Mutant Tropomyosin Research Articles

Abstract We118: Phosphoglycerate Dehydrogenase Gene Therapy for Dilated Cardiomyopathy

Introduction: Dilated cardiomyopathy (DCM) is a multifaceted cardiac disorder affecting approximately 1 in 250 to 2000 individuals and stands as a predominant cause of heart failure. Despite advancements in identifying genetic mutations associated with DCM, the precise mechanisms driving its pathogenesis are unknown, translating to the lack of disease-modifying interventions. Hypothesis: Modulating serine biosynthesis and one-carbon metabolism in cardiomyocytes by forced expression of Phosphoglycerate Dehydrogenase ( PHGDH) will ameliorate cardiac dysfunction in DCM and halt the disease progression. Methods: We developed a gene therapy vector expressing the PHGDH gene, controlled by a chicken cardiac-specific promoter (cardiac troponin T, cTnT) (AAV9.cTnT.hPHGDH). We delivered this construct via an AAV-based gene transfer approach using the cardiotropic adeno-associated virus serotype-9 in the TM54 DCM mouse – a well-established transgenic mouse that expresses a mutant tropomyosin cDNA (Tpm1 p. E54K; Tm54) under the control of the cardiac αMHC promoter. At 6 weeks old, when the animals exhibit DCM, they were treated with AAV9.cTnT.hPHGDH or control (AAV9.cTnT.GFP). Serial echocardiography was employed to assess heart function for 10 weeks. Morphometric data including heart weight, body weight, and tibia length were recorded. Subsequently, WGA and picrosirius red staining were performed to evaluate cardiomyocyte size and fibrosis, respectively. Finally, metabolic changes were assessed by untargeted metabolomics. Results: AAV9.cTnT.hPHGDH treatment of DCM TM54 mice improved the systolic function and halted the progression of DCM compared to the control mice. Furthermore, PHGDH treatment prevented the development of interstitial fibrosis and cardiomyocyte hypertrophy. Finally, these functional and structural improvements correlate with metabolic rewiring of the glucose metabolism in the DCM heart. Conclusion: These findings suggest that metabolic rewiring of the glucose metabolism by enhancing serine biosynthesis and one-carbon metabolism in the DCM heart may be cardioprotective. Thus, PHGDH gene therapy may be a novel therapeutic approach for DCM and heart failure.

Glutamate 139 of tropomyosin is critical for cardiac thin filament blocked-state stabilization

The cardiac thin filament proteins troponin and tropomyosin control actomyosin formation and thus cardiac contractility. Calcium binding to troponin changes tropomyosin position along the thin filament, allowing myosin head binding to actin required for heart muscle contraction. The thin filament regulatory proteins are hot spots for genetic mutations causing heart muscle dysfunction. While much of the thin filament structure has been characterized, critical regions of troponin and tropomyosin involved in triggering conformational changes remain unresolved. A poorly resolved region, helix-4 (H4) of troponin I, is thought to stabilize tropomyosin in a position on actin that blocks actomyosin interactions at low calcium concentrations during muscle relaxation. We have proposed that contact between glutamate 139 on tropomyosin and positively charged residues on H4 leads to blocking-state stabilization. In this study, we attempted to disrupt these interactions by replacing E139 with lysine (E139K) to define the importance of this residue in thin filament regulation. Comparison of mutant and wild-type tropomyosin was carried out using in-vitro motility assays, actin co-sedimentation, and molecular dynamics simulations to determine perturbations in troponin-tropomyosin function caused by the tropomyosin mutation. Motility assays revealed that mutant thin filaments moved at higher velocity at low calcium with increased calcium sensitivity demonstrating that tropomyosin residue 139 is vital for proper tropomyosin-mediated inhibition during relaxation. Similarly, molecular dynamic simulations revealed a mutation-induced decrease in interaction energy between tropomyosin-E139K and troponin I (R170 and K174). These results suggest that salt-bridge stabilization of tropomyosin position by troponin IH4 is essential to prevent actomyosin interactions during cardiac muscle relaxation.

In silico and in vitro models reveal the molecular mechanisms of hypocontractility caused by TPM1 M8R.

