Expression of myosin heavy chain isoforms in developing rat muscle spindles

The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein. At 17-18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19-20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to anti-slow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of myosin heavy chain isoforms in developing rat muscle spindles

Summary The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein.At 17–18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19–20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag₂ staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag₁ fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to antislow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube. At seven days after birth, the pattern of reactivity was similar to that found in the adult spindles, except for the bag₁ fiber which still expressed neonatal myosin.We show that slow tonic myosin is expressed from early development and it is a reliable marker of developing bag fibers. We suggest that muscle spindles are formed from special cell lineages of which the primary generation myotubes expressing slow tonic myosin represent the primordium of muscle spindles.     

Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers

Talmadge, Robert J., Roland R. Roy, and V. Reggie Edgerton.Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers. J. Appl.Physiol. 81(6): 2540-2546, 1996.The effects of14 days of spaceflight (SF) or hindlimb suspension (HS) (Cosmos 2044)on myosin heavy chain (MHC) isoform content of the rat soleus muscleand single muscle fibers were determined. On the basis ofelectrophoretic analyses, there was a de novo synthesis of type IIx MHCbut no change in either type I or IIa MHC isoform proportions aftereither SF or HS compared with controls. The percentage of fiberscontaining only type I MHC decreased by 26 and 23%, and the percentageof fibers with multiple MHCs increased from 6% in controls to 32% inHS and 34% in SF rats. Type IIx MHC was always found in combinationwith another MHC or combination of MHCs; i.e., no fibers contained typeIIx MHC exclusively. These data suggest that the expression of thenormal complement of MHC isoforms in the adult rat soleus muscle isdependent, in part, on normal weight bearing and that the absence ofweight bearing induces a shift toward type IIx MHC protein expression in the preexisting type I and IIa fibers of the soleus.

     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

Summary The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations.Nuclear bag₁ fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag₂ fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with antineonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag₁ fibers lacked staining for M-protein, whereas bag₂ fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag₁ fibers were less reactive and clear striations were not observed, in contrast to bag₂ and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition, (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested.Taken together, the data show that in adult rat solcus, slow tonic and neonatal myosin heavy, chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Time-dependent changes in myosin heavy chain mRNA and protein isoforms in unloaded soleus muscle of rat

Time-dependent changes in myosin heavy chain(MHC) isoform expression were investigated in rat soleus muscleunloaded by hindlimb suspension. Changes at the mRNA level weremeasured by RT-PCR and correlated with changes in the pattern of MHCprotein isoforms. Protein analyses of whole muscle revealed that MHCIdecreased after 7 days, when MHCIIa had increased, reaching a transient maximum by 15 days. Longer periods led to inductions and progressive increases of MHCIId(x) and MHCIIb. mRNA analyses of whole muscle showedthat MHCIId(x) displayed the steepest increase after 4 days andcontinued to rise until 28 days, the longest time period investigated.MHCIIb mRNA followed a similar time course, although at lower levels.MHCI mRNA, present at extremely low levels in control soleus, peakedafter 4 days, stayed elevated until 15 days, and then decayed.Immunohistochemistry of 15-day unloaded muscles revealed that MHCIwas present in muscle spindles but at low amounts also in extrafusalfibers. The slow-to-fast transitions thus seem to proceed in the orderMHCI  MHCIIa  MHCIId(x)  MHCIIb. Ourfindings indicate that MHCI is transiently upregulated in somefibers as an intermediate step during the transition from MHCI to MHCIIa.

     

Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat diaphragm muscle fibers.

