Chain Fibers Research Articles

Pulse Electropolymerization and Thermoelectrical Performances of Carbon Nanotubes/Polyaniline Composite Film

The polyaniline (PANI) films and carbon nanotubes/polyaniline (CNTs/PANI) films were obtained by the method of pulse electropolymerization on Ni substrates coated by polyaniline films in sulfuric acid system. The effects of the variables such as pulse height (φ1) and relaxation time (t2) on film morphologies and thermoelectrical performances were studied. Film morphologies were investigated using the method of scanning electron microscope (SEM) and thermoelectrical performances of these films were determined by Seebeck coefficients, resistivities, as well as power factors. Results reveal that proper pulse parameters would make composite film present uniform chain fibers and visible knot-like protrusions, which is beneficial to improve the electrical conductivity and increase the power factor. Moreover, it was found that embedding CNTs was advantageous to increase the fiber aspect ratio of PANI and further improve the thermoelectrical performances, when comparing the properties of CNTs/PANI composite films with that of pure polyaniline films synthesized at the same conditions.

Fibre typing of intrafusal fibres.

The first descriptions of muscle spindles with intrafusal fibres containing striated myofibrils and nervous elements were given approximately 150 years ago. It took, however, another 100 years to establish the presence of two types of intrafusal muscle fibres: nuclear bag and nuclear chain fibres. The present paper highlights primarily the contribution of Robert Banks in fibre typing of intrafusal fibres: the confirmation of the principle of two types of nuclear bag fibres in mammalian spindles and the variation in occurrence of a dense M-band along the fibres. Furthermore, this paper summarizes how studies from the Umeå University group (Laboratory of Muscle Biology in the Department of Integrative Medical Biology) on fibre typing and the structure and composition of M-bands have contributed to the current understanding of muscle spindle complexity in adult humans as well as to muscle spindle development and effects of ageing. The variable molecular composition of the intrafusal sarcomeres with respect to myosin heavy chains and M-band proteins gives new perspectives on the role of the intrafusal myofibrils as stretch-activated sensors influencing tension/stiffness and signalling to nuclei.

Fibre type‐specific hypertrophy mechanisms in human skeletal muscle: potential role of myonuclear addition

Fibre type‐specific hypertrophy mechanisms in human skeletal muscle: potential role of myonuclear addition

In Utero LPS Exposure Impairs Preterm Diaphragm Contractility

Preterm birth is associated with inflammation of the fetal membranes (chorioamnionitis). We aimed to establish how chorioamnionitis affects the contractile function and phenotype of the preterm diaphragm. Pregnant ewes received intra-amniotic injections of saline or 10 mg LPS, 2 days or 7 days before delivery at 121 days of gestation (term = 150 d). Diaphragm strips were dissected for the assessment of contractile function after terminal anesthesia. The inflammatory cytokine response, myosin heavy chain (MHC) fibers, proteolytic pathways, and intracellular molecular signaling were analyzed using quantitative PCR, ELISA, immunofluorescence staining, biochemical assays, and Western blotting. Diaphragm peak twitch force and maximal tetanic force were approximately 30% lower than control values in the 2-day and 7-day LPS groups. Activation of the NF-κB pathway, an inflammatory response, and increased proteasome activity were observed in the 2-day LPS group relative to the control or 7-day LPS group. No inflammatory response was evident after a 7-day LPS exposure. Seven-day LPS exposure markedly decreased p70S6K phosphorylation, but no effect on other signaling pathways was evident. The proportion of MHC IIa fibers was lower than that for control samples in the 7-day LPS group. MHC I fiber proportions did not differ between groups. These results demonstrate that intrauterine LPS impairs preterm diaphragmatic contractility after 2-day and 7-day exposures. Diaphragm dysfunction, resulting from 2-day LPS exposure, was associated with a transient activation of proinflammatory signaling, with subsequent increased atrophic gene expression and enhanced proteasome activity. Persistently impaired contractility for the 7-day LPS exposure was associated with the down-regulation of a key component of the protein synthetic signaling pathway and a reduction in the proportions of MHC IIa fibers.

