Annexins, calcium-dependent phospholipid binding proteins in irreducible heart failure.

AIM: To analyze the expression and distribution of annexins V and VI in intact human hearts and in dilated cardiomyopathy (DCM) in patients with irreducible heart failure. MATERIAL AND METHODS: The study included nine patients with DCM and irreducible heart failure. By immunoblotting and indirect immunofluorescence, the amount and location of annexins was determined using samples of left ventricular (LV) myocardium collected during orthotopic heart allotransplantation. Samples of LV myocardium from 9 individuals who died of craniocerebral trauma served as controls. RESULTS: Quantitative analysis showed an increased level of both annexins in the myocardium of DCM patients compared with normal myocardium: 122 +/- 5% (p < 0.05) and 119 +/- 5% (p < 0.05) for annexins V and VI, respectively. In the intact heart annexin V was located at the sarcolemma and intercalated discs, while in the myocardium with heart failure mainly in the interstitium. In normal hearts, annexin VI was located at the sarcolema, including T-tubules, Z-lines and intercalated discs. In heart failure, annexin V1 was also located in the interstitium. CONCLUSIONS: Redistribution of annexins V and VI in the cardiomyocytes in the interstitial space may have important functional consequences and indicates the role of myocardial protein expression disturbances in the etiology and/or pathogenesis of heart failure in pa-

Aspects of molecular mechanisms in myocardial hypertrophy, particular morphological changes and cell bioenergetic characteristics in patients with dilated cardiomyopathy.

AIM: To present the particular morphological aspects of hypertrophied myocardium by determining protein expression profile, the role of creatine kinase in cardiac cell function and mitochondrial respiration using endomyocardial biopsy samples obtained at cardiac catheterization in patients with dilated cardiomyopathy (DCM). MATERIAL AND METHODS: The study included 43 DCM patients (37 men and 6 women, mean age 34 +/- 8.6 years). Protein content in myocardial samples was determined by quantitative immunoblotting and electrophoresis, morphometric indices were evaluated with a JEM-100C electron microscope, and mitochondrial respiratory parameters were determined with a Clark electrode in an oxigrtaphic cell. RESULTS: The study of the expression of hypertrophic protein markers showed increases in the amount of tubulin on average by 42%,desmin by 33%, and skeletal myosin beta-isoform by 20%, and of smooth muscle proteins--alpha-smooth muscle actin and SM22 on average by 1.5 times. In all DCM patients C-reactive protein (CRP) level was on average 52% and that of myosin light-chain kinase (MLCK) 70% higher. The morphometric study showed variable characteristics of cardiomyocytes in the entire patient group, particularly bizarre and oversized nuclei, determining us to divide the patients into two groups. The electrophoresis analysis of total creatine kinase fraction revealed significant alterations in the expression of creatine kinase isoenzymes. Mitochondrial respiration rate after addition of creatine was very low. CONCLUSIONS: We found a high content of myosin light-chain kinase and CRP in the myocardium of patients with DCM, proving their role in hypertrophy; and this can be used in the differential diagnosis of myocardial hypertrophy as a consequence of cardiomyocyte hypertrophy and a result of the fibrotic process. Energy disturbances are significantly more pronounced in patients with DCM, with a more significant cardiomyocyte hypertrophy and an obvious increase in the size of nuclei.

The use of beta blocker Nebivolol in patients with chronic obstructive pulmonary disease in association with arterial hypertension.

There were investigated 22 patients with chronic obstructive pulmonary disease in association with arterial hypertension initially and after 5 weeks of treatment with beta-blocker Nebivolol 5 mg/day. There were not detected any negative changes on bronchial permeability after treatment. Considerable improvement of endothelial function (from 7-12% to 17-35%, p < 0.05) in parallel with decreasing of pulmonary artery, systolic (44.5 +/- 5.5 mmHg vs. 32.8 +/- 4.1 mmHg, p < 0.05) and mean pressure (26.3 +/- 4.2 mmHg vs. 21.1 +/- 2.1 mmHg, p < 0.05)--were observed after treatment. Thus, Nebilet is well-tolerated and highly effective in patients with chronic obstructive pulmonary disease in association with arterial hypertension.

