Analysis of changes in the rat cardiovascular system under the action of lead intoxication and muscular exercise

Introduction. One of the risk factors for cardiovascular diseases is the toxic metal pollution of the industrial area and the environment. Lead is the most critical of toxic metals. In industrial conditions, the body’s exposure to harmful substances is often combined with muscular work of varying severity. It has not been studied enough how these combinations influence the development of pathological processes associated with harmful exposure. Materials and methods. The subchronic experiment was carried out on white outbred male rats for six weeks. Intoxication was simulated by repeated intraperitoneal injections of lead acetate three times a week. Running was chosen to model the muscle exercise at a 25 m/min speed for 10 minutes 5 days a week. We performed biochemical and electrocardiographic studies. Blood pressure parameters were recorded. Muscle contractility was studied on isolated multicellular preparations of the right ventricular myocardium in isometric and physiological contraction modes. The ratio of myosin heavy chains was determined by the polyacrylamide gel electrophoresis. The sliding velocity of reconstituted thin filaments on myosin using an in vitro motility assay. Results. Physical exercise under lead intoxication normalized the level of calcium and the angiotensin-converting enzyme activity in the blood serum, the voltage of the isoelectric line and the amplitude of the T wave on the electrocardiogram. The combined action of lead and physical exercise showed an increase in the creatinine kinase-MB level. We found that the effect of exercise under lead intoxication on myocardial contractility was ambiguous. The maximum isotonic shortening velocity in trabeculae was normalized, but the maximum rate of strength development in the isometric mode in the papillary muscles decreased to a greater extent than under lead intoxication. The maximum sliding velocity of reconstituted thin filaments and myosin and the heavy chain ratio was partly normalized. Conclusion. In general, muscle exercise attenuated the lead cardiotoxic effects.

Investigation of the molecular motor of muscle: from generating life in a test tube to myosin structure over beers

This article traces 60 years of investigation of the molecular motor of skeletal muscle from the 1940s through the 1990s. It started with the discovery that myosin interaction with actin in the presence of ATP caused shortening of threads of actin and myosin. In 1957, structures protruding from myosin filaments were seen for the first time and called “cross bridges.” A combination of techniques led to the proposal in 1969 of the “swinging-tilting cross bridge” model of contraction. In the early 1980s, a major problem arose when it was shown that a probe attached to the cross bridges did not move during contraction. A spectacular breakthrough came when it was discovered that only the cross bridge was required to support movement in an in vitro motility assay. Next it was determined that single myosin molecules caused the movement of actin filaments in 10-nm steps. The atomic structure of the cross bridge was published in 1993, and this discovery supercharged the muscle field. The cross bridge contained a globular head or motor domain that bound actin and ATP. But the most striking feature was the long tail of the cross bridge surrounded by two subunits of the myosin molecule. This structure suggested that the tail might act as a lever arm magnifying head movement. Consistent with this proposal, genetic techniques that lengthened the lever arm resulted in larger myosin steps. Thus the molecular motor of muscle operated not by the tilting of the globular head of myosin but by tilting of the lever arm generating the driving force for contraction.

A Troponin T Variant Linked with Pediatric Dilated Cardiomyopathy Reduces the Coupling of Thin Filament Activation to Myosin and Calcium Binding

Dilated cardiomyopathy (DCM) is a significant cause of pediatric heart failure. Mutations in proteins that regulate cardiac muscle contraction can cause DCM; however, the mechanisms by which molecular-level mutations contribute to cellular dysfunction are not well-understood. Better understanding of these mechanisms might enable the development of targeted therapeutics that benefit patient subpopulations with mutations that cause common biophysical defects. We examined the molecular- and cellular-level impacts of a troponin T variant associated with pediatric-onset DCM, R134G. The R134G variant decreased calcium sensitivity in an in vitro motility assay. Using stopped-flow and steady-state fluorescence measurements, we determined the molecular mechanism of the altered calcium sensitivity: R134G decouples calcium binding by troponin from the closed-to-open transition of the thin filament and decreases the cooperativity of myosin binding to regulated thin filaments. Consistent with the prediction that these effects would cause reduced force per sarcomere, cardiomyocytes carrying the R134G mutation are hypocontractile. They also show hallmarks of DCM that lie downstream of the initial insult, including disorganized sarcomeres and cellular hypertrophy. These results reinforce the importance of multiscale studies to fully understand mechanisms underlying human disease and highlight the value of mechanism-based precision medicine approaches for DCM.

Impact of regulatory light chain mutation K104E on the ATPase and motor properties of cardiac myosin

Mutations in the cardiac myosin regulatory light chain (RLC, MYL2 gene) are known to cause inherited cardiomyopathies with variable phenotypes. In this study, we investigated the impact of a mutation in the RLC (K104E) that is associated with hypertrophic cardiomyopathy (HCM). Previously in a mouse model of K104E, older animals were found to develop cardiac hypertrophy, fibrosis, and diastolic dysfunction, suggesting a slow development of HCM. However, variable penetrance of the mutation in human populations suggests that the impact of K104E may be subtle. Therefore, we generated human cardiac myosin subfragment-1 (M2β-S1) and exchanged on either the wild type (WT) or K104E human ventricular RLC in order to assess the impact of the mutation on the mechanochemical properties of cardiac myosin. The maximum actin-activated ATPase activity and actin sliding velocities in the in vitro motility assay were similar in M2β-S1 WT and K104E, as were the detachment kinetic parameters, including the rate of ATP-induced dissociation and the ADP release rate constant. We also examined the mechanical performance of α-cardiac myosin extracted from transgenic (Tg) mice expressing human wild type RLC (Tg WT) or mutant RLC (Tg K104E). We found that α-cardiac myosin from Tg K104E animals demonstrated enhanced actin sliding velocities in the motility assay compared with its Tg WT counterpart. Furthermore, the degree of incorporation of the mutant RLC into α-cardiac myosin in the transgenic animals was significantly reduced compared with wild type. Therefore, we conclude that the impact of the K104E mutation depends on either the length or the isoform of the myosin heavy chain backbone and that the mutation may disrupt RLC interactions with the myosin lever arm domain.

