Cardiac cell survival and reversibility of myocardial ischemia.

Because of a limited capacity for cell regeneration, the cardiac tissue, when submitted to ischemic stress, may activate endogenous mechanisms of cell survival resulting in physiological conditions of adaptation to ischemia, known as myocardial stunning, ischemic preconditioning and myocardial hibernation. These conditions result from a switch in gene and protein expression, which sustains cardiac cell survival in a context of oxygen deprivation and during the stress of reperfusion. Understanding how the molecular adaptation of the cardiac myocyte during stress sustains its survival in these conditions might help to define novel mechanisms of endogenous myocardial salvage, in order to expand the conditions of maintained cellular viability and functional salvage of the ischemic myocardium. This review summarizes recent progress made in the study of the molecular pathways controlling reversible ischemic dysfunction, and the unraveling of novel genomic paradigms. We also focus on the discovery and characterization of novel genes, which further increase our knowledge of myocardial ischemia and open novel therapeutic possibilities for ischemic heart disease.

Gene program for cardiac cell survival induced by transient ischemia in conscious pigs.

Therapy for ischemic heart disease has been directed traditionally at limiting cell necrosis. We determined by genome profiling whether ischemic myocardium can trigger a genetic program promoting cardiac cell survival, which would be a novel and potentially equally important mechanism of salvage. Although cardiac genomics is usually performed in rodents, we used a swine model of ischemia/reperfusion followed by ventricular dysfunction (stunning), which more closely resembles clinical conditions. Gene expression profiles were compared by subtractive hybridization between ischemic and normal tissue of the same hearts. About one-third (23/74) of the nuclear-encoded genes that were up-regulated in ischemic myocardium participate in survival mechanisms (inhibition of apoptosis, cytoprotection, cell growth, and stimulation of translation). The specificity of this response was confirmed by Northern blot and quantitative PCR. Unexpectedly, this program also included genes not previously described in cardiomyocytes. Up-regulation of survival genes was more profound in subendocardium over subepicardium, reflecting that this response in stunned myocardium was proportional to the severity of the ischemic insult. Thus, in a swine model that recapitulates human heart disease, nonlethal ischemia activates a genomic program of cell survival that relates to the time course of myocardial stunning and differs transmurally in relation to ischemic stress, which induced the stunning. Understanding the genes up-regulated during myocardial stunning, including those not previously described in the heart, and developing strategies that activate this program may open new avenues for therapy in ischemic heart disease.

Uncoupling protein 3 transcription is regulated by peroxisome proliferator-activated receptor (alpha) in the adult rodent heart.

Relatively little is known concerning the regulation of uncoupling proteins (UCPs) in the heart. We investigated in the adult rodent heart 1) whether changes in workload, substrate supply, or cytokine (TNF-alpha) administration affect UCP-2 and UCP-3 expression, and 2) whether peroxisome proliferator-activated receptor alpha (PPARalpha) regulates the expression of either UCP-2 or UCP-3. Direct comparisons were made between cardiac and skeletal muscle. UCP-2, UCP-3, and PPARalpha expression were reduced when cardiac workload was either increased (pressure overload by aortic constriction) or decreased (mechanical unloading by heterotopic transplantation). Similar results were observed during cytokine administration. Reduced dietary fatty acid availability resulted in decreased expression of both cardiac UCP-2 and UCP-3. However, when fatty acid (the natural ligand for PPARalpha) supply was increased (high-fat feeding, fasting, and STZ-induced diabetes), cardiac UCP-3 but not UCP-2 expression increased. Comparable results were observed in rats treated with the specific PPARalpha agonist WY-14,643. The level of cardiac UCP-3 but not UCP-2 expression was severely reduced (20-fold) in PPARalpha-/- mice compared to wild-type mice. These results suggest that in the adult rodent heart, UCP-3 expression is regulated by PPARalpha. In contrast, cardiac UCP-2 expression is regulated in part by a fatty acid-dependent, PPARalpha-independent mechanism.

Differential effects of rexinoids and thiazolidinediones on metabolic gene expression in diabetic rodents.