Intrafusal myosin heavy chain expression of human masseter and biceps muscles at young age shows fundamental similarities but also marked differences

Muscle spindles are skeletal muscle mechanoreceptors that provide proprioceptive information to the central nervous system. The human adult masseter muscle has greater number, larger and more complex muscle spindles than the adult biceps. For a better knowledge of muscle diversity and physiological properties, this study examined the myosin heavy chain (MyHC) expression of muscle spindle intrafusal fibres in the human young masseter and young biceps muscles by using a panel of monoclonal antibodies (mAbs) against different MyHC isoforms. Eight MyHC isoforms were detected in both muscles-slow-tonic, I, IIa, IIx, foetal, embryonic, α-cardiac and an isoform not previously reported in intrafusal fibres, termed IIx'. Individual fibres co-expressed 2-6 isoforms. MyHC-slow tonic separated bag1, AS-bag1 and bag2 fibres from chain fibres. Typically, bag fibres also expressed MyHC-I and α-cardiac, whereas chain fibres expressed IIa and foetal. In the young masseter 98% of bag1 showed MyHC-α cardiac versus 30% in the young biceps, 35% of bag2 showed MyHC-IIx' versus none in biceps, 17% of the chain fibres showed MyHC-I versus 61% in the biceps. In conclusion, the result showed fundamental similarities in intrafusal MyHC expression between young masseter and biceps, but also marked differences implying muscle-specific proprioceptive control, probably related to diverse evolutionary and developmental origins. Finding of similarities in MyHC expression between young and adult masseter and biceps muscle spindles, respectively, in accordance with previously reported similarities in mATPase fibre type composition suggest early maturation of muscle spindles, preceding extrafusal fibres in growth and maturation.

Single-Nuclear DNA Instability Analyses by Means of Single-Cell Pulsed- Field Gel Electrophoresis - Technical Problems of the Comet Assay and Their Solutions for Quantitative Measurements

Throughout the past decade, the neutral and alkaline comet assays, which lyses cells with high salt in the absence of proteolysis, have been the most widely used methods to observe single- or double-strand breaks (SSB, DSB) in a single nucleus. These methods evaluate the degree of DNA damage based on the amount of granular fragments discharged from the origin, the so-called “comet tail,” but not of long chain DNA fibers. Meanwhile, a novel, single-cell pulsed-field gel electrophoresis (SCPFGE) method, which digested cells with trypsin, observed a different course of DNA fragmentation: first, a few large fibrous fragments were derived from a bundle of long chain fibers, then the cleavages were advanced until finally almost all the DNA was shredded to granular fragments. Some nuclear DNA binding components were tolerant for high salt and high alkalinity, but were degraded by trypsin digestion. A lack of trypsin digestion causes false negative results in both SCPFGE and comet assays. Also, most DNA fibers were still fixed with components, and the comet tail did not reflect a total amount of granular fragments, but rather those that released components. Repair of DSB is intrinsically difficult as compared to other DNA lesions, and the critical threshold is extremely low.DNA fibers that have already been shredded to numerous granular fragments may be irreparable as a result. Although 100 - 300 mmol/L NaOH was commonly used in the alkaline comet assay, the naked DNA fibers persisted in 10 mmol/L NaOH after trypsin digestion, but were shredded to granular fragments by 30 mmol/L. Neogenesis of the granular fragments by high pH did not clarify the mechanism of such a result; that is, it was unknown whether it was due to dissociation of hydrogen bonds, strand breaks through alkaline labile sites, artifactual DSB, or a combination of actions. Optimum conditions for the comet assay need to be defined to achieve quantitative measurements. DNA instability analyses by means SCPFGE is likely to serve as a fundamental step in single-cell genomics to determine the competence of the cell population provided for DNA amplification methods and is likely to play an important role in ensuring the safety of clinical regenerative medicine.

Using vertebral movement and intact paraspinal muscles to determine the distribution of intrafusal fiber innervation of muscle spindle afferents in the anesthetized cat

