Age-dependent variations in the expression of myosin isoforms and myogenic factors during the involution of the proximal sesamoidean ligament of sheep.

In ungulates the stability of the fetlock joint is dependent on several muscles, which are exposed to high stress and strain. Among those muscles, the proximal sesamoidean ligament or PSL (also known as the suspensory ligament or Ruini's elasto-tendinous organ) is organized at birth in layers of muscle fibres alternated with abundant tendinous tissue that, during the postnatal development, becomes the predominant tissue. In this study we analysed the PSL of the sheep at the age of 1, 30 and 180 days and determined the expression of several genes which either (a) are markers of muscle fibre growth and maturation, or (b) play a role as signal molecules. We observed an accelerated maturation, as indicated by the transition of MyHC isoform expression towards the slow isoforms and a reduced regenerative potential indicated by the low Pax7 expression and the altered Wnt signalling. We also found a specific myogenic expression pattern of MyoD, Myf5 and Myogenin in the developing PSL and high mRNA levels of specific fibrogenic factors, as TGF-ß1, that, undoubtedly, stimulate the growth of connective tissue. Our observations confirmed, at molecular level, the peculiarity of the fast involution observed in PSL a muscle that undergoes a very specific active differentiation process during early development, which implies myofibres involution and their replacement with connective tissue.

Structural study of skeletal muscle fibres in healthy and pseudomyotonia affected cattle.

Cattle congenital pseudomyotonia (PMT), recognized as naturally occurring animal model of human Brody disease, is an inherited recessive autosomal muscular disorder due to missense mutations in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca(2+)-ATPase protein, isoform 1 (SERCA1). PMT has been described in the Chianina and Romagnola italian cattle breeds and as a single case in Dutch improved Red and White cross-breed. The genetic defect turned out to be heterogeneous in different cattle breeds, even though clinical symptoms were homogeneous. Skeletal muscles of affected animals are characterized by a selective deficiency of SERCA1 in sarcoplasmic reticulum (SR) membranes. Recently, we provided evidence that in Chianina breed, the ubiquitin proteasome system is responsible for SERCA1 mutant premature disposal, even when the mutation does not affect the catalytic properties of the pump. Results presented here show that all SERCA1 mutants described until now, although expressed at low level, are correctly targeted to SR membranes. Ultrastructural studies confirm that in pathological muscle fibres, structure, as well as triads, is well preserved. All together these results suggest that a possible therapeutical approach based on the rescue of the defective protein at SR membranes could be hypothesized. Only fully functionally active missense mutants, whem located at the SR membrane could restore the efficient control of Ca(2+) homeostasis and prevent the appearance of the pathological signs. Moreover, these data demonstrate the increasing importance of domestic animals as genetic models of human pathologies.

Expression and identification of 10 sarcomeric MyHC isoforms in human skeletal muscles of different embryological origin. Diversity and similarity in mammalian species.

In the mammalian genome, among myosin heavy chain (MyHC) isoforms a family can be identified as sarcomeric based on their molecular structure which allows thick filament formation. In this study we aimed to assess the expression of the 10 sarcomeric isoforms in human skeletal muscles, adopting this species as a reference for comparison with all other mammalian species. To this aim, we set up the condition for quantitative Real Time PCR assay to detect and quantify MyHC mRNA expression in a wide variety of human muscles from somitic, presomitic and preotic origin. Specific patterns of expression of the following genes MYH1, MYH2, MYH3, MYH4, MYH6, MYH7, MYH8, MYH13, MYH14/7b and MYH15 were demonstrated in various muscle samples. On the same muscle samples which were analysed for mRNA expression, the corresponding MyHC proteins were studied with SDS PAGE and Western blot. The mRNA-protein comparison allowed the identification of 10 distinct proteins based on the electrophoretic migration rate. Three groups were formed based on the migration rate: fast migrating comprising beta/slow/1, alpha cardiac and fast 2B, slow migrating comprising fast 2X, fast 2A and two developmental isoforms (NEO and EMB), intermediate migrating comprising EO MyHC, slow B (product of MYH15), slow tonic (product of MYH14/7b). Of special interest was the demonstration of a protein band corresponding to 2B-MyHC in laryngeal muscles and the finding that all 10 isoforms are expressed in extraocular muscles. These latter muscles are the unique localization for extraocular, slow B (product of MYH15) and slow tonic (product of MYH14/7b).

