Myosin Heavy Chain 2X Research Articles

Effects of long-term treatment with dietary theobromine on rat skeletal muscles.

Plastic changes of skeletal muscles, such as hypertrophy and atrophy, are dependent on physiological activities and regulated by a variety of signaling pathways, including cyclic adenosine monophosphate (cAMP) pathway. The cAMP inducing agents, such as the β2-adrenergic agonist clenbuterol, are known to induce muscle hypertrophy, and has been reported to induce slow-to-fast transitions in rat soleus muscle. Theobromine, one of the active components of cacao, functions as an inhibitor of phosphodiesterase and increases cAMP. This study hypothesized that theobromine, like clenbuterol, can induce muscle hypertrophy and influence contractile properties. Male Wistar rats were fed a normal diet or a diet containing 0.05% theobromine for 20weeks. Using biochemical, anatomical, and physiological techniques, effects of dietary theobromine on skeletal muscles (soleus, extensor digitorum longus, plantaris, and gastrocnemius) were examined. There were no significant differences in body weight, serum levels of proteins and lipids, muscle weights, dry/wet ratio of muscle weights, mitochondrial oxidation enzyme activity of muscles, isometric contractile properties of muscles, and muscle fatigue between control and theobromine-fed rats. Quantitative analysis of mRNA, however, revealed upregulation of myosin heavy chain 2x and myogenic differentiation 1, as previously reported in clenbuterol-treated muscles. The long-term theobromine (0.05%) diet in rats had no effect in inducing muscle hypertrophy and in changing contractile properties, although it had some similar effects of clenbuterol on muscle gene expression.

PSXIV-33 α-chaconine induces myogenesis of bovine satellite cells but less affect adipogenesis of adipocytes

Abstract α-chaconine is a steroidal glycoalkaloid compound found in leaves, fruit and tubers of the solanaceae family such as potato, tomato, and eggplant. α-chaconine poisoning was reported in various animal models such as mice, rabbit, and chicken. However, previous study indicated that it is used to induce performance of beef cattle. We hypothesized that α-chaconine may affect myogenesis of bovine satellite cells but less affect adipogenesis of bovine adipocytes. Bovine satellite cells (BSC) were pronase-liberated from semimembranosus (SM) and longissimus dorsi (LD) muscle tissues of three new-born Hanwoo calves. Bovine preadipocytes were isolated from perirenal (PR) adipose tissues. BSC and preadipocytes were incubated with dulbecco modified eagle medium (DMEM) with 10% fetal bovine serum for proliferation. BSC induced differentiation with DMEM with 3% horse serum and preadipocytes induced differentiation with DMEM with insulin, dexamethasone, and ciglitazone. Cells were treated with various levels of α-chaconine (control, 0.001, 0.01, 0.1, 1, and 10μM). mRNA abundance for myosin heavy chain 2X (MHC2X), glucose transportor 4 (GLUT4), β2-adrenergic receptor (β2-AR), peroxisome proliferator-activated receptor γ (PPARγ), stearoyl-CoA desaturase (SCD) were measured by real-time quantitative PCR. Data were analyzed as a completely randomized design using the MIXED model, each treatment performed in triplicated. Means were considered different at P < 0.05. Relative MHC2X mRNA abundance was greater in BSC with dose-titrated chaconine treatments compared to the control (P < 0.05). Relative levels of β2-AR were greater in both BSC and adipocytes based on dose-dependent treatements (P < 0.05). The levels of PPARγ and SCD have no dose-titrated treatments in bovine PR adipocytes (P > 0.05). These result indicated that α-chaconine have a dose-dependent effect on MHC2X and β2-AR mRNA BSC and adipocytes but did not affect to adipogenesis in bovine PR adipocytes.

Comparison of Characteristics of Myosin Heavy Chain-based Fiber and Meat Quality among Four Bovine Skeletal Muscles.

