Differential effects of postconditioning on myocardial stunning and infarction: a study in conscious dogs and anesthetized rabbits

Postconditioning, i.e., brief intermittent episodes of myocardial ischemia-reperfusion performed at the onset of reperfusion, reduces infarct size after prolonged ischemia. Our goal was to determine whether postconditioning is protective against myocardial stunning. Accordingly, conscious chronically instrumented dogs (sonomicrometry, coronary balloon occluder) were subjected to a control sequence (10 min coronary artery occlusion, CAO, followed by coronary artery reperfusion, CAR) and a week apart to postconditioning with four cycles of brief CAR and CAO performed at completion of the 10 min CAO. Three postconditioning protocols were investigated, i.e., 15 s CAR/15 s CAO (n=5), 30 s CAR/30 s CAO (n=7), and 1 min CAR/1 min CAO (n=6). Left ventricular wall thickening was abolished during CAO and similarly reduced during subsequent stunning in control and postconditioning sequences (e.g., at 1 h CAR, 33+/-4 vs. 34+/-4%, 30+/-4 vs. 30+/-4%, and 33+/-4 vs. 32+/-4% for 15 s postconditioning, 30 s postconditioning, and 1 min postconditioning vs. corresponding control, respectively). We confirmed this result in anesthetized rabbits by demonstrating that shortening of left ventricular segment length was similarly depressed after 10 min CAO in control and postconditioning sequences (4 cycles of 30 s CAR/30 s CAO). In additional rabbits, the same postconditioning protocol significantly reduced infarct size after 30 min CAO and 3 h CAR (39+/-7%, n=6 vs. 56+/-4%, n=7 of the area at risk in postconditioning vs. control, respectively). Thus, contrasting to its beneficial effects on myocardial infarction, postconditioning does not protect against myocardial stunning in dogs and rabbits. Conversely, additional episodes of ischemia-reperfusion with postconditioning do not worsen myocardial stunning.     

Effect of repeated episodes of reversible myocardial ischemia on myocardial blood flow and function in humans

Nine patients with coronary artery disease and normal left ventricular (LV) function underwent two episodes of dobutamine-induced ischemia to determine whether repeated episodes of ischemia lead to cumulative stunning. Positron emission tomography (PET) and oxygen 15-labeled H(2)O was used to assess myocardial blood flow (MBF) at baseline, peak stress, and after stress for each ischemic episode. Quantitative echocardiographic assessment of global ejection fraction (EF) and regional systolic function (SF) was performed at rest and regular intervals after dobutamine. SF was assessed for regions subtended by a coronary artery with a >70% diameter stenosis. Both EF and SF were more severely impaired 45 min after the second episode of stress compared with 45 min after the first (both P < 0.01), despite no difference in duration of the two dobutamine infusions or MBF at peak stress (1.72 vs. 1.69). After both episodes of ischemia, when LV function was impaired but subsequently recovered, MBF (1.15 +/- 0.39 and 1.20 +/- 0.43, respectively) was no different to baseline MBF (1.02 +/- 0.35), confirming that repeated episodes of dobutamine-induced ischemia lead to cumulative myocardial stunning.     

Late preconditioning enhances recovery of myocardial function after infarction in conscious rabbits

It is unknown whether late preconditioning (PC) enhances the recovery of left ventricular (LV) function after a myocardial infarction. Thus 25 conscious rabbits were subjected to a 30-min coronary occlusion followed by 28 days of reperfusion after PC 24 h earlier with either ischemia or nitric oxide donor administration [S-nitroso-N-acetylpenicillamine (SNAP)]. The recovery of wall thickening (WTh) after reperfusion was significantly improved in the ischemic PC and SNAP PC groups compared with controls, both at rest and during dobutamine stress. Interestingly, neither ischemia- nor SNAP-induced late PC attenuated myocardial stunning from day 1 through day 14. Infarct size was smaller in the ischemic PC and SNAP PC groups compared with controls. In all groups, WTh at 28 days was positively and linearly related to the percentage of viable tissue in the region underlying the ultrasonic crystal (r = 0.90), indicating that the improvement in LV function after both ischemia-induced and NO donor-induced late PC can be fully explained by the reduction in infarct size; a separate effect of late PC on LV remodeling or LV contractility need not be invoked. In conclusion, in conscious rabbits late PC, induced either by ischemia or pharmacologically, not only limits infarct size but also enhances the recovery of LV function after myocardial infarction. This finding has important clinical implications and provides triphenyltetrazolium chloride-independent evidence that late PC limits myocellular death after sustained ischemia.     

