Cardiac Metabolism in Health and Disease

Introduction

Metabolism comes from the Greek word meaning ‘change,’ referring to the transformation of potential energy stored in circulating substrates such as glucose and fatty acids to adenosine triphosphate (ATP), thereby driving cardiac contraction and relaxation. Early workers delineated carbohydrate and fatty acids as two of the most important myocardial fuels. In 1914 Lovatt Evans found that about one-third of the dog heart’s energy was supplied by carbohydrate oxidation. The importance of fatty acids as a fuel source and their utilization in the absence of glucose for the mammalian heart was initially reported by Cruikshank and Kosterlitz in 1941. The oxidative metabolism of the human heart was defined by Richard Bing in a pioneering series of papers in the 1950s. In the fasted state, carbohydrates make up a relatively small part of the oxidative metabolism of the resting human heart. Focus on free fatty acids (FFA) as a major myocardial fuel started with the 1961 finding that FFA inhibited glucose oxidation in the isolated heart. The close interaction of glucose and FFA in fed and fasting conditions was delineated in the classic article describing the glucose–fatty acid cycle by Randle and his associates at Cambridge.

Since then, the key to substrate utilization has increasingly focused on the mitochondria. Major reviews have defined and refined the interaction between cardiac glucose and FFA metabolism and the complex but dominant role of FFAs in health and disease. The practical relevance of cardiac metabolism to the cardiologist, cardiovascular surgeon, and cardiovascular pathologist has constantly grown, particularly with the introduction of new drugs that directly alter energy metabolism. Furthermore the development of new techniques such as nuclear magnetic resonance spectroscopy (NMR) and positron emission tomography (PET) has enabled the non-invasive measurement of cardiac substrate metabolism. New techniques and molecular paths have led to what Taegtmeyer terms ‘the new cardiac metabolism,’ in that ‘it finally has dawned on many that metabolism is the missing link between form and function of the heart.’ ¹¹

Energy Availability

Metabolism is critical for normal cardiac contractility, and hence for the normal cardiac output that sustains life by perfusing all the vital organs. Contractile function necessitates a high turnover of ATP in the myocardium, and hence a correspondingly high rate of mitochondrial ATP production. The two major substrates for the energy metabolism of the heart are circulating glucose and free fatty acids ( Figures 2.1 and 2.2 ). After these substrates are taken up by muscle cells and transported to the cytosol, they are further broken down in mitochondria to an activated two-carbon substrate fragment (acetyl CoA in Figures 2.1, 2.2 and 2.3 ), which enters the Krebs cycle, also called the tricarboxylic acid (TCA) cycle ( Figure 2.4 ). Next comes the complex process of oxidative phosphorylation that produces mitochondrial ATP from adenosine diphosphate (ADP) and inorganic phosphate ( Figure 2.4 ). In the following step, ATP leaves the mitochondria in exchange for ADP entering, to become available for contractile work that in turn breaks down ATP to ADP and inorganic phosphate ( Figure 2.2 ), which re-enter the mitochondria to be recharged back to ATP.

FIGURE 2.4, The citrate cycle of Krebs. In reality, the following reactions are readily reversible: citrate → isocitrate; succinate → oxaloacetate via the intervening reactions. The most important potential sites of control are citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase (by regulating the supply of oxaloacetate). Of these, isocitric dehydrogenase and α-ketoglutarate dehydrogenase are calcium-sensitive, as is pyruvate dehydrogenase (PDH). These dehydrogenases respond to increased cytosolic calcium (as in inotropic stimulation) by increased activity.

FIGURE 2.2, The ATP–ADP cycle. Energy in the form of ATP is produced by the Krebs citrate cycle ( Fig. 2.3 ). Heart work utilizes the high energy liberated from ATP in the conversion to ADP which is then resynthesized together with inorganic phosphate (Pi) to produce ATP by oxidative phosphorylation.

Major Sources of Energy

The dominance of fatty acid metabolism for the fasting state has been confirmed in the human heart ( Table 2.1 ; for review see reference ).

