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.     

Release of tissue-specific proteins into coronary perfusate as a model for biomarker discovery in myocardial ischemia/reperfusion injury

Diagnosis of acute coronary syndromes is based on protein biomarkers, such as the cardiac troponins (cTnI/cTnT) and creatine kinase (CK-MB) that are released into the circulation. Biomarker discovery is focused on identifying very low abundance tissue-derived analytes from within albumin-rich plasma, in which the wide dynamic range of the native protein complement hinders classical proteomic investigations. We employed an ex vivo rabbit model of myocardial ischemia/reperfusion (I/R) injury using Langendorff buffer perfusion. Nonrecirculating perfusate was collected over a temporal profile of 60 min reperfusion following brief, reversible ischemia (15 min; 15I/60R) for comparison with irreversible I/R (60I/60R). Perfusate proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS), revealing 26 tissue-specific proteins released during reperfusion post-15I. Proteins released during irreversible I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional gel electrophoresis coupled to liquid chromatography and tandem mass spectrometry; geLC-MS) and gel-free (LC-MS/MS) methods. A total of 192 tissue-specific proteins were identified during reperfusion post-60I. Identified proteins included those previously associated with I/R (myoglobin, CK-MB, cTnI, and cTnT), in addition to examples currently under investigation in large cohort studies (heart-type fatty acid binding protein; FABPH). The postischemic release profile of a novel cardiac-specific protein, cysteine and glycine-rich protein 3 (Csrp3; cardiac LIM domain protein) was validated by Western blot analysis. We also identified Csrp3 in serum from 6 of 8 patients postreperfusion following acute myocardial infarction. These studies indicate that animal modeling of biomarker release using ex vivo buffer perfused tissue to limit the presence of obfuscating plasma proteins may identify candidates for further study in humans.     

Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systems

Brief periods of myocardial ischemia prior to timely reperfusion result in prolonged, yet reversible, contractile dysfunction of the myocardium, or "myocardial stunning". It has been hypothesized that the delayed recovery of contractile function in stunned myocardium reflects damage to one or a few key sarcomeric proteins. However, damage to such proteins does not explain observed physiological alterations to myocardial oxygen consumption and ATP requirements observed following myocardial stunning, and therefore the impact of alterations to additional functional groups is unresolved. We utilized two-dimensional gel electrophoresis and mass spectrometry to identify changes to the protein profiles in whole cell, cytosolic- and myofilament-enriched subcellular fractions from isolated, perfused rabbit hearts following 15 min or 60 min low-flow (1 mL/min) ischemia. Comparative gel analysis revealed 53 protein spot differences (> 1.5-fold difference in visible abundance) in reperfused myocardium. The majority of changes were observed to proteins from four functional groups: (i) the sarcomere and cytoskeleton, notably myosin light chain-2 and troponin C; (ii) redox regulation, in particular several components of the NADH ubiquinone oxidoreductase complex; (iii) energy metabolism, encompassing creatine kinase; and (iv) the stress response. Protein differences appeared to be the result of isoelectric point shifts most probably resulting from chemical modifications, and molecular mass shifts resulting from proteolytic or physical fragmentation. This is consistent with our hypothesis that the time course for the onset of injury associated with myocardial stunning is too brief to be mediated by large changes to gene/protein expression, but rather that more subtle, rapid and potentially transient changes are occurring to the proteome. The physical manifestation of stunned myocardium is therefore the likely result of the summed functional impairment resulting from these multiple changes, rather than a result of damage to a single key protein.     

Effect of myocardial stunning on thiol status,myofibrillar ATPase and troponin I proteolysis

To investigate the mechanism underlying postischemic contractile dysfunction (myocardial stunning) we examined myocardial sulfhydryl group content, myofibrillar Ca²⁺-dependent Mg²⁺-ATPase activity and protein profile after global ischemia and reperfusion. The Langerdorff-perfused rabbit hearts were subjected to 15 min normothermic ischemia followed by 10 min reperfusion and myofibrils were isolated from homogenates of left ventricular tissues. Depressed contractile function during reperfusion was accompanied by a decrease in total sulfhydryl group content. However, myofibrillar protein profile was unchanged and Western immunoblotting analysis showed no significant differences in troponin I immunoreactive bands between control and stunned hearts. Likewise, myofibrillar Mg²⁺-ATPase activity was unaltered after ischemia and reperfusion. We conclude that myocardial stunning is not caused by altered myofibrillar function and protein degradation but may be partly due to the oxidative modification of as yet undefined proteins.     

