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Myocardial Substrate Metabolism in the Normal and Failing Heart

Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio; Scuola Superiore Sant'Anna, Pisa, Italy; Department of Physiology, New York Medical College, Valhalla, New York; and Department of Pediatrics, University of Alberta, Edmonton, Canada

ABSTRACT

I. INTRODUCTION

II. OVERVIEW OF MYOCARDIAL SUBSTRATE METABOLISM

A. Regulation of Metabolic Pathways in the Heart

B. Carbohydrate Metabolism

C. Fatty Acid Metabolism

D. Ketone Body Metabolism

E. Interregulation of Fatty Acid and Carbohydrate Oxidation

F. Effects of Substrate Selection on Contractile Function and Efficiency

G. Role of Nitric Oxide in Regulation of Myocardial Energy Substrate Metabolism

III. REGULATION OF MYOCARDIAL METABOLIC PHENOTYPE

A. Control of the Expression of Metabolic Enzymes in the Heart

B. Cardiac Lipotoxicity

C. Regulation of Phenotype Switch from Fetal to Adult State: Implications for Heart Failure

D. Effects of Aging on Substrate Metabolism

IV. METABOLIC PHENOTYPE IN HEART FAILURE

A. Considerations Regarding the Etiology of Heart Failure

B. Electron Transport Chain and Oxidative Phosphorylation Defects in Heart Failure

C. Substrate Metabolism in Heart Failure

1. Results from HF patients

2. Results from animal models of HF

D. Alterations in Expression and Function of Metabolic Proteins in Heart Failure

V. THERAPEUTIC POTENTIAL FOR MANIPULATION OF SUBSTRATE METABOLISM

A. Short-Term Metabolic Therapy to Optimize Cardiac Function

B. Long-Term Metabolic Therapy to Slow Heart Failure Progression and Improve Function

VI. CONCLUSIONS

GRANTS

DISCLOSURES

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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Cardiovascular disease is the leading cause of death and disability in the industrialized world, and although there has been a reduction in mortality from acute myocardial infarction over the last 30 years (215), there has been a concomitant rise in mortality attributable to heart failure (HF). The syndrome of HF was described by Hippocrates over two millennia ago and presented as shortness of breath and peripheral edema (214, 215). Autopsies performed in the 17th and 18th centuries revealed an enlarged ventricular chamber and increased heart mass in HF patients (215). In the last century a myriad of structural and biochemical cardiac abnormalities were shown to be associated with HF, from defects in mitochondria to abnormal adrenergic signal transduction. At the end stages of HF, the myocardium has low ATP content due to a decreased ability to generate ATP by oxidative metabolism, and thus is unable to effectively transfer the chemical energy from the metabolism of carbon fuels to contractile work (12, 98, 213, 341). The consequences of metabolic dysfunction in HF are poorly understood, but there is growing evidence to support the concept that the alterations in substrate metabolism seen in HF contribute to contractile dysfunction and to the progression of left ventricular (LV) remodeling that are characteristic of the HF state.

Today HF is clinically defined as "a complex clinical syndrome that can result from any structural or functional cardiac disorder that impairs the ability of the ventricle to fill with or eject blood" (179). Heart failure severely reduces exercise capacity and may or may not cause fluid retention and pulmonary congestion. Many HF patients have minimal edema or pulmonary congestion, thus the term heart failure is preferred over the older term congestive heart failure (179). Approximately two-thirds of all HF patients have a history of ischemic heart disease, and the remainder do not (215). HF presents as both systolic and diastolic LV dysfunction, with diastolic dysfunction being more common in a patient with a history of hypertension and/or diabetes in the absence of myocardial ischemia (6, 14, 117). Current medical therapies for HF are aimed at suppressing neurohormonal activation (e.g., angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists, -adrenergic receptor antagonists, and aldosterone receptor antagonists), and treating fluid volume overload and hemodynamic symptoms (diuretics, digoxin, inotropic agents). These pharmacotherapies for HF can improve clinical symptoms and slow the progression of contractile dysfunction and expansion of LV chamber volume; nevertheless, there is still progression, and the prognosis for even the optimally treated patient remains poor (39, 63, 67). Moreover, there is recent evidence that intense suppression of the neurohormonal systems does not provide further benefit compared with more modest therapy (64, 385, 444). Thus there is a need for novel therapies for HF, independent of the neurohormonal axis, that can improve cardiac performance and prevent or reverse the progression of LV dysfunction and remodeling (38, 116, 381, 452).

Agents that act through optimization of cardiac substrate metabolism are particularly attractive because they could work additively with current therapies, while not exerting negative hemodynamic effects (38, 116, 381, 431). Emerging evidence suggests that disturbances in myocardial substrate utilization have adverse effects in the failing myocardium (212, 213) and that shifting the substrate preference of the heart away from fatty acids towards carbohydrate oxidation can improve pump function and slow the progression of HF (23, 27, 28, 38, 53, 116, 428). Almost a century ago the observation was made that acute ingestion of cane sugar relieved symptoms in patients with cardiac dysfunction, presumably of ischemia origin (45, 140). The optimization of cardiac substrate metabolism to improve cardiac function and slow progression in HF, without causing any direct negative hemodynamic or inotropic effects, remains a conceptually attractive therapeutic approach (38). To date, the role of myocardial substrate metabolism in the natural history of HF has not been thoroughly evaluated. Human, canine, and rodent studies show that in late-stage failure there is downregulation of myocardial fatty acid oxidation and accelerated glucose oxidation (79, 332, 359, 362, 389). However, the time course and the molecular mechanisms for this switch in substrate oxidation are not well understood (251, 388, 428).

It is important to keep in mind that HF is not a specific disease, but rather an extremely complex syndrome that is dependent on etiology, duration, underlying coronary artery disease and ischemia, endothelial dysfunction, and the co-occurrence of complicating disorders such as diabetes, hypertension, and obesity. In Europe and North America, 20–30% of HF patients are diabetic, which in itself greatly alters myocardial substrate use (434, 449, 510) and affects the development of HF and LV remodeling after myocardial infarction (424). There is tremendous heterogeneity among the published data from patients and animals models of HF that may be attributed to the etiology, severity, and duration of HF and, in the case of animal models, the species studied. Moreover, studies in animal models demonstrate that the changes in myocardial metabolism and cardiac function often occur late in the development of HF. Thus one must use caution in drawing generalities from a single time point or from a single animal model. In addition, within a given failing heart there is likely gross and micro heterogeneity in the metabolic changes within the LV.

