Calcium activation of fast striated muscle results from an opening of the regulatory N-terminal domain of fast skeletal troponin C (fsTnC), and a substantial exposure of a hydrophobic patch, essential for Ca 2؉ -dependent interaction with fast skeletal troponin I (fsTnI). This interaction is obligatory to relieve the inhibition of strong, force-generating actin-myosin interactions. We have determined intersite distances in the N-terminal domain of cardiac TnC (cTnC) by fluorescence resonance energy transfer measurements and found negligible increases in these distances when the single regulatory site is saturated with Ca Contraction of striated muscle is regulated by a group of actin-binding proteins, the troponin-tropomyosin complex located on the actin filament (1). Troponin is a heterotrimer. The subunit TnC 1 binds Ca 2ϩ , TnI binds actin and inhibits actomyosin ATPase in relaxed muscle, and troponin T anchors the three-subunit complex to tropomyosin on the actin filament. These proteins form the thin filament. Strong force-generating interactions between myosin and actin are initiated by the binding of Ca 2ϩ to the regulatory sites located in the N-terminal regulatory domain of TnC. The binding of activator Ca 2ϩ to TnC weakens or breaks the interaction between TnI and actin, thus releasing the inhibition of actomyosin ATPase and initiating force generation.The crystal structure of TnC from avian fast skeletal muscle TnC shows a dumbbell-shaped molecule with the N-and Cterminal segments folded into two globular domains connected by a long ␣-helical central helix (2, 3). Each domain has two Ca 2ϩ -binding EF-hand (helix-loop-helix) motifs. The five helices in the N-domain are labeled the N-helix and helices A-D, starting from the N terminus (Fig. 1). The four helices in the C-domain are labeled helices E-H, starting from the C-terminal end of the central helix. The N-domain of fast skeletal TnC has two Ca ). The two sites in the N-domain are the regulatory sites. Site I consists of the helix A-loop-helix B and site II the helix C-loop-helix D motif. Sites III and IV in the C-domain are believed to play a structural role and are occupied by Mg 2ϩ under physiological conditions in relaxed muscle. Site I in cardiac TnC is inactive in chelating Ca 2ϩ due to substitutions of two critical amino acids and an insertion in the binding loop; saturation of site II by Ca 2ϩ is sufficient to trigger contraction in cardiac muscle (4). The crystal structure of fsTnC contains two bound Ca 2ϩ ions in the C-domain and no bound cation in the N-domain. Based on the structure of the C-domain, an early computer model (5) (the HMJ model) suggests that Ca 2ϩ binding to the regulatory sites induces reorientations of the B and C helices relative to the A and D helices, thus exposing a hydrophobic patch located in the B helix (see Fig. 1). The exposed hydrophobic patch in this open conformation becomes available for the Ca 2ϩ -dependent interaction with TnI. This model also has been used to interpret functional and drug binding proper...

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Calcium is a key element in intracellular signaling in skeletal muscle. Changes in intracellular calcium levels are thought to mediate the fast-to-slow transformation of muscle fiber type. One factor implicated in gene regulation in adult muscle is the nuclear factor of activated T-cells (NFAT) isoform c1, whose dephosphorylation by the calcium/calmodulin-dependent phosphatase calcineurin facilitates its nuclear translocation. Here, we report that differentiated C2C12 myotubes predominantly expressing fast-type MyHCII protein undergo fast-to-slow transformation following calcium-ionophore treatment, with several transcription factors and a transcriptional coactivator acting in concert to upregulate the slow myosin heavy chain (MyHC) beta promoter. Transient transfection assays demonstrated that the calcineurin/NFATc1 signaling pathway is essential for MyHCbeta promoter activation during transformation of C2C12 myotubes but is not sufficient for complete fast MyHCIId/x promoter inhibition. Along with NFATc1, myocyte enhancer factor-2D (MEF-2D) and the myogenic transcription factor MyoD transactivated the MyHCbeta promoter in calcium-ionophore-treated myotubes in a calcineurin-dependent manner. To elucidate the mechanism involved in regulating MyHCbeta gene expression, we analyzed the -2.4-kb MyHCbeta promoter construct for cis-regulatory elements. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitation assays (ChIP), and nuclear complex coimmunoprecipitation (NCcoIP) assays, we demonstrated calcium-ionophore-induced binding of NFATc1 to a NFAT consensus site adjacent to a MyoD-binding E-box. At their respective binding sites, both NFATc1 and MyoD recruited the transcriptional coactivator p300, and in turn, MEF-2D bound to the MyoD complex. The calcium-ionophore-induced effects on the MyHCbeta promoter were shown to be calcineurin-dependent. Together, our findings demonstrate calcium-ionophore-induced activation of the beta MyHC promoter by NFATc1, MyoD, MEF-2D, and p300 in a calcineurin-dependent manner.

We have isolated cDNA clones from thyrotoxic (pMHCa) and normal (pMHC(8) adult rabbit hearts. Restriction map analysis and DNA sequence analyses show that, although there is strong homology between overlapping regions of the two clones, they are distinctly different. The two clones exhibited 78483% homology between the derived amino acid sequences and those determined by direct amino acid sequence analysis of rabbit fast skeletal muscle myosin heavy chains. The clones specify a segment of the myosin heavy chain corresponding to subfragment 2 and the COOH-terminal portions of subfragment 1. Nuclease S1 mapping was used to compare transcription of the two clones with expression of the a and (3 forms of myosin heavy chains in the ventricles of thyrotoxic, hypothyroid (propylthiouracil-treated), and normal rabbits. Thyrotoxic ventricles contained only pMHCa transcripts whereas hypothyroid ventricles contained exclusively pMHC(3 transcripts. These data correlate well with the presence of a-and (3-form myosin heavy chains. In the normal young adult rabbit, pMHC(3 transcripts predominate, agreeing with the known (3 form/a form ratio of 4: 1. We therefore conclude that pMHCa and pMHCP contain sequences of the a-and (-form myosin heavy chain genes, respectively.Myosin, a major contractile protein of skeletal and cardiac muscle, is composed of two 200,000-dalton heavy chains (HCs) and two sets oflow molecular weight light chains. The active center of myosin ATPase resides in the globular head of the heavy chain. This enzymatic activity is correlated with contractile velocity in skeletal muscle (1) and thus appears to be an important determinant of contractile function. Numerous polymorphic forms of myosin HC exist, not only in different types of muscle-e.g., fast and slow skeletal and cardiac-but also within each muscle type (2-12). Expression of these forms follows a developmental pattern (12-18) that may be altered by changes in the physiological (19-21) and hormonal (10-13) milieu ofthe cell.Cardiac ventricular muscle contains at least two formsreferred to as a and (3 (9)-of myosin HC. Electrophoresis of myosin under nondenaturing conditions reveals three bands; V1 and V3 are homodimers containing the a and 3 forms, respectively, whereas V2 is a heterodimer (9, 13). The ATPase activity of myosin with a-form HCs has been shown to be considerably higher than that of myosin with (-form .The expression of a-and (3form myosin HCs follows a defined developmental pattern that varies in different animal species. It has been shown that in rabbit the ( form/a form ratio is =3: 1 during the last halfofthe gestational period. After birth, the relative amount of the a form increases so that, during the first 2 wk postnatally, this ratio is 1: 1 (12, 14). Thereafter, the a form decreases and, in the old adult, the 3 form is present almost exclusively (12,14). In the young adult animals used in this study, the ( form/a form ratio was =4:1 (12 (27).Construction and Screening of cDNA Clones. Single-and double-stranded cDNA wa...