Muscle fiber characteristics account for meat quality and muscle fibers are mainly classified into three or more types according to their contractile and metabolic properties. However, the majority of previous studies on bovine skeletal muscle are based on myosin ATPase activity. In the present study, the differences in the characteristics of muscle fibers classified by the expression of myosin heavy chain (MHC) among four bovine skeletal muscles such as longissimus thoracis (LT), psoas major (PM), semimembranosus (SM) and semitendinosus (ST) and their relationships to beef quality were investigated. MHCs 2x, 2a and slow were identified by LC-MS/MS and IIX, IIA and I fiber types were classified. PM, which had the smallest size and highest density of fibers regardless of type, showed the highest myoglobin content, CIE L*, a*, b* and sarcomere length (p<0.05), whereas ST with the highest composition of IIX, showed high shear force and low sarcomere length (p<0.05). The correlation coefficients between muscle fiber characteristics and meat quality showed that type IIX is closely related to poor beef quality and that a high density of small-sized fibers is related to redness and tenderness. Therefore, the differences in meat quality between muscles can be explained by the differences in muscle fiber characteristics, and especially, the muscles with good quality are composed of more small-sized fibers regardless of fiber type.

FHL3 differentially regulates the expression of MyHC isoforms through interactions with MyoD and pCREB

In skeletal muscle, muscle fiber types are defined by four adult myosin heavy chain (MyHC) isoforms. Four and a half LIM domain protein 3 (FHL3) regulates myoblasts differentiation and gene expression by acting as a transcriptional co-activator or co-repressor. However, how FHL3 regulates MyHC expression is currently not clear. In this study, we found that FHL3 down-regulated the expression of MyHC 1/slow and up-regulated the expression of MyHC 2a and MyHC 2b, whereas no significant effect was found on MyHC 2x expression. MyoD and phosphorylated cAMP response element binding protein (pCREB) played important roles in the regulation of MyHC 1/slow and MyHC 2a expression by FHL3, respectively. FHL3 could interact with MyoD, CREB and pCREB in vivo. pCREB had stronger interaction with the cyclic AMP-responsive elements (CRE) of the MyHC 2a promoter compared with CREB, and FHL3 significantly affected the binding capacity of pCREB to CRE. We established a model in which FHL3 promotes the expression of MyHC 2a through CREB-mediated transcription and inhibits the expression of MyHC 1/slow by inhibiting MyoD transcription activity during myogenesis. Our data support the notion that FHL3 plays important roles in the regulation of muscle fiber type composition.

Thin-filament length correlates with fiber type in human skeletal muscle

Force production in skeletal muscle is proportional to the amount of overlap between the thin and thick filaments, which, in turn, depends on their lengths. Both thin- and thick-filament lengths are precisely regulated and uniform within a myofibril. While thick-filament lengths are essentially constant across muscles and species (∼1.65 μm), thin-filament lengths are highly variable both across species and across muscles of a single species. Here, we used a high-resolution immunofluorescence and image analysis technique (distributed deconvolution) to directly test the hypothesis that thin-filament lengths vary across human muscles. Using deltoid and pectoralis major muscle biopsies, we identified thin-filament lengths that ranged from 1.19 ± 0.08 to 1.37 ± 0.04 μm, based on tropomodulin localization with respect to the Z-line. Tropomodulin localized from 0.28 to 0.47 μm further from the Z-line than the NH(2)-terminus of nebulin in the various biopsies, indicating that human thin filaments have nebulin-free, pointed-end extensions that comprise up to 34% of total thin-filament length. Furthermore, thin-filament length was negatively correlated with the percentage of type 2X myosin heavy chain within the biopsy and shorter in type 2X myosin heavy chain-positive fibers, establishing the existence of a relationship between thin-filament lengths and fiber types in human muscle. Together, these data challenge the widely held assumption that human thin-filament lengths are constant. Our results also have broad relevance to musculoskeletal modeling, surgical reattachment of muscles, and orthopedic rehabilitation.

