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.     

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age

The age-dependent decline in skeletal muscle mass and function is believed to be due to a multi-factorial pathology and represents a major factor that blocks healthy aging by increasing physical disability, frailty and loss of independence in the elderly. This study has focused on the comparative proteomic analysis of contractile elements and revealed that the most striking age-related changes seem to occur in the protein family representing myosin light chains (MLCs). Comparative screening of total muscle extracts suggests a fast-to-slow transition in the aged MLC population. The mass spectrometric analysis of the myofibril-enriched fraction identified the MLC2 isoform of the slow-type MLC as the contractile protein with the most drastically changed expression during aging. Immunoblotting confirmed an increased abundance of slow MLC2, concomitant with a switch in fast versus slow myosin heavy chains. Staining of two-dimensional gels of crude extracts with the phospho-specific fluorescent dye ProQ-Diamond identified the increased MLC2 spot as a muscle protein with a drastically enhanced phosphorylation level in aged fibres. Comparative immunofluorescence microscopy, using antibodies to fast and slow myosin isoforms, confirmed a fast-to-slow transformation process during muscle aging. Interestingly, the dramatic increase in slow MLC2 expression was restricted to individual senescent fibres. These findings agree with the idea that aged skeletal muscles undergo a shift to more aerobic-oxidative metabolism in a slower-twitching fibre population and suggest the slow MLC2 isoform as a potential biomarker for fibre type shifting in sarcopenia of old age.     

Myofibrillar M-band proteins in rat skeletal muscles during development

Summary The distribution of three myofibrillar M-band proteins, myomesin, M-protein and the muscle isoform of creatine kinase, was investigated with immunocytochemical techniques in skeletal muscles of embryonic, fetal, newborn and four-week-old rats. Furthermore, muscles of newborn rats were denervated and examined at four weeks of age. In embryos, myomesin was present in all myotome muscle fibres of the somites, whereas M-protein was detected only in a small proportion of the myotome muscle fibres and muscle creatine kinase was not detected at all. In fetal and newborn muscles, all fibres contained all three M-band proteins. At four weeks of age, when fibre types (type 1 or slow twitch fibres and type 2 or fast twitch fibres) were clearly discernable, the pattern was changed. Myomesin and muscle creatine kinase were still observed in all fibres, whereas M-protein was present only in type 2 fibres. On the other hand, in muscle fibres denervated at birth all three M-band proteins were still detected. Our results suggest 1) that during the initial stages of myofibrillogenesis expression and incorporation of myomesin into the M-band precede that of M-protein and muscle creatine kinase; 2) that expression and incorporation of all three M-band proteins during fetal development is nerve independent and non coordinated to the expression of different forms of myosin heavy chains, and 3) that the suppression of M-protein synthesis during postnatal development is nerve dependent and reflects the maturation of slow twitch motor units.     

Myofibrillar M-band proteins in rat skeletal muscles during development.

The distribution of three myofibrillar M-band proteins, myomesin, M-protein and the muscle isoform of creatine kinase, was investigated with immunocytochemical techniques in skeletal muscles of embryonic, fetal, newborn and four-week-old rats. Furthermore, muscles of newborn rats were denervated and examined at four weeks of age. In embryos, myomesin was present in all myotome muscle fibres of the somites, whereas M-protein was detected only in a small proportion of the myotome muscle fibres and muscle creatine kinase was not detected at all. In fetal and newborn muscles, all fibres contained all three M-band proteins. At four weeks of age, when fibre types (type 1 or slow twitch fibres and type 2 or fast twitch fibres) were clearly discernable, the pattern was changed. Myomesin and muscle creatine kinase were still observed in all fibres, whereas M-protein was present only in type 2 fibres. On the other hand, in muscle fibres denervated at birth all three M-band proteins were still detected. Our results suggest 1) that during the initial stages of myofibrillogenesis expression and incorporation of myomesin into the M-band precede that of M-protein and muscle creatine kinase; 2) that expression and incorporation of all three M-band proteins during fetal development is nerve independent and non coordinated to the expression of different forms of myosin heavy chains, and 3) that the suppression of M-protein synthesis during postnatal development is nerve dependent and reflects the maturation of slow twitch motor units.     

