Carol A. Sargent

Cardioprotective profile of the cardiac-selective ATP-sensitive potassium channel opener BMS-180448

ATP-sensitive potassium channel (KATP) openers have direct protective effects on ischemic myocardium that are independent of vasorelaxation. Because reference KATP openers (e.g., cromakalim, pinacidil) are potent relaxants of smooth muscle, their utility for treating myocardial ischemia may be limited by hypotension. Efforts aimed at development of a cardioprotective KATP opener with less vasorelaxant activity led to identification of the arylcyanoguanidine analogue BMS-180448. In globally ischemic rat hearts, BMS-180448 was cardioprotective (EC25 for increasing time to contracture = 2.5 μM), with potency equal to that of cromakalim (EC25 = 4.9 μM) despite being significantly less potent as a peripheral smooth muscle relaxant (methoxamine-constricted rat aorta). The cardioprotective effects of BMS-180448 in isolated perfused rat heart were abolished by the KATP blockers glyburide and sodium 5-hydroxydecanoate, indicating KATP involvement in its mechanism of action. Further confirmation was obtained by demonstration of single KATP opening by BMS-180448 in guinea pig cardiac myocytes. In anesthetized dogs, cromakalim was > 100-fold more potent than BMS-180448 in decreasing blood pressure (BP). In anesthetized dogs subjected to 90-min coronary occlusion/reperfusion, BMS-180448 reduced in-farct size (IS) by 50% without hemodynamic effects. BMS-180448 provides the opportunity to explore the cardioprotective actions of this class of agents without the possible complications (hypotension, coronary steal) that may be caused by the currently available KATP openers.

Cardioprotective effects of the cyanoguanidine potassium channel opener P-1075

Summary P-1075 is a cyanoguanidine ATP-sensitive potassium channel opener (KATP) that relaxes smooth muscle and shortens myocardial action potential duration (APD) at concentrations in the nanomolar range. Most KATP openers have antiischemic potencies in the micromolar range. We wished to determine if the relatively high cardiac potency of P-107S could be translated into high antiischemic potency. Isolated rat hearts were pretreated with 10–300 nM P-1075 followed by 25-min global ischemia and 30-min reperfusion. Before ischemia, P-1075 had little effect on cardiac function, although it did increase coronary flow. During ischemia, P-1075 significantly increased time to contracture in a concentration-dependent manner (EC25 = 57 nM). P-1075 also improved recovery of contractile function significantly and reduced lactate dehydrogenase (LDH) release during reperfusion (at concentrations ≤60 nM). Treatment with 75 nM P-1075 both before and after ischemia did not add to the protective effects observed after preischemic treatment. Treatment with P-1075 only during reperfusion was not cardioprotective. The protective effects of P-1075 were completely abolished by the KATP blocker glyburide (100 nM). In addition, P-1075 relaxed methoxamine-constricted aorta with a higher potency relative to antiischemic potency. Thus, P-1075 has cardioprotective effects similar to that of other reference KATP openers, except that P-1075 is ~ 100-fold more potent relative to most other tested KATP openers. These results demonstrate that P-1075 is the first PATP opener that protects ischemie myocardium at nanomolar concentrations.

Protective effect of serotonin (5-HT2) receptor antagonists in ischemic rat hearts.

Summary Serotonin (5-HT) may play a role in exacerbating thrombosis and coronary spasm during myocardial ischemia, but its role in mediating myocardial damage directly is not clear. We determined the effect of the 5-HT2 receptor antagonists cinanserin (0.1–10 μM), ketanserin (0.3–10 μM), and LY 53857 (1–10 μM) on time to contracture, recovery of contractile function, and lactate dehydrogenase (LDH) release after 25-min global ischemia and 30-min reperfusion in isolated rat heart. All 5-HT2 antagonists significantly increased time to contracture in a concentration-dependent manner (EC25 = 1.6, 5.5, and 6.1 μM for cinanserin, ketanserin, and LY 53857, respectively). These compounds also significantly reduced LDH release and improved recovery of contractile function during reperfusion. 5-HT ±30 μM significantly reduced time to contracture, indicating a proischemic effect. The proischemic effect of 5-HT was abolished by ketanserin and cinanserin. Inhibition of 5-HT synthesis by parachlorophenylalanine resulted in significant cardio-protection, further indicating the involvement of 5-HT in the pathogenesis of ischemia in this model. Although cinanserin and ketanserin had α1-adrenoceptor blocking effects, LY 53857 was devoid of this activity at concentrations exhibiting cardioprotection. Therefore, 5-HT may exacerbate ischemic injury in rat heart, and this exacerbation appears to be mediated specifically by 5-HT2 receptors.

