The presence of two skeletal muscle alpha-actinins correlates with troponin-tropomyosin expression and Z-line width.

Two species of alpha-actinin from rabbit fast skeletal muscles were identified with a monospecific antisera. Designated alpha-actinin1f and alpha-actinin2f, their distribution in muscles does not correlate with histochemically defined fast fiber type. Rather, the presence of each correlates with Z-line width and with the expression of different thin filament Ca2+-regulatory complexes. alpha-Actinin1f is expressed with troponin T 1f-alpha beta tropomyosin, and alpha-actinin2f with troponin T 2f-alpha 2 tropomyosin. CNBr peptide maps show that the fast alpha-actinin species differ in primary structure. In contrast, the slow alpha-actinin is indistinguishable from alpha-actinin1f. Further evidence for the similarity of alpha-actinin1f and slow alpha-actinin comes from electron microscopic studies which show that fibers that express these species exhibit thick Z-lines. So, unlike other contractile proteins, the multiple forms of alpha-actinin do not reflect the distinction between fast- and slow-twitch muscles.

Actin from the nematode, Caenorhabditis elegans, is a single electrofocusing species.

We have prepared actin from wild type Caenorhabditis elegans animals by three procedures: a purification dependent on the ability of actin to form F-actin, affinity chromatography which preferentially binds G-actin, and co-precipitation of an actin-myosin complex by antimyosin antibodies. Each preparation yields a single electrofucsing species of actin. Comparison of actin from C. elegans embryos and animals reveals that embryos also have the same single electrofocusing species of actin.

Capillary density of skeletal muscle: a contributing mechanism for exercise intolerance in class II-III chronic heart failure independent of other peripheral alterations.

OBJECTIVES: The study was conducted to determine if the capillary density of skeletal muscle is a potential contributor to exercise intolerance in class II-III chronic heart failure (CHF). BACKGROUND: Previous studies suggest that abnormalities in skeletal muscle histology, contractile protein content and enzymology contribute to exercise intolerance in CHF. METHODS: The present study examined skeletal muscle biopsies from 22 male patients with CHF compared with 10 age-matched normal male control patients. Aerobic capacities, myosin heavy chain (MHC) isoforms, enzymes, and capillary density were measured. RESULTS: The patients with CHF demonstrated a reduced peak oxygen consumption when compared to controls (15.0+/-2.5 vs. 19.8+/-5.0 ml x kg(-1) x min(-1), p <0.05). Using cell-specific antibodies to directly assess vascular density, there was a reduction in capillary density in CHF measured as the number of endothelial cells/fiber (1.42+/-0.28 vs. 1.74+/-0.35, p = 0.02). In CHF, capillary density was inversely related to maximal oxygen consumption (r = 0.479, p = 0.02). The MHC IIx isoform was found to be higher in patients with CHF versus normal subjects (28.5+/-13.6 vs. 19.5+/-9.4, p <0.05). CONCLUSIONS: There was a significant reduction in microvascular density in patients with CHF compared with the control group, without major differences in other usual histologic and biochemical aerobic markers. The inverse relationship with peak oxygen consumption seen in the CHF group suggests that a reduction in microvascular density of skeletal muscle may precede other skeletal muscle alterations and play a critical role in the exercise intolerance characteristic of patients with CHF.

Co-operative activation of skeletal muscle thin filaments by rigor crossbridges. The effect of troponin C extraction.

When Ca2+ binds to troponin C (TnC), all 26 troponin-tropomyosin (Tn-Tm) complexes of a regulatory strand change in concert from the inactive to the active configuration. To see if the complexes respond similarly when they are activated by rigor crossbridges in the absence of Ca2+, we determined the slope (ns) of the bell-shaped pS/tension (pS = -log [MgATP], where S = MgATP2-) relationship between pS 5, where the tension is maximal, and pS 2.3, where fibers are fully relaxed. In control skinned rabbit psoas fibers the ns value is greater than 4; it progressively decreases with TnC extraction. This decrease in ns with TnC extraction is analogous to the decrease in the slope (Hill coefficient) of the pCa/tension (pCa = -log [Ca2+]) relationship with extraction. Complete TnC extraction reduces the maximum substrate-induced tension by only 25%; in contrast, it reduces the maximum Ca2+ induced tension to zero. The effects of TnC extraction on the slope of the pS/tension curve are explained by the assumptions that (1) extracted Tn-Tm complexes no longer change in concert with their neighbors but change independently of them, and (2) co-operative signals cannot cross extracted Tn-Tm complexes. The ns value, therefore, like the nH, is a direct function of the number of contiguous, intact, Tn-Tm complexes in a stretch of a regulatory strand. To describe qualitatively the bi-phasic pS/tension relationship, the mono-phasic pCa/tension relationship, and the effects of TnC extraction on them, we introduce a version of the concerted-transition formalism which includes two activating ligands, Ca2+ and rigor crossbridges.

