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European Journal of Heart Failure 2001 3(1):1-5; doi:10.1016/S1388-9842(00)00085-4
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© 2001 European Society of Cardiology

Alterations in myocardial creatinine kinase (CK) and lactate dehydrogenase (LDH) isoenzyme-distribution in a model of left ventricular dysfunction

Frank Mudersa,*, Stefan Neubauerb, Andreas Luchnera, Sabine Fredersdorfa, Guntram Ickensteina, Günter A.J. Rieggera, Michael Hornb and Dietmar Elsnera

a Klinik und Poliklinik für Innere Medizin II, Universitätsklinikum 93042 Regensburg, Germany
b Medizinische Universitätsklinik Würzburg Germany

* Corresponding author. Tel.: +49-941-944-7256; fax: +49-941-944-7213.


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
The purpose of the current study was to evaluate myocardial creatinine kinase (CK) and lactate dehydrogenase (LDH) systems in a model of epinephrine-induced cardiomyopathy in rabbits. Eight rabbits received four repetitive epinephrine infusions (300 mg/kg/60 min, i.v.) in 12-day intervals and eight untreated rabbits served as controls (CTRL). Echocardiography demonstrated a significant deterioration of LV function as well as increased LV-diameter and -mass index in catecholamine-induced cardiomyopathy. Histological examination revealed that repetitive catecholamine infusion resulted in LV fibrous areas with collagenous content and an increase in myocyte width (16.9 ± 0.8 µm vs. CTRL 12.9 ± 0.9; P < 0.05). LV dysfunction was associated with a decreased total LV lactate dehydrogenase activity (LDH; 0.43 ± 0.03 IU/mg protein vs. CTRL 0.52 ± 0.04; P < 0.05) whereas total creatinine kinase activity was unchanged (CK; 7.30 ± 0.63 IU/mg protein vs. CTRL 9.20 ± 0.49, n.s.). Furthermore, myocardial LDH isoenzymes were shifted with a decrease in LDH1 and an increase in LDH2 and LDH3 (LDH1: 84.90 ± 2.60% vs. CTRL 94.50 ± 0.40; LDH2: 7.30 ± 1.20% vs. 1.50 ± 0.13; LDH3: 5.40 ± 0.90% vs. 3.20 ± 0.25; all P < 0.05). Foetal B-CK isoenzymes were significantly increased (CK-MB 5.30 ± 0.66 vs. 2.20 ± 0.35%; P < 0.05). The current study demonstrates changes in cardiac energy metabolism including an impaired LDH activity with a shift towards anaerobic isoenzymes as well as a more efficient CK system in a model of catecholamine-induced LV dysfunction.

Key Words: Myocardium • Creatinine kinase • Lactate dehydrogenase • Isoenzymes • LV dysfunction

Received October 4, 1999; Revised April 20, 2000; Accepted April 28, 2000


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Excessive doses of catecholamines produce diffuse myocardial destruction with myocyte loss and necrosis as well as extensive fibrosis [1]. Such changes may be also noticed in patients treated with high doses of catecholamines or with pheochromocytoma, which may lead to a catecholamine-induced cardiomyopathy [2]. Various mechanisms have been postulated to be involved in the pathogenesis of this form of cardiomyopathy including highly cytotoxic radicals from the catecholamine metabolism [3], catecholamine-induced coronary vasoconstriction with radical generation resulting from ischemia [4] and most importantly deleterious high-energy phosphate deficiency by excessive activation of calcium-dependent intracellular ATPases and impairment of the phosphorylating capacity of mitochondria [5]. However, the consequences of such damage to myocardial lactate dehydrogenase (LDH) and creatinine kinase (CK) have not been studied. Therefore, the purpose of the present study was to investigate the activity and the isoenzyme profile of these two key enzymes of myocardial energy metabolism in rabbits with left ventricular (LV) dysfunction due to repetitive treatment with high doses of catecholamines. As we have previously demonstrated, this model of LV dysfunction is associated with markedly impaired cardiac function, neurohumoral activation, as well as systemic and local vasoconstriction [6].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Protocol
Sixteen male Chinchilla rabbits (3.0–3.5 kg body wt.) were used for the study. Catecholamine-induced cardiomyopathy was induced in eight animals by repetitive infusion of 300 µg/kg epinephrine i.v. over a 60-min period. This infusion was repeated at days 12, 24, and 36. Four days after the fourth and final epinephrine infusion, a 2D-guided M-Mode echocardiogram was obtained (Hewlett-Packard Sonos 1500, 5-MHz probe) to document LV dysfunction. LV end-diastolic (EDD) and end-systolic diameter (ESD) were assessed and fractional shortening (FS), a parameter of systolic LV function, was calculated by the formula FS=[(EDD–ESD)/EDD] and LV mass index by the formula of Troy. Thereafter, rabbits were killed and the hearts quickly excised to study energy metabolism and histological changes after treatment with epinephrine. Eight rabbits without catecholamine-treatment served as controls (CTRL).

