Steven Dzwonczyk

Role of myocardial ATP-sensitive potassium channels in mediating preconditioning in the dog heart and their possible interaction with adenosine A1-receptors.

BackgroundA brief period of myocardial ischemia can result in an increased resistance to subsequent, more severe episodes of ischemia. Recent studies have indicated that activation of adenosine A1-receptors may mediate this preconditioning effect. It is also known that A1-activation can lead to ATP-sensitive potassium channel (KATP) opening via a Gi protein-mediated effect. Thus, we determined whether the KATPM blocker glyburide could abolish preconditioning or the protective effects of A1-receptor activation Methods and ResultsAnesthetized dogs were subjected to 5 minutes of left circumflex coronary artery (LCx) occlusion (or sham) followed by 10 minutes of reperfusion. The hearts were then subjected to 60 minutes of LCx occlusion and 5 hours of reperfusion. Glyburide (5 μg/kg/min) or vehicle was given directly into the LCx 20 minutes before preconditioning or sham preconditioning. Preconditioning resulted in a significantly reduced infarct size compared with nonpreconditioned animals. Glyburide abolished the protective effect of preconditioning. To establish a link between KATPand A1-receptor activation, the effect of the A1-agonist R-PIA with or without glyburide on infarct size was determined. R-PIA (0.4 μg/kg/min, directly into the LCx) significantly reduced infarct size, and this protective effect was abolished by glyburide. None of the treatments described above had a significant effect on peripheral hemodynamic status or myocardial blood flow ConclusionsPreconditioning may be mediated by KATP activation, and this may be linked to A1-receptor stimulation.

Specific block of the anti-ischemic actions of cromakalim by sodium 5-hydroxydecanoate.

The potassium channel activators cromakalim and pinacidil were recently shown to have anti-ischemic properties in isolated globally ischemic rat hearts. The effects of two reported blockers of ATP-sensitive potassium channels, glibenclamide (glyburide) and sodium 5-hydroxydecanoate, on the anti-ischemic efficacy of cromakalim were determined in this model. Buffer-perfused rat hearts were subjected to 25 minutes of ischemia followed by 30 minutes of reperfusion. Pretreatment of these hearts with 60 microM cromakalim significantly decreased indexes of contractile function but caused a significant improvement of postreperfusion function and a significant decrease in release of lactate dehydroxygenase and in end-diastolic pressure. Pretreatment with glibenclamide at concentrations that reversed the preischemic effects of cromakalim (0.05 and 1.0 microM) also significantly reversed its postischemic protective effects. Sodium 5-hydroxydecanoate (100 and 300 microM) had no effect on the preischemic (negative inotropic) effects of cromakalim but completely reversed its cardioprotective effects. Sodium 5-hydroxydecanoate did not reverse the cardioprotective effects of the calcium entry blocker diltiazem. In phenylephrine-contracted rat aorta, glibenclamide (0.1-10 microM) inhibited cromakalim-induced relaxation, whereas sodium 5-hydroxydecanoate (10-1,000 microM) had no effect. Similarly, the ability of cromakalim to shorten cardiac action potential duration in guinea pig papillary muscle and to increase outward whole-cell potassium currents in isolated myocytes was inhibited by glibenclamide, whereas sodium 5-hydroxydecanoate was without effect. Thus, both glibenclamide and sodium 5-hydroxydecanoate inhibited the effects of cromakalim after reperfusion; however, sodium 5-hydroxydecanoate, unlike glibenclamide, had no effect in nonischemic preparations. These results suggest that sodium 5-hydroxydecanoate is an ischemia-selective inhibitor of ATP-sensitive potassium channels.

