Energetics of the Normal and Failing Human Heart: Focus on the Creatine Kinase Reaction

ENERGETICS OF THE NORMAL AND FAILING HUMAN HEART: FOCUS ON THE CREATINE KINASE REACTION

Joanne S . lngwall

................................................. 118 ......................................... 120 A . The Proteins and Genes .......................................... 120 B . Creatine ....................................................... 122 C . Enzyme Regulation .............................................. 123 D . In vivo Reaction Kinetics ......................................... 124 E. The Shuttle Hypothesis ........................................... 125 F. Energy Reserve and Contractile Reserve ............................. 126 111. The Creatine Kinase System in Diseased Human Myocardium . . . . . . . . . . . . . .128 A . StudyGroups .................................................. 128 B . Creatine Kinase Activity (Vmax): Tables 1 and 2 ...................... 129 C . Creatine Content ................................................ 130 D . Creatine Kinase Reaction Velocity .................................. 131 E. Creatine Kinase Isoenzyme Distribution ............................. 132 1.TheFundamentals

I1. The Creatine Kinase System

Advances in Organ Biology Volume 4A. pages 117.141 Copyright Q 1998 by JAI Press Inc All right of reproductionin any form reserved ISBN: 0-7623-0389-1

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F. Clinical Implications. ............................................

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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THE FUNDAMENTALS

Adenosine triphosphate (ATP, structure shown in Figure 1) is the high energy phosphate-containing compound directly used for excitation and contraction in muscle cells. Cleavage of the terminal phosphate (a phosphoryl bond) by ATPases releases chemical energy, which is converted into the work of contraction and relaxation, ion movements, and macromolecular synthesis. The reaction is: MgATP-2 + H,O

+ MgADP-1+ Pi-*+ H+

Chemical energy that can be used to do work is called free energy. Although the free energy of ATP hydrolysis is relatively high (-7.3 kcavmole), the concentration of ATP ([ATP]) in the cytosol of cardiac myocytes is low. Adult mammalian ventricular tissue contains about 5 pmoles per g wet weight, which is equivalent to about 10 mmolesk (mM) of intracellular water or about 33 pmoles per g cardiac protein. This amount of ATP is sufficientto maintain pump function for only about 50 beats. Thus, the cell must continuallyresynthesizeATP to maintain normal pump function and cellular viability. The distinction between the amount or concentration of ATP versus its turnover rare is central to our understanding of bioenergetics. In the normal heart, [ATP] re-

-0-

OH ATP

Figure 1.

OH Phosphocreadne

Structures of phosphocreatine and MgATP.

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mains constant but its rate of synthesis and degradation (turnover rate) varies. The energetics of increasing cardiac work illustrates this principle. As the workload of the heart increases, oxygen consumption (a good index of ATP synthesis rate) proportionately increases; yet, ATP content is essentially unchanged. Thus ATP turnover rate, but not its concentration, increases. This example also illustrates the important principle that ATP synthesis rate matches ATP utilization rate. Another important principle essential for our understanding of bioenergetics is that the chemical reactions that use ATP are “driven” by high ratios of ATP to ADP whereas ATP synthesis reactions are inhibited by high ATFVADP ratios. One expression that defines the energy state of the cell is the adenylate energy charge. It is defined as (ATP + 112 ADP) /( ATP + ADP + AMP) and distinguishes between utilizable ATP (the numerator) and total adenine nucleotide pool (the denominator). In well-perfused tissue, the cytosolic ATP, ADP, and AMP concentrations are approximately 10 mM, 30 pM, and 0.1 pM, respectively, and the energy charge is close to 1. Even when ADP and AMP concentrations increase by an order of magnitude, the energy charge does not change very much. A variation of the energy chargemore relevant to biologicalconditionsis the phosphorylation potential, defined as ATP/[ADP x Pi]. This term takes into account the ability of the end products of ATP hydrolysis, namely Pi and ADP, to inhibit ATPase activities on the one hand, and, on the other, to stimulate ATP synthesis pathways. The phosphorylation potential in well-perfused myocardium is greater than 300 mM-I(AW, ADP, and Pi concentrations of 10 mM, 30 pM, and 1 mM, respectively). Even modest increases in ADP and Pi, for example, a doubling of each at essentially constant [ATP], lowers the phosphorylation potential to about 80 mM-’.Thus the phosphorylationpotential is a sensitive marker of the energy state of the cell. It is the critical component of the thermodynamic quantity representing the free energy of ATP hydrolysis, AG. The free energy of ATP hydrolysis is the driving force for all ATP-utilizingreactions in the cell. It is calculated from the constant value for ATP hydrolysis under standard conditions, AGO, adjusted for the actual concentrations of ATP, ADP, and Pi in the cytosol. The expression is

AG = AGO - RT In [ATP]/[ADP][Pi] where AGO is the standard free energy change of ATP hydrolysis (-30.5kJ/mol under standard conditions of molarity, temperature, pH, and Mg”), R is the gas constant (8.3 J/mol.K), and T is the absolute temperature in Kelvin. The argument of the In term is the phosphorylation potential. For the heart, the values of the two terms in the equation are about the same; thus, to change AG, there must be a large change in the In term. The value of AG for pyruvate-perfused rat heart with a typical rate-pressure product of about 28,000mmHg min-’is -69.9 f2.0 kJ/mole. At the same workload for a glucose-perfused heart, which has higher ADP and Pi concentrations, AG is less negative, -57.7 0.6 kJ/mol. The less negative value means that the glucose-only perfused heart at the same workload has a lower driving force for ATP-utilizing reac-

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tions. Because it is often confusing to describe changes for a negative number, it is convenient to describe changes in AG in terms of its absolute value, I AG I . Given the critical need to maintain both a constant and high level of ATP and the ATP/[ADPxPi] ratio, it is not surprising that the cell uses many reactions and pathways to synthesize ATP and to regulate ADP and Pi levels. Primary among the ATPsynthesizing reactions are, in order of decreasing velocity, the creatine kinase (CK) re3 +), action (-10 mM s-l),oxidative phosphorylation(-1 mM s-'), glycolysis ( ~ 0 . mM and de novo synthesis (
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THE CREATINE KINASE SYSTEM A.