Prolonged ozone exposure effects behaviour, hormones and respiratory muscles in young female rats

This study evaluated behavioural and physiological (hormonal status, muscle structure) affects of prolonged ozone exposure in young females rats. Female Wistar rats were exposed at 0.12ppm of ozone during 6h per day for 15days. Prolonged ozone exposure, though not continuous, produced remarkable behavioural disturbances with an increase in drinking, grooming and resting, and a reduction of rearing, jumping-play and locomotor activities. Besides, ozone exposure was accompanied by increased plasma levels of corticosterone and free triiodothyronine (FT3). Expression of myosin heavy chain (MHC) was significantly affected in three of the five muscles studied. MHC 2B decreased significantly to the benefit of MHC 2A in diaphragm compared to control. MHC 2X increased in Anterior Digastric and decreased in Masseter Superficialis under ozone, to the benefit of MHC 2B in Masseter Superficialis. The plasma corticosterone level increase suggested that prolonged exposure to ozone was stressful. This increase could explain also the increased levels of FT3. Modified respiratory behaviour and hormonal changes both appear to be at the origin of the observed muscular adaptation.

Altered myosin heavy chain phenotype in locomotor and non‐locomotor muscles of mini‐muscle mice bred for high running capacity

Replicated selective breeding of mice for high voluntary wheel running for over 50 generations resulted in four lines of mice that display a high‐running phenotype. Individuals in two of the four lines display a smaller hindlimb muscle mass (mini‐muscle mice), inherited as a Mendelian recessive. In one line (lab designation line 3), all mice now have the mini‐muscle phenotype. Previous studies suggested that mini‐muscle mice also have differences in myosin heavy chain (MyHC) isoform content in hindlimb muscles. This study evaluated whether the changes in MyHC content were restricted to locomotor muscles. Electrophoretic analyses revealed that both locomotor (vastus lateralis, trapezius) and non‐locomotor (lingual) muscles of mini‐muscle mice had greatly reduced 2b MyHC isoform content compared to control mice. The loss in 2b MyHC isoform content in the mini‐muscle mice was compensated by a corresponding increase in 2a and 2x MyHC isoforms. The data suggest that alterations in the MyHC isoform profile in mini‐muscle mice are not restricted to locomotor muscles. Thus, the underlying gene appears to have a general influence on specifying muscle phenotype. NSF IOB‐0543429 to T.G.

DHPR α1S subunit controls skeletal muscle mass and morphogenesis

The alpha1S subunit has a dual function in skeletal muscle: it forms the L-type Ca(2+) channel in T-tubules and is the voltage sensor of excitation-contraction coupling at the level of triads. It has been proposed that L-type Ca(2+) channels might also be voltage-gated sensors linked to transcriptional activity controlling differentiation. By using the U7-exon skipping strategy, we have achieved long-lasting downregulation of alpha1S in adult skeletal muscle. Treated muscles underwent massive atrophy while still displaying significant amounts of alpha1S in the tubular system and being not paralysed. This atrophy implicated the autophagy pathway, which was triggered by neuronal nitric oxide synthase redistribution, activation of FoxO3A, upregulation of autophagy-related genes and autophagosome formation. Subcellular investigations showed that this atrophy was correlated with the disappearance of a minor fraction of alpha1S located throughout the sarcolemma. Our results reveal for the first time that this sarcolemmal fraction could have a role in a signalling pathway determining muscle anabolic or catabolic state and might act as a molecular sensor of muscle activity.

Myostatin preferentially down-regulates the expression of fast 2x myosin heavy chain in cattle

Myostatin is involved in an inhibitor of muscular growth and differentiation. Myoblasts derived from double-muscled Japanese shorthorn cattle (DM myoblasts) with absence of functional myostatin had higher abilities to proliferate and differentiate than myoblasts derived from normal-muscled cattle (NM myoblasts). In DM myoblasts, mRNA expressions of fetal myosin heavy chain (MyHC) in growth medium and of fast 2a and 2x MyHC in fusion medium were significantly greater than that in NM myoblasts. No significant difference existed in expressions of embryonic and slow MyHC mRNA between DM and NM myoblasts. The expression of MyoD mRNA was suppressed in myoblasts by administration of myostatin. Two cloned DM myoblast strains (DMc) were established. Addition of myostatin for DMc resulted in less myotube formation and suppression of mRNA expression of fast 2x MyHC. These findings suggest that the endogenous myostatin preferentially down-regulates the expression of the fast 2x MyHC and participates in differentiation of myofiber types during early bovine myogenesis.

