CalTrack: High-Throughput Automated Calcium Transient Analysis in Cardiomyocytes.

[Figure: see text].

Molecular Mechanisms of Deregulation of Muscle Contractility Caused by the R168H Mutation in TPM3 and Its Attenuation by Therapeutic Agents.

The substitution for Arg168His (R168H) in γ-tropomyosin (TPM3 gene, Tpm3.12 isoform) is associated with congenital muscle fiber type disproportion (CFTD) and muscle weakness. It is still unclear what molecular mechanisms underlie the muscle dysfunction seen in CFTD. The aim of this work was to study the effect of the R168H mutation in Tpm3.12 on the critical conformational changes that myosin, actin, troponin, and tropomyosin undergo during the ATPase cycle. We used polarized fluorescence microscopy and ghost muscle fibers containing regulated thin filaments and myosin heads (myosin subfragment-1) modified with the 1,5-IAEDANS fluorescent probe. Analysis of the data obtained revealed that a sequential interdependent conformational-functional rearrangement of tropomyosin, actin and myosin heads takes place when modeling the ATPase cycle in the presence of wild-type tropomyosin. A multistep shift of the tropomyosin strands from the outer to the inner domain of actin occurs during the transition from weak to strong binding of myosin to actin. Each tropomyosin position determines the corresponding balance between switched-on and switched-off actin monomers and between the strongly and weakly bound myosin heads. At low Ca2+, the R168H mutation was shown to switch some extra actin monomers on and increase the persistence length of tropomyosin, demonstrating the freezing of the R168HTpm strands close to the open position and disruption of the regulatory function of troponin. Instead of reducing the formation of strong bonds between myosin heads and F-actin, troponin activated it. However, at high Ca2+, troponin decreased the amount of strongly bound myosin heads instead of promoting their formation. Abnormally high sensitivity of thin filaments to Ca2+, inhibition of muscle fiber relaxation due to the appearance of the myosin heads strongly associated with F-actin, and distinct activation of the contractile system at submaximal concentrations of Ca2+ can lead to muscle inefficiency and weakness. Modulators of troponin (tirasemtiv and epigallocatechin-3-gallate) and myosin (omecamtiv mecarbil and 2,3-butanedione monoxime) have been shown to more or less attenuate the negative effects of the tropomyosin R168H mutant. Tirasemtiv and epigallocatechin-3-gallate may be used to prevent muscle dysfunction.

Genetics Matters: Voyaging from the Past into the Future of Humanity and Sustainability.

The understanding of how genetic information may be inherited through generations was established by Gregor Mendel in the 1860s when he developed the fundamental principles of inheritance. The science of genetics, however, began to flourish only during the mid-1940s when DNA was identified as the carrier of genetic information. The world has since then witnessed rapid development of genetic technologies, with the latest being genome-editing tools, which have revolutionized fields from medicine to agriculture. This review walks through the historical timeline of genetics research and deliberates how this discipline might furnish a sustainable future for humanity.

Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. METHODS: We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. RESULTS: Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. CONCLUSIONS: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.

Measurement of Myofilament-Localized Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy Mutations.

RATIONALE: Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here, we develop and characterize genetically encoded Ca2+ indicators restricted to the myofilament to directly visualize Ca2+ changes in the sarcomere. OBJECTIVE: To produce and validate myofilament-restricted Ca2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil, and levosimendan) or following cotransduction of 2 established hypertrophic cardiomyopathy disease-causing mutants (cTnT [Troponin T] R92Q and cTnI [Troponin I] R145G) that alter myofilament Ca2+ handling. METHODS AND RESULTS: When expressed in adult ventricular cardiomyocytes RGECO-TnT (Troponin T)/TnI (Troponin I) sensors localize correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca2+ on and increased Ca2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localized Ca2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Coadenoviral transduction of RGECO-TnT/TnI with hypertrophic cardiomyopathy causing thin filament mutants showed that the mutations increase myofilament [Ca2+] in systole, lengthen time to peak systolic [Ca2+], and delay [Ca2+] release. This contrasts with the effect of the same mutations on cytoplasmic Ca2+, when measured using unrestricted RGECO where changes to peak systolic Ca2+ are inconsistent between the 2 mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca2+] transient amplitude or time to peak Ca2+. CONCLUSIONS: RGECO-TnT/TnI are functionally equivalent. They visualize Ca2+ within the myofilament and reveal unrecognized aspects of small molecule and disease-associated mutations in living cells.