The PPAR-alpha activator fenofibrate fails to provide myocardial protection in ischemia and reperfusion in pigs

Rodent studies suggest that peroxisome proliferator-activated receptor-alpha (PPAR-alpha) activation reduces myocardial ischemia-reperfusion (I/R) injury and infarct size; however, effects of PPAR-alpha activation in large animal models of myocardial I/R are unknown. We determined whether chronic treatment with the PPAR-alpha activator fenofibrate affects myocardial I/R injury in pigs. Domestic farm pigs were assigned to treatment with fenofibrate 50 mg.kg(-1).day(-1) orally or no drug treatment, and either a low-fat (4% by weight) or a high-fat (20% by weight) diet. After 4 wk, 66 pigs underwent 90 min low-flow regional myocardial ischemia and 120 min reperfusion under anesthetized open-chest conditions, resulting in myocardial stunning. The high-fat group received an infusion of triglyceride emulsion and heparin during this terminal experiment to maintain elevated arterial free fatty acid (FFA) levels. An additional 21 pigs underwent 60 min no-flow ischemia and 180 min reperfusion, resulting in myocardial infarction. Plasma concentration of fenofibric acid was similar to the EC50 for activation of PPAR-alpha in vitro and to maximal concentrations achieved in clinical use. Myocardial expression of PPAR-alpha mRNA was prominent but unaffected by fenofibrate treatment. Fenofibrate increased expression of carnitine palmitoyltransferase (CPT)-I mRNA in liver and decreased arterial FFA and lactate concentrations (each P < 0.01). However, fenofibrate did not affect myocardial CPT-I expression, substrate uptake, lipid accumulation, or contractile function during low-flow I/R in either the low- or high-fat group, nor did it affect myocardial infarct size. Despite expression of PPAR-alpha in porcine myocardium and effects of fenofibrate on systemic metabolism, treatment with this PPAR-alpha activator does not alter myocardial metabolic or contractile responses to I/R in pigs.     

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.     

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.     

Lack of the effect of superoxide dismutase and catalase on Na+,K+-ATPase activity in stunned rabbit hearts

Reactive oxygen species (ROS) have been implicated in the mechanism of postischemic contractile dysfunction, known as myocardial stunning. In this study, we examined protective effects of antioxidant enzymes, superoxide dismutase (SOD) and catalase, against ischemia/reperfusion-induced cardiac dysfunction and inhibition of Na+,K+-ATPase activity. Isolated Langendorff-perfused rabbit hearts were subjected to 15 min of global normothermic ischemia followed by 10 min reperfusion. The hearts treated with SOD plus catalase did not show significant recovery of left ventricular (LV) end-diastolic pressure compared with untreated ischemic reperfused hearts. Treatment with antioxidants had no protective effects on developed LV pressure or its maximal positive and negative first derivatives (+/-LVdP/dt). Myocardial stunning was accompanied by significant loss in sarcolemmal Na+,K+-ATPase activity and thiol group content. Inhibition of enzyme activity and oxidation of SH groups were not prevented by antioxidant enzymes. These results suggest that administration of SOD and catalase in perfusate do not protect significantly against cardiac dysfunction in stunned rabbit myocardium.     

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.     