TABLE 2.1

Fuels for Oxidative Metabolism of the Human Heart: Radio-Isotopic Data

Conditions	Glucose- Glycogen (OER %)	Lactate (OER %)	Total CHO (OER %)	FFA (OER %)
Glucose infusion	25–50	27	50–75	20
Exercise, moderate	14	28	42	64
Fasting overnight	3	13	16	62

CHO, carbohydrate; FFA, free fatty acids.; Oxygen extraction ratio.

For calculation details: Opie LH, Heart Physiology, from Cell to Circulation , Lippincott, Williams & Wilkins, 2004; p. 310.

For its major sources of energy, the heart shifts its utilization between carbohydrate in the fed state and fatty acids in the fasted state ( Table 2.1 ; Figure 2.1 ). In the carbohydrate-fed state, when circulating glucose and insulin are high, circulating fatty acid levels are suppressed. The uptake of fatty acids by the heart falls, resulting in the removal of fatty acid inhibition of glycolysis, so that glucose oxidation increases. Conversely, in the fasted state, circulating free fatty acids (FFAs) are high. The high rates of FFA uptake result in their preferential use in oxidative metabolism ( Figures 2.1, 2.5 ), so that fatty acids become the major source of energy.

FIGURE 2.1, How carbohydrates and fatty acids yield energy for contractile work. Crucial are the processes whereby the fuels (glucose and fatty acids) are simplified for entry into the Krebs cycle that lies within the mitochondria. Their production of protons (H) within yields energy in the form of ATP (adenosine triphosphate) that is mostly used for contractile work.

FIGURE 2.5, Substrate metabolism in the fasted state. Blood levels of free fatty acids are high in the fasted state or poorly controlled diabetes mellitus. High levels of blood free fatty acids are oxidized by the heart in preference to glucose and lactate. Use of lipid accounts for 60–70% of the oxygen uptake of the heart, whereas use of carbohydrate accounts for less than 20%.

When the heart oxidizes fatty acids, glucose oxidation is inhibited, resulting in the utilization of glucose for glycogen conversion, which is the glucose-sparing effect of fatty acid oxidation. The energy balance between fatty acid β-oxidation and glucose oxidation can be pharmacologically modified at multiple levels of each metabolic pathway, as reviewed by the Lopaschuk group.

Fatty acid oxidation is the dominant energy source for the heart when circulating FFAs are high, as after an overnight fast ( Figure 2.5 ) or after catecholamine simulation. Free fatty acid oxidation is less energy-efficient than glucose, requiring 11–12% more oxygen for a given amount of ATP produced. With elevated circulating FFA levels, the actual amount of ATP produced by fatty acid oxidation may be much lower than expected. Thus when fatty acid oxidation was driven by supraphysiological elevation of FFAs by lipid infusions given to dogs, the myocardial oxygen uptake increased by about 26%.

Adrenergic activation increases levels of free fatty acids (FFA) by lipolysis activation in adipose tissue (see Chapter 4, Chapter 14 ). The FFA-driven metabolic component of the myocardial oxygen uptake, also called oxygen wastage, could experimentally be up to 90%, reduced by about half by an infusion of glucose. Thus, high circulating glucose concentrations could protect the heart from FFA-induced oxygen wastage, strongly supporting the concept of adverse effects of supraphysiological FFA levels. The acute FFA-induced oxygen wastage is due to an increase in the oxygen cost for both basal metabolism and excitation–contraction coupling (EC) coupling. In heart failure or myocardial infarction, high circulating levels of FFA are driven by very high circulating norepinephrine levels. The resulting excess FFA-induced oxygen wastage is countered by glucose and insulin and by promotion of glycolysis, thus closely resembling the metabolic situation after a high-carbohydrate meal ( Figure 2.6 ).

FIGURE 2.6, Substrate metabolism in the fed state. After a high carbohydrate meal or glucose feeding, blood glucose and insulin are high, and blood free fatty acids low. Glucose becomes the major fuel of the heart, and carbohydrate can account for 50–75% of the oxygen uptake. CHO, carbohydrate.

