Cardiomyocyte Metabolism

Chapter 15

Cardiomyocyte Metabolism: All Is in Flux Heinrich Taegtmeyer Department of Medicine/Cardiology, The University of Texas School of Medicine at Houston, Houston, TX

The intermediary metabolism of energy-providing substrates supports the rhythmic contraction of the heart (Figure 15.1). However, metabolism also encompasses the universal principle of nature that all is in flux. From the Krebs cycle to the cell cycle, from protein synthesis to degradation, from phosphorylation to dephosphorylation, from histone acteylation to deacetylation, all is in flux. First enunciated by Heraclitus in 513 BCE, in modern times no one has seen this principle more clearly than Hans Krebs in the discovery of the citric acid cycle (1) and Rudolph Schoenheimer in “The Dynamic State of Body Constituents” (2). This chapter highlights principles of cardiac energetics, and includes multiple newly discovered roles of intermediary metabolites that regulate cardiac cell function. The chapter also highlights emerging concepts in metabolic regulation at the transcriptional, translational, and post-translational level, and discusses the putative role of metabolic signals in the regulation of myocardial protein turnover. In the cold light of twenty-first century molecular biology metabolic pathways seem a distant memory. Yet the importance of fuel metabolism in the heart is newly appreciated in heart disease, cancer, and diabetes. A brief review of the basics is in order.

FUEL METABOLISM IN PERSPECTIVE As in any other organ of the body, it is impossible to separate function from metabolism in the heart. The bulk of the heart’s energy for contraction, ion movements, and intracellular protein turnover is provided by the metabolism of oxidizable substrates. The final product of oxidative metabolism in the mitochondria is ATP. In the simple scheme (Figure 15.1), a decrease in the flux of metabolic energy results in a decrease in ATP production, a decrease in oxygen consumption and a decrease in contractile function. Vice versa, an increase in energy demand results in increased ATP turnover, an increase in oxygen consumption and an increase in contractile function. The heart’s requirement for calcium, nutrients, and oxygen has been recognized for well over a century (3,4)

Muscle. DOI: http://dx.doi.org/10.1016/B978-0-12-381510-1.00015-6 © 2012 Elsevier Inc. All rights reserved.

and the field of cardiac metabolism has a rich history of integrative and molecular physiology (5). By measuring arteriovenous differences in the heart lung preparation (6) of the dog or in the human heart through cannulation of the coronary sinus (7), early investigators have laid the foundation for current concepts of myocardial energy substrate metabolism. The more recent reviews survey the knowledge gained through isolated heart preparations, isolated cell preparations, and isolated cell organelles (5,8 17). As will be discussed later in the chapter, the toolbox for the discovery of metabolic regulation in genetic animal models and in cardiomyocytes has grown even more dramatically than the clinically useful instrumentation.

MITOCHONDRIA AND THE DYNAMICS OF METABOLISM IN THE HEART Cardiac metabolism maintains a dynamic state of equilibrium for efficient energy transfer at the site of ATP production in the mitochondria and at the site of ATP usage in the cross-bridges. This has three consequences. Hence, the rate of energy turnover, and not the tissue content of ATP, is the main driving force of energy metabolism (10,18 20). The greater the work output, the higher the rate of ATP turnover, the higher the rate of oxygen consumption, and the higher the rate of substrate input and utilization (Figure 15.1). Secondly, energy transfer in the heart obeys the first and second laws of thermodynamics. Within a closed system, energy can only be converted from one form into another, and a process occurs spontaneously only if it is associated with an increase in randomness (or entropy) of the system. Everything else requires catalysts or enzymes. Third, when the heart’s ability to convert chemical into mechanical energy is impaired for any reason (21) the consequences manifest themselves as functional and metabolic derangements in the rest of the body, i.e. heart failure from a disease of impaired energy transfer in the heart.

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A close correlation exists among mitochondrial volume fraction, heart rate, and total body oxygen consumption, with mitochondrial volume fractions ranging from 25% humans to 38% in mice (22). Heart muscle mitochondria are not only abundant, they also contain a far greater number of cristae (the location of respiratory chain enzymes) than do mitochondria of other organs, such as liver, brain, or skeletal muscle (23). Lastly, while the term is immensely important, it is no longer sufficient to call mitochondria only the “power stations” of the cell (24). Mitochondria are dynamic cell organelles which divide and fuse, which are subject to organized destruction (mitophagy), which are the sources of cell death signals (cytochrome c) and which are a major target of metabolic dysregulation in diabetes and heart failure. Input: Substrates + O2

Metabolism

ADP + Pi

ADP

PCr

Contraction

Output: Pump action, Heat FIGURE 15.1 The heart converts substrates and oxygen (input) to pump action and heat (output). Metabolism of energy providing substrates fuels ATP production for the contractile process.

