Adult Myosin Heavy Chain Research Articles

Analysis of myosin heavy chain mRNA expression by RT-PCR.

An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

Coordinated expression of myosin heavy chain isoforms and metabolic enzymes within overloaded rat muscle fibers.

We studied the coordinated regulation of myosin heavy chains (MHC) and metabolic enzymes within individual overloaded adult rat plantaris fibers. This was done using monoclonal antibodies raised against distinct developmental and adult MHCs, and quantitative microphotometric succinate dehydrogenase (SDH) and glycerol-3-phosphate dehydrogenase (GPDH) enzyme assays. Overload shifted MHC expression in the order IIb-->IIx-->IIa-->alpha/I, with a tripling of cells coexpressing I and alpha-MHC, and a transient reexpression of two embryonic MHC and the neonatal isoform in preexisting myofibers. Overload caused a rapid, size-independent, 50% decrease in GPDH activity across all cell types, which recovered by 6 wk. Fiber SDH activities varied according to MHC composition, such that overloaded fibers coexpressing IIa MHC displayed control slow fiber SDH levels, whereas cells expressing IIx and IIb MHC displayed a transient 30% increase in SDH that recovered by 6 wk. Our results suggest that during overload, fibers adapt progressively to the new functional requirements and display more efficient cellular energy utilization and delivery characteristics. The time course of adaptations suggests a role for glycolytic enzymes in the initiation of these transformations.

Expression of fast myosin heavy chain transcripts in developing and dystrophic chicken skeletal muscle

The expression of fast myosin heavy chain (MyHC) genes was examined in vivo during fast skeletal muscle development in the inbred White Leghorn chicken (line 03) and in adult muscles from the genetically related dystrophic White Leghorn chicken (line 433). RNA dotblot and northern hybridization was employed to monitor MyHC transcript levels utilizing specific oligonucleotide probes. The developmental pattern of MyHC gene expression in the pectoralis major (PM) and the gastrocnemius muscles was similar during embryonic development with three embryonic MyHC isoform genes, Cemb1, Cemb2, and Cemb3, sequentially expressed. Following hatching, MyHC expression patterns in each muscle differed. The expression of MyHC genes was also studied in muscle cell cultures derived from 12-day embryonic pectoralis muscles. In vitro, Cvent, Cemb1, and Cemb2 MyHC genes were expressed; however, little if any Cemb3 MyHC gene expression could be detected, even though Cemb3 was the predominant MyHC gene expressed during late embryonic development in vivo. In most adult muscles other than the PM and anterior latissimus dorsi (ALD), the Cemb3 MyHC gene was the major adult MyHC isoform. In addition, two general patterns of expression were identified in fast muscle. The fast muscles of the leg expressed neonatal (Cneo) and Cemb3 MyHC genes, while other fast muscles expressed adult (Cadult) and Cemb3 MyHC genes. MyHC gene expression in adult dystrophic muscles was found to reflect the expression patterns found in corresponding normal muscles during the neonatal or early post-hatch developmental period, providing additional evidence that avian muscular dystrophy inhibits muscle maturation.

Expression of fast myosin heavy chain transcripts in developing and dystrophic chicken skeletal muscle

The expression of fast myosin heavy chain (MyHC) genes was examined in vivo during fast skeletal muscle development in the inbred White Leghorn chicken (line 03) and in adult muscles from the genetically related dystrophic White Leghorn chicken (line 433). RNA dot-blot and northern hybridization was employed to monitor MyHC transcript levels utilizing specific oligonucleotide probes. The developmental pattern of MyHC gene expression in the pectoralis major (PM) and the gastrocnemius muscles was similar during embryonic development with three embryonic MyHC isoform genes, Cemb1, Cemb2, and Cemb3, sequentially expressed. Following hatching, MyHC expression patterns in each muscle differed. The expression of MyHC genes was also studied in muscle cell cultures derived from 12-day embryonic pectoralis muscles. In vitro, Cvent, Cemb1, and Cemb2 MyHC genes were expressed; however, little if any Cemb3 MyHC gene expression could be detected, even though Cemb3 was the predominant MyHC gene expressed during late embryonic development in vivo. In most adult muscles other than the PM and anterior latissimus dorsi (ALD), the Cemb3 MyHC gene was the major adult MyHC isoform. In addition, two general patterns of expression were identified in fast muscle. The fast muscles of the leg expressed neonatal (Cneo) and Cemb3 MyHC genes, while other fast muscles expressed adult (Cadult) and Cemb3 MyHC genes. MyHC gene expression in adult dystrophic muscles was found to reflect the expression patterns found in corresponding normal muscles during the neonatal or early post-hatch developmental period, providing additional evidence that avian muscular dystrophy inhibits muscle maturation. Dev. Dyn. 208:491–504, 1997. © 1997 Wiley-Liss, Inc.

