Myosin heavy chain composition of muscle fibers in spinal muscular atrophy

Muscle biopsies from 20 cases of spinal muscular atrophy (SMA), mostly diagnosed as Werdnig–Hoffmann (W–H) disease, were examined for myosin heavy chain (HC) composition. The fetal, fast, and slow heavy chains were characterized in the isolated muscle myosin, and in myosin of single, chemically skinned fibers, by electrophoresis in SDS-6% polyacrylamide gels and by immunoblot techniques, using specific antibodies directed to each main type of myosin HC. The fiber distribution of myosin HC isozymes was further investigated on muscle cryostat sections by an indirect immunofluorescent technique. Fetal myosin HC was found to be expressed in a subpopulation of severely atrophic fibers, alone or together with the slow form of myosin HC. Triangulated fibers of intermediate size contained fetal and fast myosin or fast myosin alone. The hypertrophic fibers were characterized by the predominant expression of slow myosin HC; but in some of these fibers, also low amounts of HC fetal were found to be expressed. These findings are discussed in relation to developmental transitions of myosin heavy chains in human muscle.     

Expression of myosin heavy chain isoforms in Duchenne muscular dystrophy patients and carriers

The expression of MHC isoforms in the skeletal muscles of nine patients with Duchenne muscular dystrophy (DMD) (from 2.5 to 15 yr of age) and three DMD carriers was studied using different specific anti-MHC MAbs. We also analyzed muscle fiber size and fiber reactivity with acridine orange and/or with a surface antigen marker. One-quarter of all fibers of DMD patients, or less with age, were of normal size and contained only adult slow MHC. Half of the muscle fibers contained adult and developmental MHCs. Only half of these fibers were representative of an active regenerative process. MHC co-expression also altered the proportion of normal fast or slow fibers. Adult fast MHCs were expressed as unique MHC only in small and very small fibers in the oldest DMD patients. In DMD carrier muscles, the greatest alterations in MHC expression were observed in patients with the most reduced dystrophin expression. However, MHC changes in dystrophin-positive fibers were similar to those observed in dystrophin-free fibers. In conclusion, disruptions or delays in the switching of all genes coding for adult fast and slow MHC and developmental MHC coincided with dystrophin deletion and with perturbations in its expression.     

Development of muscle pathology in canine X-linked muscular dystrophy. I. Delayed postnatal maturation of affected and normal muscle as revealed by myosin isoform analysis and utrophin expression

Canine X-linked muscular dystrophy (CXMD) is genetically homologous to Duchenne muscular dystrophy and shares the severe myopathy and lethal clinical development of the human disease. We used immunohistochemistry to characterize the time course of postnatal expression of adult fast, adult slow and developmental myosin in the muscle of CXMD dogs, carriers and healthy controls. We also characterized the expression of utrophin and dystrophin. This detailed immunolocalization study confirmed that postnatal muscle maturation is delayed in normal dogs compared to other animals and humans, and is only achieved at around 60 days. In CXMD dogs major derangement of myosin expression became evident from about 15 days; there was a selective loss of fibers expressing fast myosin and persistence of developmental fibers compared to controls. In carriers, the proportion of dystrophin-deficient fibers, which mainly expressed fast myosin, decreased with age. In controls and carriers utrophin was absent from muscle fiber surfaces in 2-day-old animals but present between 15 and 30 days, to mostly disappear by 60 days. In dystrophic animals, sarcolemmal expression of utrophin was more marked and persistent. That immature neonatal muscle from control dogs normally contains sarcolemmal utrophin may have implications for the success of utrophin up-regulation therapy to correct the dystrophic phenotype. The data of this study provide important baseline information for further studies on the development and progression of pathological changes in the muscle of CXMD dogs. Received: 29 May 1998 / Revised, accepted: 17 August 1998     

