Increases in myocardial cyclic GMP attenuate contractile delay in myocardial stunning

We tested the hypothesis that the effects of myocardial stunning would be reduced by cyclic GMP in rabbit hearts. In three groups of anesthetized open-chest New Zealand white rabbits, myocardial stunning was produced by 15 min of occlusion of the left anterior descending coronary artery followed by 15 min of reperfusion repeated twice. Either control vehicle (saline plus 1% dimethyl sulfoxide) or 8-bromo-cyclic GMP (8-Br-cGMP (10(-4) and 10(-3) M)) was topically applied to the left ventricular surface. Hemodynamic (left ventricular and aortic pressures) and functional parameters (wall thickening, delay in onset of wall thickening, and rate of wall thickening) were determined. Coronary blood flow (microspheres) and O2 extraction (microspectrophotometry) were used to determine myocardial O2 consumption (VO2). Myocardial stunning was observed in the control group through an increased delay in onset of myocardial wall thickening (29 +/- 7 versus 55 +/- 16 ms) and decreased maximal rate of wall thickening (20 +/- 8 versus 11 +/- 3 mm x s(-1)). After treatment with 8-Br-cGMP 10(-4) and 10(-3) M, stunning did not increase the delay (37 +/- 5 versus 39 +/- 7 and 39 +/- 7 versus 28 +/- 8 ms). Myocardial stunning did not significantly alter VO2. 8-Br-cGMP 10(-3) M significantly decreased subepicardial VO2 (6.2 +/- 0.8 versus 3.7 +/- 0.6 mL O2 x min(-1) 100 g(-1)) and insignificantly decreased subendocardial VO2 (8.6 +/- 0.9 versus 6.3 +/- 1.2 mL O2 x min(-1) x 100 g(-1)) when compared with the vehicle-treated rabbits. We conclude that increasing cyclic GMP reduced the effects of myocardial stunning in the rabbit heart by ameliorating the delay in onset of wall thickening and decreasing the local O2 costs in the stunned region.     

Nitroprusside attenuates myocardial stunning through reduced contractile delay and time

We hypothesized that myocardial stunning would be reversed through increased cyclic GMP caused by nitroprusside, and that this would be accomplished through a decreased proportion of regional work during diastole. Hearts were instrumented to measure left ventricular pressure, and regional myocardial mechanics were recorded using a miniature force transducer and ultrasonic dimension crystals in eight open-chest anesthetized dogs. Following baseline (CON), the left anterior descending coronary artery (LAD) was occluded for 15 min, followed by a 30-min recovery (STUN). Then intracoronary LAD infusion of sodium nitroprusside (NP) (4 microg/kg/ min) was begun. The time delay (msec) to regional shortening increased significantly from 18+/-13 to 73+/-13 following stunning, but was reduced to 49+/-18 by NP. Total regional work (g*mm/min) at baseline (1368+/-401 CON) was unchanged with stunning (1320+/-333 STUN), but reduced (961+/-240) following NP. Time to peak force development (msec) increased significantly with stunning from 284+/-13 (CON) to 333+/-11 (STUN), but was reduced to 269+/-12 following NP. The percentage work during systole was reduced from 96%+/-2% (CON) to 77%+/-7% (STUN), but returned to 98%+/-1% with NP. Regional O2 consumption was unaffected by either treatment. Cyclic GMP was unchanged by stunning (2.9+/-0.3-2.9+/-0.4 pmol/g) but increased significantly with NP (4.6+/-0.6). These data indicated that regional myocardial stunning could be attenuated by nitroprusside, which increased cyclic GMP, decreased contractile delay, increased the proportion of work done during systole, and reduced time of shortening.     

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.     