The present study examined Ca(2+) sensitivity of diaphragm muscle (Dia(m)) fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that Dia(m) fibers expressing the MHC(slow) isoform have greater Ca(2+) sensitivity than fibers expressing fast MHC isoforms and that this fiber-type difference in Ca(2+) sensitivity reflects the isoform composition of the troponin (Tn) complex (TnC, TnT, and TnI). Studies were performed in single Triton-X-permeabilized Dia(m) fibers. The Ca(2+) concentration at which 50% maximal force was generated (pCa(50)) was determined for each fiber. SDS-PAGE and Western analyses were used to determine the MHC and Tn isoform composition of single fibers. The pCa(50) for Dia(m) fibers expressing MHC(slow) was significantly greater than that of fibers expressing fast MHC isoforms, and this greater Ca(2+) sensitivity was associated with expression of slow isoforms of the Tn complex. However, some Dia(m) fibers expressing MHC(slow) contained the fast TnC isoform. These results suggest that the combination of TnT, TnI, and TnC isoforms may determine Ca(2+) sensitivity in Dia(m) fibers.     

Developmentally regulated expression of vascular smooth muscle myosin heavy chain isoforms

Two types of smooth muscle myosin heavy chain (MHC) isoforms, SM1 and SM2, were recently identified to have different carboxyl termini (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). SM1 and SM2 are considered to be generated from a single gene through alternative RNA splicing. In this study we investigated expression of vascular MHC isoforms during development in rabbits at the mRNA, protein, and histological levels. In adults, all smooth muscle cells reacted with both anti-SM1 and anti-SM2 antibodies on immunofluorescence, suggesting the coexpression of SM1 and SM2 in a single cell. In fetal and perinatal rabbits, however, only anti-SM1 antibody consistently reacted with smooth muscles. Reactivity with anti-SM2 antibody was negative in the fetal and neonatal blood vessels and gradually increased during 30 days after birth. These developmental changes in SM1 and SM2 expression at the histological level coincided with mRNA expression of each MHC isoform as determined by S1 nuclease mapping, indicating that expression of SM1 and SM2 is controlled at the level of RNA splicing. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin from fetal and perinatal aortas revealed the presence of large amount of SM2. Interestingly, fetal SM2 did not cross-react with our anti-SM2 antibody on immunoblotting. We conclude that expression of SM1 and SM2 are differentially regulated during development and that a third type of MHC isoform may exist in embryonic and perinatal vascular smooth muscles.     

Expression of myosin heavy chain (MyHC) isoforms in rat intrafusal muscle fibres after neonatal deefferentation and subsequent denervation

The analysis of developing intrafusal fibres is not feasible in the absence of primary sensory axons, as neonatal denervation leads to the disintegration of muscle spindles. On the other hand, neonatal deefferentation does not arrest their differentiation and, moreover, it leads to the neomyogenesis of supernumerary intrafusal profiles. If the sciatic nerve was sectioned in 4-week-old rats deefferented at the birth, muscle spindles survived, the neomyogenesis proceeded and the denervated intrafusal fibres expressed the spindle specific slow tonic (STO) MyHC. The expression of MyHC pattern in individual fibres and the differentiation of the fibre type characteristics were, however, less obvious compared to the control or deefferented spindles. The newly formed intrafusal profiles (which differentiated from satellite cells in the absence of innervation) expressed the STO MyHC particularly when they developed in a spatial relation to nuclear bag fibres.     

Expression and function of COOH-terminal myosin heavy chain isoforms in mouse smooth muscle

Isoforms of the smooth muscle myosin motor, SM1 and SM2, differ in length at the carboxy terminal tail region. Their proportion changes with development, hormonal status and disease, but their function is unknown. We developed mice carrying the myosin heavy chain (MyHC) transgenes SM1, cMyc-tagged SM1, SM2, and V5-tagged SM2, and all transgenes corresponded to the SMa NH₂-terminal isoform. Transgene expression was targeted to smooth muscle by the smooth muscle -actin promoter. Immunoblot analysis showed substantial expression of the cMyc-tagged SM1 and V5-tagged SM2 MyHC protein in aorta and bladder and transgene mRNA was expressed in mice carrying unlabeled SM1 or SM2 transgenes. Despite significant protein expression of tagged MyHCs we found only small changes in the SM1:SM2 protein ratio. Significant changes in functional phenotype were observed in mice carrying unlabeled SM1 or SM2 transgenes. Force in aorta and bladder was increased (72 ± 14%, 92 ± 11%) in SM1 and decreased to 57 ± 1% and 80 ± 3% in SM2 transgenic mice. SM1 transgenic bladders had faster (1.8 ± 0.3 s) and SM2 slower (7.1 ± 0.5 s) rates of force redevelopment following a rapid step shortening. We hypothesize that small changes in the SM1:SM2 ratio could be amplified if they are associated with changes in thick filament assembly and underlie the altered contractility. These data provide evidence indicating an in vivo function for the COOH-terminal isoforms of smooth muscle myosin and suggest that the SM1:SM2 ratio is tightly regulated in smooth muscle tissues. myosin heavy chain; transgenic mice     