Molecular Mechanisms of the Deregulation of Muscle Contraction Induced by the R90P Mutation in Tpm3.12 and the Weakening of This Effect by BDM and W7.

Point mutations in the genes encoding the skeletal muscle isoforms of tropomyosin can cause a range of muscle diseases. The amino acid substitution of Arg for Pro residue in the 90th position (R90P) in γ-tropomyosin (Tpm3.12) is associated with congenital fiber type disproportion and muscle weakness. The molecular mechanisms underlying muscle dysfunction in this disease remain unclear. Here, we observed that this mutation causes an abnormally high Ca2+-sensitivity of myofilaments in vitro and in muscle fibers. To determine the critical conformational changes that myosin, actin, and tropomyosin undergo during the ATPase cycle and the alterations in these changes caused by R90P replacement in Tpm3.12, we used polarized fluorimetry. It was shown that the R90P mutation inhibits the ability of tropomyosin to shift towards the outer domains of actin, which is accompanied by the almost complete depression of troponin's ability to switch actin monomers off and to reduce the amount of the myosin heads weakly bound to F-actin at a low Ca2+. These changes in the behavior of tropomyosin and the troponin-tropomyosin complex, as well as in the balance of strongly and weakly bound myosin heads in the ATPase cycle may underlie the occurrence of both abnormally high Ca2+-sensitivity and muscle weakness. BDM, an inhibitor of myosin ATPase activity, and W7, a troponin C antagonist, restore the ability of tropomyosin for Ca2+-dependent movement and the ability of the troponin-tropomyosin complex to switch actin monomers off, demonstrating a weakening of the damaging effect of the R90P mutation on muscle contractility.

The molecular mechanism of muscle dysfunction associated with the R133W mutation in Tpm2.2.

Ghost muscle fibres reconstituted with myosin heads labeled with the fluorescent probe 1,5-IAEDANS were used for analysis of muscle fibre dysfunction associated with the R133W mutation in ß-tropomyosin (Tpm2.2). By using polarized microscopy, we showed that at high Ca2+ the R133W mutation in both αß-Tpm heterodimers and ßß-Tpm homodimers decreases the amount of the myosin heads strongly bound to F-actin and the number of switched-on actin monomers, with this effect being stronger for ßß-Tpm. This mutation also inhibits the shifting of the R133W-Tpm strands towards the open position and the efficiency of the cross-bridge work. At low Ca2+, the amount of the strongly bound myosin heads is lower for R133W-Tpms than for WT-Tpms which may contribute to a low myofilament Ca2+-sensitivity of the R133W-Tpms. It is concluded that freezing of the mutant αß- or ßß-Tpm close to the blocked position inhibits the strong binding of the cross-bridges and the switching on of actin monomers which may be the reason for muscle weakness associated with the R133W mutation in ß-tropomyosin. The use of reagents that activate myosin may be appropriate to restore muscle function in patients with the R133W mutation.

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 ß-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+.

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.

The molecular mechanisms of a high Ca2+-sensitivity and muscle weakness associated with the Ala155Thr substitution in Tpm3.12.

