Role of creatine in the regulation of cardiac protein synthesis.

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.

Specificity of creatine in the control of muscle protein synthesis.

This study provides additional evidence that creatine, an end product of contraction unique to muscle, is involved in the control of muscle protein synthesis. Creatine is shown to stimulate selectively the rate of synthesis of two major contractile proteins, actin and myosin heavy chain, in cultures of differentiating skeletal muscle. Creatine affects only the rate of synthesis and not the rate of degradation. Several creatine analogs are as effective as creatine in stimulating muscle protein synthesis, creatinine and amino acids such as arginine and glycine are not. Creatine stimulates myosin heavy chain synthesis twofold in cultures of embryonic muscle grown in either normal or dialyzed media.

Gated sodium-23 nuclear magnetic resonance images of an isolated perfused working rat heart.

Sodium-23 nuclear magnetic resonance images of phantoms and gated images of isolated perfused working rat hearts were obtained. By synchronizing the nuclear magnetic resonance process to the heartbeat, images were obtained at systole and at diastole.

A mouse model of familial hypertrophic cardiomyopathy.

A mouse model of familial hypertrophic cardiomyopathy (FHC) was generated by the introduction of an Arg 403 --> Gln mutation into the alpha cardiac myosin heavy chain (MHC) gene. Homozygous alpha MHC 403/403 mice died 7 days after birth, and sedentary heterozygous alpha MHC 403/+ mice survived for 1 year. Cardiac histopathology and dysfunction in the alpha MHC 403/+ mice resembled human FHC. Cardiac dysfunction preceded histopathologic changes, and myocyte disarray, hypertrophy, and fibrosis increased with age. Young male alpha MHC 403/+ mice showed more evidence of disease than did their female counterparts. Preliminary results suggested that exercise capacity may have been compromised in the alpha MHC 403/+ mice. This mouse model may help to define the natural history of FHC.

Acidosis during ischemia promotes adenosine triphosphate resynthesis in postischemic rat heart. In vivo regulation of 5'-nucleotidase.

Capacity for ATP resynthesis during recovery from ischemia or hypoxia is limited to the size of the adenine nucleotide pool, which is determined in part by the activity of cytosolic 5'-nucleotidase (5'-NT): AMP-->adenosine plus inorganic phosphate (Pi). To define in vivo regulation of 5'-NT, we used the tools of 31P nuclear magnetic resonance (NMR), spectroscopy and chemical assay to measure the substrates (AMP), products (Pi, adenosine, and its catabolites), and inhibitors (Pi and H+) of 5'-NT in isolated perfused rat hearts exposed to hypoxia (where pH remains near 7) and no flow, global ischemia (where pH falls to 6.1). We estimated 5'-NT reaction velocity, assessed the relative contributions of Pi and H+ to enzyme inhibition, and defined the consequences of changes in 5'-NT activity on ATP resynthesis after hypoxia and ischemia. We conclude that (a) 5'-NT is activated during hypoxia and early ischemia but is inhibited during prolonged ischemia, (b) H+ (pH < 6.2) is a potent inhibitor of 5'-NT, and (c) differences in AMP accumulation are sufficient to explain the differences in the capacity for net ATP resynthesis in ischemic and hypoxic tissue. These observations have implications for our understanding of heterogeneity of ischemic injury and myocardial protection during ischemia.

Effects of norepinephrine infusion on myocardial high-energy phosphate content and turnover in the living rat.