The thin filament of vertebrate skeletal muscle co-operatively activates as a unit.

We find that extraction of as little as one troponin C molecule per troponin-tropomyosin strand on a thin filament reduces the slope of the pCa/tension relation. We interpret this to mean that the regulatory units along a thin filament of rabbit psoas fibers are linked co-operatively so that a thin filament activates as a unit. The presence of extended co-operativity explains why the pCa/tension relation in skinned fibers has a slope much higher than predicted by binding of Ca2+ to one regulatory unit. Replacement of the extracted troponin C with purified troponin C fully reverses the effect of extraction and shows it to be the essential Ca2+ binding protein responsible for the steep slope of the pCa/tension relation.

Effect of different troponin T-tropomyosin combinations on thin filament activation.

The response of permeabilized rabbit fast skeletal muscle fibers to calcium is determined by the troponin T (TnT) and tropomyosin (Tm) isoforms they express. Fibers expressing primarily TnT2f and alpha 2 Tm exhibit steeper pCa/tension relations than those in which either TnT1f or TnT3f and alpha beta Tm predominate. Troponin C extraction studies show that lower slopes do not result from a less concerted transition on the thin filament: the Tn-Tm regulatory strand activates as a unit in all fast fibers. Because the TnT variants differ in their N-terminal segments, and this region overlaps adjacent Tms on the regulatory strand, we propose that both the end-to-end overlap of Tm and the effect of TnT on that interaction are the basis of the concerted transition of the regulatory strand to the active state that occurs in the presence of calcium. Moreover, the effect of different Tn-Tm combinations on the ratio of the affinities of TnC for calcium in the relaxed and active states appears to be a significant determinant of the contractile properties of fast fibers in vivo.

Co-operative interactions between troponin-tropomyosin units extend the length of the thin filament in skeletal muscle.

Ca2+ binding to troponin C (TnC), a subunit of the thin filament regulatory strand, activates vertebrate skeletal muscle contraction. Tension, however, increases with Ca2+ too abruptly to be the result of binding to sites on individual TnCs. Because extraction of one TnC on average per regulatory strand dramatically reduces the slope of the tension/Ca2+ relationship, we proposed that all 26 troponin-tropomyosin complexes of the regulatory strand form a co-operative system. This study of permeabilized (chemically skinned) rabbit psoas fibers analyzes the extraction time-course, the distribution of extraction sites on regulatory strands and the effects of extraction on the co-operativity of the tension/Ca2+ relationship. Two components of TnC are resolved in the time-course of extraction: a "rapidly extracting" component that can be selectively removed without affecting tension or co-operativity, and a "slow extracting" component whose loss reduces tension and co-operativity. Extraction of [14C]TnC shows that the slowly extracting component is lost randomly, so that, after removal of 5% of the TnC, most extracted strands have lost one TnC. Extraction interrupts the transmission of co-operativity by dividing the regulatory strand into smaller, independent co-operative systems; it reduces tension by preventing Ca2+ activation of TnC-depleted regulatory units. Co-operativity of the tension/Ca2+ relationship is modeled with the concerted-transition formalism for intact systems of 26 regulatory units, and for the smaller systems in extracted fibers.

Differences in skeletal muscle between men and women with chronic heart failure.

Men with chronic heart failure (CHF) have alterations in their skeletal muscle that are partially responsible for a decreased exercise tolerance. The purpose of this study was to investigate whether skeletal muscle alterations in women with CHF are similar to those observed in men and if these alterations are related to exercise intolerance. Twenty-five men and thirteen women with CHF performed a maximal exercise test for evaluation of peak oxygen consumption (VO(2)) and resting left ventricular ejection fraction, after which a biopsy of the vastus lateralis was performed. Twenty-one normal subjects (11 women, 10 men) were also studied. The relationship between muscle markers and peak VO(2) was consistent for CHF men and women. When controlling for gender, analysis showed that oxidative enzymes and capillary density are the best predictors of peak VO(2.) These results indicate that aerobically matched CHF men and women have no differences in skeletal muscle biochemistry and histology. However, when CHF groups were separated by peak exercise capacity of 4.5 metabolic equivalents (METs), CHF men with peak VO(2) >4.5 METs had increased citrate synthase and 3-hydroxyacyl-CoA dehydrogenase compared with CHF men with peak VO(2) <4.5 METs. CHF men with a lower peak VO(2) had increased capillary density compared with men with higher peak VO(2). These observations were not reproduced in CHF women. This suggests that differences may exist in how skeletal muscle adapts to decreasing peak VO(2) in patients with CHF.