Energy-wasteful total Ca(2+) handling underlies increased O(2) cost of contractility in canine stunned heart

Postischemic myocardial stunning halved left ventricular contractility [end-systolic maximum elastance (E(max))] and doubled the O(2) cost of E(max) in excised cross-circulated canine heart. We hypothesized that this increased O(2) cost derived from energy-wasteful myocardial Ca(2+) handling consisting of a decreased internal Ca(2+) recirculation, some futile Ca(2+) cycling, and a depressed Ca(2+) reactivity of E(max). We first calculated the internal Ca(2+) recirculation fraction (RF) from the exponential decay component of postextrasystolic potentiation. Stunning significantly accelerated the decay and decreased RF from 0.63 to 0. 43 on average. We then combined the decreased RF with the halved E(max) and its doubled O(2) cost and analyzed total Ca(2+) handling using our recently developed integrative method. We found a decreased total Ca(2+) transport and a considerable shift of the relation between futile Ca(2+) cycling and Ca(2+) reactivity in an energy-wasteful direction in the stunned heart. These changes in total Ca(2+) handling reasonably account for the doubled O(2) cost of E(max) in stunning, supporting the hypothesis.     

Triiodothyronine concomitantly inhibits calcium overload and postischemic myocardial stunning in diabetic rats.

Acute effects of triiodothyronine (T3) on postischemic myocardial stunning and intracellular Ca2+ contents were studied in the isolated working hearts of streptozotocin-induced diabetic rats and age-matched controls. After two weeks of diabetes, serum T3 and T4 levels were decreased to 62.5% and 33.9% of control values. Basal preischemic cardiac performance did not differ between diabetic and control rats. In contrast, during reperfusion after 20-min ischemia, diabetic rats exhibited an impaired recovery of heart rate (at 30-min reperfusion 57.5% of baseline vs. control 88.5%), left ventricular (LV) systolic pressure (44.1% vs. 89.5%), and cardiac work (23.1% vs. 66.0%). When 1 and 100 nM T3 was added before ischemia, heart rate was recovered to 77.2% and 81.8% of baseline, LV systolic pressure to 68.3% and 81.9%, and cardiac work to 50.8% and 59.0%, respectively. Diabetic rat hearts showed a higher Ca2+ content in the basal state and a further increase after reperfusion (4.96+/-1.17 vs. control 3.78+/-0.48 micromol/g, p<0.01). In diabetic hearts, H+ release was decreased after reperfusion (5.24+/-2.21 vs. 8.70+/-1.41 mmol/min/g, p<0.05). T3 administration caused a decrease in the postischemic Ca2+ accumulation (lnM T3 4.66+/-0.41 and 100 nM T3 3.58+/-0.36) and recovered the H+ release (lnM T3 16.2+/-3.9 and 100 nM T3 11.6+/-0.9). T3 did not alter myocardial O2 consumption. Results suggest that diabetic rat hearts are vulnerable to postischemic stunning, and T3 protects the myocardial stunning possibly via inhibiting Ca2+ overload.     

Cardioprotection by adenosine A2A agonists in a canine model of myocardial stunning produced by multiple episodes of transient ischemia

We sought to determine whether administration of a very low, nonvasodilating dose of a highly selective adenosine A(2A) receptor agonist (ATL-193 or ATL-146e) would be cardioprotective in a canine model of myocardial stunning produced by multiple episodes of transient ischemia. Twenty-four anesthetized open-chest dogs underwent either 4 (n=12) or 10 cycles (n=12) of 5-min left anterior descending coronary artery (LAD) occlusions interspersed by 5 or 10 min of reperfusion. Left ventricular thickening was measured from baseline through 180 min after the last occlusion-reperfusion cycle. Regional flow was measured with microspheres. In 12 of 24 dogs, A(2A) receptor agonist was infused intravenously beginning 2 min prior to the first occlusion and continuing throughout reperfusion at a dose below that which produces vasodilatation (0.01 microg x kg(-1) x min(-1)). Myocardial flow was similar between control and A(2A) receptor agonist-treated animals, confirming the absence of A(2) receptor agonist-induced vasodilatation. During occlusion, there was severe dyskinesis with marked LAD zone thinning in all animals. After 180 min of reperfusion following the last cycle, significantly greater recovery of LAD zone thickening was observed in A(2A) receptor agonist-treated vs. control animals in both the 4-cycle (91 +/- 7 vs. 56 +/- 12%, respectively; P<0.05) and the 10-cycle (65 +/- 9 vs. 8 +/- 16%, respectively; P<0.05) occlusion groups. The striking amount of functional recovery observed with administration of low, nonvasodilating doses of adenosine A(2A) agonist ATL-193 or ATL-146e supports their further evaluation for the attenuation of postischemic stunning in the clinical setting.     