Using 31P-nuclear magnetic resonance, we studied the relationship between myocardial high-energy phosphate content and flux values for the creatine kinase reaction in the living rat under inotropic states achieved during norepinephrine infusion and halothane anesthesia. Under 2% halothane anesthesia (n = 4), 1% halothane anesthesia (n = 5) and norepinephrine infusion (n = 4), rats developed rate-pressure products of 19.5 +/- 1.6, 32.0 +/- 3.5, and 48.5 +/- 2.0 X 1,000 mmHg/min, respectively. Adenosine triphosphate content was not affected by inotropic state, ranging from 24.3 +/- 1.1 to 25.6 +/- 1.1 mumol/g dry weight, but creatine phosphate content varied inversely and reversibly with cardiac performance from 45.6 +/- 6.0 under 2% halothane to 26.0 +/- 6.5 mumol/g dry weight during norepinephrine infusion. The flux values for the creatine kinase reaction were 15.4 +/- 4.6, 20.5 +/- 2.0, and 30.1 +/- 7.9 mumol/g dry weight per s under 2% halothane, 1% halothane, and 1% halothane with norepinephrine, respectively. These results suggest that the turnover of myocardial high-energy phosphate compounds, not their tissue contents, matches cardiac performance during inotropic stimulation.

Role of MgADP in the development of diastolic dysfunction in the intact beating rat heart.

Sarcomere relaxation depends on dissociation of actin and myosin, which is regulated by a number of factors, including intracellular [MgATP] as well as MgATP hydrolysis products [MgADP] and inorganic phosphate [Pi], pHi, and cytosolic calcium concentration ([Ca2+]c). To distinguish the contribution of MgADP from the other regulators in the development of diastolic dysfunction, we used a strategy to increase free [MgADP] without changing [MgATP], [Pi], or pHi. This was achieved by applying a low dose of iodoacetamide to selectively inhibit the creatine kinase activity in isolated perfused rat hearts. [MgATP], [MgADP], [Pi], and [H+] were determined using 31P NMR spectroscopy. The [Ca2+]c and the glycolytic rate were also measured. We observed an approximately threefold increase in left ventricular end diastolic pressure (LVEDP) and 38% increase in the time constant of pressure decay (P < 0.05) in these hearts, indicating a significant impairment of diastolic function. The increase in LVEDP was closely related to the increase in free [MgADP]. Rate of glycolysis was not changed, and [Ca2+]c increased by 16%, which cannot explain the severity of diastolic dysfunction. Thus, our data indicate that MgADP contributes significantly to diastolic dysfunction, possibly by slowing the rate of cross-bridge cycling.

Impairment of energy metabolism in intact residual myocardium of rat hearts with chronic myocardial infarction.

The purpose of this study was to test the hypothesis that energy metabolism is impaired in residual intact myocardium of chronically infarcted rat heart, contributing to contractile dysfunction. Myocardial infarction (MI) was induced in rats by coronary artery ligation. Hearts were isolated 8 wk later and buffer-perfused isovolumically. MI hearts showed reduced left ventricular developed pressure, but oxygen consumption was unchanged. High-energy phosphate contents were measured chemically and by 31P-NMR spectroscopy. In residual intact left ventricular tissue, ATP was unchanged after MI, while creatine phosphate was reduced by 31%. Total creatine kinase (CK) activity was reduced by 17%, the fetal CK isoenzymes BB and MB increased, while the "adult" mitochondrial CK isoenzyme activity decreased by 44%. Total creatine content decreased by 35%. Phosphoryl exchange between ATP and creatine phosphate, measured by 31P-NMR magnetization transfer, fell by 50% in MI hearts. Thus, energy reserve is substantially impaired in residual intact myocardium of chronically infarcted rats. Because phosphoryl exchange was still five times higher than ATP synthesis rates calculated from oxygen consumption, phosphoryl transfer via CK may not limit baseline contractile performance 2 mo after MI. In contrast, when MI hearts were subjected to acute stress (hypoxia), mechanical recovery during reoxygenation was impaired, suggesting that reduced energy reserve contributes to increased susceptibility of MI hearts to acute metabolic stress.

Diastolic dysfunction and altered energetics in the alphaMHC403/+ mouse model of familial hypertrophic cardiomyopathy.

