Actin-myosin Interaction Research Articles

Matching Mechanics and Energetics of Muscle Contraction Suggests Unconventional Chemomechanical Coupling during the Actin-Myosin Interaction.

The mechanical performances of the vertebrate skeletal muscle during isometric and isotonic contractions are interfaced with the corresponding energy consumptions to define the coupling between mechanical and biochemical steps in the myosin-actin energy transduction cycle. The analysis is extended to a simplified synthetic nanomachine in which eight HMM molecules purified from fast mammalian skeletal muscle are brought to interact with an actin filament in the presence of 2 mM ATP, to assess the emergent properties of a minimum number of motors working in ensemble without the effects of both the higher hierarchical levels of striated muscle organization and other sarcomeric, regulatory and cytoskeleton proteins. A three-state model of myosin-actin interaction is able to predict the known relationships between energetics and transient and steady-state mechanical properties of fast skeletal muscle either in vivo or in vitro only under the assumption that during shortening a myosin motor can interact with two actin sites during one ATP hydrolysis cycle. Implementation of the molecular details of the model should be achieved by exploiting kinetic and structural constraints present in the transients elicited by stepwise perturbations in length or force superimposed on the isometric contraction.

Double-headed binding of myosin II to F-actin shows the effect of strain on head structure

Force production in muscle is achieved through the interaction of myosin and actin. Strong binding states in active muscle are associated with Mg·ADP bound to the active site; release of Mg·ADP allows rebinding of ATP and dissociation from actin. Thus, Mg·ADP binding is positioned for adaptation as a force sensor. Mechanical loads on the lever arm can affect the ability of myosin to release Mg·ADP but exactly how this is done is poorly defined. Here we use F-actin decorated with double-headed smooth muscle myosin fragments in the presence of Mg·ADP to visualize the effect of internally supplied tension on the paired lever arms using cryoEM. The interaction of the paired heads with two adjacent actin subunits is predicted to place one lever arm under positive and the other under negative strain. The converter domain is believed to be the most flexible domain within myosin head. Our results, instead, point to the segment of heavy chain between the essential and regulatory light chains as the location of the largest structural change. Moreover, our results suggest no large changes in the myosin coiled coil tail as the locus of strain relief when both heads bind F-actin. The method would be adaptable to double-headed members of the myosin family. We anticipate that the study of actin-myosin interaction using double-headed fragments enables visualization of domains that are typically noisy in decoration with single-headed fragments.

Estimation of crossbridge-state during cardiomyocyte beating using second harmonic generation.

Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and others. Here, we established an assay system to dynamically measure second harmonic generation (SHG) anisotropy derived from myosin filaments depended on their crossbridge status in pulsating cardiomyocytes. Experiments utilizing an inheritable mutation that induces excessive myosin-actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost the force-generating ability upon myocardial differentiation. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in a Drosophila disease model. Thus, we successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on cardiomyocytes. Because genomic inspection alone may not catch the risk of cardiomyopathy in some cases, our study demonstrated herein would be of help in the risk assessment of future heart failure.

Small molecule drugs to improve sarcomere function in those with acquired and inherited myopathies.

Muscle weakness is a hallmark of inherited or acquired myopathies. It is a major cause of functional impairment and can advance to life-threatening respiratory insufficiency. During the past decade, several small-molecule drugs that improve the contractility of skeletal muscle fibers have been developed. In this review, we provide an overview of the available literature and the mechanisms of action of small-molecule drugs that modulate the contractility of sarcomeres, the smallest contractile units in striated muscle, by acting on myosin and troponin. We also discuss their use in the treatment of skeletal myopathies. The first of three classes of drugs discussed here increase contractility by decreasing the dissociation rate of calcium from troponin and thereby sensitizing the muscle to calcium. The second two classes of drugs directly act on myosin and stimulate or inhibit the kinetics of myosin-actin interactions, which may be useful in patients with muscle weakness or stiffness.NEW & NOTEWORTHY During the past decade, several small molecule drugs that improve the contractility of skeletal muscle fibers have been developed. In this review, we provide an overview of the available literature and the mechanisms of action of small molecule drugs that modulate the contractility of sarcomeres, the smallest contractile units in striated muscle, by acting on myosin and troponin.