Despite these caveats and limitations, it remains of fundamental importance to identify the abnormalities in myocardial substrate metabolism that occur over the course of the development and progression of HF, and to understand the impact they have on left ventricular function and remodeling. This review discusses 1) the metabolic changes that occur in chronic HF, with emphasis on the mechanisms that regulate the changes in the expression of metabolic genes and the function of metabolic pathways; 2) the consequences of these changes on cardiac function; 3) the role of changes in myocardial substrate metabolism in ventricular remodeling and disease progression; and 4) the therapeutic potential of acute and long-term manipulation of cardiac energy metabolism in HF.

It is important to note that this review focuses on myocardial substrate metabolism in HF, and not on the well-documented HF-induced abnormalities in the transfer of energy from mitochondrial ATP to systolic and diastolic work. The reader is referred to recent reviews on this topic (98, 190, 477).

	II. OVERVIEW OF MYOCARDIAL SUBSTRATE METABOLISM

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To understand myocardial metabolism in HF, it is important to first have a solid understanding of myocardial metabolism in the normal heart and to understand the complex pathophysiology of HF. The reader is referred to textbooks and reviews on myocardial metabolism (270, 331, 433, 448, 473) and the pathophysiology of HF (215).

A. Regulation of Metabolic Pathways in the Heart

Under nonischemic conditions almost all (>95%) of ATP formation in the heart comes from oxidative phosphorylation in the mitochondria (Fig. 1), with the remainder derived from glycolysis and GTP formation in the citric acid cycle. The heart has a relatively low ATP content (5 µmol/g wet wt) and high rate of ATP hydrolysis (0.5 µmol · g wet wt^–1 · s^–1 at rest), thus there is complete turnover of the myocardial ATP pool approximately every 10 s under normal conditions (188, 331). Approximately 60–70% of ATP hydrolysis fuels contractile shortening, and the remaining 30–40% is primarily used for the sarcoplasmic reticulum Ca²⁺-ATPase and other ion pumps (128, 440). In the healthy heart the rate of oxidative phosphorylation is exquisitely linked to the rate of ATP hydrolysis so that ATP content remains constant even with large increases in cardiac power (17, 18, 153), such as occur during intense exercise or acute catecholamine stress. Mitochondrial oxidative phosphorylation is fueled with energy from electrons that are transferred from carbon fuels by dehydrogenation reactions that generate NADH and FADH₂ produced primarily in the fatty acid -oxidation pathway, the citric acid cycle, and to a lesser extent from the pyruvate dehydrogenase reaction and glycolysis (Figs. 1 and 2). There is a stoichiometric link between the rate of oxidation of carbon fuels, NADH and FADH₂ reduction, flux through the electron transport chain, oxygen consumption, oxidative phosphorylation, ATP hydrolysis, actin-myosin interaction, and external contractile power produced by the heart (Figs. 1 and 2). Thus an increase in contractile power results in a concomitant increase in all of the components in the system.

View larger version (13K): [in this window] [in a new window]   FIG. 1. Linkages between cardiac power, ATP hydrolysis, oxidative phosphorylation, and NADH generation by dehydrogenases in metabolism. SR, sarcoplasmic reticulum.

View larger version (25K): [in this window] [in a new window]   FIG. 2. The pathways and regulatory points of myocardial substrate metabolism. CPT-I, carnitine palmitoyltransferase-I; FAT, fatty acid transporter/CD36; G 6-P, glucose 6-phosphate; GLUT, glucose transporters; MCT, monocarboxylic acid transporters; PDH, pyruvate dehydrogenase.

The rates of flux through the various metabolic pathways are controlled by both the degree of expression of key metabolic proteins (enzymes and transporters) and complex pathway regulation that is exerted by both allosteric regulation of enzymes and substrate/product relationships. The metabolic machinery in the heart is designed to generate large amounts of ATP to support high rates of external cardiac power. At maximal cardiac work loads in vivo this metabolic machinery consumes oxygen at 80–90% of the mitochondrial capacity for electron transport chain flux and oxygen consumption (315). At rest, however, the heart operates at 15–25% of its maximal oxidative capacity, thus the expression or maximal activity of a key metabolic enzyme can be greatly reduced or increased without necessarily affecting ATP production or flux through the relevant pathway under resting conditions (106, 107). This is because flux through metabolic pathways can be rapidly turned on or off by allosteric modification of regulatory enzymes, changes in the concentration of inhibitory or stimulatory metabolites, or translocation of metabolic proteins to their site of function. These mechanisms allow for the rapid adaptation to acute stresses such as exercise, ischemia, or fasting.

B. Carbohydrate Metabolism

In the well-perfused heart, 60–90% of the acetyl-CoA comes from -oxidation of fatty acids, and 10–40% comes from the oxidation of pyruvate (126, 433, 492, 493, 495) that is derived in approximately equal amounts from glycolysis and lactate oxidation (126, 433, 492, 493, 495). The glycolytic pathway converts glucose 6-phosphate and NAD⁺ to pyruvate and NADH and generates two ATP for each molecule of glucose. The NADH and pyruvate formed in glycolysis are either shuttled into the mitochondrial matrix to generate CO₂ and NAD⁺ and complete the process of aerobic oxidative glycolysis or converted to lactate and NAD⁺ in the cytosol (nonoxidative glycolysis).

The healthy nonischemic heart is a net consumer of lactate even under conditions of near-maximal cardiac power (204, 292, 426). The myocardium becomes a net lactate producer only when there is accelerated glycolysis in the face of impaired oxidation of pyruvate, such as occurs with ischemia (85, 331, 433) or poorly controlled diabetes (15, 145, 434). There is a high rate of bidirectional lactate transmembrane flux and conversion to pyruvate (125, 141, 204, 293, 492, 493). Lactate transport across the cardiac sarcolemma is facilitated by the monocarboxylic acid transporter-1 (MCT-1) (Fig. 2; Refs. 118, 203).

Glycolytic substrate is derived from exogenous glucose and glycogen stores. Glucose transport into cardiomyocytes is regulated by the transmembrane glucose gradient and the content of glucose transporters in the sarcolemma (mainly GLUT-4, and to a lesser extent GLUT-1) (Fig. 2). There is a translocation of glucose transporters from intracellular vesicles to the sarcolemmal membrane in response to insulin stimulation, increased work demand, or ischemia (433, 506, 507), which increases the membrane capacitance for glucose transport and the rate of glucose uptake. Translocation of GLUT-4 into the sarcolemma is also stimulated by activation of AMP-activated protein kinase (AMPK) (379, 506), which occurs during exercise stress in the rat heart (71). Mice with cardiac-specific overexpression of a dominant negative mutant of AMPK have depressed rates of glucose uptake (499), suggesting a critical role for AMPK in regulating basal glucose uptake in the heart. Russell et al. (380) recently demonstrated that transgenic mice expressing inactive AMPK have normal GLUT4 expression as well as baseline and insulin-stimulated cardiac glucose uptake, but fail to increase glucose uptake and glycolysis during ischemia (380), illustrating a key role for AMPK in mediating insulin-independent ischemia-induced glucose uptake.

