The role of tropomyosin isoforms and phosphorylation in force generation in thin-filament reconstituted bovine cardiac muscle fibres.

The thin filament extraction and reconstitution protocol was used to investigate the functional roles of tropomyosin (Tm) isoforms and phosphorylation in bovine myocardium. The thin filament was extracted by gelsolin, reconstituted with G-actin, and further reconstituted with cardiac troponin together with one of three Tm varieties: phosphorylated alphaTm (alphaTm.P), dephosphorylated alphaTm (alphaTm.deP), and dephosphorylated betaTm (betaTm.deP). The effects of Ca, phosphate, MgATP and MgADP concentrations were examined in the reconstituted fibres at pH 7.0 and 25 degrees C. Our data show that Ca(2+) sensitivity (pCa(50): half saturation point) was increased by 0.19 +/- 0.07 units when betaTm.deP was used instead of alphaTm.deP (P < 0.05), and by 0.27 +/- 0.06 units when phosphorylated alphaTm was used (P < 0.005). The cooperativity (Hill factor) decreased (but insignificantly) from 3.2 +/- 0.3 (5) to 2.8 +/- 0.2 (7) with phosphorylation. The cooperativity decreased significantly from 3.2 +/- 0.3 (5) to 2.1 +/- 0.2 (9) with isoform change from alphaTm.deP to betaTm.deP. There was no significant difference in isometric tension or stiffness between alphaTm.P, alphaTm.deP, and betaTm.deP muscle fibres at saturating [Ca(2+)] or after rigor induction. Based on the six-state cross-bridge model, sinusoidal analysis indicated that the equilibrium constants of elementary steps differed up to 1.7x between alphaTm.deP and betaTm.deP, and up to 2.0x between alphaTm.deP and alphaTm.P. The rate constants differed up to 1.5x between alphaTm.deP and betaTm.deP, and up to 2.4x between alphaTm.deP and alphaTm.P. We conclude that tension and stiffness per cross-bridge are not significantly different among the three muscle models.

Troponin I binds polycystin-L and inhibits its calcium-induced channel activation.

Polycystin-L (PCL) is an isoform of polycystin-2, the product of the second gene associated with autosomal dominant polycystic kidney disease, and functions as a Ca(2+)-regulated nonselective cation channel. We recently demonstrated that polycystin-2 interacts with troponin I, an important regulatory component of the actin microfilament complex in striated muscle cells and an angiogenesis inhibitor. In this study, using the two-microelectrode voltage-clamp technique and Xenopus oocyte expression system, we showed that the calcium-induced PCL channel activation is substantially inhibited by the skeletal and cardiac troponin I (60% and 31% reduction, respectively). Reciprocal co-immunoprecipitation experiments demonstrated that PCL physically associates with the skeletal and cardiac troponin I isoforms in overexpressed Xenopus oocytes and mouse fibroblast NIH 3T3 cells. Furthermore, both native PCL and cardiac troponin I were present in human heart tissues where they indeed associate with each other. GST pull-down and microtiter binding assays showed that the C-terminus of PCL interacts with the troponin I proteins. The yeast two-hybrid assay further verified this interaction and defined the corresponding interacting domains of the PCL C-terminus and troponin I. Taken together, this study suggests that troponin I acts as a regulatory subunit of the PCL channel complex and provides the first direct evidence that PCL is associated with the actin cytoskeleton through troponin I.

Role of hydration in the closed-to-open transition involved in Ca2+ binding by troponin C.

