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(Circulation. 1996;94:1894-1901.)
© 1996 American Heart Association, Inc.

Articles

Creatine Kinase System in Failing and Nonfailing Human Myocardium

Luigino Nascimben, MD; Joanne S. Ingwall, PhD; Paolo Pauletto, MD; Jan Friedrich, DPhil; Judith K. Gwathmey, VMD, PhD; Valdur Saks, PhD; Achille Cesare Pessina, MD, PhD; P.D. Allen, MD, PhD

the NMR Laboratory for Physiological Chemistry, Cardiovascular Division, Department of Medicine (L.N., J.S.I., J.F.) and Department of Anesthesia (P.D.A.), Brigham and Women's Hospital and Harvard Medical School, Boston, Mass; Istituto di Medicina Clinica, Universita di Padova (Italy) (L.N., P.P., A.C.P.); Cardiovascular Disease and Muscle Research Laboratories, Department of Medicine, Beth Israel Hospital and Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Mass (J.K.G.); and Laboratory of Bioenergetics, Research Center for Cardiology, Moscow, Russia (V.S.).

Correspondence to Luigino Nascimben, MD, NMR Laboratory for Physiological Chemistry, Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, 221 Longwood Ave, Boston, MA 02115. E-mail luigin@bustoff.bwh.harvard.edu.

	Abstract

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Background The creatine kinase (CK) reaction is important for rapid resynthesis of ATP when the heart increases its work. Studies defining the CK system in human failing and nonfailing myocardium are limited and in conflict. To resolve this conflict, we measured the activities of CK and its isoenzymes and the contents of creatine and CK-B in homogenates of human myocardium.

Methods and Results Myocardium was sampled from 23 subjects who underwent heart transplant, 36 subjects maintained in an intensive care unit before heart harvesting, 13 accident victims, and 2 patients undergoing heart surgery. Since the characteristics of myocardium of potential organ donors differed from those of myocardium of accident victims, data are presented for three groups: failing, donor, and control. CK activity was 7.7±1.9 and 6.0±1.4 IU/mg protein in left (LV) and right (RV) ventricles of failing, 9.4±2.5 and 10.7±2 IU/mg protein in LV and RV of donor, and 11.6±2.4 IU/mg protein in LV of control hearts. CK-MM and the mitochondrial isoenzyme activities were lower in failing and donor LV, and CK-MB activity and CK-B content were higher in failing and donor hearts. Creatine contents were 64±25 and 56±18.6 nmol/mg protein in LV and RV of failing, 96±30 and 110±24 nmol/mg protein in LV and RV of donor, and 131±28 nmol/mg protein in LV of control hearts.

Conclusions In failing and nonfailing donor human myocardium, there is a combined decrease of CK activity and creatine that may impair the ability to deliver ATP to energy-consuming systems.

Key Words: heart failure • cardiomyopathy • creatine kinase • creatine • transplantation

	Introduction

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Patients with DCM exhibit progressive heart failure with extensive dilation and decreased systolic function. An early clinical sign of heart failure is a diminished tolerance for exercise. The cause and pathogenesis of heart failure have not yet been clearly defined, although many hypotheses have been proposed. Among them, the hypothesis that failing myocardium is energy depleted is gaining renewed interest.^{1 2}

In hearts of several animal models of heart failure, it has been shown that the maximal velocity of the CK reaction is decreased, the relative distribution of CK isoenzymes is changed, and the contents of the substrates and products of the CK reaction, namely ATP and PCr, are decreased.^{3 4 5 6 7} Isolated perfused hearts of these animals perform less cardiac work. The importance of CK for normal contractile performance of muscle has been demonstrated in experiments designed to alter the CK reaction by either inhibiting CK activity or decreasing the concentration of creatine. The ability of hearts to increase their contractile performance is reduced when myocardial creatine is replaced with poorly hydrolyzable creatine analogues⁸ or when CK activity is acutely inhibited with sulfhydryl group modifiers.⁹ Langendorff-perfused failing hearts of cardiomyopathic hamsters and turkeys have been shown to have lower total creatine content and CK activity and to perform less cardiac work.^{5 6} In these failing hearts, both the total CK activity measured in vitro and the CK reaction velocity directly measured in vivo by ³¹P magnetization transfer were decreased. Experiments using animal models have also demonstrated that CK activity and creatine content combine to determine the capacity for resynthesizing ATP via the CK reaction and that the integrity of this pathway for ATP resynthesis is required for the recruitment of the functional reserve of heart.^{6 7 8 9} Finally, in support of these observations, it has been shown that skeletal muscle from mice that are homozygous for a "knockout" of the CK-M gene is unable to perform burst work.¹⁰