Myostatin preferentially down-regulates the expression of fast 2x myosin heavy chain in cattle

Myostatin is involved in an inhibitor of muscular growth and differentiation. Myoblasts derived from double-muscled Japanese shorthorn cattle (DM myoblasts) with absence of functional myostatin had higher abilities to proliferate and differentiate than myoblasts derived from normal-muscled cattle (NM myoblasts). In DM myoblasts, mRNA expressions of fetal myosin heavy chain (MyHC) in growth medium and of fast 2a and 2x MyHC in fusion medium were significantly greater than that in NM myoblasts. No significant difference existed in expressions of embryonic and slow MyHC mRNA between DM and NM myoblasts. The expression of MyoD mRNA was suppressed in myoblasts by administration of myostatin. Two cloned DM myoblast strains (DMc) were established. Addition of myostatin for DMc resulted in less myotube formation and suppression of mRNA expression of fast 2x MyHC. These findings suggest that the endogenous myostatin preferentially down-regulates the expression of the fast 2x MyHC and participates in differentiation of myofiber types during early bovine myogenesis.

Expression of GHR and PGC-lα in association with changes of MyHC isof orm types in longissimus muscle of Erhualian and Large White pigs (Sus scrofa) during postnatal growth

AbstractMyofibre composition in longissimus dorsi (LD) muscle of Erhualian (EHL) and Large White (LW) pigs was investigated by determining the ratios of mRNA abundances of four myosin heavy chain (MyHC) isoforms (MyHC I, MyHC 2a, MyHC 2x and MyHC 2b) using multiplex RT-PCR. The relationship between expression of growth hormone receptor (GHR) and peroxisome proliferator-activated receptor α coactivator-lα (PGC-lα) mRNAs and changes ofmyofibre type composition during postnatal growth from 3 to 180 days of age were analysed. At 3 days of age, proportions of MyHC I, 2a and 2x fibres were high while only a few MyHC 2b fibres were differentiated. Dramatic changes were observed from day 3 to day 20 with significantly decreased MyHC I, 2a and 2x fibres but abrupt increases in MyHC 2b fibres in both breeds of pigs. Breed difference was exhibited only from day 90, with higher MyHC 2b but less MyHC I and 2a expression in LW pigs. Developmental pattern of GHR mRNA expression in LD muscle coincided with that of MyHC 2b, with LW pigs expressing higher abundance of GHR mRNA. PGC-lα mRNA expression followed distinct patterns in the two breeds of pigs, a higher level of PGC-lα mRNA being expressed in LD muscle of EHL pigs, the breed that possessed more oxidative fibres (MyHC I and MyHC 2a).

Effect of glucocorticoïd receptor ligands on myosin heavy chains expression in rat skeletal muscles during controllable stress

The influence of agonist (dexamethasone) and antagonist (mifepristone) of glucocorticoïd receptor during controllable painless stress was evaluated on myosin heavy chains expression in three masticatory and two nape rat muscles: anterior digastric (AD), anterior temporalis (AT), masseter superficialis (MS), longissimus capitis (L) and rectus capitis dorsalis major (R). The relative amounts of myosin heavy chain (MHC) protein isoform contained were significantly affected in four muscles studied by dexamethasone and in three muscles studied under mifepristone, versus control during the stress procedure, after only 1 week of treatment. The control group AT muscles contained respectively 18.2% of MHC 2A, 34.5% of MHC 2X and 47.4% of MHC 2B. The effects of dexamethasone and mifepristone were opposite in this muscle: under dexamethasone, the relative proportions of the three isoforms were 14.2, 31.0 and 54.8%: consequently, MHC 2A and 2X decreased with the profit of 2B. Under mifepristone, the relative proportions were 21.1, 36.6 and 42.3% (MHC 2A and 2X increased to the detriment of 2B). The L muscle was not affected by the two treatments and MS muscle was only affected by dexamethasone. Dexamethasone increased MHC 2B to the detriment of MHC 2A in MS, AD and R. Mifepristone and dexamethasone induced the same changes in AD. The mifepristone treatment decreased the MHC 2X profile in R. Under dexamethasone, four muscles exhibited a significantly higher proportion of the more rapid isoforms than under mifepristone. A previous work showed that controllable stress induced a marked increase in the relative expression of MHC 2B in the same skeletal muscles (Martrette et al. , 1998). Our results confirm then a significant participation of glucocorticoïd in MHC isoform expression during controllable stress.