An additional source of glucose 6-phosphate for the heart is intracellular glycogen stores. The glycogen pool in the heart is relatively small (30 µmol/g wet wt compared with 150 µmol/g wet wt in skeletal muscle) (35, 331, 429) and has a relatively rapid turnover despite stable tissue concentrations (155). Glycogen concentrations are increased by an elevated supply of exogenous substrate and/or hyperinsulinemia (235, 246, 433), and glycogenolysis is activated by adrenergic stimulation (e.g., increases in cAMP and Ca²⁺), a fall in the tissue content of ATP, and a rise in inorganic phosphate such as occur with ischemia or intense exercise (133, 176, 433). Recently, there has been considerable interest focused on the role of AMPK in regulating glycogen content in the heart (9, 71, 499). Constitutively active AMPK due to a mutation in a regulatory subunit of the enzyme was recently shown to be associated with glycogen accumulation and hypertrophic cardiomyopathy (8, 9, 134). In contrast, acute activation of AMPK has been shown to activate glycogenolysis (267, 346). Clinically, patients with a mutation in the gamma-2 regulatory subunit of AMPK have Wolff-Parkinson-White syndrome and conduction system disease in the absence of cardiac hypertrophy (134, 135, 330), although the cellular mechanisms linking abnormal AMPK activity and the electrophysiological abnormalities are unclear.

Phosphofructokinase-1 (PFK-1) is a key regulatory enzyme in the glycolytic pathway and catalyzes the first irreversible step (Fig. 3). PFK-1 utilizes ATP to produce fructose 1,6-bisphosphate and is activated by ADP, AMP, and P_i and inhibited by ATP, thus accelerating flux through glycolysis when the phosphorylation potential falls. PFK-1 can also be inhibited by fructose 1,6-bisphosphate and by a fall in pH. The extent of [H⁺] inhibition of PFK-1 depends on ATP levels, with the inhibition being greatest when ATP levels are high (see Ref. 209 for review). As AMP accumulates, the sensitivity of PFK-1 to [H⁺] decreases. PFK-1 can also be stimulated by fructose 2,6-bisphosphate (F2,6BP), which is a feedforward activator of the enzyme (177). Citrate is a negative allosteric regulator of PFK-1 and links changes in mitochondrial oxidative metabolism to glycolysis. Accumulation of citrate was first proposed by Philip Randle to contribute to the decrease in glycolysis that occurs in various tissues when fatty acid oxidation increases (119, 325, 351, 352).

View larger version (29K): [in this window] [in a new window]   FIG. 3. Regulation of phosphofructokinase-1 (PFK-1) by PFK-2 and fructose 2,6-bisphosphate. PKA, protein kinase A; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate and produces the NADH molecules that originate from glycolysis. GAPDH is a major regulatory step in the glycolytic pathway, since the accumulation of NADH within the cytoplasm of cells inhibits the GAPDH reaction rate. In contrast, an increase in NAD⁺ activates GAPDH activity. High concentrations of lactate inhibit the regeneration of NAD⁺ from NADH and thus reduce the flux through GAPDH, as do high concentrations of the product of the reaction, 1,3-diphosphoglycerate. During myocardial ischemia, an accumulation of lactate and NADH can result in GAPDH becoming the main rate-controlling reaction in glycolysis. For instance, severe ischemia in heart muscle will result in the cessation of oxidative metabolism, and the subsequent accumulation of NADH in the cytosol, and accumulation of lactate within the cell.

Cell fraction studies demonstrate that glycolytic enzymes are clustered near the sarcoplasmic reticulum and sarcolemma (103, 344, 486, 500), suggesting that glycolytic reactions are not distributed throughout the cytosol, but rather occur in a subdomain outside around the perimeter of the cardiomyocyte. Further support for this concept comes from in silico studies of the transition from normal to ischemic conditions, which shows that compartmentation of glycolysis to 10% of the cytosolic space is required to simulate the burst of glycolysis that occurs with the onset of ischemia in vivo (519). Studies assessing the effect of inhibition of glycolysis suggest that glycolytically generated ATP is perferentially used by the sarcoplasmic reticulum to fuel Ca²⁺ uptake (103) and by the sarcolemma to maintain ion homeostatis (487, 488). Furthermore, inhibition of glycolysis impairs relaxation in ischemic and postischemic reperfused myocardium, suggesting that glycolytic ATP may be essential for optimal diastolic relaxation (197, 240, 486).

The pyruvate formed from glycolysis has three main fates: conversion to lactate, decarboxylation to acetyl-CoA, or carboxylation to oxaloacetate or malate. Pyruvate decarboxylation is the key irreversible step in carbohydrate oxidation and is catalyzed by pyruvate dehydrogenase (PDH) (Fig. 4) (350), a multienzyme complex located in the mitochondrial matrix. PDH is inactivated by phosphorylation on the E₁ subunit of the enzyme complex by a specific PDH kinase (PDK) and is activated by dephosphorylation by a specific PDH phosphatase (219, 350, 355, 491) (Fig. 4). PDK is inhibited by pyruvate and by decreases in the acetyl-CoA/free CoA and NADH/NAD⁺ ratios (219, 355, 490) (Fig. 4). There are four isoforms of PDK; PDK4 is the predominant form in heart, and its expression is rapidly induced by starvation, diabetes, and peroxisome proliferater activated receptor- (PPAR) ligands (36, 150, 496), suggesting that its expression is controlled by the activity of the PPAR promoter system (see sect. IIIA). High circulating lipid and intracellular accumulation of long-chain fatty acid moieties, such as occur with fasting or diabetes, enhance PPAR-mediated expression of PDK4, resulting in greater phosphorylation inhibition of PDH and less oxidation of pyruvate derived from glycolysis and lactate oxidation (172, 496). The PDH complex also contains a PDH phosphatase that dephosphorylates and activates PDH. The activity of PDH phosphatase is increased by Ca²⁺ and Mg²⁺ (295). Adrenergic stimulation of the heart increases the cytosolic Ca²⁺ transient, and mitochondrial Ca²⁺ concentration results in activation of PDH (294, 296), thus explaining the activation of PDH and greater pyruvate oxidation in response to a -adrenergic-induced increase in cardiac power (66, 126, 139, 294), despite no changes in any of the activators of PDK4 activity (416).

View larger version (28K): [in this window] [in a new window]   FIG. 4. Regulation of the oxidation of glucose and lactate by pyruvate dehydrogenase (PDH). The activity of PDH is inhibited by product inhibition from acetyl-CoA and NADH (dashed arrows), and by phosphorylation by PDH kinase and dephosphorylation by PDH phosphatase.

