ATP and Cytochrome C Oxidase in the Failing Human Heart

ATP AND CYTOCHROME C OXIDASE IN THE FAILING HUMAN HEART

Randall C. Starling, Rebecca Liebes, Denis Medeiros, and Ruth A. Altschuld

I. Myocardial Nucleotides.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 A. Regulation of Myocardial ATP Content. . . . . . . . . . . . . . . . . . . . . . . . . . . . , . 144 B. Degradation and Resynthesis of Adenine Nucleotides. . . . . . . . . . . . . . . . . . . 144 C. ATP and Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 D. Human Myocardial Tissu . . . . . . . . . . . . . . . . . . . . . . . . . . 147 E. Transvenous Endomyocardial Biopsies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 F. Surgical Specimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . 148 G . Correlation between ATP and Cardiac Function . . . . . . . . . . . . . . . . . . . .149 11. Mitochondria1 Cytochrome C Oxidase . . . . . . . . . . . . . . . . . ........ . .. . .151 A. Cytochrome C Oxidase and Copper Metabolism. . . . . . . . . . . . . . . . . . . . . . . 152 B. SOD, Copper, and Cytochrome C Oxidase in Failing Hearts . . . . . . . . . . . . . 152 111. Summary.. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 References . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Advances in Organ Biology Volume 4A, pages 143-158. Copyright 0 1998 by JAI Press Inc. All right of reproduction in any form reserved. ISBN: 0-7623-0389-1

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R.C. STARLING, R. LIEBES, D. MEDEIROS, and R.A. ALTSCHULD

I. A.

MYOCARDIAL NUCLEOTIDES Regulation of Myocardial ATP Content

Adenosine triphosphate (ATP) is the primary source of chemical energy for cardiac excitation-contraction coupling. ATP is also needed to fuel the pumps that maintain appropriate ion gradients across external and intracellular membranes. ATP is produced primarily through mitochondria1oxidative phosphorylation, but a small amount of ATP can also be generated through anaerobic glycolysis. In both metabolic systems, ATP is generated through the phosphorylation of adenosine diphosphate (ADP). Conversely, most energy-consuming reactions convert ATP to ADP and inorganic phosphate (Pi). To understand how ATP and total adenine nucleotide concentrationsmay be altered in heart failure, it is necessary to review factors that regulate the size of the intracellular ATP and total adenine nucleotide pools. The concentration of ATP within the cytoplasm of a normal heart cell is somewhat in excess of 10mh4, an amount capable of sustaining normal contractile performance for less than a minute (Chapter 7). There must therefore be a continuous resynthesis of ATP from the ADP and Piliberated during cardiac work. As discussed in Chapter 5, high energy phosphate stored in the form of phosphocreatine can help to buffer intracellular ATP. But this storage pool is also small relative to the overall energy demands of the beating heart. Thus, when the rate of myocardial ATP synthesis lags behind the normal rate of utilization, as for example during ischemia, when blood flow and the delivery of oxygen and nutrients are reduced, contractility declines, developed pressure falls, and end diastolic pressures rise.

B. Degradation and Resynthesis of Adenine Nucleotides The imbalance between ATP production and utilization during ischemia leads initially to an increase in ADP and Pi. The accumulation of ADP is limited, however, by the myokinase reaction, which converts two molecules of ADP to one ATP and one adenosine monophosphate (AMP). This helps preserve the ATP/ADP ratio, which is an important component of the phosphorylation potential (Chapter 7). Variable amounts of AMP are subsequently dephosphorylated to adenosine by 5’nucleotidase (Figure 1). Adenosine can diffuse or be transported out of the heart cell (Ford and Rovetto, 1987) and/or be degraded to inosine and hypoxanthine, leading to a net loss of intracellular adenylates. As a consequence, when the normal delivery of oxygen and metabolic substrates is restored, the cardiac cell cannot fully replenish its ATP stores because a portion of the necessary purine backbone has been washed away. Restoration of the adenine nucleotide pool, which in the heart occurs primarily through the uptake and conversion of circulating hypoxanthine to inosine monophosphate (IMP), is an exceedingly slow process: Complete recovery of intracellular ATP after a single ischemic insult can take a week ormore (Swain et al. 1982).