A Preliminary Observation Study to Evaluate Safety and Efficacy of Boheco Ease in Myalgia

One of the most frequent concerns at emergency departments and clinics across the nation is muscle soreness and fatigue. Oftentimes, severe pain and fatigue subside on their own or seem to do so in response to several medications and therapeutic methods. Excitation-contraction coupling in the muscle, and actin-myosin filament interactions, decreased function can lead to failure of voluntary muscle contraction. The two most powerful Phyto-cannabinoids found in the Cannabis sativa plant are CBD (cannabidiol) and delta-9-tetrahydrocannabinol (THC). CBD was effectively delivered trans dermally for anti-inflammatory activity in various animals. Cannabis sativa or Vijaya has Shulahara property as per Ayurveda. Muscle pain and Swelling are caused by a rise in the Ruksha and Sheeta qualities of the Vata dosha and accumulation of Kleda. Cannabis sativa, also known as Vijaya, has Ushna properties that have the power to reduce Vata dosha. It has Pachak property which helps to mitigate Kleda. Oil tends to decrease Rukshata due to Vata dosha. As a result, reducing pain and inflammation which also lessens discomfort and irritation. Methods: A preliminary survey on 30 patients was conducted and data was collected. Results: More than 90% of patients had reduced symptoms of pain and inflammation in muscles, post-application twice a day with a lasting effect of more than 3 hours. It helps in reducing Kleda present in muscles. Conclusions: Ease oil was having a better result in patients having myalgia. It was safe on use for long term.

Influence of 2,3-Butanedione Monoxime on the Actin–Myosin Interaction in Modeling Healthy Function and Congenital Myopathy in a Single Muscle Fiber

Influence of 2,3-Butanedione Monoxime on the Actin–Myosin Interaction in Modeling Healthy Function and Congenital Myopathy in a Single Muscle Fiber

Skeletal muscle single fiber force production declines early in juvenile male mice with chronic kidney disease.

Children with chronic kidney disease (CKD) frequently exhibit delayed physical development and reduced physical performance, presumably due to skeletal muscle dysfunction. However, the cellular and molecular basis of skeletal muscle impairment in juvenile CKD remains poorly understood. Cellular (single fiber) and molecular (myosin-actin interactions and myofilament properties) function was examined ex vivo in slow (soleus) and fast (extensor digitorum longus) contracting muscles of juvenile male (6 weeks old) CKD and control mice. CKD was induced by 0.2% adenine diet for 3 weeks starting at 3 weeks of age. Specific tension (maximal isometric force divided by cross-sectional area) was reduced in larger myosin heavy chain (MHC) I and IIA fibers and in all IIB fibers in juvenile male mice with CKD due to fewer strongly bound myosin-actin cross-bridges. Fiber cross-sectional area in juvenile CKD mice was unchanged in MHC I and IIB fibers and increased in MHC IIA fibers, compared to controls. CKD slowed cross-bridge kinetics (slower rate of myosin force production and longer myosin attachment time, ton ) in MHC IIA fibers, and accelerated kinetics (shorter ton ) in MHC IIB fibers, which may indicate fiber type dependent shifts in contractile velocity in juvenile CKD. Overall, our findings show that single fiber myopathy is an early event during juvenile CKD, manifesting prior to the development of cellular atrophy as reduced force generation due to fewer strongly bound myosin heads. These results warrant clinical translation and call for early interventions to preserve physical function in children with CKD.

Structural Biochemistry of Muscle Contraction.