Regionalized expression of myosin isoforms in heterotypic myotubes formed from embryonic and fetal rat myoblasts in vitro

The development of mammalian limb muscles involves the appearance and fusion of at least two separate populations of muscle precursor cells. These two populations, termed embryonic and fetal myoblasts, are first detected within the limb bud at different stages of development. We have previously demonstrated that, in the rat, each myoblast population expresses a unique pattern of myosin heavy chains (MyHCs) during differentiation in vitro (Pin and Merrifield [1993] Dev. Genet. 14:356–368). Embryonic myoblasts accumulate embryonic and slow MyHCs, whereas fetal myoblasts accumulate embryonic, neonatal, and adult fast MyHCs but not slow MyHC. To determine if the two populations can fuse with each other and whether the pattern of MyHC expression is altered in the resulting heterokaryons, embryonic and fetal myoblasts were labelled with the lipophilic dye PKH26, [3H]-thymidine, or 5-bromodeoxyuridine (BRDU) and cocultured for 24–48 hr. Our results demonstrate that fusion occurs between embryonic and fetal myoblasts in vitro. Moreover, analysis of the resulting heterokaryons revealed regionalized accumulations of MyHC around individual nuclei. Interestingly, these accumulations were typical of the default pattern of expression that individual nuclei would have normally expressed in single culture. Nuclei contributed by embryonic myoblasts were surrounded by localized accumulations of slow MyHC, whereas nuclei from fetal myoblasts were surrounded by neonatal/fast MyHC. The occurrence of such nuclear domains indicates that the myoblast-specific expression of MyHC isoforms is dictated by cis-acting factors established prior to fusion. Dev. Dyn. 208:420–431, 1997. © 1997 Wiley-Liss, Inc.

Regionalized expression of myosin isoforms in heterotypic myotubes formed from embryonic and fetal rat myoblasts in vitro.

The development of mammalian limb muscles involves the appearance and fusion of at least two separate populations of muscle precursor cells. These two populations, termed embryonic and fetal myoblasts, are first detected within the limb bud at different stages of development. We have previously demonstrated that, in the rat, each myoblast population expresses a unique pattern of myosin heavy chains (MyHCs) during differentiation in vitro (Pin and Merrifield [1993] Dev. Genet. 14:356-368). Embryonic myoblasts accumulate embryonic and slow MyHCs, whereas fetal myoblasts accumulate embryonic, neonatal, and adult fast MyHCs but not slow MyHC. To determine if the two populations can fuse with each other and whether the pattern of MyHC expression is altered in the resulting heterokaryons, embryonic and fetal myoblasts were labelled with the lipophilic dye PKH26, [3H]-thymidine, or 5-bromodeoxyuridine (BRDU) and cocultured for 24-48 hr. Our results demonstrate that fusion occurs between embryonic and fetal myoblasts in vitro. Moreover, analysis of the resulting heterokaryons revealed regionalized accumulations of MyHC around individual nuclei. Interestingly, these accumulations were typical of the default pattern of expression that individual nuclei would have normally expressed in single culture. Nuclei contributed by embryonic myoblasts were surrounded by localized accumulations of slow MyHC, whereas nuclei from fetal myoblasts were surrounded by neonatal/fast MyHC. The occurrence of such nuclear domains indicates that the myoblast-specific expression of MyHC isoforms is dictated by cis-acting factors established prior to fusion.

Short- and long-term effects of testosterone on diaphragm in castrated and normal male rats.

The effects of short- and long-term testosterone absence or treatment on the diaphragm were studied in castrated and sexually normal male rats. Compared with control rats (untreated normal males), testosterone absence or treatment did not significantly affect costal weight. In untreated castrated males, there were significant decreases in specific forces, type II fiber cross-sectional area, and myosin heavy chain (MHC) isoform 2B after 2.5 wk. In castrated males that received testosterone, there were significant increases in specific forces, type II total fiber proportional area, and relative expression of all adult diaphragm fast MHC isoforms (MHC-2all) after 2.5 wk. In normal males that received testosterone, the only significant finding was an increase in MHC-2B after 2.5 wk. Across all groups, there was close correlation between increases in maximum tetanic forces and MHC-2all. Changes in diaphragm function and composition were closely related to changes in serum testosterone levels at 2.5 wk. The lack of significant change in diaphragm function at 10 wk occurred despite changes in serum testosterone levels and diaphragm composition similar to those at 2.5 wk. These findings support our hypothesis that the effects of testosterone are dependent on basal circulating androgen levels and study duration.