ATP and Cytochrome Oxidase ATp

145 cell

membmne

ADP

GMP

1

Figure 1. Pathways for adenine nucleotide synthesis and degradation. Ado, adenosine; Ino, inosine; Hpx, hypoxanthine; Xan, xanthine; AdSuc, adenylosuccinate; and PRPP, phosphoribosylpyrophosphate.Other abbreviations are defined in the text. Note the series of reactions where AMP is converted to IMP followed by conversion of IMP to AdSuc and then AMP. These reactions constitute the purine nucleotide cycle.

Instead of dephosphorylating AMP, the heart can also deaminate AMP to IMP. This reaction is quantitatively more important in skeletal muscle, but IMP accumulation is observed in hypoxic rat myocardium, especially following a-adrenergic stimulation and activation of protein kinase C (Hu et al. 1991;Hu et al. 1993;Hohl et al. 1989). Because IMP is not membrane-permeant and can be reaminated to AMP through the purine nucleotide cycle, restoration of the rota1 adenine nucleotide pool can, in principle, occur more rapidly when AMP is degraded to IMP as opposed to adenosine. However, the adult human myocardium may have less AMP deaminase than that found in smaller mammals, and the relevance of this pathway in adult humans is unknown. C. ATP and Heart Failure

Because the failing heart exhibits many contractile abnormalities similar to those observed during ischemia, there has been speculation that energy metabolism is defective in nonischemic as well as ischemic dilated cardiomyopathles(see Bottomley, 1994; Katz, 1988; Katz, 1989 for reviews). Primary metabolic defects, such as those associated with abnormal carnitine transport (Waber et al. 1982), are well known for their ability to produce potentially reversible cardiac failure. In postviral dilated cardiomyopathy, circulating antibodies against the mitochondria1 adenine nucleotide translocase (Schultheip et al. 1983) are thought to interfere with energy metabolism

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(Schultheip et al. 1995; Schulze et al. 1989). The latter part of this chapter also discusses the possibility that changes in mitochondrial cytochrome c oxidase may contribute to human end-stage heart failure. Finally, the decline in coronary perfusion, and importantly, subendocardial blood flow, associated with heart failure could lead to a demand-type ischemia during stress, even without obstructive (hemodynamically flow-through limiting) coronary artery disease (Unverferthet al. 1981; Jones et al. 1981; Swain et al. 1982).T h ~is s an important consideration because a great many patients with nonischemic cardiomyopathy have coexistent atherosclerosis and reduced coronary blood flow (Unverferth et al., 1983). There are a number of indications that the that the failing human heart is energy starved. Noninvasive in vivo 31P-NMRstudies have typically shown declines in phosphocreatine (PCr)/ATP ratios (Bottomley, 1994).This could result from a fall in the free ATP/ADP ratio (a parameter not directly measurable by 31P-NMR)as occurs during myocardial ischemia. However, in rat models of hypertensive cardiomyopathy, the fall in PCr/ATP is due primarily to aprofound decrease in the size of the total creatine pool (Ingwall et al. 1990;Tian et al. 1996).Similar declines in total creatine have recently been reported for the failing human myocardium (Nascimben et al., 1996) and should contribute to depressed PCr/ATP ratios. Whether or not there are declines in ATP and total adenine nucleotides in the failing human myocardium is controversial. Early attempts to demonstrate ATP depletion in surgical specimens produced mixed results (Jones et al. 1981; Chidsey et al. 1966). Conflicting data were also obtained with endomyocardial biopsies from awake subjects. An early study from our laboratory showed significant declines in the ATP content of right ventricular endomyocardial biopsies from failing human myocardium (Unverferth et al. 1981) with a significant correlation between ATP depletion and diastolic dysfunction. However, a more recent study by another laboratory reported no differences between the metabolite content of biopsies from apparently normal and failing human hearts (Regitz and Fleck, 1992). We have recently reexamined the issue of the ATP content of normal and failing human myocardial tissue. To do this we: (1) examined endomyocardial biopsies from a large number of patients, (2) obtained endomyocardial biopsies from a subset of patients with pure diastolic dysfunction, and (3) had two sources of normal human myocardium. Tissue extracts were analyzed by high performance liquid chromatography (HPLC) so that ATP breakdown products could be quantified as well. With biopsies, there is the possibility that a delay between removing the tissue sample and freezing it in liquid nitrogen will allow for a substantial decline in ATP. Quantification of the ATP breakdown products-ADP, AMP, IMP, adenosine, inosine, and hypoxanthine-provided an estimate of the amount of ATP breakdown during sample handling. In most instances this was not excessive. The sum AMP + IMP + adenosine + inosine + hypoxanthine rarely exceeded that observed in freeze clamped experimental animal hearts (i.e., 2 nmoVmg protein), and when it did, the data for that particular sample were discarded. This occurred in less than 10% of the