In addition to lactate formation and oxidation by PDH, pyruvate enters the citric acid cycle (CAC) via carboxylation to either malate or oxaloacetate (442). This reaction is "anaplerotic" (127, 231) and acts to maintain the pool size of CAC intermediates and CAC function in the face of the small but constant loss of CAC intermediates through efflux of citrate and, to a lesser extent, succinate and fumarate from the heart (55, 69, 102, 244, 456, 458, 479). Pyruvate carboxylation accounts for 2–6% of the CAC flux under normal flow aerobic conditions (69, 336), and it is reduced relative to CAC flux when MO₂ is reduced in acutely hibernating swine myocardium (335). Another source of anaplerotic flux into the CAC is via transamination of glutamate to -ketoglutarate, as demonstrated by the low but persistent uptake of glutamate in the human heart (318, 457–459). Studies using ¹³C-labeled glutamate in perfused rat hearts suggest that conversion to -ketoglutarate may be critical for the regulation of the initial span of the CAC, particularly during ischemia (68). Another anaplerotic pathway is the formation of succinyl-CoA from propionyl-CoA that is generated from the terminal three carbons of odd chain length fatty acids (127, 258, 284, 290, 367). Plasma levels of propionate and other odd-chain-length fatty acids are low in humans; thus this is not a major pathway under normal conditions. However, a recent small clinical study in patients with deficiencies in long-chain fatty acid oxidation have clinical improvement and reversal of cardiac dysfunction with dietary supplementation with heptanoate, perhaps due to increased anaplerosis (369).

Pyruvate can also contribute to anaplerosis by transamination with glutamate to form alanine and the CAC intermediate -ketoglutarate. The rate of alanine output by the myocardium is relatively low in dogs, pigs, and humans (15, 142, 305, 412, 459, 495) and is unaffected by acute ischemia (142) or when glycolysis is accelerated with hyperinsulinemia and hyperglycemia (495). Studies in stable coronary artery disease patients showed elevated alanine output compared with healthy people (318).

C. Fatty Acid Metabolism

The rate of fatty acid uptake by the heart is primarily determined by the concentration of nonesterified fatty acids in the plasma (32, 270, 494), which can vary over a fourfold range in healthy humans during the course of the day (from 0.2 to 0.8 mM). Under conditions of metabolic stress, such as ischemia, diabetes, or starvation, plasma FFA concentrations can increase to much higher levels (>1.0 mM) (271). Free fatty acids are highly hydrophobic and are never truly free in vivo but rather are associated with proteins or covalently bound to coenzyme A or carnitine. They are transported in the plasma in the nonesterified form attached to albumin, or covalently bound in triglyceride, contained with chylomicrons or very-low-density lipoproteins. Plasma fatty acid concentration is regulated by their net release from triglycerides in adipocytes, which reflects the net balance between triglyceride breakdown by hormone-sensitive lipase and synthesis by glycerolphosphate acyltransferase (270). Hormone-sensitive lipase is activated by catecholamines and inhibited by insulin. Thus, with fasting, when insulin is low and catecholamines are high, the plasma FFA concentration is elevated, resulting in a high rate of fatty acid uptake and oxidation by the heart. Fatty acids are also released from triglyceride in chylomicrons and in very-low-density lipoproteins that are hydrolyzed by lipoprotein lipase bound to the outside of capillary endothelial cells and cardiomyocytes (13, 306, 501, 505).

Fatty acids enter the cardiomyocyte by either passive diffusion or by protein-mediated transport across the sarcolemma (Fig. 5) (473) involving either a fatty acid translocase (FAT), or a plasma membrane fatty acid binding protein (FABP_pm) (131, 401, 473). A specific 88-kDa FAT protein called CD36 is abundantly expressed in skeletal and cardiac muscle and appears to be the predominant form of FAT in the heart (401, 473). People with mutations in the CD36 gene have lower rates of uptake of the long-chain fatty acid analog ¹²³I-15-(p-iodophenyl)-3-methylpentadecanoic acid compared with normal people (226), suggesting that CD36 partially regulates the rate of myocardial fatty acid uptake in humans. Once transported across the sarcolemma, the nonesterified fatty acids bind to FABP and are then activated by esterification to fatty acyl-CoA by fatty acyl-CoA synthase (FACS) (Fig. 5). FABPs are abundant in the cytosol and appear to be the primary intracellular carrier of nonesterified fatty acids. Recent studies show that there are FABP and FACS protein associated with CD36 on the cytosolic side of the sarcolemmal membrane, thus raising the possibility that fatty acids transported across the membrane can also be immediately esterified to fatty acyl-CoA (401). Inhibitable fatty acid transport is greater in electrically stimulated isolated cardiomyocytes compared with unstimulated conditions, suggesting that there is a translocation of FAT/CD36 into the sarcolemma from an intracellular site in response to contraction or increased energy demand (277–279). A similar phenomenon was observed in response to insulin stimulation (279). Translocation of FAT/CD36 has not been demonstrated in response to increased cardiac energy expenditure or insulin stimulation in the intact heart (277); thus it remains to be seen if translocation of FAT/CD36 regulates myocardial fatty acid transport under physiologically relevant conditions.

View larger version (31K): [in this window] [in a new window]   FIG. 5. Schematic depiction of myocardial fatty acid metabolism. ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; CAT, carnitine acyltranslocase; CPT-I, carnitine palmitoyltransferase-I; CPT-II, carnitine palmitoyltransferase-II; FABPPM, plasma membrane fatty acid binding protein; FAT, fatty acid transporter; FFA, free fatty acids; LPL, lipoprotein lipase; MCD, malonyl-CoA decarboxylase; TG, triglyceride; VLDL, very-low-density lipoproteins.

Fatty acid -oxidation occurs primarily in the mitochondria and to a small extent in peroxisomes (238, 409). The primary products of fatty acid oxidation are NADH, FADH₂, and acetyl-CoA (Fig. 5). Before mitochondrial -oxidation, the cytoplasmic long-chain acyl-CoA must first be transported into the mitochondrial matrix. Because the inner mitochondrial membrane is not permeable to long-chain acyl-CoA, the long-chain fatty acyl moiety is transferred from the cytosol into the matrix by a carnitine-dependent transport system (220, 270). First, CPT-I catalyzes the formation of long-chain acylcarnitine from long-chain acyl-CoA in the compartment between the inner and outer mitochondrial membranes. Next, carnitine acyltranslocase transports this long-chain acylcarnitine across the inner mitochondrial membrane in exchange for free carnitine. Lastly, carnitine palmitoyltransferase II (CPT-II) regenerates long-chain acyl CoA in the mitochondrial matrix (Fig. 5). Of the three enzymes involved in the transmitochondrial membrane transport, CPT-I serves the key regulatory role in controlling the rate of fatty acid uptake by the mitochondria (220, 270).

