[Complex study of the rat heart at isoproterenol damage].

Introduction of isoproterenol (an agonist of beta-adrenoreceptors) to rats is one of the widespread experimental models of cardiac failure. It is caused by damage of cardiomyocytes with the subsequent development of substitutive fibrosis. The purpose of the given work was the complex characteristic of cardiac function by means of invasive and noninvasive (echocardiography and impedansometry) methods of research. Isoproterenol was injected twice with a daily interval in dozes 85, 120, 150 or 180 mg/kg. Echocardiographic study of the heart in 2 weeks revealed obvious attributes of cardiac failure (left ventricular dilatation, lowered ejection fraction) in the groups which have received high cumulative dozes of isoproterenol (300-360 mg/kg). The catheterization of the left ventricle in these groups has shown raised enddiastolic pressure, decreased maximal rate of pressure development and fall, and also lowered indices of myocardial contractility and relaxability. In the groups which have received smaller isoproterenol dozes, apparent decrease in relaxability parameters (constants of isovolumic and auxovolumic relaxation) has been revealed at only slightly changed parameters of contractility. A strong correlation between echocardiographic and invasive parameters of myocardial contractility has been found. The phase analysis of the cardiac cycle has shown a lengthening of isometric phases of contraction and relaxation, as well as duration of ejection due to shortening duration of filling of both ventricles. Cardiomyocytes isolated from hearts with obvious cardiac failure responded to electrostimulation by arrhythmic contractions and also by much slowed and incomplete removal of free Ca++ from the myoplasm. Results allow to conclude that relatively smaller extent of myocardial damage is accompanied by decreased relaxability at slightly changed contractility, while at greater degree of damage both processes fail, but delay of relaxation still prevails.

[Suppression of vascular endothelium hyperpermeability by cell-permeating peptide inhibitors of myosin light chain kinase].

Novel peptides originating from the peptide inhibitor of myosin light chain kinase, L-PIK (Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys), have been studied for ability to attenuate the thrombin-induced hyperpermeability of endothelial cell monolayer in culture. Peptides [NalphaMeArg1]-Lys-Lys-Tyr-Lys-Tyr-Arg-(D)Arg8-Lys and H-Arg(NO2)Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 (designated PIK2 and PIK4, respectively) appeared to be the most effective inhibitors of endothelial cell monolayer hyperpermebility, and surpassed other known peptide inhibitors of myosin light chain kinase derived from original L-PIK. Our results validate PIK2 and PIK4 as the leading molecules for the development of novel drugs intended to counteract pathological hyperpermeability of vascular endothelium.

[Molecular physiology of the endothelium and mechanisms of vascular permeability].

Endothelium of microvessels acts as selective barrier between blood and tissues and regulates transport of substances across vascular wall. In stressful situations permeability of microvessels increases acutely and leads to edema and the loss of organ function. This review describes molecular mechanisms of maintenance, loss and recovery of endothelial barrier as well as modern approaches to pharmacologic correction of vascular hyperpermeability.

[Rho-associated protein kinase is involved in establishing the contractile phenotype of cardiomyocytes].

It has been shown that Y-27632, an inhibitor of Rho-associated kinase, delays sarcomere assembly in rat neonatal cardiomyocytes pretreated with angiotensin II. Y-27632 affects the beat rate of cardiomyocytes; however, this effect is only observed at high cell density and, therefore, seems to be related to the formation of gap junctions between adjacent cardiomyocytes. Consistent with this suggestion, we established that Rho-associated kinase is localized in myofibrillar Z-discs of human myocardium and intercalated discs, the structures enriched in gap junctions. We propose that Rho-associated kinase participates in the maturation of the myocardial contractile system through phosphorylation of its molecular targets in Z-discs and intercalated discs.

[Peptide inhibitors of myosin light chain kinase. Development of peptidase resistant analogues].

Myosin light chain kinase (MLCK) is the key regulator of various forms of cell motility including endothelial and epithelial permeability in particular. One of the potential MLCK inhibitors to be used in humans is a membrane permeable peptide H-RKKYKYRRK-NH2 (L-PIK). In present work we used solid phase peptide synthesis and Fmoc-technology to produce five modifications of L-PIK. Based on (1)H NMR analysis revealed that these peptides demonstrated improved resistance to degradation in blood plasma. One of de novo synthesized peptides, L-[MeArg(1)]PIK inhibited MLCK activity in vitro with the same efficiency as L-PIK whereas other modified peptides showed reduced inhibitory activity. D-amino acid analog of PIK was the least active inhibitor. Thus, we have demonstrated the possibility to produce an effective MLCK peptide inhibitor with increased resistance to biodegradation that is suitable for further pharmacological development.