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tissue samples examined. Another potential problem is that expansion of the extracellular space through fibrosis could lead to an apparent net loss of intracellular metabolites. However, we normalized the data to total NaOH-soluble protein, as analyzed by the method of Lowry and colleagues (Lowry et al. 1951). Connective tissue is only sparingly soluble in NaOH and the Lowry method is generally assumed to measure noncollagen protein. D.

Human Myocardial Tissue Samples

We obtained tissue samples from (1) awake patients undergoing right ventricular endomyocardial biopsy, (2) cold preserved normal human donor heartsjust before allotransplantation, and (3) end-stage failing ventricles of patients without significant coronary artery disease undergoing cardiac transplantation surgery (Starling et al., 1991).

E.

Transvenous Endomyocardial Biopsies

All patients undergoing right ventricular endomyocardial biopsy were being evaluated because of (1) unexplained systolic and/or diastolic ventricular dysfunction, (2) symptoms of congestive heart failure, (3) unexplained angina, or (4) the intent to obtain baseline data before cancer chemotherapy as part of a protocol to assess anthracycline cardiotoxicity. Data from approximately three biopsies were analyzed and averaged for each subject as recommended by Brzitker and colleagues (1995). Informed consent was obtained in each instance in compliance with institutional review policies for human subject research. Significant coronary artery disease (luminal stenosis 2 50%),valvular heart disease and pericardial disease were excluded in all patients using diagnostic left and right heart catheterization and coronary angiography. Right heart catheterization and right ventricular endomyocardial biopsy (Unverferth, 1985) were performed using standard techniques (i.e., right internal jugular approach). Tissue samples were frozen in liquid nitrogen less than 15 seconds after closing of the bioptome jaws and stored at -80" C until analyzed. The normal individuals undergoing right ventricular endomyocardial biopsies consisted of 10cancer patients with normal left ventricular ejection fractions (20.55) and normal left ventricular end diastolic pressures (I13 mm Hg) prior to receiving potentially cardiotoxic chemotherapy. Forty-threepatients were diagnosed as having dilated cardiomyopathy. Mean left ventricular ejection fraction (+ standard deviation) was 0.28 +- 0.1 1, pulmonary artery wedge pressure was 18 A 9 mm Hg, and cardiac index was 2.7 0.7 L/min/m2.There were 29 men and 14 women in this group; mean age was 43 years (range 26 to 74 years). Six patients had restrictive cardiomyopathy. Mean left ventricularejection fraction was normal (0.53 +- 0.05) but pulmonary artery wedge pressure was significantly elevated (24 +- 7 mm Hg). There were five women and one man and mean age was 60 years (range 28 to 77 years).

*

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Table 1.

Metabolite Content of Normal and Failing Human Myocardial Tissue AJP

Total Adenine Nucleotides (nmollrnn protein)

NAD

39.3 f 1.9 38.2 f 2.2 24.4 f 1.3 1 2 . 0 f 3.33

51.3 f 2.6 47.4 f 2.5 34.7 f 1.5 19.2 f 4.8

6.30 f 0.40 5.89 f 0.20 4.54 f 0.17 2.46 f 0.66

right

18.8 +. 3.0

27.1 f 3.6

3 . 2 8 +_ 0.31

left epi

14.4 f 3.6 17.4 3.6 17.9 f 4.3

24.7 f 5.1 2 8 . 4 f 4.1 28.0 It 5.9

3.71 f 0.59 3.51 f 0.61 3.46 f 0.71

Normal EBX (n=12) Normal D o n o r (n=24) D C M - E B X (n=43) Restrictive EBX (n=6)

Transmural failing (n=8)

+

mid

endo Note:

EBX, endomyocardial biopsy; DCM, dilated cardiomyopathy; EPI, epicardium; MID, midmyocardium; and ENDO, endocardium. All data are meanfS.E.M. Numbers in parenthesesindicate the number of patienb in each group. Statistical comparisons among the normal EBX, normal donor, and D C M EBX groups indicated that all threevaluesforthe D C M group were significantlydepressed (p < 0.001)versus the NRL and DNR groups.