Myosin isoform transitions in four rabbit muscles during postnatal growth.

Four rabbit muscles (i.e. semimembranosus proprius, psoas major, biceps femoris and longissimus lumborum), differing in their fibre type composition in the adult, were investigated during postnatal development. Muscle samples were taken at 1, 7, 14, 21, 28, 35, 49 and 77 days of age. Complementary techniques were used to characterize myosin heavy chain (MHC) isoform transitions, i.e. SDS-PAGE, immunocytochemistry and conventional histochemistry. Good accordance was found between electrophoretic and immunocytochemical techniques. Our results show that rabbit muscles were phenotypically immature at birth. At 1 day of age, perinatal isoform represented 70-90% of the total isoform content of the muscles. Two generations of myofibres could be observed on the basis of their morphology and reaction to specific antibodies. In all muscles, primary fibres expressed slow MHC. In contrast, secondary generation of fibres never expressed slow MHC in future fast muscles, while half of them expressed slow MHC in the future slow-twitch muscle, the semimembranosus proprius. During the postnatal period, all muscles displayed a transition from embryonic to perinatal MHC isoforms, followed by a transition from perinatal to adult MHC isoforms. These transitions occured mainly during the first postnatal month. The embryonic isoform was no longer expressed after 14 days, except in longissimus where it disappeared after 28 days. On the contrary, large differences were found in the timing of disappearance of the perinatal isoform between the four muscles. The perinatal isoform disappeared between 28 and 35 days in semimembranosus proprius and 35 and 49 days in psoas and biceps femoris. Interestingly, the perinatal isoform was still present in 6% of the fibres in longissimus at 77 days, the commercial slaughter age, denoting a great delay in the maturation. Fate of each generation of fibres differed between muscles.

Altered expression of myosin mRNA and protein in rat soleus and tibialis anterior following reinnervation.

Myosin heavy chain (MHC) expression was studied in rat soleus and tibialis anterior (TA) at the mRNA and protein levels following reinnervation 8 and 32 wk after sciatic nerve injury. A sciatic nerve crush or transection injury was produced in the midthigh region of adult female Sprague-Dawley rats. A ribonuclease protection assay was developed to measure four of the adult MHCs (I, IIa, IIx, IIb) in a single sample. MHC mRNA and protein were measured and compared in the same muscles. Eight and thirty-two weeks after a crush injury, the MHC mRNA profiles were similar to control with the exception of soleus MHC IIa and TA MHC IIb, which were significantly less than control at both time points. In contrast, reinnervation of the soleus following a sciatic nerve transection injury resulted in an MHC isoform shift characterized by increases in the relative amounts of fast myosin (IIa and IIx) and a decrease in slow myosin. As expected, significant changes first occurred at the mRNA level followed by changes in protein expression. Thirty-two weeks after transection injury and repair, the primary MHC mRNA isoform in the soleus was MHC IIx. Moreover, at 32 wk, MHC IIb mRNA was detected in 50% of the reinnervated soleus following a transection injury. Reinnervation of the TA following sciatic nerve transection led to replacement of the MHC IIb isoform with MHC IIx.

Hypothyroidism alters diaphragm muscle development.

The impact of hypothyroidism (Hyp) on myosin heavy chain (MHC) isoform expression, maximum specific force (P0), fatigability, and maximum unloaded shortening velocity (V0) was determined in the rat diaphragm muscle (Dia) at 0, 7, 14, 21, and 28 days of age. Hyp was induced by treating pregnant rats with 6-n-propyl-2-thiouracil (0.05% in drinking water) beginning at gestational day 10 and was confirmed by reduced plasma levels of 3,5,3'-triiodothyronine and thyroxine. MHC isoforms were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by densitometry. Isometric P0 and fatigue resistance of the Dia were measured in vitro at 26 degrees C, and V0 was determined at 15 degrees C with the slack test. Compared with control muscles, expression of MHC-slow was higher and expression of adult fast MHC isoforms was lower in Hyp Dia at all ages. The neonatal isoform of MHC continued to be expressed in the Hyp Dia until day 28. At each age, P0 and fatigability were reduced and V0 was slower in the Hyp Dia. We conclude that Hyp-induced alterations in MHC isoform expression do not fully predict the changes in Dia contractile properties.

