Mechanical and signaling responses of unloaded rat soleus muscle to chronically elevated ß-myosin activity.

It has been reported that muscle functional unloading is accompanied by an increase in motoneuronal excitability despite the elimination of afferent input. Thus, we hypothesized that pharmacological potentiation of spontaneous contractile soleus muscle activity during hindlimb unloading could activate anabolic signaling pathways and prevent the loss of muscle mass and strength. To investigate these aspects and underlying molecular mechanisms, we used ß-myosin allosteric effector Omecamtiv Mekarbil (OM). We found that OM partially prevented the loss of isometric strength and intrinsic stiffness of the soleus muscle after two weeks of disuse. Notably, OM was able to attenuate the unloading-induced decrease in the rate of muscle protein synthesis (MPS). At the same time, the use of drug neither prevented the reduction in the markers of translational capacity (18S and 28S rRNA) nor activation of the ubiquitin-proteosomal system, which is evidenced by a decrease in the cross-sectional area of fast and slow muscle fibers. These results suggest that chemically-induced increase in low-intensity spontaneous contractions of the soleus muscle during functional unloading creates prerequisites for protein synthesis. At the same time, it should be assumed that the use of OM is advisable with pharmacological drugs that inhibit the expression of ubiquitin ligases.

N-Terminal Fragment of Cardiac Myosin Binding Protein-C Increases the Duration of Actin-Myosin Interaction.

Cardiac myosin binding protein-C (cMyBP-C) located in the C-zone of myocyte sarcomere is involved in the regulation of myocardial contraction. Its N-terminal domains C0, C1, C2, and the m-motif between C1 and C2 can bind to the myosin head and actin of the thin filament and affect the characteristics of their interaction. Measurements using an optical trap showed that the C0-C2 fragment of cMyBP-C increases the interaction time of cardiac myosin with the actin filament, while in an in vitro motility assay, it dose-dependently reduces the sliding velocity of actin filaments. Thus, it was found that the N-terminal part of cMyBP-C affects the kinetics of the myosin cross-bridge.

Effect of Cardiac Myosin-Binding Protein C on Tropomyosin Regulation of Actin-Myosin Interaction Using In Vitro Motility Assay.

We studied the modulating role of cardiac myosin-binding protein C (cMyBP-C) in tropomyosin regulation of the actin-myosin interaction. The effect of cMyBP-C on the velocity of actin-tropomyosin filament sliding over cardiac and slow skeletal myosins was evaluated using in vitro motility assay. The effect of cMyBP-C on the actin-tropomyosin filaments sliding depended on the type of myosin. The regulatory effect of cMyBP-C differs for cardiac and slow skeletal myosin because of the presence of specific essential light chain (LC1sa) in slow skeletal myosin isoform.

Investigations of Molecular Mechanisms of Actin-Myosin Interactions in Cardiac Muscle.

The functional characteristics of cardiac muscle depend on the composition of protein isoforms in the cardiomyocyte contractile machinery. In the ventricular myocardium of mammals, several isoforms of contractile and regulatory proteins are expressed - two isoforms of myosin (V1 and V3) and three isoforms of tropomyosin chains (α, ß, and κ). Expression of protein isoforms depends on the animal species, its age and hormonal status, and this can change with pathologies of the myocardium. Mutations in these proteins can lead to cardiomyopathies. The functional significance of the protein isoform composition has been studied mainly on intact hearts or on isolated preparations of myocardium, which could not provide a clear comprehension of the role of each particular isoform. Present-day experimental techniques such as an optical trap and in vitro motility assay make it possible to investigate the phenomena of interactions of contractile and regulatory proteins on the molecular level, thus avoiding effects associated with properties of a whole muscle or muscle tissue. These methods enable free combining of the isoforms to test the molecular mechanisms of their participation in the actin-myosin interaction. Using the optical trap and the in vitro motility assay, we have studied functional characteristics of the cardiac myosin isoforms, molecular mechanisms of the calcium-dependent regulation of actin-myosin interaction, and the role of myosin and tropomyosin isoforms in the cooperativity mechanisms in myocardium. The knowledge of molecular mechanisms underlying myocardial contractility and its regulation is necessary for comprehension of cardiac muscle functioning, its disorders in pathologies, and for development of approaches for their correction.

Study of regulatory effect of tropomyosin on actin-myosin interaction in skeletal muscle by in vitro motility assay.

The interaction between myosin and actin in striated muscle tissue is regulated by Ca2+ via thin filament regulatory proteins. Skeletal muscle possesses a whole pattern of myosin and tropomyosin isoforms. The regulatory effect of tropomyosin on actin-myosin interaction was investigated by measuring the sliding velocity of both actin and actin-tropomyosin filaments over fast and slow skeletal myosins using the in vitro motility assay. The actin-tropomyosin filaments were reconstructed with tropomyosin isoforms from striated muscle tissue. It was found that tropomyosins with different content of α-, ß-, and γ-chains added to actin filaments affect the sliding velocity of filaments in different ways. On the other hand, the sliding velocity of filaments with the same content of α-, ß-, and γ-chains depends on myosin isoforms of striated muscle. The reciprocal effects of myosin and tropomyosin on actin-myosin interaction in striated muscle may play a significant role in maintenance of effective work of striated muscle both during ontogenesis and under pathological conditions.