Dilated cardiomyopathy (DCM) is an inherited disorder often leading to severe heart failure. Linkage studies in affected families have revealed hundreds of different mutations that can cause DCM, with most occurring in genes associated with the cardiac sarcomere. We have developed an investigational pipeline for discovering mechanistic genotype-phenotype relationships in DCM and here apply it to the DCM-linked tropomyosin mutation TPM1 M8R. Atomistic simulations predict that M8R increases flexibility of the tropomyosin chain and enhances affinity for the blocked or inactive state of tropomyosin on actin. Applying these molecular effects to a Markov model of the cardiac thin filament reproduced the shifts in Ca2+sensitivity, maximum force, and a qualitative drop in cooperativity that were observed in an in vitro system containing TPM1 M8R. The model was then used to simulate the impact of M8R expression on twitch contractions of intact cardiac muscle, predicting that M8R would reduce peak force and duration of contraction in a dose-dependent manner. To evaluate this prediction, TPM1 M8R was expressed via adenovirus in human engineered heart tissues and isometric twitch force was observed. The mutant tissues manifested depressed contractility and twitch duration that agreed in detail with model predictions. Additional exploratory simulations suggest that M8R-mediated alterations in tropomyosin-actin interactions contribute more potently than tropomyosin chain stiffness to cardiac twitch dysfunction, and presumably to the ultimate manifestation of DCM. This study is an example of the growing potential for successful in silico prediction of mutation pathogenicity for inherited cardiac muscle disorders.

Troponin and a Myopathy-Linked Mutation in TPM3 Inhibit Cofilin-2-Induced Thin Filament Depolymerization.

Uniform actin filament length is required for synchronized contraction of skeletal muscle. In myopathies linked to mutations in tropomyosin (Tpm) genes, irregular thin filaments are a common feature, which may result from defects in length maintenance mechanisms. The current work investigated the effects of the myopathy-causing p.R91C variant in Tpm3.12, a tropomyosin isoform expressed in slow-twitch muscle fibers, on the regulation of actin severing and depolymerization by cofilin-2. The affinity of cofilin-2 for F-actin was not significantly changed by either Tpm3.12 or Tpm3.12-R91C, though it increased two-fold in the presence of troponin (without Ca2+). Saturation of the filament with cofilin-2 removed both Tpm variants from the filament, although Tpm3.12-R91C was more resistant. In the presence of troponin (±Ca2+), Tpm remained on the filament, even at high cofilin-2 concentrations. Both Tpm3.12 variants inhibited filament severing and depolymerization by cofilin-2. However, the inhibition was more efficient in the presence of Tpm3.12-R91C, indicating that the pathogenic variant impaired cofilin-2-dependent actin filament turnover. Troponin (±Ca2+) further inhibited but did not completely stop cofilin-2-dependent actin severing and depolymerization.

Functional assays reveal the pathogenic mechanism of a de novo tropomyosin variant identified in patient with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a leading cause of heart failure and a major indicator for heart transplant. Human genetic studies have identified over a thousand causal mutations for DCM in genes involved in a variety of cellular processes, including sarcomeric contraction. A substantial clinical challenge is determining the pathogenicity of novel variants in disease-associated genes. This challenge of connecting genotype and phenotype has frustrated attempts to develop effective, mechanism-based treatments for patients. Here, we identified a de novo mutation (T237S) in TPM1, the gene that encodes the thin filament protein tropomyosin, in a patient with DCM and conducted in vitro experiments to characterize the pathogenicity of this novel variant. We expressed recombinant mutant protein, reconstituted it into thin filaments, and examined the effects of the mutation on thin filament function. We show that the mutation reduces the calcium sensitivity of thin filament activation, as previously seen for known pathogenic mutations. Mechanistically, this shift is due to mutation-induced changes in tropomyosin positioning along the thin filament. We demonstrate that the thin filament activator omecamtiv mecarbil restores the calcium sensitivity of thin filaments regulated by the mutant tropomyosin, which lays the foundation for additional experiments to explore the therapeutic potential of this drug for patients harboring the T237S mutation. Taken together, our results suggest that the TPM1 T237S mutation is likely pathogenic and demonstrate how functional in vitro characterization of pathogenic protein variants in the lab might guide precision medicine in the clinic.