Increasing our knowledge regarding intrafusal fiber distribution and physiology of paraspinal proprioceptors may provide key insights regarding proprioceptive deficits in trunk control associated with low back pain and lead to more effective clinical intervention. The use of vertebral movement as a means to reliably stretch paraspinal muscles would greatly facilitate physiological study of paraspinal muscle proprioceptors where muscle tendon isolation is either very difficult or impossible. The effects of succinylcholine (SCh) on 194 muscle spindle afferents from lumbar longissimus or multifidus muscles in response to computer-controlled, ramp-and-hold movements of the L(6) vertebra were investigated in anesthetized cats. Paraspinal muscles were stretched by moving the L(6) vertebra 1.5-1.7mm in the dorsal-ventral direction. Initial frequency (IF), dynamic difference (DD), their changes (∆) following SCh injection (100-400μgkg(-1)), and post-SCh dynamic difference (SChDD) were measured. Muscle spindle intrafusal fiber terminations were classified as primary or secondary fibers as well as bag(1) (b(1)c), bag(2) (b(2)c), b(1)b(2)c, or chain (c) fibers. Intrafusal fiber subpopulations were distinguished using logarithmic transformation of SChDD and ∆IF distributions as established by previous investigators. Increases in DD indicate strength of b(1)c influence while increases in IF indicate strength of b(2)c influence. Out of 194 afferents, 46.9% of afferents terminated on b(2)c fibers, 46.4% on b(1)b(2)c fibers, 1% on b(1)c fibers, and 5.7% terminated on c fibers. Based on these intrafusal fiber subpopulation distributions, controlled vertebral movement can effectively substitute for direct tendon stretch and allow further investigation of paraspinal proprioceptors in this anatomically complex body region.

Single-Cell Pulsed-Field Gel Electrophoresis to Detect the Early Stage of DNA Fragmentation in Human Sperm Nuclei

Single-cell pulsed-field gel electrophoresis (SCPFGE) with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 µg/mL) and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra-cytoplasmic sperm injection (ICSI) usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection.

Actin nemaline myopathy mouse reproduces disease, suggests other actin disease phenotypes and provides cautionary note on muscle transgene expression.

Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ∼30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations.

Respiratory control and sternohyoid muscle structure and function in aged male rats: Decreased susceptibility to chronic intermittent hypoxia

Obstructive sleep apnoea syndrome (OSAS) is a common respiratory disorder characterized by chronic intermittent hypoxia (CIH). We have shown that CIH causes upper airway muscle dysfunction in the rat due to oxidative stress. Ageing is an independent risk factor for the development of OSAS perhaps due to respiratory muscle remodelling and increased susceptibility to hypoxia. We sought to examine the effects of CIH on breathing and pharyngeal dilator muscle structure and function in aged rats. Aged (18-20 months), male Wistar rats were exposed to alternating cycles of normoxia and hypoxia (90 s each; F(I)O(2)=5% O(2) at nadir) or sham treatment for 8h/day for 9 days. Following CIH exposure, breathing was assessed by whole-body plethysmography. In addition, sternohyoid muscle contractile and endurance properties were examined in vitro. Muscle fibre type and cross-sectional area, and the activity of key oxidative and glycolytic enzymes were determined. CIH had no effect on basal breathing or ventilatory responses to hypoxia or hypercapnia. CIH did not alter succinate dehydrogenase or glycerol phosphate dehydrogenase enzyme activities, myosin heavy chain fibre areal density or cross-sectional area. Sternohyoid muscle force and endurance were unaffected by CIH exposure. Since we have established that this CIH paradigm causes sternohyoid muscle weakness in adult male rats, we conclude that aged rats have decreased susceptibility to CIH-induced stress. We suggest that structural remodelling with improved hypoxic tolerance in upper airway muscles may partly compensate for impaired neural regulation of the upper airway and increased propensity for airway collapse in aged mammals.

Upregulation of PPARβ/δ Is Associated with Structural and Functional Changes in the Type I Diabetes Rat Diaphragm

BackgroundDiabetes mellitus is associated with alterations in peripheral striated muscles and cardiomyopathy. We examined diaphragmatic function and fiber composition and identified the role of peroxisome proliferator-activated receptors (PPAR α and β/δ) as a factor involved in diaphragm muscle plasticity in response to type I diabetes.Methodology/Principal FindingsStreptozotocin-treated rats were studied after 8 weeks and compared with their controls. Diaphragmatic strips were stimulated in vitro and mechanical and energetic variables were measured, cross bridge kinetics assessed, and the effects of fatigue and hypoxia evaluated. Morphometry, myosin heavy chain isoforms, PPAR α and β/δ gene and protein expression were also assessed. Diabetes induced a decrease in maximum velocity of shortening (−14%, P<0.05) associated with a decrease in myosin ATPase activity (−49%, P<0.05), and an increase in force (+20%, P<0.05) associated with an increase in the number of cross bridges (+14%, P<0.05). These modifications were in agreement with a shift towards slow myosin heavy chain fibers and were associated with an upregulation of PPARβ/δ (+314% increase in gene and +190% increase in protein expression, P<0.05). In addition, greater resistances to fatigue and hypoxia were observed in diabetic rats.Conclusions/SignificanceType I diabetes induced complex mechanical and energetic changes in the rat diaphragm and was associated with an up-regulation of PPARβ/δ that could improve resistance to fatigue and hypoxia and favour the shift towards slow myosin heavy chain isoforms.