Glucose Transport Regulation

The uptake of glucose from the bloodstream across the sarcolemma and into the cells of the heart is controlled by the glucose transporters GLUT 1 and especially GLUT 4 ( Figure 2.7 ). No energy is required for such glucose transport because the glucose concentration in the extracellular space is so much higher than in the cytosol. The uptake of glucose increases whenever the glucose transporter is stimulated, as during increased heart work, or by insulin as in the fed state, or during hypoxia or ischemia or by the antidiabetic drug, metformin ( Figure 2.7 ). Conversely, the uptake of glucose is inhibited by high circulating FFA concentrations ( Figure 2.7 , left side).

FIGURE 2.7, Effects of excess FFA in muscle cells. Molecular steps that lead from increased circulating FFA to insulin resistance (top left) exist together with opposing influences exerted by exercise or the antidiabetic drug metformin (top left and top right). Excess FFA entering the cell is activated to long-chain acyl-CoA, which inhibits the insulin signaling pathway. Thus there is less translocation of glucose transporter vesicles (GLUT-4 and -1) to the cell surface. The result is that muscle glucose uptake is decreased and hyperglycemia is promoted. Exercise and metformin, by stimulating the enzyme adenosine monophosphate-activated kinase (AMPK), promote the translocation of transport vesicles to the cell surface to promote glucose entry and thereby oppose insulin resistance. Protein kinase B, also called Akt, plays a key role. IRS-P, insulin receptor substrate-1; P-I-3 kinase, phosphatidyl inositol -3-kinase; PKB, protein kinase B.

Insulin is a circulating hormone whose levels rise in the fed state to increase glucose uptake by two major mechanisms. First, insulin decreases the release of FFA from adipose tissue, thereby removing the inhibitory effects of FFA on glucose uptake and glycolysis. Complex signaling paths convey the ‘message’ of insulin from the cell surface receptor to multiple internal sites of action ( Figure 2.7 ). Insulin binds to specific insulin receptors that greatly amplify the effect of insulin and in turn phosphorylate tyrosine. Tyrosine phosphorylation increases the activity of the insulin receptor substrate-1 (IRS-P in Figure 2.7 ). Thereafter the enzymes phosphatidyl inositol (PI)-3-kinase and Akt (also called protein kinase B, PKB) translocate the glucose carrier GLUT-4 and to a much lesser extent GLUT-1 from internal unavailable sites to external sarcolemmal sites to increase the influx of glucose ( Figure 2.7 ). FFAs inhibit this whole process at the level of tyrosine phosphorylation.

Beta-adrenergic stimulation acts by mobilizing FFA from adipose tissue, thereby promoting a metabolic condition similar to fasting with predominant fatty acid metabolism by the heart ( Figure 2.5 ). Intense stimulation can cause major oxygen wastage.

Pathways of Glycolysis

Glycolysis is the metabolic pathway that responds to reduced oxygen content by converting glucose to pyruvate ( Figure 2.3 ). During normal oxidative metabolism, glycolytically produced pyruvate is then oxidized in the Krebs (tricarboxylic acid (TCA)) cycle. Under anaerobic conditions, pyruvate is converted to lactate when the relatively small amounts of glycolytic ATP are of importance in preserving membrane function. Glycolysis converts glucose 6-phosphate into a compound containing two phosphate groups, fructose 1,6-diphosphate (fructose 1,6-bisphosphate) under the influence of the enzyme phosphofructokinase (PFK). Thereafter, glycolysis converts each six-carbon hexose phosphate into two three-carbon triose phosphates, using two molecules of ATP. During the next stages of glycolysis that form two molecules of pyruvate, four molecules of ATP are made independently of oxygen:

fructose 1,6 bisphosphate + 4 ADP → 2 × pyruvate + 4 ATP

FIGURE 2.3, Glycolysis simplified. G-6-P, glucose 6-phosphate; PFK, phosphofructokinase; F 1,6 bisP, fructose 1,6 bisphophate.

