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Introduction: The increasing global burden of ischemic heart disease demands a closer examination of the mechanisms by which myocardial reperfusion produces injury to initiate long-term heart failure. Reactive oxygen species (ROS) generated after ischemia reperfusion (IR), in conjunction with the dysfunction of mitochondrial metabolic enzymes, have been identified as a primary mediator of cardiac reperfusion injury. The acetylation of mitochondrial proteins, regulated by opposing actions of NAD + -dependent sirtuin deacetylases and the recently identified mitochondrial acetyltransferase GCN5L1, has emerged as a key point of intersection between nutrient status and mitochondrial protein function in cardiomyoctyes. This makes the association between acetylation and ROS production an important topic of investigation. Intriguingly, global protein acetylation was recently reported to be upregulated in the hearts of human patients with ischemic heart failure. Despite this, it remains unknown whether GCN5L1 acetyltransferase activity plays a role in the regulation of metabolic proteins during IR injury. Hypothesis: Cardiac deletion of the acetyltransferase GCN5L1 reduces the acetylation of mitochondrial proteins during IR, reducing aberrant activity and preventing ROS production. Methods: Isolated work-performing hearts from cardiac-specific inducible GCN5L1 knockout mice were subjected to global ischemia and reperfusion. Contractility (+/- dP/dT) of the left ventricle was measured throughout as an index of post IR functional recovery. Tissue damage was assessed by measuring the release of lactate dehydrogenase and post-reperfusion staining of viable tissue with triphenyltetrazolium chloride. Acetylation levels of mitochondrial proteins were measured during IR using immunoblotting of homogenized hearts, which were also used to evaluate ROS production. Results and Conclusions: Mitochondrial acetylation was decreased in GCN5L1 hearts compared to WT, coinciding with improved post-IR recovery. We therefore conclude that acetylation of mitochondrial proteins by the acetyltransferase GCN5L1 is an important regulatory mechanism of IR-induced, ROS-mediated damage.

The translation from numerous successful animal experiments on cardioprotection beyond that by reperfusion to clinical practice has to date been disappointing. Animal experiments often use reductionist approaches and are mostly performed in young and healthy animals which lack the risk factors, comorbidities, and comedications which are characteristics of patients suffering an acute myocardial infarction or undergoing cardiovascular surgery. Conceptually, it is still unclear by how much the time window for successful reperfusion is extended by preconditioning, and how long the duration of ischemia can be so that adjunct cardioprotection by postconditioning at reperfusion still protects. Experimental studies addressing long-term effects of adjunct cardioprotection beyond infarct size reduction, that is, on repair, remodeling, and mortality, are lacking. Technically, reproducibility and robustness of experimental studies are often limited. Grave faults in design and conduct of clinical trials have also substantially contributed to the failure of translation of cardioprotection to clinical practice. Cardiovascular surgery with ischemic cardioplegic arrest is only a surrogate of acute myocardial infarction and confounded by the choice of anesthesia, hypothermia, cardioplegia, and traumatic myocardial injury. Trials in patients with acute myocardial infarction have been performed on agents/interventions with no or inconsistent previous animal data and in patients who had either some reperfusion already at admission or were reperfused too late to expect any myocardial salvage. Of greatest concern is the lack of adequate phase II dosing and timing studies when rushing from promising proof-of-concept trials with surrogate end points such as infarct size to larger clinical outcome trials. Future trials must focus on interventions/agents with robust preclinical evidence, have solid phase II dosing and timing data, and recruit patients who have truly a chance to benefit from adjunct cardioprotection.

The appropriate timing of angiography to facilitate revascularization is essential to optimize outcomes in patents with ST-segment–elevation myocardial infarction and non–ST-segment–elevation acute coronary syndromes. Timely reperfusion of the infarct-related coronary artery in ST-segment–elevation myocardial infarction both with fibrinolysis or percutaneous coronary intervention minimizes myocardial damage, reduces infarct size, and decreases morbidity and mortality. Primary percutaneous coronary intervention is the preferred reperfusion method if it can be performed in a timely manner. Strategies to reduce health system–related delays in reperfusion include regionalization of ST-segment–elevation myocardial infarction care, performing prehospital ECGs, prehospital activation of the catheterization laboratory, bypassing geographically closer nonpercutaneous coronary intervention–capable hospitals, bypassing the percutaneous coronary intervention–capable hospital emergency department, and early and consistent availability of the catheterization laboratory team. With implementation of such strategies, there has been significant improvement in process measures, including door-to-balloon time. However, despite reductions in door-to-balloon times, there has been little change during the past several years in in-hospital mortality, suggesting additional factors including patient-related delays, optimization of tissue-level perfusion, and cardioprotection must be addressed to improve patient outcomes further. Early angiography followed by revascularization when appropriate also reduces rates of death, MI, and recurrent ischemia in patients with non–ST-segment–elevation acute coronary syndromes, with the greatest benefits realized in the highest risk patients. Among patients with non–ST-segment–elevation acute coronary syndromes with multivessel disease, choice of revascularization modality should be made as in stable coronary artery disease, with a goal of complete ischemic revascularization.

Rationale : Sarcomere length (SL) is a key indicator of cardiac mechanical function, but current imaging technologies are limited in their ability to unambiguously measure and characterize SL at the cell level in intact, living tissue. Objective : We developed a method for measuring SL and regional cell orientation using remote focusing microscopy, an emerging imaging modality that can capture light from arbitrary oblique planes within a sample. Methods and Results : We present a protocol that unambiguously and quickly determines cell orientation from user-selected areas in a field of view by imaging 2 oblique planes that share a common major axis with the cell. We demonstrate the effectiveness of the technique in establishing single-cell SL in Langendorff-perfused hearts loaded with the membrane dye di-4-ANEPPS. Conclusions : Remote focusing microscopy can measure cell orientation in complex 2-photon data sets without capturing full z stacks. The technique allows rapid assessment of SL in healthy and diseased heart experimental preparations.

Rationale: The function of PKN, a stress-activated protein kinase, in the heart is poorly understood. Objective: We investigated the functional role of PKN during myocardial ischemia/reperfusion (I/R). Methods and Results: PKN is phosphorylated at Thr774 in hearts subjected to ischemia and reperfusion. Myocardial infarction/area at risk (MI/AAR) produced by 45 minutes of ischemia and 24 hours of reperfusion was significantly smaller in transgenic mice with cardiac-specific overexpression of constitutively active (CA) PKN (Tg-CAPKN) than in nontransgenic (NTg) mice (15±5 versus 38±5%, P P P P 2 O 2 in cultured cardiac myocytes. Stimulation of PKN prevented, whereas inhibition of PKN aggravated, cell death induced by H 2 O 2 , suggesting that the cell-protective effect of PKN is cell-autonomous in cardiac myocytes. PKN induced phosphorylation of α B crystallin and increased cardiac proteasome activity. The infarct reducing effect in Tg-CAPKN mice was partially inhibited by epoxomicin, a proteasome inhibitor. Conclusions: PKN is activated by I/R and inhibits apoptosis of cardiac myocytes, thereby protecting the heart from I/R injury. PKN mediates phosphorylation of α B crystallin and stimulation of proteasome activity, which, in part, mediates the protective effect of PKN in the heart.

Ischemic postconditioning (IPoC) reduces infarct size following ischemia/reperfusion. Whether or not phosphorylation of RISK (reperfusion injury salvage kinases) (AKT, ERK1/2, P70S6K, GSK3β) is causal for protection by IPoC is controversial. We therefore studied the impact of RISK on IPoC in anesthetized pigs subjected to 90 minutes of left anterior descending coronary artery hypoperfusion and 120 minutes of reperfusion. In protocol 1, IPoC, by 6 cycles of 20/20 seconds of reperfusion/reocclusion (n=13), was compared with immediate full reperfusion (IFR) (n=15). In protocol 2, IPoC (n=4) or IFR (n=4) was performed with pharmacological RISK blockade by IC coinfusion of Wortmannin and U0126. Infarct size was determined by TTC staining, and the expression of phosphorylated RISK proteins by Western blot analysis in biopsies. In protocol 1, infarct size was 20±3% (percentage of area at risk; mean±SEM) with IPoC and 33±4% ( P P

Autophagy is an intracellular bulk degradation process for proteins and organelles. In the heart, autophagy is stimulated by myocardial ischemia. However, the causative role of autophagy in the survival of cardiac myocytes and the underlying signaling mechanisms are poorly understood. Glucose deprivation (GD), which mimics myocardial ischemia, induces autophagy in cultured cardiac myocytes. Survival of cardiac myocytes was decreased by 3-methyladenine, an inhibitor of autophagy, suggesting that autophagy is protective against GD in cardiac myocytes. GD-induced autophagy coincided with activation of AMP-activated protein kinase (AMPK) and inactivation of mTOR (mammalian target of rapamycin). Inhibition of AMPK by adenine 9-β- d -arabinofuranoside or dominant negative AMPK significantly reduced GD-induced autophagy, whereas stimulation of autophagy by rapamycin failed to cause an additive effect on GD-induced autophagy, suggesting that activation of AMPK and inhibition of mTOR mediate GD-induced autophagy. Autophagy was also induced by ischemia and further enhanced by reperfusion in the mouse heart, in vivo. Autophagy resulting from ischemia was accompanied by activation of AMPK and was inhibited by dominant negative AMPK. In contrast, autophagy during reperfusion was accompanied by upregulation of Beclin 1 but not by activation of AMPK. Induction of autophagy and cardiac injury during the reperfusion phase was significantly attenuated in beclin 1 +/− mice. These results suggest that, in the heart, ischemia stimulates autophagy through an AMPK-dependent mechanism, whereas ischemia/reperfusion stimulates autophagy through a Beclin 1–dependent but AMPK-independent mechanism. Furthermore, autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion.