Capillary Supply in Relation to Myosin Heavy Chain Fibre Composition of Human Intrinsic Tongue Muscles

The capillary supply and myosin heavy chain (MyHC) composition of three different intrinsic tongue muscles was analysed in the anterior and posterior regions of the human tongue with biochemical and immunohistochemical techniques. Mean capillary density for the whole tongue was 796 ± 82 cap/mm², without regional differences. The overall number of capillaries around each fibre (CAF) was higher in the posterior than in the anterior region (2.5 vs. 2.1, p = 0.009). However, correcting for regional differences in fibre size, CAF per fibre area was higher in the anterior region (4.3 vs. 3.0, p < 0.001). Muscle fibres containing fast MyHCs predominated in the anterior region (78.7%), consisting of MyHCIIa (58.5%), MyHCIIx (1.0%), MyHCIIa+MyHCIIx (11.3%) and MyHCI+MyHCIIa (7.9%). Fibres containing slow MyHC predominated in the posterior region (65.2%), consisting of MyHCI (45.5%) and MyHCI+MyHCIIa (19.7%). A minor fibre population (<2%) contained unusual MyHC isoforms, namely MyHC foetal, MyHC slow-tonic, MyHC α-cardiac or MyHC embryonic. The microvascularization of the human tongue was twice as high as in human limb muscles. Regional similarities in capillary supply, but differences in fibre phenotype composition, suggest that human tongue muscle fibres are fatigue resistant independently of MyHC content. High frequency of hybrid fibres, that is fibres co-expressing two or more MyHC isoforms, indicates a wider spectrum of fibre contractile properties than in limb muscles. In conclusion, human intrinsic tongue muscles showed internal specialization in distribution of MyHC isoforms and capillary supply, but not in the expression of unusual MyHCs.

Parapharyngeal Neurofibroma in a Pediatric patient

Parapharyngeal Neurofibroma in a Pediatric patient

Parapharyngeal Neurofibroma in a Pediatric patient

Parapharyngeal Neurofibroma in a Pediatric patient

Effects of hypothyroidism on myosin heavy chain composition and fibre types of fast skeletal muscles in a small marsupial, Antechinus flavipes

Effects of drug-induced hypothyroidism on myosin heavy chain (MyHC) content and fibre types of fast skeletal muscles were studied in a small marsupial, Antechinus flavipes. SDS-PAGE of MyHCs from the tibialis anterior and gastrocnemius revealed four isoforms, 2B, 2X, 2A and slow, in that order of decreasing abundance. After 5 weeks treatment with methimazole, the functionally fastest 2B MyHC significantly decreased, while 2X, 2A and slow MyHCs increased. Immunohistochemistry using monospecific antibodies to each of the four MyHCs revealed decreased 2b and 2x fibres, and increased 2a and hybrid fibres co-expressing two or three MyHCs. In the normally homogeneously fast superficial regions of these muscles, evenly distributed slow-staining fibres appeared, resembling the distribution of slow primary myotubes in fast muscles during development. Hybrid fibres containing 2A and slow MyHCs were virtually absent. These results are more detailed but broadly similar to the earlier studies on eutherians. We hypothesize that hypothyroidism essentially reverses the effects of thyroid hormone on MyHC gene expression of muscle fibres during myogenesis, which differ according to the developmental origin of the fibre: it induces slow MyHC expression in 2b fibres derived from fast primary myotubes, and shifts fast MyHC expression in fibres of secondary origin towards 2A, but not slow, MyHC.