Renaturation of skeletal muscle tropomyosin: implications for in vivo assembly.

The observation that the alpha beta heterodimer is the predominant species of tropomyosin in rabbit skeletal muscles has led to the suggestion that this species assembles preferentially. To understand the molecular basis of this assembly process, we have studied renaturation under conditions that favor heterodimer formation. When skeletal muscle tropomyosin composed of equal amounts of alpha and beta subunits is renatured either by cooling or by dialysis a distribution that favors homodimers is generated. In contrast, rapid renaturation by dilution from urea favors the heterodimer. Further analysis of this latter renaturation procedure with cysteine-cleavage fragments of tropomyosin using circular dichroic measurements shows that as few as 30 residues in the NH2-terminal third of each tropomyosin subunit are involved in the initial interaction that results in heterodimer formation. Based on the density of sequence substitutions between the alpha and beta subunits, that region probably includes residues 36-64.

Differences in the transient response of fast and slow skeletal muscle fibers. Correlations between complex modulus and myosin light chains.

Sinusoidal analysis of the mechanochemical properties of skinned muscle fibers under conditions of maximal activation was applied to fibers from several rabbit skeletal muscles (psoas, tibialis anterior, extensor digitorum longus, diaphragm, soleus, semitendinosus). This investigation distinguished between two general classes of fibers, which on the basis of their myosin light chain complements could be classified as fast and slow. In fast fibers (e.g., psoas) we identified the presence of at least three exponential processes (A), (B), (C) of comparable magnitudes. In slow fibers (e.g., soleus) we identified the presence of at least four exponential processes (A)-(D) of very different magnitudes; magnitudes of processes (A) and (B) are very small compared with those of (C) and (D). The apparent rate constants are 8-29-fold slower in slow fibers. Because our sinusoidal characterization takes less than or equal to 22 s and does not involve chemical denaturation or other means of disruption of the myofilament lattice, it allows the different physiological classes of fibers to be characterized and then studied further by other techniques. The perfect correlation between physiological and molecular properties as assayed by gel electrophoresis after sinusoidal analysis demonstrates this and justifies its use in distinguishing between fiber types.

Molecular heterogeneity of histochemical fibre types: a comparison of fast fibres.

The relationship between histochemical fibre type and contractile protein expression was analysed in three rabbit skeletal muscles, the erector spinae, the plantaris and the diaphragm. A procedure for staining fibre bundles was developed using the same histochemical methods as those for typing fibres in cross-section. This allowed pretyped fibres to be selected and their molecular composition to be analysed by gel electrophoresis. The balance of expression of the two predominant fast troponin species, TnT1f and TnT2f, and alpha and beta tropomyosin subunits were studied in type IIA and IIB fast fibres. Type IIA fibres exhibited a restricted pattern of thin filament expression, exhibited TnT1f and both tropomyosin subunits in all three muscles. The expression in type IIB fibres, however, ranges from predominantly TnT2f and the alpha tropomyosin subunit in the erector spinae to TnT1f with both alpha and beta subunits in the diaphragm. These results indicate that there is not a simple one-to-one relationship between the fast muscle fibre subtypes and the expression of different thin filament protein isoforms.

Troponin C modulates the activation of thin filaments by rigor cross-bridges.