F.

Surgical Specimens

A cardiac bioptome was used to obtain normal tissue from the right interventricular septa1 surface of cold preserved donor hearts immediately prior to implantation. Transmural samples from the left and right ventricular free wall were taken prior to cross clamp during cardiac transplantation surgery in eight patients with end-stage, nonischemic heart failure. All specimens were immediately frozen in liquid nitrogen. Table 1 summarizes the ATP, total adenine nucleotide, and NAD content of biopsies from the several groups of hearts. Note that the values for endomyocardial biopsies taken from normal patients and from the Cold-Preserved hearts of normal organ donors do not differ. These values are in good agreement with what we and others have reported for rapidly aspirated, snap-frozen transmural biopsies of normal swine and canine hearts (DeBoer et al. 1980; Neumar et al. 1991). They also agree with 31P-NMRdata giving a normal in vivo human myocardial ATP concentration of about 7 pmol per gram wet weight, (Bottomley, 1994), assuming a conversion factor of 0.16 mg Lowry protein per mg wet weight, and with the early data of Jones and colleagues (198 1) on the metabolite content of normal human myocardium. These values are higher, however, than those reported by Chidsey and colleagues (1966) and Regitz and Fleck (1992) for apparently normal human myocardium. In the study by Regitz and Fleck, mean values for AT” (23 nmol/mg) and total adenylates (39 nmol/mg) in samples from patients with dilated cardiomyopathy were nearly identical to those shown by us in Table 1. The

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major difference involves the normal values. In the study by Regitz and Fleck, “normal” adenine nucleotide profiles were obtained in 14 biopsies from patients in whom cardiac disease was suspected, but coronary, valvular, hypertensive, or myocardial disease was excluded. Our study obtained data from cold-preserved normal donor hearts as well as from cancer patients with normal cardiac function prior to anthracycline chemotherapy. The “normal” patients with cardiac symptoms in the study of Regitz and Fleck could have had varying degrees of diastolic dysfunction, which we have found to be associated with depressed ATP even when systolic function is normal. ATP and total adenine nucleotides were significantly lower than normal in the endomyocardial biopsies from patients with dilated cardiomyopathy (p c O.OOl), while mean ATP and total adenine nucleotides in transmural biopsies of hearts in end-stage failure were lower yet. Interestingly, there was a strong positive correlation between total adenine nucleotides and the NAD content of myocardial biopsies (r = 0.87).As mentioned above, transient episodes of myocardial ischemia could be invoked to explain the low ATP and adenine nucleotide content of some failing human hearts. Postischemic recovery of ATP and total adenine nucleotides is a slow process (DeBoer et al. 1980), one which might not keep pace with intermittent ischemic episodes. Mild ischemic insults have not typically been associated with a pronounced loss of total pyridine nucleotides, however. There of course can be a significant decline in the NADNADH ratio during ischemia, but this reverses rapidly with restoration of adequate blood flow and NADNADH ratios of about 7 were typically observed in the samples described in Table 1. While the pyridine nucleotides are known to decline following experimental coronary artery occlusion, this process occurs on a much slower time scale and is thought to result primarily from inhibition of energy-dependentNAD synthesis in the face of continuous degradation via NAD glycohydrolase activity (Klein et al. 1981). It should be noted that, while the bulk of myocardial adenine nucleotides are cytosolic, most of the pyridine nucleotides are contained in the mitochondrial compartment (Geisbuhler et al. 1984). Thus, a decline in myocardial NAD suggests a depletion of mitochondrial pyridine nucleotides due either to a decline in the intracellular volume fraction of mitochondria or to a lower NAD concentration in the mitochondrial matrix space. The strong correlation with total adenine nucleotides suggests that interactions between the cytosolic and mitochondrial compartments are important determinants of steady-state nucleotide concentrations. C.