Fine structural study of the innervation of muscle spindles in the internal oblique muscle of the abdominal wall in the adult mouse

We examined by electron microscopy the innervation of muscle spindles in the internal oblique muscle of the mouse abdominal wall. In the equatorial region, in addition to the sensory innervation on individual intrafusal muscle fibers, sensory cross terminals were often observed between nuclear chain fibers. In the area from the juxtaequatorial region to the polar region, nuclear bag fibers were supplied by trail and plate-type motor endings, while nuclear chain fibers were innervated by sensory endings, being probably secondary sensory endings. From these findings, it is clear that the innervation patterns differ between two types of intrafusal muscle fibers.

Determination of slow-tonic MyHC immunoreactivity is an important step in the evaluation of muscle spindles in porcine extraocular muscles

We have tested our hypothesis suggesting (i) that for the reliable determination and counting of muscle spindles (Msp) at the light microscopy level in extraocular muscles (EOM), analysis of the spindle specific myosin heavy chain (MyHC) immunoreactivity of intrafusal fibers, especially after staining with anti-slow-tonic MyHC antibodies, is the most convenient tool, (ii) that the number of Msp determined by the slow-tonic MyHC immunoreactivity of intrafusal fibers in EOM is much lower than that based on histological examination and (iii) that the previously reported numbers of Msp based on histological examination of EOM could be overestimated. In order to determine the number and distribution of Msp and to analyze the MyHC isoform immunoreactivity of intrafusal fibers in porcine EOM, paraffin sections of three 9-month-old pig medial (MR) and lateral rectus (LR), levator palpebrae (LP) and retractor bulbi (RB) muscles were stained histologically or using specific monoclonal antibodies (mAbs) against MyHC isoforms. Msp in recti and LP muscles studied by immunocytochemistry contained nuclear bag (NB) fiber(s) reacting with mAbs against slow-tonic, slow-twitch, α-cardiac and neonatal MyHCs, but not with the mAb against fast-twitch MyHC, which, on the contrary, stained nuclear chain (NC) fibers. Based on determination of spindle specific slow-tonic MyHC isoform immunoreactivity we have found 72 Msp in the MR and 68 Msp in the LR and 12 Msp in LP muscles, which was only 62, 55 and 32% of the Msp total counts according to histological examination, respectively. In the RB muscle, we have even found only 15 spindle-like-structures composed of encapsulated thin muscle fibers, which possessed only a reaction with anti-fast-twitch MyHC mAb, but lacked slow-tonic, slow-twitch or α-cardiac MyHCs immunoreactivity. Our analysis of porcine EOM confirmed the above suggestions, demonstrating, for the first time in the pig, the presence of “false Msp” mimicking encapsulated muscle fibers on histological sections that lack spindle specific MyHC immunoreactivity. In analogy with other muscles we suggest that “false Msp” are not innervated by sensory axons and therefore do not contribute to the physiological sensation of the muscle length changes. Our results thus show that the reliable identification of functionally effective Msp in EOM must involve immunohistochemical analysis of spindle specific MyHC isoforms of intrafusal fibers, as “false” spindles appearing on histologically stained sections as encapsulated muscle fibers could be regarded as “true” Msp and thus increase the spindle number counts in earlier studies.

Histochemical and immunohistochemical study on muscle fibers in human extraocular muscle spindles

Human extraocular muscles are unique in several ways including their endowment with proprioceptive organs. Aim of this study was to establish a classification of intrafusal muscle fibers of human extraocular muscles based on their histochemical and immunohistochemical properties and to determine their relationship to extrafusal extraocular muscle fiber types in this respect. Using light microscopy, intrafusal muscle fibers were followed on consecutive cross-sections and classified according to the localization of their myonuclei and to their enzyme- and myosin-immunohistochemical characteristics. Sixteen muscle spindles in human extraocular muscles counted as ‘true’ spindles revealed 27% nuclear chain fibers [40.1 μm ± 10.4; perimeter ± SD] and 73% anomalous fibers [44.1 μm ± 12]. Seven ‘false’ muscle spindles showed only anomalous fibers [43.8 μm ± 11.1] and entirely lacked nuclear chain fibers. Six fiber types were distinguished according to their histochemical and myosin heavy chain immunohistochemical properties. Fiber type 1 [46.3 μm ± 13.3] was made up of fast-twitch myosin heavy chain isoform. Fiber type 2 [39.5 μm ± 10] additionally expressed a developmental myosin heavy chain isoform. Fiber type 3 [42.8 μm ± 10.4] consisted of pure slow-twitch positive muscle fibers. Slow-twitch MHC and fast-twitch myosin heavy chain isoform were found in fiber type 4 [43.3 μm ± 9]. Fiber types 5 and 6 showed different myosin heavy chain patterns than fiber types 1–4. The vast majority of nuclear chain fibers displayed fiber type 2 features, but 12% of nuclear chain fibers were found to be of fiber type 1. Among anomalous fibers in true spindles the frequency of fiber type 1 was much higher than in false spindles. On the other hand, fiber type 4 was found more often in false than in true spindles. With regard to their histochemical and immunohistochemical properties intrafusal muscle fibers in human extraocular muscles differ both from intrafusal muscle fibers in other skeletal muscles and from extrafusal muscle fibers in extraocular eye muscles. These conspicuous differences to skeletal muscle spindles relate to their morphology and myosin heavy chain characteristics. In particular, the occurrence of anomalous fibers might reflect dynamic neuronal processes and might be necessary for modulating and adapting processes in advancing age, as well as maintaining proprioceptive input during the whole life.

