Effects of cycling and rigor crossbridges on the conformation of cardiac troponin C.

Abstract

The results of work by several investigators indicate that crossbridge attachment serves as a positive feedback mechanism that transiently increases the Ca2+ affinity of troponin C (TnC) during each normal heartbeat. To monitor structural changes in the cardiac isoform of TnC (cTnC) associated with Ca2+ binding and crossbridge attachment in muscle, we labeled cTnC with the sulfhydryl-specific fluorescent probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid (IAANS). When IAANS-labeled cTnC (cTnCIAANS) was substituted for endogenous TnC, the fluorescence intensity of cardiac and skeletal muscle preparations increased substantially during rigor crossbridge attachment in the absence of Ca2+ (pCa 9.2). In cardiac muscle, the fluorescence signal increased the same amount in rigor and maximal activation, whereas in skeletal muscle, it was higher in rigor (rigor: cardiac and skeletal = 1; pCa 4.0: cardiac = 0.98 +/- 0.13, skeletal = 0.59 +/- 0.05). This indicates that crossbridge attachment alone is capable of influencing the structure of cTnCIAANS. Because the relative fluorescence intensity of cTnCIAANS was more sensitive to Ca2+ than was force in both preparations (cardiac: pCa50 fluorescence = 6.05 +/- 0.05, pCa50 force = 5.51 +/- 0.11; skeletal: pCa50 fluorescence = 5.94 +/- 0.13, pCa50 force = 5.65 +/- 0.14), we measured the Ca2+ sensitivity of the strong crossbridge attachment (sinusoidal stiffness was measured by imposing 1 kHz at 0.1-0.2% muscle length) in rat trabeculae.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a contractile deficit in adult cardiac myocytes expressing hypertrophic cardiomyopathy–associated mutant troponin T proteins

“…Our results differ from a recent report showing marked reductions in maximum calcium-activated force production, as well as altered calcium sensitivity, in quail skeletal myotubes expressing I79N (10). This difference is important from a mechanistic standpoint, given that reduced maximum calcium-activated force indicates a change in the ability of the cross-bridges to interact with the thin filament, which, in itself, could account for changes in calcium sensitivity at submaximal calcium concentrations (24), as discussed below.…”

“…There is a growing appreciation that activating ligands for this thin-filament regulatory system include both calcium and myosin (24). McKillop and Geeves (26) proposed a three-state model of thin-filament activation that incorporates (a) a blocked state where stereospecific binding of myosin is prohibited; (b) a closed state where non-force-producing interactions between actin and myosin are permitted; and (c) an open state where strong-binding, force-generating myosin crossbridge formation occurs.…”

Biological Function and Site II Ca2+-induced Opening of the Regulatory Domain of Skeletal Troponin C Are Impaired by Invariant Site I or II Glu Mutations

“…Estimation of main chain order parameters (S 2 ) and conformational entropies on a per residue basis indicate that residue positions 1-3 of site I in cTnC contribute inordinately to this entropy difference. This increased rigidity is explicable in terms of the structural interactions and environments of Val 28 and Leu 29 . Val 28 , having an S 2 value of 0.83 and indicating little flexibility, is described as being largely buried and having several hydrophobic contacts with Ala 31 (position 3) and positions 6 -8 of site I. Hydrophobic contacts are also evident between Leu 29 and Ile 36 /Ser 37 (␤-sheet residues of site I).…”

“…From this perspective, the failure of cNTnC to undergo full opening upon site II Ca 2ϩ binding, as we propose for sNTnC, can be understood not in terms of its inability to bind Ca 2ϩ at site I but rather in terms of the hydrophobic nature of the amino acid alterations that lead to this loss of Ca 2ϩ binding. Pertinent to these considerations are the experiments carried out by Putkey, Sweeney, and collaborators a decade ago (66 -68) in which Ca 2ϩ binding to site I of cTnC was activated by deletion of Val 28 and substitution of residues 29 -32 (positions 1-4) of site I by their skeletal TnC counterparts. This construct was fully active as a replacement for cTnC in slow skeletal muscle fiber tension measurements and as active as wild-type cTnC in a fast skeletal muscle system.…”

The degree of activation of cardiac muscle depends on muscle length

“…In other words, how does the association of crossed bridges of myosin with actin increase the affinity of TnC to Ca 2+ and, consequently, the sensitivity of the muscle system to Ca 2+ ? Studies in which TnC was tagged with fluorescent probes by substitution of specific molecules at some of its regulatory domains furnish some evidence on this issue [31][32][33] .…”