[Apoptosis and hibernation of cardiomyocytes of periscar zone as factor of chronic cardiac aneurysm progression]. / Apoptoz i gibernatsiia kardiomiotsitov perirubtsovoi zony kak faktor progressirovaniia khronicheskoi anevrizmy serdtsa.

Tissue specimen from chronic aneurysms and adjacent myocardium obtained at aneurysmectomy and coronary bypass surgery from 46 patients were subjected to morphological study. Immunohistochemical methods and electronic microscopy were applied for detection of apoptosis and hibernation of cardiomyocytes in 11 cases and histochemical determination of activity of energetic enzymes succinate and lactate dehydrogenase was used in 5 cases. Cardiomyocytes from peri and intra scar layers of myocardium were found to be in a state of hibernation while some of them were in a state of apoptosis. Extent of apoptosis was different in aneurysms on different stages of organization. Number of altered cardiomyocytes was the greatest in immature aneurysms. Basing on these findings apoptosis of hibernating cardiomyocytes was suggested to be one of factors of expansion of sclerotic zone and aneurysm formation.

[Expression of contractile and cytoskeletal proteins in myocardium of patients with dilated cardiomyopathy.]. / Ekspressiia sokratitel'nykh i tsitoskeletnykh belkov v miokarde bol'nykh s dilatatsionnoi kardiomiopatiei

Dilated cardiomyopathy (DCM) is characterized by enlargement and dilation of all heart compartments associated with serious decrease of its contractile function. DCM hallmark is the combination of dystrophic and hypertrophic alterations of cardiomyocytes. Since the power output of cardiac cells is directly related to remodeling of their contractile machinery we investigated expression of selected contractile and cytoskeletal proteins in the left ventricle of DCM patients using immunoblotting. The content of the recognized protein markers of cardiomyocyte hypertrophy such as tubulin, desmin and slow skeletal myosin heavy chain isoform, MHCbeta, was significantly elevated in DCM compared to normal myocardium. In addition, marked increase in the content of several smooth muscle proteins (smooth muscle alpha-actin, Myosin Light Chain Kinase, Kinase Related protein SM22) that are normally expressed in embryonic myocardium, was observed in DCM hearts. Thus, cardiomyocyte hypertrophy in DCM is associated with activation of embryonic protein expression program and smooth muscle proteins could serve as markers of this process. Understanding their involvement in sarcomere assembly and pathways of their expression activation during cardiac hypertrophy may bring new insights in treatment of various forms of cardiomyopathy.

Cellular hypertrophy in cardiomyopathic patients is associated with lower creatine-stimulated mitochondrial respiration.

The mitochondrial respiration rate and morphometric indices in endomyocardial biopsy samples were measured in 43 patients with dilated cardiomyopathy selected in accordance to WHO criteria by endomyocardial biopsy studies after excluding of various forms of myocarditis, alcoholic cardiomyopathy and other specific diseases of the heart. A group of 13 patients with unusually high mean myocyte diameter, 30 +/- 4 microns, and nuclear size, 57 +/- 5 microns, was selected. The remainder of patients (n = 30) had significantly lower mean myocyte diameter and nuclear size, 23 +/- 3 and 42 +/- 6 microns, respectively, (p < 0.01). Creatine-stimulated elevation in mitochondrial respiration rate as measured in saponin-skinned fibers was found in the former group to be much lower (36 +/- 4%) as compared with the remainder (90 +/- 12%). Also, the former group of patients had higher left ventricular enddiastolic pressure and volume index with concomitantly decreased ejection fraction. The results indicate that marked nuclear and cellular hypertrophy is associated with lower creatine-stimulated mitochondrial respiration rate and more severe cardiac failure. They suggest that disorders in energy supply to myofibrils may be related to disturbances in cellular genetic apparatus.

[Cellular mechanisms of impaired cardiac energetics in patients with dilated cardiomyopathy: decrease in mitochondrial respiration and creatine kinase expression]. / Kletochnye mekhanizmy narusheniia énergetiki serdtsa u bol'nykh s dilatatsionnoi kardiomiopatiei: umen'shenie mitokhondrial'nogo dykhaniia i ékspressii kreatinkinazy.