The Proteins and Genes

ATP; creatine phosphotransferase (EC 2.7.3.2) catalyzes the transfer of a phosphoryl group between PCr and ATP: PCr-2 + MgADP-1+ H+l t)creatine + MgATP-2. The overall equilibrium position is far to the right, Keq 144, at pH 7. The transfer of the phosphoryl group between PCr and ATP is not oxygen-consuming. CK activity is highest in cells with high ATP turnover rates, namely nondividing, differentiated skeletal and cardiac muscle cells, brain cells, and rapidly dividing cells. CK is an isoenzyme family composed of five electrophoretically distinct proteins that display remarkable diversity in terms of developmental regulation, tissue specificity, species specificity, self-assembly, and intracellular localization. The five CK isoenzymes are BB, MB, MM, and two mitochondrial isoenzymes, the ubiquitous uMtCK and sarcomere-specific sMtCK isoenzymes. The isoenzymes are named based on their source: BB for brain, MM for muscle, and the mitochondrial forms because they are located in mitochondria. The B, M, and the two MtCK polypeptides are encoded by separate nuclear genes. CK isoenzymes assemble in one of two ways. The inactive monomeric B and M polypeptides randomly assemble into enzymatically active dimers, that is MM, MB, and BB. Thus, an equal number of M and B monomers in solution would assemble to form MM, MB, and BB in a ratio of 1:2:1. The sMtCK associates only with itself to form dimers that in turn form octamers (Schlegel et al., 1988). The xray crystal structure of the octameric sMtCK protein has been recently obtained (Fritz-Wolf et al., 1996). The octamer is a cube with the dimensions 93 A x 93 A x 86 A, forming a 20-A wide channel. The channel is long enough to connect the inner and outer mitochondrial membranes (Rojo et al., 1991) and to provide a means for communication between the cytosol and the mitochondrial matrix. While the

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mitochondrial isoenzymes bind only to mitochondria, the MM-CK isoenzyme is localized in organelles for ATP utilization, including both the myofibrils (MM-CK is one of the M-band proteins; Turner et al., 1973) and membranes containing cation pumps (Saks et al., 1975) as well as the cytosol. There is no evidence for localization of either BB- or MB-CK. The cDNAs for all four CK polypeptides, B, M, uMtCK, and sMtCK, have been cloned from heart (ventricular) tissue from several different species, and their transcriptional regulation and structure are under investigation (e.g., Johnson et al., 1989, Cerjesi and Olson, 1991). There is exceptional homology among these polypeptides. The homology is highest for the same polypeptide compared among species; for example, there is 87-94% homology for all B-CKs studied (VillarrealLovy et al., 1987). It is high between the cytosolic B and M-CKs: 72% homology based on nucleotide sequence and 82% homology based on predicted amino acid sequence (Hossle et al., 1986). Homology is even high among all four polypeptides isolated from the same tissue: For the four major human cardiac CKs, there is 62% sequence identity from residues 52 to 295; differences exist primarily at the amino and carboxyl terminals (Haas and Strauss, 1990).As expected for afamily of isoenzymes, the amino acid sequence in the active site of all four polypeptides is highly conserved. An important difference between the cytosolic and mitochondrial CKs is that the MtCKs contain a transit peptide required for mitochondrial import and processing. The homodimer, BB-CK, is the dominant CK isoenzyme in brain, in undifferentiated striated muscle, and in rapidly dividing cells. There is one functional B-CK gene (Van Deursen et al., 1992). Genes for mouse, rat, and human BCK are nearly identical in terms of exon-intron organization and composition. The existence of subisoforms of B-CK identified by differences in their isoelectric points in some species (chick) may be explained by alternative splicing (Wirz et al.. 1990). The cDNA encoding the B-CK predicts a molecular weight of about 42,878 (ibid). Transgenic mice overexpressing B-CK have been made. Mice have been constructed to overexpress B-CK in liver (Brosnan et al., 1990) and skeletal muscle (Brosnan et al., 1993). By attaching the cDNA for B-CK to the promoter for the rat skeletal muscle a-actin gene, skeletal muscle activity of total CK was increased by 50-loo%, and the isoenzyme distribution was changed from about 100% MM-CK to 60% MM, 32% MB, and 8% BB. 31Pnuclear magnetic resonance (NMR) spectroscopy confirmed the increase in V, for CK (ibid). The M-CK gene is transcriptionally activated by enhancers that share motifs found in regulatory regions of other muscle-specific genes (Donoviel et al., 1996). Recent evidence suggests the existence of different regulatory elements for M-CK in vivo versus in-cultured muscle cells (ibid). These same investigators presented evidence that M-CK regulation in skeletal and cardiac muscles have both elements in common and elements that appear to be tissue-specific. The successful construction of mice with the M-CK gene ablated by Wieringa and colleagues (Van Deursen et al., 1993) provides a new reagent for the study of M-CK gene and protein regulation.

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The existence of multiple nuclear genes encoding two closely related tissuespecific mitochondrial protein isoforms, uMtCK and sMtCK, is unusual (Haas and Strauss, 1990; Haas et al., 1989);its significance is unknown. uMtCK and sMtCK cDNAs have 73 % nucleotide and 80% predicted amino acid identities. Human sMtCK cDNA encodes a 419 amino acid protein. Typical of other mitochondrial proteins encoded by the nuclear genome, an amino acid transit peptide required for import into the mitochondria is located at the NH,-terminus. The first 25 residues of the MtCKs form the binding site to the mitochondnal membrane-specificlipid, cardiolipin. Predicted amino acid sequence identity for sMtCKs isolated from human heart, rat heart, and mouse skeletal muscle is 96%.The human sMtCK gene has been isolated and characterized with respect to chromosome location, number of exons, and locations of the translation start codon and of the region conferring tissue-specific expression (Klein et al., 1991). The Weiringa laboratory has also prepared mice with both the sMtCK and MCK genes ablated (Steeghs, 1995). CK isoenzyme distribution in heart is developmentally regulated. Fetal myocardium contains low levels of total CK activity and it is primarily the BB isoenzyme. Genes encoding M and sMtCK are activated by unknown mechanism(s) at different times during terminal differentiation (Ingwall et al., 1980; Ingwall et al., 1981). Adult hearts of large mammals with relatively slow metabolic rates contain 90% MM-CK, 10% (or less) sMtCK, and only small amounts of B-containing isoenzymes whereas small animal hearts contain greater isoenzyme diversity, typically up to 1% BB, 6 1 4 %MB, 25-35% sMtCK, and the balance as MM. B.