2.2. Histopathological evaluation
For histopathological evaluation, 3-mm coronal sections of the ventricles were prepared from apex to base in a plane parallel to the atrioventricular groove. Planimetric measurements were carried out to determine areas of LV chamber as well as septum and LV free wall. For histological evaluation, three tissue slices from each heart were embedded after dehydration and cleaning. Sections were stained with hematoxylin–eosin as well as with the methods according to Ladewig and Masson-Goldner to demonstrate collagen [7,8]. Each section was studied for the following pathological findings and classified according the grading described by Rona and co-workers [9]: Grade 0, no lesions; Grade 1, focal lesions of the subendocardial portion of the apex and/or the papillary muscle, composed of fibroblastic swelling or proliferation and accumulation of histiocytes; Grade 2, focal lesions extending over wider areas of the left ventricle, with right ventricular involvement; Grade 3, confluent lesions of the apex and papillary muscles, with focal lesions involving other areas of the ventricles; and Grade 4, confluent lesions throughout the heart, including infarct-like massive necrosis, with occasionally aneurysm or mural thrombi. Sections were graded and averaged to produce a single score for the entire ventricle.

Myocyte width was determined by computer-assisted quantitative image analysis (Olympus Optical Corp.). The tissue was visualised by light microscopy using a 200x magnification oil immersion objective. In randomly assigned slides only myocytes per with a visible nucleus were selected and the distance between the borders were measured (average of 100 myocytes per slide). The histological examination was performed by two independent observers who were blinded with respect to administration of epinephrine. Interobserver variability and intraobserver variability was 5% and 3%, respectively.

2.3. Myocardial enzyme measurements
LV tissue samples were homogenised in 0.1 mol/l phosphate buffer (pH 7.4) containing 1 mmol/l EGTA and 1 mmol/l betamercaptoethanol. Aliquots were taken to measure protein content by the method of Lowry [10]. Then, 0.1% Triton X was added and enzyme activities of CK, LDH and citrate synthase were measured using an Ultraspect III spectrophotometer (Pharmacia Biosystems) as previously described [11]. For the measurement of CK isoenzyme distribution the Rapid Electrophoresis System (REP, Helena Diagnostik GmbH) was used as separation unit, and the REP CK isoforms kit (Helena Diagnostika GmbH) for agarose gel and incubation solution were applied. LDH isoenzymes were determined with a Titan Gel LD Isoenzyme System (REP, Helena Diagnostika GmbH). The Electrophoresis Data Center (EDC, Helena Diagnostika GmbH) automatically quantified the separated isoenzyme bands [11].

All experiments were performed after prior institutional approval and in accordance with the principles outlined in the Declaration of Helsinki.

2.4. Statistical analysis
Statistical analysis was performed by the Wilcoxon matched pairs signed-ranks test. Statistical significance was accepted at a P-value of ≤0.05; all results are presented as means±S.E.M.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Function and morphology
On echocardiography, systolic and diastolic LV-diameters were significantly increased in epinephrine-induced cardiomyopathy (EDD: 1.41±0.04 mm vs. CTRL 1.10±0.05; ESD: 0.89±0.05 mm vs. CTRL 0.59±0.04; P<0.05) and LV fractional shortening was significantly reduced (30±5% vs. CTRL 51±4; P<0.05). In contrast, LV-mass index was elevated (0.54±0.02 vs. 0.48±0.04 g/kg body wt.; P=0.05).

Epinephrine-induced cardiac alterations included myocyte degeneration and necrosis, subsequent interstitial and perivascular fibrosis and myocardial hypertrophy especially of the left ventricle, as shown in Fig. 1. CTRL rabbits had a LV pathological score of 0.0 compared to epinephrine treated rabbits rats with 2.85±0.77. Furthermore, sections showed an increase in myocyte width (16.9±0.8 µm vs. CTRL 12.9±0.9; P<0.05), indicating myocyte hypertrophy. Morphometric measurements revealed an increase in the thickness of the LV free wall and septum (LV free wall: 153±5 mm2 vs. CTRL 133±20; septum: 96±7 mm2 vs. CTRL 80±6; P<0.05) in addition to LV volume (481±38 mm3 vs. CTRL 244±19; P<0.05).


