The control of myocardial contraction with skeletal fast muscle troponin C

The present study describes experiments on the myocardial trabeculae from the right ventricle of Syrian hamsters whose troponin C (TnC) moiety was exchanged with heterologous TnC from fast skeletal muscle of the rabbit. These experiments were designed to help define the role of the various classes of Ca2+-binding sites on TnC in setting the characteristic sensitivities for activations of cardiac and skeletal muscles. Thin trabeculae were skinned and about 75% of their troponin C extracted by chemical treatment. Tension development on activations by Ca2+ and Sr2+ was found to be nearly fully blocked in such TnC extracted preparations. Troponin C contents and the ability to develop tension on activations by Ca2+ and Sr2+ was permanently restored after incubation with 2-6 mg/ml purified TnC from either rabbit fast-twitch skeletal muscle (STnC) or the heart (CTnC, cardiac troponin C). The native (skinned) cardiac muscle is characteristically about 5 times more sensitive to activation by Sr2+ than fast muscle, but the STnC-loaded trabeculae gave response like fast muscle. Attempts were also made to exchange the TnC in psoas (fast-twitch muscle) fibers, but unlike cardiac muscle tension response of the maximally extracted psoas fibers could be restored only with homologous STnC. CTnC was effective in partially extracted fibers, even though the uptake of CTnC was complete in the maximally extracted fibers. The results in this study establish that troponin C subunit is the key in setting the characteristic sensitivity for tension control in the myocardium above that in the skeletal muscle. Since a major difference between skeletal and cardiac TnCs is that one of the trigger sites (site I, residues 28-40 from the N terminus) is modified in CTnC and has reduced affinity for Ca2+ binding, the possibility is raised that this site has a modulatory effect on activation in different tissues and limits the effectiveness of CTnC in skeletal fibers.     

Inflammatory response in human skeletal muscle cells: CXCL10 as a potential therapeutic target

Inflammatory myopathies (IMs) are systemic diseases characterized by a T helper (Th) 1 type inflammatory response and cell infiltrates within skeletal muscles. The mainstay of treatment is drugs aimed at suppressing the immune system - corticosteroids and immunosuppressants. About 25% of patients are non-responders. Skeletal muscle cells seem actively involved in the immune-inflammatory response and not only a target; understanding the molecular bases of IMs might help drug development strategies. Within muscles the interaction between the chemokine interferon (IFN)γ inducible 10 kDa protein, CXCL10 or IP-10, and its specific receptor CXCR3, present on Th1 type infiltrating cells, likely plays a pivotal role, potentially offering the opportunity for therapeutic intervention. We aimed to clarify the involvement of human skeletal muscle cells in inflammatory processes in terms of CXCL10 secretion, to elucidate the engaged molecular mechanism(s) and, finally, to evaluate muscular cell responses, if any, to some immunosuppressants routinely used in IM treatment, such as methylprednisolone, methotrexate, cyclosporin A and Infliximab. We first isolated and characterized human fetal skeletal muscle cells (Hfsmc), which expressed the specific lineage markers and showed the competence to react in the context of an in vitro alloresponse. CXCL10 protein secretion by Hfsmc was similarly induced by the inflammatory cytokines interferon (IFN)γ and tumor necrosis factor (TNF)α, above undetectable control levels, through the activation of Stat1 and NF-kB pathways, respectively; CXCL10 secretion was significantly magnified by cytokine combination, and this synergy was associated to a significant up-regulation of TNFαRII; cytokine-induced CXCL10 secretion was considerably affected only by Infliximab. Our data suggested that human skeletal muscle cells might actively self-promote muscular inflammation by eliciting CXCL10 secretion, which is known to amplify Th1 cell tissue infiltration in vivo. In conclusion, we sustain that pharmacological targeting of CXCL10 within muscular cells might contribute to keep in control pro-Th1 polarization of the immune/inflammatory response.     

The components of troponin from chicken fast skeletal muscle. A comparison of troponin T and troponin I from breast and leg muscle.

The three components of troponin were prepared from chicken breast and leg muscle. The troponin I and T components were separated by chromatography on DEAE-cellulose after citraconylation and without the use of urea-containing buffers. The troponin I and C components were similar to their counterparts from rabbit fast skeletal muscle, and a comparison of the troponin I components from breast and leg muscle by amino acid analysis, gel electrophoresis and peptide 'mapping' provides strong evidence for the identity of these proteins. The molecular weights of the troponin T components from breast and leg muscle were 33 500 and 30 500 respectively, determined by gel filtration. A comparison of these two proteins by methods similar to those used for the troponin I components suggested that they differed only in the N-terminal region of the sequence, the breast-muscle troponin T having an extra length of polypeptide chain of approx. 24 residues that is rich in histidine and alanine. The N-terminal hexapeptide sequence, however, is the same in both proteins and is (Ser,Asx,Glx)Thr-Glu-Glu. The genetic implications of these findings are considered.     