Involvement of adrenergic pathways in activation of catalase by myocardial ischemia-reperfusion

In situ rabbit hearts were subjected to 15 min of regional myocardial ischemia, and at various time points of reperfusion, antioxidant enzyme activity and mRNA expression were measured in ischemic and nonischemic myocardium. Catalase activity increased significantly in both ischemic and nonischemic myocardium, peaking at 1 h after reperfusion and then gradually returning to the control level. Northern blot analysis showed enhanced expression of catalase mRNA in both areas. There were no changes in redox status, because glutathione levels were not altered by ischemia-reperfusion (I/R). We also tested whether catalase activation in the heart results from signaling pathways that might influence not only the heart but also other organs. We found that catalase activity in the brain was increased after myocardial I/R and ischemic stress to the intestine was equipotent to myocardial I/R in catalase activation. We next sought to elucidate the possible involvement of the adrenergic system in catalase stimulation induced by ischemic stimuli. After pretreatment with the alpha-adrenergic receptor antagonist prazosin, I/R failed to increase catalase activity in the heart and brain. Intravenous norepinephrine increased catalase activity in the heart, brain, and liver. This study shows that brief I/R activates a signaling mechanism to induce catalase activation in multiple organs and the alpha-adrenergic system is involved as an intermediate pathway in this signal transmission.     

下肢骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响

目的:探讨在体情况下,骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响。方法:新西兰大白兔36只,随机分成3组(每组随机选取6只进行梗死范围的测定,另外6只进行凋亡测定):①假手术组(Sham组);②缺血/再灌注组(I/R组);③远端后处理组(RPostC组)。在缺血前、后及再灌注60 min、120 min分别抽血测定肌酸激酶(CK),乳酸脱氢酶(LDH)的活性。采用伊文思兰(evans blue)和三苯基氯化四氮唑(TTC)染色方法确定心肌缺血区范围以及心肌坏死区范围。用Tunel法检测兔心肌缺血区细胞凋亡情况,免疫组织化学方法检测心肌缺血区蛋白caspase-3、Bcl-2及Bax的表达。结果:RPostC组心肌坏死程度、再灌注末CK活性较I/R组明显减低。RPostC组缺血区心肌Tunel阳性指数显著低于I/R组(21.79%±1.07%vs35.81%±1.10%,P<0.05)。而RPostC组缺血区心肌细胞caspase-3阳性指数显著低于I/R组(25.03%±1.16%vs39%±2.43%,P<0.05)。与Sham组比较,I/R组及RPostC组Bax蛋白表达指数、Bcl-2蛋白表达指数均升高;但RPostC组的Bax/Bcl-2比值降低,而I/R组的Bax/Bcl-2比值升高。与I/R组相比较,RPostC组Bax蛋白表达指数及Bax/Bcl-2比值显著降低,Bcl-2表达指数显著升高,差异均有统计学意义。结论:远端后处理能够明显的减少缺血/再灌注心肌细胞的坏死和凋亡,其减轻心肌细胞凋亡的机制可能与抑制促凋亡基因caspase-3的活化及Bcl-2表达的上调有关。     

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.     

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 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.     

High mobility box 1 mediates neutrophil recruitment in myocardial ischemia-reperfusion injury through toll like receptor 4-related pathway

This study aimed to explore the role of high mobility box 1 (HMGB1) and its receptor toll like receptor 4 (TLR4) on neutrophils in myocardial ischemia reperfusion (I/R) injury. We constructed TLR4-mutant (C3H/HeJ) and control (C3H/HeN) mouse models of myocardial I/R injury and subjected the mice to 30min of ischemia and 6h of reperfusion. Light microscope was used to observe structural changes in the myocardium. HMGB1 levels were measured using quantitative real-time PCR and immunohistochemistry. Neutrophil accumulation, TNF-a expression and IL-8 levels were analyzed via myeloperoxidase (MPO) biochemical studies, quantitative real-time PCR and ELISA, respectively. The results demonstrated that fewer neutrophils infiltrated in the myocardium of TLR4-mutant mice after myocardial I/R and that TLR4 deficiency markedly decreased the ischemic injury caused by ischemia/reperfusion, and inhibited the expression of HMGB1, TNF-a, and IL-8, all of which were up-regulated by ischemia/reperfusion. These findings suggest that HMGB1 plays a central role in recruiting neutrophils during myocardial I/R leading to worsened myocardial I/R injury. This recruitment mechanism is possibly due to its inflammatory and chemokine functions based on the TLR4-dependent pathway.     