It has been shown that right ventricle (RV) hypertrophy involves significant compensatory vascular growth and remodeling. The objective of the present study was to determine the functional implications of the vascular growth and remodeling through a full flow analysis of arterial tree down to first capillary segments. A computer reconstruction of RV branches including the proximal right coronary artery to the posterior descending artery was established based on measured morphometric data in arrested, vasodilated porcine heart. The flows were computed throughout the reconstructed trees based on conservation of mass and momentum and appropriate pressure boundary conditions. It was found that the flow rate was significantly increased in large epicardial coronary arteries in hypertrophic as compared with control hearts but normalized in the intramyocardial coronary arteries and smaller vessels in RV hypertrophy primarily because of the significant increase in number of arterioles. Furthermore, the wall shear stress was restored to nearly homeostatic levels throughout most of the vasculature after 5 weeks of RV hypertrophy. The compensatory remodeling in RV hypertrophy functionally restores the perfusion at the arteriolar and capillary level and wall shear stress in most of larger vessels. This is the first full analysis of coronary arterial tree, with millions of vessels, in cardiac hypertrophy that reveals the compensatory adaptation of structure to function.

The modern treatment of cardiac arrest is an increasingly complex medical procedure with a rapidly changing array of therapeutic approaches designed to restore life to victims of sudden death. The 2 primary goals of providing artificial circulation and defibrillation to halt ventricular fibrillation remain of paramount importance for saving lives. They have undergone significant improvements in technology and dissemination into the community subsequent to their establishment 60 years ago. The evolution of artificial circulation includes efforts to optimize manual cardiopulmonary resuscitation, external mechanical cardiopulmonary resuscitation devices designed to augment circulation, and may soon advance further into the rapid deployment of specially designed internal emergency cardiopulmonary bypass devices. The development of defibrillation technologies has progressed from bulky internal defibrillators paddles applied directly to the heart, to manually controlled external defibrillators, to automatic external defibrillators that can now be obtained over-the-counter for widespread use in the community or home. But the modern treatment of cardiac arrest now involves more than merely providing circulation and defibrillation. As suggested by a 3-phase model of treatment, newer approaches targeting patients who have had a more prolonged cardiac arrest include treatment of the metabolic phase of cardiac arrest with therapeutic hypothermia, agents to treat or prevent reperfusion injury, new strategies specifically focused on pulseless electric activity, which is the presenting rhythm in at least one third of cardiac arrests, and aggressive post resuscitation care. There are discoveries at the cellular and molecular level about ischemia and reperfusion pathobiology that may be translated into future new therapies. On the near horizon is the combination of advanced cardiopulmonary bypass plus a cocktail of multiple agents targeted at restoration of normal metabolism and prevention of reperfusion injury, as this holds the promise of restoring life to many patients for whom our current therapies fail.

Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery evident on reperfusion. Translocation of the redox-sensitive δ-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Inhibition of this process resulted in full recovery of PDH activity. Infusion of the δPKC activator H 2 O 2 during normoxic perfusion, to mimic one aspect of cardiac reperfusion, resulted in loss in PDH activity that was largely attributable to translocation of δPKC to the mitochondria. Evidence indicates that reperfusion-induced translocation of δPKC is associated with phosphorylation of the αE1 subunit of PDH. A potential mechanism is provided by in vitro data demonstrating that δPKC specifically interacts with and phosphorylates pyruvate dehydrogenase kinase (PDK)2. Importantly, this results in activation of PDK2, an enzyme capable of phosphorylating and inhibiting PDH. Thus, translocation of δPKC to the mitochondria during reperfusion likely results in activation of PDK2 and phosphorylation-dependent inhibition of PDH.

Volume regulatory Cl − channels are key regulators of ischemic preconditioning (IPC). Because Cl − efflux must be balanced by an efflux of cations to maintain cell membrane electroneutrality during volume regulation, we hypothesize that I K1 channels may play a role in IPC. We subjected cultured cardiomyocytes to 60-minute simulated ischemia (SI) followed by 60-minute of simulated reperfusion (SR) and assessed percent cell death using trypan blue staining. Ischemic preconditioning (10-minute SI/10-minute SR) significantly ( P CM 18.0±2.1% versus control (C CM ) 48.3±1.0%]. IPC protection was not altered by overexpression of the reporter gene (enhanced green fluorescent protein, EGFP). However, overexpression of dominant-negative Kir2.1 or Kir2.2 genes using adenoviruses (AdEGFPKir2.1DN or AdEGFPKir2.2DN) encoding the reporter gene EGFP prevented IPC protection [both IPC CM +AdEGFPKir2.1DN 45.8±2.3% (mean±SEM) and IPC CM +AdEGFPKir2.2DN 47.9±1.4% versus IPC CM ; P CM + AdEGFPKir2.1DN 45.8±0.7% and C CM +AdEGFPKir2.2DN 46.2±1.3% versus C CM ; not statistically significant). Similar effects were observed in both cultured (n=5 hearts) and freshly isolated (n=4 hearts) ventricular cardiomyocytes after I K1 blockade with 20 μmol/L BaCl 2 plus 1 μmol/L nifedipine (to prevent Ba 2+ uptake). Nifedipine alone neither protected against ischemic injury nor blocked IPC protection. Our findings establish that I K1 channels play an important role in IPC protection.

The incidence and severity of primary cardiac events are inversely related to the plasma concentration of high-density lipoproteins (HDLs). We investigated whether HDLs may exert a direct cardioprotection in buffer-perfused isolated rat hearts, which underwent a 20-minute low-flow ischemia followed by a 30-minute reperfusion. The administration of HDLs at physiological concentrations (0.5 and 1.0 mg/mL) during the 10 minutes immediately before ischemia rapidly and remarkably improved postischemic functional recovery and decreased creatine kinase release in the coronary effluent. Reconstituted HDLs containing apolipoprotein A-I (apoA-I) and phosphatidylcholine, but not lipid-free apoA-I or phosphatidylcholine liposomes, were also effective in protecting the heart from ischemia-reperfusion injury. HDLs at reperfusion were less effective than when given before ischemia. HDLs caused a dose-dependent reduction of ischemia-induced cardiac tumor necrosis factor-alpha (TNF-alpha) expression and content, which correlated with the improved functional recovery. A parallel increase of TNF-alpha release in the coronary effluent was observed, due to a direct binding of cardiac TNF-alpha to HDLs. Taken together, these findings argue for a cause-effect relationship between the HDL-mediated removal of TNF-alpha from the ischemic myocardium and the HDL-induced cardioprotection. Indeed, etanercept, a recombinant TNF-alpha-blocking protein, caused a dose-dependent improvement of postischemic functional recovery. HDLs also enhanced ischemia-induced prostaglandin release, which may contribute to the cardioprotective effect. A low plasma HDL level may expose the heart to excessive ischemia-reperfusion damage, and HDL-targeted therapies may be helpful to induce immediate or delayed myocardial protection from ischemia-reperfusion injury.

