Relation of fast and slow skeletal, and atrial and ventricular myosin in various mammals

Myosin was isolated from adult mouse, rat, rabbit and cat atrial and ventricular myocardium and fast and slow skeletal muscles and examined by measuring Ca2+-ATPase activity and by electrophoretic fractionation of chymotryptic peptides and MLCs. The myosin from mouse atrial and ventricular myocardium were very similar. The properties of cat soleus muscle myosin and ventricular myocardium were also very similar (ATPase activity and electrophoretic pattern of chymotryptic peptides of myosin). The electrophoretic pattern of MLCs, however, was distinct when comparing mouse and feline muscles. These observations are consistent with the idea that atrial and ventricular alpha MHCs are closely related and that beta MHCs from ventricular myocardium and slow skeletal muscle fibres are also closely related.     

Myosin and actomyosin from human skeletal muscle.

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (+/-2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37 degrees C. Activation of the Mg-ATPase activity of Ca2+-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 muM Ca2+ concentration (CaEGTA binding constant equals 4.4 - 10(5) at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6-9 range, the Ca2+-ATPase activity of the subfragment 1 was 1.8- and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6-10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca2+-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca2+ is similar to that of rabbit fast or slow muscle.     

Role of myosin light chain kinase in muscle contraction

In resting striated muscles of the rabbit muscle in vivo, the phosphorylatable light chain is partially phosphorylated. Tetanic stimulation increased the level of phosphorylation more rapidly in fast twitch than in slow twitch muscle. In both types of muscle the rate of dephosphorylation was relatively slow. In rabbit fast twitch muscles, phosphorylation levels persisted significantly above the resting value for some time after posttetanic potentiation had disappeared. The role of myosin light chain kinase in modulating contractile response in striated muscle is uncertain. In vertebrate smooth muscle the role of myosin phosphorylation appears to be different from that in striated muscle despite the general similarity of the actomyosin system in both tissues. Although phosphorylation in vitro increases the Mg2+ -ATPase of actomyosin, a number of features imply that a somewhat complex relationship exists between the level of phosphorylation and the actin activation of the Mg2+ -ATPase in vertebrate smooth muscle. Contrary to many earlier reports, preparations of smooth muscle actomyosin can be obtained with Mg2+ -ATPase activities comparable to those of actomyosin from skeletal muscle. Preliminary evidence is presented that suggests that phosphorylation changes the Ca2+ sensitivity of the Mg2+ -ATPase of smooth muscle actomyosin.     

Effect of caffeine on active Ca2+ ion transport in a homogenate of skeletal muscles and myocardium

A Ca2-selective electrode was used to study active transport of Ca2+ by sarcoplasmic reticulum fragments of rabbit skeletal muscle and myocardium homogenates. The specific Ca2+ transport activities (mumol Ca2+/min/mg tissue) are 40 = 60 and 3 = 5 units for fast and slow muscles and the myocardium, respectively. Caffeine (5 mM) exerts a powerful inhibitory influence on Ca2+ transport in skeletal muscle homogenates. For fast muscles, the degree of inhibition exceeds 50%. The rate of Ca2+ transport in the myocardium homogenate increases in the presence of creatine phosphate. The latter produces no effect on Ca2+ transport in skeletal muscle homogenates. The high sensitivity of Ca2 transport to caffeine, a specific blocker of Ca2+ transport to the terminal cisterns of the sarcoplasmic reticulum, suggests that the terminal cisterns, apart from being a reservoir for Ca2+ needed for contraction trigger, may play an essential role in muscle relaxation.     

Early biochemical consequences of denervation in fast and slow skeletal muscles and their relationship to neural control over muscle differentiation