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 β–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 and gelsolin cooperate in actin filament severing and actomyosin motor activity

Actin is a major intracellular protein with key functions in cellular motility, signaling and structural rearrangements. Its dynamic behavior, such as polymerisation and depolymerisation of actin filaments in response to intra- and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin-binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

Impact of A134 and E218 Amino Acid Residues of Tropomyosin on Its Flexibility and Function

Tropomyosin (Tpm) is one of the major actin-binding proteins that play a crucial role in the regulation of muscle contraction. The flexibility of the Tpm molecule is believed to be vital for its functioning, although its role and significance are under discussion. We choose two sites of the Tpm molecule that presumably have high flexibility and stabilized them with the A134L or E218L substitutions. Applying differential scanning calorimetry (DSC), molecular dynamics (MD), co-sedimentation, trypsin digestion, and in vitro motility assay, we characterized the properties of Tpm molecules with these substitutions. The A134L mutation prevented proteolysis of Tpm molecule by trypsin, and both substitutions increased the thermal stability of Tpm and its bending stiffness estimated from MD simulation. None of these mutations affected the primary binding of Tpm to F-actin; still, both of them increased the thermal stability of the actin-Tpm complex and maximal sliding velocity of regulated thin filaments in vitro at a saturating Ca2+ concentration. However, the mutations differently affected the Ca2+ sensitivity of the sliding velocity and pulling force produced by myosin heads. The data suggest that both regions of instability are essential for correct regulation and fine-tuning of Ca2+-dependent interaction of myosin heads with F-actin.

Myosin-gelsolin cooperativity in actin filament severing and actomyosin motor activity

Actin is a major intracellular protein with key functions in cellular motility, signalling and structural rearrangements. Its dynamic behavior with actin filaments (F-actin) polymerising and depolymerising in response to intracellular changes, is controlled by actin-binding proteins (ABPs). Gelsolin is one of the most potent filament severing ABPs. However, myosin motors that interact with actin in the presence of ATP also produce actin filament fragmentation through motor induced shearing forces. To test the idea that gelsolin and myosin cooperate in these processes we used the in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin (5 nM) at very low [Ca2+] (free [Ca2+] ∼6.8 nM) appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM [MgATP], an effect that was increased at increased HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. As further support of myosin-gelsolin cooperativity we observed reduced sliding velocity of the HMM propelled filaments in the presence of gelsolin. Overall, the results corroborate ideas for cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity, leading among other effects to enhanced F-actin severing of possible physiological relevance.

Kinesin-14 motors drive a right-handed helical motion of antiparallel microtubules around each other

Within the mitotic spindle, kinesin motors cross-link and slide overlapping microtubules. Some of these motors exhibit off-axis power strokes, but their impact on motility and force generation in microtubule overlaps has not been investigated. Here, we develop and utilize a three-dimensional in vitro motility assay to explore kinesin-14, Ncd, driven sliding of cross-linked microtubules. We observe that free microtubules, sliding on suspended microtubules, not only rotate around their own axis but also move around the suspended microtubules with right-handed helical trajectories. Importantly, the associated torque is large enough to cause microtubule twisting and coiling. Further, our technique allows us to measure the in situ spatial extension of the motors between cross-linked microtubules to be about 20 nm. We argue that the capability of microtubule-crosslinking kinesins to cause helical motion of overlapping microtubules around each other allows for flexible filament organization, roadblock circumvention and torque generation in the mitotic spindle.

CHANGES OF MYOCARDIUM CONTRACTILITY ASSOCIATED WITH A SUBCHRONIC LEAD INTOXICATION IN RATS

Introduction. There is a high chance of a link between cardiovascular conditions and occupational or environmental exposure to lead. Taking into account the peculiarities of lead intoxication and the metal common occurrence it appeared to necessarily prove further experimental research of lead cardiotoxicity. Material and methods. After repeated intraperitoneal administration of sublethal doses of lead acetate to outbred male rats 3 times a week for 5 weeks, there was obtained the moderately pronounced subchronic lead intoxication manifested by some characteristic features. Cardiotoxic effects on myocardial contractility were studied by the analysis of the mechanical activity of isolated preparations of right ventricular trabeculae and papillary muscles contracting in isotonic and physiological modes of loading. Myocardial contractile function was also studied at the molecular level by measuring the sliding velocity of reconstructed thin filaments over myosin. Results. In papillary muscles lead intoxication led to a decrease in the maximal rate of isotonic shortening for all afterloads and a decrease in the thin filament sliding velocity in the in vitro motility assay. The same type of muscle 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 similarly exposed to lead while being treated with a Ca-containing bio protector. The amount of work produced by both muscle preparations was unchanged under lead intoxication over the entire range of afterloads, which is an evidence of adaptation to the production of adequate mechanical work despite resulting contractility disturbances. Conclusions. 1. Subchronic lead intoxication was shown to cause contractile dysfunction of rat myocardium. In papillary muscles the alterations were observed more than in trabeculae. The changes in contractile proteins corresponded with those seen in myocardium structures. 2. The reported changes were attenuated to some extent in rats being treated with a Ca-containing bio protector.