At present, characterizations of the CK system in human failing and nonfailing myocardium are incomplete and not in full agreement. Decreased total CK activity in subjects with DCM has been reported by our group in abstract form¹¹ and by Sylven et al.¹² Bristow et al,¹³ in contrast, have suggested that the total CK activities in donor and failing hearts were not different. Several groups using in vivo ³¹P NMR spectroscopy have demonstrated that the PCr/ATP ratio is lower in failing human myocardium,^{14 15 16} but PCr and ATP contents have not been determined. The creatine content of failing human myocardium has not been reported. Moreover, variables such as age, disease progression, drug therapy, regional myocardial heterogeneity, and overall heterogeneity of the population may complicate the interpretation of data for both normal and failing myocardium.

The objective of this study was to define and compare the CK systems in subjects who had heart failure due to end-stage DCM with those in subjects who had normal heart function. Subjects with normal heart function included both organ donors and accident victims. We measured the creatine content, the total CK activity, and the CK isoenzyme distribution in specimens of LV myocardium obtained at the time of surgery or autopsy. In a subset of failing and organ donor subjects, we also tested for any chamber differences. By making conservative assumptions about the fraction of the creatine pool that is phosphorylated combined with literature values for changes in ATP content in DCM hearts, we also estimated CK reaction velocity in failing and nonfailing human myocardium.

	Methods

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Subjects
Samples of human myocardium were obtained from the LV of subjects undergoing open-heart surgery for repair of mitral stenosis or atrial septal defect who had normal ventricular function (n=2), accident victims at autopsy within 3 hours of death (n=13), donor subjects at the time of organ harvesting or transplantation (n=36), and patients with DCM who underwent heart transplantation (n=23).

The subjects were divided into three groups. The control group (n=15) included the normal surgical and autopsy samples (mean age, 29.6±10.3 years; range, 17 to 53 years). Results from 9 of these subjects have been reported previously.¹⁷ The heart donor group included subjects who had no known heart disease but who had been maintained on life support treatment for 24 to 120 hours before heart harvesting (mean age, 34.4±15.9 years; range, 16 to 65 years). The heart failure group consisted of patients with DCM whose hearts were sampled at the time of cardiac transplantation (mean age, 37.2±9.0 years; range, 15 to 54 years). There were no age differences among groups. All but 2 subjects in the donor group and 1 in each of the other two groups were men.

We also collected samples from the free walls of the LA, RA, and RV from a subset of the subjects in the donor (n=6) and failure (n=17) groups for an analysis of possible regional differences.

Immediately after removal, all myocardial samples were frozen in liquid nitrogen and stored frozen until use.

Biochemical Analysis
Portions of the myocardial biopsy specimens (5 to 20 mg) were cut from frozen samples, weighed, and analyzed as previously described^{5 6 17} for noncollagen protein, CK-B content, total creatine content (PCr plus free creatine), total CK and CS activities, and the relative distribution of the CK isoenzymes (BB, MB, MM, and mitochondrial). Since the specific activities of the CK isoenzymes are similar,¹⁸ relative activities can be estimated by multiplying the relative concentrations by total CK activity.

The study design was independently approved before the study began by the Ethics Committee for Organ Transplant, University of Padova Hospital, and the Committee for the Protection of Human Subjects, Brigham and Women's Hospital, Boston, Mass.

Data Analysis
Normalizing substrates and enzyme activities by Lowry protein content minimizes the contributions of edema, fibrosis, and any change of interstitial water that could have confounding effects on enzyme and substrate concentrations. This is because extracellular proteins contain relatively low amounts of aromatic amino acids, the target for the Lowry assay. Accordingly, in this report, enzyme activities and creatine and CK-B contents were normalized to Lowry protein to better reflect the mass of viable myocytes.