Creatine

PCr (structure shown in Figure 1) is the primary high-energy phosphatecontaining molecule in heart; its cytosolic concentration is about twice that of [ATP] : about 20 mM versus 10 mM. Note that the free energy of hydrolysis is about 40%higher (-10.3 kcal/mole) than for ATP. In contrast to the constancy of the purine pool, the size of the creatine pool changes with age, hormonal status, and, as we have now found, in heart failure. Creatine, a p-amino acid (molecular weight = 131 Da) is synthesized in the liver (where the guanidino and carboxyl moieties from arginine and glycine are linked) and the ludney and pancreas (where the N-methyl group is added) and supplied to tissues via the bloodstream (blood concentration 140 pM). During development,creatine accumulatesroughly in parallel with total tissue CK activity primarily in excitable tissues with high ATP turnover rates, namely striated muscle and brain (Ingwall et al., 1980). It is transported against a large (- 100-fold) concentration gradient and is sodium-dependent. In heart, about two-thirds of the total creatine pool is rapidly phosphorylated via the CK reaction and is, hence, chemically trapped; the mechanism whereby unphosphorylated creatine is trapped remains an important unsolved issue. Creatine transport has been characterized in both heart and skeletal muscle. Modeling creatine transport measured in isolated rat hearts using radiolabeled cre-

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atine to Michaelis-Menten kinetics yields values for Km of pM and for V,,, of about 8 pM min-' (Seppet et al., 1985),values close to that found for skeletal muscle cells (Fitch et al., 1968); Na+-independent passive movements are negligible. Experiments using human primary muscle and rat skeletal muscle (L6 cell line) cells showed that the extracellular creatine concentration regulates either the number or V,, of the creatine transporter; downregulation is faster than upregulation (Loike et al., 1988).The creatine pool is lost from excitable tissues as creatinine formed via dephosphorylation and nonenzymatic dehydration of PCr. The predominant rabbit brain and muscle (i.e., fast-twitch psoas) creatine transporter has recently been cloned and characterized (Guimbal and Kilimann, 1993). Expression of this cDNA in COS-7 cells allowed its characterization as an Na- and C1-dependenttransporter with high substrate specificitywith a Km of about 35 pM, a value indistinguishable from that found for intact muscle. It is amember of a family of Na-dependent plasma membrane transportersof neurotransmittersand osmolytes that includes GABA, noradrenaline, and other p-amino acids such as taurine and betaine. The cDNA encodes a protein of 635 amino acids for a predicted molecular weight of 70,525 Da. It is most closely related to another p-amino acid transporter, taurine, but the percent homology is not impressive (52% amino acid homology).

C. Enzyme Regulation Purified CK (usually MM-CK from rabbit skeletal muscle) has been well studied in solution, and much is known about its mechanism of action as well as the structure of its active site (Kenyon and Reed, 1983; Schimerlick and Cleland, 1973; Morrison and Cleland, 1966).The in vivo concentration of CK in striated muscle is about 50-200 nM, depending on the muscle type. The in vivo concentrations of PCr and ATP are usually high enough to saturate the enzyme. The concentrations of ADP and creatine are near their apparent Km: 30-50 pM [ADPI versus a Krn,,, of about 70 pM, and 10mM [Cr] versus a Km,, of about 17 mM. Thus, one would expect large changes in reaction velocity in normal myocardium with changes in [ADP] and free [creatine]. This could occur, for example, at high workload when both [ADP] and free [creatine]increase. As we will see, in heart failure, the creatine pool is partially depleted and may fall to concentrations low enough to contribute to enzyme regulation. Based on analysis of the CK reaction in dilute solution, the form of the reaction mechanism has been deduced and the rate equation for calculating the velocity of the reaction has been formulated (ibid). The rate equation is:

where V,, is the total CK activity measured under saturating conditions; ADP is calculated using the CK equilibrium expression

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LADPI=

[ATP] [freecr] [F'Cr][H+]1.66x10Y

and D, ka,and k, are hnetic constants (ibid). Importantly, by using "P NMR spectroscopy, it is now possible to measure the kinetics of the CK reaction in perfused organs (Bittl and Ingwall, 1985) and in tissues in intact animals (Bittl et al., 1987)as well as in solution. Through the use of magnetization transfer techniques, the pseudo first-order unidirectional rate constants k, and kras well as the concentrations of PCr and ATP can be measured. The velocity of the reaction is calculated from the product of the rate constant and metabolite pool: velocity = K,, [PCr] =

[ATPI

(3)

Thus NMR spectroscopy allows measurement of the kinetics of CK in its in vivo environment, inviting comparison of CK kinetics predicted from dilute solution and actually observed in situ. D.