Amelioration of ischemia/reperfusion injury in isolated rat hearts by the ATP-sensitive potassium channel opener BMS-180448

OBJECTIVE Aims of this study were: (1) Evaluate by morphology and specific physiological and biochemical parameters, the protective effects of the cardioselective ATP-sensitive potassium channel opener BMS-180448 on ischemic/reperfused isolated rat heart, and (2) Determine the earliest time point ischemia-induced myocardial injury is observed by light microscopy. METHODS Hearts from Sprague-Dawley rats were perfused on a Langendorff apparatus. After equilibration, hearts were treated with BMS-180448 (10 micro M) or vehicle (0.04% DMSO) for 10 min before the onset of ischemia. Four hearts/group were collected following 10, 18, or 25 min of ischemia. A nonischemic control group was also evaluated. Following 25 min of ischemia, another set of hearts was reperfused with oxygenated Krebs-Hensleit solution and allowed to recover for 30 min. Light and electron microscopic changes of the myocardium were semi-quantitatively evaluated together with physiological (i.e., heart rate, left ventricular diastolic pressure, time to contracture formation) and biochemical (i.e., lactate dehydrogenase, LDH, release) endpoints. RESULTS Cardioprotective effects of BMS-180448 following ischemia/reperfusion consisted of a reduced rate of contracture formation, reduced LDH release, and enhanced recovery of contractile function during reperfusion (P < 0.05). Light microscopic evidence of myocardial damage was detected following 18 min of ischemia. Morphological changes in ischemic/reperfused hearts included interstitial edema, myofiber degeneration, and hypercontraction band formation. Ultrastructurally, swollen myofibrils, swollen mitochondria with disrupted cristae and electron-dense deposits, myofibrillar lysis, and contraction bands, were observed. Light and electron microscopic severity scores were significantly less (P < 0.05) in BMS-180448-treated hearts at the 25 min ischemic time point and in reperfused hearts, as compared to similarly-treated vehicle hearts. CONCLUSIONS BMS-180448 ameliorates morphological evidence of ischemia/reperfusion myocardial damage in the isolated rat heart model, in agreement with physiological and biochemical parameters.

Myocardial calcium-independent phospholipase A2 activity during global ischemia in isolated rabbit hearts

OBJECTIVES To study calcium-independent phospholipase A2 activity during global ischemia in isolated rabbit hearts by measuring the hydrolysis of the endogenous choline phospholipids. METHODS Langendorff perfused rabbit hearts were exposed to global ischemia for 15 or 60 min, or control perfusion for the same length of time. The hearts were then rapidly frozen in liquid nitrogen and lyophilized. Calcium-independent phospholipase A2 activity in the lyophilized tissue was studied by measuring accumulation of lysophospholipids resulting from hydrolysis of both the choline diacylphospholipid and the choline plasmalogen pool. RESULTS The calcium-independent phospholipase A2 activity showed the same pH, temperature and calcium sensitivity in control and ischemic (15 min of ischemia) lyophilized myocardial tissue. Incubation of control and ischemic tissue showed no difference in the rate of accumulation of lysophospholipids when the ischemic tissue was obtained from hearts exposed to 15 min of ischemia (107 +/- 4 vs 111 +/- 7 nmol/g dry wt x min, ischemia versus control, mean +/- s.e.m., n = 8), but a significant decrease was noticed in tissue from hearts that had been exposed to 60 min of ischemia (31 +/- 9 vs 86 +/- 18 nmol/g dry wt x min, P < 0.05, n = 4). The decreased phospholipase A2 activity in tissue exposed to 60 min of ischemia was not due to enhanced metabolism of the lysophospholipids (84 +/- 15 vs 79 +/- 8 nmol/g dry wt x min, n = 4). The calcium-independent phospholipase A2 activity was considerably lower in fresh myocardial tissue compared with lyophilized tissue, but comparison of control and ischemic fresh tissue gave results comparable to those found using lyophilized tissue. The myocardial calcium-independent phospholipase A2 activity showed no plasmalogen selectivity in either control or ischemic myocardium. CONCLUSIONS In isolated perfused rabbit hearts we found no evidence for activation of calcium-independent phospholipase A2 activity during global ischemia. With prolonged time of ischemia there was a significant decrease in calcium-independent phospholipase A2 activity.