Role of Src protein tyrosine kinases in late preconditioning against myocardial infarction

Although Src protein tyrosine kinases (PTKs) have been shown to be essential in late preconditioning (PC) against myocardial stunning, their role in triggering versus mediating late PC against myocardial infarction remains unclear. Four groups of conscious rabbits were subjected to a 30-min coronary occlusion on day 2, with or without PC ischemia on day 1. Administration of the Src PTK inhibitor lavendustin A (LD-A; 1 mg/kg iv) before the PC ischemia on day 1 (group III, n = 7) failed to block the delayed protective effect against myocardial infarction 24 h later. Late PC against infarction, however, was completely abrogated when LD-A was given 24 h after the PC ischemia, prior to the 30-min occlusion on day 2 (group IV, n = 8). We conclude that, in conscious rabbits, Src PTK activity is necessary for the mediation of late PC protection against myocardial infarction on day 2, but not for the initiation of this phenomenon on day 1. Taken together with previous studies in the setting of stunning, these findings reveal heretofore unrecognized differences in the roles of Src PTKs in late PC against stunning versus late PC against infarction.     

Reduction in postsystolic wall thickening during late preconditioning

Brief coronary artery occlusion (CAO) and reperfusion induce myocardial stunning and late preconditioning. Postsystolic wall thickening (PSWT) also develops with CAO and reperfusion. However, the time course of PSWT during stunning and the regional function pattern of the preconditioned myocardium remain unknown. The goal of this study was to investigate the evolution of PSWT during myocardial stunning and its modifications during late preconditioning. Dogs were chronically instrumented to measure (sonomicrometry) systolic wall thickening (SWT), PSWT, total wall thickening (TWT = SWT + PSWT), and maximal rate of thickening (dWT/dt(max)). Two 10-min CAO (circumflex artery) were performed 24 h apart (day 0 and day 1, n = 7). At day 0, CAO decreased SWT and increased PSWT. During the first hours of the subsequent stunning, evolution of PSWT was symmetrical to that of SWT. At day 1, baseline SWT was similar to day 0, but PSWT was reduced (-66%), while dWT/dt(max) and SWT/TWT ratio increased (+48 and +14%, respectively). After CAO at day 1, stunning was reduced, indicating late preconditioning. Simultaneously vs. day 0, PSWT was significantly reduced, and dWT/dt(max) as well as SWT/TWT ratio were increased, i.e., a greater part of TWT was devoted to ejection. Similar decrease in PSWT was observed with a nonischemic preconditioning stimulus (rapid ventricular pacing, n = 4). In conclusion, a major contractile adaptation occurs during late preconditioning, i.e., the rate of wall thickening is enhanced and PWST is almost abolished. These phenotype adaptations represent potential approaches for characterizing stunning and late preconditioning with repetitive ischemia in humans.     

Differential role of K(ATP) channels in late preconditioning against myocardial stunning and infarction in rabbits