An arginine to glutamine missense mutation at position 403 of the beta-cardiac myosin heavy chain causes familial hypertrophic cardiomyopathy. Here we study mice which have this same missense mutation (alphaMHC403/+) using an isolated, isovolumic heart preparation where cardiac performance is measured simultaneously with cardiac energetics using 31P nuclear magnetic resonance spectroscopy. We observed three major alterations in the physiology and bioenergetics of the alphaMHC403/+ mouse hearts. First, while there was no evidence of systolic dysfunction, diastolic function was impaired during inotropic stimulation. Diastolic dysfunction was manifest as both a decreased rate of left ventricular relaxation and an increase in end-diastolic pressure. Second, under baseline conditions alphaMHC403/+ hearts had lower phosphocreatine and increased inorganic phosphate contents resulting in a decrease in the calculated value for the free energy released from ATP hydrolysis. Third, hearts from alphaMHC403/+ hearts that were studied unpaced responded to increased perfusate calcium by decreasing heart rate approximately twice as much as wild types. We conclude that hearts from alphaMHC403/+ mice demonstrate work load-dependent diastolic dysfunction resembling the human form of familial hypertrophic cardiomyopathy. Changes in high-energy phosphate content suggest that an energy-requiring process may contribute to the observed diastolic dysfunction.

Cardiac hypertrophy with preserved contractile function after selective deletion of GLUT4 from the heart.

Glucose enters the heart via GLUT1 and GLUT4 glucose transporters. GLUT4-deficient mice develop striking cardiac hypertrophy and die prematurely. Whether their cardiac changes are caused primarily by GLUT4 deficiency in cardiomyocytes or by metabolic changes resulting from the absence of GLUT4 in skeletal muscle and adipose tissue is unclear. To determine the role of GLUT4 in the heart we used cre-loxP recombination to generate G4H(-/-) mice in which GLUT4 expression is abolished in the heart but is present in skeletal muscle and adipose tissue. Life span and serum concentrations of insulin, glucose, FFAs, lactate, and beta-hydroxybutyrate were normal. Basal cardiac glucose transport and GLUT1 expression were both increased approximately 3-fold in G4H(-/-) mice, but insulin-stimulated glucose uptake was abolished. G4H(-/-) mice develop modest cardiac hypertrophy associated with increased myocyte size and induction of atrial natriuretic and brain natriuretic peptide gene expression in the ventricles. Myocardial fibrosis did not occur. Basal and isoproterenol-stimulated isovolumic contractile performance was preserved. Thus, selective ablation of GLUT4 in the heart initiates a series of events that results in compensated cardiac hypertrophy.

Cardiac-specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice.

Heme oxygenase (HO)-1 degrades the pro-oxidant heme and generates carbon monoxide and antioxidant bilirubin. We have previously shown that in response to hypoxia, HO-1-null mice develop infarcts in the right ventricle of their hearts and that their cardiomyocytes are damaged by oxidative stress. To test whether HO-1 protects against oxidative injury in the heart, we generated cardiac-specific transgenic mice overexpressing different levels of HO-1. By use of a Langendorff preparation, hearts from transgenic mice showed improved recovery of contractile performance during reperfusion after ischemia in an HO-1 dose-dependent manner. In vivo, myocardial ischemia and reperfusion experiments showed that infarct size was only 14.7% of the area at risk in transgenic mice compared with 56.5% in wild-type mice. Hearts from these transgenic animals had reduced inflammatory cell infiltration and oxidative damage. Our data demonstrate that overexpression of HO-1 in the cardiomyocyte protects against ischemia and reperfusion injury, thus improving the recovery of cardiac function.

Energetics of the failing heart: new insights using genetic modification in the mouse.

Genetic modification in the mouse heart has provided new and important insights into many aspects of ATP synthesis, supply and utilization and how this changes in the failing heart. Here, three topics based on recent literature will be reviewed: direct manipulation of precursor pool for creatine; the path linking creatine, purine nucleotides and metabolic remodeling; and long-term reprogramming of ATP synthesis pathways. These examples illustrate the value of deleting, over-expressing and mutating specific proteins in our quest for understanding the energetics of the failing heart.

Progressive loss of myocardial ATP due to a loss of total purines during the development of heart failure in dogs: a compensatory role for the parallel loss of creatine.