Isomyosin distribution in skeletal muscles of normal and myotonic goats

Isoforms of myosin were examined in hindleg muscles isolated from normal and myotonic goats. The muscles studied were the soleus, gastrocnemius, gluteus accessorius, semitendinosus, semimembranosus, and adductor. The isomyosins were analyzed by pyrophosphate polyacrylamide gel electrophoresis. The relative proportions of slow and fast myosin isoforms present were determined by densitometric scanning of Coomassie Blue-stained gels. All muscles contained three fast myosin isoforms and either one or two slow myosin isoforms. In normal goat, the soleus and gastrocnemius, containing 70%-75% slow isomyosins, were representative of slow muscle. The semimembranosus, the semitendinosus, and the adductor, with more than 50% fast myosin, represented a predominantly fast muscle group. The gluteus accessorius, with approximately 45% fast myosin isoforms, was intermediate between the other two groups. In the myotonic goat, a consistent and significant increase in the proportion of fast isomyosins was observed for all the muscles studied. The largest change occurred in the gastrocnemius where the incremental increase in the percentage of fast isomyosins was over 30%. All the other muscles examined had incremental increases in fast isomyosin content which ranged from 16.1% to 22.0%. These results suggest that the abnormal action potential pattern of myotonia leads to a redistribution of the myosin isoforms.     

Heterogeneity of human skeletal muscle tropomyosin

Six polypeptides resolved by two-dimensional electrophoresis of homogenates from human skeletal muscle have been identified as tropomyosin by electrophoretic and immunochemical methods. The 6 proteins are consistently present in approximately the same abundance in normal biceps, deltoid, gastrocnemius, and quadriceps muscle. Analysis of samples from individuals with Becker's dystrophy, Duchenne dystrophy, limb girdle dystrophy, polymyositis, myopathy related to vitamin E deficiency, type II fiber deficiency, and from an infant with indistinct fiber type differentiation, however, showed quantitative variations in the tropomyosin pattern. Muscle with histochemically demonstrated type II fiber deficiency lacked two of the normal tropomyosin proteins and the type II myosin light chains. Muscles lacking type I myosin light chains were deficient in a different pair of tropomyosin proteins. The results suggest that normal human skeletal muscle contains one major type of tropomyosin protein (beta-tropomyosin) common to both fast and slow fibers, together with two other major proteins (alpha-tropomyosin and alpha'-tropomyosin), one of which is specific to fast fibers and the other to slow fibers. Preliminary data from the reaction of muscle homogenates with alkaline phosphatase indicate that 3 of the 6 tropomyosin polypeptides resolved by two-dimensional electrophoresis are phosphorylated forms of the alpha-, alpha'-, and beta-tropomyosins.     

Training-induced increase in myofibrillar ATPase intermediate fibers in human skeletal muscle

The response of human muscles triceps brachii and vastus lateralis to training, consisting of 18.5 miles a day of skiing with a backpack, 6 days a week for 8 weeks, has been investigated by means of histochemical fiber typing (myofibrillar ATPase) as well as immunohistochemical discrimination of slow and fast myosin (n = 6). A detraining period of 33 weeks was also studied. The percentage of type 2 fibers decreased by 6% in the triceps brachii during training, whereas the intermediate fiber type increased by 4%. No change was found in the distribution of fiber types in the vastus lateralis during training or in both muscles during detraining. The ATPase intermediate fibers also stained intermediately in the immunohistochemical stainings for slow and fast myosin. These results show that transformation of a fraction of the type 2 fibers into the intermediate type may occur as a response to endurace training.     

Effects of thyroid hormones on the biochemical specialization of human muscle fibers

The effects of thyrotoxicosis and of hypothyroidism on human muscle have been studied on single fiber preparations. In thyrotoxic muscle, the ratio between fibers showing the fast type of myofibrillar protein isoforms (fast fibers) and fibers showing the slow type (slow fibers) is increased, as is the percentage of fibers with incomplete segregation of fast and slow myosin (intermediate fibers). Furthermore, in fast fibers, the volume and, to a greater extent, the rate of Ca transport of sarcoplasmic reticulum (SR) are increased, without changes in the affinity for Ca2+ of the Ca-pump or in its sensitivity to the cyclic adenosine monophosphate (cAMP) dependent protein kinase system. These effects are completely reversed by the removal of thyroid hormones, as demonstrated by hypothyroid muscles. It is suggested that in human muscle cells thyroid hormones are critical for the expression of fast genes and for SR Ca transport.     