The activity of CPT-I is strongly inhibited by malonyl-CoA, which binds to CPT-I on the cytosolic side of the enzyme (Fig. 5) (220, 302, 513). There are two isoforms of CPT-I: CPT-I predominates in the liver, and CPT-I is the main isoform in the heart (300, 302). CPT-I is 30-fold more sensitive to malonyl-CoA inhibition than is CPT-I (300, 302, 485). Malonyl-CoA is a key physiological regulator of fatty acid oxidation in the heart. A fall in malonyl-CoA increases fatty acid uptake and oxidation (2, 143, 236), and an increase suppresses fatty acid oxidation (390, 430). Malonyl-CoA is formed from the carboxylation of acetyl-CoA by acetyl-CoA carboxylase (ACC) (137, 143, 145, 236, 375, 390, 454) and has a rapid rate of turnover in the heart (364, 366). Most of the acetyl-CoA in cardiomyocytes resides in the mitochondria (185); however, CPT-I activity is regulated by malonyl-CoA that is formed from carboxylation of extramitochondrial acetyl-CoA. There is indirect evidence to suggest that extramitochondrial acetyl-CoA is derived from citrate via the ATP-citrate lyase reaction (345, 395) and from the export for mitochondrial acetyl-CoA as acetylcarnitine (281, 390). More recently, results from studies with ¹³C-labeled substrates and isotopomer analysis of malonyl-CoA provided direct evidence that in the perfused rat heart malonyl-CoA is derived from acetyl-CoA formed in peroxisomal -oxidation of long-chain fatty acids (365). Furthermore, the data were consistent with long-chain fatty acids undergoing only a few cycles of -oxidation in peroxisomes, followed by the generation of C₁₂ and C₁₄ fatty acyl-CoAs that are subsequently oxidized to acetyl-CoA in the mitochondria (365).

The activity of ACC is inhibited by phosphorylation by AMPK (236, 319, 394); thus activation of AMPK can result in reduced malonyl-CoA formation and acceleration of fatty acid oxidation (94, 95). It was recently shown there is an increase in AMPK activity in rats with LV hypertrophy produced by aortic banding (462), although the metabolic consequences of this activation are unclear. As discussed in section IIB, mutations in the gamma-2 regulatory subunit of AMPK result in glycogen accumulation and hypertrophic cardiomyopathy in mice (8, 9) and Wolffe-Parkinson-White syndrome in patients (135, 135, 330). To our knowledge, the effect of HF on AMPK expression and activity are not known. As discussed above, AMPK activation in the heart also stimulates glucose transporter translocation and glucose uptake (71, 379, 499); thus activation of AMPK can effect an increase in both carbohydrate and fatty acid metabolism. Thus, when the metabolic rate of the heart is increased, as occurs during exercise, increased AMPK activity would increase acetyl-CoA production from both carbohydrates and lipids, and thus ensure an adequate supply of substrate to the mitochondria (71).

The degradation of malonyl-CoA is regulated by the activity of malonyl-CoA decarboxylase (MCD), which converts malonyl-CoA back to acetyl-CoA and CO₂ in the cytosol and mitochondrial (Fig. 5) (93, 146, 221, 225, 394). In general, situations where MCD activity is high results in low myocardial malonyl-CoA content and high rates of fatty acid oxidation (48, 92, 93). We have recently shown that pharmacological inhibition of MCD activity increases myocardial malonyl-CoA content, and (93) MCD activity regulates fatty acid oxidation. In addition, inhibition of MCD activity reduces malonyl-CoA turnover (366) and increases glucose oxidation under aerobic, ischemic, and postischemic conditions and improves postischemic recovery of contractile function (93), as has been previously observed with CPT-I inhibitors (163, 164, 427). An increase in cardiac power induced by -adrenergic receptor stimulation results in a fall in myocardial malonyl-CoA content and accelerated fatty acid uptake and oxidation; however, this effect is not due to activation of AMPK or reduced ACC activity (137, 143) but has been associated with a reduction in the K_m of MCD(137), although this has not been a consistent finding (366).

Once taken up by the mitochondria, fatty acids undergo -oxidation, a process that repeatedly cleaves off two carbon acetyl-CoA units, generating NADH and FADH₂ in the process (Fig. 5). The -oxidation process involves four reactions, with specific enzymes for each step, and each reaction has specific enzymes for long-, medium-, and short-chain length fatty intermediates (30, 32). The first step is catalyzed by acyl-CoA dehydrogenase, followed by 2-enoyl-CoA hydratase, and then 3-hydroxyacyl-CoA dehydrogenase. The final step is 3-ketoacyl-CoA thiolase (3-KAT), which regenerates acyl-CoA for another round of -oxidation and releases acetyl-CoA for the citric acid cycle. Acyl-CoA dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase generate FADH₂ and NADH, respectively, and the acetyl-CoA formed from -oxidation generates more NADH and FADH₂ in the CAC.

D. Ketone Body Metabolism

The heart extracts and oxidizes ketone bodies (-hydroxybutyrate and acetoacetate) in a concentration-dependent manner (56, 114, 152, 441). Plasma ketone bodies are formed from fatty acids in the liver, and the arterial plasma concentration is normally very low, thus they are normally a minor substrate for the myocardium. During starvation or poorly controlled diabetes, plasma ketone body concentrations are elevated secondary to low insulin and high fatty acids, and they become a major substrate for the myocardium (15, 145). As with fatty acids, the uptake and oxidation of glucose and lactate are inhibited by elevated plasma ketone bodies (32, 436, 441, 467), with the inhibitory effect presumably mediated through product inhibition on PDH (see sect. IIF).

Oxidation of ketone bodies inhibits myocardial fatty acid oxidation (56, 114, 152, 243, 261, 436, 474). Diabetic myocardium has a high rate of -hydroxybutyrate uptake and relatively low rates of fatty acid uptake (145), suggesting that in diabetic patients elevated plasma ketone concentrations can act to inhibit fatty acid uptake and oxidation. Clinical studies demonstrate that HF results in an increase in plasma ketone body concentration that appears to be secondary to elevated fatty acid levels (264–266); however, data on myocardial ketone body metabolism have not been reported from animals or patients with HF. The biochemical mechanisms responsible for inhibition of fatty acid -oxidation by ketone bodies are not well understood, but do not appear to be related to changes in malonyl-CoA or the acetyl-CoA-to-free CoA ratio (436). Elevated rates of -hydroxybutyrate and acetoacetate oxidation could inhibit fatty acid -oxidation by increasing the intramitochondrial ratio of NADH to NAD⁺, which would inhibit the ketoacyl-CoA dehydrogenase step of the fatty acid -oxidation spiral (219, 238). Hasselbaink et al. (152) recently showed that palmitate oxidation was significantly enhanced in isolated cardiomyocytes from streptozotocin diabetic rats in the absence of acetoacetate; however, when measurements were made with the addition of ketone bodies, the rate of palmitate oxidation was not affected by diabetes. They also noted greater fatty acid uptake in the myocytes from diabetic rats and suggested that ketone-induced impairment of fatty acid oxidation might be responsible for the greater triglyceride storage in the heart with diabetes (82).