We tested the hypothesis that an acute critical limitation in coronary flow reserve could rapidly recapitulate the physiological, molecular, and morphological phenotype of hibernating myocardium. Chronically instrumented swine were subjected to a partial occlusion to produce acute stunning, followed by reperfusion through a critical stenosis. Stenosis severity was adjusted serially so that hyperemic flow was severely reduced yet always higher than the preocclusion resting level. After 24 hours, resting left anterior descending coronary artery (LAD) wall thickening had decreased from 36.3±4.0% to 25.5±3.7% ( P 10%) and increased glycogen typical of hibernating myocardium in the LAD region (33±3% of myocytes from animals with hibernating myocardium versus 15±4% of myocytes from sham-instrumented animals, P

Supplemental Digital Content is available in the text., Rationale: There is mounting evidence of a higher incidence of coronary heart disease in cytomegalovirus-seropositive individuals. Objective: The aim of this study was to investigate whether acute myocardial infarction triggers an inflammatory T-cell response that might lead to accelerated immunosenescence in cytomegalovirus-seropositive patients. Methods and Results: Thirty-four patients with acute myocardial infarction undergoing primary percutaneous coronary intervention were longitudinally studied within 3 months after reperfusion (Cohort A). In addition, 54 patients with acute myocardial infarction and chronic myocardial infarction were analyzed in a cross-sectional study (Cohort B). Cytomegalovirus-seropositive patients demonstrated a greater fall in the concentration of terminally differentiated CD8 effector memory T cells (TEMRA) in peripheral blood during the first 30 minutes of reperfusion compared with cytomegalovirus-seronegative patients (−192 versus −63 cells/μL; P=0.008), correlating with the expression of programmed cell death-1 before primary percutaneous coronary intervention (r=0.8; P=0.0002). A significant proportion of TEMRA cells remained depleted for ≥3 months in cytomegalovirus-seropositive patients. Using high-throughput 13-parameter flow cytometry and human leukocyte antigen class I cytomegalovirus-specific dextramers, we confirmed an acute and persistent depletion of terminally differentiated TEMRA and cytomegalovirus-specific CD8+ cells in cytomegalovirus-seropositive patients. Long-term reconstitution of the TEMRA pool in chronic cytomegalovirus-seropositive postmyocardial infarction patients was associated with signs of terminal differentiation including an increase in killer cell lectin-like receptor subfamily G member 1 and shorter telomere length in CD8+ T cells (2225 versus 3397 bp; P

It has been assumed that all G i -coupled receptors trigger the protective action of preconditioning by means of an identical intracellular signaling pathway. To test this assumption, rabbit hearts were isolated and perfused with Krebs buffer. All hearts were subjected to a 30-minute coronary artery occlusion followed by 120 minutes of reperfusion. Risk area was measured with fluorescent particles and infarct size with triphenyltetrazolium chloride staining. Control hearts showed 29.1±2.8% infarction of the risk zone. A 5-minute infusion of acetylcholine (0.55 mmol/L) beginning 15 minutes before the 30-minute occlusion resulted in significant protection (9.2±2.7% infarction). This protection could be blocked by administration of 300 μmol/L N -2-mercaptopropionyl glycine (MPG), a free radical scavenger, or by 200 μmol/L 5-hydroxydecanoate (5-HD), a mitochondrial K ATP antagonist, for 15 minutes beginning 5 minutes before the acetylcholine infusion (35.2±3.9% and 27.8±2.4% infarction, respectively). Similar protection was observed with other known triggers, ie, bradykinin (0.4 μmol/L), morphine (0.3 μmol/L), and phenylephrine (0.1 μmol/L), and in each case protection was completely abrogated by either MPG or 5-HD. In contrast, protection by adenosine or its analog N 6 -(2-phenylisopropyl) adenosine could not be blocked by either MPG or 5-HD. Therefore, whereas most of the tested agonists trigger protection by a pathway that requires opening of mitochondrial K ATP channels and production of free radicals, the protective action of adenosine is not dependent on either of these steps. Hence, it cannot be assumed that all G i -coupled receptors use the same signal transduction pathways to trigger preconditioning.

Abstract —To elucidate the role of intracellular Na + kinetics in the mechanism for ischemic preconditioning (IPC), we measured intracellular Na + concentration ([Na + ] i ) using 23 Na–magnetic resonance spectroscopy in isolated rat hearts. IPC significantly delayed the initial [Na + ] i increase (d[Na + ] i /dt) compared with non-IPC control, resulting in attenuation of Na + accumulation (Δ[Na + ] i ) during 27 minutes of ischemia with better functional recovery. [Na + ] i in IPC, but not in control, recovered to preischemic level during a 6-minute reperfusion. The Na + -H + exchange inhibitor further suppressed d[Na + ] i /dt in both control and IPC hearts with concomitant improvement of functional recovery, suggesting little contribution to the mechanism of IPC. The mitochondrial ATP-sensitive K + (mito K ATP ) channel activator diazoxide (30 μmol/L) completely mimicked both [Na + ] i kinetics and functional recovery in IPC without any additive effects to IPC. The mito K ATP channel blocker 5-hydroxydecanoic acid (100 μmol/L) lost protective effect as well as the attenuation of d[Na + ] i /dt and [Na + ] i recovery induced by diazoxide. However, 5-hydroxydecanoic acid also lost IPC-induced protection, but incompletely abolished the alteration of d[Na + ] i /dt and the [Na + ] i recovery. The Na + /K + -ATPase inhibitor ouabain (200 μmol/L) did not change d[Na + ] i /dt in non-IPC hearts, but it abolished the IPC- or diazoxide-induced reduction of d[Na + ] i /dt and the [Na + ] i recovery, whereas IPC followed by ouabain treatment showed partial functional recovery with smaller Δ[Na + ] i than other ouabain groups. In conclusion, alteration of Na + kinetics by preserving Na + efflux via Na + /K + -ATPase mediated by mito K ATP channel activation mainly contributes to functional protection in IPC hearts. The contribution of mito K ATP channel–independent pathway relating to Na + kinetics including reduced Na + influx is limited in functional protection of IPC.

Abstract —Glucose-insulin-potassium solutions exert beneficial effects on the ischemic heart by reducing infarct size and mortality and improving postischemic left ventricular function. Insulin could be the critical protective component of this mixture, although the insulin response of the ischemic and postischemic myocardium has not been systematically investigated. The aim of this work was to study the insulin response during ischemia by analyzing insulin signaling. This was evaluated by measuring changes in activity and/or phosphorylation state of insulin signaling elements in isolated perfused rat hearts submitted to no-flow ischemia. Intracellular pH (pH i ) was measured by NMR. No-flow ischemia antagonized insulin signaling including insulin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, protein kinase B, p70 ribosomal S6 kinase, and glycogen synthase kinase-3. These changes were concomitant with intracellular acidosis. Perfusing hearts with ouabain and amiloride in normoxic conditions decreased pH i and insulin signaling, whereas perfusing at pH 8.2 counteracted the drop in pH i and the inhibition of insulin signaling by ischemia. Incubation of cardiomyocytes in normoxic conditions, but at pH values below 6.75, mimicked the effect of ischemia and also inhibited insulin-stimulated glucose uptake. Finally, the in vitro insulin receptor tyrosine kinase activity was progressively inhibited at pH values below physiological pH i , being abolished at pH 6.0. Therefore, ischemic acidosis decreases kinase activity and tyrosine phosphorylation of the insulin receptor thereby preventing activation of the downstream components of the signaling pathway. We conclude that severe ischemia inhibits insulin signaling by decreasing pH i .

Abstract —Electrical uncoupling at gap junctions during acute myocardial ischemia contributes to conduction abnormalities and reentrant arrhythmias. Increased levels of intracellular Ca 2+ and H + and accumulation of amphipathic lipid metabolites during ischemia promote uncoupling, but other mechanisms may play a role. We tested the hypothesis that uncoupling induced by acute ischemia is associated with changes in phosphorylation of the major cardiac gap junction protein, connexin43 (Cx43). Adult rat hearts perfused on a Langendorff apparatus were subjected to ischemia or ischemia/reperfusion. Changes in coupling were monitored by measuring whole-tissue resistance. Changes in the amount and distribution of phosphorylated and nonphosphorylated isoforms of Cx43 were measured by immunoblotting and confocal immunofluorescence microscopy using isoform-specific antibodies. In control hearts, virtually all Cx43 identified immunohistochemically at apparent intercellular junctions was phosphorylated. During ischemia, however, Cx43 underwent progressive dephosphorylation with a time course similar to that of electrical uncoupling. The total amount of Cx43 did not change, but progressive reduction in total Cx43 immunofluorescent signal and concomitant accumulation of nonphosphorylated Cx43 signal occurred at sites of intercellular junctions. Functional recovery during reperfusion was associated with increased levels of phosphorylated Cx43. These observations suggest that uncoupling induced by ischemia is associated with dephosphorylation of Cx43, accumulation of nonphosphorylated Cx43 within gap junctions, and translocation of Cx43 from gap junctions into intracellular pools.