Fast-twitch skeletal muscle fiber adaptation to SERCA1 deficiency in a Dutch Improved Red and White calf pseudomyotonia case.

Missense mutations in ATP2A1 gene, encoding SERCA1 protein, cause a muscle disorder designed as congenital pseudomyotonia (PMT) in Chianina and Romagnola cattle or congenital muscular dystonia1 (CMD1) in Belgian Blue cattle. Although PMT is not life-threatening, CMD1 affected calves usually die within a few weeks of age as a result of respiratory complication. We have recently described a muscular disorder in a double muscle Dutch Improved Red and White cross-breed calf. Mutation analysis revealed an ATP2A1 mutation identical to that described in CMD1, even though clinical phenotype was quite similar to that of PMT. Here, we provide evidence for a deficiency of mutated SERCA1 in PMT affected muscles of Dutch Improved Red and White calf, but not of its mRNA. The reduced expression of SERCA1 is selective and not compensated by the SERCA2 isoform. By contrast, pathological muscles are characterized by a broad distribution of mitochondrial markers in all fiber types, not related to intrinsic features of double muscle phenotype and by an increased expression of sarcolemmal calcium extrusion pump. Calcium removal mechanisms, operating in muscle fibers as compensatory response aimed at lowering excessive cytoplasmic calcium concentration caused by SERCA1 deficiency, could explain the difference in severity of clinical signs.

Inhibition of ubiquitin proteasome system rescues the defective sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) protein causing Chianina cattle pseudomyotonia.

A missense mutation in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) protein, causes Chianina cattle congenital pseudomyotonia, an exercise-induced impairment of muscle relaxation. Skeletal muscles of affected cattle are characterized by a selective reduction of SERCA1 in sarcoplasmic reticulum membranes. In this study, we provide evidence that the ubiquitin proteasome system is involved in the reduced density of mutated SERCA1. The treatment with MG132, an inhibitor of ubiquitin proteasome system, rescues the expression level and membrane localization of the SERCA1 mutant in a heterologous cellular model. Cells co-transfected with the Ca(2+)-sensitive probe aequorin show that the rescued SERCA1 mutant exhibits the same ability of wild type to maintain Ca(2+) homeostasis within cells. These data have been confirmed by those obtained ex vivo on adult skeletal muscle fibers from a biopsy from a pseudomyotonia-affected subject. Our data show that the mutation generates a protein most likely corrupted in proper folding but not in catalytic activity. Rescue of mutated SERCA1 to sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca(2+) concentration and prevent the appearance of pathological signs of cattle pseudomyotonia.

Myogenic potential of canine craniofacial satellite cells.

The skeletal fibers have different embryological origin; the extraocular and jaw-closer muscles develop from prechordal mesoderm while the limb and trunk muscles from somites. These different origins characterize also the adult muscle stem cells, known as satellite cells (SCs) and responsible for the fiber growth and regeneration. The physiological properties of presomitic SCs and their epigenetics are poorly studied despite their peculiar characteristics to preserve muscle integrity during chronic muscle degeneration. Here, we isolated SCs from canine somitic [somite-derived muscle (SDM): vastus lateralis, rectus abdominis, gluteus superficialis, biceps femoris, psoas] and presomitic [pre-somite-derived muscle (PSDM): lateral rectus, temporalis, and retractor bulbi] muscles as myogenic progenitor cells from young and old animals. In addition, SDM and PSDM-SCs were obtained also from golden retrievers affected by muscular dystrophy (GRMD). We characterized the lifespan, the myogenic potential and functions, and oxidative stress of both somitic and presomitic SCs with the aim to reveal differences with aging and between healthy and dystrophic animals. The different proliferation rate was consistent with higher telomerase activity in PSDM-SCs compared to SDM-SCs, although restricted at early passages. SDM-SCs express early (Pax7, MyoD) and late (myosin heavy chain, myogenin) myogenic markers differently from PSDM-SCs resulting in a more efficient and faster cell differentiation. Taken together, our results showed that PSDM-SCs elicit a stronger stem cell phenotype compared to SDM ones. Finally, myomiR expression profile reveals a unique epigenetic signature in GRMD SCs and miR-206, highly expressed in dystrophic SCs, seems to play a critical role in muscle degeneration. Thus, miR-206 could represent a potential target for novel therapeutic approaches.