We estimated the forward velocity (V_for) of the CK reaction in myocardium in vivo in two ways. Both methods are based on the rate equation developed to determine the velocity of the CK reaction for CK-MM in solution.¹⁹ The rate equation is

(E1)

where V_max is the total CK activity measured under saturating conditions; D, k_a, and k_ib are kinetic constants calculated from values from References 19 and 20; and ADP concentration is calculated from the CK equilibrium expression

(E2)

For the first method, we used the rate equation directly and entered the values for V_max 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 mL/g wet wt²¹ and used the ratio of mg protein per wet weight that we measured for each group. V_max at 37°C was obtained by multiplying our measured values of CK activity measured at 30°C by 1.8, a value we determined experimentally using pure CK-MM. We used [ATP] obtained by ³¹P NMR spectroscopy of healthy volunteers²² for the control group. Since Regitz and Fleck²³ reported that ATP content expressed as nmol/mg protein was the same for control and failing myocardium, we scaled the [ATP] for differences in noncollagen protein content of donor and failing groups. [PCr] and [free creatine] were obtained under the assumption that 65% of the total creatine pool was phosphorylated. [ADP] was calculated from these values and the CK equilibrium equation (Equation 2), with a pH of 7.11.

The validity of these assumptions was tested by comparison of results for the PCr/ATP ratio determined in this way with results obtained by Neubauer et al,¹⁶ who studied normal, healthy subjects and DCM patients by ³¹P NMR spectroscopy. Our calculated values of PCr/ATP of 2.0 for control and 1.0 for failing groups compare favorably to values of 2.0±0.4 for healthy volunteers and 1.4±0.5 for DCM patients classified as New York Heart Association class III to IV. It is noteworthy that three of the four patients in class III to IV of that study had a PCr/ATP ratio of 1.0, similar to the patients in the failing group (all class IV) described here.

In the second method, we estimated the relative changes in CK reaction velocity by considering only the numerator of the rate equation.⁷ By multiplying V_max by the total creatine pool, we obtain an estimate of the maximal capacity to resynthesize ATP via the CK reaction under conditions in which substrate availability is not limiting. This estimate is based on our observations that, of the parameters in the numerator of Equation 1, V_max and creatine (which is proportional to PCr) vary more than the adenylate pool (which is proportional to ADP) in DCM.⁵

Data are expressed as mean±SD. Comparisons were made by use of statistical software (Systat 5.2.1, Systat Inc, and Statview, TM 512+, Brainpower Inc). To compare results of the tests among three groups of ventricles, one-way factorial ANOVA and Tukey's highly significant difference post hoc test for multiple comparisons were used. In the regional analysis, for which samples of only two groups were available, a two-tailed unpaired Student's t test was applied to compare sections of two different groups and a two-tailed paired Student's t test to compare two sections of the same heart. A value of P=.05 was considered statistically significant. To correlate CK-MM activity with creatine content and CK-MB activity with CK-B content, we used linear regression analysis.

	Results

Top Abstract Introduction Methods Results Discussion References

 
CK Activity in the LV of Failing and Nonfailing Hearts
Values for total CK activity, CK isoenzyme distribution, and total creatine content of the LV for the three groups are summarized in Table 1. Individual data are shown in Figs 1 and 2. The content of noncollagen protein per milligram wet weight of control myocardium (0.16±0.02) was higher than that of donor myocardium (0.13±0.03, P<.001) and failing myocardium (0.12±0.02, P<.001). Because we normalized results to protein content, which was higher in the control group, we minimized any differences that could have been attributed to edema and fibrosis.

View this table: [in this window] [in a new window]   Table 1. CK System in LVs

View larger version (15K): [in this window] [in a new window]   Figure 1. Total creatine (A) and total CK activities (B) in myocardium of LVs of control, donor, and failing hearts. represents subjects with heart failure; , heart donors; , accident victims; and , two subjects who underwent heart surgery. For the statistical comparisons, these two data points were added to the accident victims to form the control group. I bars represent mean±SD. P values indicate probability that the group means are not different.

Myocardium from failing hearts contained less creatine than myocardium of control hearts (-50%, P<.001) and donor hearts (-26%, P<.001). It also contained less total CK activity than myocardium from control hearts (-34%, P<.0005) and from donor hearts (-18%, P<.005). Therefore, failing myocardium contains less total creatine and less total CK activity than both groups of nonfailing hearts.