The effect of glucocorticoids on the myosin heavy chain isoforms' turnover in skeletal muscle

The purpose of this study was to find the effect of dexamethasone on the myosin heavy chain (MyHC) isoforms' composition in different skeletal muscles and glycolytic (G) fibres in relation with their synthesis rate and degradation of MyHC isoforms by alkaline proteinases. Eighteen-week-old male rats of the Wistar strain were treated with dexamethasone (100 microg/100 g bwt) during 10 days. The forelimb strength decreased from 9.52 to 6.19 N (P<0.001) and hindlimb strength from 15.54 to 8.55 N (P<0.001). Daily motor activity decreased (total activity from 933 to 559 and ambulatory activity from 482 to 226 movements/h, P<0.001). The degradation rate of muscle contractile proteins increased from 2.0 to 5.9% per day (P<0.001), as well as the myosin heavy chain IIB isoform degradation with alkaline proteinase in fast-twitch (F-T) muscles (12 +/- 0.9%; P<0.05) and glycolytic muscle fibres (15 +/- 1.1%; P<0.001). The synthesis rate of MyHC type II isoforms decreased in Pla muscles (P<0.05) and MyHC IIA (P<0.05) and IIB in EDL muscle and G fibres (P<0.001). The relative content of MyHC IIB isoform decreased in F-T muscles (P<0.001) and in G fibres (P<0.01), and the relative content of IIA and IID isoforms increased simultaneously. Dexamethasone decreased the MyHC IIB isoform synthesis rate and increased the sensibility of MyHC IIB isoform to alkaline proteinase, which in its turn led to the decrease of MyHC IIB isoform relative content in F-T muscles with low oxidative potential and G muscle fibres.     

Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

Fast-to-Slow Transition of Skeletal Muscle Contractile Function and Corresponding Changes in Myosin Heavy and Light Chain Formation in the R6/2 Mouse Model of Huntington’s Disease

Huntington´s disease (HD) is a hereditary neurodegenerative disease resulting from an expanded polyglutamine sequence (poly-Q) in the protein huntingtin (HTT). Various studies report atrophy and metabolic pathology of skeletal muscle in HD and suggest as part of the process a fast-to-slow fiber type transition that may be caused by the pathological changes in central motor control or/and by mutant HTT in the muscle tissue itself. To investigate muscle pathology in HD, we used R6/2 mice, a common animal model for a rapidly progressing variant of the disease expressing exon 1 of the mutant human gene. We investigated alterations in the extensor digitorum longus (EDL), a typical fast-twitch muscle, and the soleus (SOL), a slow-twitch muscle. We focussed on mechanographic measurements of excised muscles using single and repetitive electrical stimulation and on the expression of the various myosin isoforms (heavy and light chains) using dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole muscle and single fiber preparations. In EDL of R6/2, the functional tests showed a left shift of the force-frequency relation and decrease in specific force. Moreover, the estimated relative contribution of the fastest myosin isoform MyHC IIb decreased, whereas the contribution of the slower MyHC IIx isoform increased. An additional change occurred in the alkali MyLC forms showing a decrease in 3f and an increase in 1f level. In SOL, a shift from fast MyHC IIa to the slow isoform I was detectable in male R6/2 mice only, and there was no evidence of isoform interconversion in the MyLC pattern. These alterations point to a partial remodeling of the contractile apparatus of R6/2 mice towards a slower contractile phenotype, predominantly in fast glycolytic fibers.     

Expression of myofibrillar proteins and parvalbumin isoforms in white muscle of dorada during development

Several polyacrylamide gel electrophoresis techniques were used to study developmental changes in myofibrillar protein composition and parvalbumin distribution in the myotomal muscle of Brycon moorei . Two myosin LC2 chains and two troponin I isoforms were successively detected. Up to four troponin T isoforms were synthesized. Slow red-muscle myofibrils from adult fish showed no common component (except actin) with larval, juvenile or adult fast white-muscle myofibrils. During growth of B. moorei , two classes of parvalbumin isoforms were sequentially expressed: larval PA I, PA IIa, and PA IIb and adult PA III. In adult fish, the content in Tn T-2 isoform decreased from the anterior to the posterior myomeres, in favour of Tn T-1 and Tn T-4. The parvalbumin content also diminished from the rostral to the caudal muscle. The fast rate of transition from larval to adult isoforms appeared to parallel the extremely fast growth of B. moorei . Sequential expression of these isoforms presumably reflected variations in the contractile properties of the muscle fibres, required by changes in physiological demands of the propulsive musculature.     

The expression of sarcomeric muscle-specific contractile protein genes in BC3H1 cells: BC3H1 cells resemble skeletal myoblasts that are defective for commitment to terminal differentiation

The BC3H1 cell line has been used widely as a model for studying regulation of muscle-related proteins, such as the acetylcholine receptor, myokinase, creatine kinase, and actin. These cells, derived from a nitrosourea-induced mouse brain neoplasm, have some of the morphological characteristics of smooth muscle and have been shown to express the vascular smooth muscle isoform of alpha-actin. To provide further information about the contractile protein phenotype of BC3H1 and to gain additional insights into the possible tissue of origin of these cells, we have examined the expression of a battery of contractile protein genes. During rapid growth, subconfluent BC3H1 cells express the nonmuscle isoform of alpha-tropomyosin (alpha-Tm) and the nonsarcomeric isoforms of myosin heavy and light chains (MHCs and MLCs, respectively), but do not express troponin T(TnT). However, when BC3H1 cells differentiate in response to incubation in serum-deprived medium or upon approaching confluence, they express TnT as well as sarcomeric muscle isoforms of MHC, MLC 2 and 3, alpha-Tm, and alpha-actin. These results suggest that BC3H1 is a skeletal muscle cell line of ectodermal origin that is defective for commitment to terminal differentiation.     