Abstract —Loss of membrane permeability caused by ischemia leads to cellular sodium accumulation and myocardial edema. This phenomenon has important implications to left ventricular structure and function in the first hours after myocardial infarction. We hypothesized that during this period of time, after prolonged coronary occlusion and complete reflow, the rate of myocardial sodium accumulation is governed by microvascular integrity. We used 3-dimensional 23 Na MRI to monitor myocardial sodium content changes over time in an in vivo closed-chest canine model (n=13) of myocardial infarction and reperfusion. Infarcts with microvascular obstruction (MO) defined by both radioactive microspheres and contrast-enhanced 1 H MRI showed a slower rate of sodium accumulation as well as lower blood flow at 20 minutes and 6 hours after reperfusion. Conversely, the absence of MO was associated with faster rates of sodium accumulation and greater blood flow restoration. In addition, infarct size by 23 Na MRI correlated best with infarct size by triphenyltetrazolium chloride and contrast-enhanced 1 H MRI at 9 hours after reperfusion. We conclude that in reperfused myocardial infarction, sodium accumulation is dependent on microvascular integrity and is slower in regions of MO compared with those with patent microvasculature. Finally, 23 Na MRI can be a useful tool for monitoring in vivo myocardial sodium content in acute myocardial infarction.

See related article, pages 98–106 Early coronary artery reperfusion is clearly the most important therapy for acute ST segment elevation myocardial infarcts. Early reperfusion reduces myocardial infarct size and in so doing helps to prevent or minimize deleterious consequences of a large myocardial infarction, including infarct expansion (thinning and dilation of the infarct), subsequent eccentric hypertrophy and dilation of the noninfarcted ventricular muscle, and global dilation of the left ventricle.1–4 These processes encompass the phenomenon of ventricular remodeling. One of the major determinants of death at 1 year after a myocardial infarction is the degree of dilation of the left ventricle (LV).5 However, suppose early reperfusion is not available. A number of manipulations and pharmacological therapies can be administered beyond the time frame of reducing myocardial infarct size and still reduce the extent of infarct expansion and LV remodeling (Table 1). Angiotensin converting enzyme inhibitors6 and angiotensin receptor blockers7 have been shown to reduce LV dilation and remodeling and in some studies reduce major cardiovascular events. Cell therapy and even some noncellular therapies (collagen, alginate) may thicken the infarct scar and prevent ventricular wall dyskinesis.8–10 Aneurysmectomy and certain suturing techniques have been attempted to prevent infarct expansion and remodeling.11 Late reperfusion—too late to reduce myocardial infarct size, but early enough to favorably affect infarct healing1—also appears to limit infarct expansion and limit LV remodeling, and is the subject of the accompanying article.12 View this table: Table 1. Table 1. Factors That May Improve or Lessen Post-MI LV Remodeling Certain features are associated with worse LV remodeling (Table 2), including a large myocardial infarct, lack of any reperfusion,1 a large zone of no reflow,13 and certain antiinflammatory agents14 (such as steroids and a host of nonsteroidal antiinflammatory agents) introduced early enough to inhibit …

Abstract —Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development and activation of the Na + -H + exchanger (NHE). Oxygen-derived free radicals are key mediators of reperfusion abnormalities, although the cellular mechanisms involved have not been fully defined. In the present study, the effects of free radicals on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute exposure of spontaneously beating myocytes to 50 μmol/L hydrogen peroxide (H 2 O 2 ) caused a sustained decrease in contraction amplitude (80% of control). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Acute exposure to H 2 O 2 (100 μmol/L, 5 minutes) resulted in sustained MAP kinase activation that persisted for 60 minutes. Catalase, but not superoxide dismutase, completely inhibited MAP kinase activation by H 2 O 2 . Pretreatment with chelerythrine (10 μmol/L, 45 minutes), a protein kinase C inhibitor, or genistein (75 μmol/L, 45 minutes) or herbimycin A (3 μmol/L, 45 minutes), tyrosine kinase inhibitors, caused significant inhibition of H 2 O 2 -stimulated MAP kinase activity (51%, 78%, and 45%, respectively, at 20 minutes). Brief exposure to H 2 O 2 also stimulated NHE activity. This effect was completely abolished by pretreatment with the MAP kinase kinase inhibitor PD 98059 (30 μmol/L, 60 minutes). These results suggest that low doses of H 2 O 2 induce MAP kinase–dependent pathways that regulate NHE activity during reperfusion injury.

Abstract Recent studies in conscious pigs and rabbits have demonstrated that a series of brief coronary occlusions renders the heart relatively resistant to myocardial “stunning” 24 hours later (late preconditioning [PC] against stunning). The mechanism of this powerful cardioprotective response is unknown. The goal of the present study was to test the hypothesis that the development of late PC against stunning is triggered by increased generation of NO during the first ischemic challenge. Conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). On day 1, rabbits received either an intravenous infusion of the NO synthase inhibitor N ω -nitro- l -arginine (L-NA, 13 mg/kg before the first occlusion) (group II, n=10) or vehicle (group I [control], n=10). In the control group, on day 1 systolic wall thickening (WTh) in the ischemic/reperfused region remained significantly depressed for 4 hours after the sixth reperfusion, indicating myocardial stunning. On days 2 and 3, however, the recovery of WTh improved markedly, so that the total deficit of WTh decreased by 60% on day 2 and 55% on day 3 compared with day 1 ( P P P

Abstract The effectiveness of the late phase of ischemic preconditioning (PC) in protecting against myocardial infarction and the concomitant contractile dysfunction after sustained ischemia remains unclear. The early and late phases of PC have not been compared using the same protocol in the same experimental model; furthermore, the late phase of PC has not been assessed in the conscious state in a large animal preparation. The goal of this study was to directly compare the effects of early and late PC on myocardial infarct size and postischemic dysfunction in chronically instrumented, conscious pigs. Four groups of pigs were subjected to a 40-minute coronary occlusion followed by 3 days of reperfusion. Group 1 (n=7) served as control. Group 2 (n=6) was subjected to ten 2-minute occlusion/2-minute reperfusion cycles 25 minutes before the 40-minute occlusion (early PC). Groups 3 (n=7) and 4 (n=4) were subjected to 10 and 25 cycles, respectively, of 2-minute occlusion/2-minute reperfusion 24 hours before the 40-minute occlusion (late PC). Infarct size averaged 45.1±5.9% of the region at risk in control pigs, was reduced by 79% (to 9.4±3.2%) in group 2, but did not differ in groups 3 (33.3±4.8%) and 4 (38.8±8.2%) versus group 1. Power analysis demonstrated that there was an 80% probability of detecting a 40% decrease in infarct size in groups 3 and 4 versus group 1. The recovery of systolic wall thickening (measured with ultrasonic crystals) after the 40-minute occlusion was poor in groups 1, 3, and 4 but markedly enhanced in group 2 throughout the 3 days of reperfusion; this beneficial effect could have been due to limitation of infarct size, alleviation of stunning, or both. Thus, a series of ten 2-minute coronary occlusions had a profound (≈80%) early infarct–limiting effect, which was associated with a marked functional benefit. This protection, however, disappeared 24 hours later and could not be reinstituted by increasing the number of PC coronary occlusions to 25. The incidence and duration of ventricular tachycardia after reperfusion was not changed by either early or late PC; no conclusions could be drawn regarding ventricular fibrillation or ischemia-induced ventricular tachycardia, since these arrhythmias did not occur in control animals. Taken together, the present results demonstrate striking differences between the early and late effects of PC: In conscious swine subjected to a sustained coronary occlusion, a PC protocol that induces powerful protection during the early phase of PC fails to induce any protection during the late phase, indicating either that a late protective effect of PC does not exist or that, if it exists, it must be weaker than the early protective effect.

Apoptosis has been observed previously in hearts subjected to either continuous ischemia or ischemia followed by reperfusion. The purpose of this study was to compare the timing and extent of apoptosis in both continuously ischemic and reperfused myocardium. We show that rats subjected to continuous coronary artery occlusion display characteristic signs of apoptosis solely in the ischemic myocardium after only 2.25 hours of ischemia, as illustrated by positive in situ end labeling (ISEL) of apoptotic cardiomyocyte nuclei in tissue sections and/or the presence of DNA “ladders” in agarose gels. In contrast, reperfusion after a 45-minute occlusion accelerated the process, with apoptosis becoming evident solely in the reperfused myocardium after only 1 hour of reperfusion. ISEL and DNA ladder intensity increased with duration of ischemia or reperfusion. The volume of myocardium in which ISEL was observed was smaller in the reperfused hearts, and the ISEL-stained nuclei represented 23% and 33% of the total nuclei in the reperfused and permanently occluded myocardium, respectively. Therefore, the data suggest that reperfusion lowers the extent of apoptosis in ischemic myocardium but, paradoxically, accelerates the residual apoptosis, possibly because of reperfusion injury. A large accumulation of neutrophils was observed in both the permanently occluded and reperfused myocardium, suggesting that the inflammatory response may have contributed to apoptosis in both settings. This study therefore confirms that both ischemic and reperfused rat myocardium can undergo apoptotic cell death. However, the data suggest that although reperfusion lowers the number of myocytes undergoing apoptosis, it accelerates apoptosis in the nonsalvageable cells.