The role of ATP-sensitive potassium (K(ATP)) channels in the late phase of ischemic preconditioning (PC) remains unclear. Furthermore, it is unknown whether K(ATP) channels serve as end effectors both for late PC against infarction and against stunning. Thus, in phase I of this study, conscious rabbits underwent a 30-min coronary occlusion (O) followed by 72 h of reperfusion (R) with or without ischemic PC (6 4-min O/4-min R cycles) 24 h earlier. Late PC reduced infarct size approximately 46% versus controls. The K(ATP) channel blocker 5-hydroxydecanoic acid (5-HD), given 5 min before the 30-min O, abrogated the infarct-sparing effect of late PC but did not alter infarct size in non-PC rabbits. In phase II, rabbits underwent six 4-min O/4-min R cycles for 3 consecutive days (days 1, 2, and 3). In controls, the total deficit of systolic wall thickening (WTh) after the sixth reperfusion was reduced by 46% on day 2 and 54% on day 3 compared with day 1, indicating a late PC effect against myocardial stunning. Neither 5-HD nor glibenclamide, given on day 2, abrogated late PC. The K(ATP) channel opener diazoxide, given on day 1, attenuated stunning, and this effect was completely blocked by 5-HD. Thus the same dose of 5-HD that blocked the antistunning effect of diazoxide failed to block the antistunning effects of late PC. Furthermore, when diazoxide was administered in PC rabbits on day 2, myocardial stunning was further attenuated, indicating that diazoxide and late PC have additive anti-stunning effects. We conclude that K(ATP) channels play an essential role in late PC against infarction but not in late PC against stunning, revealing an important pathogenetic difference between these two forms of cardioprotection.     

Elevated MnSOD is not required for exercise-induced cardioprotection against myocardial stunning

Endurance exercise provides cardioprotection against ischemia-reperfusion-induced myocardial stunning and infarction. A recent study demonstrates that an exercise-induced increase in myocardial manganese superoxide dismutase (MnSOD) activity is essential to protect the heart against infarction. It is unknown if an elevation in cardiac MnSOD is also a prerequisite to achieve exercise-induced protection against myocardial stunning. Therefore, this study determined if an exercise-induced increase in myocardial MnSOD activity is a requirement to achieve protection against myocardial stunning. Adult male rats remained sedentary or performed successive bouts of endurance exercise. Hearts were exposed to 25 min of global ischemia followed by reperfusion in an isolated working heart preparation. Postischemic recovery of cardiac external work during reperfusion was significantly higher (84 +/- 3 vs. 67 +/- 4%) in exercised animals compared with sedentary controls. Furthermore, prevention of exercise-induced expression of myocardial MnSOD via antisense oligonucleotides did not retard this exercise-induced protection against myocardial stunning. These data demonstrate that exercise-induced increases in cardiac MnSOD activity are not essential to achieve exercise-mediated protection against myocardial stunning. Therefore, we conclude that different mediators are responsible for exercise-induced cardioprotection against myocardial stunning and infarction.     

Preconditioning of salvaged myocardium in conscious rabbits with postinfarction dysfunction

Protection against postinfarction myocardial dysfunction is modest with classic preconditioning (PC). We investigated whether multiple cycles of PC could improve this protection and whether postinfarction dysfunction only depends on the amount of viable tissue. Eighteen rabbits were chronically instrumented with coronary occluders and ultrasonic crystals (segment shortening, SH) in the ischemic zone. A control group underwent 30-min coronary artery occlusion (CAO) with 72-h reperfusion (CAR). In two other groups, PC was induced by six 4-min CAO/4-min CAR cycles (PCx6) or one 5-min CAO/10-min CAR cycle (PCx1). After 72-h CAR, depression in SH was reduced in PCx1 (-68 +/- 7% from baseline) and to a greater extent in PCx6 (-18 +/- 10%) vs. control (-99 +/- 7%; all P < 0.05). Infarct sizes were reduced in PCx1 (15 +/- 2%) and to a greater extent in PCx6 (3 +/- 1%) vs. control (46 +/- 5%; P < 0.05). Contractility of salvaged myocardium was evaluated by calculating the ratio between SH at 72-h CAR and the amount of viable tissue. This index was enhanced in PCx1 (0.39 +/- 0.07, P < 0.05) and to a greater extent in PCx6 (0.82 +/- 0.09) vs. control (0.0 +/- 0.10). This differential effect of PC was not related to changes in apoptosis, endothelial nitric oxide synthase (NOS) expression, or macrophages infiltration but, rather, to blunted inducible NOS expression in PCx6 vs. control and PCx1. Thus multiple cycles of PC induced an almost complete protection against postinfarction dysfunction, potentially involving beneficial effects on salvaged myocardium.     

