Ischemic preconditioning (IPC) has become the experimental "gold standard" for reduction of infarct size. However, recently a new class of agents that inhibit the sodium/hydrogen exchanger isotype 1 (NHE-1) has demonstrated marked cardioprotective efficacy in a variety of models (1). Furthermore, we and others have demonstrated that NHE-1 inhibition and IPC do not antagonize one another but rather provide additive to greater than additive cardioprotection, suggesting completely different mechanisms (2,3).
Since IPC was first described (4), numerous factors such as the KATP channel (5), adenosine (6), and protein kinase C (PKC) (7) have been shown to contribute to its cardioprotective effect. Pharmacologic inhibition of the KATP channel with sulfonylureas or the adenosine receptor with methylxanthines has been shown to block IPC (5,6). Both classes of inhibitor are widely used clinically, sulfonylureas for the treatment of diabetes, and methylxanthines for the treatment of asthma. Recent studies have demonstrated that myocardium from patients using sulfonylureas is not able to be preconditioned (8). Furthermore, several clinical reports focusing on cardiac morbidity and mortality have demonstrated poorer outcomes in patients taking sulfonylureas (8,9). Thus, NHE-1 inhibition may represent an effective cardioprotective mechanism in patients receiving sulfonylureas and methylxanthines. To explore this hypothesis, we examined whether treatment with either a sulfonylurea or a methylxanthine attenuated the cardioprotection conferred by the new specific NHE-1 inhibitor, EMD 85131 (2), in dogs. Five isoforms of NHE have been found in mammalian cells, with cardiac cells lacking the NHE-2 to NHE-5 isoforms (10). We have previously reported that EMD 85131 is 30-fold more selective for NHE-1 than for NHE-2 and 45,000-fold more selective for NHE-1 than for NHE-3 (2). Its potency (IC50) for blocking sodium uptake in NHE-1-expressing mouse fibroblast cells is 10.4 ± 1.0 nM.
In these studies, we used the selective NHE-1 inhibitor, EMD 85131 (N-diamino-ethylene-2-methyl-5-methylsulfonyl-4-pyrrolobenzamide hydrochloride). Previous work from our laboratory has demonstrated its cardioprotective efficacy (2).
In brief, adult mongrel dogs of either sex, weighing 18-26 kg, were fasted overnight, anesthetized with sodium barbital (200 mg/kg) and sodium pentobarbital (15 mg/kg), and ventilated by a respirator with room air supplemented with 100% oxygen. Arterial blood pH, PCO2 and PO2 were monitored at selected intervals with a blood gas system (AVL 995; AVL Scientific Corp., Roswell, GA, U.S.A.). Aortic and left ventricular (LV) pressure were monitored through a double-pressure transducer-tipped catheter (PC 771; Millar Instruments, Houston, TX, U.S.A.). LV dP/dt was recorded by electronic differentiation of the LV pressure pulse, and heart rate was determined by a tachometer. The right femoral vein and artery were cannulated for drug administration and for blood gas analysis and the reference blood used to determine myocardial tissue blood flow. A left thoracotomy was performed at the fifth intercostal space, the lung was retracted, the pericardium incised, and the heart suspended in a cradle. A proximal portion of the left anterior descending coronary artery (LAD) distal to the first diagonal branch was isolated from surrounding tissue, and a mechanical occluder was placed around the vessel to produce ischemia. If the basal heart rate was <150 beats/min, the heart was paced at 150 beats/min (2). Hemodynamics, heart rate, and LAD blood flow were monitored and recorded by a polygraph (model 7; Grass Instruments, Quincy, MA, U.S.A.). The left atrium was cannulated for radioactive microsphere injection.
Twenty-eight dogs were assigned to one of four groups (n = 7 in each group). All dogs were subjected to 60 min of LAD occlusion and 3 h of reperfusion. In groups 1 and 2, either saline (control group) or 3.0 mg/kg of EMD 85131 was infused intravenously for 15 min before occlusion. In groups 3 and 4, either 3.0 mg/kg of PD 115199 or 0.3 mg/kg of glibenclamide was infused for 15 min before EMD 85131 (3.0 mg/kg). In all groups, hemodynamics and blood gases were measured before occlusion, at 30 min during the 60-min occlusion, and every hour after reperfusion. Regional myocardial blood flows were determined at 30 min during the 60-min occlusion period and at the end of the experiment.
At the end of the 3-h reperfusion period, the area at risk (AAR) and the nonischemic area were differentiated as previously described (2). The nonstained ischemic and the blue-stained normal areas were separated, and both regions incubated at 37°C for 15 min in 1% 2,3,5-triphenyltetrazolium chloride (TTC) in 0.1 M phosphate buffer adjusted to pH 7.4. After storage overnight in 10% formaldehyde, infarcted and noninfarcted tissues within the AAR were separated and determined gravimetrically. Infarct size (IS) was expressed as a percentage of the AAR. Regional myocardial blood flow was measured with the radioactive microsphere technique (6).