Functions of Glycolysis

Normally glycolysis is a bit like water flowing in and out of a tank: what goes in at the top (glucose uptake, glycogen breakdown) passes out at the bottom ( Figure 2.3 ). En route, the major enzymes that regulate the rate of flow to enter the Krebs (tricarboxylic acid (TCA)) cycle ( Figure 2.4 ) are phosphofructokinase and pyruvate dehydrogenase ( Table 2.2 ). Besides the production of protective anaerobic ATP, aerobic glycolysis provides energy for the maintenance of normal ATP-requiring membrane functions such as the sodium pump and the ATP-sensitive potassium channels. Glycolysis can also indirectly protect the mitochondria from potentially fatal calcium overload which occurs in some pathological states.

TABLE 2.2

Major Factors Controlling Glycolysis and Sites of Action

Conditions	Glucose Uptake	Glycogen Content	Activity of Phospho- fructokinase	Activity of Pyruvate Dehydro- genase
Increased heart work	+	↓	+	+
Inotropic agents	+	↓	+	+
Fed state, insulin	+	↑	+	+
Starvation, high blood fatty acids or ketones	-	↑	-	-
Hypoxia, mild ischemia	+	↓	+	-
Severe ischemia	-	↓	-	-

For further detail of glycolysis and its control by the above factors, see pages 314–321 in Opie LH, Heart Physiology, from Cell to Circulation, Lippincott, Williams & Wilkins.

+ stimulation; - inhibition; ↓ decreased content; ↑ increased content.

When glycolysis increases, the alternate and previously little emphasized hexose monophosphate bypass pathway is also stimulated. The result is an increase in the activity of the enzyme hexokinase-II. Hexokinase-II directly inhibits the mitochondrial permeability transition pore (mPTP), thereby protecting mitochondria from calcium overload. The authors of these studies found that these ‘data suggest that the glycolytic enzyme HK (hexokinase-II) is an important guardian of the mitochondrion in the beating, intact heart.’ ¹⁸

Pathways of Fatty Acid Oxidation

The chief fatty acids utilized by the human heart are long-chain fatty acids, including oleic acid, followed by palmate, of which palmitate is the better studied. In the schema shown, acyl CoA indicates both oleal CoA and palmityl CoA ( Figure 2.8 ). The oxidation of fatty acids can be summarized as follows:

1.
Extramitochondrial long-chain acyl CoA forms from long-chain FA: long-chain FA + CoA + ATP → long-chain acyl CoA + AMP + PPi;
2.
Extramitochondrial long-chain acyl (oleal and palmityl) carnitine forms from extramitochondrial long-chain acyl CoA, catalyzed by the enzyme carnitine palmityl transferase 1 (CPT-1);
3.
The enzyme carnitine acyl translocase transfers extramitochondrial long-chain acyl carnitine to within the space between outer and inner mitochondrial membranes;
4.
Mitochondrial carnitine palmityl transferase 2 (CPT-2) located on the inner membrane allows intramitochondrial oleal and palmityl carnitine to react with CoA so as to liberate intramitochondrial oleal and palmityl CoA and carnitine; the carnitine is exported out to the mitochondrial inter-membrane space;
5.
Intramitochondrial oleal and palmityl CoA enter the fatty acid β-oxidation spiral to form acetyl CoA that enters the Krebs tricarboxylic acid (TCA) cycle.
6.
During high rates of FFA uptake and subsequent metabolism by the above steps, more acetyl CoA may form than can enter the Krebs cycle. Such acetyl CoA can also react with intramitochondrial carnitine via the enzyme carnitine-acetyl transferase (CAT, Figure 2.6 ) to form acetyl carnitine. The latter is transported outwards from the mitochondria by the enzyme carnitine-acetyl translocase, and in the process cytoplasmic acetyl CoA is formed. This then undergoes transformation into malonyl CoA, which by feedback inhibition of CPT-1 helps to limit excess FFA oxidation.
7.
Also during high rates of FFA uptake and metabolism, excess acyl CoA that is not converted into malonyl CoA cannot be oxidized and forms potentially adverse myocardial triglyceride and structural lipids, the latter by changes in the degree of saturation and chain length.