BACKGROUND: Whether myocardial ATP content falls in heart failure is a long-standing and controversial issue. The mechanism(s) to explain any decrease in ATP content during heart failure have not been identified. METHODS AND RESULTS: Cardiac dysfunction, heart failure, and a prolonged steady state of heart failure were induced by chronic right ventricular pacing for 1 to 2 weeks, 3 to 4 weeks, and 7 to 9 weeks in dogs. Cardiac function and myocardial O(2) consumption (Mf1.gif" BORDER="0">O(2)) were measured with the dogs in the conscious state. ATP, total purine, and creatine were measured in biopsy specimens obtained at each stage. ATP and the total purine pool progressively fell at rates of 0.12 and 0.15 nmol. mg protein(-1). d(-1), despite an increase in Mf1.gif" BORDER="0">O(2). The rate of loss of creatine was 1.06 nmol. mg protein(-1). d(-1), 7 times faster than the depletion of total purine. CONCLUSIONS: (1) ATP contents progressively decreased during heart failure as a result of a loss of the total purine pool. The loss of purines may be due to inhibition of de novo purine synthesis. (2) Loss of creatine is an early marker of heart failure and may serve as a compensatory mechanism minimizing the reduction of the total purine pool in the failing heart.

ATP synthesis during low-flow ischemia: influence of increased glycolytic substrate.

BACKGROUND: Our goals were to (1) simulate the degree of low-flow ischemia and mixed anaerobic and aerobic metabolism of an acutely infarcting region; (2) define changes in anaerobic glycolysis, oxidative phosphorylation, and the creatine kinase (CK) reaction velocity; and (3) determine whether and how increased glycolytic substrate alters the energetic profile, function, and recovery of the ischemic myocardium in the isolated blood-perfused rat heart. METHODS AND RESULTS: Hearts had 60 minutes of low-flow ischemia (10% of baseline coronary flow) and 30 minutes of reperfusion with either control or high glucose and insulin (G+I) as substrate. In controls, during ischemia, rate-pressure product and oxygen consumption decreased by 84%. CK velocity decreased by 64%; ATP and phosphocreatine (PCr) concentrations decreased by 51% and 63%, respectively; inorganic phosphate (P(i)) concentration increased by 300%; and free [ADP] did not increase. During ischemia, relative to controls, the G+I group had similar CK velocity, oxygen consumption, and tissue acidosis but increased glycolysis, higher [ATP] and [PCr], and lower [P(i)] and therefore had a greater free energy yield from ATP hydrolysis. Ischemic systolic and diastolic function and postischemic recovery were better. CONCLUSIONS: During low-flow ischemia simulating an acute myocardial infarction region, oxidative phosphorylation accounted for 90% of ATP synthesis. The CK velocity fell by 66%, and CK did not completely use available PCr to slow ATP depletion. G+I, by increasing glycolysis, slowed ATP depletion, maintained lower [P(i)], and maintained a higher free energy from ATP hydrolysis. This improved energetic profile resulted in better systolic and diastolic function during ischemia and reperfusion. These results support the clinical use of G+I in acute MI.

Stabilization of a derangement in adenosine triphosphate metabolism during sustained, partial ischemia in the dog heart.

Severe myocardial ischemia in dogs (perfusion 10% of normal) caused progressive deterioration in adenosine triphosphate (ATP) metabolism. Between 1/2 hour and 5 hours, myocardial ATP content fell from 55 to 6% of normal, and the sum of adenine nucleotides fell from 66 to 14% of normal. Moderate ischemia (perfusion 20 to 70%) also disturbed ATP metabolism, but to a lesser degree. Moreover, there was no significant change in the concentration of any ATP metabolite between 1/2 hour and 5 hours of moderate ischemia. ATP content was 66 and 52% of normal, and adenine nucleotide content was 73 and 59% of normal at 1/2 hour and 5 hours, respectively. Trivial ischemia (perfusion 80% or greater) barely perturbed ATP metabolism at either 1/2 hour or 5 hours. Thus, in contrast to severe or trivial ischemia, prolonged moderate ischemia produced a derangement in ATP metabolism that persisted and was relatively stable for 5 hours.

