Protein kinase C signaling controls skeletal muscle fiber types

Slow myosin heavy chain 2 (MyHC2) gene expression in fetal avian skeletal muscle fibers is regulated by innervation and protein kinase C (PKC) activity. Fetal chick muscle fibers derived from the slow twitch medial adductor (MA) muscle express slow MyHC2 when innervated in vitro. The same pattern of slow MyHC2 regulation occurs in MA muscle fibers in which PKC activity is inhibited by staurosporine. To further test the function of PKC activity in the regulation of slow MyHC2 expression, wild-type and dominant-negative mutations of PKCalpha and PKCtheta were overexpressed in MA muscle fibers in vitro. Overexpression of wild-type PKCalpha and PKCtheta cDNAs resulted in increased PKC activities in muscle fibers and concomitant repression of slow MyHC2 expression under conditions that normally induced gene expression. Point mutations leading to single amino acid substitutions were generated in the ATP binding domains of PKCalpha and PKCtheta. Overexpression of CMVPKCalphaR368 and CMVPKCthetaR409 resulted in decreased PKC activities in transfected MA muscle fibers. Furthermore, transfection of CMVPKCalphaR368 and CMVPKCthetaR409 mutant constructs into MA muscle fibers did not repress the capacity of these fibers to express slow MyHC2 when cultured in medium containing staurosporine or when innervated. These results indicate that PKC activity represses slow MyHC2 expression and that PKC down-regulation, possibly in response to innervation, is required but not sufficient for slow MyHC2 expression.     

Immunohistochemical Analysis of the Effects of Cross-innervation of Murine Thyroarytenoid and Sternohyoid Muscles

This work uses cross-innervation of respiratory muscles of different developmental origins to probe myogenic and neurogenic mechanisms regulating their fiber types. The thyroarytenoid (TA) originates from the sixth branchial arch, whereas the sternohyoid (SH) is derived from somitic mesoderm. Immunohistochemical analysis using highly specific monoclonal antibodies to myosin heavy chain (MyHC) isoforms reveals that normal rat SH comprises slow, 2a, 2x, and 2b fibers, as in limb fast muscles, whereas the external division of the TA has only 2b/eo fibers coexpressing 2B and extraocular (EO) MyHCs. Twelve weeks after cross-innervation with the recurrent laryngeal nerve, the SH retained slow and 2a fibers, greatly increased the proportion of 2x fibers, and their 2b fibers failed to express EO MyHC. In the cross-innervated TA, the SH nerve failed to induce slow and 2A MyHC expression and failed to suppress EO MyHC expression in 2b/eo fibers. However, 2x fibers amounting to 4.2% appeared de novo in the external division of the TA. We conclude that although MyHC gene expression in these muscles can be modulated by neural activity, the patterns of response to altered innervation are largely myogenically determined, thus supporting the idea that SH and TA differ in muscle allotype. (J Histochem Cytochem 58:1057–1065, 2010)     

Chronic Low-Frequency Stimulation Transforms Cat Masticatory Muscle Fibers into Jaw-Slow Fibers

Cat masticatory muscle during regeneration expresses masticatory-specific myofibrillar proteins upon innervation by a fast muscle nerve but acquires the jaw-slow phenotype when innervated by a slow muscle nerve. Here, we test the hypothesis that chronic low-frequency stimulation simulating impulses from the slow nerve can result in masticatory-to-slow fiber–type transformation. In six cats, the temporalis muscle was continuously stimulated directly at 10 Hz for up to 12 weeks using a stimulator affixed to the skull. Stimulated muscles were analyzed by immunohistochemistry using, among others, monoclonal antibodies against masticatory-specific myosin heavy chain (MyHC), myosin binding protein-C, and tropomyosins. Under the electrodes, stimulation induced muscle regeneration, which generated slow fibers. Deep to the electrodes, at two to three weeks, two distinct populations of masticatory fibers began to express slow MyHC: 1) evenly distributed fibers that completely suppressed masticatory-specific proteins but transiently co-expressed fetal MyHCs, and 2) incompletely transformed fibers that express slow and masticatory but not fetal MyHCs. SDS-PAGE confirmed de novo expression of slow MyHC and β-tropomyosin in the stimulated muscles. We conclude that chronic low-frequency stimulation induces masticatory-to-slow fiber–type conversion. The two populations of transforming masticatory fibers may differ in their mode of activation or lineage of their myogenic cells.     

