A novel mutant cardiac troponin C disrupts molecular motions critical for calcium binding affinity and cardiomyocyte contractility.

Troponin C (TnC) belongs to the superfamily of EF-hand (helix-loop-helix) Ca(2+)-binding proteins and is an essential component of the regulatory thin filament complex. In a patient diagnosed with idiopathic dilated cardiomyopathy, we identified two novel missense mutations localized in the regulatory Ca(2+)-binding Site II of TnC, TnC((E59D,D75Y)). Expression of recombinant TnC((E59D,D75Y)) in isolated rat cardiomyocytes induced a marked decrease in contractility despite normal intracellular calcium homeostasis in intact cardiomyocytes and resulted in impaired myofilament calcium responsiveness in Triton-permeabilized cardiomyocytes. Expression of the individual mutants in cardiomyocytes showed that TnC(D75Y) was able to recapitulate the TnC((E59D,D75Y)) phenotype, whereas TnC(E59D) was functionally benign. Force-pCa relationships in TnC((E59D,D75Y)) reconstituted rabbit psoas fibers and fluorescence spectroscopy of TnC((E59D,D75Y)) labeled with 2-[(4'-iodoacetamide)-aniline]naphthalene-6-sulfonic acid showed a decrease in myofilament Ca(2+) sensitivity and Ca(2+) binding affinity, respectively. Furthermore, computational analysis of TnC showed the Ca(2+)-binding pocket as an active region of concerted motions, which are decreased markedly by mutation D75Y. We conclude that D75Y interferes with proper concerted motions within the regulatory Ca(2+)-binding pocket of TnC that hinders the relay of the thin filament calcium signal, thereby providing a primary stimulus for impaired cardiomyocyte contractility. This in turn may trigger pathways leading to aberrant ventricular remodeling and ultimately a dilated cardiomyopathy phenotype.

Familial hypertrophic cardiomyopathy mutations in troponin I (K183D, G203S, K206Q) enhance filament sliding.

A major cause of familial hypertrophic cardiomyopathy (FHC) is dominant mutations in cardiac sarcomeric genes. Linkage studies identified FHC-related mutations in the COOH terminus of cardiac troponin I (cTnI), a region with unknown function in Ca(2+) regulation of the heart. Using in vitro assays with recombinant rat troponin subunits, we tested the hypothesis that mutations K183Delta, G203S, and K206Q in cTnI affect Ca(2+) regulation. All three mutants enhanced Ca(2+) sensitivity and maximum speed (s(max)) of filament sliding of in vitro motility assays. Enhanced s(max) (pCa 5) was observed with rabbit skeletal and rat cardiac (alpha-MHC or beta-MHC) heavy meromyosin (HMM). We developed a passive exchange method for replacing endogenous cTn in permeabilized rat cardiac trabeculae. Ca(2+) sensitivity and maximum isometric force did not differ between preparations exchanged with cTn(cTnI,K206Q) or wild-type cTn. In both trabeculae and motility assays, there was no loss of inhibition at pCa 9. These results are consistent with COOH terminus of TnI modulating actomyosin kinetics during unloaded sliding, but not during isometric force generation, and implicate enhanced cross-bridge cycling in the cTnI-related pathway(s) to hypertrophy.

Hyalinizing spindle cell tumor with giant rosettes: a case report.

Hyalinizing spindle cell tumor with giant rosettes (HSCTGR) is characterized by both giant rosette-like structures with collagen cores sparsely distributed throughout the tumor and fibromyxoid stroma. It is a rare low-grade sarcoma with indolent behavior, and wide excision with long-term follow-up is the best treatment. Although originally considered a distinct entity, it is now regarded as a variant of low-grade fibromyxoid sarcoma. We present a case of HSCTGR arising in the deep soft tissue of the left knee in a 50-year-old woman and provide a brief review of the literature for comparison.

Ca2+ regulation of rabbit skeletal muscle thin filament sliding: role of cross-bridge number.

We investigated how strong cross-bridge number affects sliding speed of regulated Ca(2+)-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca(2+). Second, we varied the number of Ca(2+)-activatable troponin complexes at maximal Ca(2+) using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A,D63A), a mutant deficient in Ca(2+) binding at both N-terminal, low affinity Ca(2+)-binding sites (xxsTnC-sTn). Sliding speed decreased nonlinearly as the proportion of WT sTn decreased. Speed of regulated thin filaments varied with pCa when filaments contained WT sTn but filaments containing only xxsTnC-sTn did not move. pCa(50) decreased by 0.12-0.18 when either heavy meromyosin density was reduced to approximately 60% or the fraction of Ca(2+)-activatable regulatory units was reduced to approximately 33%. Third, we exchanged mixtures of sTnC and xxsTnC into single, permeabilized fibers from rabbit psoas. As the proportion of xxsTnC increased, unloaded shortening velocity decreased nonlinearly at maximal Ca(2+). These data are consistent with unloaded filament sliding speed being limited by the number of cycling cross-bridges so that maximal speed is attained with a critical, low level of actomyosin interactions.

Thin filament near-neighbour regulatory unit interactions affect rabbit skeletal muscle steady-state force-Ca(2+) relations.

The role of cooperative interactions between individual structural regulatory units (SUs) of thin filaments (7 actin monomers : 1 tropomyosin : 1 troponin complex) on steady-state Ca(2+)-activated force was studied. Native troponin C (TnC) was extracted from single, de-membranated rabbit psoas fibres and replaced by mixtures of purified rabbit skeletal TnC (sTnC) and recombinant rabbit sTnC (D27A, D63A), which contains mutations that disrupt Ca(2+) coordination at N-terminal sites I and II (xxsTnC). Control experiments in fibres indicated that, in the absence of Ca(2+), both sTnC and xxsTnC bind with similar apparent affinity to sTnC-extracted thin filaments. Endogenous sTnC-extracted fibres reconstituted with 100 % xxsTnC did not develop Ca(2+)-activated force. In fibres reconstituted with mixtures of sTnC and xxsTnC, maximal Ca(2+)-activated force increased in a greater than linear manner with the fraction of sTnC. This suggests that Ca(2+) binding to functional Tn can spread activation beyond the seven actins of an SU into neighbouring units, and the data suggest that this functional unit (FU) size is up to 10-12 actins. As the number of FUs was decreased, Ca(2+) sensitivity of force (pCa(50)) decreased proportionally. The slope of the force-pCa relation (the Hill coefficient, n(H)) also decreased when the reconstitution mixture contained < 50 % sTnC. With 15 % sTnC in the reconstitution mixture, n(H) was reduced to 1.7 +/- 0.2, compared with 3.8 +/- 0.1 in fibres reconstituted with 100 % sTnC, indicating that most of the cooperative thin filament activation was eliminated. The results suggest that cooperative activation of skeletal muscle fibres occurs primarily through spread of activation to near-neighbour FUs along the thin filament (via head-to-tail tropomyosin interactions).