Myocardium from donor hearts contained less total creatine (-26%, P<.005) and less total CK (-19%, P<.05) activity than myocardium from control hearts.

Table 2 shows estimates for the cytosolic concentrations of the substrates of the CK reaction and for the CK reaction velocity. The velocity of the CK reaction estimated from the rate equation was decreased in myocardium of failing hearts compared with both control and donor hearts (-56%, P<.0005, and -26%, P<.05, respectively) and was also decreased in donor hearts compared with controls (-40%, P<.0005).

View this table: [in this window] [in a new window]   Table 2. Cytosolic Concentrations of CK and Its Substrates and Estimates for the CK Reaction Velocity

CK Isoenzyme Distribution in the LV
The CK isoenzyme distribution, whether expressed as IU/mg protein or as percentage of the total activity, differed in myocardium of both failing and donor hearts compared with controls. Compared with the control group, mito-CK activity was decreased 45% (P<.001) in both failing and donor groups, CK-MM activity was 50% less in the failure group (P<.0005) and 32% less in the donor group (P<.0005), and CK-MB activity was increased about sevenfold in both the failure and donor groups (P<.0005). Fig 2 shows that the lower total CK activity of failing and donor myocardium is primarily a result of a decrease in CK-MM activity and, to a lesser extent, of a decrease in mito-CK activity. The increase in CK-MB in the myocardium of the LVs in both of these groups was insufficient to maintain normal total CK activity. The BB isoenzyme was detected in only some samples: 1 normal subject (3.5% of his total CK), 14 (of 36) donors (<2% of total CK on average), and 2 DCM subjects (<1% of total CK on average).

View larger version (11K): [in this window] [in a new window]   Figure 2. The distribution of CK isoenzymes CK-MB (), CK-MM (), and mito-CK () in myocardium from LVs of control, organ donor, and failing subjects. indicates the two subjects who underwent heart surgery. I bars represent mean±SD.

The percentage of the CK-MB isoenzyme activity was higher in the failure group (27.3%) than in the control (2.4%, P<.0005) and donor groups (19.7%, P<.0005). This is only partially due to the decrease of CK-MM and mito-CK isoenzymes. When CK-B was quantified directly by use of an antibody specific to CK-B, CK-B content per milligram protein was 10 times higher in the failure group (P<.0005) and 12 times higher in the donor group (P<.0005) than in the control group. Fig 3A shows the relationship between CK-B content per milligram protein and CK-MB activity in the ventricles. There is a strong correlation between MB activity and CK-B content in the control group (y=-0.01+0.43x, r=.98, P<.001) and in the failure group (y=-0.02+0.48x, r=.87, P<.001). Interestingly, there was a weaker correlation of these two variables in the donor group (y=0.36+0.44x, r=.70, P<.05). These relationships provide information about the specific activity of the B-containing CK isoenzymes in the three groups and also about the fraction of the CK-MB that is active. The specific activity of CK-MB (the inverse of the slopes) in all groups is the same, making it likely that the same protein accumulated regardless of pathology. For the control and failure groups, the y intercept was not different from zero, showing that all of the active B-containing enzyme was detected by the antibody. This was not the case in the donor group. For this group, antibody detected inactive B monomer.

View larger version (18K): [in this window] [in a new window]   Figure 3. Plot of CK-MB isoenzyme (MB-CK) against CK-B protein in LVs (A) and left and right atria (B) with regression lines. A, indicates control (y=-0.009+0.43x; r=.97); , organ donor (y=0.36+0.44x; r=.70); and , heart failure (y=-0.02+0.48x, r=.81). B, indicates organ donor (y=0.004+0.53x; r=.92); , heart failure (y=0.6+0.55x; r=.93). The shift of the regression lines of the organ donor group in both atria and ventricles indicates that some of the CK-B monomer detected by the antibody is inactive.

Fig 4 shows the relationship between total creatine content (the substrate) and CK-MM activity (the dominant enzyme) by use of the data points from all the regions of the heart and from all three groups. There is a significant relationship between CK-MM activity and the total creatine content (y=1.9+0.05x, r=.80, P<.0001). There was a weaker correlation between mito-CK activity and total creatine content (y=0.21+0.006x, r=.44, P<.0001) and no correlation between total creatine content and CK-MB.