THE HIGHWAYS OF ENERGY TRANSFER In the cardiomyocytes, complex chemical reactions proceed rapidly at a relatively low temperature and at low substrate concentrations through enzyme-catalyzed pathways, an essential part of which are cycles (e.g. the Krebs cycle, the ATP cycle), because cycles have evolved as the most efficient form of energy transfer (Figure 15.2) (5,25). The function of both extra- and intra-mitochondrial cycles requires conservation of their constituent moieties. The replenishment of intermediate moieties in a cycle requires anaplerotic pathways (26). Anaplerotic pathways of the Krebs cycle are critical for energy transfer in the heart (27). In addition to the concept of cycles, a few definitions may be useful. A metabolic pathway is defined as a series of enzyme-catalyzed reactions beginning with a flux-generating step (usually a reaction catalyzed by a non-equilibrium reaction or transport of the metabolite across a membrane) and ending with the removal of a product (28,29). Characteristic of most metabolic pathways is that, once flux has been initiated, there is a rapid and concerted response of the entire pathway. In this system of flux, metabolite levels control enzyme activities and, in turn, enzyme activities are controlled by metabolite levels. Control is the power to change metabolic flux in response to an external signal, whereas regulation is the inherent capacity of enzymes geared toward maintaining a constant internal state (30). In such a system, large changes in the flux through metabolic pathways correspond to only very small changes in myocardial metabolite concentration (31). Regulatory sites of metabolism, or pacemaker enzymes (32), have become targets for the manipulation of metabolism with drugs (33,34).

FIGURE 15.2 From the circulation of the blood to the cross-bridges of the sarcomeres energy transfer makes use of a series of moiety conserved cycles. The metabolic cycles are located in the mitochondria (red). Ca21 is considered the main regulator of both contraction and Krebs cycle flux.

Circulation Ca2+ Perfusion Krebs cycle Ca2+

NADH H+ FADH H+ H+ gradient

Ca2+

ATP PCr Mitochondria

Cardiac Muscle

Crossbridges Ca2+

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ATP production is tightly coupled to ATP utilization. Hence, substrate oxidation is also tightly coupled to cardiac work (35 37). An acute increase in work load of the isolated working rat heart results in an acute increase in myocardial oxygen consumption (measured) and CO2 production (Figure 15.4a) (predicted); and the heart uses carbohydrates (glycogen, glucose, and lactate) as fuel for respiration to meet the increased demand for chemical energy (Figure 15.4b). In other words, with an increase in

The breakdown of substrates can be divided into four stages (Figure 15.3). The first stage consists of substrate delivery and uptake by the cell. The second stage consists of intracellular pathways leading to acetyl coenzyme A (acetyl-CoA). The third stage consists of the oxidation of acetyl-CoA in the Krebs cycle; and the fourth stage consists of the reaction of reducing equivalents with molecular oxygen in the respiratory chain, where electron transfer is coupled to rephosphorylation of ADP to ATP.

Lactate Substrate uptake

GLUT G-6-P

FIGURE 15.3 The metabolism of energy-providing substrates is organized into three stages, all of which are regulated: substrate uptake, intermediary metabolism, and the oxidation of smaller carbon units in the Krebs cycle providing reducing equivalents for the respiratory chain. (With kind permission from Springer Science 1 Business Media: Cardiovascular Medicine, 3rd edition, 2007, p. 1159, eds Willerson, Cohn, Wellens, Holmes, figure 50.3.)

Free fatty acids

Glucose

Triglycerides

FFA

Glycogen ACS

Lactate

Acyl-CoA

Pyruvate

Metabolism

CPT Acyl-CoA

β Oxidation

Pyruvate PDC Acetyl-CoA

Oxidation CO2

Krebs cycle

NADH H+ ½O2 H2O NADH H+ FADH2

Respiratory chain

ATP

ATP ADP

α-MHC

ATP ADP

ADP

ATP

Ca2+

SERCA

Sarcoplasmic reticulum

7 0 6 0 5 0 4 0 5 0

5 5

measured predicted from oxidation of Exogenous Lipid + Endogenous Lipid + Exogenous Glucose + Endogenous Glucose + Exogenous Lactate + Pyruvate Release 6 6 0 5 Perfusion Time (min)

7 0

7 5

Lactate

4.0

Epinephrine

8 0

Oxidation Rate (umol/min/g dry wt.)

4.5

Epinephrine

Oxygen Consumption, measured predicted (umol.min/g dry wt.)

ATP

β-MHC

9 0

0

ATP + Pi ATP + Pi

Ca2+

Energy utilization

NAD+ FAD

3.5 3.0 2.5

Glucose

2.0

Oleate

1.5 1.0 0.5

Glycogen

0.0 −0.5

5 0

5 5

6 6 0 5 Perfusion Time (min)

7 0

7 5

FIGURE 15.4 In the isolated working heart an acute increase in work load results in an acute increase of oxygen consumption (a) and oxidation of carbohydrates (b). Note the instant, but transient, increase in glycogen oxidation, and the sustained increase in glucose and lactate oxidation. All three substrates support the increase in cardiac work. See text for further detail. (From Goodwin et al., 1998 (35).)