In vivo Reaction Kinetics

The technique of 31PNMR magnetization transfer has been successfully applied to normal human skeletal muscle (Bolinger et al., 1988) and heart (Bottomly and Hardy, 1992). Our current knowledge about pathological conditions, however, is based primarily on experiments using animal hearts. The opportunity to measure the reaction velocity of a single protein or isoenzyme family in the intact beating heart is rare, if not unique. This approach allows us to understand how this enzyme is regulated in vivo. We have compared measured reaction velocity to the rate of ATP synthesis from other pathways, to the maximal capacity of the reaction (Vmax) measured in tissue homogenatesunder saturating substrate conditions, and to the reaction velocity predicted from the rate equation developed by solution biochemists. Several conclusions can be drawn. Firstly, ATP synthesis via the CKreaction is 10-timesfaster than net ATP synthesis rate estimated from the physiological index of oxygen consumption (Bittl and Ingwall, 1985).Because its reaction velocity is high, during high demand conditions, CK effectively functions to resupply ATP and to keep [ADP] and [Pi] low thereby maintaining a high driving force for all ATPase reactions. It also contributes to buffering the intracellular hydrogen ion concentration. Secondly, the ratio of measured CK reaction velocity in the beating heart to V,, is about 0.1 (ibid). This means that most of the enzyme activity is not used. It is not known whether enzyme activity can be recruited'for use under times of high energy demand. Thirdly, comparison of measured and predicted reaction velocities are usually in good agreement (Bittl et al., 1987b). However, lack of agreement in hypoxia and ischemia when the reaction velocity measured in vivo is lower than predicted from the rate equation (Bittl et al., 1987a;Ingwall et al., 1987), and in the case of failing aged hearts that were supplied with ACE inhibitors when

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velocity is higher than predicted from the rate equation (Nascimben et al., 1995), shows that the enzyme is regulated in vivo by .mechanismsin addition to substrate control. One possible mechanism is (de)phosphorylation (Chida et al., 1990). Another is a change in reaction mechanism (McAuliffe et al., 1991).

E. The Shuttle Hypothesis The localization of CK activity in both ATP-producing and ATP-utilizing organelles in muscle cells forms the basis of the “creatine phosphate shuttle hypothesis” whereby chemical energy is exchanged withm and between microcompartments via the CK reaction. Instead of, or in addition to, diffusion of adenine nucleotides between organelles, energy is carried by PCrlcreatine. The basis for this hypothesis is as follows. sMtCK is located on the outer surface of the inner mitochondria1 membrane (Jacobus and Lehninger, 1973) and forms pores spanning the inner mitochondrial membrane space (Rojo et al., 1991).Because of its location near to the ATP:ADP translocase, sMtCK operates essentially unidirectionally synthesizing PCr from ATP produced by oxidative phosphorylation. Calculations suggest as much as 90% of the high-energy phosphate produced by rat heart mitochondria could be converted to PCr (Saks, 1980). 31Pmagnetization transfer measurements of the intact neonatal heart (Perry et al., 1988) and for CK solutions (van Dorsten et al., 1996) show that the V,, of the CK reaction increases with increasing concentration of the sMtCK isoenzyme. Thermodynamic considerations also support the existence of a functional microcompartment of sMtCK and ATP produced by oxidative phosphorylation (De Furia et al., 1980).At the other end of the shuttle, MMCK bound to the myofibrils resupplies ATP used for contraction (Walliman et al., 1984). There was a specific kinetic enhancement of the myofibrillar ATPase when ATP was regenerated by endogenous CK: The apparent I(mATP decreased sixfold from 80 pM to 14 pM (Krause and Jacobus, 1992). Furthermore, when endogenous CK was inhibited by iodoacetamide, the apparent change in kinetics could not be achieved by the addition of soluble CK. Therefore, proximity between endogenous CK and myofibrillar ATPase was required. In addition to these thermodynamic and kinetic analyses, results using protein chemistry and molecular biology tools also support the existence of the shuttle. Only sarcomere-containing,mitochondria-rich tissues accumulate high concentrations of both the sMtCK and MM-CK, the two “ends” of the shuttle. Coordinate induction of MCK and sMtCK mRNAs has been shown in at least one muscle cell model, differentiating mouse muscle cells in culture. Taken together, the evidence that CK exists in microcompartments in the myocyte is compelling. What remains uncertain is whether metabolites are exchanged between these CK-containing compartments by diffusion or by diffusion coupled with some preferred exchange. Because such issues can be addressed only in an in vivo setting, it is difficult to devise definitive experiments to test these points. It should also be noted that some of the early literature in this field discussed the CK

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shuttle in terms of an “obligatory” role for CK in energy transfer. Such a concept is not useful, if for no other reason than that the sMtCK (one essential ingredient of the shuttle) does not even accumulate in the heart until after birth.

F. Energy Reserve and Contractile Reserve By contractile reserve we mean the ability of muscle to increase its contractile performance. By analogy, we suggest that a useful term describing the capacity for increased phosphoryl synthesis and supply needed to support increased work is energy reserve. In this sense, CK is an energy reserve system. This is clearly shown for the ATP supply-demand mismatch that occurs in hypoxia and ischemia when phosphoryl transfer from PCr to ADP slows the rate of tissue ATP depletion. Experiments of the last decade have now established a link between energy reserve and contractile reserve in other settings. The link has been shown in three types of experiments designed to selectively perturb the CK reaction in intact striated muscle. Firstly, acute and selective chemical inhibition of CK activity in intact rat hearts with sulphydryl group modifiers including iodoacetamide (Hamman et al., 1995; Tian and Ingwall, 1996) and an exogenouslysupplied NO donor (Gross et al., 1996) resulted in decreased contractile reserve when the heart was inotropically stressed with either high extracellular [Ca2+]or norepinephrine. Secondly, in rat and turkey hearts in which the myocardial PCr pool was replaced with the poorly hydrolyzable guanidino analog P-guanidinopropionic, PCr content, CK reaction velocity, and heart function all decreased under high workload conditions (Zweier et al., 1991; Liao et al., 1996). Thirdly, depleting M-CK by transgenic technology reduced the ability of skeletal muscle to sustain burst work (Van Deursen, 1993). Each of these experiments showsthat decreasing energy reserve via the CK reaction limits the ability of striated muscle to increase (i.e., recruit) its contractile reserve. Moreover, the perturbations, which result in chronic impairment of CK activity, such as the M-CK knockout, elicit compensatory changes in other pathways for ATP synthesis. Most notably glycolytic capacity and the fractional cell volume of mitochondria increased in fast-twitch skeletal muscle of the M-CK knockout mouse (ibid). These results illustrate the important principle that ATP synthesis occurs by the integration of all ATP-synthesizing pathways. When one pathway is diminished or eliminated (in this case the CK reaction), compensatory increases can occur in the others so that muscle can recruit its contractile reserve. The biochemical remodeling of the myocyte also emphasizes the importance of the CK reaction for phosphoryl transfer. The characteristics of the relation between energy reserve and contractile reserve for hearts with acutely inhibited CK provide several lessons about the relation between contractile reserve and energy reserve. We have determined both the free energy of ATP hydrolysis using 31PNh4R spectroscopy and isovolumic contractile performance in isolated rat hearts challenged to perform high work by increasing perfusate [Caz+]before and after supplying iodoacetamide to inhibit CK activity.

