In vitro characterization of proliferation and differentiation of pig satellite cells

Skeletal muscle contains various muscle fiber types exhibiting different contractile properties based on the myosin heavy chain (MyHC) isoform profile. Muscle fiber type composition is highly variable and influences growth performance and meat quality, but underlying mechanisms regulating fiber type composition remain poorly understood. The aim of the present work was to develop a model based on muscle satellite cell culture to further investigate the regulation of adult MyHC isoforms expression in pig skeletal muscle. Satellite cells were harvested from the mostly fast-twitch glycolytic longissimus (LM) and predominantly slow-twitch oxidative rhomboideus (RM) muscles of 6-week-old piglets. Satellite cells were allowed to proliferate up to 80% confluence, reached after 7 day of proliferation (D7), and then induced to differentiate. Kinetics of proliferation and differentiation were similar between muscles and more than 95% of the cells were myogenic (desmin positive) at D7 with a fusion index reaching 65±9% after 4 day of differentiation. One-dimensional SDS polyacrylamide gel electrophoresis revealed that satellite cells from both muscles only expressed the embryonic and fetal MyHC isoforms in culture, without any of the adult MyHC isoforms that were expressed in vivo. Interestingly, triiodothyronine (T3) induced de novo expression of adult fast and α-cardiac MyHC in vitro making our culture system a valuable tool to study de novo expression of adult MyHC isoforms and its regulation by intrinsic and/or extrinsic factors.     

Myosin heavy chain composition of muscle spindles in human biceps brachii.

Data on the myosin heavy chain (MyHC) composition of human muscle spindles are scarce in spite of the well-known correlation between MyHC composition and functional properties of skeletal muscle fibers. The MyHC composition of intrafusal fibers from 36 spindles of human biceps brachii muscle was studied in detail by immunocytochemistry with a large battery of antibodies. The MyHC content of isolated muscle spindles was assessed with SDS-PAGE and immunoblots. Four major MyHC isoforms (MyHCI, IIa, embryonic, and intrafusal) were detected with SDS-PAGE. Immunocytochemistry revealed very complex staining patterns for each intrafusal fiber type. The bag(1) fibers contained slow tonic MyHC along their entire fiber length and MyHCI, alpha-cardiac, embryonic, and fetal isoforms along a variable part of their length. The bag(2) fibers contained MyHC slow tonic, I, alpha-cardiac, embryonic, and fetal isoforms with regional variations. Chain fibers contained MyHCIIa, embryonic, and fetal isoforms throughout the fiber, and MyHCIIx at least in the juxtaequatorial region. Virtually each muscle spindle had a different allotment of numbers of bag(1), bag(2) and chain fibers. Taken together, the complexity in intrafusal fiber content and MyHC composition observed indicate that each muscle spindle in the human biceps has a unique identity.     

Protocol for rat single muscle fiber isolation and culture

To attain a superior in vitro model of mature muscle fibers, we modified the established protocol for isolating single muscle fibers from rat skeletal muscle. Muscle fiber cultures with high viability were obtained using flexor digitorum brevis muscle and lasted for at least 7 days. We compared the expression levels of adult myosin heavy chain (MyHC) isoforms in these single muscle fibers with myotubes formed from myoblasts; isolated fibers contained markedly more abundant adult MyHC isoforms than myotubes. This muscle fiber model, therefore, will be useful for studying the various functions and cellular processes of mature muscles in vitro.     

A One-Step Immunostaining Method to Visualize Rodent Muscle Fiber Type within a Single Specimen

In this study, we present a quadruple immunostaining method for rapid muscle fiber typing of mice and rats using antibodies specific to the adult myosin heavy chain (MyHC) isoforms MyHC1, 2A, 2X, and 2B, which are common marker proteins of distinct muscle fiber types. We developed rat monoclonal antibodies specific to each MyHC isoform and conjugated these four antibodies to fluorophores with distinct excitation and emission wavelengths. By mixing the four types of conjugated antibodies, MyHC1, 2A, 2X, and 2B could be distinguished within a single specimen allowing for facile delineation of skeletal muscle fiber types. Furthermore, we could observe hybrid fibers expressing MyHC2X and MyHC2B together in single longitudinal muscle sections from mice and rats, that was not attained in previous techniques. This staining method is expected to be applied to study muscle fiber type transition in response to environmental factors, and to ultimately develop techniques to regulate animal muscle fiber types.     