Sarcomeric Thin Filament Associated Cardiomyopathic Mouse Models

Cardiomyopathies are diseases primarily associated with defects in the structure and physiological function of the heart. Hypertrophic and dilated cardiomyopathies are two common conditions associated with severe pathological abnormalities that often lead to heart failure. Studies in the early 1990’s by the Seidman laboratories linked hypertrophic cardiomyopathy with mutations in both thick and thin sarcomeric protein genes. Since then, the development and analysis of animal models of both hypertrophic and dilated cardiomyopathy has significantly advanced our knowledge of the structural, molecular, biochemical, and physiological disease processes that lead to these cardiomyopathic conditions. The focus of this article is an examination of mouse models of hypertrophic and dilated cardiomyopathies with mutations in sarcomeric thin filament protein genes (actin, tropomyosin, troponin T, I, and C) and the information these models provide in our understanding of the disease processes. Special attention addresses the significant role that tropomyosin mutation models have contributed to this information. In addition, we address how various methods have been developed to phenotypically rescue these diseased hearts with respect to their morphological and physiological functions. By thorough analysis of these mouse models, not only can we better understand the disease processes, but there is a great potential for the development of effective therapeutics to treat these severe pathological conditions.

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.

Implications of Tropomyosin Phosphorylation in Normal and Cardiomyopathic Hearts

Numerous molecular and biochemical processes regulate protein production in the cell. One of these processes, phosphorylation, allows the cell to rapidly adapt to changing physiological situations. In terminally differentiated cells, such as cardiomyocytes, phosphorylation of sarcomeric proteins controls contraction and relaxation under both normal and stressful conditions. The focus of this review is how phosphorylation of sarcomeric proteins alters physiological performance in cardiac muscle with a particular emphasis on the thin filament protein tropomyosin. This topic is addressed by the examination of tropomyosin isoform expression and its phosphorylation state from embryonic to adult murine development. Next, studies are examined which utilize in vivo model systems to express phosphorylation mimetics and de-phosphorylation genetically-altered tropomyosin transgene constructs. Results show that tropomyosin isoform expression is highly regulated, along with its phosphorylation state. Transgenic mouse hearts which express high levels of a constitutively phosphorylated tropomyosin develop a severe dilated cardiomyopathy and die within a month. A more moderate expression of this phosphorylation mimetic leads to normal systolic performance, but impaired diastolic function. When tropomyosin is dephosphorylated, the transgenic mice develop a compensated cardiac hypertrophy without systolic or diastolic alterations. Interestingly, when dephosphorylated tropomyosin is co-expressed with a hypertrophic cardiomyopathy tropomyosin mutation, the pathological phenotype is rescued with improved cardiac function and no indices of systolic or diastolic dysfunction. These studies demonstrate the functional significance of tropomyosin phosphorylation in determining cardiac performance during both normal and pathological conditions.

Loss of crossbridge inhibition drives pathological cardiac hypertrophy in patients harboring the TPM1 E192K mutation.

Hypertrophic cardiomyopathy (HCM) is an inherited disorder caused primarily by mutations to thick and thinfilament proteins. Although thin filament mutations are less prevalent than their oft-studied thick filament counterparts, they are frequently associated with severe patient phenotypes and can offer important insight into fundamental disease mechanisms. We have performed a detailed study of tropomyosin (TPM1) E192K, a variant of uncertain significance associated with HCM. Molecular dynamics revealed that E192K results in a more flexible TPM1 molecule, which could affect its ability to regulate crossbridges. In vitro motility assays of regulated actin filaments containing TPM1 E192K showed an overall loss of Ca2+ sensitivity. To understand these effects, we used multiscale computational models that suggested a subtle phenotype in which E192K leads to an inability to completely inhibit actin-myosin crossbridge activity at low Ca2+. To assess the physiological impact of the mutation, we generated patient-derived engineered heart tissues expressing E192K. These tissues showed disease features similar to those of the patients, including cellular hypertrophy, hypercontractility, and diastolic dysfunction. We hypothesized that excess residual crossbridge activity could be triggering cellular hypertrophy, even if the overall Ca2+ sensitivity was reduced by E192K. To test this hypothesis, the cardiac myosin-specific inhibitor mavacamten was applied to patient-derived engineered heart tissues for 4 d followed by 24 h of washout. Chronic mavacamten treatment abolished contractile differences between control and TPM1 E192K engineered heart tissues and reversed hypertrophy in cardiomyocytes. These results suggest that the TPM1 E192K mutation triggers cardiomyocyte hypertrophy by permitting excess residual crossbridge activity. These studies also provide direct evidence that myosin inhibition by mavacamten can counteract the hypertrophic effects of mutant tropomyosin.