Study of reciprocal effects of cardiac myosin and tropomyosin isoforms on actin-myosin interaction with in vitro motility assay.

Interaction of myosin with actin in striated muscle is controlled by Ca(2+) via thin filament associated proteins: troponin and tropomyosin. In cardiac muscle there is a whole pattern of myosin and tropomyosin isoforms. The aim of the current work is to study regulatory effect of tropomyosin on sliding velocity of actin filaments in the in vitro motility assay over cardiac isomyosins. It was found that tropomyosins of different content of α- and ß-chains being added to actin filament effects the sliding velocity of filaments in different ways. On the other hand the velocity of filaments with the same tropomyosins depends on both heavy and light chains isoforms of cardiac myosin.

Effects of cardiac myosin binding protein-C on the regulation of interaction of cardiac myosin with thin filament in an in vitro motility assay.

Modulatory role of whole cardiac myosin binding protein-C (сMyBP-C) in regulation of cardiac muscle contractility was studied in the in vitro motility assay with rabbit cardiac myosin as a motor protein. The effects of cMyBP-C on the interaction of cardiac myosin with regulated thin filament were tested in both in vitro motility and ATPase assays. We demonstrate that the addition of cMyBP-C increases calcium regulated Mg-ATPase activity of cardiac myosin at submaximal calcium. The Hill coefficient for 'pCa-velocity' relation in the in vitro motility assay decreased and the calcium sensitivity increased when сMyBP-C was added. Results of our experiments testifies in favor of the hypothesis that сMyBP-C slows down cross-bridge kinetics when binding to actin.

Study of the interaction between rabbit cardiac contractile and regulatory proteins. An in vitro motility assay.

A series of experiments was performed in an in vitro motility assay with reconstructed thin filaments to obtain pCa-force relationships for cardiac isomyosins V1 and V3. Two concentrations of each isomyosin (200 and 300 microg/ml) on the surface of a flow cell were tested. Isometric force was estimated as the amount of actin-binding protein, alpha-actinin, stopping thin filament movement. It was found that the amount of alpha-actinin stopping the movement at saturating calcium concentration for V3 was twice higher than for V1 at both concentrations of isoforms. Hill coefficients of cooperativity (h) were determined for pCa-force relationships. The value of h did not differ significantly for isoforms at 300 microg/ml of protein (h was 1.56 for V1 and 1.54 for V3). However, the Hill coefficient was higher for V3 isoform at 200 microg/ml (h = 2.00 and 1.76 for V3 and V1, respectively). Importantly, the Hill coefficient increased for both isoenzymes when their concentrations were decreased. The connection between Hill coefficient and cooperative interactions between cardiac contractile and regulatory proteins is analyzed in detail.

[Assessment of the mechanical activity of cardiac isomyosins V1 and V3 by an in vitro motility assay with regulated thin filaments].

A series of experiments in an in vitro motility assay with reconstructed thin filaments has been performed to determine the dependence of the velocity of thin filament movement on the concentration of calcium in solution (in the pCa range from 5 to 8) for rabbit cardiac isomyosins V1 and V3. The "pCa-velocity" curves had the sigmoid form. It was found for each isoform that sliding velocities of regulated thin filaments (at the saturating calcium concentration (pCa 5)) and actin filaments did not differ from each other. The Hill coefficient was 1.04 and 0.75 for isomyosins V1 and V3, respectively. The calcium sensitivity of V3 was found to be higher than that of V1. In the framework of the same method, the relationship between the velocity of thin filament sliding and the concentration of the actin-binding protein a-actinin (analog of the "force-velocity" relationship) has been estimated for each isoform V1 and V3 at the saturating calcium concentration. The results obtained suggest that the calcium regulation of the contractile activity of isomyosins V1 and V3 occurs by different mechanisms.

[Tuberculosis and HIV infection: detection, follow-up, treatment: Moscow data].

The paper describes the follow-up and treatment of patients with tuberculosis concurrent with HIV infection in Moscow in 2004-2005. Major epidemiological parameters, such as morbidity, mortality, and prevalence of this comorbidity, are given. Analysis of these indices suggests that the epidemic situation associated with tuberculosis concurrent with HIV infection became worse in the past 2 years. As compared with 2004, in 2005 the number of such patients increased from 294 to 445, including that of first detected patients rose from 123 to 174. In this group of patients, there was a preponderance of young males aged 29 to 39 years. Most patients with this pathology suffered from drug addiction and alcoholism and other concomitant diseases. The bulk of them were unemployed and disabled. In the HIV-infected, the clinical forms of tuberculosis were severe with a predominance of acute and disseminated processes; the rate of drug resistance, including multidrug resistance, was high, which made treatment difficult and resulted in high mortality.

[Results of implementation of the subprograms "Goal-oriented medical examination of the Moscow population for early tuberculosis detection"].