Na(+)/H(+) exchange subtype 1 inhibition reduces endothelial dysfunction in vessels from stunned myocardium

Myocardial ischemia and reperfusion cause myocyte and vascular dysfunction, frequently termed "stunning." We hypothesized that inhibiting the Na(+)/H(+) exchanger subtype 1 isoform (NHE(1)) during ischemia and reperfusion limits myocardial and coronary microvascular stunning. Anesthetized rats completed 2 x 10-min coronary artery occlusions separated by 5-min of reperfusion, followed by 15 or 60 min of reperfusion. Vehicle (saline) or the NHE(1) inhibitor cariporide (HOE-642) was administered 15 min before ischemia and was continued throughout each protocol. After reperfusion, hearts were excised, and the reactivity of resistance arteries (internal diameter, approximately 120 microm) was assessed. The first derivative of left ventricular (LV) pressure, LV developed pressure, and LV systolic wall thickening were depressed (P < 0.05) similarly in vehicle- and cariporide-treated rats during ischemia and after 15 or 60 min of reperfusion compared with sham-operated animals that were not exposed to ischemia (i.e., controls). In vessels obtained after 15 min of reperfusion, the maximal response to acetylcholine-induced relaxation (10(-8)-10(-4) M) was blunted (P < 0.05) in vessels from vehicle- (approximately 35%) and cariporide-treated rats (approximately 55%) compared with controls (approximately 85%). However, the percent relaxation to acetylcholine was greater (P < 0.05) in cariporide-treated rats compared with vehicle-treated rats. Maximal contractile responses to endothelin-1 (10(-11)-10(-7) M) were increased (P < 0.05) similarly in vehicle- and cariporide-treated rats compared with controls. Relaxation to sodium nitroprusside (10(-4) M) was not different among groups. Results were similar in vessels obtained from animals after 60 min of reperfusion. These findings suggest that NHE(1) inhibition before coronary occlusion lessens ischemia-induced microvascular dysfunction for 15-60 min after reperfusion but does not alter myocardial contractile function in the area at risk.     

Mechanism of reversible (99m)Tc-sestamibi perfusion defects during pharmacologically induced vasodilatation

Reversible perfusion defects on (99m)Tc-sestamibi imaging during hyperemia are thought to occur due to myocardial blood flow (MBF) "mismatch" between regions with and without stenosis. We have recently shown that myocardial blood volume (MBV) distal to a stenosis decreases during hyperemia, resulting in a reversible perfusion defect on myocardial contrast echocardiography (MCE). In this study, we hypothesized that a reversible perfusion defect on (99m)Tc-sestamibi imaging during hyperemia results from the same mechanism. We tested our hypothesis under the following conditions: 1) increases in MBF in the absence of changes in MBV by using direct intracoronary infusion of adenosine (group I, n = 10 dogs); 2) decrease in MBV despite an increase in MBF by left main infusion of adenosine proximal to a noncritical coronary stenosis placed on either coronary artery (group II, n = 13 dogs); and 3) reduction in both resting MBF and MBV by placement of a severe stenosis (group III, n = 7 dogs). In group I dogs, no difference in MBV or (99m)Tc-sestamibi uptake was found between the two coronary beds despite an up to fourfold increase in MBF in one bed with adenosine. In group II dogs, MBV distal to the stenosis decreased during hyperemia despite a twofold increase in mean MBF. A good correlation was found between (99m)Tc-sestamibi uptake and MBV ratios from the stenosed versus normal bed (r = 0.91, P < 0.001). In group III dogs, both MBF and MBV were decreased in the stenosed bed at rest with a good correlation noted between (99m)Tc-sestamibi uptake and MBV ratios from the stenosed versus normal bed (r = 0.92, P = 0.004). We conclude that reversible defects on (99m)Tc-sestamibi during vasodilator stress imaging are related to decreases in MBV distal to a stenosis and not to "flow mismatch" between beds. The decrease in MBV results in reduced (99m)Tc-sestamibi uptake during hyperemia.     