FoxO1抑制猪骨骼肌MyHC Ⅰ的表达

为研究FoxO1与骨骼肌纤维类型之间的关系,本试验以大白猪为实验材料,利用RT-PCR和Western印迹技术,检测了FoxO1与肌纤维类型标志基因MyHCⅠ、MyHCⅡa、MyHCⅡb和MyHCⅡx在特定骨骼肌中的表达规律,以及调控肌纤维类型关键基因Mef2c和NFAT的表达,并用Wortmannin处理原代培养的猪骨骼肌成肌细胞,检测了FoxO1与肌纤维类型相关基因的表达.结果显示,FoxO1在不同骨骼肌类型中mRNA表达差异不显著(P0.05),其蛋白表达与MyHC各亚型显著相关.Wortmannin处理结果显示,在处理的第3和5d,FoxO1蛋白与MyHCⅡb,MyHCⅡx和NFAT表达显著正相关,而与MyHCⅠ,MyHCⅡa和Mef2c表达显著负相关.结果表明,FoxO1通过抑制MyHCⅠ的表达调控肌纤维类型.     

A continuum of myofibers in adult rabbit extraocular muscle: force, shortening velocity, and patterns of myosin heavy chain colocalization

Extraocular muscle (EOM) myofibers do not fit the traditional fiber typing classifications normally used in noncranial skeletal muscle, in part, due to the complexity of their individual myofibers. With single skinned myofibers isolated from rectus muscles of normal adult rabbits, force and shortening velocity were determined for 220 fibers. Each fiber was examined for myosin heavy chain (MyHC) isoform composition by densitometric analysis of electrophoresis gels. Rectus muscle serial sections were examined for coexpression of eight MyHC isoforms. A continuum was seen in single myofiber shortening velocities as well as force generation, both in absolute force (g) and specific tension (kN/m(2)). Shortening velocity correlated with MyHCIIB, IIA, and I content, the more abundant MyHC isoforms expressed within individual myofibers. Importantly, single fibers with similar or identical shortening velocities expressed significantly different ratios of MyHC isoforms. The vast majority of myofibers in both the orbital and global layers expressed more than one MyHC isoform, with up to six isoforms in single fiber segments. MyHC expression varied significantly and unpredictably along the length of single myofibers. Thus EOM myofibers represent a continuum in their histological and physiological characteristics. This continuum would facilitate fine motor control of eye position, speed, and direction of movement in all positions of gaze and with all types of eye movements-from slow vergence movements to fast saccades. To fully understand how the brain controls eye position and movements, it is critical that this significant EOM myofiber heterogeneity be integrated into hypotheses of oculomotor control.     

Transitions of muscle fiber phenotypic profiles

Skeletal muscle is a complex, versatile tissue composed of a large variety of functionally diverse fiber types. The overall properties of a muscle largely result from a combination of the individual properties of its different fiber types and their proportions. Skeletal muscle fiber types, which can be delineated according to various parameters, for example, myofibrillar protein isoforms, metabolic enzyme profiles, and structural and contractile properties, are not fixed units but are capable of responding to altered functional demands and a variety of signals by changing their phenotypic profiles. This brief review summarizes our current understanding of the delineation of fiber types, modulations of their phenotypic profiles as induced under various conditions, and potential mechanisms involved in these transitions.     

Differential expression of calcineurin and SR Ca2+ handling proteins in equine muscle fibers during early postnatal growth.