Role of endothelin receptor subtype B(ET-B) in myocardial ischemia

Previous work indicated that endothelin (ET) may be involved in the pathogenesis of myocardial ischemia, although the relative importance of the ET receptor subtypes is presently not clear. The purpose of this study was to determine the role of myocardial ET-B receptors in mediating ischemic/reperfusion damage in isolated rat hearts. Saturation binding analyses were conducted with [125I]ET-1 and [125I]IRL-1620 to assess changes in ET-A and ET-B receptor binding. Total ET receptor density (Bmax) was greater in atrial versus ventricular tissue. ET-A Bmax was 8 to 10-fold greater than ET-B Bmax. In ischemic and ischemic/reperfused atrial tissue neither the equilibrium dissociation constant (Kd) nor Bmax for ET-B receptors was changed. The ET-B receptor Kd in ischemic or ischemic/reperfused ventricular tissue was also unchanged. In ischemic ventricular tissue there was a trend towards an increased ET-B Bmax, which was accentuated after ischemia/reperfusion. No changes were found in ET-A Bmax or Kd in ischemic ventricular or atrial tissue. The physiological importance of this receptor subtype in ischemic myocardium was determined using the selective ET-B agonist, sarafotoxin S6c. In non-ischemic tissue no effect on coronary flow or function were observed with sarafotoxin S6c. Furthermore, no changes were seen in ischemic time to contracture or any of the reperfusion indexes of myocardial damage. The sarafotoxin S6c utilized was active as it inhibited [125I]ET-3 binding to ET-B receptors (Ki = 0.1 nM). Thus, the pro-ischemic effect of ET-1 seems to be mediated by ET-A receptors. ET-B receptors do not appear to play a role in the pathogenesis of myocardial ischemia.

Glyburide-reversible cardioprotective effects of calcium-independent phospholipase A2 inhibition in ischemic rat hearts

OBJECTIVES A myocardial calcium-independent PLA2 has been described that is activated during myocardial ischemia and this enzyme may modulate ATP-sensitive potassium channels (KATP). The aim of this study was to determine the effect of an inhibitor of this enzyme, a bromoenol lactone, in isolated globally ischemic rat hearts. METHODS Isolated rat hearts were treated for 10 min with 0.3-6 microM bromoenol lactone and then subjected to 25 min ischemia and 30 min reperfusion. RESULTS The bromoenol lactone significantly increased coronary flow in nonischemic myocardium, and slightly reduced cardiac function at 6 microM. During global ischemia, time to contracture was significantly increased from vehicle group values in the presence of the bromoenol lactone (EC50 = 1.2 microM). During reperfusion, a concentration-dependent increase in function and a reduction in LDH release were observed for the PLA2 inhibitor. The concentrations of the PLA2 inhibitor which were significantly cardioprotective, inhibited this enzyme in membrane fractions of rat myocardium (IC50 = 0.87 microM). The KATP blocker sodium 5-hydroxydecanoate (5-HD) inhibited the increase in time to contracture observed for the bromoenol lactone. During reperfusion, 5-HD abolished the protective effects of the bromoenol lactone on cardiac function and LDH release. Glyburide had similar effects on the cardioprotective activity of the bromoenol lactone, although it only partially abolished the LDH reducing effect of this agent. CONCLUSIONS The bromoenol lactone protects ischemic myocardium at concentrations which also inhibit calcium-independent PLA2. This cardioprotection can be attenuated by blockers of KATP, suggesting a potential mechanism for modulation of myocardial KATP.