Prior transient episodes of ischemia (“ischemic preconditioning”) reduce lactate accumulation and attenuate acidosis during a subsequent prolonged ischemic insult. The mechanisms responsible for attenuated glycolytic catabolite accumulation have not been established but may include earlier exhaustion of glycogen stores, slowed glycogenolysis before complete glycogen depletion, and/or inhibition of glycolysis. Simultaneous repeated measures of myocardial glycogen and the rates of glycolysis, glycogenolysis, glucose utilization, and glycolytic ATP production were obtained during total ischemia by 13 C nuclear magnetic resonance spectroscopy in control and ischemia-preconditioned isolated rat hearts. Both [ 13 C]glycolytic and [ 13 C]glycogenolytic rates were significantly lower during total ischemia in preconditioned compared with control hearts (0.77±0.04 versus 1.06±0.06 μmol/min per gram wet weight [ P P 13 C]glycogen were still present. Importantly, the reduction in the rate of glycogenolysis was larger and out of proportion to the reduction in glycolysis and occurred despite an increase in glucose utilization in preconditioned hearts (2.23±0.15 versus 1.5±0.10 μmol/min per gram wet weight at 1.25 minutes, P a or “active” form was less in preconditioned than in control hearts (29.1±2.6% versus 41.2±9.8%, respectively; P

Activation of both ATP-sensitive K + (K ATP ) channels and the enzyme protein kinase C (PKC) has been associated with the cardioprotective response of ischemic preconditioning. We recently showed that at low cytoplasmic ATP (≤50 μmol/L), PKC inhibits K ATP channel activity. This finding is surprising, as both K ATP channels and PKC are activated during preconditioning. However, PKC also altered ATP binding to the channel, changing the Hill coefficient from ≈2 to ≈1. This apparent change in stoichiometry would lead to a PKC-induced activation of K ATP channels at more physiological (millimolar) levels of ATP. The aim of the present study was to determine whether PKC activates cardiac K ATP channels at millimolar levels of ATP. The effects of PKC on single K ATP channels were studied at millimolar internal ATP levels using excised inside-out membrane patches from rabbit ventricular myocytes. Application of purified constitutively active PKC (20 nmol/L) to the intracellular surface of the patches produced an approximately threefold increase in the channel open probability. The specific PKC inhibitor peptide PKC(19-31) prevented this increase. Heat-inactivated PKC had no effect on K ATP channel properties. K ATP channel activity spontaneously returned to control levels after washout of PKC. This spontaneous reversal did not occur in the presence of 5 nmol/L okadaic acid, suggesting that the reversal of PKC's action is dependent on activity of a membrane-associated type 2A protein phosphatase (PP2A). In the presence of exogenous PP2A (7.5 nmol/L), PKC had no effect. We conclude that the PKC-induced increase in K ATP channel activity at millimolar ATP results from a crossing of the ATP concentration-response curves for inhibition of the phosphorylated and nonphosphorylated forms of the channel. This identifies a mechanism by which PKC activates K ATP channels at near physiological levels of ATP and thus could link these two components in a signaling pathway that induces ischemic preconditioning.

Although it may be debatable whether preconditioning will ever fulfill its clinical expectations, the hope that a new therapeutic modality may emerge from this fascinating phenomenon has given great impetus to the search for its underlying mechanism. Preconditioning is unquestionably a powerful phenomenon, it is readily and consistently demonstrable under many experimental conditions, and it has just celebrated its 10th birthday.1 In view of all this, it is perhaps surprising that the precise mechanism of preconditioning remains elusive and recently has become the subject of controversy. An observer of the preconditioning literature could, at first sight, be forgiven for concluding that the precise mechanism is established and that protein kinase C (PKC) activation is a pivotal common factor that links a spectrum of receptor-mediated triggers of preconditioning. Downey and colleagues, now supported by other investigators (reviewed in References 2 and 3),2 3 have developed the compelling hypothesis that stimulation of a variety of G protein–coupled receptors (eg, adenosine A1, α1-adrenergic, muscarinic, bradykinin, and endothelin-1 receptors) results in the activation of PKC. This, in turn, leads to the physical translocation of PKC from the cytoplasm to the sarcolemma, where it phosphorylates a substrate protein (possibly the ATP-sensitive K+ [KATP] channel4 ) and thereby confers resistance to ischemia. Support for this attractive hypothesis comes from a wealth of studies (most of which rely upon indirect evidence in the absence of PKC activity measurements) with activators and inhibitors of PKC or its translocation process, with receptor agonists and antagonists, and with agents that interfere with the signaling pathways between various receptors and PKC (reviewed in References 5 and 6). Considering the substantial body of evidence supporting the involvement of PKC activation in the preconditioning phenomenon, the observer could again be forgiven for wondering …

The delay of infarct size development by ischemic preconditioning involves the activation of protein kinase C in rats and rabbits. In dogs the role of protein kinase C in ischemic preconditioning is controversial. We investigated whether or not the activation of protein kinase C is a prerequisite for ischemic preconditioning in swine. Swine were used, since they are large mammals and since infarct development in this species, due to the lack of an innate collateral circulation, is similar to that in humans. In 20 enflurane-anesthetized swine, the proximal left anterior descending coronary artery was cannulated and perfused from an extracorporeal circuit. The impact of continuous intracoronary infusion of 10 −7 mol/L staurosporine, a potent protein kinase C inhibitor, on global and regional myocardial function (sonomicrometry), subendocardial blood flow (ENDO, microspheres), and infarct size (IS, triphenyltetrazolium chloride staining after 120 minutes of reperfusion) was analyzed. Staurosporine (10 −7 mol/L) abolished the 1.6-fold increase in coronary arterial resistance in response to 10 −6 mol/L IC 4β-phorbol 12-myristate 13-acetate, a potent protein kinase C activator. In the presence of staurosporine, 90 minutes of low-flow ischemia at an ENDO of 0.05±0.04 (mean±SD) mL·min −1 ·g −1 resulted in an IS of 12.5±8.6% (n=10) of the area at risk. Also, in the presence of staurosporine, ischemic preconditioning by a cycle of 10 minutes of low-flow ischemia followed by 15 minutes reperfusion before the 90 minutes sustained ischemic period (ENDO, 0.05±0.03 mL·min −1 ·g −1 ) reduced IS to 3.3±3.4% (n=10, P

It has recently been recognized that cellular stresses activate certain members of the mitogen-activated protein kinase (MAPK) superfamily. One role of these “stress-activated” MAPKs is to increase the transactivating activity of the transcription factors c-Jun, Elk1, and ATF2. These findings may be particularly relevant to hearts that have been exposed to pathological stresses. Using the isolated perfused rat heart, we show that global ischemia does not activate the 42- and 44-kD extracellular signal–regulated (protein) kinase (ERK) subfamily of MAPKs but rather stimulates a 38-kD activator of MAPK-activated protein kinase-2 (MAPKAPK2). This activation is maintained during reperfusion. The molecular characteristics of this protein kinase suggest that it is a member of the p38/reactivating kinase (RK) group of stress-activated MAPKs. In contrast, stress-activated MAPKs of the c-Jun N-terminal kinase (JNK/SAPKs) subfamily are not activated by ischemia alone but are activated by reperfusion following ischemia. Furthermore, transfection of ventricular myocytes with activated protein kinases (MEKK1 and SEK1) that may be involved in the upstream activation of JNK/SAPKs induces increases in myocyte size and transcriptional changes typical of the hypertrophic response. We speculate that activation of multiple parallel MAPK pathways may be important in the responses of hearts to cellular stresses.

