Changes in the Cardiotoxic Effects of Lead Intoxication in Rats Induced by Muscular Exercise.

Exposure to lead is associated with an increased risk of cardiovascular diseases. Outbred white male rats were injected with lead acetate intraperitoneally three times a week and/or were forced to run at a speed of 25 m/min for 10 min 5 days a week. We performed noninvasive recording of arterial pressure, electrocardiogram and breathing parameters, and assessed some biochemical characteristics. Electrophoresis in polyacrylamide gel was used to determine the ratio of myosin heavy chains. An in vitro motility assay was employed to measure the sliding velocity of regulated thin filaments on myosin. Isolated multicellular preparations of the right ventricle myocardium were used to study contractility in isometric and physiological modes of contraction. Exercise under lead intoxication normalized the level of calcium and activity of the angiotensin-converting enzyme in the blood serum, normalized the isoelectric line voltage and T-wave amplitude on the electrocardiogram, increased the level of creatine kinase-MB and reduced the inspiratory rate. Additionally, the maximum sliding velocity and the myosin heavy chain ratio were partly normalized. The effect of exercise under lead intoxication on myocardial contractility was found to be variable. In toto, muscular loading was found to attenuate the effects of lead intoxication, as judged by the indicators of the cardiovascular system.

Comparative structural and functional studies of low molecular weight tropomyosin isoforms, the TPM3 gene products.

Tropomyosin (Tpm) is an actin-associated protein and key regulator of actin filament structure and dynamics in muscle and non-muscle cells where it participates in many vital processes. Human non-muscle cells produce many Tpm isoforms; however, little is known yet about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five low molecular weight Tpm isoforms (Tpm3.1, Tpm3.2, Tpm3.4, Tpm3.5, and Tpm3.7), the products of TPM3 gene, which significantly differ by alternatively spliced internal exon 6 (6a or 6b) and C-terminal exon 9 (9a, 9c or 9d). Our results clearly demonstrate that the properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. These differences can be important in further studies to explain why these Tpm isoforms play a key role in organization and dynamics of the cytoskeleton.

Cardioinotropic Effects in Subchronic Intoxication of Rats with Lead and/or Cadmium Oxide Nanoparticles.

Subchronic intoxication was induced in outbred male rats by repeated intraperitoneal injections with lead oxide (PbO) and/or cadmium oxide (CdO) nanoparticles (NPs) 3 times a week during 6 weeks for the purpose of examining its effects on the contractile characteristics of isolated right ventricle trabeculae and papillary muscles in isometric and afterload contractions. Isolated and combined intoxication with these NPs was observed to reduce the mechanical work produced by both types of myocardial preparation. Using the in vitro motility assay, we showed that the sliding velocity of regulated thin filaments drops under both isolated and combined intoxication with CdO-NP and PbO-NP. These results correlate with a shift in the expression of myosin heavy chain (MHC) isoforms towards slowly cycling ß-MHC. The type of CdO-NP + PbO-NP combined cardiotoxicity depends on the effect of the toxic impact, the extent of this effect, the ratio of toxicant doses, and the degree of stretching of cardiomyocytes and muscle type studied. Some indices of combined Pb-NP and CdO-NP cardiotoxicity and general toxicity (genotoxicity included) became fully or partly normalized if intoxication developed against background administration of a bioprotective complex.

Myosin from the ventricle is more sensitive to omecamtiv mecarbil than myosin from the atrium.

Omecamtiv mecarbil (OM), an activator of cardiac myosin, strongly affects contractile characteristics of the ventricles and, to a much lesser extent, the characteristics of atrial contraction. We compared the molecular mechanism of action of OM on the interaction of atrial and ventricular myosin with actin using an optical trap and an in vitro motility assay. In concentrations up to 0.5 µM, OM did not affect the step size of a myosin molecule but reduced it at a higher OM level. OM substantially prolonged the interaction of both isoforms of myosin with actin. However, the interaction characteristics of ventricular myosin with actin were more sensitive to OM than those of atrial myosin. Our results, obtained at the level of isolated proteins, can explain why the impact of OM in therapeutic concentrations on the contractile function of the atrium is less significant as compared to those of the ventricle.