Figure 1
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  		Fig. 1 Masson-Goldner staining of rabbit myocardium (magnification 100-fold): typical photomicrographs of the left ventricular myocardium of a control rabbit (a) and after repetitive catecholamine treatment (b). While myocytes stain red/brown, fibrotic tissue stains green.

 
3.2. Myocardial CK and LDH system
Total LDH activity (Table 1) was significantly decreased in epinephrine-induced cardiomyopathy. The LDH isoenzymes showed a significant reduction of the LDH1 isoenzyme, while the LDH2 and LDH3 isoenzymes were increased. Total CK activity showed a tendency to decrease in epinephrine-induced cardiomyopathy but this trend did not reach statistical significance. The relative distribution of the four CK isoenzymes BB, MB, MM and mitochondrial CK was altered in epinephrine-induced cardiomyopathy. Whereas the relative amount of CK-MM did not change, CK-MB isoenzymes significantly increased in rabbits with epinephrine-induced cardiomyopathy. Although the decrease in mitochondrial CK and citrate synthase activity were not significantly changed, they both tended to decrease (by 17% and 14%, respectively).


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  		Table 1 Energy metabolism in rabbits with catecholamine induced cardiomyopathya

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, histological and echocardiographic measurements revealed that repeated infusions with high doses of epinephrine in rabbits reproducibly induce a cardiomyopathy with significant left ventricular deterioration as well as changes in two key enzymes of myocardial energy metabolism — the CK and LDH system.

Rabbits with epinephrine-induced cardiomyopathy were characterised by decreased myocardial LDH activity, a mechanism that might contribute to an energy-deficiency and play a key role in the pathophysiology of LV dysfunction. Since in the current study, total LDH activity was normalised to non-collagenous protein, we believe that this finding reflects truly decreased enzyme activity rather than a phenomenon associated with myocyte loss. Decreased LDH activity was also accompanied by a different pattern in LDH isoenzymes. Specifically, we observed a relative decrease in LDH1, an aerobic isoenzyme, and a relative increase in LDH2 and LDH3, two more anaerobic isoenzymes. This finding might suggest a shift of LDH activity towards more anaerobic metabolism in LV dysfunction and extends previous studies in rats with myocardial infarction or pressure-overload hypertrophy towards a larger animal species [11,12].

The fraction of left ventricular LDH1 isoenzyme was over 90% in the current studies. These results are similar to previous studies, which have also demonstrated a high myocardial LDH1 isoenzyme fraction of over 90% in rabbit myocardium [13]. In contrast, measurements of LDH1 isoenzyme fraction in humans and rats showed values of approximately 30% and 50%, respectively [14,15]. Therefore, these differences of cardiac LDH1 distribution in rats, humans and rabbits might be most likely due to species differences. However, systematic data are still missing.

The current study also demonstrates a significant shift of myocardial CK isoenzymes towards the foetal B enzymes and suggests re-expression of foetal CK isoenzymes as an important adaptation in LV dysfunction. Indeed, Ingwall and co-workers demonstrated that during the development from foetal to adult myocardium, total CK activity and mitochondrial CK isoenzyme increase, whereas the foetal isoenzymes BB and MB decrease [16]. Since the foetal B isoenzyme has a higher affinity for phosphocreatine than the M isoenzyme, phosphoryl transfer to ATP is more efficient by the MB and BB isoenzymes. Therefore, re-expression of foetal isoenzymes MB and BB may be an important adaptive mechanism to improve cardiac energy metabolism in LV dysfunction. Again, the current study extends similar findings in rats with pressure overload left ventricular hypertrophy and heart failure to a model of LV dysfunction in larger animals [12,17].

Unlike in other heart failure models in rats [15], myocardial activity of citrate synthase, a marker enzyme for mitochondrial mass, was unaltered in our current studies. This finding suggests, that mitochondrial mass, estimated as citrate synthase activity, does not change after epinephrine treatment and that decreased mitochondrial mass does not contribute to the pathophysiology of LV dysfunction in this model.

In summary, left ventricular dysfunction is accompanied by a decrease in LDH activity and a shift of myocardial LDH isoenzymes towards an anaerobic metabolism as well as an increase in foetal B-CK isoenzymes, suggesting a compensatory mechanism towards a more efficient CK system. The model of epinephrine-induced cardiomyopathy in rabbits is suitable to further study this mechanism which has also been suggested as an important biochemical adaptation in human chronic heart failure [17,18].


   Acknowledgements
 
This study was supported by the NOVARTIS-Stiftung für therapeutische Forschung.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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