View larger version (16K): [in this window] [in a new window]   Figure 4. Graph showing the relation between the content of total creatine and the activity of the MM isoenzyme of creatine kinase (CK-MM). The three groups include all the samples from left ventricles of the control group and from the atria and the ventricles of the failing and organ donor groups. indicates control (n=15); , organ donor (n=54); and , heart failure (n=74). Regression line: y=1.9+0.05x; r=.80.

Regional Characteristics of CK System in Failing and Donor Hearts
Table 3 presents the results of the biochemical assays for RV, LA, and RA in subgroups of the donor and failing hearts. RV, LA, and RA from the donor and failure groups contained a similar amount of noncollagen protein per milligram wet weight, which was not different from what was found in their LVs.

View this table: [in this window] [in a new window]   Table 3. CK System in Myocardium of RVs and Atria of Donor and Failing Groups

As was found in the LV, the total creatine content and total CK activity in the RV of the failure group were lower (-49% and -39%, respectively) than in the RV of the donor group. Likewise, CK-MM and mito-CK activities were decreased (-50% and -70%, respectively) in the RV of the failure group compared with the donor group.

Total creatine content and total CK activity in LA and RA were lower than in their respective ventricles for both groups (compare Table 3 and Table 1). There were no differences between LA and RA of either group or between atria in donor and failing groups. CK-MM and CK-MB activities were also not different between the two groups, but mito-CK activity was 65% lower in the failure group than in the donor group. Myocardium from the RV and from both atria of donor hearts contained about 50% and 40% more CK-B protein than myocardium from failing hearts, respectively. Fig 3B shows the relationship between the content of CK-B protein and CK-MB enzyme activity for all atrial samples in the donor and failure groups. As was seen in the LV, the higher CK-B content in the donor hearts was not accompanied by a comparable increase in CK-MB activity.

Mitochondrial Enzyme Activities by Region and by Group
Changes of mito-CK could reflect either a specific reduction in the amount of the mito-CK activity in otherwise normal mitochondria or a nonspecific decrease in the fractional cell volume of mitochondria. To distinguish between these possibilities, we also measured the activity of CS, which is proportional to mitochondrial cell volume, and calculated the ratio of mito-CK to CS activities for the LV of all three groups and the RV, LA, and RA of the donor and the failing groups. Values for the LV were the same for all three groups (0.49±0.12, 0.45±0.11, and 0.48±0.09 IU/mg protein, P=NS) and were indistinguishable from values for RV of the donor and failing groups (0.47±0.05 and 0.44±0.06 IU/mg protein, respectively). Atria of donor hearts contained the same CS activity (0.48±0.06 IU/mg protein for both LA and RA) as the ventricles. However, CS activity in the LA (0.36±0.09 IU/mg protein) and RA (0.35±0.09 IU/mg protein) of failing hearts was lower than in atria of the donor group (P<.005) and in ventricles of the failing group (P<.005).

Taking into account possible differences in the fractional cell volume of mitochondria as estimated by CS activity, the calculated mito-CK/CS ratio was lower in ventricles of failing (1.4±0.6) and donor (1.4±0.7) myocardium compared with controls (2.2±1.1, P<.05) (Table 1). This shows that the relative concentration of mito-CK on the mitochondria is lower in failing and donor hearts. There were no regional differences in this ratio for either the donor or the failing group (Table 3).

	Discussion

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Total Creatine Content and Total CK Activity in Failing Hearts
In this study, we found that the creatine pool is decreased in all four chambers of the failing human myocardium. Ventricles but not atria from the failing hearts also have a lower total CK activity. The consequence of a decreased substrate pool coupled with a decreased enzyme activity is a reduction in the capacity of the CK system to synthesize ATP when there is an increased workload. We suggest that the decreased capacity to resynthesize ATP in the failing heart be described as decreased energy reserve. It is important to emphasize that, on the basis of known reaction kinetics, the effect of decreases in both substrate and enzyme activities on the capacity of the CK reaction is not just additive but rather multiplicative. The product of V_max and total creatine pool was highest for control and lowest for failing hearts. This result was confirmed by calculating CK reaction velocity from the rate equation.