Cardiomyopathy-associated mutations in tropomyosin differently affect actin-myosin interaction at single-molecule and ensemble levels.

In the heart, mutations in the TPM1 gene encoding the α-isoform of tropomyosin lead, in particular, to the development of hypertrophic and dilated cardiomyopathies. We compared the effects of hypertrophic, D175N and E180G, and dilated, E40K and E54K, cardiomyopathy mutations in TPM1 gene on the properties of single actin-myosin interactions and the characteristics of the calcium regulation in an ensemble of myosin molecules immobilised on a glass surface and interacting with regulated thin filaments. Previously, we showed that at saturating Ca2+ concentration the presence of Tpm on the actin filament increases the duration of the interaction. Here, we found that the studied Tpm mutations differently affected the duration: the D175N mutation reduced it compared to WT Tpm, while the E180G mutation increased it. Both dilated mutations made the duration of the interaction even shorter than with F-actin. The duration of the attached state of myosin to the thin filament in the optical trap did not correlate to the sliding velocity of thin filaments and its calcium sensitivity in the in vitro motility assay. We suppose that at the level of the molecular ensemble, the cooperative mechanisms prevail in the manifestation of the effects of cardiomyopathy-associated mutations in Tpm.

The interchain disulfide cross-linking of tropomyosin alters its regulatory properties and interaction with actin filament.

Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein that plays a key role in the Ca2+-regulated contraction of striated muscles. Two chains of Tpm can be cross-linked by formation of a disulfide bond between Cys-190 residues. Normally, the SH-groups of these residues in cardiac muscle are in reduced state but in heart pathologies the interchain cross-linking of Tpm was shown to occur. Previous studies have shown that this cross-linking increases the thermal stability of the C-terminal part of the Tpm molecule. However it was unclear how this affects its functional properties. In the current work, we studied functional features of cross-linked Tpm at the level of isolated proteins. The results have shown that the cross-linking greatly decreases affinity of Tpm for F-actin and stability of the Tpm-F-actin complex. It also increases sliding velocity of regulated thin filaments in an in vitro motility assay. This last effect was mostly pronounced when cardiac isoforms of myosin and troponin were used instead of skeletal ones. The results indicate that cross-linking significantly affects properties of Tpm and actin-myosin interaction and can explain, at least partly, the role of the interchain disulfide cross-linking of cardiac Tpm in human heart diseases.

Cooperativity of myosin interaction with thin filaments is enhanced by stabilizing substitutions in tropomyosin.

Muscle contraction is powered by myosin interaction with actin-based thin filaments containing Ca2+-regulatory proteins, tropomyosin and troponin. Coiled-coil tropomyosin molecules form a long helical strand that winds around actin filament and either shields actin from myosin binding or opens it. Non-canonical residues G126 and D137 in the central part of tropomyosin destabilize its coiled-coil structure. Their substitutions for canonical ones, G126R and D137L, increase structural stability and the velocity of sliding of reconstructed thin filaments along myosin coated surface. The effect of these stabilizing mutations on force of the actin-myosin interaction is unknown. It also remains unclear whether the stabilization affects single actin-myosin interactions or it modifies the cooperativity of the binding of myosin molecules to actin. We used an optical trap to measure the effects of the stabilization on step size, unitary force and duration of the interactions at low and high load and compared the results with those obtained in an in vitro motility assay. We found that significant prolongation of lifetime of the actin-myosin complex under high load observed at high extent of tropomyosin stabilization, i.e. with double mutant, G126R/D137L, correlates with higher force in the motility assay. Also, the higher the extent of stabilization of tropomyosin, the fewer myosin molecules are needed to propel the thin filaments. The data suggest that the effects of the stabilizing mutations in tropomyosin on the myosin interaction with regulated thin filaments are mainly realized via cooperative mechanisms by increasing the size of cooperative unit.