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These experiments are described in full elsewhere (Tian and Ingwall, 1996).Figure 2 shows linear fits of the relation between isovolumic contractile performance assessed as the rater-pressure product (RPP) and the absolute value of AG for normal hearts with 100% CK activity and hearts with only about 1% CK activity. Also shown on the x-axis are literature values for AG 1 s determined for the primary ATPases of the cell, namely myosin, the sodium pump, and the calcium pump. Four points can be made. Firstly, at low workloads, CK-inhibited hearts operate at a lower AG 1 ’thannormal hearts. This means that the CK-inhibited hearts have less free energy available for ATP hydrolysis. The difference between 59 kJ/mole for control hearts and 57 kJ/mole for CK inhibited hearts is due to substantial changes in metabolite pool size: ATP levels were not maintained and fell from about 11.4 to 9.9 mM and ADP increased from about 60 to 110 pM while Pi remained about the same (-6 mM). Thus the ATP/ADP ratio fell from 192 to 90 and ATP/[ADP x Pi] fell from 32 to 15 &-I. Secondly, the increase in RPP achieved in CK-inhibited hearts perfused with the same high CaZ+challenge was much less than for normal hearts. These experiments show that reducing energy reserve of the heart by inhibiting CK activity limits the contractile reserve of the heart. Thirdly, the decrease in the slope of the RPP-AG relation for the CK-inhibited hearts compared to normal hearts shows that there is a large change in free energy of ATP hydrolysis for a small

I

I

‘I 80

myosin ATPase

SR Ca2 + ATPase

k + l K+ ATPase

Free Energy Release from ATP Hydrolysis (kJ/mol)

Figure2. Relationship between rate pressure product (RPP), (10’ mm Hghin) and the absolute value of the free energy release from ATP hydrolysis (kl/mol) for hearts with 100%(solid line) and 1% (dashed line) creatine kinase activity. See text for explanation of numbers and arrows. Redrawn from Tian and Ingwall, 1996

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change in RPP. There is less work output in CK-inhibited hearts for a similar energy expenditure. Fourthly, for both normal and CK-inhibited hearts, essentially no data points fell beyond the value for I AG I for the sarcoplasmic reticularcalcium pump. At least under these conditions, there appears to be alimiting threshold for I AG I .If the I AG I were to go below 52 kJ/mole, the hearts would suffer calcium overload. If this interpretation is supported by other experiments (Tian and Ingwall, 1996), it would provide a rational basis for the link between energy starvation, or at least an energy limitation, and abnormal ion homeostasis. With this background, we will now analyze the significance of the changes in the CK system in diseased human myocardium.

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THE CREATINE KINASE SYSTEM IN DISEASED H U M A N MYOCARDIUIM

In this section, we present a summary primarily of our work analyzing the CK system using biopsy specimens obtained from human heart. This work has been described in full (Ingwall et al., 1985; Nascimben et al., 1996). A.

Study Groups

Samples of human myocardium were obtained from the left ventricles of the following: subjects undergoing open-heart surgery for repair of mitral stenosis or atrial septa1 defect that had normal ventricular function (n=2); accident victims at autopsy (n=13); donor subjects at the time of organ harvesting or transplantation (organ donors, n=36); and patients with aortic stenosis (AS, n=l l), coronary artery Qsease (CAD, n=9), both aortic stenosis and coronary artery disease (AS +CAD, n=5); or dilated cardiomyopathy who underwent heart transplantation (DCM, n=23). Thus we studied three groups of normal subjects and four groups of patients each witb a different pathology. The three groups of putatively normal subjectsdiffered by the length of time before biopsy specimens were harvested and by how heart function was maintained in vivo: Samples were obtained either during open heart surgery, within three hours of accidental demise, or after subjects were maintained on inotropic and mechanical support for one to seven days. For the four patient groups, left ventricular mass was substantially increased in the AS and AS+CAD groups, confirming the presence of left ventricular hypertrophy. The CAD and the AS+CAD groups demonstrated wall motion abnormalities associated with coronary artery disease. The DCM group consisted of patients who were in severe failure, NYHA Class IV. Immediately after removal, all myocardial samples were frozen in liquid nitrogen and stored in liguid nitrogen until analyzed. Portions of the myocardial biopsy specimens (5-20 mg) were homogenized and aliquots were removed for measurement of protein (Lowry et al., 1951), total creatine content (i.e., PCr plus free creatine; Kammemeier,l973), total CK activity (Rosalki, 1967), citrate synthase

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activity (Srere et al., 1963), the relative distribution of the CK isoenzymes determined electrophoretically (Hall and DeLuca, 1976), and B-CK content using a commercial radioimmunoassay kit.