1. One week after denervation several biochemical characteristics of the fast extensor digitorum longus and slow soleus muscles from adult rats were investigated and compared with the characteristics of the corresponding unoperated contralateral muscles. 2. After these short periods of denervation-induced atrophy, the isolated myosins showed unchanged ATPase (adenosine triphosphatase) activities, but there was the expected difference between fast and slow muscle. 3. The specific activities of several soluble enzymes and their characteristic patterns were found to be only slightly modified in both the extensor and soleus muscles after denervation, as were most of the activities measured in the isolated mitochondria. 4. The most significant modifications were in the isolated sarcoplasmic reticulum, and appeared to be specific to either slow or fast muscle. 5. Denervation of slow muscle led to a marked increase of Ca(2+)-transport rates, and of the specific activity of the Mg(2+)-activated K(+)-modulated Ca(2+)-stimulated ATPase, together with changes in the polyacrylamide-electrophoretic profiles of the microsomal membrane protein. Transformation of these several properties of slow muscle sarcoplasmic reticulum to those of fast muscle sarcoplasmic reticulum was further substantiated by electron-microscopic analysis after negative staining. Control experiments with tenotomized soleus muscle gave negative results. 6. The isolated sarcoplasmic reticulum from fast muscle showed a slight diminution of ATPase-linked Ca(2+)-transport activity and a selective increase of rotenone-insensitive NADH-cytochrome c reductase activity, in addition to a greater emphasis on slow-type electrophoretic components of the structural membrane protein. 7. The significance of these results in relation to specific differentiating influences from motor nerves is discussed.     

Properties of C protein from skeletal and cardiac muscles of the ground squirrel Citellus undulatus at various stages of hibernation

Changes in the molecular weight and functional properties of the C and X proteins from skeletal muscles and the C protein from the cardiac muscle of hibernating ground squirrels Citellus undulatus at different stages of the hibernation were studied. A decrease in the molecular weight of the C protein from fast fibers of skeletal muscles of hibernating ground squirrels compared with awakening and active animals was revealed. The appearance of shorter molecules of the C protein upon hibernation was accompanied by a lowering of its capacity to enhance the actin-activated ATPase activity of control myosin and by the inhibition of its Ca(2+)-sensitivity. No similar changes were observed for the skeletal X protein and the cardiac C protein. The influence of the skeletal C protein on the main functional properties of myosin allows one to draw a conclusion about its contribution to the inhibition of contractile activity of skeletal muscles upon hibernation. The physiological significance of the changes in the C protein upon hibernation is discussed in connection with similar changes in some cardiomyopathies.     

Relationship of primary and secondary myogenesis to fiber type development in embryonic chick muscle.

The formation of fast and slow myotubes was investigated in embryonic chick muscle during primary and secondary myogenesis by immunocytochemistry for myosin heavy chain and Ca2(+)-ATPase. When antibodies to fast or slow isoforms of these two molecules were used to visualize myotubes in the posterior iliotibialis and iliofibularis muscles, one of the isoforms was observed in all primary and secondary myotubes until very late in development. In the case of myosin, the fast antibody stained virtually all myotubes until after stage 40, when fast myosin expression was lost in the slow myotubes of the iliofibularis. In the case of Ca2(+)-ATPase, the slow antibody also stained all myotubes until after stage 40, when staining was lost in secondary myotubes and in the fast primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis. In contrast, the antibodies against slow muscle myosin heavy chain and fast muscle Ca2(+)-ATPase stained mutually exclusive populations of myotubes at all developmental stages investigated. During primary myogenesis, fast Ca2(+)-ATPase staining was restricted to the primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis, whereas slow myosin heavy chain staining was confined to all of the primary myotubes of the slow region of the iliofibularis. During secondary myogenesis, the fast Ca2(+)-ATPase antibody stained nearly all secondary myotubes, while primaries in the slow region of the iliofibularis remained negative. Thus, in the slow region of the iliofibularis muscle, these two antibodies could be used in combination to distinguish primary and secondary myotubes. EM analysis of staining with the fast Ca2(+)-ATPase antibody confirmed that it recognizes only secondary myotubes in this region. This study establishes that antibodies to slow myosin heavy chain and fast Ca2(+)-ATPase are suitable markers for selective labeling of primary and secondary myotubes in the iliofibularis; these markers are used in the following article to describe and quantify the effects that chronic blockade of neuromuscular activity or denervation has on these populations of myotubes.     

Roles of troponin isoforms in pH dependence of contraction in rabbit fast and slow skeletal and cardiac muscles.