Role of nucleoside transport and purine release in a rabbit model of myocardial stunning

Previously, we have demonstrated the role of nucleoside transport and purine release in post-ischemic reperfusion injury (myocardial stunning) in several canine models of ischemia. Since rabbits are deficient of xanthine oxidase, it is not known whether selective blockade of purine release is beneficial in a rabbit model of coronary artery occlusion and reperfusion (stunning). Therefore, we determined the hemodynamic and metabolic correlates in response to myocardial stunning in the presence or absence of selective nucleoside transport blocker (p-nitrobenzylthioinosine, NBMPR) and adenosine deaminase inhibitor (erythro-9-(2-hydroxy-3-nonyl)adenine, EHNA).Sixty adult anaesthetized rabbits were surgically prepared for hemodynamic measurements. After stabilization period, the left anterior descending coronary artery was occluded for 15 min and reperfused for 30 min. Transmural myocardial biopsies were obtained from the ischemic LAD area and from the non-ischemic posterior (circumflex, CFX) segment of the myocardium.Rabbits (n = 60) were randomly assigned to either the control or the EHNA/NBMPR-treated group (n = 30 each). Each group was further divided to either functional or metabolic groups (n = 15 each subgroup). Each animal received intravenously 30 ml of either a vehicle solution or 100 M EHNA and 25 M NBMPR 10 min before ischemia.Although administration of EHNA/NBMPR did not affect the heart rate, it did cause mild hypotension (about 20-30%). Fifteen minutes of LAD occlusion resulted in significant ATP depletion and concomitant accumulation of nucleosides in both groups (p < 0.05 vs. baseline and non-ischemic CFX segment). AMP was higher in the LAD compared to the CFX segment. Significant accumulation of adenosine was observed in the treated group compared to the control group.It is concluded that EHNA/NBMPR induced site specific entrapment of adenosine of nucleoside transport in the rabbit heart, in vivo.     

Myofibrillar disruption in hypocontractile myocardium showing perfusion-contraction matches and mismatches

Chronically instrumented dogs underwent 2- or 5-h regional reductions in coronary flow that were followed, respectively, by balanced reductions in myocardial contraction and O(2) consumption ("hibernation") and persistently reduced contraction despite normal myocardial O(2) consumption ("stunning"). Previously unidentified myofibrillar disruption developed during flow reduction in both experimental models and persisted throughout the duration of reperfusion (2-24 h). Aberrant perinuclear aggregates that resembled thick filaments and stained positively with a monoclonal myosin antibody were present in 34 +/- 3.8% (SE) and 68 +/- 5.9% of "hibernating" and "stunned" subendocardial myocytes in areas subjected to flow reduction and in 16 +/- 2.5% and 44 +/- 7.4% of subendocardial myocytes in remote areas of the same ventricles. Areas of myofibrillar disruption also showed glycogen accretion and unusual heterochromatin clumping adjacent to the inner nuclear envelope. The degrees of flow reduction employed were sufficient to reduce regional myofibrillar creatine kinase activity by 25-35%, but troponin I degradation was not evident. The observed changes may reflect an early, possibly reversible, phase of the myofibrillar loss characteristic of hypocontractile myocardium in patients undergoing revascularization.     