Stabilizing the central part of tropomyosin increases the bending stiffness of the thin filament.

A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness.

The lifetime of the actomyosin complex in vitro under load corresponding to stretch of contracting muscle.

During eccentric contraction, muscle is lengthening so that the actin-myosin cross-bridges bear a load that exceeds the force they generate during isometric contraction. Using the optical trap technique, we simulated eccentric contraction at the single molecule level and investigated the effect of load on the skeletal actomyosin lifetime at different ATP concentrations. The range of the loads was up to 17 pN above the isometric level. We found that the frequency distribution of the lifetime of the actin-bound state of the myosin molecule was biphasic: it quickly rose and then decreased slowly. The rate of the slow phase of this distribution increased with both the load and the ATP concentration. The fast phase accelerated sharply with the load, but it was independent of ATP concentration. The presence of the fast phase demonstrates that some transition(s) in the actomyosin complex occur before the myosin head becomes able to bind ATP and detach from actin. Its high sensitivity to the load indicates that the transition is load-dependent.

Pseudo-phosphorylation of essential light chains affects the functioning of skeletal muscle myosin.

The work aimed to investigate how the phosphorylation of the myosin essential light chain of fast skeletal myosin (LC1) affects the functional properties of the myosin molecule. Using mass-spectrometry, we revealed phosphorylated peptides of LC1 in myosin from different fast skeletal muscles. Mutations S193D and T65D that mimic natural phosphorylation of LC1 were produced, and their effects on functional properties of the entire myosin molecule and isolated myosin head (S1) were studied. We have shown that T65D mutation drastically decreased the sliding velocity of thin filaments in an in vitro motility assay and strongly increased the duration of actin-myosin interaction in optical trap experiments. These effects of T65D mutation in LC1 observed only with the whole myosin but not with S1 were prevented by double T65D/S193D mutation. The T65D and T65D/S193D mutations increased actin-activated ATPase activity of S1 and decreased ADP affinity for the actin-S1 complex. The results indicate that pseudo-phosphorylation of LC1 differently affects the properties of the whole myosin molecule and its isolated head. Also, the results show that phosphorylation of LC1 of skeletal myosin could be one more mechanism of regulation of actin-myosin interaction that needs further investigation.

Molecular mechanisms of mechanical function changes of the rat myocardium under subchronic lead exposure.

A moderate degree of lead intoxication was observed in male rats after repeated intraperitoneal injections with two doses of lead acetate three times a week during 5 (12.5 mg of Pb per kg body mass) and 6 (6.01 mg of Pb per kg body mass) weeks. Using an in vitro motility assay, we investigated the impact of this intoxication on the characteristics of actin-myosin interaction and its regulation in the atria, right, and left ventricles. Both lead doses exposure decreased the maximum sliding velocity of reconstituted thin filaments over myosin and fraction of motile filaments in all heart chambers, caused the myosin isoforms shift towards slower ß-myosin heavy chains in ventricles and decreased regulatory light chain phosphorylation in atria. No statistically significant difference was found in force and calcium regulation of actin-myosin interaction. A dose-dependent effect of lead on myosin functional characteristics was found in all heart chambers, but the degree of this effect varied depending on the heart chamber.

Force-velocity characteristics of isolated myocardium preparations from rats exposed to subchronic intoxication with lead and cadmium acting separately or in combination.

This investigation continues our study of the effects of Pb-Cd poisoning on the heart, extending the enquiry from isometric to auxotonic contractions, thereby examining the effect on the ability of myocardial tissues to perform mechanical work. Different shifts were revealed in myocardial force-velocity relations following subchronic exposure of rats to lead acetate and cadmium chloride acting separately, in combination, or in combination with a bioprotective complex (BPC). The experiments were conducted on isolated preparations of trabecules and papillary muscles of the right ventricle in physiological loading conditions and on isolated heart muscle contractile proteins examined by the in vitro motility assay. The results of the latter correlate with the shifts in the ratio of cardiac myosin isoforms. The amount of work performed by the myocardium was calculated on the basis of the tension-shortening loop area and was found to be similar in the preparations from all experimental groups. This fact presumably reflects adaptive capacity of the myocardial function even when contractility is damaged due to the metallic intoxication of a moderate severity. Some characteristics of rat myocardium altered by the impact of lead-cadmium intoxication became fully or partly normalized if intoxication developed against background administration of a bioprotective complex (BPC). Together with previously reported results obtained in the isometric mode of contractility, all these results strengthen the scientific foundations of risk assessment and risk management projects in the occupational and environmental conditions characterized by human exposure to lead and/or cadmium.

