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.

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.

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.

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.

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.

Activation of nitric oxide synthase by ischaemia in the perfused heart.

OBJECTIVE: Recent data have indicated that the activity of nitric oxide synthase (NO synthase), the enzyme producing NO from L-arginine, could be modified by ischaemia. The aim of the present work was therefore to study whether ischaemia activated NO synthase. METHODS: NO synthase activity was measured by the conversion of radioactive arginine into citrulline in extracts of isolated perfused rabbit hearts submitted to low-flow ischaemia and reperfusion. RESULTS: When measured in heart homogenates, NO synthase activity was significantly increased during ischaemia. This activation was already detectable after 5 min of ischaemia and was maintained during the whole ischaemic period. After cell fractionation, NO synthase was recovered in cytosolic and membrane fractions. The increase in NO synthase activity by ischaemia was related to an activation of the cytosolic activity, while the membrane-bound NO synthase activity remained constant. CONCLUSION: NO synthase activity in the heart is rapidly stimulated by ischaemia and this stimulation is maintained during the whole ischaemic episode. This activation is found only in the cytosolic fraction, whereas the particulate activity is not affected by ischaemia.

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.

Cyclic GMP in the perfused rat heart. Effect of ischaemia, anoxia and nitric oxide synthase inhibitor.

Working rat hearts perfused with 5.5 mM glucose were submitted to a 10-min period of no-flow ischaemia or anoxia. Both conditions stimulated glycogenolysis, activated phosphorylase and increased cyclic GMP content, although the time course of these changes differed in anoxia and ischaemia. Changes in cyclic GMP content were not correlated with glycogenolysis or phosphorylase activation. Perfusion with 1 microM L-nitroarginine methylester, an inhibitor of nitric oxide synthase, decreased cGMP concentration under normoxic conditions and abolished the ischaemia-induced increase in cGMP. The inhibitor decreased the coronary flow without affecting the overall working performance of the hearts under normoxic conditions.

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.

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.

Correlation of functional recovery with myocardial blood flow, glucose uptake, and morphologic features in patients with chronic left ventricular ischemic dysfunction undergoing coronary artery bypass grafting.

OBJECTIVE: Our objective was to investigate the influence of preoperative myocardial ultrastructure and metabolism on recovery of contractile function after coronary artery bypass grafting in patients with coronary artery disease and left ventricular dysfunction. METHODS: Dynamic positron emission tomography with 13N-ammonia and 18F-deoxyglucose was used to assess myocardial perfusion and glucose uptake in 53 patients scheduled for coronary revascularization because of coronary artery disease and left ventricular dysfunction. The degree of tissue fibrosis and the presence of potentially reversible alterations of cardiomyocytes (loss of myofilaments and accumulation of glycogen) were quantified from transmural biopsy specimens. These were harvested from the center of the dysfunctional area during the operation and analyzed with a light microscope. The recovery of contractile performance was assessed from the changes in left ventricular function at contrast ventriculography or echocardiography before and 6 months after the operation. RESULTS: According to postoperative changes in regional wall motion, left ventricular function was considered to have improved in 34 patients, whereas dysfunction persisted in 19 patients. In patients with improved wall motion, ejection fraction rose by 12% and end-systolic volume decreased by 28%. By contrast, in patients with persistent dysfunction, ejection fraction decreased by 6% and end-systolic volume increased by 25%. Before revascularization, myocardium with reversible dysfunction displayed higher levels of absolute myocardial blood flow, higher myocardial glucose uptake, less tissue fibrosis, and more altered cardiomyocytes than myocardium with persistent dysfunction. Significant correlations were found between regional blood flow and the surface of the biopsy specimen covered by fibrosis, as well as between glucose uptake and the density of altered cardiomyocytes. CONCLUSION: In patients with left ventricular ischemic dysfunction, the recovery of regional and global left ventricular function after surgical revascularization is associated with higher preoperative blood flow and glucose uptake, with less tissue fibrosis and a higher amount of viable cardiomyocytes in the dysfunctional area. The current study thus confirms the value of noninvasive preoperative metabolic imaging for identification of residual viable myocardium and for prediction of the functional outcome after revascularization.

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.

Chronic ischemic viable myocardium in man: Aspects of dedifferentiation.

Histologic analysis of biopsies derived from patients with chronic dysfunctional but viable (hibernating) myocardium showed characteristic cell alterations. These changes consisted of a partial to complete loss of sarcomeres, accumulation of glycogen, and disorganization and loss of sarcoplasmic reticulum. Most of the adaptive changes that these affected cells undergo are suggestive of dedifferentiation. In the present study the expression and organizational pattern of contractile and cytoskeletal proteins such as titin, cardiotin, and α-smooth muscle actin were assessed in hibernating and normal myocardium because the expression and organization of these constituents have been related to certain stages of cardiomyocyte differentiation. In normal cells titin shows a cross-striated staining pattern, whereas cardiotin displays a fibrillar array, parallel to the sarcomeres. α-Smooth muscle actin is not expressed in adult cardiomyocytes. The expression of titin in a punctated pattern and the marked decrease to virtual absence of cardiotin in hibernating cardiomyocytes speak in favor of an embryonic phenotype of these cells. The re-expression of α-smooth muscle actin in hibernating cells strongly supports this hypothesis. The observations on three different structural proteins of heart muscle suggest that hibernating myocardium acquired aspects of muscle cell dedifferentiation.

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.