Abstract Myofibrillar proteins (MPs) were extracted from isolated and perfused rat hearts subjected to different periods of ischemia to investigate the occurrence of protein degradation and/or the association of cytosolic proteins with the myofibrillar pellet. A 23-kD band was detected by SDS-PAGE of MPs after 5 minutes of ischemia, with its density gradually increasing to a plateau after 20 minutes. Longer periods of ischemia were associated with the appearance of a 39-kD band. Irrespective of the duration of ischemia, both these bands persisted during reperfusion. A partial proteolytic degradation of troponin T (TnT) and troponin I (TnI) has been claimed to be responsible for the generation of these peptides. However, the N-terminal sequence of the 39-kD band was identical to that of GAPDH, whereas Edman sequencing after pepsin digestion showed that the 23 kD is αB-crystallin. The binding of the two cytosolic proteins to myofibrils was confirmed by immunofluorescence analysis on cryosections of ischemic hearts. In vitro studies showed that acidosis was sufficient to induce the binding of αB-crystallin, whereas the inhibition of ATP depletion prevented the binding of GAPDH. Thiol oxidation is unlikely to promote GAPDH binding, since perfusion with iodoacetate under aerobic conditions or treatment of homogenates with N -ethylmaleimide or diamide failed to induce GAPDH association with the myofibrils. These changes of the myofibrillar proteins could be considered as intracellular markers of the evolution of the ischemic damage. In addition, the binding of the 23-kD peptide might be involved in alterations of contractility.

Brief intermittent episodes of ischemia and reperfusion, at the onset of reperfusion after a prolonged period of ischemia, confer cardioprotection, a phenomenon termed “ischemic postconditioning” (Postcond). We hypothesized that this phenomenon may just represent a modified form of reperfusion that activates the reperfusion injury salvage kinase (RISK) pathway. Isolated perfused rat hearts were subjected to: (a) 35 minutes of ischemia and 120 minutes of reperfusion, and infarct size was determined by tetrazolium staining; or (b) 35 minutes of ischemia and 7 minutes of reperfusion, and the phosphorylation states of Akt, endothelial NO synthase (eNOS), and p70S6K were determined. Postcond reduced infarct size from 51.2±3.4% to 31.5±4.1% ( P P P =NS). Inhibiting phosphatidylinositol 3-kinase (PI3K) at reperfusion using LY or Wortmannin (Wort) during the first 15 minutes of reperfusion completely abolished Postcond-induced protection (31.5±4.1% with Postcond versus 51.7±4.5% with Postcond+LY, P P

Abstract It has been proposed that NO may function as an endogenous cardioprotectant. We have investigated whether modulation of NO levels (detected in coronary effluent by chemiluminescence) by a blocker of its synthesis, by supplementation of its precursor, and by administration of an NO donor can influence reperfusion arrhythmias in the isolated rat heart. Rat hearts were perfused with modified Krebs’ solution and subjected to 5, 35, or 60 minutes of left regional ischemia followed by 10 minutes of reperfusion. N G -Nitro- l -arginine methyl ester (L-NAME), which blocks NO synthase, increased the incidence of reperfusion-induced ventricular fibrillation (VF) from 5% in the control condition to 35% after 60 minutes of ischemia (n=20, P l -arginine (an NO precursor) but persisted in hearts coperfused with d -arginine (1 mmol/L). L-NAME did not increase VF susceptibility in hearts reperfused after 5 or 35 minutes of ischemia. L-NAME caused sinus bradycardia (264±10 versus 309±5 bpm in control groups, P −1 · g −1 tissue in controls, P −1 · g −1 ( P l -arginine (10 344±1730 pmol · min −1 · g −1 , P P =NS). The NO donor sodium nitroprusside (10 μmol/L) significantly increased coronary flow 1 minute before ischemia (15.4±1.1 versus 9.2±0.6 mL · min −1 · g −1 tissue and coronary effluent NO levels (from 1122±122 to 4093±1466 pmol · min −1 · g −1 , P

Abstract Ischemic preconditioning (IP) and myocardial hibernation (MH) are both adaptive phenomena during acute myocardial ischemia, characterized by preserved myocardial viability and attenuated alterations of energy metabolism. Recent data from isolated buffer-perfused rabbit hearts pointed to a further link between IP and MH, in that an initial stimulus of no-flow ischemia was required to permit the development of MH during subsequent sustained low-flow ischemia. In the present study, we therefore investigated in the in situ pig heart whether a brief episode of no-flow ischemia enhances the myocardial tolerance to subsequent sustained low-flow ischemia. By blocking ATP-dependent potassium channels, we attempted to further determine whether such increased tolerance to ischemia is related to IP or MH, since blockade of ATP-dependent potassium channels abolishes the cardioprotection achieved by IP but not by MH. In 8 enflurane-anesthetized pigs serving as controls (group 1), the inflow into the cannulated left anterior descending coronary artery was reduced to achieve a 90% reduction in the anterior myocardial work index (sonomicrometry) for 90 minutes. In 15 pigs (group 2), a 10-minute no-flow ischemic episode preceded 80 minutes of sustained ischemia at a blood flow reduction identical to that in pigs of group 1. In 8 additional pigs (group 3), glibenclamide was administered before the 10-minute no-flow ischemic episode. In all pigs after 120 minutes of reperfusion, infarct size (IS, percentage of area at risk) was determined by triphenyltetrazolium chloride staining. In group 2, IS was reduced (6.8±6.0% [mean±SD], P −1 · g −1 . This blood flow was lower than that in groups 1 (0.11±0.04 mL · min −1 · g −1 , P −1 · g −1 , P =NS), indicating an increased ischemic tolerance of the myocardium in pigs of group 2. Conclusions are as follows: (1) A brief episode of no-flow myocardial ischemia without intermittent reperfusion increases the tolerance to sustained low-flow ischemia. (2) The cardioprotective effect is mediated by activation of ATP-dependent potassium channels and therefore relates to IP rather than to MH.

Abstract To elucidate the abnormalities of excitation-contraction coupling in stunned myocardium, we measured [Ca 2+ ] i and force in thin fura 2–loaded ventricular trabeculae from control or stunned (20 minutes ischemia followed by 20 minutes reflow at 37°C) rat hearts. At any given [Ca 2+ ] o , force development was significantly lower in the stunned trabeculae than in control trabeculae. In contrast, there was no difference in the amplitude of Ca 2+ transients between the two groups. The steady state force-[Ca 2+ ] i relationship, assessed by tetanization in the presence of ryanodine, revealed both a decrease in maximal Ca 2+ -activated force and an increase in the [Ca 2+ ] i required for 50% activation in stunned trabeculae. Postischemic myocardium also exhibited an accelerated rate of diastolic relaxation that was not due to changes in the rate of Ca 2+ transient decay. Destabilization of attached cross-bridges in a quantitative model of cardiac myofibrils accurately reproduced the salient systolic and diastolic features of the stunned phenotype, suggesting an abnormality of the thin filaments. In response to supraphysiological increases in [Ca 2+ ] o , diastolic [Ca 2+ ] i and diastolic tone increased much more in stunned trabeculae than in controls, with the frequent occurrence of aftercontractions. This novel experimental model lends further support to the hypothesis that the primary lesion of excitation-contraction coupling resides at the level of the contractile proteins. The finding of enhanced susceptibility to calcium overload helps to rationalize the functional deterioration of stunned myocardium during intense inotropic stimulation and additionally suggests that stunned myocardium may represent a favorable substrate for triggered arrhythmias.

Specific rabbit anti-cardiolipin (anti-CL) antibodies were used to investigate the hypothesis that cardiolipin, associated with mitochondrial membrane proteins, binds C1 and facilitates activation of the complement cascade following reperfusion of ischemic myocardium. By immunoelectron microscopy, anti-CL localized to subsarcolemmal mitochondria, emerging through breaks in membranes of damaged cardiac myocytes. Anti-CL reacted with > 15 mitochondrial constituents, most of which comigrated with the proteins that bind C1q in transblots of subsarcolemmal mitochondria, fractionated by polyacrylamide gel electrophoresis under reducing conditions in the presence of sodium dodecyl sulfate. A subset of the C1q-binding proteins > 24 to 37 kDa served as stable sites for assembly of C3, C5, and C9. Cardiac lymph, collected during the first hour after reperfusion of ischemic myocardium, contained proteins of diverse size that reacted with both anti-CL and C1q. Cardiac lymph, collected before occlusion and 4 to 5 hours after reperfusion, in comparison, had few if any C1q or anti-CL reactive proteins. Treatment with phospholipase suppressed the C1q-binding activity and anti-CL reactivity of the proteins in reperfusion lymph and those with similar properties in mitochondrial extracts. Our data suggest that during ischemia, mitochondria, extruded through breaks in the sarcolemma, unfold and release membrane fragments in which cardiolipin and protein are intimately associated. By binding C1 and supplying sites for the assembly of later-acting complement components, these fragments provide the means to disseminate the complement-mediated inflammatory response to ischemic injury.