Changes in rat myocardium contractility under subchronic intoxication with lead and cadmium salts administered alone or in combination.

Subchronic intoxications induced in male rats by repeated intraperitoneal injections of lead acetate and cadmium chloride, administered either alone or in combination, are shown to affect the biochemical, cytological and morphometric parameters of blood, liver, heart and kidneys. The single twitch parameters of myocardial trabecular and papillary muscle preparations were measured in the isometric regime to identify changes in the heterometric (length-force) and chronoinotropic (frequency-force) contractility regulation systems. Differences in the responses of these systems in trabecules and papillary muscles to the above intoxications are shown. A number of myocardium mechanical characteristics changing in rats under the effect of a combined lead-cadmium intoxication and increased proportion of α-myosin heavy chains were observed to normalize fully or partially if such intoxication was induced against background administration of a proposed bioprotective complex. Based on the experimental results and literature data, some assumptions are suggested concerning the mechanisms of the cardiotoxic effects produced by lead and cadmium.

The effects of cardiomyopathy-associated mutations in the head-to-tail overlap junction of α-tropomyosin on its properties and interaction with actin.

Tropomyosin (Tpm) plays a crucial role in the regulation of muscle contraction by controlling actin-myosin interaction. Tpm coiled-coil molecules bind each other via overlap junctions of their N- and C-termini and form a semi-rigid strand that binds the helical surface of an actin filament. The high bending stiffness of the strand is essential for high cooperativity of muscle regulation. Point mutations M8R and K15N in the N-terminal part of the junction and the A277V one in the C-terminal part are associated with dilated cardiomyopathy, while the M281T and I284V mutations are related to hypertrophic cardiomyopathy. To reveal molecular mechanism(s) underlying these pathologies, we studied the properties of recombinant Tpm carrying these mutations using several experimental approaches and molecular dynamic simulation of the junction. The M8R and K15N mutations weakened the interaction between the N- and C-termini of Tpm in the overlap junction and reduced the Tpm affinity for actin. These changes possibly led to a reduction in the regulation cooperativity. The C-terminal mutations caused only small and controversial changes in properties of Tpm and its complex with actin. Their involvement in disease phenotype is possibly caused by interaction with other sarcomere proteins.

Further analysis of rat myocardium contractility changes associated with a subchronic lead intoxication.

A moderate subchronic lead intoxication was observed in male rats after repeated intraperitoneal injections of lead acetate. Right ventricular trabeculae and papillary muscles were isolated for in vitro studying of the contraction-relaxation cycle under isotonic and physiological loading. The contractile function of the myocardium was also assessed by measuring the velocity of thin filament movement over myosin. Lead intoxication led in papillary muscles to a decrease in the maximal rate of isotonic shortening for all afterloads and a decrease in the thin filament sliding velocity. Papillary muscles from lead-exposed rats displayed marked changes in most of the main characteristics of afterload contraction-relaxation cycles, but in trabeculae these changes were less pronounced. The reported changes were attenuated to some extent in rats treated with a Ca-containing bioprotector. The amount of work produced by both types of heart muscle preparations was not changed by lead. Only in papillary muscles the load-dependent relaxation index was significantly increased in the lead-treated groups. Thus subchronic lead intoxication affects the peak rate of force development and relaxation properties of cardiac muscle contracting in isotonic/physiological regimes rather than the total amount of mechanical work, which may reflect adaptive changes in the myocardial function under decreased contractility.