The mitochondrial functional characteristics were assessed in the biopsy specimens from patients with various Functional Classes dilated cardiomyopathy (DCMP). The assessment was made by using endomyocardial biopsy specimens weighing 2-4 mg which had been taken from 39 patients aged 19-64 years during coronary ventriculography and cardiac transplantation. The status of mitochondria and the efficiency of mitochondrial creatine kinase functioning were evaluated by recording the respiration of saponin-skinned muscular fibers. The maximum mitochondrial respiration rate calculated on a dry weight basis was not substantially different in all functional classes of DCMP, while the acceptor control index (Vmax/V0) and the level of creatine-activated respiration decreased with an increase in the functional class of DCMP. The findings show a good positive correlation between ejection fraction and creatine-stimulated respiration values and a linear negative correlation between this parameter and end-diastolic pressures. Thus, the respiratory parameters of mitochondria in the endomyocardial biopsy specimens may be used to assess the severity of cardias lesions.

The creatine kinase system and cardiomyopathy.

Changes in the creatine kinase system, cellular energetics, regulation of respiration and alterations in parameters of contractility in experimental animals (myopathic hamsters), and in patients with dilated cardiomyopathy were studied. 31P-NMR methods were used to show that cardiomyopathic hearts are characterized by decreased work index, lower tissue ATP, phosphocreatine, and total creatine contents and diminished creatine kinase activity and energy fluxes. In isolated mitochondria, only the creatine kinase activity was decreased. Both in cardiomyopathic hamsters and human hearts a share of mitochondrial creatine kinase in the total tissue enzyme activity was decreased from 33% to 18% and that of BB elevated from 5% in control to 20%, at an unchanged relative level of MM. In saponins-skinned cardiac fibers on cardiomyocytes creatine (Cr, 25 mM) decreased Km for ADP in regulation of respiration from 133 +/- 20 to 20 +/- 4 microM due to activation of coupled mitochondrial creatine kinase-oxidative phosphorylation reactions in control hamster hearts. In the case of cardiomyopathy it decreased Km for ADP only to 81 +/- 13 microM. In endocardial biopsy samples from the hearts of patients with dilated cardiomyopathy taken during angiography, creatine stimulated respiration was decreased by 36% of control value, which correlated well with increase of end-diastolic pressure and fall in ejection fraction. Thus, changes in mitochondrial creatine kinase expression diminished the efficiency of cellular regulation of respiration in cardiomyopathic hearts that may have functional consequences for hemodynamics or may be adaptive alterations in response to decreased contractility.

Phosphocreatine pathway for energy transport: ADP diffusion and cardiomyopathy.

Chemically skinned (by treatment with saponin, 40 micrograms/ml) isolated cardiomyocytes were used to study the intracellular diffusion of ADP and creatine (Cr). Stimulation of respiration was studied in these cardiomyocytes without intact sarcolemma and in isolated heart mitochondrial by addition of ADP and Cr in the presence of 0.2 mM ATP (via mitochondrial creatine kinase reaction: Cr + MgATP = MgADP + PCr). The Michaelis constant (Km) for Cr was similar in both cases, 5.67 +/- 0.11 (SD) mM in skinned myocytes and 6.9 +/- 0.2 mM in mitochondria, showing that there is no significant restriction to the diffusion of this substrate. However, the apparent Km for external ADP increased from 17.6 +/- 1.0 microM for mitochondria to 250 +/- 38 microM for skinned cardiomyocytes, showing decreased diffusivity of ADP as a result of binding to cellular structures. In the presence of 25 mM Cr, the Km for ADP for myocytes decreased to 35.6 +/- 5.6 microM due to the coupling of the creatine kinase and oxidative phosphorylation reactions. Provision of substrate for the creatine kinase reaction amplified the weak ADP signal in the regulation of respiration. The activity of the mitochondrial creatine kinase was decreased by a factor of two in cardiomyopathic hamsters and human hearts and was associated with a twofold decrease in creatine-stimulated respiration. These data show a potentially key role of mitochondrial creatine kinase in the regulation of cellular respiration and the possible importance of changes in its activity for the functional disturbances of the cardiomyopathic heart.