Parathyroid hormone-related peptide improves contractile function of stunned myocardium in rats and pigs

The effect of synthetic parathyroid hormone (PTH)-related peptide [PTHrP(1-34)] on regional myocardial function was studied in 11 anesthetized pigs. Intracoronary infusion of PTHrP (cumulative dose: 14 +/- 1 microg) decreased coronary resistance to 33 +/- 2% of baseline (P < 0.05) and regional myocardial function to 90 +/- 3% of baseline (not significant). Ischemia-reperfusion alters the activity of several kinases and therefore possibly the myocardial effects of PTHrP. In stunned myocardium, induced by 20-min ischemia and 30-min reperfusion, the dose of PTHrP reducing coronary resistance to a minimum of 29 +/- 2% was decreased to 8 +/- 2 microg (P < 0.05). Regional myocardial function was no longer decreased but increased to 132 +/- 9% (P < 0.05). The increase in regional myocardial function during PTHrP was inversely related to baseline function at 30-min reperfusion in vivo (r = 0.9) as well as in myocytes isolated from stunned pig hearts (r = 0.7). In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, blockade of endogenous PTHrP by d-Trp(12)-Tyr(34)-PTH(7-34) attenuated the recovery of left ventricular developed pressure by 30 +/- 14% (P < 0.05). Thus endogenous and exogenous PTHrP impact on the function of stunned myocardium.     

Biochemical mechanism(s) of stunning in conscious dogs

The mechanism(s) underlying contractile dysfunction in cardiac stunning is not completely understood. The expression and/or the phosphorylation state of cardiac Ca(2+) homoeostasis-regulating proteins might be altered in stunning. We tested this hypothesis in a well-characterized model of stunning. Conscious dogs were chronically instrumented, and the left anterior descending artery (LAD) was occluded for 10 min. Thereafter, reperfusion of the LAD was initiated. Tissues from reperfused LAD (stunned) and Ramus circumflexus (control) areas were obtained when left ventricular regional wall thickening fraction had recovered by 50%. Northern and Western blotting revealed no differences in the expression of the following genes: phospholamban, calsequestrin, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, and the inhibitory subunit of troponin I (TnI). However, the phosphorylation state of TnI and phospholamban were reduced in the LAD area. Fittingly, cAMP levels were reduced by 28% (P < 0.05). It is concluded that the contractile dysfunction in cardiac stunning might be mediated in part by decreased levels of cAMP and subsequently a reduced phosphorylation state of phospholamban and TnI.     

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.     

Is reduced SERCA2a expression detrimental or beneficial to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a knockout mice

Recent studies have demonstrated that increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions, including myocardial ischemia-reperfusion (I/R). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in stunned hearts or in isolated myocardium following I/R. The goal of this study was to test how decreases in SERCA pump level/activity affect cardiac function following I/R. To address this question, we used a heterozygous SERCA2a knockout (SERCA2a+/-) mouse model with decreased SERCA pump levels and studied the effect of myocardial stunning (20-min ischemia followed by reperfusion) and infarction (30-min ischemia followed by reperfusion) following 60-min reperfusion. Our results demonstrate that postischemic myocardial relaxation was significantly impaired in SERCA2a+/- hearts with both stunning and infarction protocols. Interestingly, postischemic recovery of contractile function was comparable in SERCA2a+/- and wild-type hearts subjected to stunning. In contrast, following 30-min ischemia, postischemic contractile function was reduced in SERCA2a+/- hearts with significantly larger infarction. Rhod-2 spectrofluorometry revealed significantly higher diastolic intracellular Ca2+ in SERCA2a+/- hearts compared with wild-type hearts. Both at 30-min ischemia and 2-min reperfusion, intracellular Ca2+ levels were significantly higher in SERCA2a+/- hearts. Electron paramagnetic resonance spin trapping showed a similar extent of postischemic free-radical generation in both strains. These data provide direct evidence that functional SERCA2a level, independent of oxidative stress, is crucial for postischemic myocardial function and salvage during I/R.     