Correlation between ATP and Cardiac Function

In dilated cardiomyopathy, both systolic and diastolic function are often impaired. In the cardiomyopathic patients, whose myocardial metabolites are summarized in Table 1 , mean ventricular ejection fractions were low while end diastolic pressures were high. Thus, whether a decline in ATP has effects primarily on systolic or diastolic function obviously cannot be determined from such a

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group where both inotropy and lusitropy are abnormal. Data from patients with primary diastolic dysfunction are more informative (Starling et al. 1992). As a group, these individuals have normal ejection fractions but lower mean ATP, total adenine nucleotides, and NAD in their right ventricular endomyocardial biopsies than even those individuals with markedly depressed ejection fractions. Thus, when data from all patients were analyzed, there was a significant negative correlation between ATP and pulmonary capillary wedge pressure (Figure 2). By contrast, there was no significant correlation between ATP and left ventricular ejection fraction (not shown). A number of studies have shown good correlations between myocardial ATP and end diastolic pressures (Unverferth et al. 1984; Starling et al. 1992), and it has been argued on theoretical grounds that relaxation should be more sensitive than contraction to energy status (Katz, 1988).ATP has arelaxing effect on cardiac myofibrils, and studies with permeabilized heart cells have shown that varying MgATP over the 1-10 mM range greatly alters myofibrillar calcium sensitivity. That is, at each controlled level of free calcium, when the concentration of MgATP is decreased, there is an increase in isotonic shortening (Altschuld et al. 1985).Calcium sequestration by the sarcoplasmic reticulum is also quite sensitive to ATP and to ATP/ADPratios, and these nucleotideeffects are readily observed in permeabilized myocytes incubated with physiologically relevant concentrations of ATP (Hohl et al. 1992; Wimsatt et al. 1990).Thus, the rate at which the sarcoplasmic reticulum removes calcium from the myofibrillar space during diastole should decline when

50 C .-

$ 0

40

L

n 30

10 E

c 20 U

a I-

Q

< X

x

n

10

r=-0.43

x x x

p=0.0006

x x xxx %xx x x

xX

x

xx

x

x,X

X

x

x

X

xx

X

X

"0

10

20

30

40

50

rnm Hg Pulmonary Arte ry Wedge Pressure

Figure 2. Relationship between ATP content of endomyocardial biopsies and pulmonary artery wedge pressure. The figure includesdata from all of the biopsy patients described in Table 1.

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ATP falls and/or when ADP rises. This, along with an increased myofibrillar calcium sensitivity, could lead to decreased diastolic compliance.

II.

MITOCHONDRIAL CYTOCHROME C OXIDASE

Cytochrome c oxidase, a key regulatory enzyme of cardiac energy metabolism, has been reported to be reduced in humans with cardiomyopathy (Zeviani et al. 1986; Buchwald et al. 1990; Schwartzkopff et al. 1991). Deletions in nucleotide base pairs coding for the mitochondrial encoded cytochrome c oxidase subunits have been reported by DiMauro and colleagues (1988). Mitochondria1cytochrome c oxidase is a multisubunit peptide and represents the terminal oxidative step in energy metabolism. In eukaryotic cells, this enzyme is located in the mitochondrial inner membrane and inserted into the lipid bilayer (Capaldi et al, 1983). In mammalian tissues, the enzyme is composed of 13 different subunits and each subunit has been fully sequenced (Hood 1990;Zhang et al. 1991).Subunits I through I11 are encoded by mitochondrial DNA and the remaining ten subunits are encoded by nuclear DNA (Hood 1990; Zhang et al. 1991). The mitochondrial encoded subunits have catalytic properties such as electron transport and proton translocation whereas the nuclear-encoded subunits are thought to have regulatory functions (Hood 1990) and exist as tissue-specific isoforms (Kuhn-Nentwigand Kadenbach 1985). The mammalian subunits of cytochrome c oxidase are designated as I, 11,111, IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, and VIII (Zhang et al. 1991). Genes for subunits I, 11, and I11 are all located within the mitochondrial genome; genes for all other subunits are located in the nucleus of the cell (Capitanio et al. 1994).Subunits I, 11, and I11 compose the catalytic core of this enzyme and are responsible for oxygen reduction and proton pumping. Subunit I is the largest of all the subunits and binds two heme groups (Calhoun et al. 1994). Heme a3is associated with copper and referred to as CUB(Calhoun et al. 1994).Subunit I1contains the binding site for cytochrome c, which donates the electrons to subunit IT (Capitanio et al., 1994). There are two copper atoms present that form amixed valence center known as CuA found in subunit 11. There is also a magnesium atom located on this subunit (Tsukihara et al. 1995). The center of subunit I1 accepts the electrons from cytochrome c and passes them along to subunit I where they will eventually be used to reduce molecular oxygen to water and facilitate proton pumping (Calhoun et al. 1994; Capitanio et al. 1994), an essential step in mitochondrial ATP synthesis. The precise role of the nuclear encoded subunits is speculative. Possible roles may be to facilitate assembly of the functional cytochromec oxidase enzyme, stabilize the holoenzyme, and/or regulate activity of the functional oxidase (Capitanio et al. 1994).The nuclear encoded subunits are synthesizedin the cytoplasm as precursors carrying N-terminal basic presequences (Planner and Newport 1987; Kadenbach et al. 1987). The precursor proteins are released as water-soluble forms and