Isolation and mapping of two porcine skeletal muscle myosin heavy chain isoforms

The present paper describes the isolation and linkage mapping of two isoforms of skeletal muscle myosin heavy chain in pig. Two partial cDNAs (pAZMY4 and pAZMY7), coding for the porcine myosin heavy chain-2B and -β respectively, have been isolated from a pig skeletal muscle cDNA library. Four RFLPs were detected with the putative porcine skeletal myosin heavy chain-2B probe (pAZMY4) and one RFLP was identified with the putative myosin heavy chain-β probe (pAZMY7). Two myosin heavy chain loci were mapped by linkage analysis performed with the five RFLPs against the PiGMaP linkage consortium ResPig database: the MYH1 locus, which identifies the fast skeletal muscle myosin heavy chain gene cluster, was located at the end of the map of porcine chromosome 12, while the MYH7 locus, which identifies the myosin heavy chain-α/-β gene cluster, was assigned to the long arm of porcine chromosome 7.     

Age effect on expression of myosin heavy and light chain isoforms in suspended rat soleus muscle.

This study was designed to test the hypothesis that myosin heavy (MHC) and light chain (MLC) plasticity resulting from hindlimb suspension (HS) is an age-dependent process. By using an electrophoretic technique, the distribution of MHC and MLC isoforms was quantitatively evaluated in the soleus muscles from 3- or 12-wk-old rats after 1-3 wk of HS treatment was maintained. In normal 12- and 15-wk-old rats, the soleus muscles contained a predominance of MHCI ( approximately 94%) with small amounts of MHCIIa, but not MHCIId or MHCIIb. The suspended muscles of adult rats were characterized by the appearance of MHCIIb and MHCIId, the latter reaching approximately 6% after 3 wk of HS treatment. In contrast to changes in MHC, HS did not induce a transition in the MLC pattern in the soleus muscles from adult rats. Compared with adult rats, in juveniles HS had a much more pronounced effect on the shift toward faster MHC and MLC isoform expression. The soleus muscles of 6-wk-old rats after 3 wk of HS were composed of 37.0% MHCI, 19.1% MHCIIa, 23.7% MHCIId, and 20.2% MHCIIb. Changes in MLC isoforms consisted of an increase in MLC1f and MLC2f concomitant with a decrease in MLC2s. These results indicate the existence of a differential effect of HS on MHC and MLC transitions that appears to be age dependent. They also suggest that the suspended soleus muscles from young rats may acquire the intrinsic contractile properties that are intermediate between those in the normal soleus and typical fast-twitch skeletal muscles.     

Smooth muscle-type myosin heavy chain isoforms in bovine smooth muscle and non-muscle tissues

Summary— The distribution of smooth muscle (SM)-type myosin heavy chain isoforms in several bovine muscular and non-muscular (NM) tissues was evaluated by immunofluorescence tests using monoclonal antibodies SM-E7, reactive with 204 (SM1) and 200 (SM2) kDa isoforms, and SM-F11, specific for SM2 isoform. SM-E7 reacted equally with vascular, respiratory and intestinal SM tissues, whereas SM-F11 stained heterogeneously SM cells in the various muscular systems examined and in some peculiar tissues was unreactive (perisinusoidal cells of hepatic lobule, pulmonary interstitial cells and intestinal muscularis mucosae) or uniquely reactive (nerve cells). On the whole, our findings indicate that SM1 and SM2 isoforms are unequally distributed at the cellular level in various SM and NM tissues and support previous results obtained with tissue extracts and electrophoretic procedures.     