Substitution of Ala for Thr residue in 155th position in γ-tropomyosin (Tpm3.12) is associated with muscle weakness. To understand the mechanisms of this defect, we studied the Ca2+-sensitivity of thin filaments in solution and multistep changes in mobility and spatial arrangement of actin, Tpm, and myosin heads during the ATPase cycle in reconstituted muscle fibres, using the polarized fluorescence microscopy. It was shown that the Ala155Thr (A155T) mutation increased the Ca2+-sensitivity of the thin filaments in solution. In the absence of the myosin heads in the muscle fibres, the mutation did not alter the ability of troponin to switch the thin filaments on and off at high and low Ca2+, respectively. However, upon the binding of myosin heads to the thin filaments at low Ca2+, the mutant Tpm was found to be markedly closer to the open position, than the wild-type Tpm. In the presence of the mutant Tpm, switching on of actin monomers and formation of the strong-binding state of the myosin heads were observed at low Ca2+, which indicated a higher myofilament Ca2+-sensitivity. The mutation decreased the amount of myosin heads bound strongly to actin at high Ca2+ and increased the number of these heads at relaxation. It is suggested that direct binding of myosin to Tpm may be one оf the reasons for muscle weakness associated with the A155T mutation. The use of reagents that decrease the Ca2+-sensitivity of the troponin complex may not be adequate to restore muscle function in patients with the A155T mutation.

The reason for the low Ca2+-sensitivity of thin filaments associated with the Glu41Lys mutation in the TPM2 gene is "freezing" of tropomyosin near the outer domain of actin and inhibition of actin monomer switching off during the ATPase cycle.

The E41K mutation in TPM2 gene encoding muscle regulatory protein beta-tropomyosin is associated with nemaline myopathy and cap disease. The mutation results in a reduced Ca2+-sensitivity of the thin filaments and in muscle weakness. To elucidate the structural basis of the reduced Ca2+-sensitivity of the thin filaments, we studied multistep changes in spatial arrangement of tropomyosin (Tpm), actin and myosin heads during the ATPase cycle in reconstituted fibers, using the polarized fluorescence microscopy. The E41K mutation inhibits troponin's ability to shift Tpm to the closed position at high Ca2+, thus restraining the transition of the thin filaments from the "off" to the "on" state. The mutation also inhibits the ability of S1 to shift Tpm to the open position, decreases the amount of the myosin heads bound strongly to actin at high Ca2+, but increases the number of such heads at low Ca2+. These changes may contribute to the low Ca2+-sensitivity and muscle weakness. As the mutation has no effect on troponin's ability to switch actin monomers on at high Ca2+ and inhibits their switching off at low Ca2+, the use of reagents that increase the Ca2+-sensitivity of the troponin complex may not be appropriate to restore muscle function in patients with this mutation.

The Primary Causes of Muscle Dysfunction Associated with the Point Mutations in Tpm3.12; Conformational Analysis of Mutant Proteins as a Tool for Classification of Myopathies.

Point mutations in genes encoding isoforms of skeletal muscle tropomyosin may cause nemaline myopathy, cap myopathy (Cap), congenital fiber-type disproportion (CFTD), and distal arthrogryposis. The molecular mechanisms of muscle dysfunction in these diseases remain unclear. We studied the effect of the E173A, R90P, E150A, and A155T myopathy-causing substitutions in γ-tropomyosin (Tpm3.12) on the position of tropomyosin in thin filaments, and the conformational state of actin monomers and myosin heads at different stages of the ATPase cycle using polarized fluorescence microscopy. The E173A, R90P, and E150A mutations produced abnormally large displacement of tropomyosin to the inner domains of actin and an increase in the number of myosin heads in strong-binding state at low and high Ca2+, which is characteristic of CFTD. On the contrary, the A155T mutation caused a decrease in the amount of such heads at high Ca2+ which is typical for mutations associated with Cap. An increase in the number of the myosin heads in strong-binding state at low Ca2+ was observed for all mutations associated with high Ca2+-sensitivity. Comparison between the typical conformational changes in mutant proteins associated with different myopathies observed with α-, ß-, and γ-tropomyosins demonstrated the possibility of using such changes as tests for identifying the diseases.

Myopathy-causing Q147P TPM2 mutation shifts tropomyosin strands further towards the open position and increases the proportion of strong-binding cross-bridges during the ATPase cycle.