Troponin C (TnC) is the Ca(2+)-binding subunit of the troponin complex of vertebrate skeletal muscle. It consists of two structurally homologous domains, N and C, connected by an exposed alpha-helix. The C-domain has two high-affinity sites for Ca(2+) that also bind Mg(2+), whereas the N-domain has two low-affinity sites for Ca(2+). Previous studies using isolated N- and C-domains showed that the C-domain apo form was less stable than the N-domain. Here we analyzed the stability of isolated N-domain (F29W/N-domain) against urea and pressure denaturation in the absence and in the presence of glycerol using fluorescence spectroscopy. Increasing the glycerol concentration promoted an increase in the stability of the protein to urea (0-8 M) in the absence of Ca(2+). Furthermore, the ability to expose hydrophobic surfaces normally promoted by Ca(2+) binding or low temperature under pressure was partially lost in the presence of 20% (v/v) glycerol. Glycerol also led to a decrease in the Ca(2+) affinity of the N-domain in solution. From the ln K(obs) versus ln a(H)2(O), we obtained the number of water molecules (63.5 +/- 8.7) involved in the transition N <=>N:Ca(2) that corresponds to an increase in the exposed surface area of 571.5 +/- 78.3 A(2). In skinned fibers, the affinity for Ca(2+) was also reduced by glycerol, although the effect was much less pronounced than in solution. Our results demonstrate quantitatively that the stability of this protein and its affinity for Ca(2+) are critically dependent on protein hydration.

Myofibrillar determinants of rate of relaxation in skinned skeletal muscle fibers.

The influence of Ca2+ dissociation rate from TnC and decreased cross-bridge detachment rate on the time course of relaxation induced by flash photolysis of diazo-2 in rabbit skinned psoas fibers was investigated at 15 degrees C. A TnC mutant (M82Q TnC) that exhibited increased Ca2+ sensitivity caused by a decreased Ca2+ dissociation rate in solution also increased the Ca2+ sensitivity of force and decreased the rate of relaxation in fibers approximately 2-fold. In contrast, a TnC mutant (NHdel TnC) with decreased Ca2+ sensitivity caused by an increased Ca2+ dissociation rate in solution decreased Ca2+ sensitivity of force but did not accelerate relaxation. Decreasing the rate of cross-bridge kinetics by reducing [Pi] slowed relaxation -2-fold and led to two phases of relaxation, a linear phase followed by an exponential phase. In fibers, M82Q TnC further slowed relaxation in low [Pi] approximately 2-fold whereas NHdel TnC had no significant effect on relaxation. These results are consistent with the interpretation that the Ca2+ dissociation rate and cross-bridge detachment rate are similar in fast twitch skeletal muscle such that decreasing either rate slows relaxation but accelerating Ca2+ dissociation has little effect on relaxation.

Determinants of relaxation rate in rabbit skinned skeletal muscle fibres.

The influence of Ca(2+)-activated force, the rate of dissociation of Ca(2+) from troponin C (TnC) and decreased crossbridge detachment rate on the time course of relaxation induced by flash photolysis of diazo-2 in rabbit skinned psoas fibres was investigated at 15 degrees C. The rate of relaxation increased as the diazo-2 chelating capacity (i.e. free [diazo-2]/free [Ca(2+)]) increased. At a constant diazo-2 chelating capacity, the rate of relaxation was independent of the pre-photolysis Ca(2+)-activated force in the range 0.3-0.8 of maximum isometric force. A TnC mutant that exhibited increased Ca(2+) sensitivity caused by a decreased Ca(2+) dissociation rate in solution (M82Q TnC) also increased the Ca(2+) sensitivity of steady-state force and decreased the rate of relaxation in fibres by approximately twofold. In contrast, a TnC mutant with decreased Ca(2+) sensitivity caused by an increased Ca(2+) dissociation rate in solution (NHdel TnC) decreased the Ca(2+) sensitivity of steady-state force but did not accelerate relaxation. Decreasing the rate of crossbridge kinetics by reducing intracellular inorganic phosphate concentration ([P(i)]) slowed relaxation by approximately twofold and led to two phases of relaxation, a slow linear phase followed by a fast exponential phase. In fibres, M82Q TnC further slowed relaxation in low [P(i)] conditions by approximately twofold, whereas NHdel TnC had no significant effect on relaxation. These results are consistent with the interpretation that the Ca(2+)-dissociation rate and crossbridge detachment rate are similar in fast-twitch skeletal muscle, such that decreasing either rate slows relaxation, but accelerating Ca(2+) dissociation has little effect on relaxation.