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imported by an import receptor. Chaperonin or Heat Shock Proteins are involved in the transport of these proteins to the mitochondrial membrane. The precursors are translocated to the inner membrane by an energy-dependent process, whereby a peptidase cleaves the terminal end in the matrix of the mitochondria (Kadenbach et al. 1987). A.

Cytochrome C Oxidase and Copper Metabolism

Activity of cytochrome c oxidase has been shown to be depressed as a result of dietary copper deficiency. Studies have shown the depression of cytochrome c oxidase activity in spleen, thymus, liver, kidney, intestine, brain (Prohaska, 1991),and heart (Medeiros et al. 1993b,Chao et al. 1993). Presumably the decrease in activity is attributed to a lack of Cu atoms for the CuA and CUBmetallic centers of subunits I and 11. Recently, copper-deficientrats have been reported to have decreased levels of the nuclear encoded subunits of cytochrome c oxidase (Medeiros et al., 1993, Chao et al., 1994;Liao et al., 1995a and b). Copper-deficiencyis known to result in a wide variety of cardiovascular defects including cardiac hypertrophy, increased mitochondrial volume density with vacuolization and disruption of fine structure, myofibrillar disarray, and increased deposition of lipid droplets and glycogen granules. Aberrant ECG patterns have been reported on numerous occasions and include notching within the QRS complex, inverted T waves, and significant alterations in P wave amplitude and duration. Animals die within a few weeks of copper restriction, usually from aneurysms or hemothorax (Medeiros, 1991a; 1992, 1993b). A key aspect of copper deficiency hypertrophy may be the decreased nuclear encoded subunits of cytochrome c oxidase. There typically is vacuolization of the mitochondrial cristae, which could result from the absence of these subunits because they are a fundamental part of the inner mitochondrial membrane. Another biochemical measure of Cu status is the activity of Cu, Zn, superoxide dismutase (SOD), which declines markedly with dietary Cu deficiency. This enzyme protects against superoxide radicals converting them to hydrogen peroxide.

B.

SOD, Copper, and Cytochrome C Oxidase in Failing Hearts

To assess whether copper has any role in human heart disease, cardiac explants and control heart samples of the left ventricular outer wall were assayed for SOD activity, cytochrome c oxidase subunit peptide levels, and copper content to determine if there was a connection between copper and cytochrome c oxidase. Cardiomyopathic hearts had significantly higher than normal SOD, which averaged (k SD) 10,357 k 680 U/g tissue (n=9) in failing hearts compared to 6,244 f 1271 (n=7) for the noncardiomyopathiccontrols (p < 0.05). Subsequent copper analyses revealed no significant difference by group (1.4 mg Cu/g k 0.6 for cardiomyopathc vs. 1.7 mg C d g 0.7 for non-cardiomyopathic controls). Western blots for cyto-