Fiber types in rat laryngeal muscles and their transformations after denervation and reinnervation.

The intrinsic laryngeal muscles cricothyroid (CT) and thyroarythenoid (TA) differ in myosin expression. CT expresses limb myosin heavy chains (MyHCs) and TA expresses an MyHC found in extraocular (EO) muscles, in addition to limb isoforms. We used immunohistochemical (IHC) analyses with highly specific monoclonal antibodies (MAbs) against various MyHCs to study muscle fiber types in rat CT and TA and to investigate whether nerves to laryngeal muscles control MyHC expression. CT was found to have the full complement of limb fiber types. TA had three major fiber types: 2b/eo, co-expressing 2B and EO MyHCs, 2x/2b, co-expressing 2X and 2B MyHCs, and 2x, expressing 2X MyHC. Type 2a and slow fibers were absent. TA consisted of two divisions: the external division (TA-X), which is homogeneously 2b/eo, and the vocalis division (TA-V), composed principally of 2x and 2b/eo fibers with a minority of 2x/2b fibers. TA-V had two compartments that differ in fiber type composition. At 4 weeks after cutting and re-uniting the recurrent laryngeal nerve (RLN), many 2b/eo fibers in the TA-X began to express 2X MyHC, while EO and 2B MyHC expression in these fibers progressively declined. By 12 weeks, up to 16.5% of fibers in the TA-X were of type 2x. These findings suggest that nerve fibers originally innervating 2x fibers in TA-V and other muscles have randomly cross-innervated 2b/eo fibers in the TA-X and converted them into 2x fibers. We conclude that CT and TA are distinct muscle allotypes and that laryngeal muscle fibers are subject to neural regulation.

Method of isolation, rate of postmortem metabolism, and myosin heavy chain isoform composition influence ATPase activity of isolated porcine myofibrils

The objective of this study was to determine the influence of myofibril isolation procedures and myosin heavy chain (MyHC) isoform composition on myofibrillar ATPase activity as related to postmortem muscle metabolism. Myofibrils from the red (RST) and white (WST) portions of semitendinosus muscles were isolated using two different methods (A and B) at 3 min and 24 h postmortem in control (NS) and electrically stimulated (ES) pork carcasses. Comparison of the relative MyHC isoform profiles between the two different myofibril isolation methods and myosin extracts from the RST and WST at 3 min showed that method B myofibrils were more similar to the myosin extract than method A. Myofibrillar ATPase activity remained constant or increased ( P<0.01) from 3 min to 24 h postmortem in NS carcasses and decreased ( P<0.0001) in ES carcasses. From the RST, method A myofibrils had higher ( P<0.0001) ATPase activity compared to method B across sampling time and carcass treatment. In the WST, method A myofibrils had lower ( P<0.01) activity at 3 min, were not different at 24 h in NS carcasses, but had higher ( P<0.05) activity at 24 h in ES carcasses versus method B myofibrils. Compared to method B, isolation method A biased the isoform profile of myofibril samples more towards faster MyHC (2A and 2X) in the RST and towards MyHC 2X in the WST. Results suggest that the ATPase activity and MyHC isoform profile of isolated myofibril samples are influenced by method of myofibril isolation, postmortem sampling time, and the rate of postmortem metabolism. Thus, differences in MyHC isoform profile and method of myofibril isolation must be taken into account to determine accurately the relationship between myofibrillar ATPase activity and rate of postmortem metabolism.

