Myopathy-causing mutation R91P in the TPM3 gene drastically impairs structural and functional properties of slow skeletal muscle tropomyosin γß-heterodimer.

Tropomyosin (Tpm) is a regulatory actin-binding protein involved in Ca2+ activation of contraction of striated muscle. In human slow skeletal muscles, two distinct Tpm isoforms, γ and ß, are present. They interact to form three types of dimeric Tpm molecules: γγ-homodimers, γß-heterodimers, or ßß-homodimers, and a majority of the molecules are present as γß-Tpm heterodimers. Point mutation R91P within the TPM3 gene encoding γ-Tpm is linked to the condition known as congenital fiber-type disproportion (CFTD), which is characterized by severe muscle weakness. Here, we investigated the influence of the R91P mutation in the γ-chain on the properties of the γß-Tpm heterodimer. We found that the R91P mutation impairs the functional properties of γß-Tpm heterodimer more severely than those of earlier studied γγ-Tpm homodimer carrying this mutation in both γ-chains. Since a significant part of Tpm molecules in slow skeletal muscle is present as γß-heterodimers, our results explain why this mutation leads to muscle weakness in CFTD.

N-Terminal Fragment of Cardiac Myosin Binding Protein C Modulates Cooperative Mechanisms of Thin Filament Activation in Atria and Ventricles.

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C is located on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both kinetics of the ATP hydrolysis cycle and lifetime of the cross-bridge, as well as calcium regulation of the actin-myosin interaction, thereby modulating contractile function of myocardium. The role of cMyBP-C in atrial contraction has not been practically studied. We examined effect of the N-terminal C0-C1-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in an in vitro motility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of the actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of ATP and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less than ventricular myosin, and the C0-C2 fragment makes these differences in the myosin isoforms more pronounced.

Structural and Functional Properties of Kappa Tropomyosin.

In the myocardium, the TPM1 gene expresses two isoforms of tropomyosin (Tpm), alpha (αTpm; Tpm 1.1) and kappa (κTpm; Tpm 1.2). κTpm is the result of alternative splicing of the TPM1 gene. We studied the structural features of κTpm and its regulatory function in the atrial and ventricular myocardium using an in vitro motility assay. We tested the possibility of Tpm heterodimer formation from α- and κ-chains. Our result shows that the formation of ακTpm heterodimer is thermodynamically favorable, and in the myocardium, κTpm most likely exists as ακTpm heterodimer. Using circular dichroism, we compared the thermal unfolding of ααTpm, ακTpm, and κκTpm. κκTpm had the lowest stability, while the ακTpm was more stable than ααTpm. The differential scanning calorimetry results indicated that the thermal stability of the N-terminal part of κκTpm is much lower than that of ααTpm. The affinity of ααTpm and κκTpm to F-actin did not differ, and ακTpm interacted with F-actin significantly worse. The troponin T1 fragment enhanced the κκTpm and ακTpm affinity to F-actin. κκTpm differently affected the calcium regulation of the interaction of pig and rat ventricular myosin with the thin filament. With rat myosin, calcium sensitivity of thin filaments containing κκTpm was significantly lower than that with ααTpm and with pig myosin, and the sensitivity did not differ. Thin filaments containing κκTpm and ακTpm were better activated by pig atrial myosin than those containing ααTpm.

Novel Mutation Glu98Lys in Cardiac Tropomyosin Alters Its Structure and Impairs Myocardial Relaxation.

We characterized a novel genetic variant c.292G > A (p.E98K) in the TPM1 gene encoding cardiac tropomyosin 1.1 isoform (Tpm1.1), found in a proband with a phenotype of complex cardiomyopathy with conduction dysfunction and slow progressive neuromuscular involvement. To understand the molecular mechanism by which this mutation impairs cardiac function, we produced recombinant Tpm1.1 carrying an E98K substitution and studied how this substitution affects the structure of the Tpm1.1 molecule and its functional properties. The results showed that the E98K substitution in the N-terminal part of the Tpm molecule significantly destabilizes the C-terminal part of Tpm, thus indicating a long-distance destabilizing effect of the substitution on the Tpm coiled-coil structure. The E98K substitution did not noticeably affect Tpm's affinity for F-actin but significantly impaired Tpm's regulatory properties. It increased the Ca2+ sensitivity of the sliding velocity of regulated thin filaments over cardiac myosin in an in vitro motility assay and caused an incomplete block of the thin filament sliding at low Ca2+ concentrations. The incomplete motility block in the absence of Ca2+ can be explained by the loosening of the Tpm interaction with troponin I (TnI), thus increasing Tpm mobility on the surface of an actin filament that partially unlocks the myosin binding sites. This hypothesis is supported by the molecular dynamics (MD) simulation that showed that the E98 Tpm residue is involved in hydrogen bonding with the C-terminal part of TnI. Thus, the results allowed us to explain the mechanism by which the E98K Tpm mutation impairs sarcomeric function and myocardial relaxation.