Muscles are essential for movement and heart function. Contraction and relaxation of muscles rely on the sliding of two types of filaments-the thin filament and the thick myosin filament. The thin filament is composed mainly of filamentous actin (F-actin), tropomyosin, and troponin. Additionally, several other proteins are involved in the contraction mechanism, and their malfunction can lead to diverse muscle diseases, such as cardiomyopathies. We review recent high-resolution structural data that explain the mechanism of action of muscle proteins at an unprecedented level of molecular detail. We focus on the molecular structures of the components of the thin and thick filaments and highlight the mechanisms underlying force generation through actin-myosin interactions, as well as Ca2+-dependent regulation via the dihydropyridine receptor, the ryanodine receptor, and troponin. We particularly emphasize the impact of cryo-electron microscopy and cryo-electron tomography in leading muscle research into a new era.

Human induced pluripotent stem cell line YCMi007-A generated from a dilated cardiomyopathy patient with a heterozygous dominant c.613C > T (p. Arg205Trp) variant of the TNNT2 gene

Cardiac muscle troponin T protein binds to tropomyosin and regulates the calcium-dependent actin–myosin interaction on thin filaments in cardiomyocytes. Recent genetic studies have revealed that TNNT2 mutations are strongly linked to dilated cardiomyopathy (DCM). In this study, we generated YCMi007-A, a human induced pluripotent stem cell (hiPSC) line from a DCM patient with a p. Arg205Trp mutation in the TNNT2 gene. The YCMi007-A cells show high expression of pluripotent markers, normal karyotype, and differentiation into three germ layers. Thus, YCMi007-A—an established iPSC—could be useful for the investigation of DCM.

Skeletal muscle fiber force declines with chronic kidney disease in juvenile and adult mice.

Juvenile and adult chronic kidney disease (CKD) patients frequently have compromised physical performance, which worsens with disease severity. Examining the underlying mechanisms behind this contractile dysfunction can be difficult to impossible in humans, especially in juveniles, but can be accomplished using animal models. We characterized CKD-induced changes in cellular (single fiber force) and molecular (myosin-actin interactions, myofilament properties) function in soleus and extensor digitorum longus muscles of 6-week-old (juvenile, control = 5, CKD = 6) and 16-week-old (adults, control = 10, CKD = 12) mice. CKD was induced by 0.2% adenine diet for 3 weeks (early-stage CKD) for juveniles and 8 weeks (late-stage CKD) for adults. Maximally activated (pCa 4.5, 25°C) specific tension (maximal isometric force divided by cross-sectional area) was reduced in all myosin heavy chain (MHC) I, IIA, and IIB fibers in juvenile (23-30%) and adult (36-51%) mice with CKD due, in a large part, to a reduction in strongly-bound myosin-actin cross-bridges. Additionally, myofilament stiffness was reduced in MHC IIA fibers from juveniles and adults as well as MHC IIB fibers from adults, potentially reducing force transmission. For adults, myosin-actin interactions were slower in MHC I and IIA fibers. In juveniles, myosin-actin interactions were slower only in MHC IIA fibers but faster in MHC IIB fibers. Fiber cross-sectional area with CKD was increased or unchanged in all fiber types, except for a decrease in MHC IIB fibers from adults. Overall, juvenile early-stage and adult late-stage CKD showed similar molecular mechanisms leading to reduced force production and cross-bridge kinetics, although alterations were generally larger in adult late-stage CKD. Thus, physical function in juvenile early-stage and adult late-stage CKD is being impaired on the molecular level due to intrinsic contractile deficits independent of skeletal muscle atrophy.

Effects of blebbistatin on force production, actin motility and the force-velocity relation produced by myosin II filaments and myofibrils.