Innervation regulates myosin heavy chain isoform expression in developing skeletal muscle fibers

The influence of innervation on primary and secondary myogenesis and its relation to fiber type diversity were investigated in two specific wing muscles of quail embryo, the posterior (PLD) and anterior latissimus dorsi (ALD). In the adult, these muscles are composed almost exclusively of pure populations of fast and slow fibers, respectively. When slow ALD and fast PLD muscles developed in ovo in an aneurogenic environment induced after neural tube ablation, the cardiac ventricular myosin heavy chain (MHC) isoform was not expressed. The adult slow MHC isoform, SM2, appeared by embryonic day 7 (ED 7) in normal innervated slow ALD but was not expressed in denervated muscle. Analysis of in vitro differentiation of myoblasts from fast PLD and slow ALD muscles isolated from ED 7 control and neuralectomized quail embryos showed no fundamental differences in the pattern of MHC isoform expression. Newly differentiated fibers accumulated cardiac ventricular, embryonic fast, slow SM1 and SM3 MHC isoforms. Nevertheless, the expression of slow SM2 isoform in myotubes formed from slow ALD myoblasts only occurred when myoblasts were cultured in the presence of embryonic spinal cord. Our studies demonstrate that the neural tube influences primary as well as secondary myotube differentiation in avian forelimb and facilitates the expression of different MHC, particularly slow SM2 MHC gene expression in slow myoblasts.

Inherited muscular disorder in mutant Japanese quail ( Coturnix coturnix japonica): an immunohistochemical study

Cryostat sections of myofibres from the Musculus pectoralis thoracicus of a newly established mutant strain (LWC) of Japanese quail with a myotonic dystrophy-like myopathy were labelled with antibody against myosin heavy chain (MHC) isoforms and neural cell adhesion molecule (N-CAM). The characteristic lesions found in sections of muscle of LWC quail stained with haematoxylin and eosin were type 2B fibre atrophy, sarcoplasmic masses, and ring fibres. Immunohistochemical examination failed to distinguish type 2A and 2B fibres in the LWC quail. Antibody to adult fast MHC, which reacted only with type 2A fibres in normal quail, reacted in LWC quail with type 2B fibres, and to a limited degree with type 2A fibres. Sarcoplasmic masses reacted with both fast and slow MHC antibodies. Some masses also reacted with NCAM antibody, but apparently independently of similar reactions in fibres. These findings suggest that the changes observed in the myofibres of the LWC quail were not neurogenic but represented defects in both the plasma membrane and intermediate filaments.

Adaptive range of myosin heavy chain expression in regenerating soleus is broader than in mature muscle

In adult rat muscles experimentally exposed to various patterns of activation, expression of myosin heavy chain isoforms changes, but only within a certain adaptive range. It is characteristic and different in fast or slow muscles. This may be due either to different intrinsic properties of the myogenic cells of the two types of muscles or to extrinsic factors. To test these assumptions, either rat soleus or extensor digitorum longus muscles were injured and transplanted to the bed of the extensor digitorum longus muscle. They regenerated and were reinnervated by the extensor digitorum longus nerve. Expression of myosin heavy chain isoforms was demonstrated immunohistochemically and by in situ hybridization, and analysed by SDS-gel electrophoresis. Three months after cross-transplantation, regenerated soleus expressed all adult myosin heavy chain isoforms, including the myosin heavy chain-2B. The latter was detected in about 50% of muscle fibres and contributed about 10-20% of all myosin heavy chains. The same percentage of myosin heavy chain-2B was found in regenerated extensor digitorum longus. In this regard therefore, the adaptive range of the regenerated soleus muscle was not significantly different from that of the extensor digitorum longus regenerating under the same conditions. This indicates that restriction of the adaptive range in a mature soleus muscle is not due to intrinsic properties of its myogenic cells. It is probably imposed by an extrinsic factor leading to irreversible shut-down of individual myosin heavy chain genes. On the other hand, myosin heavy chain-1 expression was significantly greater in the regenerated soleus than in the extensor digitorum longus innervated by the same nerve. Myosin heavy chain-1 and myosin heavy chain-2B were co-expressed in some regenerated soleus muscle fibres.