Effects of in vivo applications of peripheral blood-derived mesenchymal stromal cells (PB-MSCs) and platlet-rich plasma (PRP) on experimentally injured deep digital flexor tendons of sheep.

Tendon injuries, degenerative tendinopathies, and overuse tendinitis are common in races horses. Novel therapies aim to restore tendon functionality by means of cell-based therapy, growth factor delivery, and tissue engineering approaches. This study examined the use of autologous mesenchymal stromal cells derived from peripheral blood (PB-MSCs), platelet-rich plasma (PRP) and a combination of both for ameliorating experimental lesions on deep digital flexor tendons (DDFT) of Bergamasca sheep. In particular, testing the combination of blood-derived MSCs and PRP in an experimental animal model represents one of the few studies exploring a putative synergistic action of these treatments. Effectiveness of treatments was evaluated at 30 and 120 days comparing clinical, ultrasonographic, and histological features together with immunohistochemical expression of collagen types 1 and 3, and cartilage oligomeric matrix protein (COMP). Significant differences were found between treated groups and their corresponding controls (placebo) regarding tendon morphology and extracellular matrix (ECM) composition. However, our results indicate that the combined use of PRP and MSCs did not produce an additive or synergistic regenerative response and highlighted the predominant effect of MSCs on tendon healing, enhanced tissue remodeling and improved structural organization.

Pseudomyotonia in Romagnola cattle caused by novel ATP2A1 mutations.

BACKGROUND: Bovine congenital pseudomyotonia (PMT) is an impairment of muscle relaxation induced by exercise preventing animals from performing rapid movements. Forms of recessively inherited PMT have been described in different cattle breeds caused by two independent mutations in ATP2A1 encoding a skeletal-muscle Ca2+-ATPase (SERCA1). We observed symptoms of congenital PMT in four related Romagnola beef cattle from Italy and evaluated SERCA1 activity and scanned ATP2A1 for possible causative mutations. RESULTS: We obtained four PMT affected Romagnola cattle and noted striking clinical similarities to the previously described PMT cases in other cattle breeds. The affected animals had a reduced SERCA1 activity in the sarcoplasmic reticulum. A single affected animal was homozygous for a novel complex variant in ATP2A1 exon 8 (c.[632 G>T; 857 G>T]). Three out of four cases were compound heterozygous for the newly identified exon 8 variant and the exon 6 variant c.491 G>A(p. Arg146Gly), which has previously been shown to cause PMT in Chianina cattle. Pedigree analysis showed that the exon 8 double mutation event dates back to at least 1978. Both nucleotide substitutions are predicted to alter the SERCA1 amino acid sequence (p.[(Gly211Val; Gly284Val)]), affect highly conserved residues, in particular the actuator domain of SERCA1. CONCLUSION: Clinical, biochemical and DNA analyses confirmed the initial hypothesis. We provide functional and genetic evidence that one novel and one previously described ATP2A1 mutation lead to a reduced SERCA1 activity in skeletal muscles and pseudomyotonia in affected Romagnola cattle. Selection against these mutations can now be used to eliminate the mutant alleles from the Romagnola breed.

Crystal structure of sarcoplasmic reticulum Ca2+-ATPase (SERCA) from bovine muscle.

The SERCA pump, a membrane protein of about 110kDa, transports two Ca(2+) ions per ATP hydrolyzed from the cytoplasm to the lumen of the sarcoplasmic reticulum. In muscle cells, its ability to remove Ca(2+) from the cytosol induces relaxation. The transport mechanism employed by the enzyme from rabbit muscle has been extensively studied, and several crystal structures representing different conformational states are available. However, no structure of the pump from other sources is known. In this paper we describe the crystal structure of the bovine enzyme, crystallized in the E1 conformation and determined at 2.9Å resolution. The overall molecular model is very similar to that of the rabbit enzyme, as expected by the high amino acid sequence identity. Nevertheless, the bovine enzyme has reduced catalytic activity with respect to the rabbit enzyme. Subtle structural modifications, in particular in the region of the long loop that protrudes into the SR lumen connecting transmembrane α-helices M7 and M8, may explain the difference.