Divalent cation binding properties of slow skeletal muscle troponin in comparison with those of cardiac and fast skeletal muscle troponins

1. New methods of preparing troponins from slow skeletal and cardiac muscle of the chicken have been developed. The electrophoretic mobilities of slow skeletal muscle troponin subunits were different from those of the corresponding fast skeletal muscle subunits. 2. A new method for determining the amount of divalent cations bound to troponin was developed. The principle of the method is to immobilize troponin by conjugating it with Sepharose 4B resin, thus making it readily sedimentable. 3. The numbers of Sr and Ca ions bound to slow muscle troponin at concentrations sufficient to produce maximum contraction were 1.73 and 1.36 mol per mol, respectively, being nearly equal to those of cardiac troponin but half of those of fast muscle troponin. 4. The concentrations of Sr and Ca ions giving half-maximal ion binding to slow muscle troponin (K50%) were 5.5 X 10(-6) M and 4.6 X 10(-7) M, respectively. 5. K50% for Sr of cardiac troponin was significantly higher than that of slow muscle troponin. Although K50% for Sr of cardiac troponin was the same as that of fast muscle troponin, cardiac troponin bound more Sr ions than fast muscle troponin at lower Sr ion concentrations. The mechanism underlying the high sensitivity of cardiac muscle contraction to Sr ions is discussed in comparison with that of slow muscle.     

Conformational change of skeletal muscle troponin

The fluorescence titration curve of skeletal muscle troponin containing TnI with 2-[4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid-labeled Cys-48 and/or Cys-64 was composed of two transition curves. One transition occurred at the pCa region higher than 8.0, and the other between pCa 8.0 and 6.0. The transition at the lower pCa region had a midpoint of pCa 6.85, and the midpoint did not depend on Mg2+. The time course of the fluorescence change subsequent to the rapid pCa-jump of the solution was biphasic. The fast phase was due to the transition at the lower pCa region, and the rate constant of the process was characteristic of the conformational change of the protein induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC. The slow phase was from the transition at the higher pCa region, and its rate constant was characteristic of the conformational change of the protein induced by Ca2+ binding to the high affinity Ca2+-binding sites of TnC. Therefore we can conclude that the fluorescence probe bound to Cys-48 and/or Cys-64 of TnI detects the conformational change of the Tn complex induced by Ca2+ binding to both the low and high affinity Ca2+-binding sites of TnC. The fluorescence probe bound to Cys-133 of TnI or Met residues of TnT detected the conformational change of the Tn complex induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC.     

Adult stem cells: the therapeutic potential of skeletal muscle

Embryonic stem cells have revolutionised our understanding of normal and deregulated growth and development. The potential to produce cells and tissues as needed offers enormous therapeutic potential. The use of these cells, however, is accompanied by ongoing ethical, religious and biomedical issues. The expansion potential and plasticity of adult stem cells have therefore received much interest. Adult skeletal muscle is highly adaptable, responding to both the hypertrophic and degenerative stresses placed upon it. This extreme plasticity is in part regulated by resident stem cells. In addition to regenerating muscle, if exposed to osteogenic or adipogenic inducers, these cells spontaneously form osteoblasts or adipocytes. The potential for and heterogeneity of muscle stem cells is underscored by the observation that CD45+ muscle side population cells are capable of reconstituting bone marrow in lethally irradiated mice and of contributing to neo-vascularisation of regenerating muscle. Finally, first attempts to replace infarcted myocardium relied on injection of skeletal myoblasts into the heart. Cells successfully engrafted and cardiac function was improved. Harnessing their differentiation/trans-differentiation capacity provides enormous potential for adult stem cells. In this review, current understanding of the different stem cells within muscle will be discussed as will their potential utility for regenerative medicine.     

Receptor modification as a therapeutic approach against viral diseases

Poliovirus causes flaccid paralysis through the destruction of motor neurons in the CNS. Susceptibility to its infection is mainly due to the interaction in between the surface capsid proteins and its receptors on the host cell surface, important for binding, penetration and other necessary events during early infection. Receptor modification is a new approach to treat viral diseases by the modification of target proteins structure. Binding domains are modified in an effective way to make it difficult for the virus to recognize it. In this study, tolerant and intolerant induced mutations in the poliovirus receptor, VP1 and VP2 were identified and substituted in the seed sequence to get the modified versions. Substitutions causing changes in initial folding were short listed and further analyzed for high level folding, physiochemical properties and interactions. Highest RMSD values were observed in between the seed and the mutant K90F (3.265 Å) and Q130W (3.270Å) respectively. The proposed substitutions were found to have low functional impact and thus can be further tested and validated by the experimental researchers. Interactions analyses proved most of the substitutions having decreased affinity for both the VP1 and VP2 and thus are of significant importance against poliovirus. This study will play an important role for bridging computational biology to other fields of applied biology and also will provide an insight to develop resistance against viral diseases. It is also expected that same approach can also be applicable against other viruses like HCV, HIV and other in near future.     