Biochemical and immunocytochemical analysis in chronic proximal spinal muscular atrophy

Immunocytochemical and biochemical analyses were carried out on patients affected by chronic SMA. Three groups of patients were identified. In group I, the muscle presented a fascicular atrophy; a high percentage of atrophic type II fibers; and fibers expressing fast, slow, embryonic, and fetal myosin isoforms. In group II, the muscle was characterized by atrophic fibers and normal/hypertrophic fibers expressing only slow myosin isoforms. In group III, the muscle was characterized by fiber type grouping and fibers coexpressing fast and slow myosin isoforms but never embryonic or fetal MHC isoforms. The muscles of groups I and III contained both fast and slow myosins whereas group II muscles were predominantly slow by immunocytochemical analysis or only slow by biochemical analysis. In view of these results, immunocytochemical and histochemical analyses could help to classify chronic SMA and help to understand the different pathogenic processes which seem to be related to the maturational stage of the muscle at the age of onset of the disease. © 1994 John Wiley & Sons, Inc.     

Myosin light chain components in single muscle fibers of duchenne muscular dystrophy

Single muscle fibers were prepared from biopsy specimens of Duchenne muscular dystrophy (DMD), normal, and neuromuscular disease controls. Single muscle cells were classified as type 1, type 2, or intermediate by the skinned fiber method. The intermediate fiber was most abundant in DMD, comprising 29% of fibers examined. The fiber type of single muscle fibers was contrasted to the composition of myosin light chain (MLC) components, which was analyzed by micro two-dimensional gel electrophoresis. In DMD, each of the components exhibited the same electrophoretic mobility as those in the controls. Type 1 fibers of DMD were more diverse in the composition of MLC than those of controls; 55% of type 1 fibers of DMD contained distinct fast-type MLC 3. Some intermediate fibers contained all five MLC components, but in others the composition was not different from usual type 1 or type 2 fibers. The diversity of MLC composition in DMD muscle cells might reflect the abundance of young muscle fibers in the tissue due to active muscle regeneration andsol;or retardation of maturation.     

Muscle after spinal cord injury

The morphological and contractile changes of muscles below the level of the lesion after spinal cord injury (SCI) are dramatic. In humans with SCI, a fiber‐type transformation away from type I begins 4–7 months post‐SCI and reaches a new steady state with predominantly fast glycolytic IIX fibers years after the injury. There is a progressive drop in the proportion of slow myosin heavy chain (MHC) isoform fibers and a rise in the proportion of fibers that coexpress both the fast and slow MHC isoforms. The oxidative enzymatic activity starts to decline after the first few months post‐SCI. Muscles from individuals with chronic SCI show less resistance to fatigue, and the speed‐related contractile properties change, becoming faster. These findings are also present in animals. Future studies should longitudinally examine changes in muscles from early SCI until steady state is reached in order to determine optimal training protocols for maintaining skeletal muscle after paralysis. Muscle Nerve, 2009     

Distinct contractile protein profile in congenital myotonic dystrophy and X-linked myotubular myopathy.

The contractile proteins present in muscle biopsies taken from infants suffering either from congenital myotonic dystrophy or X-linked myotubular myopathy were compared using biochemical and immunocytochemical techniques. Two-dimensional gel analysis has revealed that in all cases of X-linked myotubular myopathy the pattern of expression of myosin light chains, tropomyosin and troponin was roughly similar to that of normal age matched control muscle. However, biopsies from infants affected by congenital myotonic dystrophy demonstrated a predominance of most fast contractile protein isoforms. Non-denaturing gel electrophoresis confirmed the presence of both fast and slow myosin isoforms in X-linked myotubular myopathy. Fetal myosin was also present but in amounts higher than that found in normal muscles of the same age. In congenital myotonic dystrophy fetal and fast myosin were the predominant isoforms detected by native gel electrophoresis. These results were confirmed by immunocytochemistry and Western blot analysis using antibodies specific for the different myosin isoforms.     

Effect of denervation of neonatal rat sciatic nerve on the differentiation of myosin in a single muscle fiber

Myosin light chains in normal and neonatally denervated rat muscle were studied to examine the neural effect on the differentiation of myosin molecules. Those of fast- or slow-twitch muscle were identified by single fiber gel electrophoresis. Myosin light chains of rat soleus and extensor digitorum longus (EDL) muscles were converted to the fast type after neonatal denervation. In denervated EDL muscle, some hypertrophied intermediate fibers remained even after 30 days. Single fiber gel electrophoresis showed that slow and fast types of myosin light chains coexisted in these hypertrophied fibers.     