Blebbistatin is a myosin II-specific inhibitor that is often used to study the relation between force generation and Pi release. The goal of this study was to investigate the effects of blebbistatin on force production and the actin motility while interacting with arrays of myosin motors. We used myosin filaments isolated from mammalian smooth muscles, rabbit psoas and the anterior byssus retractor (ABRM) muscles extracted from fresh Mytilus edulis in different experiments. We also used whole myofibrils isolated from the rabbit psoas. The force during myosin-actin interactions was measured with a system of micro-fabricated cantilevers, and the sliding velocity of actin was measured with a standard in-vitro motility assay. The force-velocity relation was measured in myofibrils with atomic force cantilevers. The force produced by the myosin filaments upon interaction with actin decreased with increasing concentrations of blebbistatin (5 μM-12.5 μM). The force produced by smooth and skeletal muscle myosin filaments were 95.93 ± 12.05 pN/μm before blebbistatin treatment, 71.45 ± 5.94 pN/μm with 5 μm blebbistatin, 62.05 ± 9.44 pN/μm with 7.5 μM blebbistatin, 38.28 ± 8.77 pN/μm with 10 μm blebbistatin and dropped to zero with 12.5 μM blebbistatin. All these differences were significantly different (p<0.0001). Sliding velocity of actin filaments also decreased consistently with increasing concentrations of blebbistatin. Before treatment, filaments slid over HMM at 3.68 ± 0.06 μm/s, decreasing to 2.30 ± 0.056 μm/s with 1 μM, 1.27 ± 0.043 μm/s with 5 μM, and 1.44 ± 0.032 μm/s with 30 μM. The maximal velocity decrease was observed at 5 μM of blebbistatin. These differences were significantly different (P<0.0001). There was a downward shift in the force-velocity relation in myofibrils treated with blebbistatin. These results suggest that blebbistatin regulates the mechanics of myosin motors working collectively in different experimental preparations.

Pharmacological interventions improve myosin-actin cross-bridge function in mice with advanced chronic kidney disease.

Skeletal muscle fiber force production in adult mice with late-stage chronic kidney disease (CKD) is significantly impaired, although by different myofilament and myosin-actin cross-bridge disease responses in the various fiber types. To reverse some of the CKD-induced contractile deficits, we examined the effects of the ATP analog, 2-deoxyadenosine triphosphate (dATP), to enhance cross-bridge kinetics and the troponin sensitizer, Tirasemtiv (2 µM), to increase cross-bridge formation. Single fiber force production, myosin-actin interactions and myofilament properties were measured at 25°C in fibers from soleus and extensor digitorum longus muscles of 16-week-old CKD mice (N=5) and controls (N=5). Late-stage CKD was induced by 0.2% adenine diet for 8 weeks. dATP in myosin heavy chain (MHC) I and IIA fibers with CKD increased cross-bridge kinetics, returning them to control values or faster, leading to more strongly bound cross-bridges for maximal Ca2+-activation (pCa 4.5, pH 7.0, 5 mM Phosphate) and fatiguing (pCa 4.5, pH 6.2, 30 mM Phosphate) conditions. Specific tensions in CKD were also improved 12-21% under maximal and fatiguing conditions, except in maximally Ca2+-activated MHC I fibers, but fell short of control values. Notably, other studies have found that improvements of 13-25% in knee extensor strength in CKD patients improved their physical function, suggesting changes of this magnitude are clinically meaningful. At submaximal Ca2+-activation (pCa 6.0), Tirasemtiv in MHC IIB fibers with CKD increased myofilament stiffness 520%, number of strongly bound cross-bridges 705%, and specific tension 370%, returning specific tension back to control values. This study provides evidence that cellular and molecular contractile function in CKD may be enhanced through use of dATP or Tirasemtiv, which should be tested to potentially improve physical function and overall quality of life in late-stage CKD patients.

Mechanistic study of mechanical stretch induced increase in Ca2+ sensitivity of troponin C.