Relationships of myosin heavy chain fibre types to meat quality traits in traditional and modern pigs

Porcine skeletal muscle fibres were molecularly classified, using in situ hybridisation and immunocytochemistry, into four types, according to the isoform of myosin heavy chain (MyHC) that was present in each fibre (MyHC slow/I, MyHC 2a, MyHC 2x and MyHC 2b). The relationship between MyHC fibre types and meat quality traits between two phenotypically divergent muscles [longissimus dorsi (LD) and psoas], and between the same muscles of different breeds (traditional Berkshire and Tamworth, and modern Duroc-based and Large White-based) were examined. We found that the greater abundance of fast oxidative-glycolytic MyHC 2a and 2x fibres in the psoas was associated with superior meat quality traits, and that the greater presence of fast glycolytic MyHC 2b fibres in the LD could account for less favourable quality traits, both in terms of pH, drip loss, grain, colour, yield force and work done. Although significant correlations were found between specific fibre types and quality traits, within either the psoas or LD muscle of some breeds, no consistent correlation was found across both muscles and all breeds. This finding was in line with the view that a given fibre type could have considerable differences in phenotype between breeds, and between muscles. The observed inverse compositional and functional-meat quality relationship between MyHC 2b and 2x fibres, and MyHC 2b and 2a fibres could form a basis of fibre type manipulation to improve meat quality.

Expression of insulin growth factor-1 splice variants and structural genes in rabbit skeletal muscle induced by stretch and stimulation.

1. Skeletal muscle is a major source of circulating insulin growth factor-1 (IGF-1), particularly during exercise. It expresses two main isoforms. One of the muscle IGF-1 isoforms (muscle L.IGF-1) is similar to the main liver IGF-1 and presumably has an endocrine action. The other muscle isoform as a result of alternative splicing has a different 3' exon sequence and is apparently designed for an autocrine/paracrine action (mechano-growth factor, MGF). Using RNase protection assays with a probe that distinguishes these differently spliced forms of IGF-1, their expression and also the expression of two structural genes was measured in rabbit extensor digitorum longus muscles subjected to different mechanical signals. 2. Within 4 days, stretch using plaster cast immobilization with the limb in the plantar flexed position resulted in marked upregulation of both forms of IGF-1 mRNA. Electrical stimulation at 10 Hz combined with stretch (overload) resulted in an even greater increase of both types of IGF-1 transcript, whereas electrical stimulation alone, i.e. without stretch, resulted in no significant increase over muscle from sham-operated controls. Previously, it was shown that stretch combined with electrical stimulation of the dorsiflexor muscles in the adult rabbit results in a marked increase in muscle mass involving increases in both length and girth, within a few days. The expression of both systemic and autocrine IGF-1 growth factors provides a link between the mechanical signal and the marked increase in the structural gene expression involved in tissue remodelling and repair. 3. The expression of the beta actin gene was seen to be markedly upregulated in the stretched and stretched/stimulated muscles. It was concluded that the increased expression of this cytoskeletal protein gene is an indication that the production of IGF-1 may initially be a response to local damage. 4. Switches in muscle fibre phenotype were studied using a specific gene probe for the 2X myosin heavy chain gene. Type 2X expression was found to decrease markedly with stimulation alone and when electrical stimulation was combined with stretch. Unlike the induction of IGF-1 and beta actin, the decreased expression of the 2X myosin mRNA was less marked in the 'stretch only' muscles. This indicates that the interconversion of fibre type 2X to 2A may in some situations be commensurate with, but not under the control of IGF-1.

Expression of Myosin Heavy Chain mRNA in Rat Laryngeal Muscles

The composition of myosin heavy chain mRNA was analysed quantitatively in 5 intrinsic laryngeal muscles of rats, using a competitive polymerase chain reaction. Intrinsic laryngeal muscles with the fastest contraction times, e.g. ventricular thyroarytenoid muscle, lateral cricoarytenoid muscle, and vocalis muscle, contained 2 fast isoforms, comprising mainly type 2B myosin heavy chains (52.1, 44.6 and 8.2%, respectively) and type 2X myosin heavy chains (21.9, 37.6 and 80.8%, respectively). Conversely, muscles with slower contraction times, such as posterior cricoarytenoid muscle and cricothyroid muscle, contained more than 85% of 2 fast isoforms; mainly type 2X myosin heavy chains (52.4-72.1%, respectively) and type 2A myosin heavy chains (34.6-25.2%, respectively). The results show a strong correlation between the composition of fast myosin heavy chain isoforms and muscle contraction times. Type 2L myosin heavy chain transcripts specific for laryngeal muscles and extra-ocular muscles were expressed in the order of ventricular thyroarytenoid (9.5%)>lateral cricoarytenoid (4.8%)>vocalis (2.5%)>posterior cricoarytenoid muscle (0.9%), but were not expressed in cricothyroid muscle. Neonatal myosin heavy chain was also expressed in all laryngeal muscles, ranging from 0.04 to 3%, but embryonic myosin heavy chain was expressed in ventricular thyroarytenoid, posterior cricoarytenoid and cricothyroid muscle at very low levels. These results suggest that intrinsic laryngeal muscles have different expression patterns for myosin heavy chain isoforms and may have different regulatory roles related to their functional requirement.