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energy demand the heart readily adds a second substrate for energy provision. Reduced flavin adenine dinucleotide (FADH2) and NADH are generated, and the reducing equivalents enter the electron transport chain producing an electrochemical gradient across the inner mitochondrial membrane that drives ATP synthesis in the presence of molecular oxygen, ADP, and inorganic phosphate. The exact mechanism by which respiration is coupled to energy expenditure in vivo is, however, still not known (38 40). It can be stated with certainly that for a given physiologic environment the heart oxidizes the most efficient substrate.

TRACING METABOLIC PATHWAYS EX VIVO AND IN VIVO The tracing of metabolic pathways can be accomplished qualitatively owing to the development of new, nondestructive imaging techniques such as nuclear magnetic resonance spectroscopy or positron emission tomography (PET). Both techniques also permit the assessment of regional metabolic processes in the beating heart both ex vivo and in vivo (41 48). Analysis of energy-rich phosphates in the beating heart by nuclear magnetic resonance spectroscopy of 31P lends further support to the view that over a relatively wide range of work output, tissue content of ATP is not correlated with rates of ATP turnover, which can be assessed by oxygen consumption or contractile performance of the heart (49). The recent adaptation of isotopomer analysis of 13C natural abundance or 13C-labeled compounds allows the analysis of flux through specific pathways, especially the citric acid cycle and glycogen turnover, to be studied quantitatively as serial spectra are obtained (48,50 52). More recently, the use of hyperpolarization of 13C biomolecules (53) has made it possible to assess Krebs cycle metabolism with magnetic resonance spectroscopy (MRS) (54). Collectively, MRS signals from 31P, 13C, and other stable isotopes hold the promise to provide further insight into the dynamics of heart metabolism (55). Short-lived positron-emitting tracers have been so successful in their clinical application because they permit a visual assessment of regional differences in metabolic activity of the heart (47,56). Two types can be distinguished. The first approach entails uptake and retention of a tracer analogue, such as [18F-]-2-deoxy, 2-fluoroglucose (FDG). The second approach entails the uptake and clearance of 11C tracers (fatty acids, glucose, or lactate) where the rapid phase of clearance from the tissue represents their rate of oxidation or, in the case of [11C]acetate, oxidation in the citric acid cycle alone (56). Uptake and retention of the tracer analogue FDG increase linearly

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with time (57), whereas the clearance of a labeled fatty acid after an initial peak in tissue activity is biexponential (56,58,59). Both types of approaches have been used clinically to assess substrate metabolism in normal and ischemic myocardium. Enhanced glucose uptake (assessed with FDG) is used to identify reversible ischemia in “hibernating” myocardium where heart muscle remodels metabolically and the mismatch of perfusion and metabolism is an indicator of myocardial viability (60). Assessment of myocardial metabolism with [11C] tracers, which are oxidized by the heart, suggests that patients with idiopathic dilated cardiomyopathy exhibit changes in substrate preference characterized by decreased fatty acid metabolism and increased glucose metabolism (61) in keeping with the constitutive expression of the fetal metabolic gene program in the failing human heart (62).

GENETIC MODELS FOR THE ELUCIDATION OF CARDIAC METABOLISM Redirecting energy metabolism is largely orchestrated by proteins that are involved in regulating flux through entire pathways. Accordingly, much has been learned from genetic models in which specific proteins are either mutated, overexpressed, or deleted. Examples are given in the sections on substrate metabolism below (63 66).

MAJOR ENERGY-PROVIDING SUBSTRATES A legitimate question is: Why is it important to know metabolic pathways in detail? Metabolic pathways can be likened to a power grid, and the heart can be likened to a light bulb. Fuels are the various sources of energy that are all converted to electricity. A simple explanation for this redundancy is that the grid uses those fuels that are most readily available, but all fuels are converted to the same form of energy. The same principle applies to the heart: For a given environment the heart uses the most efficient source of fuel. However, the fuel distribution system is far more complex than an electrical power grid.

Glucose, Lactate, and Glycogen Although long chain fatty acids are the predominant fuel for energy provision to the post-natal heart, carbohydrates are the fuel for the fetal heart (67) and also for the stressed adult heart in the state of exercise or pressure overload (68,69). The reasons for such a “hierarchy” of fuels can be deduced from a number of observations:

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1. In the normal, i.e. the non-diabetic, mammalian organism glucose levels in the blood are tightly regulated at around 5 mM or (90 mg%). 2. When we exercise and blood lactate levels rise, lactate replaces all other substrates as fuel for respiration of the heart (68). 3. When the normal heart is stressed, it oxidizes first glycogen, then glucose and lactate (35,70). 4. Glucose is an anaplerotic substrate for the citrate cycle (37,71) and glucose is essential for the initiation of fatty acid oxidation in heart (72). Isotopic tracer studies have demonstrated that the normal human heart produces lactate at the same time it oxidizes lactate (35,73). It appears that a large portion of exogenous glucose, when taken up by the cardiomyocyte, is shunted into first glycogen before it is oxidized (74,75).