[Novel peptide inhibitors of the myosin light chain kinase suppress hyperpermeability of vascular endothelium].

The ability of novel cell-permeating peptide molecules derived from the peptide inhibitor of the myosin light chain kinase (MLCK) L-PIK (Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys) to inhibit this kinase in vitro and attenuate the thrombin-induced hyperpermeability of endothelial cell monolayer in culture has been studied. It was found that the compounds [NalphaMeArg1]-L-PIK and [Cit1]-L-PIK possess the inhibitory activity towards MLCK comparable to that of L-PIK and the ability to suppress the hyperpermeability of endothelium, whereas other modifications of L-PIK were less effective. Thus, among de novo synthesized peptides, [NalphaMeArg1]-L-PIK and [Cit1]-L-PIK demonstrate the inhibitory properties of the original peptide L-PIK and additionally surpass it by stability in blood plasma. These peptides may be used in the design of novel antiedemic drugs.

[Myosin phosphorylation as the main way of regulating smooth muscle contractions].

Rho-kinase is a key enzyme of the receptor-dependent signal cascades and is regarded today as the most prospective target for pharmacological therapy of smooth muscle contractility disorders.

[Penetrating peptide inhibitor of the myosin light chain kinase suppresses hyperpermeability of vascular endothelium].

Nonapeptide H-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 corresponding to a modified sequence of autoinhibitory region of myosin light chain kinase (MLCK) was synthesized from L-amino acids and from D-amino acids. Using nuclear magnetic resonance spectroscopy it has been demonstrated that D-peptide is significantly more stable in human blood plasma than its L-enantiomer. D-peptide accumulated in cultured human umbilical vein endothelial cells suppressed development of hyperpermeability in endothelial monolayer induced by thrombin addition. Following intravenous administration D-peptide decreased the extent of lung oedema in rats induced by infusion of oleic acid in bloodstream. Thus, the peptide molecules based on an autoinhibitory peptide of MLCK may serve as a prototype for development of a novel antioedematous drugs that directly affect the MLCK-dependent motile processes in vascular endothelium.

[Molecular mechanisms of cardiomyogenesis and perspectives of cardiomyocyte regeneration in cardiac failure].

This review analyzes the current state in investigations of molecular, genetic and cellular mechanisms of cardiac development as well as perspectives to use this knowledge for treatment of cardiac failure by means of replenishing cardiomyocytes in damaged myocardium.

[The myosin-activating protein kinases in human myocardium: localization and content].

Using the immunofluorescence approach, we have determined that the recently detected protein kinases, among which are RhoA-activated kinase, integrin-linked kinase, zipper interacting protein kinase, and death-associated protein kinase, which are capable of phosphorylating myosin, are localized in the Z-lines sarcomeres of human myocardium. Additionally, we studied the content of integrin-linked and zipper interacting protein kinases in human embryonic myocardium, as well as in normal and hypertrophic adult human heart. The content of these protein kinases in adult normal myocardium increases in comparison with the embryonic heart. The content of integrin-linked and zipper interacting protein kinases in hypertrophic myocardium is higher compared with the normal adult heart. The data obtained suggest the involvement of these protein kinases in the development and hypertrophy of human heart.

[KRP/telokin differentially regulates filament assembly and phosphorylation of light chains of non-muscle myosin II in fibroblasts].

Transgenic 3T3 fibroblasts have been generated that express either the wild-type KRP or its truncated mutant lacking the C-terminal domain, which primarily contributes to myosin binding of KRP. It was found that KRP-expressing cells display a significantly increased content of myosin filaments and a reduced level of rMLC phosphorylation, whereas the mock transfected cells or cells expressing the C-terminally truncated KRP do not. Our results suggest that (1) KRP promotes the polymerization of myosin II and reduces the rMLC phosphorylation level in cells, (2) KRP acts through direct binding to myosin II, and (3) transgenic 3T3 fibroblasts stably expressing KRP represent a useful and versatile model to study the role of myosin II filament dynamics in cell motility.

[Content of myosin-activating protein kinases in myocardium of patients with dilated cardiomyopathy and in the animal heart].

The skeletal myosin light chain kinase (skMLCK) was identified in human and chicken embryo myocardium but not in embryo and adult rat heart using western blotting. The content of skMLCK and myosin-activating protein kinases: RhaA-activated protein kinase (ROCK), integrin-linked protein kinase (ILK), and zipper-interacting protein kinase (ZIPK) was compared in normal human myocardium and the hearts of patients with dilated cardiomyopathy (DCM). It was demonstrated that the content of skMLCK, ROCK and ILK increases in DCM whereas the content of ZIPK decreases. The results obtained may reflect compensatory processes in cardiomyocytes in DMC, which are aimed at increasing their viability and contractility.