Long-term beta-blocker treatment prevents chronic creatine kinase and lactate dehydrogenase system changes in rat hearts after myocardial infarction.

OBJECTIVES: We tested the hypothesis that long-term beta-blocker treatment with bisoprolol prevents creatine kinase (CK) and lactate dehydrogenase system changes that occur after chronic myocardial infarction. BACKGROUND: The mechanism of the beneficial effect of beta-blocker therapy is still unclear. METHODS: Six groups of rats were studied. Sham operated (sham) and hearts with ligated left anterior descending coronary artery (myocardial infarction) were untreated, treated early (beginning 30 min after infarction) or treated late (beginning 14 days after infarction). After 8 weeks, hearts were isolated and buffer perfused isovolumetrically. With a left ventricular balloon, mechanical function was recorded at an end-diastolic pressure of 10 mm Hg. Biopsy samples of noninfarcted left ventricular tissue were taken. Enzyme activities were measured spectrophotometrically; isoenzymes were separated by agar gel electrophoresis; and total creatine levels were measured with high performance liquid chromatography. RESULTS: The decrease in left ventricular developed pressure in untreated hearts (120 +/- 9 vs. 104 +/- 5 mm Hg [mean +/- SE], p < 0.05, sham vs. myocardial infarction) after myocardial infarction was prevented by early treatment (118 +/- 9 vs. 113 +/- 4 mm Hg). Late treatment failed to improve mechanical function. Reduction of CK activity occurring in untreated infarcted hearts (6.4 +/- 0.3 vs. 5.1 +/- 0.3 IU/mg protein, p < 0.05, sham vs. myocardial infarction) was prevented by early beta-blocker therapy. The increase in CK isoenzyme BB and MB levels, decrease in mitochondrial CK isoenzyme levels and increase in anaerobic lactate dehydrogenase isoenzyme levels in untreated infarcted hearts did not occur during bisoprolol treatment. The decrease in total creatine levels after myocardial infarction (74.2 +/- 4.9 vs. 54.9 +/- 3.3 nmol/mg protein, p < 0.05, sham vs. myocardial infarction) was prevented by bisoprolol treatment. Early treatment was more effective than late therapy in preventing CK and lactate dehydrogenase system changes. In addition, in sham hearts, a 40% increase of creatine levels above normal levels was detected. CONCLUSIONS: Bisoprolol prevented changes in CK and lactate dehydrogenase system that occur after myocardial infarction. These observations may be related to the beneficial effects of long-term beta-blocker treatment in patients with chronic myocardial infarction.

Comparison of twitch force and calcium handling in papillary muscles from right ventricular pressure overload hypertrophy in weanling and juvenile ferrets.

OBJECTIVE: The aim was to compare differences in peak twitch force occurring despite similar degrees of right ventricular hypertrophy in ferrets with pulmonary artery banding at either weanling or juvenile age. METHODS: After inducing pressure overload hypertrophy by banding the pulmonary artery of weanling and juvenile age ferrets, mechanical function (that is, isometric twitch force and passive stiffness), intracellular [Ca2+] using the calcium indicator aequorin, markers of myocardial energy supply, and quantified connective tissue content were studied. RESULTS: It was previously found that there was a reduced peak isometric twitch force despite normal [Ca2+]i in juvenile banded ferrets age 10-12 weeks with right ventricular pressure overload hypertrophy (POHj). In the present study we report findings in banded weanling ferrets (POHw) age 7 weeks. POHw animals showed a similar degree of hypertrophy to that found in the POHj. However, there was a greater peak twitch force in hypertrophied muscles at higher [Ca2+]o. There was no difference in peak [Ca2+]i: -3.1(SEM 0.1) v -3.1(0.3) (log fractional luminescence) at 16 mM [Ca2+]o for control and POHw, respectively. Connective tissue content for control animals was 10(1)% versus 10(2)% in POHw. Despite a lack of quantitative change in connective tissue content or resting [Ca2+]i in POHw, passive stiffness in papillary muscles was increased. Retrospective analysis of tissue from POHj revealed a connective tissue content of 24(6.8)% (P << 0.001). Thus the decreased peak twitch force reported in POHj might in part be due to an increase in fibrous connective tissue. In this study, lactate dehydrogenase was significantly higher (38%) in POHw animals. In contradistinction, total creatine kinase activity and total creatine content were significantly less (22%) in hearts from POHj animals, indicating differences in myocyte remodelling despite similar degrees and durations of hypertrophy. CONCLUSIONS: Comparison of POHw and POHj showed that, when there is restructuring of the extracellular space in terms of increased fibrosis, there is also molecular remodelling in the myocyte, as demonstrated by a decrease in the creatine kinase system.