The effects of the tropomyosin cardiomyopathy mutations on the calcium regulation of actin-myosin interaction in the atrium and ventricle differ.

The molecular mechanisms of pathogenesis of atrial myopathy associated with hypertrophic (HCM) and dilated (DCM) mutations of sarcomeric proteins are still poorly understood. For this, one needs to investigate the effects of the mutations on actin-myosin interaction in the atria separately from ventricles. We compared the impact of the HCM and DCM mutations of tropomyosin (Tpm) on the calcium regulation of the thin filament interaction with atrial and ventricular myosin using an in vitro motility assay. We found that the mutations differently affect the calcium regulation of actin-myosin interaction in the atria and ventricles. The DCM E40K Tpm mutation significantly reduced the maximum sliding velocity of thin filaments with ventricular myosin and its Ca2+-sensitivity. With atrial myosin, its effects were less pronounced. The HCM I172T mutation reduced the Ca2+-sensitivity of the sliding velocity of filaments with ventricular myosin but increased it with the atrial one. The HCM L185R mutation did not affect actin-myosin interaction in the atria. The results indicate that the difference in the effects of Tpm mutations on the actin-myosin interaction in the atria and ventricles may be responsible for the difference in pathological changes in the atrial and ventricular myocardium.

Properties of Cardiac Myosin with Cardiomyopathic Mutations in Essential Light Chains.

The effects of cardiomyopathic mutations E56G, M149V, and E177G in the MYL3 gene encoding essential light chain of human ventricular myosin (ELCv), on the functional properties of cardiac myosin and its isolated head (myosin subfragment 1, S1) were investigated. Only the M149V mutation upregulated the actin-activated ATPase activity of S1. All mutations significantly increased the Ca2+-sensitivity of the sliding velocity of thin filaments on the surface with immobilized myosin in the in vitro motility assay, while mutations E56G and M149V (but not E177G) reduced the sliding velocity of regulated thin filaments and F-actin filaments almost twice. Therefore, despite the fact that all studied mutations in ELCv are involved in the development of hypertrophic cardiomyopathy, the mechanisms of their influence on the actin-myosin interaction are different.

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.

Effects of myopathy-causing mutations R91P and R245G in the TPM3 gene on structural and functional properties of slow skeletal muscle tropomyosin.

Tropomyosin (Tpm) is an actin-binding protein that plays a crucial role in the regulation of muscle contraction. Numerous point mutations in the TPM3 gene encoding Tpm of slow skeletal muscles (Tpm 3.12 or γ-Tpm) are associated with the genesis of various congenital myopathies. Two of these mutations, R91P and R245G, are associated with congenital fiber-type disproportion (CFTD) characterized by hypotonia and generalized muscle weakness. We applied various methods to investigate how these mutations affect the structural and functional properties of γγ-Tpm homodimers. The results show that both these mutations lead to strong structural changes in the γγ-Tpm molecule and significantly impaired its functional properties. These changes in the Tpm properties caused by R91P and R245G mutations give insight into the molecular mechanism of the CFTD development and the weakness of slow skeletal muscles observed in this inherited disease.

Mechanisms of disturbance of the contractile function of slow skeletal muscles induced by myopathic mutations in the tropomyosin TPM3 gene.

Several congenital myopathies of slow skeletal muscles are associated with mutations in the tropomyosin (Tpm) TPM3 gene. Tropomyosin is an actin-binding protein that plays a crucial role in the regulation of muscle contraction. Two Tpm isoforms, γ (Tpm3.12) and ß (Tpm2.2) are expressed in human slow skeletal muscles forming γγ-homodimers and γß-heterodimers of Tpm molecules. We applied various methods to investigate how myopathy-causing mutations M9R, E151A, and K169E in the Tpm γ-chain modify the structure-functional properties of Tpm dimers, and how this affects the muscle functioning. The results show that the features of γγ-Tpm and γß-Tpm with substitutions in the Tpm γ-chain vary significantly. The characteristics of the γγ-Tpm depend on whether these mutations located in only one or both γ-chains. The mechanism of the development of nemaline myopathy associated with the M9R mutation was revealed. At the molecular level, a cause-and-effect relationship has been established for the development of myopathy by the K169E mutation. Also, we described the structure-functional properties of the Tpm dimers with the E151A mutation, which explain muscle weakness linked to this substitution. The results demonstrate a diversity of the molecular mechanisms of myopathy pathogenesis induced by studied Tpm mutations.