During early postnatal development, the myosin heavy chain (MyHC) expression pattern in equine gluteus medius muscle shows adaptation to movement and load,resulting in a decrease in the number of fast MyHC fibers and an increase in the number of slow MyHC fibers. In the present study we correlated the expression of MyHC isoforms to the expression of sarcoplasmic(endo)reticulum Ca2+-ATPase 1 and 2a (SERCA), phospholamban (PLB), calcineurin A (CnA), and calcineurin B (CnB). Gluteus medius muscle biopsies were taken at 0, 2, 4, and 48 weeks and analyzed using immunofluorescence. Both SERCA isoforms and PLB were expressed in almost all fiber types at birth. From 4 weeks of age onward, SERCA1 was exclusively expressed in fast MyHC fibers and SERCA2a and PLB in slow MyHC fibers. At all time points, CnA and CnB proteins were expressed at a basal level in all fibers, but with a higher expression level in MyHC type 1 fibers. From 4 weeks onward, expression of only CnA was also higher in MyHC type 2a and 2ad fibers. We propose a double function of calcineurin in calcium homeostasis and maintenance of slow MyHC fiber type identity. Although equine muscle is already functional at birth, expression patterns of the monitored proteins still show adaptation, depending on the MyHC fiber type.     

Evidence for three fast myosin heavy chain isoforms in type II skeletal muscle fibers in the adult llama (Lama glama).

Skeletal muscle fiber types classified on the basis of their content of different myosin heavy chain (MHC) isoforms were analyzed in samples from hindlimb muscles of adult sedentary llamas (Lama glama) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry, and quantitative histochemistry of fiber metabolic and size properties. The immunohistochemical technique allowed the separation of four pure (i.e., expressing a unique MHC isoform) muscle fiber types: one slow-twitch (Type I) and three fast-twitch (Type II) phenotypes. The same four major fiber types could be objectively discriminated with two serial sections stained for mATPase after acid (pH 4.5) and alkaline (pH 10.5) preincubations. The three fast-twitch fiber types were tentatively designated as IIA, IIX, and IIB on the basis of the homologies of their immunoreactivities, acid denaturation of their mATPase activity, size, and metabolic properties expressed at the cellular level with the corresponding isoforms of rat and horse muscles. Acid stability of their mATPase activity increased in the rank order IIA>IIX>IIB. The same was true for size and glycolytic capacity, whereas oxidative capacity decreased in the same rank order IIA>IIX>IIB. In addition to these four pure fibers (I, IIA, IIX, and IIB), four other fiber types with hybrid phenotypes containing two (I+IIA, IIAX, and IIXB) or three (IIAXB) MHCs were immunohistochemically delineated. These frequent phenotypes (40% of the semitendinosus muscle fiber composition) had overlapped mATPase staining intensities with their corresponding pure fiber types, so they could not be delineated by mATPase histochemistry. Expression of the three fast adult MHC isoforms was spatially regulated around islets of Type I fibers, with concentric circles of fibers expressing MHC-IIA, then MHC-IIX, and peripherally MHC-IIB. This study demonstrates that three adult fast Type II MHC isoproteins are expressed in skeletal muscle fibers of the llama. The general assumption that the very fast MHC-IIB isoform is expressed only in small mammals can be rejected.     

Dietary Fat Influences the Expression of Contractile and Metabolic Genes in Rat Skeletal Muscle