Isolation and localization of the skeletal myosin heavy chain 2X gene on pig chromosome 12q1.4-q1.5.

Isolation and localization of the skeletal myosin heavy chain 2X gene on pig chromosome 12q1.4-q1.5.

Maximum shortening velocity and coexistence of myosin heavy chain isoforms in single skinned fast fibres of rat skeletal muscle

Myosin heavy chain composition of a large number (288) of single fibres from slow (soleus), and fast (superficial part of tibialis anterior, and plantaris) muscles of adult (3-5-month-old) Wistar rats was determined. A combination of SDS-PAGE and monoclonal antibodies against myosin heavy chains allowed to identify four myosin heavy chain isoforms (1, 2A, 2X, and 2B) and to detect myosin heavy chain coexistence. Four groups of fibres containing only one myosin heavy chain (1 myosin heavy chain, 2A myosin heavy chain, 2X myosin heavy chain, and 2B myosin heavy chain), and five groups containing more than one myosin heavy chain (1 and 2A myosin heavy chains, 2A and 2X myosin heavy chains, 2X and minor amounts of 2B (2X-2B fibres), 2B and minor amounts of 2X (2B-2X fibres), and 2A, 2X, and 2B myosin heavy chain were identified and their relative percentages were assessed. Coexistence of fast myosin heavy chain isoforms was found to be very frequent (50% of the fibres in plantaris, and 30% in tibialis anterior), whereas coexistence of slow and fast (2A) myosin heavy chain was very rare. Maximum shortening velocity (V0) was determined using the slack-test procedure in a subset of 109 fast fibres from the above population. The values of V0 formed a continuum extending from 2A to 2X to 2X-2B to 2B-2X to 2B fibres. 2A fibres had the lowest value of V0 and 2B fibres the highest. Only the differences between 2A and 2B and 2A and 2B-2X fibres were statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Type 2X-myosin heavy chain is coded by a muscle fiber type-specific and developmentally regulated gene.

We have previously reported the identification of a distinct myosin heavy chain (MyHC) isoform in a major subpopulation of rat skeletal muscle fibers, referred to as 2X fibers (Schiaffino, S., L. Gorza, S. Sartore, L. Saggin, M. Vianello, K. Gundersen, and T. Lømo. 1989. J. Muscle Res. Cell Motil. 10:197-205). However, it was not known whether 2X-MyHC is the product of posttranslational modification of other MyHCs or is coded by a distinct mRNA. We report here the isolation and characterization of cDNAs coding a MyHC isoform that is expressed in type 2X skeletal muscle fibers. 2X-MyHC transcripts differ from other MyHC transcripts in their restriction map and 3' end sequence and are thus derived from a distinct gene. In situ hybridization analyses show that 2X-MyHC transcripts are expressed at high levels in the diaphragm and fast hindlimb muscles and can be coexpressed either with 2B- or 2A-MyHC transcripts in a number of fibers. At the single fiber level the distribution of each MyHC mRNA closely matches that of the corresponding protein, determined by specific antibodies on serial sections. In hindlimb muscles 2X-, 2A-, and 2B-MyHC transcripts are first detected by postnatal day 2-5 and display from the earliest stages a distinct pattern of distribution in different muscles and different fibers. The emergence of type 2 MyHC isoforms thus defines a distinct neonatal phase of fiber type differentiation during muscle development. The functional significance of MyHC isoforms is discussed with particular reference to the velocity of shortening of skeletal muscle fibers.