MUSCLE FIBER REGENERATION IMPAIRED IN DYSTROPHIN/GUANIDINOACETATE METHYLTRANSFERASE-DEFICIENT MICE COMPARED WITH MDX MICE.

Duchenne muscular dystrophy (DMD) is characterized by mutations in a gene located on the X chromosome, which codes for the dystrophin protein. These mutations result in loss of functional dystrophin protein, making the muscle fragile and susceptible to damage. The mdx mouse is a well-studied model of human DMD because it also lacks dystrophin protein. However, the mdx phenotype is benign compared with that of human boys. In an attempt to better characterize and understand human DMD, this lab has generated a mouse model believed to be more representative of the human DMD phenotype. A putative modifier gene coding for guanidinoacetate methyltransferase or GAMT (a key enzyme involved in creatine synthesis), which is up-regulated in mdx mice and down-regulated in boys with DMD, may be important in the generation of the human phenotype. The mouse generated by this lab, the double null (dn) mouse, is lacking dystrophin and GAMT and is more crippled than the mdx mouse. Pathologically, the appearance of dn muscles is more similar to human DMD muscles as well. With immunofluorescent and histologic techniques, we find that the dn mouse has these three characteristics, all suggesting less successful regeneration: (1) the dn has 62% central nucs versus 70% in mdx, (2) the dn mouse has no embryonic myosin heavy chain (MHC) fibers versus 6% in mdx, and (3) the dn mouse has smaller fibers cross-sectional areas 3,000 square microns/fiber versus 3,500 in mdx. It seems that although its degeneration pattern is similar to that of the mdx mouse, because of its dystrophin and GAMT null state, the dn mouse is not able to regenerate muscle fibers as well as the mdx mouse. Acknowledgments: NIH NIAMS, Sharp Family Foundation, The Young Fund.

Myosin heavy chain fibre type composition in foals: analyses at the mRNA and protein level

An optimal developed musculoskeletal system is vital for the performance of the horse. Previously, we showed that in the m. gluteus medius from adult untrained horses, identical mRNA and protein expression patterns were found in the majority of fibres. However, co-expression of IIa and IId/x myosin heavy chain (MyHC) was substantially more common at the protein than at the mRNA level, suggesting a transcriptionally controlled fine-tuning of these 2 genes. To analyse the MyHC transcripts and proteins (including the cardiac alpha isoform) in the same muscle during post natal development when the muscle is adapting to movement and load. Biopsies were taken from the m. gluteus medius of 2 Dutch Warmblood foals at 0, 2, 4, 22 and 48 weeks of age. mRNA was compared to protein expression on a fibre-to-fibre basis using in situ hybridisation and immunofluorescence. The MyHC slow (I), alpha, IIa and IId/x isoforms were analysed. At all ages the expression of the mRNA and protein MyHC isoforms was almost identical. Surprisingly, coexpression of the IIad isoform was also detected at the mRNA level especially early in life. The transcript of the alpha isoform was only detectable at young age, indicating silencing of the gene around birth. During the first year of life, MyHCs are continuously adapting at the mRNA and protein level. Additionally, the regulation of hybrid fibres is different from that in adult fibres. We postulate that interfering in this process by e.g. early training will be levelled out by the maturation of the muscle.