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.

Comparison of Functional Characteristics of Myosin in Fast and Slow Skeletal Muscles.

Myosins of fast and slow skeletal muscles differ by the isoform composition of the heavy and light chains. We compared functional characteristics of myosin from the fast (m. psoas) and slow (m. soleus) muscles of rabbits. The parameters of single actin-myosin interaction were measured in an optical trap, and the characteristics of the Ca2+ regulation of actin-myosin interaction were studied using an in vitro motility assay. The duration of interaction of myosin from the fast muscle with actin was shorter and the filament sliding velocity over this myosin was higher than the corresponding parameters for myosin from the slow muscle. The dependence pCa-velocity for myosin from the fast muscle was less sensitive to Ca2+ than that of slow muscle myosin. Thus, functional properties of myosin determine not only mechanical and kinetic characteristics of muscle contraction, but also the peculiarities of its Ca2+ regulation.

Effect of Interchain Disulfide Crosslinking in the Tropomyosin Molecule on Actin-Myosin Interaction in the Atrial Myocardium.

Tropomyosin (Tpm) is one of the main regulatory proteins in the myocardium. In some heart pathologies, interchain disulfide crosslinking in the Tpm molecule occurs. In the ventricle, this change in the structural properties of the Tpm molecule affects calcium regulation of the actin-myosin interaction. Using an in vitro motility assay, we found that Tpm crosslinking does not affect the actin-myosin interaction in the atria. We assume that the intramolecular crosslinking of Tpm in the atrium does not play such a crucial role in the pathogenesis of heart failure as it plays in the heart ventricles.

Hydrogen Peroxide Treatment of Muscle Fibres Inhibits the Formation of Myosin Cross-Bridges.

The molecular mechanism of violation of the contractile function of skeletal muscles caused by oxidative damage to myosin is not fully understood. Using permeabilized fibres from fast (m. psoas) and slow (m. soleus) rabbit muscles, we studied the effect of myosin oxidation on the mechanism of force generation and its calcium regulation. It was found that this treatment simultaneously reduces the maximum force and fibers stiffness without affecting their calcium sensitivity. This suggests that the mechanism of oxidation-related impairment the force-generating ability of fibers consists in suppression of myosin cross-bridges formation and does no affect the characteristics of actin-myosin interaction.

The Effect of Experimental Hyperthyroidism on Characteristics of Actin-Myosin Interaction in Fast and Slow Skeletal Muscles.

The molecular mechanism of the failure of contractile function of skeletal muscles caused by oxidative damage to myosin in hyperthyroidism is not fully understood. Using an in vitro motility assay, we studied the effect of myosin damage caused by oxidative stress in experimental hyperthyroidism on the actin-myosin interaction and its regulation by calcium. We found that hyperthyroidism-induced oxidation of myosin is accompanied by a decrease in the sliding velocity of the regulated thin filaments in the in vitro motility assay, and this effect is increased with the duration of the pathological process.

Carbonylation of atrial myosin prolongs its interaction with actin.

Carbonylation induced by hyperthyroidism suppresses force generation of skeletal myosin and sliding velocity of actin filaments in an in vitro motility assay. However, its effects on cardiac myosin at the molecular level have not been studied. Hyperthyroidism induces a change in expression of myosin heavy chains in ventricles, which may mask the effect of oxidation. In contrast to ventricular myosin, expression of myosin heavy chains in the atrium does not change upon hyperthyroidism and enables investigation of the effect of oxidation on cardiac myosin. We studied the influence of carbonylation, a type of protein oxidation, on the motor function of atrial myosin and Ca2+ regulation of actin-myosin interaction at the level of isolated proteins and single molecules using an in vitro motility assay and an optical trap. Carbonylation of atrial myosin prolonged its attached state on actin and decreased maximal sliding velocity of thin filaments over this myosin but did not affect the calcium sensitivity of the velocity. The results indicate that carbonylation of atrial myosin induced by hyperthyroidism can be a rate-limiting factor of atrium contractility and so participates in the genesis of heart failure in hyperthyroidism.

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.

Effect of Cardiomyopathic Mutations in Tropomyosin on Calcium Regulation of the Actin-Myosin Interaction in Skeletal Muscle.

Tropomyosin plays an important role in the regulation of actin-myosin interaction in striated muscles. Mutations in the tropomyosin gene disrupt actin-myosin interaction and lead to myopathies and cardiomyopathies. Tropomyosin with mutations in the α-chain is expressed in both the myocardium and skeletal muscles. We studied the effect of mutations in the α-chain of tropomyosin related to hypertrophic (D175N and E180G) and dilated cardiomyopathies (E40K and E54K) on calcium regulation of the actin-myosin interaction in skeletal muscles. We analyzed the calcium-dependent sliding velocity of reconstructed thin filaments containing F-actin, troponin, and tropomyosin over myosin surface in an in vitro motility assay. Mutations D175N and E180G in tropomyosin increased the sliding velocity and its calcium sensitivity, while mutation E40K reduced both these parameters. E54K mutation increased the sliding velocity of thin filaments, but did not affect its calcium sensitivity.