Extraction of troponin C (TnC) from skinned muscle fibers reduces maximum Ca2+ and rigor cross-bridge (RXB)-activated tensions and reduces cooperativity between neighboring regulatory units (one troponin-tropomyosin complex and the seven associated actins) of thin filaments. This suggests that TnC has a determining role in RXB, as well as in Ca(2+)-dependent activation processes. To investigate this possibility further, we replaced fast TnC (fTnC) of rabbit psoas fibers with either CaM[3,4TnC] or cardiac TnC (cTnC) and compared the effects of these substitutions on Ca2+ and RXB activation of tension. CaM[3,4TnC] substitution has the same effect on Ca(2+)- and RXB-activated tensions; they are reduced 50%, and cooperativity between regulatory units is reduced 40%. cTnC substitution also reduces the maximum Ca(2+)-activated tension and cooperativity. But with RXB activation the effects on tension and cooperativity are opposite; cTnC substitution potentiates tension but reduces cooperativity. We considered whether tension potentiation could be explained by increased activation by cycling cross-bridges (CXBs), but the concerted transition formalism predicts fibers will fail to relax in high substrate and high pCa when CXBs are activator ligands. It predicts resting tension, which is not observed in either control or cTnC-substituted fibers. Rather, it appears that cTnC facilitates RXB activation of fast fibers more effectively than fTnC. The order of RXB-activated tension facilitation is cTnC > fTnC > CaM[3,4TnC] > empty TnC-binding sites. Comparison of the structures of fTnC, CaM[3,4TnC], and cTnC indicates that the critical region for this property lies in the central helix or N-terminal domain, including EF hand motifs 1 and 2.

Biochemical comparison of fast- and slow-contracting squid muscle.

The myofilament protein compositions of muscle fibres from the transverse muscle mass of the tentacles and the transverse muscle mass of the arms of the loliginid squid Sepioteuthis lessoniana were compared. These two muscle masses are distinct types, differing in their ultrastructural and behavioural properties. The transverse muscle of the tentacles consists of specialized muscle fibres that exhibit cross-striation and unusually short sarcomeres and thick filaments. The transverse muscle of the arms consists of obliquely striated muscle fibres that are typical of cephalopod skeletal muscle in general. The specialization of the tentacle muscle results in a high shortening speed and reflects its role in creating rapid elongation of the tentacles during prey capture. Comparison of samples of myofilament preparations of the two muscle fibre types using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and peptide mapping of myosin heavy chains from the two muscle fibre types, however, showed little evidence of differences in contractile protein isoforms. Thus, specialization for high shortening speed appears to have occurred primarily through changes in the dimensions and arrangement of the myofilament lattice, rather than through changes in biochemistry. The thick filament core protein paramyosin was tentatively identified in the squid muscle fibres. This protein was less abundant in the short thick filament cross-striated tentacle muscle cells than in the obliquely striated arm cells.

Heterogeneity of contractile proteins. Differences in tropomyosin in fast, mixed, and slow skeletal muscles of the rabbit.

The subunit composition and dimeric species of tropomyosin with respect to its alpha and beta subunits have been analyzed from several physiologically different types of skeletal muscle of the rabbit using one- and two-dimensional gel electrophoresis. The findings indicate that there are two types of tropomyosin distributions in rabbit muscles: one in which alpha 2 is the prevalent species and another in which alpha beta is prevalent. While the alpha 2-prevalent pattern is characteristic of some fast muscles, notably longissimus dorsi and psoas, it is not the only pattern found in fast muscles. Plantaris, which is histochemically indistinguishable from longissimus dorsi, exhibits an alpha beta-prevalent distribution. This observation shows that there are at least two types of fast white muscle fibers which differ in their tropomyosin composition and species. The alpha beta-pattern is also found in fast red, slow, and mixed muscles. The fast and slow alpha beta-prevalent patterns can be distinguished electrophoretically because of heterogeneity of their subunits. Finally, the relative abundance of the different forms of tropomyosin in each of the muscles studied indicates that the tropomyosin dimer does not assemble randomly from alpha and beta subunits, but that alpha beta is assembled preferentially in vivo.

Heterogeneity of contractile proteins. A continuum of troponin-tropomyosin expression in mammalian skeletal muscle.

Comparison of the myofibrillar proteins from several adult rabbit skeletal muscles has led to the identification of multiple forms of fast and slow troponin T. In Briggs et al. (Briggs, M. M., Klevit, R., and Schachat, F. H. (1984) J. Biol. Chem. 259, 10369-10375) two species of rabbit fast skeletal muscle troponin T (TnT), TnT1f and TnT2f, were characterized. Here, the distribution of these fast TnT species and the alpha- and beta- tropomyosin (Tm) subunits is characterized in fast muscles and in single muscle fibers. Evidence is also presented for two forms of slow skeletal muscle TnT. The presence of each fast TnT species is associated with the presence of a different Tm dimer: TnT1f with alpha beta-Tm and TnT2f with alpha 2-Tm. Histochemical analysis shows that expression of the fast TnT-Tm combinations is not due to differences in the distribution of fast-twitch glycolytic and fast-twitch oxidative-glycolytic fiber types. The absence of a correlation between histochemical typing and the composition of the thin filament Ca2+-regulatory complex is more apparent in individual fast muscle fibers where both fast TnT-Tm combinations appear to be expressed in a continuum. The implications of these observations for mammalian skeletal muscle fiber diversity are discussed.

