Paul G. Sleph

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.

Pharmacologic profile of the dihydropyrimidine calcium channel blockers SQ 32,547 and SQ 32,946

SQ 32,926 and SQ 32,547, two dihydropyrimidine calcium channel blockers, were characterized as potent inhibitors of depolarization-induced contractions of isolated smooth muscle preparations. In rat aorta, the IC50 values were 5.5 nM for SQ 32,547 and 8.1 nM for SQ 32,926, values which compare favorably with that of 2.9 nM for nifedipine. The dihydropyrimidines were also tested in a model of stable angina: pacing-induced ischemia in dogs. Both SQ 32,547 and SQ 32,926 reduced the ST-segment elevation observed in vehicle-treated animals. No significant changes in hemodynamic status were detected, suggesting that a reduction in cardiac work secondary to afterload reduction was probably not a major contributor to the protective effects. Neither was increased coronary blood flow important for the antiischemic outcome because no significant effects of the dihydropyrimidines were observed on ischemic regional blood flow. SQ 32,547 was also studied in globally ischemic, isolated rat hearts. In this model, SQ 32,547 was protective because it significantly reduced contracture formation and lactate dehydrogenase (LDH) release. Washing out the effect of SQ 32,547 in isolated hearts and smooth muscles was difficult, presumably due to its lipophilicity. In the smooth muscle preparations, the effects of both nifedipine and SQ 32,926 were much more easily washed out. As with other calcium channel blockers, increasing the antiischemic effects of SQ 32,547 was associated with a higher cost in terms of cardiac function. In summary, the two novel dihydropyrimidines, SQ 32,547 and SQ 32,926, showed antiischemic properties in animal models.

Selective thyroid hormone receptor-β activation: A strategy for reduction of weight, cholesterol, and lipoprotein (a) with reduced cardiovascular liability

Few treatments for obesity exist and, whereas efficacious therapeutics for hyperlipidemia are available, further improvements are desirable. Thyroid hormone receptors (TRs) regulate both body weight and cholesterol levels. However, thyroid hormones also have deleterious effects, particularly on the heart. The TRβ subtype is involved in cholesterol lowering and possibly elevating metabolic rate, whereas TRα appears to be more important for control of heart rate (HR). In the current studies, we examined the effect of TRβ activation on metabolic rate and HR with either TRα1–/– mice or the selective TRβ agonist KB-141 in mice, rats, and monkeys. 3,5,3′-triiodi-l-thyronine (T3) had a greater effect on increasing HR in WT than in TRα–/– mice (ED15 values of 34 and 469 nmol/kg/day, respectively). T3 increased metabolic rate [whole body oxygen consumption (MVO2)] in both WT and TRα–/– mice, but the effect in the TRα1–/– mice at the highest dose was half that of the WT mice. Thus, stimulation of MVO2 is likely due to both TRα and -β. T3 had equivalent potency for cholesterol reduction in WT and TRα–/– mice. KB-141 increased MVO2 with selectivities of 16.5- and 11.2-fold vs. HR in WT and TRα1–/– mice, respectively. KB-141 also increased MVO2 with a 10-fold selectivity and lowered cholesterol with a 27-fold selectivity vs. HR in rats. In primates, KB-141 caused significant cholesterol, lipoprotein (a), and body-weight reduction (up to 7% after 1 wk) with no effect on HR. TRβ-selective agonists may constitute a previously uncharacterized class of drugs to treat obesity, hypercholesterolemia, and elevated lipoprotein (a).

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.

Pharmacologic profile of cromakalim in the treatment of myocardial ischemia in isolated rat hearts and anesthetized dogs.