The Frank-Starling Law of the heart is a direct consequence of myofilament length dependent activation (LDA) whereby increased sarcomere length (SL) leads to both increased myocardial Ca2+ sensitivity and maximum force. The molecular mechanisms underlying LDA remain elusive. Since troponin complex binds Ca2+ and regulates myosin-actin interaction during muscle contraction, it has been considered the key element involved LDA. This study aims to explore the molecular mechanism underlying sarcomere LDA and to investigate the functional role of each troponin protein in the LDA regulation. A mutant troponin-I, in which the SP-region was replaced with a G-linker (cTnI-ΔSP), was constructed to study the molecular interaction between troponin-I, troponin-C, and myosin during LDA regulation. The cTnI-ΔSP was used to prevent the functional effect from crossbridge (XB) on the SL-induced Ca2+ sensitivity changes of troponin complex. The SL-induced Ca2+ sensitivity change was monitored by in situ FRET which represents the Ca2+-induced cTnC structural change. Our results show that there are no significant changes of the SL-induced pCa50 shift of troponin in the absence of strong XB, suggesting that besides the XB feedback, the LDA of thin filament may have an alternative pathway involving troponin itself. To further understand the mechanism underlying LDA, recombinant cTnT and cTnI mutants carrying an enzyme cutting site at one end of the IT arm region, respectively, were used in our study. By selectively breaking T1 and T2 of cTnT or separating the C-domain of cTnI from the N-domain, we were able to examine the mechanistic pathway of SL-stretch on the Ca2+-induced structural change of cTnC. Our results provide novel information on the molecular mechanism of how troponin-Ca2+ activation senses myocardial sarcomere stretches to promote myosin-actin interactions at the diastolic level of Ca2+.

Iron supplementation improves skeletal muscle contractile properties in mice with chronic kidney disease.

Adult chronic kidney disease (CKD) patients commonly display decreased physical function, leading to increased mortality, which is exacerbated with iron deficiency and anemia. Interventions to mitigate CKD myopathy are scarce, potentially because the underlying contractile deficits have not been fully investigated. We examined single fiber force production, myosin-actin interactions and myofilament properties in soleus and extensor digitorum longus muscles of 16-week-old CKD mice without (CKD, N=12) and with iron supplementation (CKDFE, N=6, weekly injections of 0.2 mg/kg iron dextran) and controls (N=10). Late-stage CKD was induced by 0.2% adenine diet for 8 weeks. CKD with iron supplementation (CKDFE) improved anemia status compared to CKD, but not back to control levels. Maximally activated (pCa 4.5, 25°C) specific tension (maximal isometric force divided by cross-sectional area) was reduced 36-51% with CKD due, in a large part, to a reduction in strongly-bound myosin-actin cross-bridges in all myosin heavy chain (MHC) I, IIA, and IIB fibers. Iron supplementation improved specific tension and number of strongly-bound cross-bridges in MHC IIA and IIB fibers. Myosin-actin cross-bridge kinetics were slower with CKD in MHC I and IIA fibers, with iron supplementation restoring kinetics closer to control values in MHC IIA fibers. Higher hemoglobin levels were correlated to improvements in specific tension and strongly-bound cross-bridges in MHC IIA and IIB fibers, and with faster cross-bridge kinetics in MHC IIA fibers. However, CKD induced decreases in myofilament lattice stiffness for MHC IIA and IIB fibers were not improved with iron supplementation. Our findings show that iron supplementation improved various aspects of the cellular and molecular CKD-induced myopathy in fast-contracting fiber types. Thus, correction of iron imbalance and/or anemia may be an effective countermeasure to reduce contractile deficits and improve quality of life in late-stage CKD patients.

Structural changes in myosin affect chemo-mechanical properties of the myosin-actin interaction.