E. Interregulation of Fatty Acid and Carbohydrate Oxidation

The primary physiological regulator of flux through PDH and the rate of glucose oxidation in the heart is the rate of fatty acid oxidation (Fig. 4). High rates of fatty acid oxidation inhibit PDH activity via an increase in mitochondrial acetyl-CoA/free CoA and NADH/ NAD⁺, which activates PDH kinase causing phosphorylation and inhibition of PDH (Fig. 4). In addition, in isolated heart mitochondria elevated rates of fatty acid oxidation inhibit flux through PDH at a given PDH phosphorylation state (148). Conversely, inhibition of fatty acid oxidation increases glucose and lactate uptake and oxidation by 1) decreasing citrate levels and inhibition of PFK and 2) lowering acetyl-CoA and/or NADH levels in the mitochondrial matrix, thereby relieving the inhibition of PDH (165) (Figs. 4 and 5). This effect has been demonstrated with 1) lowering plasma free fatty acid concentration by administering an inhibitor of lipolysis in adipocytes (229, 230, 245, 438), 2) inhibition of CPT-I (51, 164, 165), 3) inhibition of malonyl-CoA decarboxylase (which elevates malonyl-CoA content and inhibits CPT-I activity)(93, 366), and 4) direct inhibitors of fatty acid -oxidation (207, 297). It is important to note that partial inhibitors of myocardial fatty acid oxidation have been shown to lessen ischemic dysfunction and tissue damage in animal models of ischemia and reperfusion and have clear benefits in clinical trials in patients with chronic stable angina (26, 50, 78, 195, 340, 406, 445); this effect has been attributed to increased pyruvate oxidation and less lactate accumulation and efflux, and decreased proton accumulation (29, 164, 165, 427, 433).

F. Effects of Substrate Selection on Contractile Function and Efficiency

Several lines of evidence suggest that the contractile performance of the heart at a given MO₂ is greater when the heart is oxidizing more glucose and lactate, and less fatty acids (47, 183, 227, 232, 243, 309, 310, 421). Studies in isolated rat hearts demonstrate that the mechanical power of the LV is less at a given rate of oxygen consumption when fatty acids rather than glucose are the sole exogenous substrate (47). Classic studies by Ole Mjøs in closed-chest dogs demonstrated that increasing the rate of fatty acid uptake by the heart with an infusion of heparin and triglyceride emulsion resulted in a 26% increase in myocardial oxygen consumption without changing the mechanical power of the LV (309, 310). A similar decrease in cardiac mechanical efficiency with elevated plasma FFA concentration was observed in healthy humans (421) and pigs (232), as well as during ischemia of moderate severity in dogs (227, 311). Furthermore, inhibition of fatty acid -oxidation by 4-bromocrotonic acid decreased MO₂ and improved mechanical efficiency of the LV of the working rat heart (183); a similar response was observed following acute administration of the fatty acid oxidation inhibitor ranolazine in dogs with HF (53). Korvald et al. (232) compared the relationship between MO₂ and the LV pressure-volume loop over a wide range of work loads in anesthetized pigs. Treatment with insulin and glucose resulted in a 39% reduction in noncontractile basal MO₂ [estimated from the intercept of the MO₂-pressure-volume loop relationship (232)] compared with pigs subjected to high plasma fatty acids. Importantly, they did not observe a difference in the slope of this relationship, suggesting that under these conditions fatty acids did not affect excitation-contraction coupling or the ATP requirement for contractile power.

The mechanisms for impaired mechanical efficiency with high fatty acid oxidation are unclear. On a theoretical basis, fatty acid oxidation requires a greater rate of oxygen consumption for a given rate of ATP synthesis than do carbohydrates (428). The theoretical ATP-to-oxygen ratio for glucose or lactate are 3.17 and 3.00, respectively, while for palmitate and oleate the values are 2.80 and 2.86, respectively (209, 428). The actual values in vivo may be much lower due to constitutive leakage of protons across the inner mitochondrial membrane (37, 370, 371, 439). Fatty acid concentrations uncouple oxidative phosphorylation (decrease the P/O) and cause wasting of O₂ by mitochondria (34, 348). This would require a greater MO₂ for a given rate of ATP formation by oxidative phosphorylation when fatty acids are the substrate (34, 348). These effects would alter the MO₂ requirement for ATP production for both basal metabolism and for generating contractile power and relaxation (i.e., ATP hydrolysis to support Ca²⁺ uptake into the sarcoplasmic reticulum). In addition, high concentrations of long-chain fatty acids can also activate sarcolemmal Ca²⁺ channels, which would increase the entry of extracellular Ca²⁺ into the cytosol and increase the rate of ATP hydrolysis required to maintain normal cytosolic Ca²⁺ cycling (173).

It has been proposed that fatty acids waste ATP (and hence O₂) through the extrusion of long-chain fatty acids out of the mitochondria via uncoupling protein 3 (UCP3) (166, 408). In this scheme high rates of intramitochondrial fatty acyl-CoA production would result in formation of FFA by mitochondrial thioesterase-1, which would be transported out of the mitochondria by UCP3 and reesterified by FACS to long-chain fatty acyl-CoA in the cytosol (a reaction that consumes two ATP) (166, 178). Garlid and co-workers (120–123, 193, 198–201) observed that UCP3 can translocate the FA^– out of the mitochondrial matrix; once in the intramembranous space, the FA^– can associate with a proton. The resulting neutral FA species is able to "flip-flop" back into the mitochondrial matrix, where it relinquishes the proton. The net effect is a leak of protons, as with classic uncoupling, but with no net flux of fatty acids (120–123, 193, 198–201). While this clearly occurs, it may not play a major role in the energy-wasting effects of fatty acid observed in vivo, as many studies show no effect of UCP3 content on the P/O in isolated mitochondria (60, 62, 222, 408, 478). Studies in isolated skeletal muscle mitochondria show that lipid substrate (palmitoylcarnitine or palmitoyl-CoA) causes a UCP3-dependent decrease in state 4 respiration with no change in state III respiration or P/O, an effect that is not observed with nonlipid substrates (222). In addition, skeletal muscle mitochondria isolated from mice lacking UCP3 show a decreased in state 4 respiration (478) and mice overexpressing UCP3 show an increase in state 4 respiration (60) with no effect on state 3 respiration (60, 478). Thus exposure of the myocardium to high plasma concentrations of FFA could waste ATP via this UCP3-mediated futile cycle.