Regulation of Glucose Metabolism There are three energy-yielding stages of glucose metabolism: the glycolytic pathway leading to pyruvate, oxaloacetate, and lactate; the Krebs cycle; and the respiratory chain. Each state is regulated by its own set of checkpoints, so that overall flux through the pathways (which may be assessed externally on a second-by-second timescale with the glucose tracer analogue FDG) (57) proceeds at a rate just sufficient to satisfy the heart’s beat-to-beat needs for ATP. We discuss the regulatory sites of metabolism in more detail because dysregulation of one of these steps can affect the rate and efficacy of energy transfer and, hence, contractile function of the heart.

Glucose Entry into the Myocyte Glucose uptake by the cardiomyocyte follows classical Michaelis Menten kinetics, and the transport of glucose occurs along a steep concentration gradient and is regulated by specific transporters (76). The stereospecificity of the transporter for sugars of the carbon configuration is not matched by the same degree of selectivity, and various tracer analogues, including 2-deoxyglucose and FDG, are transported in the same way as glucose. Glucose transport, the rate-limiting step in myocardial glucose utilization (77), requires facilitative glucose transporters. The family of glucose transporters (GLUT) is conserved over a wide range of organisms, suggesting a common evolutionary origin (78 81). GLUT-1 and GLUT-4 are the major glucose transporter isoforms expressed in the heart (62,82). GLUT-4, the insulinsensitive transporter, is also expressed by skeletal muscle and in adipose tissue (78). Recruitment of GLUT-4 from a microsomal cytosolic pool to the sarcolemma by insulin (or ischemia or adrenergic stimulation) (83 86) increases

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the maximal velocity of glucose transport. Alphaadrenergic stimulation uses the same signaling pathway as insulin to promote glucose uptake (87), whereas the effects of ischemia, beta-receptor stimulation, and insulin on glucose uptake are additive (88). Cardiomyocytes also express the GLUT-1 transporter isoform, which is presumably independent of insulin regulation and predominates in fetal, hypertrophied, atrophied and failing myocardium (62,79,89). GLUT-1 is the first gene whose transcription is dually stimulated in response to hypoxia and inhibition of oxidative phosphorylation (90), and overexpression of GLUT-1 prevents the functional decline of hypertrophied heart (91). Both transporters have a Km for glucose (i.e. the concentration at which the rate of glucose transport is half maximal) that is in the range of plasma glucose concentrations under fasting conditions (92). The normal heart also expresses a low amount of GLUT-3, which has a Km below the normal plasma glucose concentration (93). Over the years a number of novel GLUTs have been identified in heart muscle including GLUT-8, GLUT-11, and GLUT-12 (94). A role of these proteins in myocardial glucose metabolism has not yet been defined. Phosphorylation of glucose by hexokinase becomes rate-limiting for glycolysis at high rates of glucose transport. Rates of glucose phosphorylation measured in vitro are more than twice as high as the maximal measured rates of glucose utilization by the heart at a physiologic workload and with glucose as the only substrate (95). However, intracellular glucose concentrations rise during starvation, in diabetes, and with the concomitant oxidation of fatty acids, ketone bodies, or lactate, which indicates inhibition of the phosphorylation step. This is most likely to be due to accumulation of glucose-6-phosphate, which is an allosteric inhibitor of hexokinase II, the cardiac isoform of hexokinase. Reduction of hexokinase II levels results in decreased cardiac function and altered remodeling after ischemia and reperfusion (96). Glucose-6-phosphate is at the branch point of four distinct pathways: 1. degradation via the Embden Meyerhof pathway (also termed the glycolytic pathway when it entails metabolism of glucose to lactate only); 2. conversion to glycogen via the glycogen synthase reaction; 3. metabolism (oxidation) via the pentose-phosphate pathway, which yields ribose and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) (97); 4. entry into the hexosamine biosynthetic pathway (98). While the latter two pathways are of quantitatively lesser importance in heart muscle than are the former two, they provide glycogen.

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Glycogen Glycogen metabolism was the first paradigm for the molecular basis of hormone action (99). In this pathway the control of enzyme activity by an allosteric regulator (activation of phosphorylase by adenosine monophosphate [AMP]) was first described (100), enzyme regulation by covalent modification was discovered (101), and the molecular basis of hormone action by signal transduction was elucidated by the discovery of cyclic adenosine monophosphate (cAMP) (102). Although the enzyme glycogen synthase kinase 3β (GSK3β), which phosphorylates and inhibits glycogen synthesis, is a well-known regulator of cardiac growth, its role in intermediary metabolism must not be overlooked. Hypertrophic stimuli inhibit GSK3β to regulate changes in metabolism, gene expression, and cytoskeletal integrity needed to promote cell growth (103). The role of glycogen metabolism in the heart is still not completely understood (104). Here two points should be made. Firstly, the vast amount of glycogen in fetal cardiac muscle probably allows the heart to maintain its contractile activity in the face of severe hypoxia (105) during birth. Secondly, glycogen and glycogen phosphorylase are closely associated with the sarcoplasmic reticulum (106) and in skeletal muscle a decreased glycogen content results in a reversible reduction in force, Ca21 release from the sarcoplasmic reticulum, and contractile protein function (107).