Mutations in the genes encoding striated muscle myosins lead to a variety of diseases including hypertrophic (HCM) and dilated (DCM) cardiomyopathies (MYH7) and distal arthrogryposis syndromes (MYH3 and MYH8). We have used a muscle myoblast cell line to express and purify recombinant human beta-cardiac myosin and human embryonic skeletal muscle myosins, comparing the WT versions of these proteins with an array of disease-causing variants. HCM-causing mutations in the beta-cardiac motor domain often destabilize a folded back, autoinhibited state of myosin, leading to an increase in the number of heads available within the sarcomere to interact with actin and produce force, which in turn results in the hypercontractility characteristic of HCM. Destabilization is seen for mutations that alter residues involved in the regions of myosin important for forming the intramolecular contacts in the folded back state. However, mutations affecting residues remote from these interfaces can also increase the number of available heads by destabilizing the pre-power stroke state of myosin that enables formation of the folded back state. HCM mutations that affect only myosin motor function without affecting the stability of the folded back state are less common. In contrast, we have studied a variety of DCM-causing mutations and found that most DCM-mutant myosins exhibit decreases in one or more aspects of motor function, including actin-activated ATPase activity and in vitro gliding velocity. Current efforts focus on correlating the effects of distal arthrogryposis syndrome-causing mutations on MYH3 biomechanical function and contrasting the effects of these mutations with paralogous mutations in MYH7.

Drug discovery for heart failure: Targeting the myosin-binding protein C interactions with actin.

We are using novel fluorescence detection technology to seek small-molecule drugs targeting the interaction of myosin-binding protein C (MyBP-C) with actin. Myosin-targeting drugs have been FDA-approved to treat heart failure (HF) and obstructive hypertrophic cardiomyopathy (HCM), to correct contractile dysfunction. However, cardiomyopathies are diverse, and a variety of drugs may be needed in order to provide adequate treatment options for patients. Cardiac MyBP-C (cMyBP-C) modulates actin-myosin interactions, and these interactions are regulated by phosphorylation. HF patients often have decreased cMyBP-C phosphorylation, and phosphorylation in model systems appears to be cardioprotective for HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic phosphorylation and/or perturb its interactions with actin or myosin. We previously used a fluorescence lifetime-based assay to screen a 1280-compound small-molecule library (LOPAC) and identified the first three cMyBP-C binding compounds that inhibit interactions with actin. In the present study, we have used a novel dual-wavelength time-resolved fluorescence resonance energy transfer (FRET)-based high-throughput screen (HTS) to discover small molecules that can specifically bind to cMyBP-C and affect its interactions with actin or myosin. We have labeled actin at C374 with fluorescein-5-maleimide (donor) and the C0-C2 fragment of cMyBP-C at C249 with tetramethyl rhodamine (acceptor), and then monitored FRET between the donor and acceptor in the presence and absence of compounds. The plates of samples were then re-screened for effects of the compounds on the environmentally-sensitive fluorescence lifetime of the acceptor, indicative of binding of compounds to C0-C2. We screened a 3000-compound FDA-approved library and identified several compounds that affected the interactions of cMyBP-C with actin or myosin in secondary assays. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF/HCM.

Conformational changes linked to ADP release from human cardiac myosin bound to actin-tropomyosin.

Following binding to the thin filament, β-cardiac myosin couples ATP-hydrolysis to conformational rearrangements in the myosin motor that drive myofilament sliding and cardiac ventricular contraction. However, key features of the cardiac-specific actin-myosin interaction remain uncertain, including the structural effect of ADP release from myosin, which is rate-limiting during force generation. In fact, ADP release slows under experimental load or in the intact heart due to the afterload, thereby adjusting cardiac muscle power output to meet physiological demands. To further elucidate the structural basis of this fundamental process, we used a combination of cryo-EM reconstruction methodologies to determine structures of the human cardiac actin-myosin-tropomyosin filament complex at better than 3.4 Å-resolution in the presence and in the absence of Mg2+·ADP. Focused refinements of the myosin motor head and its essential light chains in these reconstructions reveal that small changes in the nucleotide-binding site are coupled to significant rigid body movements of the myosin converter domain and a 16-degree lever arm swing. Our structures provide a mechanistic framework to understand the effect of ADP binding and release on human cardiac β-myosin, and offer insights into the force-sensing mechanism displayed by the cardiac myosin motor.