Our data showing decreased total creatine in failing hearts are supported by ³¹P NMR spectroscopy findings that the in vivo PCr/ATP ratio is 25% lower in failing human hearts^{14 15 16} than in healthy subjects. Because CK functions to maintain a constant PCr/creatine ratio, we would expect that our finding of a 50% decrease in creatine would be accompanied by a 50% decrease in PCr concentration. 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 human²⁴ and in animal models,^{5 6} and the magnitude of the decrease is sufficient to reconcile these results. The results presented in the present study (Table 2) are in accord with these observations.

Our results showing decreased total CK activity are in good agreement with the reports of Sylven et al¹² comparing hearts from patients with DCM and heart donors. They found that CK activity in the LV of failing hearts was 44% lower, whereas we found a 34% decrease. Bristow et al¹³ reported that CK activity in the LV from patients with DCM was 25% lower than for heart donors. They attributed this difference to a 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, our results in subjects >14 years of age show that the decrease seen in DCM is primarily due to pathology.

The importance of the decreases in the creatine pool and in CK activity on myocardial energetics can be explained by analysis of the role of the CK system in excitable tissues. Because ATP resynthesis via the CK reaction is an order of magnitude faster than ATP synthesis via oxidative phosphorylation,²⁰ the CK system guarantees constant ATP levels. This is especially important for the myocardium, in which ATP turnover rates are high even at baseline and can be rapidly increased more than fivefold under conditions of increased workload, such as occurs with exercise. Because the CK activity under saturating conditions determines the V_max 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, they combine to determine the maximal capacity of ATP resynthesis through the CK reaction. Our results suggest that the capacity for ATP resynthesis via the CK system is 83% lower for failing myocardium than for control myocardium (Table 2). They also show that the decreases in creatine content and CK activity (in particular CK-MM) are coordinate (Fig 4).

Changes in the CK Activity and Creatine Content in Donor Hearts
A surprising finding of this report is that myocardium of heart donors has a lower creatine pool and lower CK activity than does myocardium obtained from the LVs of healthy accident victims. We cannot fully explain this finding, but it seems possible that the clinical conditions in which the subjects were maintained before heart harvesting had an effect on both the expression of CK isoenzymes in the myocardium and the size of the creatine pool. These subjects were mechanically ventilated and maintained under intensive life support treatment while in a coma for 24 to 120 hours. Subjects maintained on life support after a traumatic event have increased production of stress hormones and receive multiple drugs before organ harvest. Several observations in human and animal models of disease support the hypothesis that an increased adrenergic drive can modulate the contents of creatine and CK in the heart. Cultures of adult rat cardiac muscle cells²⁵ and isolated perfused rat hearts²⁶ lose CK when norepinephrine is supplied. Interestingly, these changes can be prevented with pretreatment with parasympathetic cholinergic agents.²⁶ Turkeys with furazolidone-induced cardiomyopathy also have a decreased creatine content and a decreased total CK activity, which are prevented by long-term oral administration of propranolol and atenolol.²⁷ Most importantly, human heart failure is characterized by activation of the sympathetic nervous system, and it has been found that the serum level of catecholamines correlates with the degree of heart failure.²⁸

CK Isoenzyme Distribution
Among the many physiological and metabolic changes associated with myocardial development are progressive increases of PCr (and creatine) content, increased synthesis of CK-M and mito-CK, and a relative decrease in CK-B. With the development of cardiac hypertrophy and evolution to failure, an opposite pattern takes place, with a decrease in the size of the creatine pool and a decrease in the expression of CK-M and usually an increase in the expression of CK-B. It is still unclear whether the increase of CK-B is part of a general reexpression of immature-type proteins such as is seen with troponin T²⁹ or whether it reflects a specific adaptive change to utilize PCr more effectively. If the latter, this would be especially important in DCM, in which the size of the creatine pool is decreased. The lower K_m of the MB isoenzyme compared with CK-MM indicates a higher affinity for the substrates of the reaction³⁰ and supports the hypothesis of an adaptive change.