Regulation of Jaw-specific Isoforms of Myosin-binding Protein-C and Tropomyosin in Regenerating Cat Temporalis Muscle Innervated by Limb Fast and Slow Motor Nerves

Cat jaw-closing muscles are a distinct muscle allotype characterized by the expression of masticatory-specific myofibrillar proteins. Transplantation studies showed that expression of masticatory myosin heavy chain (m-MyHC) is promoted by fast motor nerves, but suppressed by slow motor nerves. We investigated whether masticatory myosin-binding protein-C (m-MBP-C) and masticatory tropomyosin (m-Tm) are similarly regulated. Temporalis muscle strips were transplanted into limb muscle beds to allow innervation by fast or slow muscle nerve during regeneration. Regenerated muscles were examined postoperatively up to 168 days by peroxidase IHC using monoclonal antibodies to m-MyHC, m-MBP-C, and m-Tm. Regenerates in both muscle beds expressed fetal and slow MyHCs, m-MyHC, m-MBP-C, and m-Tm during the first 4 weeks. Longer-term regenerates innervated by fast nerve suppressed fetal and slow MyHCs, retaining m-MyHC, m-MBP-C, and m-Tm, whereas fibers innervated by slow nerve suppressed fetal MyHCs and the three masticatory-specific proteins, induced slow MyHC, and showed immunohistochemical characteristics of jaw-slow fibers. We concluded that expression of m-MBP-C and m-Tm is coregulated by m-MyHC and that neural impulses to limb slow muscle are capable of suppressing masticatory-specific proteins and to channel gene expression along the jaw-slow phenotype unique to jaw-closing muscle. (J Histochem Cytochem 58:989–1004, 2010)     

Fiber content and myosin heavy chain composition of muscle spindles in aged human biceps brachii.

The present study investigated potential age-related changes in human muscle spindles with respect to the intrafusal fiber-type content and myosin heavy chain (MyHC) composition in biceps brachii muscle. The total number of intrafusal fibers per spindle decreased significantly with aging, due to a significant reduction in the number of nuclear chain fibers. Nuclear chain fibers in old spindles were short and some showed novel expression of MyHC alpha-cardiac. The expression of MyHC alpha-cardiac in bag1 and bag2 fibers was greatly decreased in the A region. The expression of slow MyHC was increased in nuclear bag1 fibers and that of fetal MyHC decreased in bag2 fibers whereas the patterns of distribution of the remaining MyHC isoforms were generally not affected by aging. We conclude that aging appears to have an important impact on muscle spindle composition. These changes in muscle spindle phenotype may reflect an age-related deterioration in sensory and motor innervation and are likely to have an impact in motor control in the elderly.     

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.     

Related expression of MyoD and Myf5 with myosin heavy chain isoform types in bovine adult skeletal muscles

Skeletal muscles are characterized as fast and slow muscles, according to the expression pattern of myosin heavy chain (MyHC) isoforms in the muscle fibers. To investigate the relationships between MyHC isoforms and myogenic regulatory factors (MRFs) including MyoD, Myf5, myogenin, and MRF4 in adult skeletal muscles, expressions of these MRFs in the ten muscles of three cows were analyzed by a semi-quantitative RT-PCR. The results showed that MyoD expression was significantly lower in the lingual muscles (TN), masseter (MS) and diaphragm (DP), which lack MyHC-2x (fast glycolytic) expression and abound with MyHC-slow (slow oxidative) and/or MyHC-2a (fast oxidative), than it was in the pectoralis (PP), psoas major (PM), longissimus thoracis (LT), spinnalis (SP), semitendinosus (ST), semimembranosus (SM), and biceps femoris (BF). In contrast, the Myf5 expression in TN, MS, and DP was significantly higher than in PM, LT, ST, SM, and BF. No significant difference was observed in myogenin and MRF4 expression among the muscles tested. The results suggest that MyoD and Myf5 influence the MyHC isoform expression, although the effects are not decisive in specifying the phenotypes of adult muscles.     

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.     

Expression of SERCA2a is independent of innervation in regenerating soleus muscle