The implementation of the subprograms "Goal-oriented medical examination of the Moscow population for early tuberculosis visage a differential screening of the population in relation to the risk of tuberculosis; fitting of health care facilities with current digital fluorographic equipment; improvement of an epidemiological screening of patients with tuberculosis. Over 7 years of implementation of the Program, advances were made: the population's coverage with preventive examinations increased by 11%; that of detively; the active detection of patients with respiratory abnormalities and those with pulmonary tuberculosis increased by 2.0 and 2.4 times, respectively; the proportion of tuberculosis patients actively detected rose by 1.5 times; 151 digital fluorographs and 11 X-ray units were purchased; a program for machine fluorography study accounting was worked out.

[The in vitro motility assay to study the calcium-mechanical relationship in skeletal and cardiac muscles].

In a series of experiments on regulated contractile systems (i.e., in vitro mobile systems with reconstructed thin filaments), the velocities of the movement of a thin filament on the surface covered by either rabbit skeletal or rat cardiac myosin at various concentrations of calcium ions in solution (in the pCa range from 4 to 8) were assessed. The corresponding "pCa-velocity" relationships were plotted, which proved to be of the sigmoid form. It was found that, at a saturating calcium concentration (pCa 4), the velocity of regulated thin filaments was 65% higher than for unregulated ones in the case of skeletal myosin and 87% higher than for unregulated thin filaments in the case of cardiac myosin. It was also found that the Hill coefficient was 1.95 and 2.5 for skeletal and cardiac myosins, respectively. The difference in the Hill coefficients for skeletal and cardiac myosins is discussed in terms of the difference in contribution of cooperativity mechanisms of contractile and regulatory proteins in the regulation of contraction in these types of muscles.

Effects of mechanical interaction between two rabbit cardiac muscles connected in parallel.

The hypothesis that myocardium mechanical inhomogeneity produces a substantial effect on mechanical function was tested. Muscle inhomogeneity was studied in isolated papillary muscles or trabeculae excised from rabbit right ventricle and connected in a parallel duplex. Each muscle was placed in a separate perfusion bath. One end of each muscle was fastened to an individual force transducer and the other to the common lever of a servomotor. This arrangement allowed both muscles, being excited independently, to pull jointly a load applied to the lever. Separate electrodes for each perfusion bath allowed to stimulate muscles with a time delay. Tension developed in the individual muscles and their interaction were studied. Developed tension was critically dependent on the timing and sequence of excitation. Using mathematical modeling, patterns of tension distribution experimentally observed in parallel duplexes were simulated. These results suggest that changes both in Ca(2+) transients and in the time course of Ca(2+)-troponin complexion due to the duplexed muscles interaction offset the effect of mechanical inhomogeneity.

Mathematical modelling of the contribution of mechanical inhomogeneity in the myocardium to contractile function.

Earlier we developed a mathematical model of the cardiac muscle that allowed for inactivation through the effects of cooperativity of contractile proteins. In the present work we used the model to analyze the mechanical function of an inhomogeneous myocardium. To simulate the latter we chose, as the simplest sytstem, a duplex in which muscles with different mechanical properties were connected in series and in parallel. Numerical experiments showed that the basic effect due to the inhomogeneity consists in the non-additivity of the mechanical characteristics of the muscle, e.g., of the relationship between end-systolic length and end-systolic force (Les - Pes). As a rule, non-additivity consists in a negative inotropic effect. The analysis showed that the cause of non-additivity is redistribution of loads between muscles (in a parallel duplex), redistribution of lengths (in a serial duplex), changes in the rate of contraction of each muscle compared to contraction that when working separately, shifts in time to Les. Also, the model predicts that additional inactivation of contractile proteins in a muscle within a duplex against isolation is the substantial mechanism of enhanced non-additivity. Among the factors of inhomogeneity studied the basic determinants are difference in amplitudes between isometric tensions developed by each muscle in isolation and the asynchronism in the development of these tensions.

[A device for automatic determination, calculation and storage of parameters in cardiointervalography]. / Pribor dlia avtomaticheskogo opredeleniia, rascheta i khraneniia parametrov v kardiointervalografii.

Cardiointervalography is a highly informative tool of functional diagnosis and for its performance it is better to apply special devices. This paper outlines a device for automatic determination, calculation and storage of parameters in cardiointervalography and presents its technical data.

[The characteristics of the effect of parathyroid hormone on the mechanical activity of the myocardium in rats with a deficiency of Ca and Mg in the drinking water]. / Osobennosti vliianiia paratireoidnogo gormona na mekhanicheskuiu aktivnost' miokarda krys pri defitsite Ca i Mg v pitevoi vode.

Inotropic and lusitropic effects of the parathyroid hormone (PTH) upon the rat heart ventricle myocardium were studied in conditions of calcium and magnesium deficit in drinking water. The control rats revealed two phases of the hormone effect: the positive and negative ones, whereas the experimental rats only revealed the negative inotropic effect. Both the negative and positive inotropic effects were followed by an acceleration of the relaxation.