[A new actin-binding area of the myosin light chains' high-molecular kinase]. / Novyi aktinsviazivaiushchii uchastok vysokomolekuliarnoi kinazy legkikh tsepei miozina.

High-molecular weight isoform of myosin light chain kinase (MLCK-210) contains a unique N-terminal domain not present in a low-molecular weight isoform, MLCK-108. Functional features of this domain are poorly studied. We provide evidence that N-terminal area of the unique MLCK-210 domain incorporates previously unrecognized actin-binding region that could be involved in the interaction of MLCK-210 with the actin cytoskeleton.

[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.

[Isoproterenol induced changes of protein expression and myocardial ultrastructure]. / Izmeneniia ékspressii belkov i ul'trastruktury serdechnoi myshtsy pod vliianiem izoproterenola.

AIM: To elucidate alterations in myocardial ultrastructure and protein expression caused by isoproterenol. METHODS: Biochemical, immunohistochemical and electron microscopic studies of rat myocardium were carried out 2 hours and 3 weeks after single injections of isoproterenol (50 and 10 mg/kg). Relative content of myospecific proteins (KRP - kinase-related protein, desmin), cytoskeletal proteins (tubulin, vinculin, and myosin light chain kinase - MLCK) and extracellular matrix protein, fibronectin, was determined by immunoblotting. RESULTS: In 2 hours after injection of 50 mg/kg of isoproterenol destruction of some cardiomyocytes, contracture of myofibrils, and mild edema of intercellular space occurred; the content of KRP decreased by l6%, and that of tubulin, vinculin and fibronectin - by 27-29%. Reduced level of these proteins and also of MLCK persisted until 3 weeks after injection and was associated with altered cardiomyocyte ultrastructure. Glycogen granules were sparse, mitochondria contained arrow-like inclusions characteristic for calcium overload, huge mitochondria connected by specialized intermitochondrial contacts were present. Enlarged intercellular space contained areas of fibrosis with increased amount of type I and II collagens and fibronectin. Lower dose of isoproterenol (10 mg/kg) did not cause noticeable damaging action in the acute period, but in 3 weeks thickening of extracellular matrix occurred accompanied by increases of KRP and tubulin contents (by 26-32% compared with control level). Similar rise in expression of these proteins, and also of MLCK was observed after addition of isoproterenol to culture of chicken cardiomyocytes. CONCLUSION: These results indicate that even single injection of isoproterenol causes long lasting structural alterations in cardiac muscle accompanied by increased expression of extracellular matrix proteins as well as sarcoplasmic proteins apparently involved in the hypertrophic response of the cardiomyocytes.

[Effect of adriamycin on expression and content of myocardial structural and regulatory proteins]. / Izmeneniia ékspressii strukturnykh i reguliatornykh belkov v miokarde pri deistvii adriamitsina.

The biochemical and morphological study of the cytoskeleton and extracellular matrix of rat heart was carried out after single injection of adriamycin (2.2 or 0.44 mg/kg). Hearts were taken for the study after 2 hours and 3 weeks after injection. The light and electronic microscopy, immunohistochemical determination of type I, III and IV collagens and fibronectin using specific antibodies were implied for morphological study; electrophoresis and immunoblotting were implied for the determination of the content of some proteins of cardiomyocytes (KRP or telokin, desmin, tubulin and vinculin), and extracellular matrix (fibronectin) and vascular smooth muscle cells (MLCK, myosin light chain kinase). Adriamycin injection in the dose 2.2 mg/kg which is close to therapeutic and known to alter intracellular membranes approximately in the half of cardiomyocytes, did not influence the relative volume and structure of collagen network but distinctly reduced the density of fibronectin-distribution. The content of tubulin, fibronectin, MLCK and KRP was significantly decreased by 18-24%, while contents of desmin and vinculin were changed insignificantly. After 3 weeks, an increased density and extension of collagen network indicating the development of diffuse fibrosis were observed. Contents of tubulin and KRP were increased above control level by 50 and 20%, respectively. Similar hyperrestitution of tubulin, fibronectin and KRP content by 15-25% was determined after smaller dose of adriamycin (0.44 mg/kg). Only content of MLCK out of proteins studied remained at lower level in both groups by 25-34%. Isolated chick embryo cardiomyocytes subjected to adriamycin responded by increased level of KRP expression by 20% in 4 days while the level of tubulin expression remained unchanged. Results showed that damage of cardiomyocytes and extracellular matrix after single injection of adriamycin in the dose close to therapeutic was followed by increased expression of some proteins of cytoskeleton and extracellular matrix. KRP seems to play active role in this reparative response while the steadily reduced level of MLCK expression may disturb the control of coronary vessels.