The molecular mechanisms of skeletal muscle dysfunction in congenital myopathies remain unclear. The present study examines the effect of a myopathy-causing mutation Q147P in ß-tropomyosin on the position of tropomyosin on troponin-free filaments and on the actin­myosin interaction at different stages of the ATP hydrolysis cycle using the technique of polarized fluorimetry. Wild-type and Q147P recombinant tropomyosins, actin, and myosin subfragment-1 were modified by 5-IAF, 1,5-IAEDANS or FITC-phalloidin, and 1,5-IAEDANS, respectively, and incorporated into single ghost muscle fibers, containing predominantly actin filaments which were free of troponin and tropomyosin. Despite its reduced affinity for actin in co-sedimentation assay, the Q147P mutant incorporates into the muscle fiber. However, compared to wild-type tropomyosin, it locates closer to the center of the actin filament. The mutant tropomyosin increases the proportion of the strong-binding myosin heads and disrupts the co-operation of actin and myosin heads during the ATPase cycle. These changes are likely to underlie the contractile abnormalities caused by this mutation.

The reason for a high Ca2+-sensitivity associated with Arg91Gly substitution in TPM2 gene is the abnormal behavior and high flexibility of tropomyosin during the ATPase cycle.

Substitution of Arg for Gly residue in 91th position in ß-tropomyosin caused by a point mutation in TPM2 gene is associated with distal arthrogryposis, characterized by a high Ca2+-sensitivity of myofilament and contracture syndrome. To understand the mechanisms of this defect, we studied multistep changes in mobility and spatial arrangement of tropomyosin, actin and myosin heads during the ATPase cycle in reconstituted ghost fibres, using the polarized fluorescence microscopy. The mutation was shown to markedly decrease the bending stiffness of ß-tropomyosin in the thin filaments. In the absence of the myosin heads the mutation did not alter the ability of troponin to shift tropomyosin to the blocked position and to switch actin monomers off at low Ca2+. During the ATPase cycle the movement of the mutant tropomyosin is restrained, it is located near the open position, which allows strong binding of the myosin heads to actin even at low Ca2+. This may be the reason for both high Ca2+-sensitivity and contractures associated with the Arg91Gly mutation. The use of reagents that decrease the Ca2+sensitivity of the troponin complex may not be appropriate to restore muscle function in patients with this mutation.

Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca(2+)-sensitivity and suppress the modulation of Ca(2+)-sensitivity by troponin I phosphorylation.

We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca(2+)-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca(2+)-sensitivity when compared with donor heart troponin and the Ca(2+)-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca(2+)-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca(2+)-sensitivity and was also completely uncoupled. Five TnT mutations (Δ14, Δ28 + 7, ΔE160, S179F and K273E) studied in recombinant troponin increased Ca(2+)-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca(2+)-sensitisation together with uncoupling in vitro is the usual response and both factors may contribute to the HCM phenotype. We also found that Epigallocatechin-3-gallate (EGCG) can restore coupling to all uncoupled HCM-causing TnT mutations. In fact the combination of Ca(2+)-desensitisation and re-coupling due to EGCG completely reverses both the abnormalities found in troponin with a TnT HCM mutation suggesting it may have therapeutic potential.

Does extracellular cardiac troponin I play a pathogenic role independently of autoantibodies?

Cardiac troponin I (cTnI) is a key component of the Ca2+-regulatory mechanism of cardiac contractility. It is released into the circulation upon ischaemia and has become established as one of the principal diagnostic biomarkers of myocardial damage. The release of cTnI results in the generation of autoantibodies, and these have been suggested to play a pathogenic role. However, in this Edition of Clinical Science, Han, Y. et al suggests that cTnI can act independently of immunological involvement, with the protein being found to increase infarct size caused by ischaemia/reperfusion (I/R) prior to the development of cTnI antibody. In vitro work shows that cTnI can induce increases in vascular cell adhesion molecule 1 (VCAM-1) expression and cell adhesion, with toll-like receptor 4 (TLR4) and nuclear factor kappa beta (NF-κB) involved in the downstream signalling.