The properties of the actin-myosin interaction in the heart muscle depend on the isoforms of myosin but not of α-actin.

In myocardium of mammals there are two isoforms of myosin heavy chains, α and ß. In ventricle, together with ventricular isoforms of light chains they form two isomyosins: V1 and V3, homodimers of α- and ß-heavy chains, respectively. In atria, α- and ß-heavy chains together with atrial light chains form A1 (αα) and A2 (ßß) isomyosins. Besides in myocardium two isoforms of α-actin, skeletal and cardiac, are expressed. We assume that the differences in the amino acid sequence of cardiac and skeletal actin may affect its interaction with myosin. To test this hypothesis, we investigated characteristics of actin-myosin interactions of cardiac and skeletal isoforms of α-actin with the isoforms of cardiac myosin using an optical trap technique and an in vitro motility assay. It was found that the mechanical and kinetic characteristics of the interactions of the isoforms of cardiac myosin with actin depend on the isoforms of myosin not α-actin.

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.

Stabilization of the Central Part of Tropomyosin Molecule Alters the Ca2+-sensitivity of Actin-Myosin Interaction.

We show that the mutations D137L and G126R, which stabilize the central part of the tropomyosin (Tm) molecule, increase both the maximal sliding velocity of the regulated actin filaments in the in vitro motility assay at high Са(2+) concentrations and the Са(2+)-sensitivity of the actin-myosin interaction underlying this sliding. Based on an analysis of the recently published data on the structure of the actin-Tm-myosin complex, we suppose that the physiological effects of these mutations in Tm can be accounted for by their influence on the interactions between the central part of Tm and certain sites of the myosin head.

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.

Tripeptide Pro-Gly-Pro prevents disturbances in osmotic resistance of rat erythrocytes under conditions of inflammation.

The development of inflammation (experimental model of peritonitis induced by administration of sodium thioglycolate) was accompanied by a decrease in osmotic resistance of erythrocytes. Changes in osmotic resistance of erythrocytes associated with preliminary (15 min before induction of inflammation) administration of peptide Pro-Gly-Pro were significantly weaker, and the percentage of hemolyzed cells was reduced. The peptide injected against the background of developed inflammation (1 h 45 min after induction) had no corrective effect on osmotic resistance. During in vitro experiments, Pro-Gly-Pro did not affect hemolysis of intact erythrocytes. These results support the assumption that prophylactic administration of the peptide protects erythrocyte membranes and increases their osmotic resistance.

Effects of Prolyl-Glycyl-Proline (PGP) peptide on disorders in rat mesenteric lymphatic vessel function induced by injection of substance 48/80.

Injection of substance 48/80 to rats led to dysfunction of mesenteric lymphatic microvessels, in particular inhibition of their contractility and modification of their reaction to norepinephrine. Injection of PGP peptide before and after substance 48/80 alleviated these disorders. The results indicated the possibility of peptide correction of lymphatic vessel dysfunction.

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.

Effect of acute and moderate repeated stress on disturbances in reactivity of mesenteric lymphatic vessels during inflammation in rats.

We studied the effect of acute (single immobilization for 1 h) and repeated (daily immobilization for 1 min, 5 days) moderate stress on disturbances in contractility of mesenteric lymphatic vessels in rats with experimental peritonitis. Acute stress was shown to potentiate, while moderate repeated stress attenuate the effect of inflammatory stimulus. It can be hypothesized that moderate repeated stress improves adaptive capacities of the organism, which manifests in reduction or prevention of dysfunction in contractile activity of lymphatic vessels.

Protective and therapeutic effects of glyprolines in psychoemotional stress induced by cholecystokinin-4 injection.

Experiments on outbred albino male rats showed that psychoemotional stress induced by intraperitoneal injection of cholecystokinin-4 (100 microg/kg) increased anxiety, impaired orientation and exploration activities in the elevated plus-maze and hole-board tests, and increased the level of depression of Porsolt test. Preliminary intranasal administration of glyprolines (15 min before cholecystokinin) in a dose of 3.7 micromol/kg prevented the development of stress-induced behavioral disturbances. Administration of peptides 30 min after cholecystokinin-4, i.e., to rats with developed behavioral disturbances, almost completely abolished these disturbances.

[A comparative analysis of distribution of glyprolines administered by various routes].

The distribution of the glyprolines Pro-Gly-Pro and Thr-Lys-Pro-Arg-Pro-Gly-Pro (Selanc) was analyzed and compared in tissues of rat organs after different ways of their administration using the peptides uniformly labeled with tritium. Comparative data on changes in concentrations of the peptides in the rat organs after their intraperitoneal, intranasal, intragastric, and intravenous administration are given. The intranasal administration of both peptides was shown to be optimal for the delivery of glyproline molecules in the CNS. A high affinity of the studied glyprolines for gastric tissues was found for all the ways of their administration. We suggest that a high efficiency of action of glyprolines on homeostasis of the gastric mucous tunic was partially provided by accumulation of these peptides (to high concentrations) in gastric tissues.

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.