Troponin C isoform composition determines differences in Sr(2+)-activation characteristics between rat diaphragm fibers

Single fibers of rat diaphragm containing different naturally occurring combinations of myofibrillar protein isoforms were used to evaluate the contribution of troponin C (TnC) isoforms to fiber type-related differences with respect to sensitivity to Sr²⁺ of the contractile system. Mechanically skinned fibers were studied for their isometric force vs. Sr²⁺ concentration ([Sr²⁺]) relationships and then analyzed electrophoretically for myofibrillar protein isoform composition. Our data demonstrate that fiber-type differences in Sr²⁺ dependence of contractile activation processes are primarily determined by the TnC isoform composition, with the slow isoform conferring on average a sevenfold greater sensitivity to Sr²⁺ than the fast isoform. Moreover, the ratio of TnC isoforms determined functionally from the force-pSr (–log₁₀ [Sr²⁺]) curves is tightly (r² = 0.97) positively correlated with that estimated electrophoretically. Together, these results validate the use of Sr²⁺ activation characteristics to distinguish fibers containing different proportions of fast and slow TnC isoforms and to study the mechanisms by which divalent cations activate the contractile apparatus. We also found that the functionally and electrophoretically determined ratios of TnC isoforms present in a fiber display similar sigmoidal relationships with the ratio of myosin heavy chain (MHC) isoform types expressed. These relationships 1) offer further insight in the functional and molecular expression of TnC in relation to the molecular expression of MHC isoform types and 2) may provide the basis for predicting sensitivity to Sr²⁺, TnC, and MHC isoforms in pure and hybrid skeletal muscle fibers. muscle contraction; skeletal muscle; myofibrillar proteins; single fiber; sensitivity to strontium; sensitivity to calcium     

Fiber type conversion alters inactivation of voltage-dependent sodium currents in murine C2C12 skeletal muscle cells

Each skeletal muscle of the body contains a unique composition of "fast" and "slow" muscle fibers, each of which is specialized for certain challenges. This composition is not static, and the muscle fibers are capable of adapting their molecular composition by altered gene expression (i.e., fiber type conversion). Whereas changes in the expression of contractile proteins and metabolic enzymes in the course of fiber type conversion are well described, little is known about possible adaptations in the electrophysiological properties of skeletal muscle cells. Such adaptations may involve changes in the expression and/or function of ion channels. In this study, we investigated the effects of fast-to-slow fiber type conversion on currents via voltage-gated Na⁺ channels in the C₂C₁₂ murine skeletal muscle cell line. Prolonged treatment of cells with 25 nM of the Ca²⁺ ionophore A-23187 caused a significant shift in myosin heavy chain isoform expression from the fast toward the slow isoform, indicating fast-to-slow fiber type conversion. Moreover, Na⁺ current inactivation was significantly altered. Slow inactivation less strongly inhibited the Na⁺ currents of fast-to-slow fiber type-converted cells. Compared with control cells, the Na⁺ currents of converted cells were more resistant to block by tetrodotoxin, suggesting enhanced relative expression of the cardiac Na⁺ channel isoform Na_v1.5 compared with the skeletal muscle isoform Na_v1.4. These results imply that fast-to-slow fiber type conversion of skeletal muscle cells involves functional adaptation of their electrophysiological properties. muscle plasticity; myosin heavy chain expression; sodium channel expression     

Increased expression of the nicotinic acetylcholine receptor in stimulated muscle

Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts.     