Skinned fibers prepared from rabbit fast and slow skeletal and cardiac muscles showed acidotic depression of the Ca2+ sensitivity of force generation, in which the magnitude depends on muscle type in the order of cardiac>fast skeletal>slow skeletal. Using a method that displaces whole troponin-complex in myofibrils with excess troponin T, the roles of Tn subunits in the differential pH dependence of the Ca2+ sensitivity of striated muscle were investigated by exchanging endogenous troponin I and troponin C in rabbit skinned cardiac muscle fibres with all possible combinations of the corresponding isoforms expressed in rabbit fast and slow skeletal and cardiac muscles. In fibers exchanged with fast skeletal or cardiac troponin I, cardiac troponin C confers a higher sensitivity to acidic pH on the Ca2+ sensitive force generation than fast skeletal troponin C independently of the isoform of troponin I present. On the other hand, fibres exchanged with slow skeletal troponin I exhibit the highest resistance to acidic pH in combination with either isoform of troponin C. These results indicate that troponin C is a determinant of the differential pH sensitivity of fast skeletal and cardiac muscles, while troponin I is a determinant of the pH sensitivity of slow skeletal muscle.     

Two fast-type fibers in claw closer and abdominal deep muscles of the Australian freshwater crustacean, Cherax destructor, differ in Ca2+ sensitivity and troponin-I isoforms

One type of fast fiber and two types of slow (slow-twitch, S1 and slow-tonic, S2) fibers are found in decapod crustacean skeletal muscles that differ in contractile properties and myofibrillar protein isoform compositions. In this study the structural characteristics, protein isoform compositions, and Ca2+-activation properties of fast fibers in the claw closer (F1) and abdominal deep flexor (F2) muscles of Cherax destructor were analyzed. For comparison, myofibrillar protein isoform compositions of slow (long-sarcomere) fibers from claw and abdomen were also determined; our results indicate that the slow fibers in the claw closer were the slow-twitch (S1) type and those in the abdominal superficial flexor were primarily slow-tonic (S2) type. F1 fibers had shorter resting sarcomere lengths (2.93 microm in unstretched fibers and 3.06 microm in stretched fibers) and smaller fiber diameter (256 microm) than F2 fibers (sarcomere lengths 3.48 microm in unstretched and 3.46 microm in stretched; 747 microm diameter). Moreover, F1 fibers showed a narrower range in sarcomere lengths than F2 fibers (2.81 to 3.28 microm vs. 2.47 to 4.05 micro m in unstretched fibers). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the fast fibers from claw and abdomen differed in troponin-I composition; F1 fibers expressed two isoforms of troponin-I (TnI1 and TnI2) in approximately equal amounts, whereas F2 fibers expressed primarily TnI3 and lower levels of TnI1. F1 fibers were more sensitive to Ca2+, as shown by higher pCa values at threshold activation (pCa(10)=6.50+/-0.07) and at 50% maximum force (pCa(50)=6.43+/-0.07) than F2 fibers (pCa(10)=6.12+/-0.04 and pCa(50)=5.88+/-0.03, respectively). F1 fibers also had a greater degree of co-operativity in Ca2+ activation, as shown by a higher maximum slope of the force-pCa curve (n(Ca)=12.98+/-2.27 vs. 4.34+/-0.64). These data indicate that there is a greater fast fiber-type diversity in crustacean muscles than was previously supposed. Moreover, the differences in activation properties suggest that the TnI isoform composition influences the Ca2+ sensitivity of the contractile mechanism.     

Purification and biochemical characteristics of myosin from rat malignant sarcoma-45

Myosin was purified from rat tumour sarcoma-45 whose properties (effects of cations on ATPase activity, substrate specificity, temperature- and pH-optima, thermal stability, sensitivity of Mg2(+)-ATPase to F-actin, molecular mass, subunit composition) are similar to those of fast skeletal muscle myosin. Some parameters of the protein, namely, the levels of Ca2(+)- and K+, EDTA-ATPase activity, relative content of myosin light chains with Mr 16500 and the degree of tumoural myosin Mg2(+)-ATPase activation by F-actin, were significantly lower than those of skeletal muscle myosin.     