B. Creatine Kinase Activity (Vmax): Tables 1 and 2 Total CK activity normalized to cardiac protein (Table 1) for the three groups of normal subjects was similar. The relatively small decrease for ventricular tissue obtained for organ donors (15-20% depending on the comparison) is significant. Tissue from the AS, AS+CAD, and DCM groups, but not the CAD alone, had lower CK activity-typically 30 to 42%lower than for normal subjects. The lowest values were obtained for patients in the DCM group. It is important to point out that the method used to normalize biochemical data determines the magnitude of any observed change. This is illustrated by comparing results obtained for the four groups described by Ingwall and colleagues (1985) for two enzyme activities measured using aliquots obtained from the same tissue homogenate, each normalized by wet weight and by Lowry protein content determined using aliquots of the same tissue homogenates (Table 2). In the case of CK Table 1. The Creatine Kinase System in Normal and Diseased Human Myocardium

Normal fresh biopsy < 3 hr 1-7 days

n

Creatine Kinase Activity W m g protein)

Creatine ( n m o h g protein)

Vmax x Cr

2 13 36

11.0 11.7 f 0.7 9.3 f 0.4

128 131 f 8.4 96 f 5.0

1408 1533 a93

11 9

8.2 zk 0.6 11.2 f 0.8 6.8 f 1.7 7.7 f 0.4

Disease AS CAD AS CAD DCM

+

5

23

77 f 7.0 83 f 12.0 68 f 10.0 63 f 5.4

631 930 462 485

Table 2. Creatine Kinase and Citrate Synthase Activities in Normal and Diseased Human Myocardium Creatine Kinase Activity

(I U/mgww)

(I U/mg

Citrate Synthase Activity

(mlU/mgww)

protein)

Normal (9)

1.99 k 0.1 1 1.08 f 0.08 1.46 f 0.10 AS + CAD (5) 0.78 f 0.15 AS (11) CAD (9)

12.0 k 0.57 8.2 f 0.60 11.2 f 0.78 6.8 k 1.73

77*3 61 f 3 59 f 3 43f6

(mlU/mg protein) 445 f 14 460 f 35 456 f 32 373 f 70

(mg protein/ mgww) 0.1 7 f 0.01 0.1 3 f 0.01 0.1 3 f 0.01 0.12 f 0.01

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activity, expressing the data per mg wet weight supports the conclusion that heart tissue from each of the disease states studied has lower CK activity. When expressed as mg Lowry protein, we find that CK activity in the CAD group was not different from that for normal subject, and that the magnitude of the apparent decreases in the AS and AS+CAD groups was greatly diminished. Results for citrate synthase activity are even more dramatic. Where expressed per mg wet weight, differences in all three disease states compared to normal tissue were found, but when normalized to Lowry protein, none of the values are different from each other. This illustration shows that normalizing enzyme activities (as well as substrate contents) by noncollagen protein content minimizes any confounding contributions from overall edema, fibrosis, and any change of interstitial water. Any protein assay (or even expressing data as dry weight) eliminates any contributions due to edema. Only the Lowry protein assay minimizes the contribution of extracellular matrix proteins. This is because the targets for the Lowry assay are aromatic side chains (tyrosine, tryptophan, and phenylamine), which are found in lower abundance in s is particularly important for failing myoextracellular matrix proteins. T h ~ point cardium that has substantial fibrosis. Our results showing a decreased total CK activity in failing myocardium are in good agreement with the reports of Sylven and colleagues (1993) comparing hearts from patients with cardiomyopathyand heart donors. Sylven and colleagues (1993) found that CK activity in the left ventricles of failing hearts was 44% lower while we found a 34% decrease. Bristow and colleagues (1991) reported that CK activity in the left ventricular myocardium from patients with DCM was 25% lower than for heart donors. They attributed this difference to the lower age of some subjects in the donor group; however, the mean age difference (5 years) was not significant. Although CK activity may well be decreased by aging (Fetters et al., 1985), these results show that the decrease seen in failing myocardium is primarily due to pathology. Note that the decrease in CK activity is about the same for the AS with or without the CAD and DCM groups. Thus, all of these uncompensated hearts have lower CK activity.

C. Creatine Content Unless biopsy specimens are obtained from the beating heart with a drill or other biopsy tool allowing for very rapid collection and freezing, PCr and ATP contents cannot be reliably measured. Total creatine content can, however, be reliably measured in tissue if frozen rapidly within minutes. Results in Table 1 show that the total creatine pool is lower (-27%) in organ donors compared to the other normal groups. Creatine content is also lower in each of the disease states compared to the normal subjects. The greatest decrease is for these uncompensated groups where the total creatine pool is only about 50% of normal. These results showing decreased total creatine in failing hearts are supported by the 31PNMR spectroscopy findings that the in vivo PCr/ATF’ ratio is 25% lower in

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failing human hearts (Conway et al., 1991; Hardy et al., 1991; Neubauer et al., 1992) compared to healthy subjects. Because CK functions to maintain a constant PCr/Cr ratio, we would expect that a 50%decrease in creatine would be accompanied by a 50% decrease in [PCr]. To reconcile this prediction with the NMR observations would require that the ATP pool also be depleted. A reduction in cytosolic ATP concentration in failing hearts has been observed both in human (Bashore et al., 1987) and in animal models (Nascimben et al., 1995; Liao et al., 1996), and the magnitude of the decrease (-25%) is sufficient to reconcile these results. D. Creatine Kinase Reaction Velocity

We can estimate the velocity of the CKreaction in human myocardium in vivo in two ways. Both methods are based on the rate equation developed to determine the velocity of the CK reaction for MM-CK in solution. To use the rate equation, we input the values for V, and the substrates for the CK reaction into equation 1. To convert substrate amounts and enzyme activities to cytosolic concentrations, we assumed that the cytosolic volume of blood-perfused myocardium is 0.65 mug wet weight (Bittl et al., 1987) and used the mg proteidwet weight ratio measured for each group. We used [ATP] obtained by 31PNMR spectroscopy of healthy volunteers (Weiss et al., 1990) for the control group. Values for [ATP] for patients are sparse, but some information is available. Swain and colleagues (1982) have shown that [ATP] in AS and CAD is likely to be nearly as high as for normal subjects. Regitz and colleagues (1992) have reported that ATP content expressed as nmollmg protein was the same for normal and failing myocardium. Since the protein content in DCM samples is lower, [ATP] must be proportionally lower. [PCr] and free [Cr] were obtained by assuming that 65% of the total creatine pool was phosphorylated. [ADP] was calculated from these values and the CKequilibrium (equation 2), using a pH of 7.1. The validity of these assumptions can be tested by comparing results for the PCr/ATP ratio determined in this way to results obtained by Neubauer and colleagues (1992) who studied normal healthy subjects and DCM patients using 31P NMR spectroscopy. Our calculated values of PCr/ATP of about 2 for normal subjects and about 1 for Class IV heart failure patients compare favorably to their values. In the second method, we estimate the relative changes in CK reaction velocity by multiplying V, by the total creatine pool (Tian et al., 1996). CK activity under saturating conditions determines the V,,, of the CK reaction, and the total creatine content sets the amount of PCr (as well as free creatine) that is available for the CK reaction. Therefore the content of creatine and the activity of CK combine to determine the maximal capacity of ATP resynthesis through the CKreaction. It is important to emphasize that the decreases in substrate and enzyme activity are not just additive, but multiplicative. This parameter estimates velocity by considering the two terms in the numerator of equation 1 that have been found to vary the most in disease states.