Engineering competitive magnesium binding into the first EF-hand of skeletal troponin C.

The goal of this study was to examine the mechanism of magnesium binding to the regulatory domain of skeletal troponin C (TnC). The fluorescence of Trp(29), immediately preceding the first calcium-binding loop in TnC(F29W), was unchanged by addition of magnesium, but increased upon calcium binding with an affinity of 3.3 microm. However, the calcium-dependent increase in TnC(F29W) fluorescence could be reversed by addition of magnesium, with a calculated competitive magnesium affinity of 2.2 mm. When a Z acid pair was introduced into the first EF-hand of TnC(F29W), the fluorescence of G34DTnC(F29W) increased upon addition of magnesium or calcium with affinities of 295 and 1.9 microm, respectively. Addition of 3 mm magnesium decreased the calcium sensitivity of TnC(F29W) and G34DTnC(F29W) approximately 2- and 6-fold, respectively. Exchange of G34DTnC(F29W) into skinned psoas muscle fibers decreased fiber calcium sensitivity approximately 1.7-fold compared with TnC(F29W) at 1 mm [magnesium](free) and approximately 3.2-fold at 3 mm [magnesium](free). Thus, incorporation of a Z acid pair into the first EF-hand allows it to bind magnesium with high affinity. Furthermore, the data suggests that the second EF-hand, but not the first, of TnC is responsible for the competitive magnesium binding to the regulatory domain.

Single mutation (A162H) in human cardiac troponin I corrects acid pH sensitivity of Ca2+-regulated actomyosin S1 ATPase.

In contrast to skeletal muscle, the efficiency of the contractile apparatus of cardiac tissue has long been known to be severely compromised by acid pH as in the ischemia of myocardial infarction and other cardiac myopathies. Recent reports (Westfall, M. V., and Metzger, J. M. (2001) News Physiol. Sci. 16, 278-281; Li, G., Martin, A. F., and Solaro, R. J. (2001) J. Mol. Cell. Cardiol. 33, 1309-1320) have indicated that the reduced Ca(2+) sensitivity of cardiac contractility at low pH (

Kinetic studies of calcium and cardiac troponin I peptide binding to human cardiac troponin C using NMR spectroscopy.

Ca2+ and human cardiac troponin I (cTnI) peptide binding to human cardiac troponin C (cTnC) have been investigated with the use of 2D [1H,15N] HSQC NMR spectroscopy. The spectral intensity, chemical shift, and line-shape changes were analyzed to obtain the dissociation ( K(D)) and off-rate ( k(off)) constants at 30 degrees C. The results show that sites III and IV exhibit 100-fold higher Ca2+ affinity than site II ( K(D(III,IV)) approximately 0.2 microM, K(D(II)) approximately 20 microM), but site II is partially occupied before sites III and IV are saturated. The addition of the first two equivalents of Ca2+ saturates 90% of sites III and IV and 20% of site II. This suggests that the Ca2+ occupancy of all three sites may contribute to the Ca2+-dependent regulation in muscle contraction. We have determined a k(off) of 5000 s(-1) for site II Ca2+ dissociation at 30 degrees C. Such a rapid off-rate had not been previously measured. Three cTnI peptides, cTnI(34-71), cTnI(128-147), and cTnI(147-163), were titrated to Ca2+-saturated cTnC. In each case, the binding occurs with a 1:1 stoichiometry. The determined K(D) and k(off) values are 1 microM and 5 s(-1) for cTnI(34-71), 78+/-10 microM and 5000 s(-1) for cTnI(128-147), and 150+/-10 microM and 5000 s(-1) for cTnI(147-163), respectively. Thus, the dissociation of Ca2+ from site II and cTnI(128-147) and cTnI(147-163) from cTnC are rapid enough to be involved in the contraction/relaxation cycle of cardiac muscle, while that of cTnI(34-71) from cTnC may be too slow for this process.