Dietary fat plays a major role in obesity, lipid metabolism, and cardiovascular diseases. To determine whether the intake of different types of dietary fats affect the muscle fiber types that govern the metabolic and contractile properties of the skeletal muscle, we fed male Wistar rats with a 15% fat diet derived from different fat sources. Diets composed of soybean oil (n-6 polyunsaturated fatty acids (PUFA)-rich), fish oil (n-3 PUFA-rich), or lard (low in PUFAs) were administered to the rats for 4 weeks. Myosin heavy chain (MyHC) isoforms were used as biomarkers to delineate the skeletal muscle fiber types. Compared with soybean oil intake, fish oil intake showed significantly lower levels of the fast-type MyHC2B and higher levels of the intermediate-type MyHC2X composition in the extensor digitorum longus (EDL) muscle, which is a fast-type dominant muscle. Concomitantly, MyHC2X mRNA levels in fish oil-fed rats were significantly higher than those observed in the soybean oil-fed rats. The MyHC isoform composition in the lard-fed rats was an intermediate between that of the fish oil and soybean oil-fed rats. Mitochondrial uncoupling protein 3, pyruvate dehydrogenase kinase 4, and porin mRNA showed significantly upregulated levels in the EDL of fish oil-fed rats compared to those observed in soybean oil-fed and lard-fed rats, implying an activation of oxidative metabolism. In contrast, no changes in the composition of MyHC isoforms was observed in the soleus muscle, which is a slow-type dominant muscle. Fatty acid composition in the serum and the muscle was significantly influenced by the type of dietary fat consumed. In conclusion, dietary fat affects the expression of genes related to the contractile and metabolic properties in the fast-type dominant skeletal muscle, where the activation of oxidative metabolism is more pronounced after fish oil intake than that after soybean oil intake.     

Quantifying the temporospatial expression of postnatal porcine skeletal myosin heavy chain genes.

Postnatal skeletal muscle fiber type is commonly defined by one of four major myosin heavy chain (MyHC) gene isoforms (slow/I, 2a, 2x, and 2b) that are expressed. We report on the novel use of combined TaqMan quantitative real-time RT-PCR and image analysis of serial porcine muscle sections, subjected to in situ hybridization (ISH) and immunocytochemistry (IHC), to quantify the mRNA expression of each MyHC isoform within its corresponding fiber type, termed relative fiber type-restricted expression. This versatile approach will allow quantitative temporospatial comparisons of each MyHC isoform among muscles from the same or different individuals. Using this approach on porcine skeletal muscles, we found that the relative fiber type-restricted expression of each postnatal MyHC gene showed wide spatial and temporal variation within a given muscle and between muscles. Marked differences were also observed among pig breeds. Notably, of the four postnatal MyHC isoforms, the 2a MyHC gene showed the highest relative fiber type-restricted expression in each muscle examined, regardless of age, breed, or muscle type. This suggests that although 2a fibers are a minor fiber type, they may be disproportionately more important as a determinant of overall muscle function than was previously believed.     

Immunohistochemical Analysis of Myosin Heavy Chain Expression in Laryngeal Muscles of the Rabbit, Cat, and Baboon

We studied myosin heavy chain (MyHC) expression and fiber type distribution in laryngeal muscles in the rabbit, cat, and baboon using immunohistochemistry with highly MyHC-specific antibodies. Two types of variation in MyHC expression were found: between muscles of different function within species and within specific muscles between species. Within species, thyroarytenoid (Ta), an adductor, had faster MyHCs and fiber type profiles than the abductor, posterior cricoarytenoid (PCA), which expressed faster MyHCs than the vocal fold tensor, cricothyroid (CT). Between species, laryngeal muscles generally expressed faster MyHCs in small animals than in larger ones: extraocular (EO) MyHC was expressed in the Ta and PCA of the rabbit but not in the cat and baboon, whereas 2B MyHC was expressed in these muscles of the cat but not of the baboon. The CT expressed only MyHC isoforms and fiber types found in the limb muscles of the same species. These results are discussed in light of the hypothesis that the between-species variations in laryngeal muscle fiber types are evolutionary adaptations in response to changes in body mass and respiratory frequency. Within-species variations in fiber types ensure that protective closure of the glottis is always faster than movements regulating airflow during respiration. (J Histochem Cytochem 56:929–950, 2008)     

Fiber type and myosin heavy chain compositions of adult pretarsal orbicularis oculi muscle