FIGURE 2.8, Long-chain free fatty acid (FFA) metabolism. Effects of excess FFA supply. Blood borne FFA is taken into the cytosol, where the acyl carnitine carrier on the mitochondrial membrane transfers activated fatty acid (acyl CoA) into the mitochondrial space for oxidation in the fatty acid oxidation cycle. ACS/HF, acute coronary syndrome, heart failure; acyl CoA, long-chain acyl CoA compounds; CPT, carnitine palmityl CoA transferase; acyl carnitine, long-chain acyl carnitine compounds; CAT, carnitine acyl translocase; FFA, free fatty acids; GIK, glucose-insulin-potassium; MM, mitochondrial PDH, pyruvate dehydrogenase.

Fatty Acid Toxicity and Oxidation Inhibitors

In view of these adverse metabolic inhibitory effects of excess uptake and metabolism of high circulating FFA, it is noteworthy that therapeutic inhibition with practical clinical application can be achieved by infusions of glucose–insulin–potassium (GIK therapy) ( Figure 2.8 ) and by the drugs trimetazidine and perhexiline that act by inhibition of myocardial carnitine palmityltransferase-1 ( Figure 2.8 ).

Krebs Tricarboxylic Acid (TCA)/Citrate Cycle

When the Nobel prize winner Sir Hans Krebs was an unknown biochemist, he presented his concept and data of the citrate cycle to the prestigious journal Nature which politely rejected it. This classic then found its way to a minor journal, Experientia . Despite the concept’s initial struggle, the Krebs cycle remains at the basis of production of essential energy in the form of ATP ( Figure 2.4 ).

The breakdown of ATP is the only immediate source of energy for contraction, the maintenance of ion gradients, and other vital functions. The complex metabolic pathways already described transform the major fuels (glucose, free fatty acids, and lactate) to acetyl CoA, which enters the citrate cycle to produce NADH ₂ (NADH + H ⁺ ). NADH ₂ is the reduced form of the cofactor nicotinamide adenine dinucleotide. The 2H units, in turn, yield the protons pumped across the mitochondrial membrane, and the electrons that flow along the cytochrome chain, with resulting conversion of ADP into ATP by oxidative phosphorylation. Once produced in the mitochondria, ATP is transported outward to the cytosol by the ATP/ADP transport system for use in the cytoplasm, chiefly in contraction. As cytosolic ATP is used, ATP is synthesised from ADP that occurs in the mitochondria. The rates of synthesis and breakdown of ATP are, therefore, closely linked.

The rate at which the Krebs’ citrate cycle operates is a major factor controlling the rate of ATP production by the heart. The standard dogma is that each turn of the Krebs cycle produces 12 molecules of ATP; in reality, allowing for technical factors often ignored it is more accurately estimated that each cycle produces closer to 10 ATP ( Table 2.3 ). The citrate cycle accelerates with increased heart work. Conversely, decreased rates of operation of the cycle occur during states of oxygen deprivation, such as hypoxia or ischemia or during cardioplegic arrest.

TABLE 2.3

Comparative Energy Yields of Various Fuels Per Molecule Fully Oxidized

Molecule	ATP Yield Per Molecule		ATP Yield Per Carbon Atom		ATP Yield Per Oxygen Atom Taken up to (P/O ratio) ^a
	‘Old’	‘New’	‘Old’	‘New’	‘Old’	‘New’
Glucose	38 [CR]	32 [CR]	6.3	5.2	3.17	2.58
Lactate	18	14.75	6.0	4.9	3.00	2.46
Pyruvate	15	12.25	5.0	4.1	3.00	2.50
Palmitate ^b	130	105	8.1	6.7	2.83	2.33

‘old’, conventional; ‘new’, revised. See Hinkle et al. Biochemistry 1991;30:3576.

a P/O, phosphorylation/oxidation;

b For palmitate details see Brand, The Biochemist, Aug–Sept 1994, p20;

c 36 via mitochondria, 2 via glycolysis;

d 30 via mitochondria, 2 via glycolysis.

When the heart must suddenly increase ATP production as during a sudden jump in work demand, it is controversial as to which one of several potential factors limits oxidative phosphorylation. Such potential factors that limit oxidative phosphorylation include the rate of ATP formation from ADP, the supply of oxygen from the coronary circulation, and the rate of the cycle (malate–aspartate) that transports cytosolic NADH ₂ to the mitochondria, thereby helping to drive the citrate cycle.

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