% versus 26.0 +/- 6.2%; P < 0.05). However, IPC did not significantly protect the myocardium if a 2 h reperfusion period occurred between the initial IPC stimulus and the subsequent prolonged (60 min) ischemic challenge (IS/AAR: 22.5 +/- 4.8%: P > 0.05). Furthermore, hearts treated with IPC followed by 2 h of reperfusion were resistant to an additional IPC stimulus administered just prior to the subsequent 60 min. occlusion period (IS/AAR: 22.9 +/- 3.2%: P > 0.05). In contrast, administration of the NHE-1 inhibitor EMD 85131 (IS/AAR: 7.4 +/- 2.5%: P < 0.05) or the K(ATP) channel opener bimakalim (IS/AAR: 11.8 +/- 2.4%: P < 0.05) both afforded significant cardioprotection when administered at 2 h of reperfusion in previously preconditioned canine hearts resistant to IPC., Conclusions: IPC resistance occurs in this canine model of ischemia-reperfusion injury. However, in spite of IPC resistance, hearts can still be pharmacologically protected by direct application of the K(ATP) channel opener bimakalim or the NHE inhibitor EMD 85131.

We have previously reported that genetic reduction of the Na,K-ATPase alpha1 isoform (alpha1(+/-)) results in a hypocontractile cardiac phenotype. This observation was surprising and unexpected. In order to determine if calcium overload contributes to the depressed phenotype, cardiac performance was examined by perfusing the hearts with buffer containing 2 or 1.5 mM calcium. At 2 mM calcium, +dP/dt for the alpha1(+/-) hearts (1374 +/- 180) was significantly less than that of wild-type (2656 +/- 75, P < 0.05). At 1.5 mM calcium, a larger decrease in +dP/dt occurred (vs. 2 mM calcium) for the alpha1(+/-) hearts (517 +/- 92) compared to wild-type (2238 +/- 157). At 2 mM calcium, -dP/dt was 50% lower in alpha1(+/-) hearts (-1903 +/- 141) than wild-type (-982 +/- 143). At 1.5 mM calcium relaxation was further reduced in alpha1(+/-) compared to wild-type (-443 +/- 56 vs. - 1691 +/- 109). We also tested whether the compensatory upregulation of the Na,K-ATPase alpha2 isoform in the alpha1(+/-) hearts contributes to the hypocontractile phenotype. At 8 x 10(-6) M ouabain, that would completely inhibit the alpha2 isoform, a 30% increase in contractility was obtained in alpha1(+/-) hearts compared to no ouabain treatment, while a 63% faster time-to-peak (TTP) and 67% faster half-time-to-relaxation (RT(1/2)) were observed in alpha1(+/-) hearts treated with ouabain. These results suggest that upregulation of the alpha2 isoform may play a role in slower TTP and RT(1/2) in the alpha1(+/-) hearts. Furthermore, lowering extracellular calcium in the perfusate did not alleviate the depressed contractile phenotype in the alpha1(+/-) hearts and resulted in further depressed cardiac contractility suggesting that these hearts are not calcium overloaded.

Objective: Sarcoplasmic reticulum dysfunction may contribute to calcium (Ca2+) overload during myocardial reperfusion. The aim of this study was to investigate its role in reperfusion injury., Methods: Open chest dogs undergoing 15 min of left anterior descending coronary artery occlusion and 3 h of reperfusion were randomized to intracoronary infusions of 0.9% saline, vehicle, or the Ca2+ channel antagonist, nifedipine (50 micrograms/min from 2 minutes before to 5 minutes after reperfusion). After each experiment, transmural myocardial biopsies were removed from ischemic/reperfused and nonischemic myocardium in the beating state and analyzed for (i) sarcoplasmic reticulum protein content (Ca2+ ATPase, phospholamban, and calsequestrin) by immunoblotting and (ii) Ca2+ uptake by sarcoplasmic reticulum vesicles with and without 300 micromolar ryanodine or the Ca2+ ATPase activator, antiphospholamban (2D12) antibody., Results: Contractile function did not recover in controls and vehicle-treated dogs after ischemia and reperfusion (mean systolic shortening, -2 +/- 2%), but completely recovered in nifedipine-treated dogs (17 +/- 2%, p = NS vs. baseline, p < 0.01 vs. control). Ventricular fibrillation occurred in 50% of controls and vehicle dogs and 0% of nifedipine-treated dogs (p < 0.01). Ca2+ uptake by the sarcoplasmic reticulum vesicles was severely reduced in ischemic/reperfused myocardium of controls and vehicle dogs (p < 0.01 vs. nonischemic). Ryanodine and the 2D12 antibody improved, but did not reverse the low Ca2+ uptake. Protein content was similar in ischemic/reperfused and nonischemic myocardium. In contrast, Ca2+ uptake and the responses to ryanodine and 2D12 antibody were normal in ischemic/reperfused myocardium from nifedipine-treated dogs., Conclusion: Dysfunction of the sarcoplasmic reticulum Ca2+ ATPase pump correlates with reperfusion injury. Reactivation of Ca2+ channels at reperfusion contributed to Ca2+ pump dysfunction. Ca2+ pump injury may be a critical event in myocardial reperfusion injury.