Glycolysis Glucose is special among the energy-providing substrates for the heart in its ability to provide a small, but significant, amount of ATP through substrate-level phosphorylation in the glycolytic pathway. This occurs especially in the setting of hypoxia and ischemia when flux through the glycolytic pathway is enhanced resulting in the formation of lactate as well as alanine (108). Other stimulants of flux through the glycolytic pathway are increases in cardiac work, either acutely with exercise (35,109) or chronically with pressure overload without (110) or with hypertrophy (69,111). Both hypertrophy and atrophy are associated with increased glucose oxidation rates in the face of decreased insulin responsiveness of the heart (112). In ischemia the accumulation of glycolytic intermediates may worsen contractile function (113) and acute hyperglycemia may abolish ischemic preconditioning in vivo (114) while provision of glucose together with insulin and potassium improves contractile function in the acutely ischemic, reperfused myocardium (115 118). The first step committing glucose to the glycolytic pathway is 6-phospho-fructo-1-kinase (PFK-1), which catalyzes the phosphorylation of fructose-6-phosphate to fructose 1,6-bisphosphate. Because of the complex

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allosteric regulation of PFK, this is a rate-limiting step (pacemaker enzyme) for glycolysis (119). ATP, citrate, and protons are negative allosteric effectors, whereas AMP and fructose 1,6-bisphosphate are positive effectors (120 122). Fructose 2,6-bisphosphate is the main activator of PFK-1 in normoxic heart (123). Further down in the glycolytic pathway, the oxidation of the triose-phosphate glyceraldehydes 3-phosphate to 1,3-diphsophoglycerate couples in the energy-conserving step in the glycolytic pathway that leads to the nonoxidative formation of ATP with high cardiac work (124) or ischemia (125) when PFK becomes strongly activated, glycolysis is controlled further downstream, at the triosephosphate dehydrogenase step.

Pyruvate Metabolism: An Branch Point The last glycolytic intermediate, pyruvate, is substrate for another branchpoint in metabolism. Pyruvate can be reduced to lactate (which completes the glycolytic pathway), transaminated to alanine (108), carboxylated to oxaloacetate or malate (126,127), or, most importantly, oxidized to acetyl-CoA. In well-oxygenated, working heart muscle, however, the bulk of pyruvate enters the mitochondrion through a transporter which can be inhibited by 4-hydroxy-alpha-cyanocinnamate (128). Once inside the mitochondrial matrix, pyruvate is either decarboxylated to acetyl-CoA or carboxylated to oxaloacetate (127). The capture of metabolically produced carbon dioxide from the pyruvate dehydrogenase reaction to form oxaloacetate is an example for the efficient use of one substrate supplying two precursors for citrate synthesis and for the efficient recycling of carbon dioxide. Oxidative decarboxylation of pyruvate is highly regulated by activation and inactivation of the pyruvate dehydrogenase complex (PDC) (129). The conversion of pyruvate to acetyl-CoA requires the sequential action of three different enzymes: pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. The reaction also requires five different coenzymes or prosthetic groups: thiamine pyrophosphate, lipoic acid, uncombined coenzyme A (CoA SH), FAD1, and NAD1. These enzymes and coenzymes are organized into a multienzyme cluster. Much work was done in the 1970s on the regulation of PDC by covalent modification through a phosphorylation and dephosphorylation cycle (8). Multi-site phosphorylation of the pyruvate dehydrogenase component of the complex provides an indirect means by which the entire complex is regulated by the relative activities of the PDC kinase and phosphatase reactions. Of the metabolite pairs ATP ADP, acetyl-CoA CoA SH, NADH NAD1, and lactate pyruvate, the first member either activates the kinase or serves as substrate, and the

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second member inhibits the enzyme. Ca21 and Mg21 both inhibit the kinase and activate the phosphatase reaction (i.e. lead to PDC-activation). The effects of fatty acids or ketone body oxidation are likely to be mediated by the increase in acetyl-CoA because the primary effect or the inhibition-inactivation of PDC by fatty acids or ketone bodies in the acetyl-CoA COA SH ratio (130). Conversely, an increase in cardiac work may inhibit the PDC kinase owing to a decrease in NADH, acetyl-CoA, and ATP, leading to activation of PDC (131).