Отличительные особенности регуляторной функции тропомиозина при различных вариантах наследственной миопатии

Congenital myopathies are a group of clinically and genetically heterogeneous diseases, which are united by primary lesions of skeletal muscles and are characterized by progressive muscle weakness and hypotension, as well as morphological changes in muscle tissue. The range of variants of congenital myopathies is quite wide, and the signs are heterogeneous, therefore, the diagnosis is often difficult. Until now, there is no effective therapy for myopathies - only symptomatic treatment is used to improve metabolism and blood microcirculation in the muscles. At the same time, many of these diseases significantly reduce the quality and duration of human life. The accumulation of scientific knowledge necessary for the early diagnosis of congenital myopathies of human and for the development of approaches to effective treatment of diseases of muscle tissue dysfunction is one of the urgent tasks of biology and medicine. Most recently, a series of works has appeared in which an attempt is made to characterize the molecular mechanisms of the onset and development of a number of myopathies caused by gene mutations. It seems extremely important to analyze the published data and, on the basis of this, highlight critical changes in the conformational state of muscle proteins that can be used as tests for the differential diagnosis of myopathies. This review article is devoted to the analysis and generalization of literature and original data obtained by the method of polarized microfluorimetry on the molecular mechanisms of muscle contraction regulation by mutant tropomyosins appearing in muscle tissue in various human musculoskeletal diseases in order to identify molecular targets for the therapeutic effects.

M8R tropomyosin mutation disrupts actin binding and filament regulation: The beginning affects the middle and end

Dilated cardiomyopathy (DCM) is associated with mutations in cardiomyocyte sarcomeric proteins, including α-tropomyosin. In conjunction with troponin, tropomyosin shifts to regulate actomyosin interactions. Tropomyosin molecules overlap via tropomyosin-tropomyosin head-to-tail associations, forming a continuous strand along the thin filament. These associations are critical for propagation of tropomyosin's reconfiguration along the thin filament and key for the cooperative switching between heart muscle contraction and relaxation. Here, we tested perturbations in tropomyosin structure, biochemistry, and function caused by the DCM-linked mutation, M8R, which is located at the overlap junction. Localized and nonlocalized structural effects of the mutation were found in tropomyosin that ultimately perturb its thin filament regulatory function. Comparison of mutant and WT α-tropomyosin was carried out using in vitro motility assays, CD, actin co-sedimentation, and molecular dynamics simulations. Regulated thin filament velocity measurements showed that the presence of M8R tropomyosin decreased calcium sensitivity and thin filament cooperativity. The co-sedimentation of actin and tropomyosin showed weakening of actin-mutant tropomyosin binding. The binding of troponin T's N terminus to the actin-mutant tropomyosin complex was also weakened. CD and molecular dynamics indicate that the M8R mutation disrupts the four-helix bundle at the head-to-tail junction, leading to weaker tropomyosin-tropomyosin binding and weaker tropomyosin-actin binding. Molecular dynamics revealed that altered end-to-end bond formation has effects extending toward the central region of the tropomyosin molecule, which alter the azimuthal position of tropomyosin, likely disrupting the mutant thin filament response to calcium. These results demonstrate that mutation-induced alterations in tropomyosin-thin filament interactions underlie the altered regulatory phenotype and ultimately the pathogenesis of DCM.