Aberrant movement of ß-tropomyosin associated with congenital myopathy causes defective response of myosin heads and actin during the ATPase cycle.

We have investigated the effect of the E41K, R91G, and E139del ß-tropomyosin (TM) mutations that cause congenital myopathy on the position of TM and orientation of actin monomers and myosin heads at different mimicked stages of the ATPase cycle in troponin-free ghost muscle fibers by polarized fluorimetry. A multi-step shifting of wild-type TM to the filament center accompanied by an increase in the amount of switched on actin monomers and the strongly bound myosin heads was observed during the ATPase cycle. The R91G mutation shifts TM further towards the inner and outer domains of actin at the strong- and weak-binding stages, respectively. The E139del mutation retains TM near the inner domains, while the E41K mutation captures it near the outer domains. The E41K and R91G mutations can induce the strong binding of myosin heads to actin, when TM is located near the outer domains. The E139del mutation inhibits the amount of strongly bound myosin heads throughout the ATPase cycle.

The E117K mutation in ß-tropomyosin disturbs concerted conformational changes of actomyosin in muscle fibers.

The effect of the skeletal myopathy-causing E117K mutation in human ß-tropomyosin on actomyosin structure during the ATPase cycle was studied using fluorescent probes bound to actin subdomain 1 and the myosin head. Multistep changes in flexural rigidity of actin filament and in spatial arrangement of actin subdomain 1 and myosin SH1 helix in troponin-free ghost muscle fibers were revealed. During the ATPase cycle E117K tropomyosin inhibited the rotation of subdomain 1 by 46% and the tilt of the SH1 helix by 49% compared with wild-type. At strong-binding stages the proportion of strong binding sub-states in the actomyosin population is decreased by the mutation. At weak-binding stages abnormally high numbers of switched-on actin monomers were observed, thus indicating a disturbance in concerted conformational changes of actomyosin. These structural alterations are likely to underlie the contractile deficit observed with this mutation.

Gly126Arg substitution causes anomalous behaviour of α-skeletal and ß-smooth tropomyosins during the ATPase cycle.

To investigate how TM stabilization induced by the Gly126Arg mutation in skeletal α-TM or in smooth muscle ß-TM affects the flexibility of TMs and their position on troponin-free thin filaments, we labelled the recombinant wild type and mutant TMs with 5-IAF and F-actin with FITC-phalloidin, incorporated them into ghost muscle fibres and studied polarized fluorescence at different stages of the ATPase cycle. It has been shown that in the myosin- and troponin-free filaments the Gly126Arg mutation causes a shift of TM strands towards the outer domain of actin, reduces the number of switched on actin monomers and decreases the rigidity of the C-terminus of α-TM and increases the rigidity of the N-terminus of ß-TMs. The binding of myosin subfragment-1 to the filaments shifted the wild type TMs towards the inner domain of actin, decreased the flexibility of both terminal parts of TMs, and increased the number of switched on actin monomers. Multistep alterations in the position of α- and ß-TMs and actin monomers in the filaments and in the flexibility of TMs and F-actin during the ATPase cycle were observed. The Gly126Arg mutation uncouples a correlation between the position of TM and the number of the switched on actin monomers in the filaments.

The effect of the Asp175Asn and Glu180Gly TPM1 mutations on actin-myosin interaction during the ATPase cycle.

Hypertrophic cardiomyopathy (HCM), characterized by cardiac hypertrophy and contractile dysfunction, is a major cause of heart failure. HCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). To understand how the HCM-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin affect on actin-myosin interaction during the ATPase cycle, we labeled the SH1 helix of myosin subfragment-1 and the actin subdomain-1 with the fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphtylo)ethylenediamine. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. Wild-type α-tropomyosin increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of the work of cross-bridges. Both mutant TMs increase the proportion of the strong-binding sub-states, with the effect of the Glu180Gly mutation being greater than that of Asp175Asn. It is suggested that the alteration in the concerted conformational changes of actomyosin is likely to provide the structural basis for the altered cardiac muscle contraction.