Cytokines as potential therapeutic targets for inflammatory skin diseases

A network of pro-inflammatory cytokines is a central feature in the pathophysiology of cutaneous inflammatory diseases. Thus, the delineation of precise roles for particular cytokines and the development of cytokine-directed therapeutics have become areas of intense investigation. While anti-TNF therapeutics have proven to be effective for the treatment of psoriasis, clinical investigations have now begun with other cytokine-directed therapies, such as those targeting IFN-g, IL-12p40, and IL-18. In addition to therapeutics that target cytokines directly, strategies that target cytokine signaling pathways are in development too. In this short review, we summarize key findings from a recent workshop on cytokines as potential therapeutic targets for inflammatory skin diseases.     

The roles of carnosine in aging of skeletal muscle and in neuromuscular diseases

Skeletal muscles undergo specific alterations that are related to the aging process. The incidence of several neuromuscular diseases (e.g., amyotrophic lateral sclerosis (ALS), myasthenia gravis, polymyositis, drug-induced myopathies, late-onset mitochondrial myopathy) is age-related. The increased sensitivity to disease of aging muscle represents an additional age-related negative influence in the presence of existing risk factors (such as a genetic predisposition). The potential significance of carnosine lies on one hand in its possible influence on specific physiological changes in muscle associated with the aging process, and on the other in its effect on oxidative stress and the antioxidative system in specific neuromuscular diseases such as ALS or polymyositis.     

Isolation and sequence of a cDNA clone for rabbit fast skeletal muscle troponin C. Homology with calmodulin and parvalbumin

The binding of Ca2+ to troponin C (TnC) regulates skeletal muscle contraction. We have isolated a full-length cDNA clone for fast skeletal muscle TnC from a neonatal rabbit skeletal muscle library and determined its nucleic acid sequence. The amino acid sequence deduced from this clone matches the previously reported amino acid sequence (Collins, J. H., Greaser, M. L., Potter, J. D., and Horn, M. J. (1977) J. Biol. Chem. 252, 6356-6362) except at the amino terminus. According to the nucleotide sequence, the first 2 residues of TnC are threonine-aspartic acid, which is the reverse of the order reported previously. The isolation of the adult form of TnC from a neonatal library suggests that there may be no developmental isoforms of fast TnC. The protein coding region of the fast TnC clone has 67% homology with the reported nucleotide sequence for chicken slow TnC (Putkey, J. A., Carroll, S. L., and Means, A. R. (1987) Mol. Cell. Biol. 7, 549-1553). The homologies between the nucleotide sequences of TnC, calmodulin, and parvalbumin provide evidence that all three proteins were derived from a common precursor molecule which had four Ca2+-binding sites.     

Structural characterization of the human fast skeletal muscle troponin I gene (TNNI2)

Three troponin I genes have been identified in vertebrates that encode the isoforms expressed in adult cardiac muscle (TNNI3), slow skeletal muscle (TNNI1) and fast skeletal muscle (TNNI2), respectively. While the organization and regulation of human cardiac and slow skeletal muscle genes have been investigated in detail, the fast skeletal troponin I gene has to date only been examined in birds. Here, we describe the structure and complete sequence of the human fast skeletal muscle troponin I gene (TNNI2) and identify putative regulatory elements within both the 5' flanking region and the first intron. In particular, a region containing MEF-2, E-box, CCAC and CAGG elements was identified in intron 1 that closely resembles the fast internal regulatory element (FIRE) of the quail intronic enhancer. We have previously shown that the fast skeletal muscle troponin I gene is located at 11p15.5 and noted potential close linkage with the fast skeletal muscle troponin T gene (TNNT3). Here, we have isolated two independent human PAC genomic clones that contain either TNNI2 or TNNT3 and demonstrate by interphase FISH mapping that they are less than 100 kb apart in the genome. The results demonstrate that the human TNNI2 gene is closely related to its avian counterparts with conserved elements within both the putative promoter and first intron. Our data further confirm close physical linkage of TNNI2 and TNNI3 on 11p15.5.     