Overexpression of the Na+/H+ exchanger and ischemia-reperfusion injury in the myocardium

In the myocardium, the Na(+)/H(+) exchanger isoform-1 (NHE1) activity is detrimental during ischemia-reperfusion (I/R) injury, causing increased intracellular Na(+) (Na(i)(+)) accumulation that results in subsequent Ca(2+) overload. We tested the hypothesis that increased expression of NHE1 would accentuate myocardial I/R injury. Transgenic mice were created that increased the Na(+)/H(+) exchanger activity specifically in the myocardium. Intact hearts from transgenic mice at 10-15 wk of age showed no change in heart performance, resting intracellular pH (pH(i)) or phosphocreatine/ATP levels. Transgenic and wild-type (WT) hearts were subjected to 20 min of ischemia followed by 40 min of reperfusion. Surprisingly, the percent recovery of rate-pressure product (%RPP) after I/R improved in NHE1-overexpressing hearts (64 +/- 5% vs. 41 +/- 5% in WT; P < 0.05). In addition, NMR spectroscopy revealed that NHE1 overexpressor hearts contained higher ATP during early reperfusion (levels P < 0.05), and there was no difference in Na(+) accumulation during I/R between transgenic and WT hearts. HOE642 (cariporide), an NHE1 inhibitor, equivalently protected both WT and NHE1-overexpressing hearts. When hearts were perfused with bicarbonate-free HEPES buffer to eliminate the contribution of HCO(3)(-) transporters to pH(i) regulation, there was no difference in contractile recovery after reperfusion between controls and transgenics, but NHE1-overexpressing hearts showed a greater decrease in ATP during ischemia. These results indicate that the basal activity of NHE1 is not rate limiting in causing damage during I/R, therefore, increasing the level of NHE1 does not enhance injury and can have some small protective effects.     

Intermittent hypobaric hypoxia improves postischemic recovery of myocardial contractile function via redox signaling during early reperfusion

Intermittent hypobaric hypoxia (IHH) protects hearts against ischemia-reperfusion (I/R) injury, but the underlying mechanisms are far from clear. ROS are paradoxically regarded as a major cause of myocardial I/R injury and a trigger of cardioprotection. In the present study, we investigated whether the ROS generated during early reperfusion contribute to IHH-induced cardioprotection. Using isolated perfused rat hearts, we found that IHH significantly improved the postischemic recovery of left ventricular (LV) contractile function with a concurrent reduction of lactate dehydrogenase release and myocardial infarct size (20.5 ± 5.3% in IHH vs. 42.1 ± 3.8% in the normoxic control, P < 0.01) after I/R. Meanwhile, IHH enhanced the production of protein carbonyls and malondialdehyde, respective products of protein oxidation and lipid peroxidation, in the reperfused myocardium and ROS generation in reperfused cardiomyocytes. Such effects were blocked by the mitochondrial ATP-sensitive K(+) channel inhibitor 5-hydroxydecanoate. Moreover, the IHH-improved postischemic LV performance, enhanced phosphorylation of PKB (Akt), PKC-ε, and glycogen synthase kinase-3β, as well as translocation of PKC-ε were not affected by applying H(2)O(2) (20 μmol/l) during early reperfusion but were abolished by the ROS scavengers N-(2-mercaptopropionyl)glycine (MPG) and manganese (III) tetrakis (1-methyl-4-pyridyl)porphyrin. Furthermore, IHH-reduced lactate dehydrogenase release and infarct size were reversed by MPG. Consistently, inhibition of Akt with wortmannin and PKC-ε with εV1-2 abrogated the IHH-improved postischemic LV performance. These findings suggest that IHH-induced cardioprotection depends on elevated ROS production during early reperfusion.     

