While calcium binding to troponin C (TnC) triggers the contraction of both skeletal and cardiac muscle, there is clear evidence that different mechanisms may be involved. For example, activation heart myofilaments occurs with a single regulatory site on TnC, whereas fast two sites. The physiological difference between not understood at molecular level due lack structural details for response TnC calcium. We determined solution structures apo calcium-saturated domain human by using...

Abstract The backbone resonance assignments have been completed for the apo ( 1 H and 15 N) calcium‐loaded H, N, 13 C) regulatory N‐domain of chicken skeletal troponin‐C (1–90), using multidimensional homonuclear heteronuclear NMR spectroscopy. chemical‐shift information, along with detailed NOE analysis 3 J HNHα coupling constants, permitted determination quantification Ca 2+ ‐induced secondary structural change in TnC. For both structures, 5 helices 2 short β‐strands were found, as was...

Nebulin is a giant filamentous F-actin-binding protein ( approximately 800 kDa) that binds along the thin filament of skeletal muscle sarcomere. one least well understood major proteins. Although nebulin usually viewed as structural protein, here we investigated whether plays role in contraction by using skinned fiber bundles from knock-out (NEB KO) mouse model. We measured force-pCa (-log[Ca(2+)]) and force-ATPase relations, rate tension re-development (k(tr)) tibialis cranialis fibers. To...

The functional consequences of the R92Q mutation in cardiac troponin T (cTnT), linked to familial hypertrophic cardiomyopathy humans, are not well understood. We have studied steady- and pre-steady-state mechanical activity detergent-skinned fiber bundles from a transgenic (TG) mouse model which 67% total cTnT heart was replaced by mutant cTnT. TG fibers were more sensitive Ca 2+ than nontransgenic (NTG) [negative logarithm half maximally activating molar (pCa 50 ) = 5.84 ± 0.01 6.12 for NTG...

During β-adrenergic stimulation of the heart, there is a decrease in myofilament Ca2+ sensitivity mediated by protein kinase A-(PKA-) induced phosphorylation troponin I (cTnI). Phosphorylation, which occurs at Ser 23 and 24 an amino-terminal extension unique to cTnI, decreases affinity regulatory site cardiac C (cTnC). In view antiparallel organization cTnI−cTnC complex [Krudy, G. A., Kleerekoper, Q., Guo, X., Howarth, J. W., Solaro, R. J., Rosevear, P. (1994) Biol. Chem. 269, 23731−23735],...

How different mutations in cardiac troponin T (cTnT) lead to distinct secondary downstream cellular remodeling familial hypertrophic cardiomyopathy (FHC) remains elusive. To explore the molecular basis for impact of cTnT on myocytes, we studied mechanical activity detergent-skinned muscle fiber bundles from lines transgenic (TG) mouse hearts that express wild-type (WTTG), R92W cTnT, R92L and Delta-160 (deletion amino acid 160). The amount mutant is ∼50% total myocellular both TG ∼35% hearts....

Although myostatin negatively regulates skeletal muscle growth, its function in heart is virtually unknown. Herein we demonstrate that it inhibits basal and IGF-stimulated proliferation differentiation also modulates cardiac excitation-contraction (EC) coupling. Loss of induced eccentric hypertrophy enhanced responsiveness to beta-adrenergic stimulation vivo. This was due null ventricular myocytes having larger [Ca(2+)](i) transients contractions responding more strongly than wild-type...

Phosphorylation of two adjacent serine residues in the unique N-terminal extension cardiac muscle troponin I (cTnI) is known to decrease Ca2+-sensitivity myofilaments. To probe structural significance extension, we have constructed cTnI mutants each containing a single cysteine: (1) full-length mutant (S5C/C81I/C98S) and (2) truncated (S9C/C50I/C67S) which 32 amino acid were deleted. We determined apparent binding constants for complex formation between IAANS-labeled C (cTnC) mutants. The...

To describe the dynamics of constantly activated cardiac muscle, we propose that length affects force via both recruitment and distortion myosin cross bridges. This hypothesis was quantitatively tested for descriptive explanative validity. Skinned muscle fibers from animals expressing primarily alpha-myosin heavy chain (MHC) (mouse, rat) or beta-MHC (rabbit, ferret) were with solutions pCa 6.1 to 4.3. Activated subjected small-amplitude perturbations [deltaL(t)] rich in frequency content...