G. Role of Nitric Oxide in Regulation of Myocardial Energy Substrate Metabolism

In 1989, Brune and Lapetina demonstrated that nitric oxide (NO) can enhance ADP-ribosylation of a 37-kDa cytosolic protein (43), later identified as GAPDH (90, 91, 313, 475), a key enzyme of the glycolytic pathway. Zhang and Snyder documented a similar action of NO in neuronal cells (517). These seminal studies on the direct effects of NO on glucose metabolism were followed by others that described inhibitory actions of NO on phosphofructokinase of pancreatic islets (465) and an indirect stimulatory action of NO on 6-phosphofructo-2-kinase in neurons (4). Although the mechanisms are still poorly defined, convincing evidence has been provided that NO also plays an important role in the regulation of myocardial substrate metabolism. Depre et al. (83) observed a decreased glucose uptake in isolated hearts during 8-bromo-cGMP infusion. They concluded that NO, via its second messenger cGMP, probably inhibits glucose transport into cardiomyocytes and that this could explain their previous findings of a cardioprotective action of NO synthase (NOS) inhibition during myocardial ischemia (84, 87), when cardiac function is highly dependent on glycolytic flux. In hearts isolated from endothelial cell NOS knockout mice, Tada et al. (447) found that basal cardiac glucose uptake is markedly increased and can be inhibited by 8-bromo-cGMP. It is possible that all of these findings in vitro were affected by nonphysiological conditions of substrate and oxygen supply. This was not the case, however, since we found that cardiac glucose uptake and oxidation are increased after systemic NOS inhibition in conscious dogs (360, 361). Conversely, under the same experimental conditions, fatty acid uptake and oxidation are reduced. On the other hand, we did not observe this shift in substrate oxidation with NOS inhibition in isolated rat hearts (239). An interesting paradox is that NO/cGMP exerts opposite effects on glucose transport and metabolism in skeletal muscle (512). Additional research is needed to elucidate the mechanisms underlying the regulation of myocardial substrate metabolism by NO.

It has been suggested that the activity of cardiac NOS during metabolic stress is regulated by a possible feedback mechanism involving AMPK (57). Chen et al. (57) found that AMPK coimmunoprecipitates with cardiac eNOS and activated it by phosphorylating Ser-1177, but only in the presence of Ca²⁺-calmodulin, both in tissue homogenates and in ischemic isolated rat hearts. Because NO is a main regulator of vascular tone and cardiac function, these findings lead to the intriguing hypothesis that the stimulatory effect exerted by AMPK on eNOS may represent a link between metabolic adaptations and cardiovascular function under conditions of stress. Li et al. (255) also observed coprecipitation of AMPK and eNOS and demonstrated that activation of AMPK with 5-amino-4-imidazole-1--carboxamide ribofuranoside (AICAR) results in activation of eNOS, GLUT4 translocation and greater glucose uptake in isolated papillary muscles. AICAR treatment also results in increased glucose uptake (255), which is in contrast with previous studies showing decreased glucose uptake with NOS inhibition or 8-bromo-cGMP (83, 84, 87, 360, 361). Additional research is needed to elucidate the mechanisms underlying the complex interactions between myocardial substrate metabolism and NO.

	III. REGULATION OF MYOCARDIAL METABOLIC PHENOTYPE

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Myocardial metabolic phenotype can be defined as the substrate preference by the heart at a given metabolic milieu (e.g., arterial concentrations of glucose, lactate, fatty acids, insulin, catecholamines, oxygen), hemodynamic condition (heart rate, preload, afterload, coronary blood flow), and inotropic state. This phenotype is primarily dependent on the content of the proteins (enzymes and transporters) that facilitate flux through the metabolic pathways and the structure and integrity of key cellular organelles, such as mitochondria, that are responsible for energy metabolism. To affect metabolic phenotype, however, it is important that the protein that is modified exerts a key regulatory role in metabolism. Many metabolic enzymes in a pathway are redundant or expressed in great excess and therefore do not regulate flux through the pathway. It is therefore possible to have dramatic changes in expression of some proteins without much effect on substrate flux. In other words, changes in flux cannot simply be inferred from changes in enzyme activity or expression. Therefore, in evaluating the effects of heart failure on myocardial substrate metabolism, it is important to assess several aspects of the metabolic pathway of interest, specifically: 1) the rate of flux through metabolic pathways under physiologically relevant conditions; 2) the expression, activity, and characteristics of key regulatory proteins (e.g., V_max, K_m, allosteric modification of an enzyme); and 3) the tissue content of regulatory metabolites.

There have been a variety of investigations into the metabolic phenotype changes that occur in response to chronic cardiac stress like cardiac hypertrophy and HF. These studies have used a variety of methodological approaches but have mainly been aimed at the assessment of flux through the metabolic pathway [either in vivo using invasive (30, 31, 338, 493, 494) or noninvasive techniques, e.g., positron emission tomography (PET)] (79, 81, 237, 453, 480), or in isolated perfused hearts (24, 224, 269), or they have sampled the myocardium to evaluate mRNA levels using real-time quantitative polymerase chain reaction (86, 356, 357, 509) or gene chip microarrays (307, 451). It is more complex to assess changes in the protein expression, activity, and rate of turnover. Nevertheless, in studying the effects of HF on metabolic phenotype, the goal has been to understand the relative importance of changes in the function of selected proteins on the flux through various metabolic pathways, on cardiac function, and on the progression of HF. At present, much emphasis is placed on understanding the mechanisms that signal changes in the expression of genes encoding proteins that regulate substrate metabolism and, in turn, affect cardiac function and progression in HF. However, as discussed, care should be taken in interpreting these data without parallel measurements of metabolic flux.

A. Control of the Expression of Metabolic Enzymes in the Heart

Regulation of the expression of the multitude of enzymes and transporters involved in myocardial energy metabolism is complex and not well understood. In HF, recent interest has focused on altered expression of both glycolytic and mitochondrial enzymes. In general, the overall capacity for oxidative metabolism in a cell is dependent on the volume density and composition of mitochondria. However, over the last decade it has become clear that mitochondrial fuel selection can be altered by differential expression of enzymes of the fatty acid oxidation pathway relative to enzymes that oxidize pyruvate (PDH and PDK) or acetyl-CoA (e.g., citrate synthase), or those in the electron transport chain. Mitochondria have a circular DNA genome that encodes for the 13 subunits of the respiratory complexes I, III, IV, and V (400). The remaining respiratory subunits and all of the proteins required for carbon substrate metabolism are encoded by nuclear genes. It is becoming clear that both nuclear and mitochondrial transcription alterations are important in the metabolic phenotype changes observed in HF (124, 477).