Effects of ischemic preconditioning and adenosine pretreatment on myocardial function and energetics in a clinically relevant model

Preconditioning (PC) is a potential approach to myocardial protection. We hypothesize that brief ischemia or adenosine given prior to an extended period of warm ischemia may prevent myocardial stunning by altering myocardial metabolism. Using a global ischemia model, 19 dogs were subjected to no PC(control), two episodes of ischemia (2 min of global ischemia followed by 3 min of reperfusion) (IPC), or 30 min of pulmonary artery adenosine infusion (AP), to a maximum of 350 microg/kg/min, followed by 20 min of global warm ischemia on cardiopulmonary bypass. Left ventricular pressure-volume loops and myocardial oxygen consumption (MVO(2)) were measured at baseline and after 60 min of reperfusion, on right heart bypass. All data were compared between baseline and reperfusion. Load independent left ventricular function, defined as preload recruitable stroke work (PRSW), decreased in control and IPC groups (72+/-7%, 71+/-12%, respectively). AP blunted the decrease in PRSW (45+/-9%, p<.05 compared to control). Myocardial energetic conversion efficiency, defined as the slope of the MVO(2)-Stroke work relationship was not significantly changed for controls (2.17+/-0.47 to 1.84+/-0.68) and IPC (2.99+/-0.45 to 2.16+/-0.65), but was for AP (1.16+/-0.88 to 5.71+/-1.66, p<0.04). IPC did not prevent ventricular stunning or alter myocardial energetics. AP reduced ventricular stunning but resulted in worsened myocardial energy efficiency. The benefits to ventricular function of the adenosine pretreatment protocol used in this study were only possible at a cost of higher metabolic requirements.     

Protein tyrosine kinase signaling is necessary for NO donor-induced late preconditioning against myocardial stunning

Although protein tyrosine kinases (PTKs) signaling has been implicated in the late phase of ischemic preconditioning (PC), it is unknown whether PTK signaling is necessary for the development of nitric oxide (NO) donor-induced late PC. Thus conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles followed by a 5-h recovery period of reperfusion for 3 consecutive days (days 1, 2, and 3). On day 0 (24 h before the 6 O/R cycles on day 1), rabbits received no treatment (control), the NO donor diethylenetriamine (DETA)/NO (DETA/NO), the PTK inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), or DETA/NO plus PP2 (DETA/NO + PP2). In control rabbits (n = 6), the six O/R cycles on day 1 resulted in delayed functional recovery, indicating severe myocardial stunning. In rabbits pretreated with DETA/NO (n = 5) on day 1, myocardial stunning caused by the six O/R cycles on day 1 was markedly attenuated, with a significant reduction ( approximately 60%) in the total deficit of wall thickening (WTh) compared with controls, indicating that DETA/NO induced a late PC effect against stunning. However, in rabbits pretreated with DETA/NO + PP2 (n = 5), the total deficit of WTh was significantly greater than that in rabbits treated with DETA/NO alone and was similar to that in controls, indicating that PP2 prevented the development of DETA/NO-induced late PC. In rabbits pretreated with PP2 on day 0 (n = 4), the total deficit of WTh was similar to that in controls, indicating that PP2 does not affect myocardial stunning in itself. We conclude that a PTK-dependent signaling mechanism is necessary for the development of NO donor-induced late PC against myocardial stunning in conscious rabbits.     

Use of Spin Traps in Intact Animals Undergoing Myocardial Ischemia/Reperfusion: A New Approach to Assessing the Role of Oxygen Radicals in Myocardial “Stunning”