A monoclonal antibody to the Ca2+-ATPase of cardiac sarcoplasmic reticulum cross-reacts with slow type I but not with fast type II canine skeletal muscle fibers: an immunocytochemical and immunochemical study

Ca2+-ATPase of the sarcoplasmic reticulum was localized in cryostat sections from three different adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with monoclonal antibodies to the Ca2+-ATPase. Type I (slow) myofibers were strongly labeled for the Ca2+-ATPase with a monoclonal antibody (II D8) to the Ca2+-ATPase of canine cardiac sarcoplasmic reticulum; the type II (fast) myofibers were labeled at the level of the background with monoclonal antibody II D8. By contrast, type II (fast) myofibers were strongly labeled for Ca2+-ATPase of rabbit skeletal sarcoplasmic reticulum. The subcellular distribution of the immunolabeling in type I (slow) myofibers with monoclonal antibody II D8 corresponded to that of the sarcoplasmic reticulum as previously determined by electron microscopy. The structural similarity between the canine cardiac Ca2+-ATPase present in the sarcoplasmic reticulum of the canine slow skeletal muscle fibers was demonstrated by immunoblotting. Monoclonal antibody (II D8) to the cardiac Ca2+-ATPase binds to only one protein band present in the extract from either cardiac or type I (slow) skeletal muscle tissue. By contrast, monoclonal antibody (II H11) to the skeletal type II (fast) Ca2+-ATPase binds only one protein band in the extract from type II (fast) skeletal muscle tissue. These immunopositive proteins coelectrophoresed with the Ca2+-ATPase of the canine cardiac sarcoplasmic reticulum and showed an apparent Mr of 115,000. It is concluded that the Ca2+-ATPase of cardiac and type I (slow) skeletal sarcoplasmic reticulum have at least one epitope in common, which is not present on the Ca2+-ATPase of sarcoplasmic reticulum in type II (fast) skeletal myofibers. It is possible that this site is related to the assumed necessity of the Ca2+-ATPase of the sarcoplasmic reticulum in cardiac and type I (slow) skeletal myofibers to interact with phosphorylated phospholamban and thereby enhance the accumulation of Ca2+ in the lumen of the sarcoplasmic reticulum following beta-adrenergic stimulation.     

Effects of monensin on Na+/K(+)-ATPase and Ca(++)-AtPase activities in chick skeletal muscle and myocardium after subacute treatment

Chicks were treated at 2 weeks of age with 4,15, 40, 100 and 150 mg/kg of monensin, an ionophore used for its anticoccidial and growth-promoting properties. In the present immunohistochemical study, the expressions and distribution of Na+/K(+)-ATPase and Ca(++)-ATPase were studied in myocardium and skeletal muscles (pectoral and quadriceps femoris). We detected an increase of Na+/K(+)-ATPase immunostaining with prominent staining of the sarcolemma and a slight increase of Ca(+)-ATPase with prominent staining of the sarcoplasma.     

Cooperative interactions between the contractile proteins of cardiac and skeletal muscle.

The calcium activation of the ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of cardiac actomyosin reconstituted from bovine cardiac myosin and a complex of actin-tropomyosin-troponin extracted from bovine cardiac muscle at 37 degrees C was studied and compared with similar proteins from rabbit fast skeletal muscle. The proteins of the actin complex were identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Half-maximal activation of the cardiac actomyosin was seen at a calcium concentration of 1.2 +/- 0.002 (S.E. of mean) muM. A hybridized reconstituted actomyosin made with cardiac myosin and the actin-tropomyosin-troponin complex extracted from rabbit skeletal muscle was also activated by calcium but the half-maximal value was shifted to 0.65 +/- 0.02 (S.E. of mean) muM Ca2+. Homologous rabbit skeletal actomyosin showed half-maximal activation at 0.90 +/- 0.01 (S.E. of mean) muM Ca2+ and the value for a hybridized actomyosin made with rabbit skeletal myosin and the actin-complex from cardiac muscle was found at 1.4 +/- 0.03 (S.E. of mean) muM Ca2+ concentration. Kinetic analysis of the Ca2+ activated ATPase activity of reconstituted bovine cardiac actomyosin indicated some degree of cooperativity with respect to calcium. Double reciprocal plots of reconstituted actomyosins made with bovine cardiac actin complex were curvilinear and significantly different than those of reconstituted actomyosins made with the rabbit fast skeletal actin complex. The Ca2+-dependent cooperativity was of a mixed type as determined from Hill plots for homologous reconstituted bovine cardiac and rabbit fast skeletal actomyosin. The results show that cooperative interactions in reconstituted actomyosins were greater when the actin-tropomyosin-troponin complex was derived from cardiac than skeletal muscle.     