Looking for Targets to Restore the Contractile Function in Congenital Myopathy Caused by Gln147Pro Tropomyosin.

We have used the technique of polarized microfluorimetry to obtain new insight into the pathogenesis of skeletal muscle disease caused by the Gln147Pro substitution in β-tropomyosin (Tpm2.2). The spatial rearrangements of actin, myosin and tropomyosin in the single muscle fiber containing reconstituted thin filaments were studied during simulation of several stages of ATP hydrolysis cycle. The angular orientation of the fluorescence probes bound to tropomyosin was found to be changed by the substitution and was characteristic for a shift of tropomyosin strands closer to the inner actin domains. It was observed both in the absence and in the presence of troponin, Ca2+ and myosin heads at all simulated stages of the ATPase cycle. The mutant showed higher flexibility. Moreover, the Gln147Pro substitution disrupted the myosin-induced displacement of tropomyosin over actin. The irregular positioning of the mutant tropomyosin caused premature activation of actin monomers and a tendency to increase the number of myosin cross-bridges in a state of strong binding with actin at low Ca2+.

Hemodynamic Effects of Alpha-Tropomyosin Mutations Associated with Inherited Cardiomyopathies: Multiscale Simulation

The effects of two cardiomyopathy-associated mutations in regulatory sarcomere protein tropomyosin (Tpm) on heart function were studied with a new multiscale model of the cardiovascular system (CVS). They were a Tpm mutation, Ile284Val, associated with hypertrophic cardiomyopathy (HCM), and an Asp230Asn one associated with dilated cardiomyopathy (DCM). When the molecular and cell-level changes in the Ca2+ regulation of cardiac muscle caused by these mutations were introduced into the myocardial model of the left ventricle (LV) while the LV shape remained the same as in the model of the normal heart, the cardiac output and arterial blood pressure reduced. Simulations of LV hypertrophy in the case of the Ile284Val mutation and LV dilatation in the case of the Asp230Asn mutation demonstrated that the LV remodeling partially recovered the stroke volume and arterial blood pressure, confirming that both hypertrophy and dilatation help to preserve the LV function. The possible effects of changes in passive myocardial stiffness in the model according to data reported for HCM and DCM hearts were also simulated. The results of the simulations showed that the end-systolic pressure–volume relation that is often used to characterize heart contractility strongly depends on heart geometry and cannot be used as a characteristic of myocardial contractility.

Molecular Mechanisms of Muscle Weakness Associated with E173A Mutation in Tpm3.12. Troponin Ca2+ Sensitivity Inhibitor W7 Can Reduce the Damaging Effect of This Mutation.

Substitution of Ala for Glu residue in position 173 of γ-tropomyosin (Tpm3.12) is associated with muscle weakness. Here we observe that this mutation increases myofilament Ca2+-sensitivity and inhibits in vitro actin-activated ATPase activity of myosin subfragment-1 at high Ca2+. In order to determine the critical conformational changes in myosin, actin and tropomyosin caused by the mutation, we used the technique of polarized fluorimetry. It was found that this mutation changes the spatial arrangement of actin monomers and myosin heads, and the position of the mutant tropomyosin on the thin filaments in muscle fibres at various mimicked stages of the ATPase cycle. At low Ca2+ the E173A mutant tropomyosin shifts towards the inner domains of actin at all stages of the cycle, and this is accompanied by an increase in the number of switched-on actin monomers and myosin heads strongly bound to F-actin even at relaxation. Contrarily, at high Ca2+ the amount of the strongly bound myosin heads slightly decreases. These changes in the balance of the strongly bound myosin heads in the ATPase cycle may underlie the occurrence of muscle weakness. W7, an inhibitor of troponin Ca2+-sensitivity, restores the increase in the number of myosin heads strongly bound to F-actin at high Ca2+ and stops their strong binding at relaxation, suggesting the possibility of using Ca2+-desensitizers to reduce the damaging effect of the E173A mutation on muscle fibre contractility.