Mismatch of local blood flow and oxidative metabolism in stunned myocardium

Myocardial blood flow is spatially heterogeneous, reflecting nonuniform oxygen supply. Also, myocardial oxidative metabolism is spatially heterogeneous. The effects of acute ischemia and reperfusion on the relationship between local myocardial blood flow (LMF) and oxidative metabolism are still unknown. LMF was measured in isolated, blood-perfused rabbit hearts using colored microspheres and oxidation water labeled with 18O2 (H2(18)O). Three protocols were performed: 18O2-perfusion during normoxia (N; n=7), during early reperfusion (ER; 10 min, n=6), and late reperfusion (LR; 40 min, n=6) following 20 min no-flow ischemia. LMF and local H2(18)O residues were determined within defined myocardial samples (105+/-15 mg). For interindividual comparison, values were normalized to the mean of the individual experiment and expressed as percentages. LMF ranged from 18 to 193% (N), 12 to 250% (ER), and 11 to 180% (LR). The H2(18)O tissue residue ranged from 63 to 132% (N), 73 to 142% (ER) and 32 to 148% (LR). The correlation between LMF and local oxidative metabolism during N (r=0.77; n=56) was lost in the postischemic heart during ER and LR. LMF during N and ER were only weakly correlated (r=0.24; n=48), whereas LMF during N and LR correlated well (r=0.87; n=48). It is concluded that the heterogeneous LMF pattern at baseline is maintained in the stunned myocardium whereas that of local oxidative metabolism is not. Apart from the established mechanisms underlying myocardial stunning, a mismatch between local flow and oxidative metabolism might also contribute.     

Coronary smooth muscle reactivity to muscarinic stimulation after ischemia-reperfusion in porcine myocardial infarction.

This study tested whether ischemia-reperfusion alters coronary smooth muscle reactivity to vasoconstrictor stimuli such as those elicited by an adventitial stimulation with methacholine. In vitro studies were performed to assess the reactivity of endothelium-denuded infarct-related coronary arteries to methacholine (n = 18). In addition, the vasoconstrictor effects of adventitial application of methacholine to left anterior descending (LAD) coronary artery was assessed in vivo in pigs submitted to 2 h of LAD occlusion followed by reperfusion (n = 12), LAD deendothelization (n = 11), or a sham operation (n = 6). Endothelial-dependent vasodilator capacity of infarct-related LAD was assessed by intracoronary injection of bradykinin (n = 13). In vitro, smooth muscle reactivity to methacholine was unaffected by ischemia-reperfusion. In vivo, baseline methacholine administration induced a transient and reversible drop in coronary blood flow (9.6 +/- 4.6 to 1.9 +/- 2.6 ml/min, P < 0.01), accompanied by severe left ventricular dysfunction. After ischemia-reperfusion, methacholine induced a prolonged and severe coronary blood flow drop (9.7 +/- 7.0 to 3.4 +/- 3.9 ml/min), with a significant delay in recovery (P < 0.001). Endothelial denudation mimics in part the effects of methacholine after ischemia-reperfusion, and intracoronary bradykinin confirmed the existence of endothelial dysfunction. Infarct-related epicardial coronary artery shows a delayed recovery after vasoconstrictor stimuli, because of appropriate smooth muscle reactivity and impairment of endothelial-dependent vasodilator capacity.     

Determinants of contractile reserve in viable, chronically dysfunctional myocardium

There is considerable variability in the sensitivity of inotropic reserve to identify viability in chronically dysfunctional myocardium. This is partially related to the underlying pathophysiology, with more frequent contractile reserve in chronically stunned (with normal resting perfusion) than hibernating myocardium (with reduced flow). This study was undertaken to determine the physiological responses to transient and graded stimulation in chronically stunned and hibernating myocardium to define the relative roles of acute catecholamine desensitization and biphasic responses. Pigs were chronically instrumented with a fixed left anterior descending artery stenosis that resulted in chronically stunned myocardium after 2 mo. One month later, hibernating myocardium was confirmed by regional dysfunction (wall thickening, 3.2 +/- 0.3 vs. 5.5 +/- 5 mm in remote, P=0.01) with reduced resting flow (0.70 +/- 0.07 vs. 0.92 +/- 0.09 ml x min(-1) x g(-1) in remote, P=0.01) without infarction. Wall thickening in dysfunctional regions significantly increased during both graded and transient epinephrine stimulation in chronically stunned (from 3.6 +/- 0.3 to 5.6 +/- 0.5 and 4.9 +/- 0.5 mm, respectively) and hibernating myocardium (from 3.3 +/- 0.3 to 5.4 +/- 0.6 and 5.0 +/- 0.7 mm, respectively) and returned to baseline within 15 min. Although a biphasic response during graded stimulation was common, the subsequent decrement in function was small and similar in both groups (stunned, 0.7 +/- 0.2 mm; hibernating, 1.1 +/- 0.3 mm, P=0.25). We conclude that 1) the extent of contractile reserve during beta-adrenergic stimulation is similar in chronically stunned and hibernating myocardium, 2) there are no significant differences between the responses to transient compared with graded catecholamine stimulation, and 3) submaximal catecholamine stimulation does not induce additional stunning in either chronically stunned or hibernating myocardium.     