Patterns of troponin T expression in mammalian fast, slow and promiscuous muscle fibres.

The distribution of troponin T (TnT) species in typed single muscle fibres was analysed using one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) and a monoclonal antibody specific for fast TnT. Fibres taken from erector spinae (Es), plantaris (Plt), diaphragm (Dia) and soleus (Sol) muscles of adult rabbits were pretyped as fast-twitch-glycolytic (FG), fast-twitch-oxidative-glycolytic (FOG), slow-twitch-oxidative (SO) or promiscuous (P) using a combination of histochemical staining and PAGE. Although none of the four size classes of TnT was either muscle or fibre type specific, their pattern of expression differed in each muscle and between the fibre types. FG fibres expressed TnT1f or TnT2f as predominant species, depending on the muscle; TnT3f and TnT4f were minor components. In contrast, all size classes of TnT were expressed in varying proportions in FOG fibres from Es and Plt, while those from Dia resembled FG fibres, expressing TnT1f as their major species. P fibres from Es, Plt, and Sol exhibited a distinctive pattern of fast TnT expression, TnT3f being the predominant species. Dia differed from the other muscles as TnT1f was the dominant fast TnT species in its P fibres as it is in the Dia fast fibres. Quantitative analysis of one- and two-dimensional gels revealed that the P fibres could be divided into two classes, those that exhibited discoordinate expression of fast and slow TnTs, myosin light chains and myosin heavy chains and those in which their expression was coordinate. In addition low levels of TnT4f were detected in SO fibres and of slow TnT in fast fibres.

Heterogeneity of contractile proteins. Purification and characterization of two species of troponin T from rabbit fast skeletal muscle.

Two species of troponin T have been purified by ion-exchange chromatography from erector spinae, the major fast white muscle of the rabbit back, and from a pool of the fast hindlimb muscles gastrocnemius and plantaris. Designated Tn-T1f and Tn-T2f, they can be resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, with apparent molecular weights of 37,500 and 37,000 respectively. Their amino acid compositions are similar and correlate well with that reported for troponin T from fast muscle (Pearlstone, J. R., Carpenter, M. R., and Smillie, L. B. (1977) J. Biol. Chem. 252, 971-977). Tn-T2f most likely corresponds to the previously studied troponin T; further characterization was undertaken to determine how the newly identified Tn-T1f differs from Tn-T2f. Phosphorylation of alkaline phosphatase-treated troponin demonstrated that Tn-T1f and Tn-T2f are not interconverted by a change in phosphorylation state. Comparison of the CNBr fragments of Tn-T1f and Tn-T2f by SDS-gel electrophoresis and reverse phase high-performance liquid chromatography revealed similar but not identical peptide patterns. The major difference occurs in the amino-terminal CNBr peptides corresponding to CB3. Since both Tn-T1f and Tn-T2f have blocked amino termini, the difference does not result from proteolysis at the amino terminus of one of the proteins. These observations indicate that the two species of troponin T do not result from a known post-translational modification, but rather from differences in the amino acid sequence, suggesting that they arise either from the expression of different genes or a single gene from which different mRNAs are transcribed.

Changes in myosin and C-protein isoforms proceed independently of the conversion to singly innervated neuromuscular junctions in developing pectoral muscle.

Changes in contractile protein expression during myogenesis are usually categorized as developmentally programmed or neuronally dependent. Studies on aneurogenic chick embryos indicated that the neuronally dependent phase begins at about Embryonic Day 15, immediately prior to the fetal transition in myosin and C-protein expression. The prime candidate for the neuronal event that induces the fetal transition is the conversion to the adult form of singly innervated neuromuscular junctions (NMJs), which occurs contemporaneously with the fetal transition. Using curare to inhibit the conversion to focal innervation, we find that the fetal transition proceeds unimpaired, demonstrating that there is no causal link between the fetal transition and the conversion to focal innervation. Furthermore, because the doses of curare used inhibit motor activity by more than 80%, the fetal transition can occur in the absence of normal levels of motor activity. These observations show that the fetal transition in ovo is not induced by either a specific change in innervation or use. Rather, the dependence on innervation seems to be a consequence of the need for muscle activity to prevent atrophy, and the fetal transition appears to have characteristics more like the preprogrammed contractile protein transitions that precede it.