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 Effect of Tropomyosin Mutations on Actin-Tropomyosin Binding: In Search of Lost Time

The initial binding of tropomyosin onto actin filaments and then its polymerization into continuous cables on the filament surface must be precisely tuned to overall thin-filament structure, function, and performance. Low-affinity interaction of tropomyosin with actin has to be sufficiently strong to localize the tropomyosin on actin, yet not so tight that regulatory movement on filaments is curtailed. Likewise, head-to-tail association of tropomyosin molecules must be favorable enough to promote tropomyosin cable formation but not so tenacious that polymerization precedes filament binding. Arguably, little molecular detail on early tropomyosin binding steps has been revealed since Wegner’s seminal studies on filament assembly almost 40 years ago. Thus, interpretation of mutation-based actin-tropomyosin binding anomalies leading to cardiomyopathies cannot be described fully. In vitro, tropomyosin binding is masked by explosive tropomyosin polymerization once cable formation is initiated on actin filaments. In contrast, in silico analysis, characterizing molecular dynamics simulations of single wild-type and mutant tropomyosin molecules on F-actin, is not complicated by tropomyosin polymerization at all. In fact, molecular dynamics performed here demonstrates that a midpiece tropomyosin domain is essential for normal actin-tropomyosin interaction and that this interaction is strictly conserved in a number of tropomyosin mutant species. Elsewhere along these mutant molecules, twisting and bending corrupts the tropomyosin superhelices as they “lose their grip” on F-actin. We propose that residual interactions displayed by these mutant tropomyosin structures with actin mimic ones that occur in early stages of thin-filament generation, as if the mutants are recapitulating the assembly process but in reverse. We conclude therefore that an initial binding step in tropomyosin assembly onto actin involves interaction of the essential centrally located domain.

Congenital myopathy-related mutations in tropomyosin disrupt regulatory function through altered actin affinity and tropomodulin binding.

Tropomyosin (Tpm) binds along actin filaments and regulates myosin binding to control muscle contraction. Tropomodulin binds to the pointed end of a filament and regulates actin dynamics, which maintains the length of a thin filament. To define the structural determinants of these Tpm functions, we examined the effects of two congenital myopathy mutations, A4V and R91C, in the Tpm gene, TPM3, which encodes the Tpm3.12 isoform, specific for slow-twitch muscle fibers. Mutation A4V is located in the tropomodulin-binding, N-terminal region of Tpm3.12. R91C is located in the actin-binding period 3 and directly interacts with actin. The A4V and R91C mutations resulted in a 2.5-fold reduced affinity of Tpm3.12 homodimers for F-actin in the absence and presence of troponin, and a two-fold decrease in actomyosin ATPase activation in the presence of Ca2+ . Actomyosin ATPase inhibition in the absence of Ca2+ was not affected. The Ca2+ sensitivity of ATPase activity was decreased by R91C, but not by A4V. Invitro, R91C altered the ability of tropomodulin 1 (Tmod1) to inhibit actin polymerization at the pointed end of the filaments, which correlated with the reduced affinity of Tpm3.12-R91C for Tmod1. Molecular dynamics simulations of Tpm3.12 in complex with F-actin suggested that both mutations reduce the affinity of Tpm3.12 for F-actin binding by perturbing the van der Waals energy, which may be attributable to two different molecular mechanisms-a reduced flexibility of Tpm3.12-R91C and an increased flexibility of Tpm3.12-A4V.