Summary: The detailed antiischemic pharmacology of the potassium channel activator cromakalim was determined in isolated globally ischemic rat hearts and a canine model of coronary occlusion and reperfusion. Cromakalim significantly improved reperfusion function in rat hearts starting at a concentration of 1 μM; this effect peaked at 7μM. No cardiodepressant effects were observed in nonischemic tissue with cromakalim until a concentration of 100 μM was achieved, and this effect was reversed by glyburide. The antiischemic effect of 7 μM cromakalim was also completely reversed by glyburide and the novel ATP-sensitive potassium channel blocker sodium 5-hydroxydecanoate (5-HD). Glyburide did not reverse the antiischemic effects of 1 μM diltiazem. Cromakalim not only improved reperfusion contractile function in rat hearts, but improved the functional reserve and efficiency of O2 utilization. In anesthetized dogs, intracoronary cromakalim (0.1 μg/kg/min given throughout ischemia and reperfusion) significantly reduced infarct size in hearts subjected to 90-min coronary occlusion and 5-h reperfusion. Along with this reduced infarct size, the frequency of ectopic beats and the proportion of animals fibrillating during reperfusion were significantly reduced by cromakalim. In isolated globally ischemic and reperfused rat hearts, cromakalim was significantly profibrillatory. Thus, cromakalim is significantly cardioprotective, and may have the propensity for profibrillatory activity, although this is not true under all conditions.

Reduction of Skeletal Muscle Atrophy by a Proteasome Inhibitor in a Rat Model of Denervation

The ubiquitin-proteasome system is the primary proteolytic pathway implicated in skeletal muscle atrophy under catabolic conditions. Although several studies showed that proteasome inhibitors reduced proteolysis under catabolic conditions, few studies have demonstrated the ability of these inhibitors to preserve skeletal muscle mass and architecture in vivo. To explore this, we studied the effect of the proteasome inhibitor Velcade (also known as PS-341 and bortezomib) in denervated skeletal muscle in rats. Rats were given vehicle or Velcade (3 mg/kg po) daily for 7 days beginning immediately after induction of muscle atrophy by crushing the sciatic nerve. At the end of the study, the rats were euthanized and the soleus and extensor digitorum longus (EDL) muscles were harvested. In vehicle-treated rats, denervation caused a 33.5 ± 2.8% and 16.2 ± 2.7% decrease in the soleus and EDL muscle wet weights (% atrophy), respectively, compared to muscles from the contralateral (innervated) limb. Velcade significantly reduced denervation-induced atrophy to 17.1 ± 3.3% in the soleus (P < 0.01), a 51.6% reduction in atrophy associated with denervation, with little effect on the EDL (9.8 ± 3.2% atrophy). Histology showed a Preservation of muscle mass and preservation of normal cellular architecture after Velcade treatment. Ubiquitin mRNA levels in denervated soleus muscle at the end of the study were significantly elevated 120 ± 25% above sham control levels and were reduced to control levels by Velcade. In contrast, testosterone proprionate (3 mg/kg sc) did not alleviate denervation-induced skeletal muscle atrophy but did prevent castration-induced levator ani atrophy, while Velcade was without effect. These results show that proteasome inhibition attenuates denervation-induced muscle atrophy in vivo in soleus muscles. However, this mechanism may not be operative in all types of atrophy.

Glyburide-reversible cardioprotective effect of BMS-180448 is independent of action potential shortening

OBJECTIVE We determine if action potential duration (APD) shortening and cardioprotection are separable phenomena in ATP-sensitive potassium channel (KATP) openers which protect ischemic myocardium via a glyburide-reversible mechanism. METHODS We determined the effect of the weakly vasodilating KATP opener, BMS-180448, and the less cardiac-selective cromakalim, on APD in normal, hypoxic or ischemic guinea pig papillary muscles or isolated hearts and compared their APD effects with their cardioprotective activity in isolated guinea pig hearts. RESULTS In isolated ischemic guinea pig hearts, cromakalim and BMS-180448 had similar cardioprotective potencies (EC25 of 3.2 and 3.3 microM, respectively, for increasing time to the onset of contracture). At 10 microM, BMS-180448 produced no APD shortening, yet was equally protective at this concentration compared to cromakalim, which produced profound APD shortening under either hypoxic or ischemic conditions. The cardioprotective effects of both compounds at 10 microM were abolished by 0.3 microM glyburide. CONCLUSIONS APD shortening is not correlated with cardioprotective activity for BMS-180448 and cromakalim while their cardioprotective effects are abolished by glyburide. These results suggest the possibility of reduced proarrhythmic activity in some KATP openers and that their cardioprotective activity is not associated with sarcolemmal KATP opening.