Numerous studies have indirectly, suggested that oxygen-derived free radicals play an important path-ogenetic role in the prolonged depression of contractile function observed in myocardium reperfused after reversible ischemia (myocardial “stunning”). In order to provide direct evidence for the oxy-radical hypothesis of stunning, we administered the spin trap, α-phenyl N-tert-butyl nitrone (PBN), to open-chest dogs undergoing a 15-min coronary artery occlusion followed by reperfusion. Plasma of local coronary venous blood was analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR signals characteristic of radical adducts of PBN appeared during ischemia and increased dramatically in the first few minutes after reperfusion. After this initial burst, the production of adducts abated but did not cease, persisting up to 3 h after reflow. The production of PBN adducts after reperfusion was inversely related to collateral flow during ischemia. PBN itself enhanced recovery of contractile function. indicating that the radicals trapped may play a pathogenetic role in myocardial stunning. Superoxide dismutase plus catalase attenuated PBN adduct production and, at the same time, improved recovery of contractile function. Antioxidant therapy given 1 min before reperfusion suppressed PBN adduct production and improved contractile recovery; however, the same therapy given 1 min after reperfusion did not suppress early radical production and did not attenuate contractile dysfunction. After i.v. administration, the elimination half-life of PBN was estimated to be approximately 4–5 h. The results demonstrate that 1) free radicals are produced in the stunned myocardium in intact animals; 2) inhibition of free radical production results in improved contractile recovery; and 3) the free radicals important in causing dysfunction are produced in the first few minutes of reperfusion. Taken together, these studies provide cogent evidence supporting the oxy-radical hypothesis of stunning in open-chest dogs. It is now critical to determine whether these results can be reproduced in conscious animal preparations.     

Dissociation between myocardial relaxation and diastolic stiffness in the stunned heart: its prevention by ischemic preconditioning

The effects of myocardial stunning and ischemic preconditioning on left-ventricular developed pressure and end-diastolic pressure (diastolic stiffness) as well as on coronary-perfusion pressure were examined in isolated isovolumic rabbit hearts. The isovolumic relaxation was evaluated, and the time constant of pressure decay during the isovolumic period was calculated. Our experimental protocol comprised: 1) myocardial stunning-global ischemia (15 min) followed by reperfusion (30 min); 2) myocardial stunning-global ischemia (20 min) followed by reperfusion (30 min); and 3) ischemic preconditioning — a single cycle of brief global ischemia and reperfusion (5 min each), before a second ischemic period, of 20-min duration. There was no effect upon systolic and diastolic parameters when 15 and 20 minutes of ischemia were evaluated. In both stunned groups the left ventricular developed pressure first recovered to near control values, but then stabilized at only 60% of the control values. Whereas the isovolumic relaxation time constant was increased after 5 min of reperfusion, and return to control values at late reperfusion, the end diastolic pressure remained elevated during the entire period. Values of dP/dV calculated at common pressure levels, were used as a second index of diastolic stiffness. They were increased after stunning, as also was the coronary perfusion pressure. When the heart was preconditioned with a single episode of ischemia, the systolic and diastolic alterations were completely abolished. We thus concluded that diastolic abnormalities incurred by myocardial stunning consist in both an increase in diastolic stiffness and an early impairment of isovolumic relaxation. The increase in stiffness cannot result from incomplete relaxation since these two parameters become temporally dissociated during the reperfusion period.     

Molecular mechanism of preconditioning

During the last 20 years, since the appearance of the first publication on ischemic preconditioning (PC), our knowledge of this phenomenon has increased exponentially. PC is defined as an increased tolerance to ischemia and reperfusion induced by previous sublethal period ischemia. This is the most powerful mechanism known to date for limiting the infract size. This adaptation occurs in a biphasic pattern (i) early preconditioning (lasts for 2-3 h) and (ii) late preconditioning (starting at 24 h lasting until 72-96 h after initial ischemia). Early preconditioning is more potent than delayed preconditioning in reducing infract size. Late preconditioning attenuates myocardial stunning and requires genomic activation with de novo protein synthesis. Early preconditioning depends on adenosine, opioids and to a lesser degree, on bradykinin and prostaglandins, released during ischemia. These molecules activate G-protein-coupled receptor, initiate activation of K(ATP) channel and generate oxygen-free radicals, and stimulate a series of protein kinases, which include protein kinase C, tyrosine kinase, and members of MAP kinase family. Late preconditioning is triggered by a similar sequence of events, but in addition essentially depends on newly synthesized proteins, which comprise iNOS, COX-2, manganese superoxide dismutase, and possibly heat shock proteins. The final mechanism of PC is still not very clear. The present review focuses on the possible role signaling molecules that regulate cardiomyocyte life and death during ischemia and reperfusion.     