Endogenous catecholamines are not necessary for ischaemic preconditioning in the isolated perfused rat heart

OBJECTIVE The mechanism of the protective effect of ischaemic preconditioning in the myocardium is not yet known. The aim of this study was to test the hypothesis that endogenous myocardial catecholamines may be mediators of preconditioning. METHODS To test whether endogenous catecholamines are involved in preconditioning, experiments were performed in hearts from rats which had been catecholamine depleted with either reserpine or 6-hydroxydopamine. Experiments were also done to determine if noradrenaline can mimic preconditioning. RESULTS Catecholamine depletion with either reserpine or 6-hydroxydopamine had no effect on preischaemic coronary flow or cardiac function. Ischaemic preconditioning (four episodes of 5 min global ischaemia and 5 min reperfusion) resulted in a significant increase in postischaemic cardiac function and a 50% decrease in lactate dehydrogenase (LDH) release following 30 min ischaemia and 30 min reperfusion compared with non-preconditioned hearts. Reserpine pretreatment did not affect the response to ischaemia or to preconditioning, although LDH release tended to be greater than in normal hearts, especially in the non-preconditioned group. Although 6-hydroxydopamine significantly increased postischaemic cardiac function in the preconditioned group, no other index of ischaemic damage (for example, LDH release, left ventricular end diastolic pressure) was affected. Further studies showed that 10 nmol.min-1 noradrenaline did not affect the severity of ischaemia, indicating that it does not mimic preconditioning. CONCLUSIONS Endogenous catecholamines are not necessary for ischaemic preconditioning in isolated rat hearts and play little or no role in the functional responses to ischaemia.

In vitro effects of capsaicin: antiarrhythmic and antiischemic activity

The antiarrhythmic effects of vehicle (0.1% dimethyl sulfoxide: DMSO) or capsaicin were evaluated in isolated perfused rat and guinea pig heart preparations. In the rat, capsaicin reduced ischemic ventricular tachycardia from 100% in control to 0%, and ischemic ventricular fibrillation from 60% in control to 0% at 30 microM, and diltiazem reduced the incidence of ischemic ventricular tachycardia and ventricular fibrillation to 55% and 0%, respectively. Reperfusion ventricular fibrillation was reduced from 90% to 20% and 33% for capsaicin and diltiazem, respectively, at these concentrations. In isolated perfused globally ischemic rat hearts, antiischemic efficacy was assessed as a significant extension (36% and 50%) in time to contracture with 30 microM capsaicin and 1 microM diltiazem, respectively. Capsaicin reduced left ventricular developed pressure by 35% in non-ischemic rat hearts, and increased coronary flow by 40%. The increased time to contracture for either compound was not blocked by glyburide (0.1 microM) suggesting a lack of any involvement of ATP-sensitive K+ channels. In isolated guinea pig hearts subjected to global ischemia, capsaicin and diltiazem reduced reperfusion ventricular fibrillation from 100% to 10% and 0% at 30 and 3 microM, respectively. Electrophysiologic evaluation in guinea pig papillary muscles using standard microelectrode techniques demonstrated significant (P < 0.05) action potential durations at 90% repolarization shortening at 1 Hz by 9%, 28% and 39%, and 23%, 37% and 51% at 10, 30, and 100 microM of capsaicin or diltiazem, respectively. Unlike diltiazem, no changes in action potential duration were observed with capsaicin (up to 100 microM) at faster stimulation rates (5 Hz). In conclusion, capsaicin displays both antiarrhythmic and antiischemic efficacy. These data suggest that the effects of capsaicin are mediated primarily through block of Ca2+ channels in these preparations.

The KATP blocker sodium 5-hydroxydecanoate does not abolish preconditioning in isolated rat hearts

Blockers of ATP-sensitive K+ channels (KATP) abolish preconditioning in several species. Glyburide does not abolish preconditioning in rat hearts, but this may be due to a loss of its activity during ischemia. We determined the effect of a KATP blocker, which is more active during ischemia (sodium 5-hydroxydecanoate, 5-HD), on preconditioning in isolated rat hearts. Rat hearts were subjected to 4 periods of 5 min global ischemia followed by 30 min of global ischemia and reperfusion. Preconditioning significantly enhanced post-ischemic recovery of function and reduced lactate dehydrogenase (LDH) release vs. sham. 5-HD (100 microM) did not abolish preconditioning. Cromakalim (20 microM) was protective in this ischemic model and this was abolished by 5-HD. This is further evidence that KATP opening is not the mechanism of preconditioning in rats.

