Calcium binding to the thin filament protein troponin is required for cardiac and skeletal muscles to contract, and several studies indicate that this regulation involves shifting the tropomyosin position on the actin filament. When the regulatory sites of troponin do not have bound Ca 2ϩ , tropomyosin is located on the actin outer domain. In this position tropomyosin sterically interferes with much more of the myosin-binding site than it does in the presence of Ca 2ϩ , and therefore contraction is inhibited at low Ca 2ϩ concentrations. This regulatory scheme is supported by three-dimensional helical reconstructions of thin filaments examined by electron microscopy with negative staining (1, 2) or unstained in vitreous ice (3), and by modeling of x-ray diffraction patterns of muscle (4). Furthermore, it is consistent with the solution kinetics of myosin S1-thin filament binding in the absence of ATP (5). However, it is unclear how troponin affects the position of tropomyosin on actin, and more generally the inhibitory action of troponin is not well understood at a structural level, as opposed to better understandings of tropomyosin.Troponin consists of a relatively globular domain (including TnI 1 and TnC) and an elongated tail region, the NH 2 terminus of TnT (6, 7). The inhibitory actions of troponin have long been attributed to the TnI subunit. Skeletal muscle TnI inhibits the actin-myosin ATPase rate in the absence of the other troponin subunits TnC and TnT (8 -10), and this effect requires lower TnI concentrations in the presence rather than in the absence of tropomyosin (11). Cardiac TnI has similar properties, although the inhibition is less effective (12-14). The inhibitory effects of skeletal muscle TnI can be mimicked by the so-called inhibitory peptide, residues 96 -116 (10, 15), or identically by the corresponding cardiac peptide (14). The reversal of inhibition is related to Ca 2ϩ -dependent TnI-TnC interactions, elucidated in part at the atomic level (16). An additional TnI region, approximately 130 -150 residues, has also been implicated in inhibition (17)(18)(19). These and other data are consistent with an inhibitory mechanism consisting primarily of a TnI-actin interaction that is reversed by Ca 2ϩ , i.e. a localized actin-troponin interaction tethers the much longer tropomyosin on the actin outer domain in the absence, but not in the presence, of Ca 2ϩ . Indeed, our own electron microscope results show that Ca 2ϩ causes a decrease in troponin density in contact with actin (20). However, no high resolution data exists for these interactions, or for the assembled thin filament, and it remains possible that other mechanisms are also important for regulation and for determining the shifting positions of tropomyosin on the actin surface.This report describes new and unexpected attributes of the troponin tail, i.e. the NH 2 terminus of TnT. In the absence of all other portions of troponin, including TnI, cardiac TnT-(1-153) inhibited the interaction of myosin with actin-tropomyosin filaments. Helica...

Cooperative myosin binding to the thin filament is critical to regulation of cardiac and skeletal muscle contraction. This report delineates and fits to experimental data a new model of this process, in which specific tropomyosin-actin interactions are important, the tropomyosin-tropomyosin polymer is continuous rather than disjointed, and tropomyosin affects myosin-actin binding by shifting among three positions as in recent structural studies. A myosin- and tropomyosin-induced conformational change in actin is proposed, rationalizing the approximately 10,000-fold strengthening effect of myosin on tropomyosin-actin binding. Also, myosin S1 binding to regulated filaments containing mutant tropomyosins with internal deletions exhibited exaggerated cooperativity, implying an allosteric effect of tropomyosin on actin and allowing the effect's measurement. Comparisons among the mutants suggest the change in actin is promoted much more strongly by the middle of tropomyosin than by its ends. Regardless of calcium binding to troponin, this change in actin facilitates the shift in tropomyosin position to the actin inner domain, which is required for tight myosin-actin association. It also increases myosin-actin affinity 7-fold compared with the absence of troponin-tropomyosin. Finally, initiation of a shift in tropomyosin position is 100-fold more difficult than is its extension from one actin to the next, producing the myosin binding cooperativity that underlies cooperative activation of muscle contraction.