The intermediary metabolite pyruvate attenuates stunning and reduces infarct size in in vivo porcine myocardium

The intermediary metabolite pyruvate has been shown to exert significant beneficial effects in in vitro models of myocardial oxidative stress and ischemia-reperfusion injury. However, there have been few reports of the ability of pyruvate to attenuate myocardial stunning or reduce infarct size in vivo. This study tested whether supraphysiological levels of pyruvate protect against reversible and irreversible in vivo myocardial ischemia-reperfusion injury. Anesthetized, open-chest pigs (n = 7/group) underwent 15 min of left anterior descending coronary artery (LAD) occlusion and 3 h of reperfusion to induce stunning. Load-insensitive contractility measurements of regional preload recruitable stroke work (PRSW) and PRSW area (PRSWA) were generated. Vehicle or pyruvate (100 mg/kg i.v. bolus + 10 mg x kg(-1) x min(-1) intra-atrial infusion) was administered during ischemia and for the first hour of reperfusion. In infarct studies, pigs (n = 6/group) underwent 1 h of LAD ischemia and 3 h of reperfusion. Group I pigs received vehicle or pyruvate for 30 min before and throughout ischemia. In group II, the infusion was extended through 1 h of reperfusion. In the stunning protocol, pyruvate significantly improved the recovery of PRSWA at 1 h (50 +/- 4% vs. 23 +/- 3% in controls) and 3 h (69 +/- 5% vs. 39 +/- 3% in controls) reperfusion. Control pigs exhibited infarct sizes of 66 +/- 1% of the area at risk. The pyruvate I protocol was associated with an infarct size of 49 +/- 3% (P < 0.05), whereas the pyruvate II protocol was associated with an infarct size of 30 +/- 2% (P < 0.05 vs. control and pyruvate I). These findings suggest that pyruvate attenuates stunning and decreases myocardial infarction in vivo in part by reduction of reperfusion injury. Metabolic interventions such as pyruvate should be considered when designing the optimal therapeutic strategies for limiting myocardial ischemia-reperfusion injury.     

A highly potent peptide analgesic that protects against ischemia-reperfusion-induced myocardial stunning

We recently discovered an opioid peptide analgesic, 2',6'-dimethyltyrosine (Dmt)-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA), that can protect against ischemia-induced myocardial stunning. In buffer-perfused hearts, 30-min global ischemia followed by reperfusion resulted in a significant increase in norepinephrine (NE) overflow immediately upon reperfusion and significant decline in contractile force (45%). Pretreatment with [Dmt(1)]DALDA before ischemia completely abolished myocardial stunning and significantly reduced NE overflow (68%). In contrast, pretreatment with morphine before ischemia only provided brief protection against myocardial stunning and no reduction in NE overflow. [Dmt(1)]DALDA inhibited [(3)H]NE uptake into cardiac synaptosomes in vitro (IC(50) = 3.9 microM), whereas morphine had no effect. Surprisingly, protection against myocardial stunning was apparent even when hearts were perfused with [Dmt(1)]DALDA only upon reperfusion, whereas reperfusion with morphine had no effect. Binding studies with [(3)H][Dmt(1)]DALDA revealed no high-affinity specific binding in cardiac membranes, suggesting that the cardioprotective actions of [Dmt(1)]DALDA are not mediated via opioid receptors. These findings suggest that [Dmt(1)]DALDA is a potent analgesic that may be useful for myocardial stunning resulting from cardiac interventions or myocardial ischemia.     

The effect of delayed preconditioning on connexin 43 in ischemic myocardium.