Type 2 iodothyronin deiodinase transgene expression in the mouse heart causes cardiac-specific thyrotoxicosis.

Type 2 iodothyronine deiodinase (D(2)) catalyzes intracellular 3, 5, 3' triiodothyronine (T(3)) production from thyroxine (T(4)), and its messenger RNA mRNA is highly expressed in human, but not rodent, myocardium. The goal of this study was to identify the effects of D(2) expression in the mouse myocardium on cardiac function and gene expression. We prepared transgenic (TG) mice in which human D(2) expression was driven by the alpha-MHC promoter. Despite high myocardial D(2) activity, myocardial T(3) was, at most, minimally increased in TG myocardium. Although, plasma T(3) and T(4), growth rate as well as the heart weight was not affected by TG expression, there was a significant increase in heart rate of the isolated perfused hearts, from 284 +/-12 to 350 +/- 7 beats/min. This was accompanied by an increase in pacemaker channel (HCN2) but not alpha-MHC or SERCA II messenger RNA levels. Biochemical studies and (31)P-NMR spectroscopy showed significantly lower levels of phosphocreatine and creatine in TG hearts. These results suggest that even mild chronic myocardial thyrotoxicosis, such as may occur in human hyperthyroidism, can cause tachycardia and associated changes in high energy phosphate compounds independent of an increase in SERCA II and alpha-MHC.

Nuclear magnetic resonance imaging: potential cardiac applications.

During the past several years, the production of high resolution images of organs in intact animals and human beings using nuclear magnetic resonance (nmr) has generated much interest and raised the possibility that the technique could be usefully applied to clinical problems. Because the images are derived from biochemical as well as structural information, valuable data relating to the metabolic status of the tissues and organs may be obtained. Furthermore, nuclear magnetic resonance imaging involves no potentially hazardous ionizing radiation. The technology of the technique is complex and much work remains to be done defining the biochemical and physiologic basis of such images, but the potential rewards of defining the metabolic state of organs such as heart and brain in the intact animal and human justify continued research.

Effects of reduced coronary flow on thallium-201 accumulation and release in an in vitro rat heart preparation.

To study the relation between myocardial thallium-201 (TI-201) uptake, TI-201 release, and reduced coronary flow, isolated Langendorff rat hearts (n = 8) were perfused for 3 hours at constant flows ranging from physiologic (12 ml/min) to severely ischemic (1.5 ml/min); thallium activity was monitored with a scintillation probe. Each heart was perfused for 1 hour with thallium buffer, followed by 2 hours with thallium-free buffer at the same flow rate. Accumulation curves for all 4 flows were monoexponential. However, release curves during the 2 hours of washout with thallium-free buffer demonstrated a biexponential configuration. The early fast release component decreased with reductions in coronary flow, and the later slow release component did not vary significantly with flow. These data show that thallium clearance has at least 2 components: a rapid (possibly extracellular) component related to coronary flow and a slow (possibly intracellular) component independent of coronary flow. These findings should be useful in providing a better understanding of thallium redistribution observed clinically.