Acidosis modifies effects of phosphorylated tropomyosin on the actin-myosin interaction in the myocardium.

Phosphorylation of α-tropomyosin (Tpm1.1), a predominant Tpm isoform in the myocardium, is one of the regulatory mechanisms of the heart contractility. The Tpm 1.1 molecule has one site of phosphorylation, Ser283. The degree of the Tpm phosphorylation decreases with age and also changes in heart pathologies. Myocardial pathologies, in particular ischemia, are usually accompanied by pH lowering in the cardiomyocyte cytosol. We studied the effects of acidosis on the structural and functional properties of the pseudo-phosphorylated form of Tpm1.1 with the S283D substitution. We found that in acidosis, the interaction of the N- and C-ends of the S283D Tpm molecules decreases, whereas that of WT Tpm does not change. The pH lowering increased thermostability of the complex of F-actin with S283D Tpm to a greater extent than with WT Tpm. Using an in vitro motility assay with NEM- modified myosin as a load, we assessed the effect of the Tpm pseudo-phosphorylation on the force of the actin-myosin interaction. In acidosis, the force generated by myosin in the interaction with thin filaments containing S283D Tpm was higher than with those containing WT Tpm. Also, the pseudo-phosphorylation increased the myosin ability to resist a load. We conclude that ischemia changes the effect of the phosphorylated Tpm on the contractile function of the myocardium.

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

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

Myopathic mutations in the ß-chain of tropomyosin differently affect the structural and functional properties of ßß- and αß-dimers.

Tropomyosin (Tpm) is an actin-binding protein that plays a vital role in the regulation of muscle contraction. Fast skeletal muscles express 2 Tpm isoforms, α (Tpm 1.1) and ß (Tpm 2.2), resulting in the existence of 2 forms of dimeric Tpm molecule: αα-homodimer and αß-heterodimer. ßß-Homodimer is unstable and absent in the native state, despite which most of the studies of myopathy-relating Tpm mutations have been performed on the ßß-homodimer. Here, we applied different methods to investigate the effects of myopathic mutations R133W and N202K in the ß-chain of Tpm on properties of αß-heterodimers and to compare them with the features of ßß-homodimers with the same mutations. The results show that properties of αß-Tpm and ßß-Tpm with substitutions in the ß-chain differ significantly, and this indicates that the effects of myopathic mutations in the Tpm ß-chain should be studied on the Tpm αß-heterodimer.-Bershitsky, S. Y., Logvinova, D. S., Shchepkin, D. V., Kopylova, G. V., Matyushenko, A. M. Myopathic mutations in the ß-chain of tropomyosin differently affect the structural and functional properties of ßß- and αß-dimers.

Functional outcomes of structural peculiarities of striated muscle tropomyosin.

Tropomyosin is a dimer coiled-coil actin-binding protein. Adjacent tropomyosin molecules connect each other 'head-to-tail' via an overlap junction and form a continuous strand that winds around an actin filament and controls the actin-myosin interaction. High cooperativity of muscle contraction largely depends on tropomyosin characteristics. Here we summarise experimental evidence that local peculiarities of tropomyosin structure have long-range effects and determine functional properties of the strand, including changes in its bending stiffness and interaction with actin and myosin. Point mutations and posttranslational modifications help to probe the roles of the conserved 'non-canonical' residues, clusters of stabilising and destabilising core residues, and core gap in tropomyosin function. The data suggest that tropomyosin structural lability including a diversity of homo- and heterodimers of different isoforms provide a balance of stiffness, flexibility, and strength of interaction with partner sarcomere proteins necessary for fine-tuning of Ca2+ regulation in various types of striated muscles.

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

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

Structural and functional properties of αß-heterodimers of tropomyosin with myopathic mutations Q147P and K49del in the ß-chain.

Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein that plays a key role in the Ca2+-regulated contraction of striated muscles. Two Tpm isoforms, α (Tpm 1.1) and ß (Tpm 2.2), are expressed in fast skeletal muscles. These Tpm isoforms can form either αα and ßß homodimers, or αß heterodimers. However, only αα-Tpm and αß-Tpm dimers are usually present in most of fast skeletal muscles, because ßß-homodimers are relatively unstable and cannot exist under physiologic conditions. Nevertheless, the most of previous studies of myopathy-causing mutations in the Tpm ß-chains were performed on the ßß-homodimers. In the present work, we applied different methods to investigate the effects of two myopathic mutations in the ß-chain, Q147P and K49del (i.e. deletion of Lys49), on structural and functional properties of Tpm αß-heterodimers and to compare them with the properties of ßß-homodimers carrying these mutations in both ß-chains. The results show that the properties of αß-Tpm heterodimers with these mutations in the ß-chain differ significantly from the properties of ßß-homodimers with the same substitutions in both ß-chains. This indicates that the αß-heterodimer is a more appropriate model for studying the effects of myopathic mutations in the ß-chain of Tpm than the ßß-homodimer which virtually does not exist in human skeletal muscles.