Both retinoid X receptor (RXR)-selective agonists (rexinoids) and thiazolidinediones (TZDs), PPAR (peroxisome proliferator-activated receptor)-gamma-specific ligands, produce insulin sensitization in diabetic rodents. In vitro studies have demonstrated that TZDs mediate their effects via the RXR/PPAR-gamma complex. To determine whether rexinoids lower hyperglycemia by activating the RXR/PPAR-gamma heterodimer in vivo, we compared the effects of a rexinoid (LG100268) and a TZD (rosiglitazone) on gene expression in white adipose tissue, skeletal muscle, and liver of Zucker diabetic fatty rats (ZDFs). In adipose tissue, rosiglitazone decreased tumor necrosis factor-alpha (TNF-alpha) mRNA and induced glucose transporter 4 (GLUT4), muscle carnitine palmitoyl-transferase (MCPT), stearoyl CoA desaturase (SCD1), and fatty acid translocase (CD36). In contrast, LG100268 increased TNF-alpha and had no effect or suppressed the expression of GLUT4, MCPT, SCD1, and CD36. In liver, the rexinoid increased MCPT, SCD1, and CD36 mRNAs, whereas rosiglitazone induced only a small increase in CD36. In skeletal muscle, rosiglitazone and LG100268 have similar effects; both increased SCD1 and CD36 mRNAs. The differences in the pattern of genes induced by the rexinoids and the TZDs in diabetic animals found in these studies suggests that these compounds may have independent and tissue-specific effects on metabolic control in vivo.

Streptozotocin-induced changes in cardiac gene expression in the absence of severe contractile dysfunction.

UNLABELLED: Diabetes mellitus alters energy substrate metabolism and gene expression in the heart. It is not known whether the changes in gene expression are an adaptive or maladaptive process. To answer this question, we determined both the time-course and the extent of the alteration of gene expression induced by insulin-deficient diabetes. Transcript analysis with real-time quantitative polymerase chain reaction (PCR) was performed in rat hearts 1 week (acute group) or 6 months (chronic group) after administration of streptozotocin (55 mg/kg). In the acute group, insulin-dependent diabetes induced a 55-70% decrease of both glucose transporter 1 (GLUT1) and GLUT4 transcripts, a slight decrease of liver-specific carnitine palmitoyltransferase I (CPT I), and no change in muscle-specific CPT I. The uncoupling protein UCP-3 increased three-fold, with no change in UCP-2. These metabolic alterations were accompanied by an isoform switching from the normally expressed alpha myosin heavy chain (MHC) to the fetal isoform betaMHC mRNA, by a 50% decrease of cardiac alpha-actin mRNA, a 30% decrease of the sarcoplasmic Ca++-ATPase mRNA, and a 50% decrease of muscle creatine kinase (P<0.01 v controls). All genomic changes were also present in the chronic group. Genomic markers of ventricular dysfunction [tumor necrosis factor alpha (TNF-alpha), inducible nitric oxide synthase, cyclo-oxygenase-2] were not affected by chronic diabetes. In both groups, there were no changes in resting left ventricular function by echocardiography. CONCLUSION: The heart adapts to insulin-deficient diabetes by a rapid and simultaneous response of multiple genes involved in cardiac metabolism and function. This genomic adaptation resembles the adaptation of cardiac hypertrophy, remains stable over time, and does not lead to major contractile dysfunction.

Time course of functional recovery after coronary artery bypass graft surgery in patients with chronic left ventricular ischemic dysfunction.

Chronic left ventricular (LV) ischemic dysfunction, a condition often referred to as myocardial hibernation, is associated in humans with ultrastructural alterations of the myocytes, including the loss of myofilaments and the accumulation of glycogen. Given the severity of these structural changes, contractile function is unlikely to resume immediately upon revascularization. Therefore, the aim of the present study was to assess the time course of functional improvement after successful revascularization as well as its potential structural correlates. We studied 32 patients with coronary disease and chronic LV ischemic dysfunction who underwent bypass surgery. Dynamic positron emission tomography with N-13 ammonia and F-18 deoxyglucose to assess myocardial perfusion and glucose metabolism was performed in 29 patients. In all patients, a transmural biopsy was harvested from the center of the dysfunctional area, to quantify the increase in extracellular matrix and the presence of structurally altered cardiomyocytes. LV function was serially measured by digitized 2-dimensional echocardiography before and at 10 days, 2 months, and 6 months after revascularization. The time course of recovery of regional function was estimated from the monoexponential decrease in dysfunctional wall motion score. At follow-up, 19 patients had improved LV function, whereas 13 patients showed persistent dysfunction. Before revascularization, reversibly dysfunctional segments had higher myocardial blood flow (82 +/- 29 vs 53 +/- 21 ml. (min. 100 g)(-1), p = 0.044), higher glucose uptake (40 +/- 16 vs 21 +/- 9 micromol. (min. 100 g)(-1), p = 0.001), and less increase in extracellular matrix (25 +/- 15% vs 46 +/- 17%, p = 0.0008) than segments with persistent dysfunction. The extent to which function recovered was positively correlated with myocardial blood flow and negatively correlated with the increase in the extracellular matrix. In patients with reversible dysfunction, the return of segmental function was progressive and followed a monoexponential time course with a median time constant of 23 days (range 6 to 78). The rate of recovery correlated best with the proportion of altered cardiomyocytes in the biopsy. The present study thus indicates that the recovery of regional and global LV function after successful revascularization is progressive and follows a monoexponential time course that is influenced by the extent of the structural changes affecting cardiomyocytes.