Endothelin (ET) has been shown to be elevated under conditions of cardiac pathology and to produce diverse cardiac effects, including coronary constriction and a positive inotropic influence. We characterized the concentration- and time-dependent effects of the most potent of the ET isoforms, ET-1 (0.4, 2, and 4 nmol/L), on myocardial contractility and coronary resistance and assessed its effects on the ischemic and reperfused heart. Because ET-1 has been shown to activate the Na(+)-H+ exchanger in cardiac myocytes, we determined the contribution of the antiport by examining the effects of ET-1 in the presence of the Na(+)-H+ exchange inhibitor methylisobutyl amiloride (MIA). At all three concentrations, ET-1 produced an initial positive inotropic effect that was reversed with continued perfusion, the degree of the reversal being dependent on ET-1 concentration. With 0.4 nmol/L, contractility returned to pre-ET-1 values, whereas after 75 minutes of perfusion with 4 nmol/L ET-1, contractility was depressed by 75%. At all concentrations, ET-1 produced a coronary-constricting effect, whereas an elevation in resting tension was observed only with 4 nmol/L ET-1. MIA significantly prevented the positive inotropic effect of ET-1 but had no effect on loss in function or elevation in resting tension produced by 4 nmol/L ET-1. Furthermore, MIA partially, but not significantly, attenuated the constricting effects of all ET-1 concentrations. In the ischemic heart, 0.4 nmol/L ET-1 appeared to delay the loss in contractility produced by cessation of flow, although the effect was not significant. Higher concentrations of ET-1 were without effect on ischemia-induced contractile depression, although their presence produced a marked elevation in resting tension during ischemia that was attenuated by MIA. Recovery in contractility was reduced by all concentrations of ET-1, although the effects of the lowest concentration were associated primarily with defective relaxation. The depressant effects of ET-1 either in normal or ischemic/reperfused hearts were irreversible. The inhibitory effects of ET-1 on contractile recovery were associated with diminished tissue glycogen and elevated lactate levels. High-energy phosphates after reperfusion were depressed in hearts treated with 4 nmol/L ET-1. The attenuation in contractile recovery and alterations in metabolite content were prevented by MIA. These results provide evidence that ET-1 produces complex effects on heart function that are likely mediated via different mechanisms and demonstrate its ability to aggravate ischemic and reperfusion injury through a mechanism possibly involving Na(+)-H+ exchange activation.

A number of studies show the relation between oxygen-derived free radicals and cardiac ischemia/reperfusion injury. However, little is known about oxidative stress in the human heart, which can be measured by oxidized glutathione (GSSG) and malondialdehyde (MDA) formation. Furthermore, data on MDA production by rat hearts are controversial, possibly because of the use of the aspecific thiobarbituric acid assay. Therefore, GSSG and MDA were measured, with colorimetric and high-performance liquid chromatographic assays, respectively, in buffer-perfused explanted human hearts and normal rat hearts made temporarily ischemic. Human hearts received cardioplegia; rat hearts were studied in a control and an ischemic group with or without cardioplegia. Baseline GSSG release was < 0.01 nmol.min-1.g wet wt-1 in both species. During reperfusion, GSSG release from human hearts and from ischemic and cardioplegic/ischemic rat hearts peaked at 0.24 +/- 0.12, 1.1 +/- 0.4, and 0.19 +/- 0.04 nmol.min-1.g-1, respectively. MDA was undetectable (< 0.02 nmol.min-1.g-1) in the effluent of both species and in human hearts (< 4 nmol/g protein). Rat heart reduced glutathione levels decreased 32% as a consequence of cardioplegia and ischemia. Cardioplegia induced a 41% (P = .08) decrease in rat heart MDA content, whereas cumene hydroperoxide increased it 3.6 times (P < .01). Thus, after ischemia human and rat hearts release GSSG, indicating that oxidative stress has occurred. Apparently, lipid peroxidation takes place in normal rat hearts, decreases after cardioplegia, but does not increase after ischemia/reperfusion. Human hearts lack MDA under normoxic and ischemic conditions. This novel finding seems to reflect a low MDA-forming potential in both situations.(ABSTRACT TRUNCATED AT 250 WORDS)

Preconditioning may find ready applicability in humans facing scheduled global cardiac ischemia-reperfusion (IR) during bypass or transplantation, where such a maneuver is feasible before arrest. Our objective was to delineate and exploit the endogenous preconditioning mechanism triggered by transient ischemia (TI) and thereby attenuate myocardial postischemic mechanical dysfunction by clinically acceptable means. Preconditioning by 2 minutes of TI followed by 10 minutes of normal perfusion protected isolated rat left ventricle function assessed after 20 minutes of global, 37 degrees C ischemia and 40 minutes of reperfusion. Final recovery of developed pressure (DP) was improved (91.5 +/- 1.9% of equilibration DP versus unconditioned IR control, 57.4 +/- 2.4%, P < .01) and was accompanied by increased contractility (+/- dP/dt). Norepinephrine release increased after TI, and reserpine pretreatment abolished TI preconditioning. This suggests that endogenous norepinephrine mediates functional preconditioning in rat. Brief pretreatment (2 minutes) with exogenous norepinephrine reproduced the protection (89.1 +/- 1.4%) of postischemic function. Functional protection persisted after the hemodynamic effects had resolved. Norepinephrine-induced preconditioning was simulated by phenylephrine and blocked by alpha 1-adrenergic receptor antagonist. TI preconditioning was similarly lost after selective alpha 1-adrenergic receptor blockade. We conclude that transient ischemic preconditioning is mediated by the sympathetic neurotransmitter release and alpha 1-adrenergic receptor stimulation. Although the postreceptor mechanism remains unclear, functional protection after IR does not seem related to the magnitude of ATP depletion and elevation of resting pressure during ischemia. Rather, the endogenous mechanisms facilitate both recovery of mechanical function and ATP repletion during reperfusion.

The recovery of systolic and diastolic function during unstable angina may be modified by the repetition of brief episodes of ischemia and by the presence of left ventricular hypertrophy (LVH). We studied the effects of six consecutive 5-minute cycles of no-flow ischemia and reperfusion followed by 25 minutes of recovery in isovolumic red blood cell-perfused hearts from aortic-banded rats with chronic LVH (n = 8) and sham-operated control rats (n = 8). At baseline (left ventricular end-diastolic pressure [LVEDP], 10 mm Hg), left ventricular developed pressure (123 +/- 5 versus 114 +/- 5 mm Hg/g) and coronary flow [2.5 +/- 0.3 versus 2.2 +/- 0.2 (mL/min)/g] were similar in LVH versus control rats. Repetitive ischemia was associated with progressive depression of postischemic recovery of left ventricular systolic function, and the recovery of left ventricular developed pressure after the final 25-minute reperfusion period was similar in LVH versus control rats (61 +/- 6% versus 72 +/- 4% of baseline, P = NS). Although there was no increase in isovolumic LVEDP during the initial cycle of transient ischemia, both groups showed a rapid and similar rise in LVEDP during subsequent ischemic cycles (delta 82 +/- 8 versus delta 89 +/- 7 mm Hg/g in response to the final ischemia cycle for LVH versus control rats, respectively; P = NS). The control hearts showed complete restoration of LVEDP to baseline during final reperfusion, whereas the LVH hearts showed prolonged and severe postischemic diastolic dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study tested the hypothesis that nitric oxide production in coronary resistance vessels is an important mechanism affecting the regulation of myocardial perfusion in unanesthetized dogs. We inhibited nitric oxide synthesis with the arginine analogue N omega-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg) and maintained the compressive determinants of myocardial blood flow constant by ventricular pacing. L-NAME did not affect resting coronary blood flow and reduced the receptor-mediated increase in flow to intracoronary acetylcholine (100 micrograms/min IC) from 143 +/- 20% (mean +/- SEM) under control conditions to 31 +/- 10% after L-NAME (P < .001). Coronary autoregulatory relations were determined as steady-state coronary pressure was reduced by inflating a hydraulic occluder. Initial resistance adjustments over the autoregulatory plateau were not affected by L-NAME. Closed-loop autoregulatory gain was 0.84 +/- 0.09 under control conditions versus 0.78 +/- 0.07 after L-NAME (P = NS). As coronary pressure was reduced further, however, the critical pressure at which myocardial ischemia began (lower autoregulatory break point) increased from 45 +/- 3 mm Hg under control conditions to 61 +/- 2 mm Hg (P < .001) after L-NAME. In addition, the slope of the coronary pressure-flow relation below the autoregulatory break point was reduced (1.0 +/- 0.2 versus 0.58 +/- 0.09 mL.min-1.mm Hg-1 after L-NAME, P < .05), reflecting a reduction in the maximal conductance recruitable during ischemia. In concert with the effects of L-NAME on autoregulatory responses during ischemia, peak reactive hyperemic flow to a 30-second coronary occlusion was also reduced (from 200 +/- 22 to 166 +/- 24 mL/min after L-NAME, P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant desulfatohirudin (HI), a potent and specific thrombin inhibitor, was compared with heparin (HE) as an adjunct to streptokinase thrombolysis. In pentobarbital-anesthetized dogs, an occlusive thrombus (whole blood+thrombin) was introduced into the left anterior descending coronary artery (LAD) with superimposed endothelial damage and distal high-grade stenosis. Intravenous infusion of saline (vehicle), HI (0.3 mg/kg followed by 0.3 mg/kg per hour, 1 mg/kg followed by 1 mg/kg per hour, or 2 mg/kg followed by 2 mg/kg per hour), or HE (60 units/kg followed by 40 units/kg per hour or 100 units/kg followed by 60 units/kg per hour) was initiated 15 minutes before streptokinase (750,000 units for 60 minutes) administration. Vessel patency was monitored for 180 minutes after streptokinase administration with a volume flow probe on the proximal LAD. In dogs treated with no adjunctive agent (saline control), none of the vessels were recanalized with streptokinase. Both HI and HE promoted reperfusion, inhibited reocclusion, and reduced the residual thrombus mass in a dose-dependent fashion. However, at comparable levels of therapeutic anticoagulation (activated partial thromboplastin time [APTT] = 1.5-2.0 times baseline) HI exhibited a higher incidence of reperfusion (eight of eight dogs [100%] versus one of eight dogs [12%]), a shorter time to reperfusion (33 +/- 6 versus 59 minutes), a longer duration of initial reperfusion (106 +/- 21 versus 10 minutes), and a smaller residual thrombus mass than did HE. Likewise, the slope of the relation between the APTT prolongation and the total reperfusion time ("anticoagulation/antithrombosis profile") was almost five times higher for the combined HI data than for the HE data. Our results indicate that HI is more effective than HE in enhancing and sustaining coronary recanalization with streptokinase at a HI dose that modestly prolongs coagulation time and does not alter bleeding times.