Influence of neonatal motor denervation on expression of myosin heavy chain isoforms in rat muscle spindles

In order to evaluate the effects of fusimotor elimination on the expression of myosin heavy chain (MHC) proteins in intrafusal fibres, we compared the muscle spindles in hind limb muscles of 3- to 6-week-old rats de-efferented at birth with those of their litter-mate controls. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal MHC isoforms, against synaptophysin, the neurofilament 68 kD subunit and laminin. We found that de-efferented intrafusal fibres differentiated, as in normal spindles, into nuclear bag and bag fibres both containing predominantly slow MHC, and nuclear chain fibres that contained fast and neonatal MHC. In both de-efferented and control intrafusal fibres the same MHCs were stained; the degree and extent of staining, however, varied. Both types of de-efferented bag fibres displayed a high content of slow tonic and slow twitch MHC along most of the fibre length, in contrast to the prominent regional variation in control bag fibres. In their encapsulated regions, the de-efferented bag fibres were more similar to each other in their reactivity to anti-fast twitch and anti-neonatal MHC antibodies than the control bag fibres. In these aspects they resembled more closely the bag fibres of newborn rats. The differences might be due to an arrest of "specialization" in the regional expression of the different MHC isoforms. Chain fibres developed MHC patterns identical to those of control spindles with all the antibodies used, even though they differentiated from the beginning in the absence of motor innervation. The structural differentiation of the capsule and sensory innervation in de-efferented muscle spindles, as shown by anti-laminin, anti-synaptophysin and anti-neurofilament staining, did not differ from the controls. We conclude, in agreement with previous studies, that the sensory innervation plays a key role in inducing and supporting the differentiation of intrafusal fibres and the specific expression of their MHC. However, we also show that motor innervation and/or muscle function seem to be necessary for the diversity in the expression and distribution of different slow and fast MHC isoforms in the bag and bag fibres.     

Influence of neonatal motor denervation on expression of myosin heavy chain isoforms in rat muscle spindles

Summary In order to evaluate the effects of fusimotor elimination on the expression of myosin heavy chain (MHC) proteins in intrafusal fibres, we compared the muscle spindles in hind limb muscles of 3- to 6-week-old rats de-efferented at birth with those of their litter-mate controls. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal MHC isoforms, against synaptophysin, the neurofilament 68 kD subunit and laminin. We found that de-efferented intrafusal fibres differentiated, as in normal spindles, into nuclear bag₁ and bag₂ fibres both containing predominantly slow MHC, and nuclear chain fibres that contained fast and neonatal MHC. In both de-efferented and control intrafusal fibres the same MHCs were stained; the degree and extent of staining, however, varied. Both types of de-efferented bag fibres displayed a high content of slow tonic and slow twitch MHC along most of the fibre length, in contrast to the prominent regional variation in control bag fibres. In their encapsulated regions, the de-efferented bag fibres were more similar to each other in their reactivity to anti-fast twitch and anti-neonatal MHC antibodies than the control bag fibres. In these aspects they resembled more closely the bag fibres of newborn rats. The differences might be due to an arrest of specialization in the regional expression of the different MHC isoforms. Chain fibres developed MHC patterns identical to those of control spindles with all the antibodies used, even though they differentiated from the beginning in the absence of motor innervation.The structural differentiation of the capsule and sensory innervation in de-efferented muscle spindles, as shown by anti-laminin, anti-synaptophysin and anti-neurofilament staining, did not differ from the controls.We conclude, in agreement with previous studies, that the sensory innervation plays a key role in inducing and supporting the differentiation of intrafusal fibres and the specific expression of their MHC. However, we also show that motor innervation and/or muscle function seem to be necessary for the diversity in the expression and distribution of different slow and fast MHC isoforms in the bag₁ and bag₂ fibres.