A missense mutation in the skeletal muscle chloride channel 1 (CLCN1) as candidate causal mutation for congenital myotonia in a New Forest pony.

A 7-month-old New Forest foal presented for episodes of recumbency and stiffness with myotonic discharges on electromyography. The observed phenotype resembled congenital myotonia caused by CLCN1 mutations in goats and humans. Mutation of the CLCN1 gene was considered as possible cause and mutation analysis was performed. The affected foal was homozygous for a missense mutation (c.1775A>C, p.D592A) located in a well conserved domain of the CLCN1 gene. The mutation showed a recessive mode of inheritance within the reported pony family. Therefore, this CLCN1 polymorphism is considered to be a possible cause of congenital myotonia.

GDNF family ligand RET receptor in the brain of adult zebrafish.

RET is a tyrosine kinase receptor, and transduces signaling by family of glial cell line-derived neurotrophic factor ligands (GFLs). RET is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons. RET exists in two main isoforms originated by differential splicing, RET9 and RET51; phylogenetic studies have shown that the RET gene is conserved across vertebrates. The aim of this study was to investigate the RET expression within the brain of zebrafish, using immunohistochemistry, western blotting and RT-PCR. In homogenate brains both RET protein and mRNA were observed. RET immunoreactivity was widespread in neurons and neural processes of all the major regions of the brain. These results demonstrate the occurrence of RET and suggest an involvement of GDNF family ligands in the brain of adult zebrafish.

Pseudomyotonia, a muscle function disorder associated with an inherited ATP2A1 (SERCA1) defect in a Dutch Improved Red and White cross-breed calf.

A Dutch Improved Red and White cross-breed heifer calf was evaluated for a muscular disorder resulting in exercise induced muscle stiffness. Clinical findings included generalized exercise-induced muscle spasms with normal response to muscle percussion. Electromyography showed no myotonic discharges, thus ruling out myotonia. Whereas histological examination of muscle tissue was unremarkable, Ca(2+)-ATPase activity of sarcoplasmatic reticulum membranes (SERCA1) was markedly decreased compared to control animals. Mutation analysis revealed the presence of a missense mutation in the ATP2A1 gene encoding the SERCA1 protein (p.Arg559Cys). The present case presents similarities to human Brody's disease, but also to pseudomyotonia and congenital muscular dystonia previously described in different cattle breeds.

Cryopreservation does not affect the stem characteristics of multipotent cells isolated from equine peripheral blood.

Mammalian adult stem cells show, in vitro, extensive differentiative ability and may represent a versatile tool for tissue regenerative purposes, even after long-term storage. Multipotent stem cells isolated from horse blood have been shown to possess the capacity to differentiate into diverse mesenchymal lineages although their full characterization is still at an early stage. The aim of this study was to examine the effects of cryopreservation on stemness characteristics of adult equine mesenchymal stem cells isolated from peripheral blood (ePB-MSC). Each sample of ePB-MSC was analyzed immediately and then after being frozen in liquid nitrogen for 10-12 months. After cryopreservation, cells conserved their morphology, alkaline phosphatase positivity, telomerase activity, karyotype profile, proliferation rate, and CD expression pattern. We characterized ePB-MSC as cells expressing CD44, CD90, CD117, and CD13, but not CD34 and CD45. Finally, freezing and storing ePB-MSC did not change their adipogenic, osteogenic, and myogenic differentiative potential, as analyzed by histochemistry, immunofluorescence, and polymerase chain reaction expression analyses. Overall, our results demonstrate that cryopreservation of ePB-MSC provides a convenient tool for in vitro applications, because cryopreserved cells possess the same stem characteristics as freshly isolated cells. Moreover, the feasibility of maintaining stem cell features of ePB-MSC after long-term storage has important implications for autologous cellular-based therapy in veterinary medicine.

A defective SERCA1 protein is responsible for congenital pseudomyotonia in Chianina cattle.