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.

Preconditioning in rat hearts is independent of mitochondrial F1F0ATPase inhibition

Mitochondrial F1F0adenosinetriphosphatase (ATPase) is responsible for the majority of ATP synthesis during normoxic conditions, but under ischemic conditions it accounts for significant ATP hydrolysis. A previous study showed that preconditioning in isolated rat hearts is mediated by inhibition of this ATPase during ischemia. We tested this hypothesis in our isolated rat heart model of preconditioning. Preconditioning was accomplished by three 5-min periods of global ischemia separated by 5 min of reperfusion. This was followed by 20 min of global ischemia and 30 min of reperfusion. Preconditioning significantly enhanced reperfusion contractile function and reduced lactate dehydrogenase release but paradoxically reduced the time to onset of contracture during global ischemia. Myocardial ATP was depleted at a faster rate during the prolonged ischemia in preconditioned than in sham-treated hearts, which is consistent with the reduced time to contracture. ATP during reperfusion was repleted more rapidly in preconditioned hearts, which is consistent with their enhanced contractile function. Preconditioning significantly reduced lactate accumulation during the prolonged ischemia. We were not able to demonstrate that mitochondrial F1F0ATPase (measured in submitochondrial particles) was inhibited by preconditioning before or during the prolonged ischemia. The mitochondrial ATPase inhibitor oligomycin significantly conserved ATP during ischemia and increased the time to the onset of contracture, which is consistent with inhibition of the mitochondrial ATPase. Our results show that preconditioning in rat hearts can be independent of mitochondrial ATPase inhibition as well as ATP conservation.

Nicorandil improves postischemic contractile function independently of direct myocardial effects

We determined whether any of the antiischemic effects of nicorandil were due to direct cardioprotective effects such as potassium channel activation or to its peripheral hemodynamic effects. Nicorandil was administered either intravenously (i.v.) or directly into the ischemic coronary artery (i.c.) and compared with i.c. cromakalim (a potassium channel activator previously shown to improve reperfusion function directly in rat hearts) or vehicle for their ability to improve postischemic contractile function as measured by ultrasonic crystals in anesthetized dogs or in isolated perfused rat hearts. In a model of 25-min global ischemia and reperfusion in isolated perfused rat hearts, nicorandil (10–100 μM) did not improve reperfusion function or decrease LDH re-lease, although 300 μM nicorandil did protect the hearts. Cromakalim (7 μM)significantly improved reperfusion function and reduced lactate dehydrogenase (LDH) re-lease. In the dog studies, the left anterior descending coronary artery (LAD) was occluded for 15 min and was reperfused for 3 h. Nicorandil improved reperfusion function only when administered i.v., although i.c. cromakalim was efficacious in improving function. Neither nicorandil nor cromakalim improved collateral flow, although cromakalim significantly improved preischemic and reperfusion blood flows, particularly in the subepicardial region. Although i.c. treatment with cromakalim and nicorandil did not result in significant changes in peripheral hemodynamic status, i.v. nicorandil reduced both preload and afterload. Thus, at the dose used, nicorandil does not appear to have direct myocardial protective effects and the beneficial effects of nicorandil do not appear to be related to potassium channel activation in the myocardium. Potassium channel activation by cromakalim does result in direct cardioprotective effects whereas nicorandil appears to be dependent on peripheral actions for its efficacy.