New insights into skeletal muscle fibre types in the dog with particular focus towards hybrid myosin phenotypes

Electrophoresis, immunoblots, immunohistochemistry and image analysis methods were applied to characterise canine trunk and appendicular muscle fibres according to their myosin heavy chain (MyHC) composition and to determine, on a fibre-to-fibre basis, the correlation between contractile [MyHC (s), myofibrillar ATPase (mATPase) and sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) isoforms], metabolic [succinate dehydrogenase (SDH) and glycerol-3-phosphate dehydrogenase (GPDH) activities and glycogen and phospholamban (PLB) content] and morphological (cross-sectional area and capillary and nuclear densities) features of individual myofibres. An accurate delineation of MyHC-based fibre types was obtained with the developed immunohistochemical method, which showed high sensitivity and objectivity to delineate hybrid fibres with overwhelming dominance of one MyHC isoform. Phenotypic differences in contractile, metabolic and morphological properties seen between fibre types were related to MyHC content. All canine skeletal muscle fibre types had a relatively high histochemical SDH activity but significant differences existed in the order IIA>I>IIX. Mean GPDH was ranked according to fibre type such that I<IIA<IIX. Type IIA fibres were the smallest, type IIX fibres the largest and type I of intermediate size. Capillary and nuclear density decreased in the order IIA>I>IIX. Hybrid fibres, which represented nearly one third of the whole pool of skeletal muscle fibres analysed, had mean values intermediate between their respective pure phenotypes. Slow fibres expressed the slow SERCA isoform and PLB, whereas type II fibres expressed the fast SERCA isoform. Discrimination of myofibres according to their MyHC content was possible on the basis of their contractile, metabolic and morphological features. These intrafibre interrelationships suggest that myofibres of control dogs exhibit a high degree of co-ordination in their physiological, biochemical and morphological characteristics. This study demonstrates that canine skeletal muscle fibres have been misclassified in numerous previous studies and offers useful baseline data and new prospects for future work on muscle-fibre-typing in canine experimental studies.     

Myosin heavy chain isoform composition in striated muscle after denervation and self-reinnervation

The total content of myosin heavy chains (MHC) and their isoform pattern were studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (extensor digitorum longus) muscles of adult rat during atrophy after denervation and recovery after self-reinnervation. The pattern of fibre types, in terms of ultrastructure, was studied in parallel. After denervation, total MHC content decreased sooner in the slow-twitch muscle than in the fast-twitch. The ratio of MHC-1 and the MHC-2B isoforms to the MHC-2A isoform decreased in the slow and the fast denervated muscles, respectively. After reinnervation of the slow muscle, the normal pattern of MHC recovered within 10 days and the type 1 isoform increased above the normal. In the reinnervated fast muscle, the 2B/2A isoform ratio continued to decrease. Traces of the embryonic MHC isoform, identified by immunochemistry, were found in both denervated and reinnervated slow and fast muscles. A shift in fibre types was similar to that found in the MHC isoforms. Within 2 months of recovery a tendency to normalization was observed. The results show that (a) MHC-2B isoform and the morphological characteristics of the 2B-type muscle fibres are susceptible to lack of innervation, similar to those of type 1, (b) during muscle recovery induced by reinnervation the MHC isoforms and muscle fibres shift transiently to type 1 in the soleus and to type 2A in the extensor digitorum longus muscles, and (c) the embryonic isoform of MHC may appear in the adult skeletal muscles if innervation is disturbed.     

Determinants of the contractile properties in the embryonic chicken gizzard and aorta

Smooth muscle is generally grouped into two classes of differing contractile properties. Tonic smooth muscles show slow rates of force activation and relaxation and slow speeds of shortening (V(max)) but force maintenance, whereas phasic smooth muscles show poor force maintenance but have fast V(max) and rapid rates of force activation and relaxation. We characterized the development of gizzard and aortic smooth muscle in embryonic chicks to identify the cellular determinants that define phasic (gizzard) and tonic (aortic) contractile properties. Early during development, tonic contractile properties are the default for both tissues. The gizzard develops phasic contractile properties between embryonic days (ED) 12 and 20, characterized primarily by rapid rates of force activation and relaxation compared with the aorta. The rapid rate of force activation correlates with expression of the acidic isoform of the 17-kDa essential myosin light chain (MLC(17a)). Previous data from in vitro motility assays (Rover AS, Frezon Y, and Trybus KM. J Muscle Res Cell Motil 18: 103-110, 1997) have postulated that myosin heavy chain (MHC) isoform expression is a determinant for V(max) in intact tissues. In the current study, differences in V(max) did not correlate with previously published differences in MHC or MLC(17a) isoforms. Rather, V(max) was increased with thiophosphorylation of the 20-kDa regulatory myosin light chain (MLC(20)) in the gizzard, suggesting that a significant internal load exists. Furthermore, V(max) in the gizzard increased during postnatal development without changes in MHC or MLC(17) isoforms. Although the rate of MLC(20) phosphorylation was similar at ED 20, the rate of MLC(20) dephosphorylation was significantly higher in the gizzard versus the aorta, correlating with expression of the M130 isoform of the myosin binding subunit in the myosin light chain phosphatase (MLCP) holoenzyme. These results indicate that unique MLCP and MLC(17) isoform expression marks the phasic contractile phenotype.