We previously described an increase in the content of CK-B (usually seen as an increase in CK-MB) in the myocardium of several animal models of cardiac hypertrophy and failure² and in human myocardium of subjects with LV hypertrophy, coronary artery disease, and mitral regurgitation.¹⁷ This observation has been confirmed by others.^{12 31 32 33 34} The data presented here support the hypothesis that the synthesis of CK-B also increases in the failing human myocardium. Our data also show large variability of the content of CK-MB in all groups (ranges are 0% to 16%, 5.2% to 31%, and 13.5% to 37.1% for control, donor, and failure groups, respectively).

The increase of CK-B and the decrease of CK-M and mito-CK in donor hearts suggest the existence of rapid mechanisms for regulation of synthesis of CK isoenzymes. A rapid increase in expression of CK-B has also been reported in the dog heart upon coronary artery occlusion³⁴ and in the rat uterus in response to estrogens.³⁵ CK-M mRNA decreased 40% in myocardium of dogs and rabbits³³ within 2 hours after either coronary ligation or heat stress.

The variability of CK-B content in human myocardium and its rapid modulation suggest that CK-B expression may be mediated by one of the local or systemic neurohormonal factors that are activated during stress conditions. In support of this hypothesis, our data show that in both atria and ventricles of donor hearts, the amount of CK-B exceeds the amount predicted by the activity of CK-MB and CK-BB. Since it is unlikely that the CK-B antibody cross-reacts with some other protein only in the donor group, it is likely that these hearts synthesize an excess of CK-B and that the assembly into an active CK enzyme is not complete.

Mito-CK has been reported to be decreased³ or unchanged⁵ in animal models of DCM. We found decreased mito-CK activity in LV of failing and donor hearts compared with controls. This decrease is not a result of loss in mitochondrial mass, because the activity of CS, another mitochondrial protein, is the same in all groups. In contrast to these results for ventricular tissue, the mito-CK/CS ratio is similar in failing and donor atria, suggesting that the fractional cell volume of mitochondria of failing atria is decreased.

Clinical Implications
The results presented here from a study of human myocardium suggest that the failing heart is "energy starved,"¹ at least with respect to its capacity to resynthesize ATP via the CK system. On the basis of experiments using animal models showing a relationship between energy reserve via the CK system and contractile reserve of both heart and skeletal muscles,^{6 7 8 9} 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 ability of DCM patients to exercise.

Our data also suggest that the information collected from the myocardium of organ donors, at least as it relates to the CK system, may not be representative of a population of healthy subjects. This conclusion may also apply to other biochemical systems measured in hearts of organ donors.

We do not know whether the loss of either CK activity or creatine content in the failing or donor hearts is an irreversible process. Moreover, it is not known whether the loss of CK and creatine has an impact on the outcome of the transplant or the functional capacity of the recipient patients. We can speculate, however, that any pharmacological or other intervention that maintains the capacity of the CK system in the organ donors by decreasing the loss of creatine content and CK activity would be beneficial.

Study Limitations
First, clinical characterization of the organ donors and accident victims is incomplete with respect to hemodynamic parameters and functional capacity. This limits our ability to relate biochemical and functional parameters. Second, it is possible that drugs that were administered to either the failing or donor subjects could have modulated the expression of the CK isoenzymes. Our study groups are too small to identify any specific interactions. Finally, the mechanisms that determine the biochemical remodeling of the CK system in failing and donor hearts remain to be elucidated.

	Selected Abbreviations and Acronyms

 

CK = creatine kinase CS = citrate synthase DCM = dilated cardiomyopathy LA = left atrium LV = left ventricular, left ventricle mito-CK = mitochondrial CK PCr = phosphocreatine RA = right atrium RV = right ventricle

	Acknowledgments

 
This study was supported in part by National Institutes of Health grant HL-52320. Dr Nascimben was a recipient of a fellowship award from Merck Sharp & Dohme Italy Spa. The authors thank Ilana Reis, MSc, and Martha Kramer, PhD, for technical assistance; the surgeons and the pathologists of the Cardiothoracic Departments of Brigham and Women's Hospital, Boston, Mass; Padova University Hospital, Italy; and NDRI for their cooperation during the collection of the samples; and Helena Laboratories, Beaumont, Tex, for technical assistance and for the use of a Cardio-Rep Instrument for automated electrophoretic analysis.

	Footnotes

 
Presented in part at the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 8-11, 1993.

Received January 18, 1996; revision received April 18, 1996; accepted May 1, 1996.

	References

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