Insulin-like growth factor-1 (IGF-1) plays a role in the regulation of myocardial structure and function. IGF-1 is capable of improving cardiac muscle survival, growth, calcium signaling, and differentiation.1 Among various actions of IGF-1 on heart, the ability of IGF-1 to counteract apoptosis of cardiomyocytes has drawn significant attention in recent years. IGF-1 suppresses myocardial apoptosis and improves myocardial function in various models of experimental cardiomyopathy.2 3 4 5 Compared with other growth factors, the survival effect of IGF-1 on myocardium is rather unique. Using an ex vivo model of perfused heart isolated from IGF-1 overexpressing transgenic mice, the study by Yamashita et al6 in this issue of Circulation Research demonstrated that disrupting the activation of Akt abolished the antiapoptotic effects of autocrine/paracrine IGF-1 during ischemia/reperfusion injury. Activation of p38 mitogen-activated protein (MAP) kinase has been previously implicated in myocardial injury during ischemia/reperfusion. These investigators confirmed previous studies that blocking p38 MAP kinase reduced myocardial apoptosis in wild-type heart. However, in this study, inhibiting p38 MAP kinase activation was accompanied by significant suppression of Akt activation during ischemia/reperfusion and increased apoptosis in the IGF-1 transgenic heart. The authors concluded that, on inhibiting p38 MAP kinase activation, the reduction of Akt activation in the transgenic myocardium disrupted the spatial balance between p38 MAP kinase and Akt activation and thus triggered more apoptosis during ischemia/reperfusion. In this study, inhibition of p38 MAP kinase was achieved with chemical inhibitor SB203580. One may argue that chemical inhibitors are not impeccably specific; thus the attenuation of Akt activation by SB203580 might have been attributable to cross-inhibition. Furthermore, we cannot …

Pharmacological modulation of [K+]o accumulation and action potential changes during acute myocardial ischemia is under evaluation as a promising new antiarrhythmic and cardioprotective strategy during myocardial ischemia and reperfusion. We studied the effects of cromakalim, a K+ channel opener that activates ATP-sensitive K+ channels, in isolated arterially perfused rabbit interventricular septa subjected to ischemia and reperfusion and, through use of the patch clamp technique, in inside-out membrane patches excised from guinea pig ventricular myocytes. During aerobic perfusion, 5 microM cromakalim shortened action potential duration (APD) from 217 +/- 7 to 201 +/- 10 msec, had no effect on [K+]o, and reduced tension by 17 +/- 3% (n = 11). During ischemia, pretreatment with 5 microM cromakalim resulted in 1) more rapid APD shortening (71 +/- 9 versus 166 +/- 7 msec at 10 minutes and 63 +/- 12 versus 122 +/- 8 msec at 30 minutes), 2) similar [K+]o accumulation after 10 minutes (8.9 +/- 0.3 versus 9.6 +/- 0.5 mM) but a trend toward increased [K+]o accumulation after 30 minutes (11.0 +/- 1.7 versus 9.6 +/- 1.0 mM), and 3) similar times for tension to decline to 50% of control (2.14 +/- 0.16 versus 2.14 +/- 0.19 minutes) but shorter time to fall to 20% of control (4.34 +/- 0.33 versus 4.90 +/- 0.22 minutes; p = 0.003). After 60 minutes of reperfusion following 30 minutes of ischemia, recovery of function was similar, with a trend toward better recovery of developed tension (to 58 +/- 9% versus 39 +/- 10% of control; p = 0.18) and tissue ATP levels in cromakalim-treated hearts but no differences in APD or rest tension. Thus, 5 microM cromakalim had mild effects in normal heart but greatly accelerated APD shortening during ischemia without markedly increasing [K+]o accumulation, possibly because the more rapid APD shortening reduced the time-averaged driving force for K+ efflux through ATP-sensitive K+ channels. A significant cardioprotective effect during 30 minutes of ischemia plus 60 minutes of reperfusion could not be demonstrated in this model. In excised membrane patches studied at room temperature, the ability of cromakalim to activate ATP-sensitive K+ channels was significantly potentiated by 100 microM but not 15 microM cytosolic ADP, suggesting that in addition to the modest fall in cytosolic ATP during early ischemia, the rapid increases in cytosolic ADP may further sensitize cardiac ATP-sensitive K+ channels to activation by cromakalim.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid washout of extracellular H+ accumulated during preceding ischemia (i.e., the abrupt restoration of extracellular pH) has been implicated as an arrhythmogenic factor during reperfusion. Therefore, we hypothesized that by limiting the rate at which extracellular pH was restored during early reperfusion it should be possible to protect against reperfusion-induced arrhythmias. To test this, we used isolated rat hearts (n = 12 per group) and a novel dual coronary perfusion cannula that permitted the induction of regional ischemia (10 minutes) and the selective reperfusion (8 minutes) of the ischemic zone with modified solutions. We examined the antiarrhythmic efficacy of 1) acidic (pH 6.6) reperfusion with stepwise restoration of extracellular pH to 7.4 (stepped pH) and 2) transient (2-minute) acidic (pH 7.1, 6.8, 6.6, or 6.4) reperfusion with subsequent abrupt restoration of extracellular pH to 7.4. Hearts in two contemporary control groups were reperfused with solution at pH 7.4 throughout. In all groups, 100% of hearts exhibited ventricular tachycardia (VT) on reperfusion. VT degenerated into ventricular fibrillation (VF) in 100% of hearts in the control group but in only 42% of hearts in the stepped-pH group (p < 0.05). In the groups subjected to transient acidic reperfusion, there was a pH-dependent prolongation of VT cycle length (measured at 15 seconds of reperfusion), which was 47.1 +/- 3.9, 51.1 +/- 5.5, 56.0 +/- 1.9, 60.4 +/- 2.8 (p < 0.05), and 68.8 +/- 5.0 (p < 0.05) msec in the pH 7.4 (control), 7.1, 6.8, 6.6, and 6.4 groups, respectively. In these groups, VT degenerated into VF in 92%, 92%, 83%, 42% (p < 0.05), and 33% (p < 0.05) of hearts, respectively. In conclusion, limiting the rate at which extracellular pH is restored during early reperfusion does not affect the rapid induction of VT but inhibits the degeneration of VT into VF and promotes spontaneous reversion to normal sinus rhythm. This is consistent with a major arrhythmogenic role, during uncontrolled reperfusion, for the rapid washout of extracellular H+.