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Results in Table 1 show that the product of V,,, and total creatine pool was high for normal groups, intermediate for organ donors, and lowest for AS+CAD and DCM hearts. The result showing that CKreaction velocity is low in the DCM group was confirmed by calculating CK reaction velocity using the full rate equation (Nascimben et al., 1996). We and our colleagues have observed decreased creatine and PCr contents, decreasedV,,, and decreasedCK reaction velocity in several animal models of heart failure, each with a different etiology: the 18-month old failing spontaneously hypertensive rat heart (Bittl and Ingwall, 1987),the 10-monthold failing hamster heart (Nascimben et al., 1995;Tian et al., 1996),the rat heart eight weeks followingmyocardial infarction (Neubaueret al., 1995), and the furazolidone-treatedturkey poult c i a o et al., 1996).Consistentwith depressed cardiacperformance in congestive heart failure in man and in other animal models of failure, isovolumic contractile performance measured as the FWP in hearts isolated from severely failing animals was lower (typically 50-70% lower) than for hearts isolated from age-matched non-failing animals perfused under the same conditionsof load and coronary flow. Thus in concert with the decrease in contractile performance, the tissue content of PCr, the capacity for ATP synthesisvia the CK reaction measured as tissue enzyme activity (V),, and the rate of phosphoryl transfer directly measured as CK reaction velocity in vivo using magnetization transfer are all lower in the severely failing mammalian myocardium. From these measurements,we concludethat the CK phenotype in failingmyocardium is characterized by decreasesin both total CK activity (V,,) and the creatinepool and that these decreases combine to limit CK reaction velocity. These decreases are independent of species and etiology,strongly suggestingthat this is a property of failingmyocardium. The mechanisms underlying the apparent downregulationof CK synthesis(primarily M-CK) and loss of creatineremain to be defined. Little is known at present. The development of transgenic mice with CK genes ablated and upregulated should be particularly useful in this regard. Several observations in human and animal models of disease supportthe hypothesis that an increased adrenergicdrive can modulatethe content of creatine and CK in the heart. Cultures of adult rat cardiac muscle cells and isolated perfused rat hearts lose CK when norepinephrine is added to the culture media (Mann et al., 1992; Waldenstrom et al., 1994). These changes can be prevented with pretreatment of parasympathetic cholinergic agents. Turkeys with furazolidoneinduced cardiomyopathy also have a decreased creatine content and a decreased total CK activity that can be prevented following chronic oral administration of propranolol and atenolol (Chapados et al., 1992). Finally, human heart failure is characterized by activation of the sympatheticnervous system, and it has been found that the serum level of catecholaminescorrelates with the degree of heart failure (Cohn et al., 1984).

E. Creatine Kinase lsoenzyme Distribution Human myocardium contains MB, MM, and the mitochondrial isoenzymes; BB is present in trace amounts. The amount of each isoenzyme varies in disease.

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MB-CK

Figure 3 shows results for percent MB-CK obtained for each biopsy specimen analyzed. Data on the left side of the figure are for normal subjects and on the right side for the four disease states. We detected no MB-CK activity in the two biopsies obtained during surgery. For the 13 samples harvested within a few hours following demise, values ranged from “not detectable” to 3.6% except for one subject-a teenage female with 17.5%MB-CK. The overall average is 2.3% (calculatedby assigning a minimum-detectable value to the samples with no detectable activity). A threshold value could reasonably be set at less than 4%. A surprising finding is that myocardium of organ donors had high and highly variable percent MB-CK. The group mean was 19.8%,but a histogram (not shown but easily inferred from Figure 3 ) of the re-

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Figure 3. Distribution of % MB-CK determined electrophoreticallyusing homogenates of biopsy specimens obtained from normal subjects, organ donors, and patients with either aortic stenosis (AS), coronary artery disease (CAD), both aortic stenosis and coronary artery disease (AS CAD), or dilated cardiomyopathy (DCM). The crosses are the arithmetic means.

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sults from this group suggests the presence of two populations, one centered near 6% and another between 20-30%. Biopsy specimens from each of the four disease states StudiedcontainedhighpercentMB-CK: 15+2% for AS, 18+1% forCAD, 22fl% for AS+CAD, and 27+1% for DCM. Histograms of the AS and DCM groups (with n=l 1 and n=23) showed near-Gaussian distributions,with values for 10 out of 11 AS biopsies falling between 8-20% and 19 out of 23 DCM falling between 20-32%. DCM samples had more MB than any other group. In addition to assessing the relative distribution of the CK isoenzymes, one must consider whether the increase is due to an absolute increase in B-CK protein or whether the percent is higher because MM-CK and sMtCK decrease. There are two ways to assess this. Firstly, using an RIA specific to B-CK, we measured the amount of B-CK in all samples. In spite of optimizing the assay for low B-CK, no protein could be detected in 10 of the 15 normal samples. Of the remaining five samples, ng B-CWmg protein ranged from 0.05 to 0.25 for the four samples with less than 4% MB-CK and was 1.03 for the 17-year-old female with 17.5% MB-CK. Although highly variable, each of the other groups (organ donors and the four disease states) averaged about 1 ng B-CWmg protein. The range for all samples in these five groups encompassed an order of magnitude, 0.19 to 1.9 ng protein. If one assumes that only the B polypeptide is detected by the assay, these amounts should be multiplied by two to yield amount of MB-CK. Secondly, because the specific activities of the CK isoenzymes in human tissues are likely to be similar (as they are in the dog; Basson et al., 1985), multiplying the percent distribution by the total CK activity yields the activity of that isoenzyme. Results of this estimate also show that the mean value for MB-CK in normal human myocardium is low and that diseased myocardium contains an order of magnitude more than MB-CK. Thus, by both activity estimates and RIA of protein amounts, we conclude that the absolute amount of MB-CK is high in organ donors and in each of the disease states studied and very low in normal subjects. Our initial results showing that normal human myocardium contained little or no detectable MB-CK was met by some with skepticism (Roberts, 1985). However, these results have been independently confirmed by others (Sylven et al., 1991; Sylven et al., 1993; van der Laarse et al., 1992). Indeed, Sylven and colleagues (1991) analyzed 10 regions of normal, freshly explanted human hearts and found uniformly low MB-CK activity throughout the left and right ventricles. The concern raised by Roberts (1985) that the biopsy specimens from normal subjects had lost MB-CK is highly unlikely based on the biochemical profile of those samples: The samples from normal subjects had the highest CK activity, the highest creatine content, and the highest protein content. Thus both low and high molecular weight molecules were well preserved. These points were made by Ingwall and colleagues (1985). The observations that normal human myocardium contains low amounts of MB-CK do not conflict with everyday clinical observations that MB-CK is high in serum of patients with heart disease, especially CAD. It is rare that acute myocardial infarction would occur in a healthy heart. Our observations would explain the