Fatty Acids Fatty acids, esterified as triglycerides, are the heart’s predominant fuel for respiration (7,132,133). In spite of their preeminence as a source for energy, fatty acids are also the only fuel capable of uncoupling oxidative phosphorylation (134,135), which lowers the efficiency of fatty acids as energy substrates. The predominant forms of fatty acids in the blood stream are the monounsaturated long chain fatty acid oleate (C18:1) and the saturated fatty acid palmitate (C16:0). The pathway of long chain fatty acid oxidation starts with the liberation of fatty acids from triglycerides and ends with the entry of acetyl-CoA into the citric acid cycle (Figure 15.3). Fatty acids cross the plasma membrane with the help of carrier proteins (136), are bound by a heart-specific fatty acid-binding protein (h-FABP) (137 139) and ushered to mitochondria (140) via activation to fatty-acyl coenzyme A (acyl-CoA). Deletion of fatty acyl-CoA synthetase in the heart causes hypertrophy, most likely by rerouting glucose metabolism (141). AcylCoA is transesterified with carnitine for transport across the inner mitochondrial membrane in exchange for carnitine, re-esterification with CoA SH, beta-oxidation, and finally, oxidation of acetyl-CoA. A variable amount of FFA taken up by heart muscle is also esterified with glycerol in the cytosol to form triglycerides. Increased net triglyceride synthesis by the heart has been observed with starvation, diabetes, ischemia, and in heart failure (142). At present it is not known whether increased triglyceride levels are the result of increased rates of esterification or a decreased rate of lipolysis (143).

Carnitine Palmitoyl Transferase and a Critical Role for Malonyl-CoA Transfer of long chain fatty acyl-CoA into mitochondria is rate-limiting for β-oxidation of long chain fatty acids and requires a three-step membrane transport process. The first step in this sequence is the transfer of the acyl group from CoA to carnitine, catalyzed by the enzyme carnitine palmitoyl transferase I (CPT I).

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The CPT I system is inhibited by physiologic concentrations of malonyl-CoA, the product of acetyl-CoA carboxylation, and was first characterized in the liver, where malonyl-CoA serves as a signal regulating the relative rates of fatty acid oxidation, ketogenesis, and triglyceride synthesis (144) and subsequently found in the heart (145), where the inhibition of cardiac CPT I by malonyl-CoA was also demonstrated (146). A tissue-specific acetylCoA carboxylase has been identified (147) and so has malonyl-CoA decarboxylase (148 150). Because of the importance of malonyl-CoA for the regulation of fatty acid oxidation, the regulation of ACC and MCD is intensely investigated. Specifically, ACC is inhibited by phosphorylation through AMP kinase. This inhibition results in lower malonyl-CoA levels and increased rates of fatty acid oxidations. Not surprisingly, AMP kinase has earned its name as “fuel gauge” of the cell (151). The carnitine-acyl unit traverses the inner mitochondrial membrane and is transferred to CoA SH inside the mitochondrial matrix by a second carnitine acyl-CoA transferase (CPT II) located in the inner surface of the inner mitochondrial membrane. Once inside the mitochondria, acyl-CoA is committed to oxidation by the system of β-oxidation (Figure 15.3). The β-oxidation pathway is controlled by fluctuations in the concentrations of substrates (acyl-CoA, NAD1, and FAD1) (152) or, expressed in more physiologic terms, by workload and oxygen supply of the heart. It is not clear how the rate of oxidation of FFA in the intact heart is related to citric acid cycle activity and the rate of oxidative phosphorylation, but it is clear that the accumulation of long chain acyl-CoA esters in ischemia inhibits the adenine nucleotide translocase in mitochondria (153). This inhibition provides a possible explanation for contractile dysfunction. To explore the gene regulatory mechanisms involved in the metabolic control of long chain fatty acid oxidation in the heart, the expression of muscle-type CPT-1 (mCPT-1) has been characterized in primary cardiac myocytes after incubation with oleate, which regulates mCPT-1 expression via the peroxisome proliferators-activated receptor alpha (PPAR-α) (154). PPAR-α belongs to a nuclear receptor family with multiple functions (155). Malonyl-CoA decarboxylase (MCD) is transcriptionally regulated by PPAR-α (150) and MCD inhibition protects the ischemic heart most likely by inhibiting fatty acid oxidation (156) and promoting glucose oxidation (157).

Transcriptional Regulation of Myocardial Fatty Acid Metabolism One way by which the heart responds to increased plasma fatty acid levels is by increasing the expression of various proteins involved in fatty acid utilization (154,158 160)

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including uncoupling protein 3 (135). The mechanism by which fatty acids activate the transcription of these genes is through activation of the nuclear receptor PPAR-α which heterodimerizes with 9-cis retinoic acid receptor (RXR) and activates the transcription of genes whose promoter contains the PPAR response element (PPRE). Of the co-factors that bind to the PPAR-α/RXR heterodimer, PCG-1α is highly expressed in the heart (161,162) and plays a key role in mitochondrial biogenesis (163), as discussed in another chapter.