The speed of contraction of a skeletal muscle largely depends on the myosin heavy chain isoforms (MyHC), whereas the relaxation is initiated and maintained by the sarcoplasmic reticulum Ca²⁺-ATPases (SERCA). The expression of the slow muscle-type myosin heavy chain I (MyHCI) is entirely dependent on innervation, but, as we show here, innervation is not required for the expression of the slow-type sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) in regenerating soleus muscles of the rat, although it can play a modulator role. Remarkably, the SERCA2a level is even higher in denervated than in innervated regenerating soleus muscles on day 7 when innervation is expected to resume. Later, the level of SERCA2a protein declines in denervated regenerated muscles but it remains expressed, whereas the corresponding mRNA level is still increasing. SERCA1 (i.e., the fast muscle-type isoform) expression shows only minor changes in denervated regenerating soleus muscles compared with innervated regenerating controls. When the soleus nerve was transected instead of the sciatic nerve, SERCA2a and MyHCI expressions were found to be even more uncoupled because the MyHCI nearly completely disappeared, whereas the SERCA2a mRNA and protein levels decreased much less. The transfection of regenerating muscles with constitutively active mutants of the Ras oncogene, known to mimic the effect of innervation on the expression of MyHCI, did not affect SERCA2a expression. These results demonstrate that the regulation of SERCA2a expression is clearly distinct from that of the slow myosin in the regenerating soleus muscle and that SERCA2a expression is modulated by neuronal activity but is not entirely dependent on it. slow type sarcoplasmic reticulum Ca²⁺ pump; MyHCI; nerve influence     

Irx4 forms an inhibitory complex with the vitamin D and retinoic X receptors to regulate cardiac chamber-specific slow MyHC3 expression

The slow myosin heavy chain 3 gene (slow MyHC3) is restricted in its expression to the atrial chambers of the heart. Understanding its regulation provides a basis for determination of the mechanisms controlling chamber-specific gene expression in heart development. The observed chamber distribution results from repression of slow MyHC3 gene expression in the ventricles. A binding site, the vitamin D response element (VDRE), for a heterodimer of vitamin D receptor (VDR) and retinoic X receptor alpha (RXR alpha) within the slow MyHC3 promoter mediates chamber-specific expression of the gene. Irx4, an Iroquois family homeobox gene whose expression is restricted to the ventricular chambers at all stages of development, inhibits AMHC1, the chick homolog of quail slow MyHC3, gene expression within developing ventricles. Repression of the slow MyHC3 gene in ventricular cardiomyocytes by Irx4 requires the VDRE. Unlike VDR and RXR alpha, Irx4 does not bind directly to the VDRE. Instead two-hybrid and co-immunoprecipitation assays show that Irx4 interacts with the RXR alpha component of the VDR/RXR alpha heterodimer and that the amino terminus of the Irx4 protein is required for its inhibitory action. These observations indicate that the mechanism of atrial chamber-specific expression requires the formation of an inhibitory protein complex composed of VDR, RXR alpha, and Irx4 that binds at the VDRE inhibiting slow MyHC3 expression in the ventricles.     

Arginine promotes skeletal muscle fiber type transformation from fast-twitch to slow-twitch via Sirt1/AMPK pathway

This study investigated the effect of arginine on skeletal muscle fiber type transformation in mice and in C2C12 myotubes. Our data showed that dietary supplementation of arginine in mice significantly up-regulated the slow myosin heavy chain (MyHC), troponin I-SS, sirtuin1 (Sirt1) and peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) protein expressions, as well as significantly down-regulated the fast MyHC protein expression. In C2C12 myotubes, arginine significantly increased the protein level of slow MyHC and the number of slow MyHC-positive cells, as well as significantly decreased the protein level of fast MyHC and the number of fast MyHC-positive cells. We also showed that arginine increased the activities of succinic dehydrogenase and malate dehydrogenase and decreased the activity of lactate dehydrogenase in mice and in C2C12 myotubes. Here we found that AMP-activated protein kinase (AMPK) was activated by arginine in mice and in C2C12 myotubes. However, inhibition of AMPK activity by compound C significantly attenuated the effects of arginine on slow MyHC and fast MyHC expressions in C2C12 myotubes. Finally, we showed that inhibition of Sirt1 expression by EX527 attenuated arginine-induced increase in the protein levels of phospho-AMPK and slow MyHC, the mRNA level of nitric oxide synthase (NOS) and the contents of NOS and NO, as well as decrease in fast MyHC protein level. Together, our findings indicated that arginine promotes skeletal muscle fiber type switching from fast-twitch to slow-twitch via Sirt1/AMPK pathway.     