Slow/cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban mRNAs are expressed in chronically stimulated rabbit fast-twitch muscle

Fast-twitch extensor digitorum longus muscles of the rabbit were subjected to chronic low-frequency stimulation during different time periods. Changes in the relative amounts of mRNAs encoding fast and slow/cardiac Ca2+-ATPase isoforms were assessed through the use of an RNase-protection assay. Stimulation-induced increases in slow cardiac Ca2+-ATPase and phospholamban mRNAs were quantified by mRNA hybridization. Prolonged stimulation resulted in an exchange of the fast with the slow/cardiac Ca2+-ATPase isoform mRNAs. The exchange was complete after 72 d of stimulation as compared with normal slow-twitch soleus muscle. The tissue content of phospholamban mRNA reached levels similar to that found in normal slow-twitch soleus muscle by the same time. The conversion of the sarcoplasmic reticulum coincided with the fast-to-slow troponin C isoform transition, previously investigated in the same muscles.     

Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin.

A new technique for obtaining a myofibril-like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate-gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin-like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+-sensitivity in tropomyosin-free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.     

Myosin and actomyosin from human skeletal muscle

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (±2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37°C. Activation of the Mg-ATPase activity of Ca²⁺-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 μM Ca²⁺ concentration (CaEGTA binding constant = 4.4 · 10⁵ at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6–9 range, the Ca²⁺-ATPase activity of the subfragment 1 was 1.8-and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6–10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca²⁺-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca²⁺ is similar to that of rabbit fast or slow muscle     

Expression and functional properties of four slow skeletal troponin T isoforms in rat muscles

We investigated the expression and functional properties of slow skeletal troponin T (sTnT) isoforms in rat skeletal muscles. Four sTnT cDNAs were cloned from the slow soleus muscle. Three isoforms were found to be similar to sTnT1, sTnT2, and sTnT3 isoforms described in mouse muscles. A new rat isoform, with a molecular weight slightly higher than that of sTnT3, was discovered. This fourth isoform had never been detected previously in any skeletal muscle and was therefore called sTnTx. From both expression pattern and functional measurements, it appears that sTnT isoforms can be separated into two classes, high-molecular-weight (sTnT1, sTnT2) and low-molecular-weight (sTnTx, sTnT3) isoforms. By comparison to the apparent migration pattern of the four recombinant sTnT isoforms, the newly described low-molecular-weight sTnTx isoform appeared predominantly and typically expressed in fast skeletal muscles, whereas the higher-molecular-weight isoforms were more abundant in slow soleus muscle. The relative proportion of the sTnT isoforms in the soleus was not modified after exposure to hindlimb unloading (HU), known to induce a functional atrophy and a slow-to-fast isoform transition of several myofibrillar proteins. Functional data gathered from replacement of endogenous troponin complexes in skinned muscle fibers showed that the sTnT isoforms modified the Ca(2+) activation characteristics of single skeletal muscle fibers, with sTnT2 and sTnT1 conferring a similar increase in Ca(2+) affinity higher than that caused by low-molecular-weight isoforms sTnTx and sTnT3. Thus we show for the first time the presence of sTnT in fast muscle fibers, and our data show that the changes in neuromuscular activity on HU are insufficient to alter the sTnT expression pattern.     

Ca2+ movements in sarcoplasmic reticulum of rat soleus fibers after hindlimb suspension.

The functional capacity of skeletal muscle sarcoplasmic reticulum (SR) was examined in the slow soleus of rats submitted to 15 days of disuse produced by hindlimb suspension (HS). By using caffeine-induced contractions of single skinned fibers, Ca2+ uptake, Ca2+ release, and passive Ca2+ leakage through the SR membrane were investigated. In the SR of atrophied muscles, the amounts of Ca2+ uptake and Ca2+ release were significantly higher than in the control muscles and were close to those found for a fast muscle, the plantaris. Moreover, the study of the Ca2+ leakage showed that the time required to empty the SR previously loaded with Ca2+ was reduced by a factor of two after HS. Such disturbances of the Ca2+ movements in the SR suggested that alterations of the SR membrane occurred after HS. The results supported the idea that after hindlimb unweighting the slow soleus muscle acquired SR properties that were very much like those of a faster muscle.     

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.     

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.