Matrix metalloproteinases and collagen ultrastructure in moderate myocardial ischemia and reperfusion in vivo

Severe ischemic injury or infarction of myocardium may cause activation of matrix metalloproteinases (MMPs) and damage the interstitial matrix. However, it is unknown whether MMP activation and matrix damage occur after moderate ischemia and reperfusion that result in myocardial stunning without infarction, and if so whether such changes contribute to postischemic myocardial expansion and contractile dysfunction. To address these questions, open-chest anesthetized pigs underwent 90 min of regional ischemia (subendocardial blood flow 0.4 +/- 0.1 ml. g(-1). min(-1)) and 90 min of reperfusion. After ischemia plus reperfusion, histological and ultrastructural examination revealed no myocardial infarction or inflammatory cell infiltration. Myocardial MMP-9 content increased threefold with a fourfold increase in the active form (P < 0.001). Myocardial collagenase content doubled (P < 0.01) but remained in latent form. MMP-2 and tissue inhibitors of metalloproteinases were unaffected. Despite increases in MMPs, collagen ultrastructure (assessed by cell maceration scanning electron microscopy) was unaltered. Intracoronary administration of the MMP inhibitor GM-2487 did not prevent or attenuate myocardial expansion (assessed by regional diastolic dimensions at near-zero left ventricular pressure) or contractile dysfunction. We conclude that although moderate ischemia and reperfusion alter myocardial MMP content and activity, these effects do not result in damage to interstitial collagen, nor do they contribute to myocardial expansion or contractile dysfunction.     

Early activation of IKKbeta during in vivo myocardial ischemia

We have demonstrated that in vitro brief ischemia activates nuclear factor (NF)-kappaB in rat myocardium. We report in vivo ischemia-reperfusion (I/R)-induced NF-kappaB activation, IkappaB kinase -beta (IKKbeta) activity, and IkappaBalpha phosphorylation and degradation in rat myocardium. Rat hearts were subjected to occlusion of the coronary artery for up to 45 min or occlusion for 15 min followed by reperfusion for up to 3 h. Cytoplasmic and nuclear proteins were isolated from ischemic and nonischemic areas of each heart. NF-kappaB activation was increased in the ischemic area (680%) after 10 min of ischemia and in the nonischemic area (350%) after 15 min of ischemia and remained elevated during prolonged ischemia and reperfusion. IKKbeta activity was markedly increased in ischemic (1,800%) and nonischemic (860%) areas, and phosphorylated IkappaBalpha levels were significantly elevated in ischemic (180%) and nonischemic (280%) areas at 5 min of ischemia and further increased after reperfusion. IkappaBalpha levels were decreased in the ischemic (45%) and nonischemic (36%) areas after 10 min of ischemia and remained low in the ischemic area during prolonged ischemia and reperfusion. The results suggest that in vivo I/R rapidly induces IKKbeta activity and increases IkappaBalpha phosphorylation and degradation, resulting in NF-kappaB activation in the myocardium.     

Short-term exercise improves myocardial tolerance to in vivo ischemia-reperfusion in the rat.

These experiments examined the independent effects of short-term exercise and heat stress on myocardial responses during in vivo ischemia-reperfusion (I/R). Female Sprague-Dawley rats (4 mo old) were randomly assigned to one of four experimental groups: 1) control, 2) 3 consecutive days of treadmill exercise [60 min/day at 60-70% maximal O2 uptake (VO2 max)], 3) 5 consecutive days of treadmill exercise (60 min/day at 60-70% VO2 max), and 4) whole body heat stress (15 min at 42 degrees C). Twenty-four hours after heat stress or exercise, animals were anesthetized and mechanically ventilated, and the chest was opened by thoracotomy. Coronary occlusion was maintained for 30-min followed by a 30-min period of reperfusion. Compared with control, both heat-stressed animals and exercised animals (3 and 5 days) maintained higher (P < 0.05) left ventricular developed pressure (LVDP), maximum rate of left ventricular pressure development (+dP/dt), and maximum rate of left ventricular pressure decline (-dP/dt) at all measurement periods during both ischemia and reperfusion. No differences existed between heat-stressed and exercise groups in LVDP, +dP/dt, and -dP/dt at any time during ischemia or reperfusion. Both heat stress and exercise resulted in an increase (P < 0.05) in the relative levels of left ventricular heat shock protein 72 (HSP72). Furthermore, exercise (3 and 5 days) increased (P < 0.05) myocardial glutathione levels and manganese superoxide dismutase activity. These data indicate that 3-5 consecutive days of exercise improves myocardial contractile performance during in vivo I/R and that this exercise-induced myocardial protection is associated with an increase in both myocardial HSP72 and cardiac antioxidant defenses.