Myosin heavy chain profile of cat soleus following chronic reduced activity or inactivity.

To determine the role that normal neuromuscular activity plays in maintaining the myosin heavy chain (MHC) profile of adult cat soleus muscles, the spinal cords of 4 cats were transected (ST) and 8 cats were spinal isolated (SI) for 6 months. Nine nonoperated cats served as controls. Electrophoresis demonstrated that the soleus from control cats contained 98% type I, and 2% IIa MHCs. Both ST and SI resulted in decreased type I and increased IIa MHC, as well as de novo expression of IIb MHC. Immunohistochemistry with MHC-specific antibodies demonstrated that the soleus from control cats contained 99% type I, 1% IIa, and < 1% hybrid fibers (containing both type I and II MHCs). Following ST there were 67% type I, 17% IIa, 3% IIb, and 13% hybrid fibers. After SI, 48% of the fibers were type I, 11% were IIa, 1% were IIb, 25% were hybrid, and 15% contained embryonic MHC. Thus, normal levels of neuromuscular activity appear to be necessary for maintenance of the normal adult MHC profile in some fibers. Complete inactivation results in developmental MHC isoform expression in some fibers. Therefore, the dependence of a fiber on activity as a source of MHC modulation differs substantially among fibers even in a relatively homogeneous muscle.

Hypothyroid-mediated changes in adult rat diaphragm muscle contractile properties and MHC isoform expression.

The purpose of the present study was to examine the effect of acute hypothyroidism on myosin heavy chain (MHC) isoform composition and contractile properties in the adult rat diaphragm muscle. Hypothyroidism was induced by the addition of propylthiouracil (0.05%) in the drinking water for a period of 3 wk. MHC isoform composition of control and hypothyroid diaphragm muscles was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In vitro isometric contractile properties of midcostal diaphragm muscle segements were measured at 26 degrees C, whereas the maximal unloaded shortening velocity was measured at 15 degrees C with the "slack test" method. Serum triiodothyronine and thyroxine values were significantly lower in the hypothyroid compared with the control group. A small but significant increase in the percentage of slow MHC isoform in the diaphragm was observed with acute hypothyroidism, whereas the percentage of the fast MHC isoforms (2A, 2X, and 2B) did not significantly differ between groups. Peak twitch force did not differ between groups. However, twitch contraction and half-relaxation times were significantly prolonged in the hypothyroid group compared with control. Maximal specific force was reduced in the hypothyroid compared with the control group, averaging 15.7 and 19.8 N/cm2, respectively (P < 0.05). The maximal unloaded shortening velocity averaged 4.3 and 8.2 muscle lengths/s in the hypothyroid and control groups, respectively (P < 0.05). We conclude that acute hypothyroidism results in alterations in adult diaphragm muscle contractile properties that cannot be attributed solely to changes in MHC isoform composition.

SV40 Large T Antigen Interferes with Adult Myosin Heavy Chain Expression, but Not with Differentiation of Human Satellite Cells

The growth of muscle fibers during late development as well as in regeneration following muscle injury is the result of the proliferation and differentiation of satellite cells. However, all human cells, including satellite cells, show a limit in their proliferation. In order to define a cellular system with enhanced proliferative capacity, human satellite cells were transfected with a construct containing large T antigen from SV40 under the control of the human vimentin promoter. Vimentin is normally expressed during proliferation, and its expression is down-regulated as differentiation proceeds. In transfected cells, the construct is regulated like the endogenous vimentin gene. The effect of exogenous T antigen expression on both the proliferation and differentiation of human satellite cells was investigated. T antigen expression reduced the doubling time of human satellite cells from 36 to 20 h and increased the final proliferative capacity from 46 to 69 mean population doublings. When differentiation was triggered, although T antigen did not prevent the formation of myotubes, fusion was delayed. A similar delay was observed in the appearance of myogenin protein, one of the HLH regulatory factors, but not in the corresponding mRNA. Finally, T antigen has an effect on adult myosin isoform expression, since both adult slow and fast isoforms were only detected in myotubes negative for T antigen. These results led us to propose a model of the possible interactions between T antigen and muscle-specific factors.