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.     

Cyclic GMP Reduces Ventricular Myocyte Stunning after Simulated Ischemia–Reperfusion

We tested the hypothesis that the second messenger activated by nitric oxide, cyclic GMP, would reduce the effects of myocyte stunning following simulated ischemia-reperfusion and that this was related to cyclic GMP protein kinase. Ventricular cardiac myocytes were isolated from New Zealand White rabbits (n = 8). Cell shortening was measured by a video edge detector and protein phosphorylation was determined autoradiographically after SDS gel electrophoresis. Cell shortening data were acquired at: (i) baseline followed by 8-Bromo-cGMP 10(-6) M (8-Br-cGMP) and then KT 5823 10(-6) M (cyclic GMP protein kinase inhibitor) and (ii) simulated ischemia (20 min of 95% N(2)-5% CO(2) at 37 degrees C) followed by simulated reperfusion (reoxygenation) with addition of 8-Br-cGMP 10(-6) M followed by KT 5823 10(-6) M, (iii) addition of 8-Br-cGMP prior to ischemia followed by the addition of KT 5823 10(-6) M after 30 min of reoxygenation. In the control group, 8-Br-cGMP 10(-6) M decreased percentage shortening (%short) (5.0 +/- 0.6 vs 3.8 +/- 0. 4) and the maximum velocity (V(max), microm/s) (48.6 +/- 6.9 vs 40.2 +/- 6.4). KT 5823 10(-6) M added after 8-Br-cGMP partially restored %short (4.6 +/- 0.5) and V(max) (46.6 +/- 8.0). After stunning, baseline myocytes had decreased %short (3.4 +/- 0.2) and V(max) (36. 0 +/- 4.2). After the addition of 8-Br-cGMP, the %short (2.7 +/- 0. 2) and V(max) (27.6 +/- 2.5) decreased further. The addition of KT 5823 did not change either the %short or the V(max). The myocytes with 8-Br-cGMP during ischemia had increased %short (4.2 +/- 0.2) and V(max) (37.2 +/- 3.4) when compared to the stunned group. The addition of KT 5823 did not significantly alter %short (3.3 +/- 0.4) or V(max) (29.2 +/- 5.0) in the myocytes pretreated with 8-Br-cGMP. Protein phosphorylation was increased by 8-Br-cGMP in control and stunned myocytes. KT 5823 blocked this effect in control but not stunned myocytes, suggesting some change in the cyclic GMP protein kinase. Ischemia-reperfusion produced myocyte stunning that was reduced when 8-Br-cGMP was added prior to but not after ischemia.     

Endothelial nitric oxide synthase (NOS3) knockout decreases NOS2 induction, limiting hyperoxygenation and conferring protection in the postischemic heart