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.

Hemodynamic and cardiac effects of BMS-180448, a novel K+ATP opener, in anesthetized dogs and isolated rat hearts.

Hemodynamic and cardiac effects of BMS-180448 (0.3-10 mg/kg i.v.) or cromakalim (0.01-0.3 mg/kg i.v.) were evaluated in anesthetized open-chest dogs and isolated perfused rat hearts. In the canine studies, heart rate (HR), mean arterial pressure and left ventricular pressure were measured as well as electromagnetic blood flows recorded from the aortic, renal, coronary and femoral vascular beds. BMS-180448 was 187-fold less potent than cromakalim in lowering blood pressure (ED-20 values of 7.84, and 0.042 mg/kg for BMS-180448 and cromakalim, respectively). Both compounds increased HR. Effects of BMS-180448 occurred at doses higher than those of cromakalim, but at doses slightly lower than those needed to cause hypotension (ED(HR)/ED(MABP) ratio of 0.18 for BMS-180448). BMS-180448 had no effect on myocardial contractility or relaxation over the doses studied, whereas cromakalim significantly increased +dP/dt and lowered -dP/dt. Effects on +dP/dt were associated with a decrease in blood pressure. Although BMS-180448 reduced total peripheral resistance (ED-25 = 5.75 mg/kg), it had little effect on specific vascular beds, with the exception of the coronary bed. BMS-180448, unlike cromakalim which caused more general vasodilating effects, appeared to be relatively selective in dilating the coronary vascular bed. In isolated perfused rat hearts, BMS-180448, 10-fold more potent as a cardioprotectant (EC25 = 2.7 microM) than as a cardiodepressant (ED-25 = 27.8 microM), had no effect on HR, suggesting a lack of effect of BMS-180448 on myocardial conduction. In conclusion, BMS-180448, a recently developed K+ATP opener, exerted less hypotensive and more selective vascular effects than did cromakalim. These results suggest that BMS-180448, at doses previously reported to give cardioprotection, should have a safe hemodynamic profile.

The effect of α2-adrenoceptor antagonists in isolated globally ischemic rat hearts

The α2-adrenoceptor antagonist, yohimbine, has been reported to protect hypoxic myocardium. Yohimbine has several other activities, including 5-HT receptor antagonism, at the concentrations at which protection was found. Therefore we designed a study to determine if yohimbine was protecting ischemic myocardium via antagonism of α2-adrenoceptors. In isolated globally ischemic rat hearts, the effects of two structurally distinct classes of α2-adrenoceptor antagonists, the indole alkaloids (yohimbine and rauwolscine) and the imidazolines (idozoxan and tolazoline) were investigated. Pretreatment with yohimbine (1–10 μM) caused a concentration-dependent increase in reperfusion left ventricular developed pressure and a reduction in end diastolic pressure and lactate dehydrogenase release. The structurally similar compound rauwolscine (10 μM) also protected the ischemic myocardium. In contrast, idozoxan (0.3–10 μM) or tolazoline (10 μM) had no protective effects. The cardioprotective effects of yohimbine were partially reversed by 30 μM 5-HT. These results indicate that the mechanism for the cardioprotective activity of vohimbine may involve 5-HT receptor antagonistic activity.

Effect of the Potassium Channel Opener EMD 56431 on Globally Ischemic Rat Hearts

The effect of the ATP-sensitive potassium channel opener EMD 56431 on coronary vasodilation and cardioprotection in isolated rat hearts was investigated. EMD 56431 caused a significant increase in pre-ischemic coronary flow. Time to contracture and reperfusion function were significantly increased at 3, 10 and 30 microM concentrations. LDH release was significantly reduced at 10 and 30 microM concentrations. 1 microM glyburide completely abolished the protective effects found with 10 microM EMD 56431. When given during reperfusion only, 10 microM EMD 56431 showed no cardioprotection. Thus, EMD 56431 appeared to reduce the severity of ischemia/reperfusion injury. The vasorelaxant versus cardioprotective effects for EMD 56431 are similar to other potassium channel openers, such as cromakalim.