Striated muscle contraction is regulated by Ca2؉ binding to troponin, which has a globular domain and an elongated tail attributable to the NH 2 -terminal portion of the bovine cardiac troponin T (TnT) subunit. Truncation of the bovine cardiac troponin tail was investigated using recombinant TnT fragments and subunits TnI and TnC. Progressive truncation of the troponin tail caused progressively weaker binding of troponin-tropomyosin to actin and of troponin to actin-tropomyosin. A sharp drop-off in affinity occurred with NH 2 -terminal deletion of 119 rather than 94 residues. Deletion of 94 residues had no effect on Ca 2؉ -activation of the myosin subfragment 1-thin filament MgATPase rate and did not eliminate cooperative effects of Ca 2؉ binding. Troponin tail peptide TnT1-153 strongly promoted tropomyosin binding to actin in the absence of TnI or TnC. The results show that the anchoring function of the troponin tail involves interactions with actin as well as with tropomyosin and has comparable importance in the presence or absence of Ca 2؉. Residues 95-153 are particularly important for anchoring, and residues 95-119 are crucial for function or local folding. Because striated muscle regulation involves switching among the conformational states of the thin filament, regulatory significance for the troponin tail may arise from its prominent contribution to the protein-protein interactions within these conformations.Troponin is a striated muscle regulatory protein (see reviews in Refs. 1-4) that is located at periodic, 38-nm spacing along muscle thin filaments. This spacing is due to the 1:1 complex formation of troponin with tropomyosin, an elongated coiledcoil protein that stretches along seven actin monomers. Ca 2ϩ binding to troponin triggers conformational changes in the thin filament, thereby allowing actin and myosin to interact to produce force and movement. Troponin contains two domains: a globular region, which is composed of subunits TnC, 1 TnI, and the COOH-terminal portion of TnT, and a highly extended region, or tail, containing the remainder of TnT (5, 6). The globular region has a central role in regulation, because it is the site of calcium binding. In contrast, the tail region of troponin, which is the subject of this report, has an uncertain role in conformational changes of the thin filament. One possibility is that it has little direct effect on regulation, acting instead as a calcium-insensitive anchor that holds troponin onto tropomyosin (7,8). However, the details of the interactions of troponin with actin and tropomyosin are unknown, in any of the conformations of the thin filament. Moreover, there is increasing evidence that the structure of the troponin tail can alter thin filament function in a complex manner (9 -16). To better understand the troponin tail region, the present study reports the properties of a series of troponin complexes containing progressively less of this region. Cardiac TnC plus TnI was reconstituted with either cardiac TnT or a series of recombinant NH 2 -termina...

Missense mutations in the cardiac thin filament protein troponin T (TnT) are a cause of familial hypertrophic cardiomyopathy (FHC). To understand how these mutations produce dysfunction, five TnTs were produced and purified containing FHC mutations found in several regions of TnT. Functional defects were diverse. Mutations F110I, E244D, and COOH-terminal truncation weakened the affinity of troponin for the thin filament. Mutation ⌬E160 resulted in thin filaments with increased calcium affinity at the regulatory site of troponin C. Mutations R92Q and F110I resulted in impaired troponin solubility, suggesting abnormal protein folding. Depending upon the mutation, the in vitro unloaded actin-myosin sliding speed showed small increases, showed small decreases, or was unchanged. COOH-terminal truncation mutation resulted in a decreased thin filament-myosin subfragment 1 MgATPase rate. The results indicate that the mutations cause diverse immediate effects, despite similarities in disease manifestations. Separable but repeatedly observed abnormalities resulting from FHC TnT mutations include increased unloaded sliding speed, increased or decreased Ca 2؉ affinity, impairment of folding or sarcomeric integrity, and decreased force. Enhancement as well as impairment of contractile protein function is observed, suggesting that TnT, including the troponin tail region, modulates the regulation of cardiac contraction. Familial hypertrophic cardiomyopathy (FHC)1 can be caused by dominant mutations in genes encoding any one of several proteins of the cardiac contractile apparatus: myosin, C-protein, tropomyosin, or troponin (1-5). These molecules have central roles in contraction or its regulation, suggesting that abnormal contraction of the heart leads to the clinical, histological, and morphological manifestations of FHC. Strong support for this conclusion comes from three related types of observations: (i) purified mutant proteins retain many of their basic functions but display kinetic or other abnormalities that would be expected to alter muscle contraction (6 -11); (ii) altered contraction occurs in isolated muscle fibers or cells expressing the mutant protein (12-15); and (iii) transgenic animals with FHC-linked mutations exhibit altered cardiac function (15-18).One of the more commonly affected genes in FHC is that for the tropomyosin binding subunit of troponin, troponin T (1). Like the abnormalities in other genes that cause this syndrome, the troponin T mutations occur widely throughout the sequence, are generally missense or point deletions, and occasionally are premature truncations. Although there is less severe hypertrophy in kindreds with the troponin T mutations than is observed in other FHC patients, these kindreds show a high incidence of sudden death and have mortality rates as high as those accompanying the most severe mutations in the myosin heavy chain gene (1). This raises the possibility that the contraction abnormalities resulting from troponin T mutations may differ from those found in FHC more gener...