Defective Fatty Acid Metabolism as Cause for Heart Failure Inborn errors in myocardial fatty acid metabolism are causes of cardiomyopathy and sudden death in children (164 166). The advent of tandem mass spectrometry has greatly advanced the screening for such metabolic cardiomyopathies (166). Perhaps best identified are the defects of the acyl-CoA dehydrogenase enzyme family, which catalyzes the initial reactions in mitochondrial β-oxidation of fatty acids (167 169). Enzymatic defects involving long and very long chain fatty acids (C14 or greater) or defects in cellular carnitine import of enzymes involved in the carnitine shuttle cause more severe heart failure than defects involving the metabolism of shorter-chain fatty acids (170). Impaired β-oxidation and accumulation of triglycerides in cardiomyocytes are hallmarks of contractile dysfunction of the heart from obese Zucker rats (171). In Zucker diabetic fatty rats lipotoxicity and contractile dysfunction are reversed by the administration of agents that lower plasma triglyceride and fatty acid levels (172,173). Reactivation of PPAR-α in adapted hypertrophied or hibernating myocardium results in severe contractile dysfunction (174,175).

Ketone Bodies Even in physiologic situations, such as short-term starvation or exercise, ketone body concentrations in the plasma can rise by up to 50-fold (176) and a key feature of myocardial ketone body metabolism is their concentrationdependent uptake in vivo (177). The ketone bodies, acetoacetate and beta-hydoxybutyrate, have ready access to the enzymes of the citric acid cycle in the heart (178). Ketone bodies do not sustain the full work output of the heart in vivo (179) and their rate of oxidation in the citric acid cycle is insufficient to meet the energy needs of the heart ex vivo (37). The relative inhibition of the citric acid cycle at the level of 2-oxoglutarate dehydrogenase provides a rare example of a defect in metabolism that causes reversible contractile dysfunction in the heart (37,180), which is relieved by anaplerosis (i.e. the provision of oxaloacetate or other citric acid cycle

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intermediates in addition to acetyl-CoA (127)). Pyruvate carboxylation in the in vivo heart accounts for at least 3 6% of citric acid cycle flux despite considerable variations in flux through pyruvate decarboxylase (181), supporting the principle that the heart functions best when it oxidizes several substrates simultaneously.

Amino Acids Although they are the building blocks of all proteins, amino acids are also an integral part of myocardial energy metabolism. Under physiologic circumstances one of the main functions of transaminases in heart muscle is the provision of carbon skeletons for the citric acid cycle (182). Furthermore, the amino acids aspartate and glutamate play an important role in the transfer of reducing equivalents across the mitochondrial membrane for oxidation of cytosolic NADH by the mitochondrial electron transport chain (183). In the malate aspartate cycle, cytosolic NADH, which cannot cross the inner mitochondrial membrane, is oxidized by the reduction of oxaloacetate to malate. Malate enters the mitochondrion and is oxidized to oxaloacetate, which is transaminated with glutamate to form 2-oxoglutarate and aspartate. The aspartate and 2-oxoglutarate leave the mitochondrion. Transmission of these metabolites regenerates oxaloacetate and glutamate in the cytosol, the net effect being a transfer of hydrogen ions across the mitochondrial membrane (184 186). A second postulated function for the malate aspartate shuttle is the delivery of intermediates (malate) to the citric acid cycle, serving as a modulator of cycle activity (187). Myocardial amino acid metabolism during hypoxia, ischemia, and reperfusion has been studied in the intact and isolated heart muscle (188 190). The amino acid alanine is like lactate, an end product of anaerobic glucose breakdown, arising through transamination of glutamate in the reaction: glutamate 1 pyruvate-2-oxoglutarate 1 alanine Where alanine leaves the cell, 2-oxoglutarate is further metabolized and decarboxylated to succinate via substrate level phosphorylation in the citric acid cycle (191): 2-oxoglutarate 1 NAD1 1 GDP 1 Pi -succinate 1 CO2 1 NADH2 1 GTP: GTP is readily transphosphorylated to ATP; this reaction is a source of anaerobic energy independent of lactate formation. Enhanced glutamate uptake in vivo has been shown by 13N accumulation from labeled glutamate in ischemic heart muscle (192) and glutamate enrichment has become a successful strategy to lessen the effects of ischemia with blood cardioplegia (193).

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METABOLIC REMODELING

intracellular long chain fatty acids are also highly regulated. Consequently, there is a balance between transport of fatty acids from the interstitial space into the cell, transport from the cytosol into mitochondria and subsequent oxidation. This balance is regulated mainly by malonyl-CoA, which inhibits transport of fatty acids into mitochondria. Prolonged inhibition of CPT I promotes intramyocellular lipid accumulation and insulin resistance in rats (196). Conversely, leptin inhibits malonyl-CoA synthesis in skeletal muscle leading to greater rates of fatty acid oxidation (197). Metabolic dysregulation, especially in the situation where the rate of fatty acid uptake exceeds the rate of fatty acid oxidation (198,199) leads to a wide range of disturbances that have been attributed to mitochondrial dysfunction (200,201) and which include triglyceride accumulation (143,202). Like a sentinel, lipid accumulation also is a feature of the failing human heart (Figure 15.5) (142) and is associated with a broad range of cellular and metabolic derangements collectively called lipotoxicity (143,203). Possible mechanisms causing a decline in contractile function of the “lipotoxic heart” include the inhibition of the adenine nucleotide translocator by fatty-acylCoA, the upregulation of TNF-α, increased ceramide levels, ROS accumulation, iNOS induction, chronic activation

Changes in metabolic fluxes are the first responders to changes in the physiologic environment of the heart. It follows that metabolic remodeling triggers and sustains functional and structural remodeling (194). Many of the acute changes in metabolic flux are brought about by the same signal transduction cascades believed to be involved in the adaptation to the heart’s environment, e.g. phosphotidylinositol 3-kinase (87), diacylglycerol, Ca21, and protein kinase C (195). Thus, metabolic signals are an integral part of cardiac adaptation to its environment (194).