Effects of Aficamten on cardiac contractility in a feline translational model of hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiac disease in humans and cats and lacks efficacious pharmacologic interventions in the preclinical phase of disease. LV outflow tract obstruction (LVOTO) is commonly observed in HCM-affected patients and is a primary driver of heart failure symptoms and reduced quality of life. Novel small-molecule cardiac myosin inhibitors target actin-myosin interactions to alleviate overactive protein interactions. A prospective, randomized, controlled cross-over study was performed to evaluate pharmacodynamic effects of two doses (0.3 and 1 mg/kg) of a next-in-class cardiac myosin inhibitor, aficamten (CK-3773274, CK-274), on cardiac function in cats with the A31P MYBPC3 mutation and oHCM. Dose-dependent reductions in LV systolic function, LVOT pressure gradient, and isovolumetric relaxation times compared to baseline were observed. Promising beneficial effects of reduced systolic function warrant further studies of this next-in-class therapeutic to evaluate the benefit of long-term administration in this patient population.

De Novo Asp219Val Mutation in Cardiac Tropomyosin Associated with Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM), caused by mutations in thin filament proteins, manifests as moderate cardiac hypertrophy and is associated with sudden cardiac death (SCD). We identified a new de novo variant, c.656A>T (p.D219V), in the TPM1 gene encoding cardiac tropomyosin 1.1 (Tpm) in a young SCD victim with post-mortem-diagnosed HCM. We produced recombinant D219V Tpm1.1 and studied its structural and functional properties using various biochemical and biophysical methods. The D219V mutation did not affect the Tpm affinity for F-actin but increased the thermal stability of the Tpm molecule and Tpm-F-actin complex. The D219V mutation significantly increased the Ca2+ sensitivity of the sliding velocity of thin filaments over cardiac myosin in an in vitro motility assay and impaired the inhibition of the filament sliding at low Ca2+ concentration. The molecular dynamics (MD) simulation provided insight into a possible molecular mechanism of the effect of the mutation that is most likely a cause of the weakening of the Tpm interaction with actin in the "closed" state and so makes it an easier transition to the “open” state. The changes in the Ca2+ regulation of the actin-myosin interaction characteristic of genetic HCM suggest that the mutation is likely pathogenic.

Impact of Troponin in Cardiomyopathy Development Caused by Mutations in Tropomyosin

Tropomyosin (Tpm) mutations cause inherited cardiac diseases such as hypertrophic and dilated cardiomyopathies. We applied various approaches to investigate the role of cardiac troponin (Tn) and especially the troponin T (TnT) in the pathogenic effects of Tpm cardiomyopathy-associated mutations M8R, K15N, A277V, M281T, and I284V located in the overlap junction of neighboring Tpm dimers. Using co-sedimentation assay and viscosity measurements, we showed that TnT1 (fragment of TnT) stabilizes the overlap junction of Tpm WT and all Tpm mutants studied except Tpm M8R. However, isothermal titration calorimetry (ITC) indicated that TnT1 binds Tpm WT and all Tpm mutants similarly. By using ITC, we measured the direct KD of the Tpm overlap region, N-end, and C-end binding to TnT1. The ITC data revealed that the Tpm C-end binds to TnT1 independently from the N-end, while N-end does not bind. Therefore, we suppose that Tpm M8R binds to TnT1 without forming the overlap junction. We also demonstrated the possible role of Tn isoform composition in the cardiomyopathy development caused by M8R mutation. TnT1 dose-dependently reduced the velocity of F-actin-Tpm filaments containing Tpm WT, Tpm A277V, and Tpm M281T mutants in an in vitro motility assay. All mutations impaired the calcium regulation of the actin-myosin interaction. The M281T and I284V mutations increased the calcium sensitivity, while the K15N and A277V mutations reduced it. The Tpm M8R, M281T, and I284V mutations under-inhibited the velocity at low calcium concentrations. Our results demonstrate that Tpm mutations likely implement their pathogenic effects through Tpm interaction with Tn, cardiac myosin, or other protein partners.