Deficiency of muscle alpha-actinin-3 is compatible with high muscle performance

Long-distance runners generally have a remarkably high proportion of slow type I fibers in their lower muscle groups. However, the transformation of type II fast fibers to slow type I fibers as a result of exercise has not been demonstrated clearly. We report the analysis of muscle type composition on m. vastus lateralis from six endurance athletes through the expression of fast, slow, and developmental myosin isoforms, and α-actinin-3 (ACTN3) protein. Only one among the marathon runners presented evident type I fiber predominance, and surprisingly, a second athlete showed a deficiency of ACTN3. The deficiency of ACTN3 in the muscle tissue of endurance athletes confirmed the redundancy of this protein for muscle function, even in muscles that are highly required.     

Myosin expression in hypertrophied fast twitch and slow tonic muscles of normal and dystrophic chickens.

Disruption of the development program of myosin gene expression has been reported in chicken muscular dystrophy. In the present report, the relationship between muscular dystrophy and the ability of muscle to respond to an increased work load with a transition in the myosin phenotype has been investigated. Hypertrophy of slow tonic anterior latissimus dorsi (ALD) and fast twitch patagialis (PAT) muscles was induced by overloading for 35 days and myosin expression was analyzed by electrophoresis and immunocytochemistry. Normal and dystrophic chicken ALD muscles have nearly identical proportions of SM-1 and SM-2 isomyosins and both exhibit an age-related repression of the SM-1 isomyosin which is enhanced and accelerated by overloading. Immunocytochemistry with anti-myosin heavy chain (MHC) antibodies demonstrates the appearance of nascent myofibers in overloaded ALD muscles from both normal and dystrophic chickens. A minor fast twitch fiber population is also identified which doubles in number with overloading in normal ALD muscles. There are only half as many fast twitch fibers in control dystrophic ALD muscles and this number does not increase with overloading. In contrast to ALD muscles, the isomyosin profile of normal and dystrophic PAT muscles is quite different. There is significantly more FM-3 and significantly less FM-1 isomyosin in the dystrophic PAT muscle. However, both normal and dystrophic PAT muscles exhibit an overload-induced accumulation of the FM-3 isomyosin. Immunocytochemistry reveals that, unlike the normal PAT muscle, the dystrophic PAT muscle contains a population of myofibers which express slow MHCs. As in the ALD muscle, overload-induced hypertrophy is associated with a repression of the SM-1 MHC in these fibers. Nascent myofiber formation does not occur in either normal or dystrophic overloaded PAT muscles.     

Evolution of muscle specific proteins in Werdnig-Hoffman's disease.

The pattern of expression of desmin, vimentin, titin and different myosin isoforms expressed in atrophic and hypertrophic type I and type II muscle fibers was investigated in 7 biopsies from patients of various ages all diagnosed as suffering from Werdnig-Hoffman's disease. The results revealed that there was a progressive atrophy affecting both type I and type II muscle fibers. The proportion of atrophic type II fibers increased with age. These atrophic fibers expressed predominantly fast MHC together with variable amounts of embryonic and fetal abnormal concentrations of desmin, vimentin and titin were also observed in some of these fibers. Hypertrophic type I fibers expressed exclusively slow MHC. These results are in good agreement with the hypothesis that Werdnig-Hoffman's disease is associated with a persistence of slow twitch type I motor units and a loss of phasic type II motor units. They also confirm that the atrophic fibers were frequently immature although embryonic MLC was never detected in these muscles. In addition we have demonstrated that the hypertrophic fibers were not completely normal since they frequently contained abnormal concentrations of desmin and titin at their periphery.     

Myosin light and heavy chains in muscle regenerating in absence of the nerve: Transient appearance of the embryonic light chain

We examined myosin of fast and slow skeletal rat muscles regenerating after ischemia and bupivacaine injection in denervated limbs. Four days after injury two-dimensional gel electrophoresis revealed the presence of the embryonic light chain in the myosin isolated from the portion of muscle showing a homogeneous population of new small fibers by histological examination. Two weeks after injury this subunit was absent, whereas the two light chains, LC1F and LC2F, became prominent. One month after injury the still denervated soleus muscle maintained this light chain pattern. Gel electrophoresis in native condition of the myosin and peptide mapping of electrophoretically purified heavy chains confirmed that the muscle regenerating in absence of the nerve accumulated a myosin that had the general features of a fast, not slow, myosin but contained definite differences from the former.     