Although it has been shown that endothelial nitric oxide synthase (eNOS)-derived nitric oxide downregulates mitochondrial oxygen consumption during early reperfusion, its effects on inducible NOS (iNOS) induction and myocardial injury during late reperfusion are unknown. Wild-type (WT) and eNOS(-/-) mice were subjected to 30 min of coronary ligation followed by reperfusion. Expression of iNOS mRNA and protein levels and peroxynitrite production were lower in postischemic myocardium of eNOS(-/-) mice than levels in WT mice 48 h postreperfusion. Significantly improved hemodynamics (+/-dP/dt, left ventricular systolic pressure, mean arterial pressure), increased rate pressure product, and reduced myocardial infarct size (18 +/- 2.5% vs. 31 +/- 4.6%) were found 48 h after reperfusion in eNOS(-/-) mice compared with WT mice. Myocardial infarct size was also significantly decreased in WT mice treated with the specific iNOS inhibitor 1400W (20.5 +/- 3.4%) compared with WT mice treated with PBS (33.9 +/- 5.3%). A marked reperfusion-induced hyperoxygenation state was observed by electron paramagnetic resonance oximetry in postischemic myocardium, but Po(2) values were significantly lower from 1 to 72 h in eNOS(-/-) than in WT mice. Cytochrome c-oxidase activity and NADH dehydrogenase activity were significantly decreased in postischemic myocardium in WT and eNOS(-/-) mice compared with baseline control, respectively, and NADH dehydrogenase activity was significantly higher in eNOS(-/-) than in WT mice. Thus deficiency of eNOS exerted a sustained beneficial effect on postischemic myocardium 48 h after reperfusion with preserved mitochondrial function, which appears to be due to decreased iNOS induction and decreased iNOS-derived peroxynitrite in postischemic myocardium.     

Physiological insulin concentrations protect against ischemia-induced loss of cardiac function in rats

This study determined whether insulin at pre- (fasting) and post-prandial concentrations increases coronary blood flow and improves cardiac function after acute ischemia during a situation of myocardial stunning. The experiments were performed using an isolated, erythrocyte perfused, working rat heart model. To the perfusate we added erythrocytes and 1.5% bovine serum albumin to improve clinical relevance. The following protocol was used: 8 min baseline performance assessment, 10 min pre-ischemic treatment, 12 min global ischemia, 20 min post-ischemic treatment and 8 min recovery assessment. Vehicle, 10 mIU l(-1) and 100 mIU l(-1) human insulin were tested (all n=6). No significant vasodilator response to insulin was observed either pre- or post-ischemically. After the 12-min ischemic insult, cardiac function returned dose-dependently to pre-ischemic values (function loss with 100 mIU l(-1) insulin: -0.2+/-0.4% vs. vehicle: 10.7+/-0.8%). This study clearly shows that in our clinically relevant model of moderate ischemia (stunning), insulin is highly cardioprotective at physiological concentrations. This may be explained primarily by higher glucose uptake, improving the myocardial energetic state during ischemia. Therefore, insulin should be considered for use when the myocardium is at acute risk for ischemic incidents.     

Acetaminophen is cardioprotective against H2O2-induced injury in vivo

Here we report our ongoing investigation of the cardiovascular effects of acetaminophen, with emphasis on oxidation-induced canine myocardial dysfunction. The objective of the current study was to investigate whether acetaminophen could attenuate exogenous H(2)O(2)-mediated myocardial dysfunction in vivo. Respiratory, metabolic, and hemodynamic indices such as left ventricular function (LVDP and +/-dP/dt(max)), and percent ectopy were measured in anesthetized, open-chest dogs during intravenous administration of 0.88 mM, 2.2 mM, 6.6 mM H(2)O(2). Following 6.6 mM H(2)O(2), tissue from the left ventricle was harvested for electron microscopy. Left ventricular function did not vary significantly between vehicle and acetaminophen groups under baseline conditions. Acetaminophen-treated dogs regained a significantly greater fraction of baseline function after high concentrations of H(2)O(2) than vehicle-treated dogs. Moreover, the incidence of H(2)O(2)-induced ventricular arrhythmias was significantly reduced in the acetaminophen-treated group. Percent ectopy following 6.6 mM concentrations of H(2)O(2) was 1 +/- 0.3 vs. 0.3 +/- 0.1 (P < 0.05) for vehicle- and acetaminophen-treated dogs, respectively. Additionally, electron micrograph images of left ventricular tissue confirmed preservation of tissue ultrastructure in acetaminophen-treated hearts when compared to vehicle. We conclude that, in the canine myocardium, acetaminophen is both functionally cardioprotective and antiarrhythmic against H(2)O(2)-induced oxidative injury.