Effect of Calmodulin and Protein Kinase C Inhibitors on Globally Ischemic Rat Hearts

Summary Several calmodulin inhibitors have been reported to be cardioprotective, but the ability of these compounds to inhibit protein kinase C (PKC) suggests that calmodulin inhibition may not be the sole mechanism responsible. To distinguish between the effects, we determined the cardioprotective activity of several calmodulin inhibitors with differing PKC inhibitory potencies in isolated globally ischemic rat hearts. Twenty-five minutes of global ischemia caused significant myocardial dysfunction, contracture formation, and lactate dehydrogenase (LDH) release on reperfusion in vehicle-treated hearts. The calmodulin inhibitors trifluoperazine, W-7, calmidazolium, W-13, and CGS 9343B improved postischemic contractile function and/or reduced LDH release. They also reduced preischemic cardiac function, although cardioprotection did not appear to be correlated with cardio-depression. Calmodulin inhibitors increased preischemic coronary flow (CF) and decreased heart rate (HR), but controlling these parameters did not affect the cardioprotection. Pretreatment of ischemic hearts with trifluoperazine was associated with preservation of myocardial ATP. Pretreatment of ischemic rat hearts with the PKC inhibitors staurosporine, calphostin C, polymyxin B, and H-7 did not result in cardioprotection. Thus, calmodulin inhibition causes cardioprotection that appears to be independent of PKC inhibition.

Effect of endothelin antagonists with or without BQ 788 on ET-1 responses in pithed rats.

Summary: The overall effects of endothelin-1 (ET-1) on blood pressure are caused by a composite activation of constrictor ETA and ETB receptors and dilator ETB receptors. Therefore, it is difficult to accurately compare the ETA activity of selective ETA receptor antagonists (BQ 123 and BMS 182874) with mixed ETA/ETB antagonists (SB 209670 and bosentan) on the cumulative dose-response curve to ET-1. The development of a selective ETB antagonist (BQ 788), which inhibits both the dilator and constrictor ETB receptors, offered the opportunity to investigate the role of ETB receptors in the response to exogenous ET-1 in the pithed rat. BQ 788 (3 mg/kg) resulted in an eightfold leftward shift in the ET-1 doseresponse curve, suggesting a significant involvement of ETB dilator receptors. In the absence or presence of BQ 788, each ET antagonist evoked a rightward shift from vehicle. With the exception of BMS 182874, BQ 788 increased the magnitude of the shifts. Furthermore, the profile of the shifts changed from nonparallel to parallel in the presence of BQ 788. The inclusion of BQ 788 also altered the rank order of the ET antagonists tested. The results presented describe an in vivo system that accurately characterizes the ETA activity of ET antagonists.

Sarafotoxin S6c elicits a non-ETA or non-ETB-mediated pressor response in the pithed rat

Summary: Sarafotoxin S6c (Sx6c) is reported to evoke depressor and pressor effects via activation of endothelin (ET) ETB receptors located on the endothelium and smooth muscle, respectively. We have examined the effects of the selective ETB receptor antagonist BQ 788 on the fall and rise in diastolic blood pressure induced by Sx6c (1 nmol/kg, i.v.) in pithed rats. A dose-dependent reduction in both depressor and pressor response was observed. BQ 788 completely ablated the Sx6c-mediated fall in blood pressure at 1 mg/kg. In contrast, the pressor response was not completely abolished by 10 mg/kg BQ 788 (10 mg/kg: 16.5 < 3.0 mm Hg vs. control 49.0 ± 2.5 mm Hg). Co-administration of the ETA receptor antagonist BQ 123 (1.5 mg/kg) with BQ 788 produced no further antagonism of the Sx6c-mediated pressor response. BQ 788 plus BQ 123 (1.5 mg/kg) totally blocked the pressor response to ET-1 (0.1 nmol/kg, i.v.) suggesting a difference in the mechanism of action between the two agonists. In conclusion, a portion of the Sx6c-induced pressor response is resistant to blockade by known ETA and ETB receptor antagonists. Whether Sx6c acts on a novel ET receptor or produces a nonspecific effect remains to be determined.