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Figure 3. Western blot of cytochrome c oxidase from explants of subjects with or without cardiomyopathy. C, control; D, dilated cardiomyopathy; m, low molecular eight marker; and Ox, bovine cytochrome c oxidase marker. Individual subunits are indicated on the right of panel.

chrome c oxidase showed that in all of the cardiomyopathic samples, the nuclear encoded subunits of cytochrome c oxidase were significantly decreased (P < 0.05) compared to the noncardiomyopathic controls (Figure 3). (Liebes and Medeiros, 1997). Furthermore, the mitochondria1encoded subunits appeared not to differ by heart disease group as determined by densitometry analysis (Table 2). The cytochrome c oxidase pattern was strikingly similar to that reported for copper-deficient rats as discussed above, but the elevated SOD activity among the cardiomyopathic heart samples suggested that copper “status” was more than adequate. A possible interpretation of these results could be that the cardiomyopathic patients had been experiencing ischemic episodes and developed a clinical profile similar to experimentally induced preconditioning. Preconditioning affords the heart protection from ischemidreperfusion, in part through upregulation of antioxidant defenses such as glutathione peroxidase, Mn-SOD, and Cu, Zn SOD (Yellon et al., 1992; Maulik et al., 1993). It may be that when Cu,Zn SOD activity levels increase, the available copper (and zinc) within the myocyte are directed toward SOD rather than cytochrome c oxidase synthesis. Thus, a functional deficiency of copper within the rnyocyte relative to the need for adequate cytochrome c oxidase activity and subunit synthesis appears likely. In the cardiomyopathic hearts, all nine had significant declines in the nuclear encoded subunits of cytochrome c oxidase whereas none of the controls did.

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Table 2. Peptide Subunits of Cytochrome C Oxidase in Samples from the Left Ventricular Free Wall of Humans with Dilated Cardiomyopathy Compared to Samples from Noncardiomyopathic Subjects as Determined by Reflectance Densitometry of Western Blots. Subunit Cardiomyopathic (n =9) NonCardiomyopathic (n = 7) Percent S.D. I II IV' V a and b' VI a,b, and c' Note:

7.7f 6.9 8.4f 5.5 0.6+_ 0.2 3.0f 3.9 7.4 5.5

*

9.6f 7.8 12.0f 17.1 29.5f 23.8 12.8f12.9 42.3 f 26.4

'Differences by group are statistically significant (P < 0.05) as determined by Student t-test.

111.

SUMMARY

There are metabolic abnormalities in the failing human heart that can have a negative impact on ventricular function. Total creatine and adenine and pyridine nucleotide concentrations are significantly lower, on the average, in failing, as compared with normal, human hearts. Depressed ATP is strongly associated with diastolic dysfunction, a relationship most apparent in patients with pure restrictive cardiomyopathies. An ischemic basis for altered metabolite levels, even in the absence of significant coronary artery disease, is suggested by the increased amount of superoxide dismutase found in end-stage failing hearts. The increase in superoxide dismutase, a copper-containing enzyme, is accompanied by decreases in the nuclear-encoded subunits of cytochrome c oxidase-a phenomenon similar to that observed in severe dietary copper deficiency. The role such metabolic changes play in the progressive cardiac deterioration seen in congestive heart failure is not yet known.

REFERENCES Altschuld, R. A,, Wenger, W. C., Lamka, K.G., Kindig, 0. R., Capen, C. C.. Mizuhira, V., Vander Heide, R. S.,and Brierley, G. P. (1985).Structural and functional properties of adult rat heart myocytes lysed with digitonin. J. Biol. Chem. 260,14325-14334. B@tker,H.E.,Kimose, H. H., Thomassen, A. R., and Nielsen, T. T. (1995). Applicability of small endomyocardial biopsies for evaluation of high energy phosphates and glycogen in the heart. J. Mol. Cell Cardiol.27,2081-2089. Bottomley, P. A. (1994).MR spectroscopyofthe human heart: The status and thechallenges. [Review]. Radiology 191,593-612. Buchwald, A,, Till,H., Unterberg, C., Oberschmidt,R.. Figulla. H. R., and Wiegand, V. (1990).Alterations of the mitochondrial respiratory chain in human dilated cardiomyopathy.Eur. Heart I. 11,509-516.

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