Effects of growth hormone injection to embryos on growth and myosin heavy chain isoforms in growing turkeys ( Meleagris gallopavo)

Effects of embryonic imprinting with growth hormone (GH) on growth and myosin heavy chain (MyHC) isoforms in pectoralis muscle were determined by injecting turkey embryos with ovine growth hormone (oGH) at a dose of 10 μg three times a day. Injections were made on days 20 and 26 (Treatment 1), days 14 and 20 (Treatment 2) or days 14 and 26 (Treatment 3) of incubation. In Treatement 1 poults, plasma GH concentrations were elevated at 3 days posthatch and in Treatment 3 poults, plasma GH concentrations were elevated at 15 days posthatch, as compared to control poults. At 4 weeks of age, in males, body weights, shank length and weights of pectoralis, gastrocnemius and sartorius muscles were increased in Treatment 3, and in females, body weights, shank length and weights of gastrocnemius muscle of female turkeys were increased in Treatment 1. The growth rate of female turkeys from 4 weeks through 16 weeks was increased by Treatment 1. Treatment 1 resulted in a delay in the transition from the embryonic MyHC isoform to the neonatal MyHC isoform and to the adult MyHC isoform. Treatment 3 induced an earlier appearance of the adult MyHC isoform. No effects on body and muscle growth and MyHC isoforms were observed by Treatment 2.

Influence of a dextran derivative on myosin heavy chain expression during rat skeletal muscle regeneration

We recently described dextran derivatives (RGTA) which stimulate tissue repair in several in vivo models. One of them, RGTA11, has been shown to accelerate crush-induced regeneration and reinnervation of rat EDL and Soleus muscles. In this study we wanted to know if RGTA11 alters the pattern of myosin heavy chain expression during regeneration. In both EDL and Soleus muscles, RGTA11 injected at the moment of the crush, was found to accelerate the shift from neonatal to adult myosin heavy chain isoforms within 2 weeks. The proportion of slow fibers increased considerably, especially in the Soleus where RGTA11 induced a precocious and permanent expression of slow myosin isoform, thus confirming that a more efficient innervation had occurred in the presence of RGTA11. These results illustrate the interesting potential pharmacological use of such dextran derivatives in neuromuscular disease.

Expression of myosin heavy chain and of myogenic regulatory factor genes in fast or slow rabbit muscle satellite cell cultures.

We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch muscle; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous myotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.

Smooth muscle myosin heavy chain isoforms are developmentally regulated in male fetal and neonatal sheep.

Adult vascular smooth muscle expresses 204-kD (SM1) and 200-kD (SM2) myosin heavy chain (MHC) isoforms. Fetal vascular smooth muscle expresses another 200-kD isoform, MHC-B, that appears to be developmentally regulated. The ontogeny of expression of these MHC isoforms in vascular and nonvascular smooth muscles is not fully understood and may differ. In the present report we examined the ontogeny of these isoforms in aortic and bladder smooth muscle from male fetal (n = 12, 119-140-d gestation; term 145 +/- 5 d) and neonatal (n = 12, 1-33 d) sheep. Tissues were analyzed for total and soluble protein contents. Actin, MHC, and MHC isoforms were analyzed by SDS-PAGE using 3-20% and 4% polyacrylamide gels, respectively. The expression of the adult and fetal 200-kD MHC isoforms were determined by Western analysis. Between 119 d gestation and 33 d neonatal, age-dependent increases (p < 0.02) occurred in bladder actin (16 +/- 0.8 versus 22 +/- 1.4 micrograms/mg of wet weight), MHC (6.5 +/- 0.2 versus 9.7 +/- 1.1) and both soluble (71 +/- 2.9 versus 92 +/- 6.3) and total protein (78 +/- 3.9 versus 103 +/- 5.5). Aortic smooth muscle actin (8.5 +/- 0.7 versus 17 +/- 1.1), MHC (3.1 +/- 0.4 versus 5.2 +/- 0.5), and soluble (44 +/- 2.3 versus 61 +/- 3.0) and total protein (87 +/- 5.8 versus 108 +/- 3.2) also increased (p < 0.01). Aortic SM1 increased (r = 0.79, p < 0.001) during this time, whereas expression of the 200-kD MHC fell (r = -0.79, p < 0.001). In contrast, bladder SM1 fell (r = -0.88, p < 0.001) as the 200-kD MHC rose (r = 0.88, p < 0.001). The type of 200-kD MHC isoform expressed also differed between tissue types; bladder expressed SM2 and little or no MHC-B throughout this phase of development, whereas fetal aorta appeared to express primarily MHC-B, which decreased as adult SM2 expression rose after birth. Expression of smooth muscle proteins and MHC isoforms are developmentally regulated and tissue-dependent, the latter perhaps reflecting developmental differences in organ growth and/or function.