The objective of this study is to investigate the effects of preconditioning on the restoration and distribution of connexin 43 (Cx43) in ischemic myocardium in dogs. In this study, 40 dogs were randomly divided into 5 groups of 8 as follows: control, 0hI-R (ischemia followed by 0 h reperfusion), 6hI-R (ischemia followed by 6 h reperfusion), 24hI-R (ischemia followed by 24 h reperfusion), and 48hI-R (ischemia followed by 48 h reperfusion). Four dogs in each group were preconditioned with brief episodes of ischemia prior to the respective treatments and were referred as the PC groups, while the other 4 were not preconditioned and were referred as the nonPC groups. The myocardial ischemia was induced by ligation of the left anterior descending coronary artery. The expression and distribution of Cx43 within the ischemic myocardium were measured by Western blot analysis and studied using laser confocal microscopy using a double-label immunohistochemistry technique. Compared with the control group, there was a significant reduction in Cx43 content within ischemic myocardium of all test groups both with and without PC (P < 0.01, P < 0.05). Within the 0hI-R, 6hI-R, and 24hI-R groups, an insignificant difference was found in the expression and distribution of Cx43 within the ischemic region between the PC and the nonPC groups. However, in the 48hI-R group, the area and intensity of Cx43 staining within the ischemic region of the PC dogs were significantly larger and more intense than those of the nonPC dogs (P < 0.01), and the ratio of Cx43 pixel density in intercalated disk areas to that in side-to-side junction areas in the PC dogs was significantly greater than that in nonPC dogs (P < 0.01). Our results suggest that preconditioning has a significant effect on the restoration and distribution of Cx43 in the ischemic myocardium in dogs after 48 h. Hence, preconditioning may be a plausible cause for the observed reductions in cardiac arrhythmias.     

Loss of exercise-induced cardioprotection after cessation of exercise.

Endurance exercise provides cardioprotection against ischemia-reperfusion (I/R) injury. Exercise-induced cardioprotection is associated with increases in cytoprotective proteins, including heat shock protein 72 (HSP72) and increases in antioxidant enzyme activity. On the basis of the reported half-life of these putative cardioprotective proteins, we hypothesized that exercise-induced cardioprotection against I/R injury would be lost within days after cessation of exercise. To test this, male rats (4 mo) were randomly assigned to one of five experimental groups: 1). sedentary control, 2). exercise followed by 1 day of rest, 3). exercise followed by 3 days of rest, 4). exercise followed by 9 days of rest, and 5). exercise followed by 18 days of rest. Exercise-induced increases (P < 0.05) in left ventricular catalase activity and HSP72 were evident at 1 and 3 days postexercise. However, at 9 days postexercise, myocardial HSP72 and catalase levels declined to sedentary control values. To evaluate cardioprotection during recovery from I/R, hearts were isolated, placed in working heart mode, and subjected to 20.5 min of global ischemia followed by 30 min of reperfusion. Compared with sedentary controls, exercised animals sustained less I/R injury as evidenced by maintenance of a higher (P < 0.05) percentage of preischemia cardiac work during reperfusion at 1, 3, and 9 days postexercise. The exercise-induced cardioprotection vanished by 18 days after exercise cessation. On the basis of the time course of the loss of cardioprotection and the return of HSP72 and catalase to preexercise levels, we conclude that HSP72 and catalase are not essential for exercise-induced protection during myocardial stunning. Therefore, other cytoprotective molecules are responsible for providing protection during I/R.     

Delta-opioid receptor-induced late preconditioning is mediated by cyclooxygenase-2 in conscious rabbits

Although activation of delta-opioid receptors is known to induce both early and late preconditioning (PC) against myocardial infarction, the mechanisms for this salubrious effect are unclear. Furthermore, it is unknown whether delta-opioid receptors can also induce late PC against myocardial stunning. By using conscious rabbits (n = 120) in this study, we found that the delta-opioid receptor agonist (+/-)-4-[(alpha-R*)-alpha-[(2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide (BW-373U86) induced late PC against myocardial stunning 24 h after treatment and that this effect was abolished by the selective cyclooxygenase-2 (COX-2) inhibitors N-[2-(cyclohexyloxy)4-nitrophenyl]methanesulfonamide (NS-398) and celecoxib. This protective effect was also abrogated by the selective delta(1)-opioid receptor antagonist 7-benzylidenenaltrexone, indicating that the delta(1)-opioid receptor is necessary for BW-373U86-induced late PC. BW-373U86 did not induce early PC against stunning. In addition, BW-373U86 induced late PC against infarction, which was blocked by NS-398. At 24 h after BW-373U86 administration, myocardial COX-2 protein expression and PGE(2) and 6-keto-PGF(1alpha) levels were significantly increased. These results demonstrate that activation of delta-opioid receptors induces late PC against both stunning and infarction via a COX-2-dependent mechanism.