Metabolic aspects of programmed cell survival and cell death in the heart.

Normal cardiac function requires a tight interaction between metabolism, contractile function and gene expression. The main perturbation challenging this equilibrium in vivo is ischemia, which alters energy flux through the control of key enzymes. The review highlights metabolic imprints and energetic aspects of programmed cell survival, programmed cell death, and of necrosis. When sustained and severe, ischemia leads to a total collapse of energy transfer, to the accumulation of metabolic endproducts, and to the development of myocardial necrosis. When moderate, ischemia results in a coordinated cellular response including enhanced anaerobic glucose metabolism, a modification of cardiac gene expression, and the development of specific mechanisms for programmed cell survival (preconditioning, stunning, hibernation). Repetitive stress results in a decrease of contractile function, a downregulation of gene expression and an impairment of energy transfer, which eventually cause the heart to fail. When the failing heart becomes energy-depleted, the programs of cell survival are no longer operational and programmed cell death ensues. To define the point of departure from programmed cell survival to cell death remains a major challenge.

Morphological analysis of atherosclerotic plaque retrieved by coronary atherectomy.

The development of atherectomy catheters and their use in clinical practice during percutaneous revascularization procedures permitted the analysis of the pathophysiology of obstructive coronary disease in vivo. The various clinical presentations of coronary disease are related to distinct morphological aspects of the culprit coronary stenosis as assessed by angiography, angioscopy or intravascular ultrasound imaging. Analysis of plaque fragments revealed the underlying histopathology. Restenotic lesions following various mechanical interventions have been studied in detail both in native coronary arteries and in bypass conduits. The biological reaction to implantation of endovascular stents involves inflammation around the stent wires as well as smooth muscle cell proliferation. Specific processes such as nitric oxide production or the activity of intramural proteases can be characterized and contribute to identify targets for future pharmacological therapy.

Expression of inducible nitric oxide synthase in human coronary atherosclerotic plaque.

OBJECTIVE: Macrophages in atherosclerotic plaque may express the inducible isoform of NO synthase (iNOS), which produces large amounts of NO. On one hand, the production of NO can be protective by its vasodilatory, antiaggregant and antiproliferative effects. On the other hand, the formation of peroxynitrite from NO may favour vasospasm and thrombogenesis. In this study, we investigated whether iNOS is present in human coronary atherosclerotic plaque, and we correlated these data with the clinical instability of the patients. METHODS: Fragments were retrieved by coronary atherectomy from 24 patients with unstable angina and 12 patients with stable angina. The presence of macrophages, and the production of TNF alpha, iNOS and nitrotyrosine were detected by immunocytochemistry. RESULTS: Macrophage clusters were found in 67% of stable patients and 87% of patients with unstable angina (NS). TNF alpha was expressed in about 50% of cases in both groups. iNOS was not expressed in fragments from stable patients but was found in macrophages from 58% of unstable patients (P < 0.001). The expression of iNOS was associated with the presence of nitrotyrosine residues, a marker of peroxynitrite formation. Expression of iNOS was correlated both with complaints of angina at rest (P < 0.05) and with the presence of thrombus at morphological examination (P < 0.001). CONCLUSION: The expression of iNOS may be induced in human coronary atherosclerotic plaque and is associated with different factors of instability.

Transcriptional adaptation of the heart to mechanical unloading.

Novel strategies in the treatment of heart failure include mechanical unloading with a left ventricular assist device. Although first considered as a bridge to cardiac transplantation, this surgical treatment may improve cardiac function in patients with heart failure, even after removal of the device. The molecular adaptation of the heart to unloading remains largely unknown. Most of the enzymes involved in the regulation of myocardial energetics (including contractile proteins, ion pumps, and metabolic enzymes)exist in "fetal" and "adult" isoforms. It is known that cardiac hypertrophy due to increased work load in vivo involves a switching from the normally expressed adult isoform to the fetal isoform. Our work has now shown that the same pattern occurs in the unloaded heart. In both conditions, this switching is accompanied by the reexpression of growth factors and proto-oncogenes. The functional improvement of the failing heart after mechanical unloading may in part be the result of a reexpression of fetal genes.