All values are expressed as mean ± SEM. Differences between groups in hemodynamics and blood gases were compared by use of a two-way analysis of variance (ANOVA) with repeated measures and Fisher's least significant difference test. Differences between groups in tissue blood flows, AAR, and infarct size were compared by one-way ANOVA, and comparisons between groups were made with Fisher's test. Differences between groups were significant if the probability value was p < 0.05.
Twenty-eight dogs were initially used. Three dogs in the control group fibrillated; one in the EMD 85131 group, two in the PD 115199 group, and three in the glibenclamide group fibrillated on reperfusion; however, all were successfully defibrillated and used in data analysis.
Table 1 summarizes the hemodynamic data. There were no differences between groups. There were also no differences in pH and blood gases between groups at the times studied.
Figure 1 and Table 2 summarize the effect of treatment with glibenclamide and PD 115199 on EMD 85131-induced cardioprotection. Treatment with neither glibenclamide nor PD 115199 at doses previously shown to abolish IPC-induced cardioprotection (1,2) resulted in significant reduction of IS, expressed as a percentage of the AAR (IS/AAR) produced by EMD 85131 (Control, 24.2 ± 3.6%; EMD, 6.4 ± 2.3%; PD, 6.6 ± 2.4; glibenclamide, 3.5 ± 1.2%). There were no significant differences in LV weight, AAR, AAR/LV, or transmural collateral blood flow between groups (Fig. 1 and Table 2).
Pharmacologically separate cardioprotection by IPC and NHE-1 inhibition
Work first reported from our laboratory clearly demonstrated that IPC is mediated through effects on the KATP channel (5). Furthermore, work also conducted in our laboratory has demonstrated that pharmacologic antagonism of the adenosine A1 receptor blocks IPC (6). The mechanism of adenosine-induced cardioprotection appears to occur through KATP channel opening. Thus, the predominant mediator of IPC is the KATP channel, which is inhibited by sulfonylurea agents used clinically in the treatment of type 2 diabetes mellitus. However, recently NHE-1 inhibitors have demonstrated marked cardioprotective efficacy in a variety of animal models (1). The major mechanism thought to be responsible for the protective effect of NHE-1 inhibition during ischemia and/or reperfusion is a reduction in intracellular sodium and subsequently a decrease in calcium overload. During IPC it has been demonstrated that intracellular pH decreases and that intracellular Na+ increases. Because the primary activator of NHE is acidosis (10,11), it would be expected that NHE activity would increase with IPC. Based on these observations, one might predict that blocking NHE-1 would antagonize IPC.
Further to investigate whether IPC and NHE-1 inhibition convey protection through distinct or similar mechanisms, we examined the effect of known inhibitors of IPC on the cardioprotection conferred by NHE-1 inhibition. In a canine infarct model, neither the KATP channel antagonist glibenclamide, a sulfonylurea, nor the adenosine receptor antagonist PD 115199, a methylxanthine, attenuated NHE inhibitor-mediated cardioprotection at doses previously shown to inhibit IPC-mediated cardioprotection (5,6). Although comparable results have been reported in vitro in rabbit (12) and rat (13) models with an inhibitor of PKC, a major signaling molecule also thought to be involved in IPC-mediated cardioprotection, the current study represents the first demonstration that in vivo, NHE-1 inhibition and IPC do not overlap pharmacologically.
Sulfonylureas such as tolbutamide and glibenclamide are the primary therapy used in patients with non-insulin-dependent diabetes mellitus (8,9). Similarly, theophylline, a methylxanthine, is used widely in the treatment of asthma. Several earlier clinical studies have demonstrated that in patients with non-insulin-dependent diabetes mellitus, sulfonylureas are associated with adverse outcomes in those with cardiac disease (9,14), although a more recent report suggests that no excess mortality has been observed in patients treated with glibenclamide (15). In contrast, results from a recent study have demonstrated that myocardium from patients treated over the long term with oral hypoglycemic agents is not able to be preconditioned (8). Similarly, a recent retrospective analysis demonstrated that sulfonylurea use is associated with an increased risk of in-hospital mortality among diabetic patients undergoing coronary angioplasty for acute myocardial infarction (9). In light of our study and the experimental and clinical data reviewed earlier, it appears that in animal models and probably in humans, sulfonylureas and methylxanthines may abrogate IPC. As such, cardioprotective agents based on the mechanisms of IPC may not prove beneficial in these patients. However, NHE-1 inhibition may have clinical efficacy in patients with acute coronary syndromes that are concurrently treated with methylxanthines or sulfonylureas. This hypothesis deserves further study.
Acknowledgment: This study was supported by NIH grant HL-08311 and a grant from Merck KGaA. We thank Jeannine Moore and Anna Hsu for their excellent technical assistance.
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