An important nuclear gene transcription control that regulates the capacity for myocardial mitochondrial fatty acid oxidation is regulated by the ligand-activated transcription factors, named peroxisome proliferator-activated receptors, or PPARs (25, 180). These transcription factors control gene expression by first forming heterodimers with retinoid X receptors and then binding to specific response elements (PPAR response elements, or PPREs) located within promoter regions of many genes encoding metabolic enzymes (Fig. 6). In addition, the PPAR/RXR complex is positively regulated by the cofactor PPAR coactivator-1 (PGC-1) (25, 180, 476). Cardiac overexpression of PGC-1 increases the mRNA of numerous mitochondrial genes and triggers mitochondrial biogenesis (181, 250, 476). Once bound to the PPRE, the PPAR/RXR/PGC-1 complex increases the rate of transcription of fatty acid oxidation genes and PDK-4 (the inhibitory kinase of PDH) (129, 150, 172, 251). In addition, stimulation of gene transcription is inhibited by the binding of several cofactors such as COUP and SP1 (251). The activity of PPAR/RXR heterodimers is increased by fatty acids and eicosanoids; thus PPAR/RXR heterodimers act as lipid sensors in the cell, resulting in a greater capacity for fatty acid catabolism in response to a greater cell exposure to lipid (Fig. 6) (25, 180).

View larger version (29K): [in this window] [in a new window]   FIG. 6. Regulation of expression of metabolic genes in cardiomyocytes by stimulation of the peroxisome proliferator activated receptor (PPAR). Note that PPAR has similar, but less well described, effects on gene expression (see text). ACC, acetyl-CoA carboxylase, mCPT-I, muscle isoform of carnitine palmitoyltransferase-I; FABP, fatty acid binding protein; LCAD, long-chain acyl-CoA dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase; MCD, malonyl-CoA decarboxylase; MTE1, mitochondrial thioesterase 1; PDK4, pyruvate dehydrogenase kinase 4; PPRE, peroxisome proliferator activated receptor response element; RXR, retinoid X receptor ; UCP3, uncoupling protein 3.

PPAR [also referred to as PPAR/ (129)] is also expressed in cardiomyocytes and stimulates the expression of proteins in the fatty acid oxidation pathway in a manner similar to PPAR (58, 129). Gilde et al. (129) showed that exposure of isolated neonatal rat cardiomyocytes to PPAR agonists results in upregulation of the mRNA for fatty acid oxidation enzymes and increased fatty acid oxidation. Cheng et al. (58) recently showed that mice with a cardiomyocyte-restricted deletion of PPAR downregulated the expression of the mRNA and protein for fatty acid oxidation enzymes and had reduced myocardial oxidation of fatty acids. The mice had progressive LV dysfunction and hypertrophy, but had myocardial lipid accumulation and increased mortality late in life. These recent findings suggest that PPAR may play a role that is similar to PPAR in the regulation of cardiac fatty acid metabolism.

PPAR mRNA is expressed at very low levels in cardiomyocytes, and at higher rates in a broad range of tissues including skeletal muscle, colon, small and large intestines, kidney, pancreas, and spleen. Although PPAR does not appear to play a direct role regulating fatty acid oxidation in the heart (129, 216), it can indirectly regulate fatty acid oxidation by decreasing the fatty acid concentration to which the heart is exposed. The effective insulin-sensitizing agents, the thiazolidinediones, are PPAR ligands and act to sequester fatty acids in adipocytes, lower circulating fatty acids, and triglycerides and therefore reduce the exposure of the myocardium to fatty acids (25). Thus the thiazolidinediones ("PPAR agonists") can decrease myocardial fatty acid oxidation in vivo by decreasing plasma fatty acid levels and thus myocardial fatty acid uptake and oxidation. Furthermore, the action of PPAR agonists on adipocytes likely reduces ligand stimulation of the PPAR/RXR complex in cardiomyocytes, and thus reduces the expression of proteins regulating fatty acid uptake and oxidation in the heart.

Recent studies in isolated neonatal cardiomyocytes suggest that the orphan nuclear receptor estrogen-related receptor (ERR) interacts with PGC-1 and binds to the PPRE to increase the expression of PPAR-regulated genes and increased fatty acid uptake, accumulation, and oxidation (181, 182). Overexpression of ERR also resulted in an increase in the mRNA for proteins that are not regulated by PPAR, including contractile proteins and enzymes involved in carbohydrate metabolism and mitochondrial respiration (182). The endogenous ligand(s) for ERR have not been identified.

B. Cardiac Lipotoxicity

Recent evidence from animal studies demonstrates that obesity and elevated plasma fatty acid and triglycerides can result in a cardiac specific "lipotoxicity," characterized by accumulation of neutral lipids (triglycerides) and ceramides, which are associated with increased apoptosis, and contractile dysfunction (109, 301, 402, 468, 469, 501, 505, 520). For instance, Zhou et al. (520) showed that mature obese Zucker diabetic rats develop cardiac dilatation and reduced contractility that correspond with elevated myocardial triglycerides, ceramide, and DNA laddering, an index of apoptosis. Suppression of plasma triglyceride with the PPAR agonist troglitazone lowers myocardial triglyceride and ceramide content, which was associated with complete prevention of DNA laddering and loss of cardiac function. Cardiac overexpression of FACS results in lipid accumulation, cardiac hypertrophy, gradual development of LV dysfunction, and premature death (59). Cardiac overexpression of human lipoprotein lipase with a glycosylphosphatidylinositol anchoring sequence that localizes the enzyme to the surface of cardiomyocytes causes LV chamber enlargement and impaired contractile function compared with wild-type mice (501). The mechanism for lipid-induced cardiac remodeling and dysfunction in this model is unclear but could be due to apoptotic cell loss (162, 260, 333, 425, 520) and/or a decrease in cardiac mechanical efficiency with very high rates of fatty acid oxidation and impaired carbohydrate oxidation (227, 232, 309, 310, 421).

The toxic effects of lipid accumulation in the heart can be demonstrated in small animal models; however, the clinical significance of these findings is not yet clear in type 2 diabetes, obesity, and HF. Epidemiological studies show that obese people have a decrease in life expectancy and greater mortality from cardiovascular disease (113, 174) and a greater risk for developing HF (218). However, once a patient is diagnosed with HF, there is a paradoxical reduction in the rate of mortality in obese compared with lean patients (80, 171, 247, 248). These observations are complicated by the fact that cachexia is a positive predictor of mortality in HF, and weight loss is strongly associated with poor outcome (7, 80). The clinical complexities o