Isolated working heart: description of models relevant to radioisotopic and pharmacological assessments.

Isolated heart preparations are used to study physiological and metabolic parameters of the heart independently of its environment. Several preparations of isolated perfused heart are currently used, mainly the retrograde perfusion system and the working heart model. Both models allow investigations of the metabolic regulation of the heart in various physiological conditions (changes in workload, hormonal influences, substrate competition). These systems may also reproduce different pathological conditions, such as ischemia, reperfusion and hypoxia. Quantitation of metabolic activity can be performed with specific radioactive tracers. Finally, the effects of various drugs on cardiac performance and resistance to ischemia can be studied as well. Heart perfusion also revealed efficient methods to determine the tracer/tracee relation for radioisotopic analogues used with Positron Emission Tomography.

Unloaded heart in vivo replicates fetal gene expression of cardiac hypertrophy.

The cardiac response to increased work includes a reactivation of fetal genes. The response to a decrease in cardiac work is not known. Such information is of clinical interest, because mechanical unloading can improve the functional capacity of the failing heart. We compared here the patterns of gene expression in unloaded rat heart with those in hypertrophied rat heart. Both conditions induced a re-expression of growth factors and proto-oncogenes, and a downregulation of the 'adult' isoforms, but not of the 'fetal' isoforms, of proteins regulating myocardial energetics. Therefore, opposite changes in cardiac workload in vivo induce similar patterns of gene response. Reactivation of fetal genes may underlie the functional improvement of an unloaded failing heart.

Pathology of restenosis in saphenous bypass grafts after long-term stent implantation.

The implantation of saphenous vein grafts on the coronary arterial tree eventually leads to graft narrowing, which can be treated by the implantation of intravascular stents. However, long-term restenosis after stent implantation occurs in at least 30% of cases. Ten saphenous bypass grafts, in which a total of 12 stents had been implanted for an average of 32 months, were retrieved at least 10 months after implantation for angiographic diagnosis of reocclusion or severe restenosis. The metal struts were removed after macroscopic inspection of the vein, and the grafts were examined by light microscopy. Angiography revealed total occlusion in 9 stents and severe narrowing in 3. Pathologic examination revealed graft occlusion due to cellular hyperplasia in 4 cases and to recent thrombus formation in 5. Progression of atherosclerotic plaque was the cause of restenosis in the 3 severely narrowed grafts. In 2 of 5 grafts implanted with Palmaz-Schatz stents, the metallic struts had induced a local inflammatory reaction. Therefore, the long-term reocclusion of saphenous bypass grafts after stent implantation may be due to atherosclerotic plaque or fibromuscular hyperplasia. However, thrombus formation may still occur several years after implantation. In specific cases, stent implantation also induces inflammation around the stent struts.

Inhibition of myocardial glucose uptake by cGMP.

Guanosine 3',5'-cyclic monophosphate (cGMP), a second messenger of nitric oxide (NO), regulates myocardial contractility. It is not known whether this effect is accompanied by a change in heart metabolism. We report here the effects of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), a cGMP analog, on regulatory steps of glucose metabolism in isolated working rat hearts perfused with glucose as the substrate. When glucose uptake was stimulated by increasing the workload, addition of the cGMP analog totally suppressed this stimulation and accelerated net glycogen breakdown. 8-BrcGMP did not affect pyruvate dehydrogenase activity but activated acetyl-CoA carboxylase, the enzyme that produces malonyl-CoA, an inhibitor of long-chain fatty acid oxidation. To test whether glucose metabolism could also be affected by altering the intracellular concentration of cGMP, we perfused hearts with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, or with S-nitroso-N-acetylpenicillamine (SNAP), a NO donor. Perfusion with L-NAME decreased cGMP and increased glucose uptake by 30%, whereas perfusion with SNAP resulted in opposite effects. None of these conditions affected adenosine 3',5'-cyclic monophosphate concentration. Limitation of glucose uptake by SNAP or 8-BrcGMP decreased heart work, and this was reversed by adding alternative oxidizable substrates (pyruvate, beta-hydroxybutyrate) together with glucose. Therefore, increased NO production decreases myocardial glucose utilization and limits heart work. This effect is mediated by cGMP, which is thus endowed with both physiological and metabolic properties.

Cyclic AMP suppresses the inhibition of glycolysis by alternative oxidizable substrates in the heart.