Summary Pretarsal orbicularis oculi muscle (POOM) is an important structure of eyelid movement in human. The aim of this study was to investigate fiber histomorphology and myosin heavy chain (MyHC) isoform composition of adult POOM, and to clarify their age-related changes. Eyelid specimens from 58 subjects (age range, 21 to 91 years) were collected during upper blepharoplasty procedures. Serial cross sections of POOM were ATPase-stained and examined under miscroscope. Quantitative measures of muscle fiber size and fiber type distribution were obtained in 35 subjects with adequate fiber cross sections. Relative MyHC isoform contents of POOM were retrieved by gel electrophoresis in all 58 subjects. Examination of the histochemical staining revealed an abundance of type II fiber ( >85%) in human POOM, with more type IIX than IIA fibers. Decreased mean area of all fibers and type IIA fibers were noted in the old group when compared to the young. As for MyHC analysis, the relative content of MyHC isoforms exhibited an order of IIX > IIA > I, and the relative MyHC IIA content showed a negative correlation with age. Comparing with previous studies of limb or masticatory muscles, adult POOM exhibits a unique fiber and MyHC composition, as well as a different aging pattern.     

Monospecific antibodies against the three mammalian fast limb myosin heavy chains

Skeletal muscle fibres in mammalian limb muscles are of four types: slow, 2A, 2X, and 2B, each characterized by a distinct myosin heavy chain (MyHC) isoform. Existing monoclonal antibodies (mabs) against fast MyHCs lack fibre-type specificity across species and could not positively identify 2X fibres. In this work, mabs were raised against each of the fast MyHCs. These mabs were shown to be monospecific by Western blots and immunohistochemistry in the rat. The advantages of using these mabs for identifying the three fast fibre types and hybrid fibres expressing multiple isoforms were illustrated using rat tibialis anterior muscle. Immunohistochemical analyses confirmed the monospecificity of these mabs in the following additional species: mouse, guinea pig, rabbit, cat, and baboon. 2B fibres were absent in limb muscles of the cat and baboon. These mabs constitute a set of powerful tools for studying muscle fibre types and their transformations.     

Mutation of the IIB myosin heavy chain gene results in muscle fiber loss and compensatory hypertrophy

The fast skeletal IIb gene is the source of most myosin heavy chain (MyHC) in adult mouse skeletal muscle. We have examined the effects of a null mutation in the IIb MyHC gene on the growth and morphology of mouse skeletal muscle. Loss in muscle mass of several head and hindlimb muscles correlated with amounts of IIb MyHC expressed in that muscle in wild types. Decreased mass was accompanied by decreases in mean fiber number, and immunological and ultrastructural studies revealed fiber pathology. However, mean cross-sectional area was increased in all fiber types, suggesting compensatory hypertrophy. Loss of muscle and body mass was not attributable to impaired chewing, and decreased food intake as a softer diet did not prevent the decrease in body mass. Thus loss of the major MyHC isoform produces fiber loss and fiber pathology reminiscent of muscle disease.     

Sarco(endo)plasmic reticulum calcium pump isoforms in paralyzed rat slow muscle

To assess the influence of paralysis on the expression of phenotypic protein isoforms related to muscle relaxation, the effects of spinal cord transection (ST) on sarco(endo)plasmic reticulum calcium ATPase (SERCA) pump isoform protein levels in the slow rat soleus were measured. Western blotting using SERCA isoform specific antibodies demonstrated a rapid up-regulation (7 days post ST) of the fast fiber type-specific isoform (SERCA1). In contrast, the slow fiber type-specific isoform, SERCA2, was decreased with a slower time-course. The up-regulation of SERCA1 protein preceded the up-regulation of fast myosin heavy chain (MyHC) (i.e., MyHC-II). Immunohistochemical analyses of single muscle fibers showed that 15 days after ST there was a pronounced increase in the proportion of slow MyHC fibers with SERCA1 confirming that SERCA1 was up-regulated in the slow fibers of the soleus prior to MyHC-II. These data suggest that the expression of the SERCA isoforms (particularly SERCA1) may serve as more sensitive markers of phenotypic adaptation in response to altered levels of contractile activity than the MyHC isoforms. In addition, since the expression of SERCA isoforms was dissociated from MyHC isoforms, regulation of gene expression for these two different protein systems must involve different signaling events and/or synthetic processes.     

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.