Engineered Thin Filament Mutation to Increase Calcium Sensitivity of Force in Tropomyosin Mutation of Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is a leading cause of heart failure, which is quickly rising in prevalence in the United States. In roughly 20% of DCM cases, mutations in sarcomeric proteins, such as troponin (Tn), tropomyosin (Tm) and titin, are considered the cause. Decreased tension and calcium sensitivity of force are characteristics of sarcomere-linked DCM. Currently there are no therapies that directly target the underlying condition and typically treatments target only the symptoms. Here we are exploring treatment of DCM by genetically altering Ca2+ binding in the sarcomere. Specifically, we countered a DCM mutation in cardiac Tm, D230N, by introducing an engineered mutation in cardiac TnC (cTnC), L48Q, that binds Ca2+ more strongly than native cTnC. Mice with the D230N-Tm mutation were crossed with L48Q-cTnC mice (DTG). At 4-6 months of age, when DCM is well established in D230N mice, triton X-100 skinned left ventricular trabeculae were mounted between a force transducer and motor. Ca2+ sensitivity (pCa50) of force was determined at two sarcomere lengths (SL), 2.0μm and 2.3μm. As expected, pCa50 is lower in D230N-Tm versus WT mice at both SL 2.0μm (5.373 ± 0.033 vs 5.655 ± 0.088) and 2.3μm (5.476 ± 0.037 vs 5.751 ± 0.079), and L48Q-cTnC mice show increased pCa50 (2.0μm: 5.784 ± 0.055; 2.3μm: 5.962 ± 0.095). Importantly, trabeculae from DTG mice showed increased pCa50 (2.0μm: 5.486 ± 0.034; 2.3μm: 5.582 ± 0.022) compared to D230N-Tm mice. No changes were seen in maximal Ca2+-activated force. We will next examine Ca2+ handling in isolated cardiomyocytes. In conclusion, introducing a mutation with stronger Ca2+ binding can successfully overcome the decrease in Ca2+ sensitivity at SL 2.0 and 2.3μm caused by the D230N-Tm DCM mutation.

Effects of cardiomyopathy-linked mutations K15N and R21H in tropomyosin on thin-filament regulation and pointed-end dynamics.

Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used Förster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca2+-dependent regulation of cardiac thin filaments. We found that the K15N mutation desensitizes thin filaments to Ca2+ and slows the kinetics of structural changes in troponin induced by Ca2+ dissociation from troponin, while the R21H mutation has almost no effect on these parameters. Expression of the K15N mutant in cardiomyocytes decreases leiomodin’s thin-filament pointed-end assembly but does not affect tropomodulin’s assembly at the pointed end. Our in vitro assays show that the R21H mutation causes a twofold decrease in tropomyosin’s affinity for F-actin and affects leiomodin’s function. We suggest that the K15N mutation causes DCM by altering Ca2+-dependent thin-filament regulation and that one of the possible HCM-causing mechanisms by the R21H mutation is through alteration of leiomodin’s function.

CONGENITAL MYOPATHIES: NEMALINE AND TITINOPATHIES: P.236Congenital fiber type disproportion with mutations in tropomyosin 3 (TPM3) gene presenting as respiratory failure

Congenital fiber type disproportion(CFTD) is a rare subtype of congenital myopathy. CFTD is clinically heterogenous and histologically marked by hypotrophy of type I fibers compared with type 2 fibers. CFTD has been related with mutations in ACTA1, SEPN1, RYR1 and TPM3 genes. Particularly, TPM3 mutation was identified to the most frequent cause of CFTD and was also detected in cap myopathy and nemaline myopathy. We report autosomal dominant TPM3 missense mutations with CFTD in a family over two-generations. The two patients, the brother and sister, experienced first symptoms of muscle weakness in infancy with delayed motor milestones and followed a slowly progressive course. They presented generalized muscular hypotrophy, neck flexor muscle and distal limb muscle weakness, elongated face, retrognathia, high arched palate and scoliosis. Respiratory function became critical in two patients despite relatively good limb strength. The brother presented in respiratory arrest during sleep. The sister did not complain of difficulty on walking and climbing stairs but presented in respiratory failure needing ventilator. The finding of electromyography was compatible with myopathy and muscle biopsy showed type 1 fiber atrophy with predominance in the absence of other notable pathological findings such as nemaline rods and cap structures. TPM3 missense mutation c.502C>T (p.R168C) in exon 5 was identified by a target NGS. We confirmed CFTD caused by TPM3 mutation in this family. CFTD patients with mutation in TPM3 have been previously reported that many patients required nocturnal noninvasive ventilatory support despite remaining ambulant and several patients of these abruptly presented in respiratory failure. All patients in our family also showed sudden respiratory failure with relatively spared limb weakness. Therefore, it is clinically important for monitoring respiratory function regularly in CFTD patients with TPM3 mutation.