In normoxic conditions, myocardial glucose utilization is inhibited when alternative oxidizable substrates are available. In this work we show that this inhibition is relieved in the presence of cAMP, and we studied the mechanism of this effect. Working rat hearts were perfused with 5.5 mM glucose alone (controls) or together with 5 mM lactate, 5 mM beta-hydroxybutyrate, or 1 mM palmitate. The effects of 0.1 mM chlorophenylthio-cAMP (CPT-cAMP), a cAMP analogue, were studied in each group. Glucose uptake, flux through 6-phosphofructo-1-kinase, and pyruvate dehydrogenase activity were inhibited in hearts perfused with alternative substrates, and addition of CPT-cAMP completely relieved the inhibition. The mechanism by which CPT-cAMP induced a preferential utilization of glucose was related to an increased glucose uptake and glycolysis, and to an activation of phosphorylase, pyruvate dehydrogenase, and 6-phosphofructo-2-kinase, the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, the well-known stimulator of 6-phosphofructo-1-kinase. In vitro phosphorylation of 6-phosphofructo-2-kinase by cAMP-dependent protein kinase increased the Vmax of the enzyme and decreased its sensitivity to the inhibitor citrate. Therefore, in hearts perfused with various oxidizable substrates, cAMP induces a preferential utilization of glucose by a concerted stimulation of glucose transport, glycolysis, glycogen breakdown, and glucose oxidation.

Mechanisms of control of heart glycolysis.

This review focuses on the mechanisms of control of heart glycolysis under conditions of normal and reduced oxygen supply. The kinetic properties and the biochemical characteristics of control steps (glucose transporters, hexokinase, glycogen phosphorylase and phosphofructokinases) in the heart are reviewed in the light of recent findings and are considered together to explain the control of glycolysis by substrate supply and availability, energy demand, oxygen deprivation and hormones. The role of fructose 2,6-bisphosphate in the control of glycolysis is analysed in detail. This regulator participates in the stimulation of heart glycolysis in response to glucose, workload, insulin and adrenaline, and it decreases the glycolytic flux when alternative fuels are oxidized. Fructose 2,6-bisphosphate integrates information from various metabolic and signalling pathways and acts as a glycolytic signal. Moreover, a hierarchy in the control of glycolysis occurs and is evidenced in the presence of adrenaline or cyclic AMP, which relieve the inhibition of glycolysis by alternative fuels and stimulate fatty acid oxidation. Insulin and glucose also stimulate glycolysis, but inhibit fatty acid oxidation. The mechanisms of control underlying this fuel selection are discussed. Finally, the study of the metabolic adaptation of glucose metabolism to oxygen deprivation revealed the implication of nitric oxide and cyclic GMP in the control of heart glucose metabolism.

Pathology of unstable plaque: correlation with the clinical severity of acute coronary syndromes.

OBJECTIVES: The aim of this study was to relate the various clinical presentations of acute coronary syndromes to the underlying plaque morphology as assessed from histopathologic analysis of plaque fragments obtained by directional coronary atherectomy (DCA). BACKGROUND: Autopsy studies have shown that unstable angina and infarction are related to plaque instability and involve events such as fissure or rupture of the fibrous cap, thrombosis and inflammation. The clinical severity and prognosis of acute coronary syndromes can be estimated by the Braunwald classification of unstable angina. Whether plaque morphology can be related to the Braunwald classification has not been evaluated. METHODS: Plaque fragments were obtained by DCA in 75 patients: 38 with unstable angina, 19 with stable angina and 18 with no symptoms after infarction. The presence of fibrous tissue, thrombus, high cellularity, inflammatory cells, atheroma, neovessels and "stellar-shaped" smooth muscle cells was evaluated in 7-micron thick sections by appropriate staining. The patients were classified according to clinical presentation without knowledge of the results of pathologic examination, and a plaque instability score was assigned. The risk of further cardiac events was classified as low, medium or high. RESULTS: Increasing severity of the score of unstable angina was associated with increasing prevalence of thrombus, high cellularity, atheroma and neovessels. Plaque from patients with unstable angina considered to be at low risk of further events appeared very similar to that of patients with stable angina, whereas the specific morphologic characteristics of plaque instability were more frequently observed as the clinical score and the risk of further events increased. After thrombolyzed infarction, plaque morphology depends on the delay between the acute event and DCA. Within 1 week after infarction, plaque still showed the morphologic characteristics of instability, whereas late DCA provided samples with morphologic features similar to those observed in patients with stable angina. CONCLUSIONS: The morphologic features of plaque fragments vary at different stages of acute coronary disease. The specific features of plaque instability correlate with the clinical scoring system of the Braunwald classification.