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 Closed State of the Thin Filament Is Not Occupied in Fully Activated Skeletal Muscle.

Muscle contraction is powered by actin-myosin interaction controlled by Ca2+ via the regulatory proteins troponin (Tn) and tropomyosin (Tpm), which are associated with actin filaments. Tpm forms coiled-coil dimers, which assemble into a helical strand that runs along the whole ∼1 µm length of a thin filament. In the absence of Ca2+, Tn that is tightly bound to Tpm binds actin and holds the Tpm strand in the blocked, or B, state, where Tpm shields actin from the binding of myosin heads. Ca2+ binding to Tn releases the Tpm from actin so that it moves azimuthally around the filament axis to a closed, or C, state, where actin is partially available for weak binding of myosin heads. Upon transition of the weak actin-myosin bond into a strong, stereo-specific complex, the myosin heads push Tpm strand to the open, or O, state allowing myosin binding sites on several neighboring actin monomers to become open for myosin binding. We used low-angle x-ray diffraction at the European Synchrotron Radiation Facility to check whether the O- to C-state transition in fully activated fibers of fast skeletal muscle of the rabbit occurs during transition from isometric contraction to shortening under low load. No decrease in the intensity of the second actin layer line at reciprocal radii in the range of 0.15-0.275 nm-1 was observed during shortening suggesting that an azimuthal Tpm movement from the O- to C-state does not occur, although during shortening muscle stiffness is reduced compared to the isometric state, and the intensities of other actin layer lines demonstrate a ∼2-fold decrease in the fraction of myosin heads strongly bound to actin. The data show that a small fraction of actin-bound myosin heads is sufficient for supporting the O-state and, therefore the C-state is not occupied in fully activated skeletal muscle that produces mechanical work at low load.

Structural and Functional Effects of Cardiomyopathy-Causing Mutations in the Troponin T-Binding Region of Cardiac Tropomyosin.

Hypertrophic cardiomyopathy (HCM) is a severe heart disease caused by missense mutations in genes encoding sarcomeric proteins of cardiac muscle. Many of these mutations are identified in the gene encoding the cardiac isoform of tropomyosin (Tpm), an α-helical coiled-coil actin-binding protein that plays a key role in Ca2+-regulated contraction of cardiac muscle. We employed various methods to characterize structural and functional features of recombinant human Tpm species carrying HCM mutations that lie either within the troponin T-binding region in the C-terminal part of Tpm (E180G, E180V, and L185R) or near this region (I172T). The results of our structural studies show that all these mutations affect, although differently, the thermal stability of the C-terminal part of the Tpm molecule: mutations E180G and I172T destabilize this part of the molecule, whereas mutation E180V strongly stabilizes it. Moreover, various HCM-causing mutations have different and even opposite effects on the stability of the Tpm-actin complexes. Studies of reconstituted thin filaments in the in vitro motility assay have shown that those HCM-associated mutations that lie within the troponin T-binding region of Tpm similarly increase the Ca2+ sensitivity of the sliding velocity of the filaments and impair their relaxation properties, causing a marked increase in the sliding velocity in the absence of Ca2+, while mutation I172T decreases the Ca2+ sensitivity and has no influence on the sliding velocity under relaxing conditions. Finally, our data demonstrate that various HCM mutations can differently affect the structural and functional properties of Tpm and cause HCM by different molecular mechanisms.

The isoforms of α-actin and myosin affect the Ca2+ regulation of the actin-myosin interaction in the heart.

Myocardium of mammals contains a wide range of isoforms of proteins that provides contractile function of the heart. These are two isoforms of ventricular and two of atrial myosin, α- and ß-tropomyosin, and two isoforms of α-actin: cardiac and skeletal. We believe that the difference in the amino acid sequence of α-actin can affect the calcium regulation of the actin-myosin interaction. To test this hypothesis, we investigated effects of the isoforms of α-actin, cardiac and skeletal, and the isoforms of cardiac myosin on the calcium regulation of the actin-myosin interaction in an in vitro motility assay using reconstructed regulated thin filaments. The results show that isoforms of α-actin and the ratio of α/ß-chains of Tpm differently affect the calcium regulation of the actin-myosin interaction in myocardium in dependence on cardiac myosin isoforms.