Evidence for myoblast-extrinsic regulation of slow myosin heavy chain expression during muscle fiber formation in embryonic development

Vertebrate muscles are composed of an array of diverse fast and slow fiber types with different contractile properties. Differences among fibers in fast and slow MyHC expression could be due to extrinsic factors that act on the differentiated myofibers. Alternatively, the mononucleate myoblasts that fuse to form multinucleated muscle fibers could differ intrinsically due to lineage. To distinguish between these possibilities, we determined whether the changes in proportion of slow fibers were attributable to inherent differences in myoblasts. The proportion of fibers expressing slow myosin heavy chain (MyHC) was found to change markedly with time during embryonic and fetal human limb development. During the first trimester, a maximum of 75% of fibers expressed slow MyHC. Thereafter, new fibers formed which did not express this MyHC, so that the proportion of fibers expressing slow MyHC dropped to approximately 3% of the total by midgestation. Several weeks later, a subset of the new fibers began to express slow MyHC and from week 30 of gestation through adulthood, approximately 50% of fibers were slow. However, each myoblast clone (n = 2,119) derived from muscle tissues at six stages of human development (weeks 7, 9, 16, and 22 of gestation, 2 mo after birth and adult) expressed slow MyHC upon differentiation. We conclude from these results that the control of slow MyHC expression in vivo during muscle fiber formation in embryonic development is largely extrinsic to the myoblast. By contrast, human myoblast clones from the same samples differed in their expression of embryonic and neonatal MyHCs, in agreement with studies in other species, and this difference was shown to be stably heritable. Even after 25 population doublings in tissue culture, embryonic stage myoblasts did not give rise to myoblasts capable of expressing MyHCs typical of neonatal stages, indicating that stage-specific differences are not under the control of a division dependent mechanism, or intrinsic "clock." Taken together, these results suggest that, unlike embryonic and neonatal MyHCs, the expression of slow MyHC in vivo at different developmental stages during gestation is not the result of commitment to a distinct myoblast lineage, but is largely determined by the environment.     

The Effect of Passive Movement on Denervated Soleus Highlights a Differential Nerve Control on SERCA and MyHC Isoforms

The sarco-endoplasmic reticulum Ca²⁺ ATP-ase (SERCA) and myosin heavy chain (MyHC) levels were measured in hindlimb-denervated and selectively denervated rat soleus muscles. Selective denervation allowed passive movement of the soleus, whereas hindlimb denervation rendered it to passivity. To minimize chronic effects, we followed the changes only for 2 weeks. Selective denervation resulted in less muscle atrophy, a faster slow-to-fast transition of MyHC isoforms, and less coordinated expressions of the slow vs fast isoforms of MyHC and SERCA. Generally, expression of the slow-twitch type SERCA2a was found to be less dependent, whereas the slow-twitch type MyHC1 was the most dependent on innervation. Our study shows that passive movement is able to ameliorate denervation-induced atrophy of the soleus and that it also accentuates the dyscoordination in the expression of the corresponding slow and fast isoforms of MyHC and SERCA. (J Histochem Cytochem 56:1013–1022, 2008)     

Muscle type-specific myosin isoforms in crustacean muscles

Differential expression of multiple myosin heavy chain (MyHC) genes largely determines the diversity of critical physiological, histochemical, and enzymatic properties characteristic of skeletal muscle. Hypotheses to explain myofiber diversity range from intrinsic control of expression based on myoblast lineage to extrinsic control by innervation, hormones, and usage. The unique innervation and specialized function of crayfish (Procambarus clarkii) appendicular and abdominal musculature provide a model to test these hypotheses. The leg opener and superficial abdominal extensor muscles are innervated by tonic excitatory motoneurons. High resolution SDS-PAGE revealed that these two muscles express the same MyHC profile. In contrast, the deep abdominal extensor muscles, innervated by phasic motoneurons, express MyHC profiles different from the tonic profiles. The claw closer muscles are dually innervated by tonic and phasic motoneurons and a mixed phenotype was observed, albeit biased toward the phasic profile seen in the closer muscle. These results indicate that multiple MyHC isoforms are present in the crayfish and that differential expression is associated with diversity of muscle type and function.     

Synapse elimination by fiber type and maturational state in rabbit soleus muscle

We have compared the development of fast and slow motor innervation in the neonatal rabbit soleus, a muscle which contains two distinct motor unit types during the early period of polyneuronal innervation. The innervation state of individual muscle fibers was ascertained using an intracellular electrode; a fluorescent dye was then injected into particular fibers to permit subsequent identification of histochemical type. We found no significant difference in the time course of synapse elimination for fast and slow motor units as judged by the percentage of fibers remaining polyneuronally innervated at two ages: 7-8 days, when most fibers are multiply innervated, and 10-11 days, when the level of polyinnervation is low. In a second experiment, we examined a phenomenon in which compound end-plate potentials were occasionally seen in muscle fibers at an age (17-23 days) well past the major episode of synapse elimination. We present evidence that this apparent polyinnervation in fact derives from an electrode-induced electrical coupling artifact and that genuinely polyinnervated fibers are very rare at this stage, if present at all.