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absence of elevated MB-CK in a patient with acute myocardial infarction not caused by CAD. An example would be a young patient with acute myocardial infarction due to spasm precipitated by drug overdose. Sylven and colleagues (1993) have also found an increase in percent MB (along with decrease in percent MM-CK) in failing human myocardium. An increase in the content of the B-CK (usually seen as an increase in MB-CK) has been described in the myocardium of several animal models of cardiac hypertrophy and failure (see Ingwall, 1993). An important observation is the rapidity with which B-CK can be upregulated. In a “Letter to the Editor” to the New England Journal of Medicine regarding findings of Ingwall and colleagues (1983, Kaye and colleagues (1986) pointed out that mRNA for B-CK and B-CK protein levels increased within an hour of stimulation in estrogen-stimulated rat uterus (Reiss and Kaye, 1981). Using a canine model of CAD, Mehta and colleagues (1988) showed a 40% decrease in M-CK mRNA within hours after coronary artery occlusion. Sharkey and colleagues (1989) reported a rapid increase of MB-CK activity in the myocardium of dogs after coronary occlusion. A rapid increase in expression of B-CK has also been reported in bone cells in response to parathyroid hormone and prostaglandin E, (Somjen et al., 1985). Our new data for organ donors suggesting a large and variable induction of B-CK synthesis within hours to days on inotropic support are in full accord with these observations. Myocardial differentiation is characterized by a progressive increase of PCr (and creatine) levels and preferential synthesis of M-CK and Mito-CK coupled with a relative decrease in B-CK. With the development of cardiac hypertrophy and evolution to failure, an opposite pattern takes place, with adecrease in the size of the creatine pool, decrease in the expression of M-CK, and usually an increase in the expression of B-CK. It is still unclear whether such an increase of B-CK, and therefore MB-CK isoenzyme, is part of an a general re-expression of immature-type proteins such as is seen with lactate dehydrogenase (Fox and Reed, 1969; Bishop and Altschuld, 1970), myosin (Lompre et al., 1979), troponin T (Anderson et al., 1991), and ANF (Takahashi et al., 1992), or whether it reflects a specific adaptive change to utilize PCr more effectively. If it is the latter, this would be especially important in heart failure where the size of the creatine pool is decreased. The lower K, of the MB isoform compared to MM implies a higher affinity for the substrates of the reaction (Wong and Smith, 1976; Szasz and Gruber, 1978) and supports the hypothesis of an adaptive change. The observation of increased B-CK amount and activity in so many different tissue types and in response to so many diverse pathophysiological stimuli strongly suggests that B-CK is rapidly upregulated in response to stress. The variability of B-CK content in human myocardium and its rapid modulation further suggest that B-CK expression may be mediated by one of the local or systemic neurohonnonal factors that are activated during stress conditions. This remains to be fully tested.

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MM-CK and sMtCK

Wh l e the increase in MB-CK observed in failing myocardium is large, it is not sufficient to compensate for the decrease in MM-CK that occurs. Loss of MM-CK activity is the dominant contributor to the observed decrease in total CK. At present, it is not known whether the cytosolic pools and the bound pools are equally depleted. In some (Bittl and Ingwall, 1987; Khuchua et al., 1989) but not all (Tian et al., 1996) animal models of DCM, sMtCK decreases in addition to MM-CK. sMtCK activity decreases in ventricular myocardium of failing human hearts compared to normal subjects. This decrease is not a result of loss in mitocondrial mass because citrate synthase activity, another mitochondrial protein, is unchanged.

F.

Clinical Implications

The results summarized here using human and animal myocardium suggest that the failing heart is “energy starved” (Katz, 1991) at least with respect with its capacity to resynthesize ATP via the CK system. Based on experiments using animal models showing a relationship between energy reserve via the CK system and contractile reserve of both heart and skeletal muscles, it seems likely that the decreased energy reserve of the human failing heart has a similar functional correlate. Decreased ability to rapidly resynthesize ATP would be particularly important under conditions of acute increases in workload. Thus, decreased energy reserve may explain, at least in part, the decreased tolerance to exercise of failing patients.

ACKNOWLEDGMENTS I wish to acknowledge my many colleagues who have contributed to this work over the past 15 years: Paul D. Allen, John A. Bittl, Jan Friedrich, Julie K. Fetters,Judith K. Gwathmey, Baron Hamman, William E. Jacobus, Martha F. Kramer, Ronglih Liao, Luigino Nascimben, Stefan Neubauer, Paulo Pauletto, Ilana Reis, and Rong Tian. In particular, I thank Luigino Nascimben and Rong Tian, on whose work this chapter is focused.

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