1 The heterogenic nature of familial hypertrophic cardiomyopathy (FHC) in humans suggests a link between the type mutation and patho-physiological alterations cardiac myocytes. Exactly how FHC-associated mutations troponin T (cTnT) lead to impaired function is unclear. 2 We measured steady-state isometric force ATPase activity detergent-skinned fibre bundles from three transgenic (TG) mouse hearts which 50, 92 6 % native cTnT was replaced by wild (WT) cTnT, R92Q mutant (R92Q) C-terminal...

A spectral probe mutant (F29W) of chicken skeletal muscle troponin C (TnC) has been prepared in which Phe-29 substituted by Trp. Residue 29 is at the COOH-terminal end helix immediately adjacent to Ca2+ binding loop site I (residues 30-41) regulatory N domain. Since this protein naturally devoid Tyr and Trp, features can be assigned unambiguously single The fluorescent quantum yield 336 nm increased almost 3-fold going from Ca(2+)-free state 4Ca2+ with no change wavelength maximum emission....

Ca(2+) binding to cardiac troponin C (cTnC) triggers contraction in heart muscle. In failure, myofilaments response are often altered and compounds that sensitize the possess therapeutic value this syndrome. One of most potent selective sensitizers is thiadiazinone derivative EMD 57033, which increases myocardial contractile function both vivo vitro interacts with cTnC vitro. We have determined NMR structure 1:1 complex between Ca(2+)-saturated C-domain human (cCTnC) 57033. Favorable...

Protein kinase (PK)C-induced phosphorylation of cardiac troponin (cTn)I has been shown to regulate contraction.Characterize functional effects increased PKC-induced cTnI and identify underlying mechanisms using a transgenic mouse model (cTnI(PKC-P)) expressing mutant (S43E, S45E, T144E).Two-dimensional gel analysis showed 7.2+/-0.5% replacement endogenous with the form. Experiments included: mechanical measurements (perfused isolated hearts, papillary muscles, skinned fiber preparations),...

Motivated by the need for an analytical tool that can be used routinely to analyze data collected from isolated, detergent-skinned cardiac muscle fibers, we developed a mathematical model representing force response step changes in length (i.e., quick stretch and release). Our proposed is reasonably simple, consisting of only five parameters representing: (1) rate constant which change–induced distortion elastic elements dissipated; (2) stiffness fiber; (3) amplitude length-mediated...

Coordinated expression of species-specific myosin heavy chain (MHC) and troponin (Tn) isoforms may bring about a dynamic complementarity to match muscle contraction speed with heart rates. Contractile system function force-length measurements were made in fibers from mouse rat hearts after reconstitution either recombinant homologous Tn or orthologous Tn. The rate constants length-mediated cross-bridge (XB) recruitment (b) tension redevelopment (k(tr)) significantly faster than those fibers....

The two globular N and C domains of chicken troponin (TnC) are connected by an exposed alpha-helix (designated D/E; residues 86-94). Recombinant (residues 1-90) 88-162) containing either F29 or W29 F105 W105 have been engineered expressed in Escherichia coli. These termination initiation sites were chosen to minimize disruption side-chain interactions between the D/E helix other residues. served as useful spectral probes for monitoring Ca(2+)-induced structural transitions domains,...

Ca2+ binding to the N-domain of skeletal muscle troponin C (sNTnC) induces an "opening" structure [Gagné, S. M., et al. (1995) Nat. Struct. Biol. 2, 784−789], which is typical Ca2+-regulatory proteins. However, recent structures E41A mutant (E41A sNTnC) (1997) Biochemistry 36, 4386−4392] and cardiac (cNTnC) [Sia, K., J. Chem. 272, 18216−18221] reveal that both these proteins remain essentially in "closed" conformation their Ca2+-saturated states. Both are modified Ca2+-binding site I, albeit...

Protein kinase C (PKC)-mediated phosphorylation of cardiac troponin I (cTnI) and T (cTnT) has been shown to diminish maximum activation myofilaments. The functional role cTnI investigated. However, the impact cTnT on myofilament force is not well studied. We tested effect endogenous PKC steady-state tension development Ca 2+ sensitivity in skinned fiber bundles from transgenic (TG) mouse hearts expressing fast skeletal TnT (fsTnT), which naturally lacks sites present cTnT. 12-...

The regulatory function of cardiac troponin I (cTnI) involves three important contiguous regions within its C-domain: the inhibitory region (IR), (RR), and mobile domain (MD). Within these regions, dynamics regional structure kinetics transitions in dynamic state are believed to facilitate signaling. This study was designed use fluorescence anisotropy techniques acquire steady-state kinetic information on C-domain cTnI reconstituted thin filament. A series single cysteine mutants generated,...