Background-We report hypertrophic cardiomyopathy (HCM) in a Spanish-American family caused by a novel ␣-tropomyosin (TPM1) mutation and examine the pathogenesis of the clinical disease by characterizing functional defects in the purified mutant protein. Methods and Results-HCM was linked to the TPM1 gene (logarithm of the odds [LOD] score 3.17). Sequencing andrestriction digestion analysis demonstrated a TPM1 mutation V95A that cosegregated with HCM. The mutation has been associated with 13 deaths in 26 affected members (11 sudden deaths and 2 related to heart failure), with a cumulative survival rate of 73Ϯ10% at the age of 40 years. Left ventricular wall thickness (mean 16Ϯ6 mm) and disease penetrance (53%) were similar to those for the ␤-myosin mutations L908V and G256E previously associated with a benign prognosis. Left ventricular hypertrophy was milder than with the ␤-myosin mutation R403Q, but the prognosis was similarly poor. With the use of recombinant tropomyosins, we identified several functional alterations at the protein level. The mutation caused a 40% to 50% increase in calcium affinity in regulated thin filament-myosin subfragment-1 (S1) MgATPase assays, a 20% decrease in MgATPase rates in the presence of saturating calcium, a 5% decrease in unloaded shortening velocity in in vitro motility assays, and no change in cooperative myosin S1 binding to regulated thin filaments. Conclusions-In contrast to other reported TPM1 mutations, V95A-associated HCM exhibits unusual features of mild phenotype but poor prognosis. Both myosin cycling and calcium binding to troponin are abnormal in the presence of the mutant tropomyosin. The genetic diagnosis afforded by this mutation will be valuable in the management of HCM. Key Words: cardiomyopathy Ⅲ genetics Ⅲ death, sudden Ⅲ prognosis H ypertrophic cardiomyopathy (HCM) is characterized by left ventricular (LV) hypertrophy (LVH) and myocyte disarray. Clinical characteristics of HCM vary from a benign asymptomatic course to severe heart failure and sudden death. Molecular genetic studies [1][2][3][4][5][6][7] have shown that HCM may be caused by mutations in several sarcomeric genes, including TPM1, that may determine clinical outcome. Most ␤-myosin heavy chain gene (MYH7) mutations, such as R403Q, are associated with high disease penetrance and a poor prognosis. 4 In contrast, the few MYH7 mutations that have a benign prognosis, such as L908V and G256E, are associated with a low disease penetrance. 5 HCM caused by myosin-binding protein-C gene (MYBPC3) is characterized by low disease penetrance (Ϸ60%), mild cardiac phenotype in young subjects, and a favorable prognosis. 1-3 HCM due to cardiac troponin-T gene (TNNT2) is usually associated with relatively low disease penetrance (Ϸ80%) and mild LVH but a high incidence of sudden death. 6 The TPM1 gene consists of 14 exons and 4 isoforms (␣-and ␤-tropomyosins, tropomyosin-4, and tropomyosin-30). 1,8 The cardiac isoform is generated from 10 exons, is expressed in both myocardium and fast skeletal muscle fibers, and ...