Dysregulated Fatty Acid Metabolism A case in point are long chain fatty acids. In addition to their role as energy-providing substrates, fatty acids serve as mediators of signal transduction and as ligands for the nuclear receptor PPAR-α. Changes in substrate supply to the heart are reflected in changes of flux through fatty acid metabolizing pathways, which are both transcriptionally and post-transcriptionally regulated. In other words, redirecting energy metabolism is largely orchestrated by proteins that are, in turn, transcriptionally or post-transcriptionally regulated by metabolites. Levels of

Oil Red O Staining in Failing Human Heart

Low

Moderate

High

30%

44% 26%

Low

N = 27

Arbitrary units

High

Moderate

Oil Red O

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

*#

NF

HF

HF+O

HF+DM

FIGURE 15.5 Triglyceride accumulation in the failing heart. Note the high prevalence of intracellular fat droplets (stained with oil red O), especially in patients showing signs of metabolic dysregulation (obesity and type 2 diabetes). See text for further detail. (From Sharma et al., 2004 (142).)

196

of PKCs, myosin isoform switches (204), downregulation of MEF2C regulated genes (205), cytochrome C release (206), and apoptosis (172). Interestingly, the heart’s ability to secrete lipoproteins is inversely related to pathologic lipid accumulation (207). In addition, the ability to secrete lipoproteins protects the heart against the development of cardiomyopathy in a diabetes model (208). Lastly, in rodent models of lipotoxicity the administration of leptin decreases non-adipose tissue lipid accumulation and ameliorates lipotoxicity (209). Thus, a wide array of potentially reversible disturbances of fatty acid and lipid metabolism are involved in the phenomenon of lipotoxicity.

Dysregulated Glucose Metabolism Glucose and its metabolites also have multiple functions in the cardiac myocyte. Failure to adequately control levels of intracellular glucose metabolites has been implicated in the development of insulin resistance and in the generation of reactive oxygen species (ROS). Compared to the liver (210), relatively little is known about the effects of glucose metabolites on gene expression in the heart. Through investigations of the glucose/ carbohydrate response elements (GIRE/ChoRE) in the promoter regions of various glucose-regulated genes, a number of candidate transcription factors have been identified that are believed to be involved in glucosemediated gene expression. In the liver and in fat cells upstream stimulatory factor (USF), stimulatory protein 1 (Sp1), and sterol regulatory element binding protein 1 (SREBP1) play a role in glucose sensing (211), and it is reasonable to assume that the same transcription factors play similar roles in the heart (194). Excessive glucose metabolic accumulation is also associated with various cardiac pathologies. Although studied mainly in the vasculature, the four main mechanisms proposed for hyperglycemia induced diabetic complications (209) are likely to be operative in the cardiomyocyte as well. The four hypotheses are increased polyol pathway flux (via aldose reductase), increased intracellular formation of advanced glycation end products (AGE) and AGEinduced ROS generation activation of protein kinase C mostly through activation by the lipid second messenger diacylglycerol (DAG), and increased flux through the hexosamine biosynthetic pathway (209). As mentioned, many of these hypotheses have thus far been tested only in vascular tissue and await further confirmation in cardiomyocytes. However, it is already known that O-linked β-N-acetylglucosamine (O-GlcNAc) modifies many different nuclear and cytoplasmic proteins and that O-GlcNAc plays an important role in signal transduction (212). The basis is a “spillover” of intermediates of glucose metabolism into the hexosamine

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biosynthetic pathway and into the pentose phosphate pathway (194).

CONCLUSIONS Myocardial metabolism is an integral part of the function of the heart as consumer and provider of energy and has pleiotropic roles. The bulk of the energy for contraction of the heart comes from oxidative phosphorylation of ADP. A vast network of highly regulated metabolic pathways matches demand and supply with precision. The complexity of intermediary metabolism is also a rich source of speculation on impaired energy transfer as either cause or consequence of impaired cardiac function. Not surprisingly, restoration of efficient energy substrate metabolism as target for the treatment of the failing heart has thus far been an elusive goal for most investigators. All is in flux.

ACKNOWLEDGMENTS I thank Roxy Ann Tate for help with the preparation of this manuscript as well as past and present members of my laboratory for many discussions. Work in my lab is supported by the National Heart, Lung and Blood Institute of the US Public Health Service.

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