Background: Antibodies to selected neutrophil or endothelial cell adhesion molecules decrease myocardial infarct size in vivo. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is an immunoglobulin gene superfamily member expressed constitutively on neutrophils and endothelium. F(ab')2 fragments of antibody against PECAM-1 inhibit transendothelial migration of neutrophils in several in vivo models of acute inflammation. Therefore, we examined the effect of F(ab')2 fragments of anti-PECAM-1 antibody in a rat model of myocardial infarction., Methods and Results: F(ab')2 fragments of the anti-PECAM-1 antibody SEW16 and control normal rabbit IgG (NRIgG) were administered at 5 mg/kg to male Wistar rats, and the rats were subjected to a 30-minute coronary artery occlusion followed by 2 hours of reperfusion. At the completion of each experiment, the area at risk, infarct size (IS), and myeloperoxidase (MPO) activity were determined. Compared with untreated (n = 8; IS, 57 +/- 5%) or NRIgG-treated (n = 10; IS, 62 +/- 3%) control rats, SEW16-treated rats (n = 15; IS, 28.5 +/- 4%) displayed a 54% decrease in myocardial infarct size (P < .001). Hemodynamic parameters, leukocyte counts, total left ventricular weight, and area-at-risk weights did not differ significantly between the treatment groups. However, measurement of MPO activity revealed that neutrophil accumulation was reduced 83% (NRIgG, 975 +/- 55 mU/g; SEW16, 167 +/- 62 mU/g)., Conclusions: These results demonstrate that blocking PECAM-1 exerts a significant protective effect in a rat model of myocardial ischemia-reperfusion injury via blockade of neutrophil accumulation in the myocardium.

Objectives: This study sought to establish the effect of amiloride on stunned myocardium and to determine the role of hemodynamic alterations and inhibition of sodium/proton (Na+/H+) exchange and L-type cytosolic calcium (Ca2+) channels., Background: Amiloride is a nonspecific agent that may reduce reperfusion injury, but its effect on reversible dysfunction or stunned myocardium is unclear., Methods: Ninety-seven open chest dogs undergoing 15 min of left anterior descending coronary artery occlusion and 3 h of reperfusion with monitoring of hemodynamic variables, systolic shortening and myocardial blood flow were randomized to seven intracoronary infusions: control dogs (5% dextrose, n = 16); low dose amiloride (1 mg/min, n = 14); high dose amiloride (5 mg/min) with (n = 12) and without (n = 16) atrial pacing; sodium nitroprusside (20 micrograms/min, n = 16); hexamethylene amiloride (a specific inhibitor of Na+/H+ exchange, 60 micrograms/min, n = 14); and nifedipine (a specific inhibitor of L-type Ca2+ channels, 5 micrograms/min, n = 9). Drug infusions were started 40 min before occlusion and stopped at 30 min after reperfusion., Results: Forty-three dogs were excluded because of ventricular fibrillation or high collateral flow. The incidence of ventricular fibrillation was similar in all groups to that in control dogs. Systolic shortening completely recovered (p = NS vs. baseline; p < 0.01 vs. control group) by 2 h after reperfusion in the low dose amiloride group and 30 min in the high dose group (p < 0.01 vs. low dose). High dose amiloride increased myocardial blood flow and had positive inotropic and negative chronotropic effects (p < 0.05 vs. control group). Atrial pacing did not attenuate recovery. The only effect of low dose amiloride was increased myocardial blood flow after reperfusion. Systolic shortening did not deteriorate after washout of drug effects. Sodium nitroprusside and nifedipine similarly increased myocardial blood flow, but systolic shortening never recovered. Hexamethylene amiloride had no hemodynamic effects, and systolic shortening never recovered., Conclusions: Amiloride prevented the contractile dysfunction of myocardial stunning but did not prevent arrhythmias. Hemodynamic alterations, increased myocardial blood flow and inhibition of Na+/H+ exchange or L-type Ca2+ channels alone did not account for the improved function. Inhibition of Na+/Ca2+ exchange may be the mechanism of improved postischemic function.