[Differences in phorbol-dependent phosphorylation of regulatory proteins and contraction of phasic and tonic smooth muscle]. / Razlichia v forbolzavisimom fosforilirovanii regliatornykh belkov i sokrashchenii faznykh i tonicheskikh gladkikh myshts.

Smooth muscles are divided into slowly contracting tonic and relatively fast phasic muscles. In both cases Ca2+ is a key mediator of the contractile response. However, the appearance of a tonic component during sphincter or arterial muscle contraction and its absence in contracting visceral smooth muscle is characteristic of their difference. We have found that in chicken tissues phorbol 12,13-dibutyrate (PDBu) induces a sustained contraction in carotid arterial muscle, but provokes no contraction in phasic gizzard smooth muscle. Next we were aimed to find differences in PDBu-induced phosphorylation of the key proteins involved in regulation of smooth muscle contraction, i.e. caldesmon, myosin light chain kinase (MLCK), and the myosin light chain kinase-related protein (KRP, also known as telokin). Two correlative differences were observed. 1. PDBu stimulated phosphorylation of MLCK in tonic smooth muscle and had no effect on the level of MLCK phosphorylation in phasic muscle. Phosphopeptide mapping suggests the involvement of mitogen-activated protein (MAP) kinases in phosphorylation of MLCK in situ. 2. PDBu induced phosphorylation of MAP-kinase sites in caldesmon in both types of smooth muscle, but this phosphorylation had no significant effect on caldesmon functional activity in vitro. For the first time we have shown that in gizzard PDBu also stimulates a yet unknown transitory caldesmon-kinase different from protein kinase, C, Ca2+/calmodulin-dependent kinase II and casein kinase CK2. 3. No significant difference was found in the kinetics of PDBu-dependent phosphorylation of KRP in tonic and phasic smooth muscles. KRP was also demonstrated to be a major phosphoprotein in smooth muscle phosphorylated in vivo at several sites located within its N-terminal sequence. Protein kinases able to phosphorylate these sites were identified in vitro. Among them, MAP-kinase was suggested to phosphorylate a serine residue homologous to that phosphorylated in MLCK. 4. p42erk2 and p38 MAP-kinases were found in phasic and tonic smooth muscles. Both were responsive to PDBu in cultured chicken aortic smooth muscle cells, and their role in phosphorylation of MLCK and low molecular weight isoform of caldesmon was evaluated.

[Functional properties and intracellular localization of high molecular weight isoforms of ligh chain myosin kinase]. / Funktsional'nye svoistva i vnutrikletochnaia lokalizatiia vysokomolekuliarnoi izoformy kinazy legkikh tsepei miozina.

The vertebrate genetic locus, coding for a Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK), the key regulator of smooth muscle contraction and cell motility, reveals a complex organization. Two MLCK isoforms are encoded by the MLCK genetic locus. Recently identified M(r) 210 kDa MLCK contains a sequence of smooth muscle/non-muscle M(r) 108 kDa MLCK and has an additional N-terminal sequence (Watterson et al., 1995. FEBS Lett. 373 : 217). A gene for an independently expressed non-kinase product KRP (telokin) is located within the MLCK gene (Collinge et al., 1992. Mol. Cell. Biol. 12 : 2359). KRP binds to and regulates the structure of myosin filaments (Shirinsky et al., 1993. J. Biol. Chem. 268 : 16578). Here we compared biochemical properties of MLCK-210 and MLCK-108 and studied intracellular localization of MLCK-210. MLCK-210 was isolated from extract of chicken aorta by immunoprecipitation using specific antibody and biochemically analysed in vitro. MLCK-210 phosphorylated myosin regulatory light chain and heavy meromyosin. The Ca(2+)-dependence and specific activity of MLCK-210 were similar to that of MLCK-108 from turkey gizzard. Using sedimentation assay we demonstrated that MLCK-210 as well as MLCK-108 binds to both actin and myosin filaments. MLCK-210 was localized in smooth muscle cell layers of aortic wall and was found to co-localize with microfilaments in cultured aortic smooth muscle cells.

[Molecular mechanisms of regulation of the contractile apparatus of smooth muscles]. / Molekuliarnye mekhanizmy reguliatsii sokratitel'nogo apparata gladkikh myshts.

This review describes the main ultrastructural features, molecular organization and regulation of smooth muscle contractile machinery. Possible molecular mechanisms of smooth muscle sensitization to Ca2+, Ca(2+)-independent contraction and latch state are discussed.