cDNA clone and expression analysis of rodent fast and slow skeletal muscle troponin I mRNAs

We have characterized the structure and expression of rodent mRNAs encoding the fast and slow skeletal muscle isoforms of the contractile regulatory protein, troponin I (TnIfast and TnIslow). TnIfast and TnIslow cDNA clones were isolated from mouse and rat muscle cDNA clone libraries and were used as isoform-specific probes in Northern blot and in situ hybridization studies. These studies showed that the TnIfast and TnIslow mRNAs are expressed in skeletal muscle, but not cardiac muscle or other tissues, and that they are differentially expressed in individual muscle fibers. Fiber typing on the basis of in situ hybridization analysis of TnI isoform mRNA content showed an excellent correlation with fiber type as assessed by myosin ATPase histochemistry. These results directly demonstrate that the differential expression of skeletal muscle TnI isoforms in the various classes of vertebrate striated muscle cells is based on gene regulatory mechanisms which control the abundances of specific TnI mRNAs in individual muscle cells. Both TnIfast and TnIslow mRNAs are expressed, at comparable levels, in differentiated cultures of rat L6 and mouse C2 muscle cell lines. Thus, although neuronal input has been shown to be an important factor in determining fast versus slow isoform-specific expression in skeletal muscle, both TnIfast and TnIslow genes can be expressed in muscle cells in the absence of nerve. Comparison of the deduced rodent TnI amino acid sequences with previously determined rabbit protein sequences showed that residues with potential fast/slow isoform-specific function are present in several discrete clusters, two of which are located near previously identified actin and troponin C binding sites.     

Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity.

A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities.     

Crystallographic data for troponin C from turkey skeletal muscle

Crystals of troponin C from turkey skeletal muscle suitable for high resolution X-ray studies have been grown from a Ca2+-containing solution. These crystals diffract to at least 2.5 A resolution, and have space group P3(1)21 or P3(2)21 and the following cell dimensions: a = b = 66.6 A, c = 61.0 A, alpha = beta = 90 degrees, gamma = 120 degrees.     

Identification of a region of fast skeletal troponin T required for stabilization of the coiled-coil formation with troponin I

We have previously identified evolutionarily conserved heptad hydrophobic repeat (HR) domains in all isoprotein members of troponin T (TnT) and troponin I (TnI), two subunits of the Ca(2+)-regulatory troponin complex. Our suggestion that the HR domains are involved in the formation of a coiled-coil heterodimer of TnT and TnI has been recently confirmed by the crystal structure of the core domain of the human cardiac troponin complex. Here we studied a series of recombinant deletion mutants of the fast skeletal TnT to determine the minimal sequence required for stable coiled-coil formation with the HR domain of the fast skeletal TnI. Using circular dichroism spectroscopy, we measured the alpha helical content of the coiled-coil formed by the various TnT peptides with TnI HR domain. Sedimentation equilibrium experiments confirmed that the individual peptides of TnT were monomeric but formed heterodimers when mixed with HR domain of TnI. Isothermal titration calorimetry was then used to directly measure the affinity of the TnT peptides for the TnI HR domain. Surprisingly we found that the HR regions alone of the fast skeletal TnT and TnI, as defined earlier, were insufficient to form a coiled-coil. Furthermore we showed that an additional 14 amino acid residues N-terminal to the conserved HR region (TnT residues 165-178) are essential for the stable coiled-coil formation. We discuss the implication of our finding in the fast skeletal troponin isoform in the light of the crystal structure of the cardiac isoform.     

N-terminal amino acid sequences of three functionally different troponin T isoforms from rabbit fast skeletal muscle

The different isoforms of fast skeletal muscle troponin T (TnT) are generated by alternative splicing of several 5' exons in the fast TnT gene. In rabbit skeletal muscle this process results in three major fast TnT species, TnT1f, TnT2f and TnT3f, that differ in a region of 30 to 40 amino acid residues near the N terminus. Differential expression of these three isoforms modulates the activation of the thin filament by calcium. To establish a basis for further structure-function studies, we have sequenced the N-terminal region of these proteins. TnT2f is the fast TnT sequenced by Pearlstone et al. The larger species TnT1f contains six additional amino acid residues identical in sequence and position to those encoded by exon 4 in the rat fast skeletal muscle TnT gene. TnT3f also contains that sequence but lacks 17 amino acid residues spanning the region encoded by exons 6 and 7 of the rat gene. These three TnTs appear to be generated by discrete alternative splicing pathways, each differing by a single event. Comparison of these TnT sequences with those from chicken fast skeletal muscle and bovine heart shows that the splicing pattern resulting in the excision of exon 4 is evolutionarily conserved and leads to a more calcium-sensitive thin filament.