Role of fructose 2,6-bisphosphate in the control of glycolysis. Stimulation of glycogen synthesis by lactate in the isolated working rat heart.

Fructose 2,6-bisphosphate (Fru-2,6-P2) is the most potent stimulator of 6-phosphofructo-1-kinase (PFK-1), a key enzyme of glycolysis. We studied whether this regulator is involved in the changes of glycolysis that can be induced experimentally in the isolated working rat heart. The glycolytic flux was assessed by the rate of detritiation of [2-3H]- and [3-3H]glucose, by lactate output and by the changes in glycogen content. A 20-40% increase in Fru-2,6-P2 content was observed when glycolysis was stimulated by increasing either the workload (by increasing both preload and afterload) or the concentration of glucose (from 2 to 11 mM), or by adding 7 microM insulin. Anoxia decreased the external work developed by the heart, stimulated glycolysis by activating glycogenolysis, but did not increase Fru-2,6-P2. The increase of Fru-2,6-P2 content observed after insulin, high workload or glucose load might be related to a stimulation of glucose transport, and/or an activation of 6-phosphofructo-2-kinase (PFK-2), the enzyme responsible for the synthesis of Fru-2,6-P2. Addition to the perfusate of 0.5 to 10 mM lactate, which is a preferred substrate for the heart, with pyruvate in a 10:1 ratio, induced a dose-dependent inhibition of the glycolytic flux through PFK-1, with a maximal inhibition of 75% at 5 mM lactate. The accumulation of hexose 6-phosphates without change in fructose 1,6-bisphosphate and triose-phosphates concentrations confirmed that the inhibition of glycolysis was mainly exerted on PFK-1. This inhibition resulted from a doubling of the citrate concentration, an inhibitor, and from 75% decrease in Fru-2,6-P2. Despite the inhibition of glycolysis, glucose phosphorylation was barely affected by lactate, suggesting a change in glucose metabolism. Indeed, lactate induced a dose-dependent increase in glycogen content, which doubled at 5 mM lactate, reaching the level obtained after addition of 7 microM insulin. Increased glycogen synthesis was explained by the accumulation of UDP glucose, the substrate, and glucose 6-phosphate, a stimulator of glycogen synthase. We conclude that, during aerobiosis, Fru-2,6-P2 can be regarded as a glycolytic signal which is switched on by glucose availability, workload and insulin, and which is switched off by the availability of alternative oxidative substrates such as lactate. The latter also controls glucose metabolism by diverting glucose from glycolysis to glycogen synthesis.

Inhibition of glycogenolysis by a glucose analogue in the working rat heart.

The effects of BAY o 1248, an inhibitor of alpha-amylo-1, 6-glucosidase, on glycogenolysis and post-ischemic functional recovery were investigated in isolated perfused rat hearts. Working rat hearts were perfused during 30 min with 11 mm glucose (controls) and, in some hearts, with 1 microM insulin or 5 mM lactate to increase their glycogen concentration. The hearts were then submitted to 10 min of no-flow ischemia and reperfused during 15 min with 11 mM glucose alone. Glycogen content was increased by 50% in hearts perfused with insulin or lactate. During ischemia, glycogen breakdown was similar in the control and lactate groups, but was abolished in the insulin-group. At reperfusion, functional recovery was improved in glycogen-loaded hearts compared to controls. When hearts were perfused with 1 mM BAY o 1248, added before ischemia, glycogenolysis was inhibited in the three groups and functional recovery was hampered in both the control and lactate groups. In the insulin group, however, the functional recovery was barely affected by BAY o 1248. We conclude that: (i) BAY o 1248 is an inhibitor of heart glycogen breakdown; (ii) the consequences of inhibition of ischemic glycogenolysis on post-ischemic functional recovery depend on the conditions; and (iii) the protective effect of insulin does not result from ischemic glycogenolysis.

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.

Neovascularization in human coronary atherosclerotic lesions.

Neovessels within human coronary atherosclerotic lesions are frequently observed, but their pathophysiological significance is still subject to debate. Also, the origin of these vessels and their pathways in the arterial wall are not well-known. In this study, we describe the transmural pathway and the frequency of neovessels both in vivo and in autopsy cases. Atherosclerotic coronary arteries were obtained during autopsy in 10 subjects without previous cardiovascular symptoms. In 25 patients undergoing percutaneous intervention for either stable or unstable angina, plaque fragments were retrieved by directional coronary atherectomy. In the autopsy study, at least one coronary artery in each case showed some degree of neointimal proliferation, characterized by smooth muscle cells in a dense extracellular matrix. A neovascularization process was seen in 17.5% of the 40 samples analyzed. In 2 cases, the transmural pathway of the neovessels could be tracked: serial sections revealed the emergence of an arteriole from the adventitia of the coronary artery, its transmedial course as a capillary, and its opening into the coronary arterial lumen. In symptomatic patients who underwent atherectomy, neovessels were found in 1 of 9 patients with stable angina (11%) and in 8 of 16 patients with unstable angina (50%, P < 0.05). Mostly, the neovessels appeared as capillaries cut in their short axis. In 2 cases, however, the capillary was seen in its longitudinal axis, and its pathway could be traced through the atherosclerotic lesion to its opening in the coronary lumen, as in the autopsy study. Therefore, neovessels frequently develop in the atherothrombotic plaque, both in asymptomatic and anginal patients. In the latter group, the proliferation of neovessels is more frequent in acute coronary syndromes. These findings have several implications, in particular for percutaneous coronary angioplasty and related procedures, such as local drug delivery.

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.