Higher amount of MyHC IIX in a wrist flexor in tetraplegic compared to hemiplegic cerebral palsy

Spastic cerebral palsy can be divided into diagnostic groups by the relative severity of the arm impairment. This study investigates if hemiplegic, tetraplegic or diplegic cerebral palsy (CP) results in different patterns of myosin heavy chain (MyHC) expression in the flexor carpi ulnaris muscle from 17 young patients with CP. Using enzyme-immunohistochemistry and gel electrophoresis techniques we found a higher percentage of fibers expressing fast MyHC IIx (52%) in tetraplegic CP compared to hemiplegic patients (32%), (p<0.05). Tetraplegic CP also resulted in a lower amount of fibers expressing slow MyHC I (18%) compared to hemiplegic CP (40%), (p<0.005). The proportion of muscle fibers containing fetal MyHC was higher in tetraplegic CP compared to other groups, (p<0.005). Taken together theses results indicate that tetraplegic CP is associated with a shift from slow to fast myosins and that regenerative events are more prominent in tetraplegic CP compared with milder brain damage.     

Activity-independent neural influences on cat soleus motor unit phenotypes

The physiological and phenotypic properties of motor units in the cat soleus muscle were studied after 4 months of inactivity induced by spinal cord isolation (SI). The soleus of some SI cats were stimulated for 30 min/day during an isometric (SI-I), shortening (SI-S), or lengthening (SI-L) phase of a simulated step cycle. Mean maximum tetanic tensions were approximately 15, 26, 32, and 51% of the control in the SI, SI-S, SI-L, and SI-I groups. Mean time-to-peak tension was approximately 50% shorter than the control in all SI groups. One motor unit was glycogen-depleted in each muscle via repetitive stimulation. Eighteen physiologically slow and 9 fast motor units from the spinal cord-isolated groups consisted of fibers that contained only slow myosin heavy chain (MHC) and sarco(endo)plasmic reticulum calcium-adenotriphosphatase (SERCA) isoforms. Two motor units (physiologically fast) consisted primarily of fibers that contained both fast and slow MHC and SERCA. These data reflect a dissociation between isometric speed-related properties and MHC and SERCA isoforms following inactivity. The predominance of fibers containing both fast and slow MHC and SERCA isoforms in 2 motor units demonstrates a strong motoneuronal influence on the muscle-fiber phenotype even when the motoneurons are silent.     

A rapid and reversible muscle fiber transformation in the rat

Muscle fibers (in untreated muscles) undergo physiologic and metabolic changes in response to nearby operative procedures. To elucidate this phenomenon the following experiments in rats were conducted: (a) excision of one sternohyoid superior muscle by a midline incision of the skin and the underlying superficial cervical fascia, (b) incision of skin and fascia [as in (a)], (c) incision of the skin [as in (a)] alone, and (d) application of a single anesthesia without surgical manipulations. At 3-day intervals the muscles were removed and serial sections were incubated for myosin ATPase and with antibodies recognizing fast (FM) or slow (SM) myosin, respectively. All these procedures induced a conspicuous muscle fiber transformation from fast to slow type within 6 to 9 days. The occurrence of transformed muscle fibers displayed an undulating time course attaining nearly control values within 3 days after the initial increase. These experiments demonstrated that a muscle fiber transformation can be brought about within a short time and by a variety of indirect stimuli.     

Changes in the distribution of the components of the troponin complex in muscle fibers after cross-innervation

Monospecific antibodies to the fast and slow skeletal muscle forms of the components of the troponin complex were used to follow the changes that occur in troponin I, troponin C, and troponin T during cross-innervation in rabbit skeletal muscles. During the period of transition after either soleus muscle was innervated by a fast nerve or when extensor digitorum longus and tibialis anterior muscles were innervated with slow nerves, most of the fibers contained both fast and slow forms of the components of the troponin complex. About 15 weeks after surgery the transformation of physiologic properties was complete in the soleus. The fully transformed soleus muscle consisted of about 10% of fibers containing only the slow troponin complex; the other 90% of fibers contained only the fast troponin complex. With extensor digitorum longus and tibialis anterior muscles transformation of fiber type was complete in 22 weeks when more than 90% of the innervated fibers contained only the slow troponin complex and the remaining fibers only the fast troponin complex. The results suggest that the synthesis of the forms of the components of the troponin complex appropriate to the activity resulting from the imposed innervation was under some type of coordinated control as was the degradation of the troponin components that were replaced as a consequence of the cross-innervation.