Recently, a muscular disorder defined as "congenital pseudomyotonia" was described in Chianina cattle, one of the most important Italian cattle breeds for quality meat and leather. The clinical phenotype of this disease is characterized by an exercise-induced muscle contracture that prevents animals from performing muscular activities. On the basis of clinical symptoms, Chianina pseudomyotonia appeared related to human Brody's disease, a rare inherited disorder of skeletal muscle function that results from a sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) deficiency caused by a defect in the ATP2A1 gene that encodes SERCA1. SERCA1 is involved in transporting calcium from the cytosol to the lumen of the sarcoplasmic reticulum. Recently, we identified the genetic defect underlying Chianina cattle pseudomyotonia. A missense mutation in exon 6 of the ATP2A1 gene, leading to an R164H substitution in the SERCA1 protein, was found. In this study, we provide biochemical evidence for a selective deficiency in SERCA1 protein levels in sarcoplasmic reticulum membranes from affected muscles, although mRNA levels are unaffected. The reduction of SERCA1 levels accounts for the reduced Ca(2+)-ATPase activity without any significant change in Ca(2+)-dependency. The loss of SERCA1 is not compensated for by the expression of the SERCA2 isoform. We believe that Chianina cattle pseudomyotonia might, therefore, be the true counterpart of human Brody's disease, and that bovine species might be used as a suitable animal model.

Masticatory myosin unveiled: first determination of contractile parameters of muscle fibers from carnivore jaw muscles.

Masticatory myosin heavy chain (M MyHC) is a myosin subunit isoform with expression restricted to muscles derived from the first branchial arch, such as jaw-closer muscles, with pronounced interspecies variability. Only sparse information is available on the contractile properties of muscle fibers expressing M MyHC (M fibers). In this study, we characterized M fibers isolated from the jaw-closer muscles (temporalis and masseter) of two species of domestic carnivores, the cat and the dog, compared with fibers expressing slow or fast (2A, 2X, and 2B) isoforms. In each fiber, during maximally calcium-activated contractions at 12 degrees C, we determined isometric-specific tension (P(o)), unloaded shortening velocity (v(o)) with the slack test protocol, and the rate constant of tension redevelopment (K(TR)) after a fast shortening-relengthening cycle. At the end of the mechanical experiment, we identified MyHC isoform composition of each fiber with gel electrophoresis. Electrophoretic migration rate of M MyHC was similar in both species. We found that in both species the kinetic parameters v(o) and K(TR) of M fibers were similar to those of 2A fibers, whereas P(o) values were significantly greater than in any other fiber types. The similarity between 2A and M fibers and the greater tension development of M fibers were confirmed also in mechanical experiments performed at 24 degrees C. Myosin concentration was determined in single fibers and found not different in M fibers compared with slow and fast fibers, suggesting that the higher tension developed by M fibers does not find an explanation in a greater number of force generators. The specific mechanical characteristics of M fibers might be attributed to a diversity in cross-bridge kinetics.

Identification of a missense mutation in the bovine ATP2A1 gene in congenital pseudomyotonia of Chianina cattle: an animal model of human Brody disease.

Congenital pseudomyotonia in Chianina cattle is a muscle function disorder very similar to that of Brody disease in humans. Mutations in the human ATP2A1 gene, encoding SERCA1, cause Brody myopathy. The analysis of the collected Chianina pedigree data suggested monogenic autosomal recessive inheritance and revealed that all 17 affected individuals traced back to a single founder. A deficiency of SERCA1 function in skeletal muscle of pseudomyotonia affected Chianina cattle was observed as SERCA1 activity in affected animals was decreased by about 70%. Linkage analysis showed that the mutation was located in the ATP2A1 gene region on BTA25 and subsequent mutation analysis of the ATP2A1 exons revealed a perfectly associated missense mutation in exon 6 (c.491G>A) leading to a p.Arg164His substitution. Arg164 represents a functionally important and strongly conserved residue of SERCA1. This study provides a suitable large animal model for human Brody disease.

Myostatin shows a specific expression pattern in pig skeletal and extraocular muscles during pre- and post-natal growth.

Myogenesis is driven by an extraordinary array of cellular signals that follow a common expression pattern among different animal phyla. Myostatin (mstn) is a secreted growth factor that plays a pivotal role in skeletal muscle mass regulation. The aim of the present study was to investigate mstn expression in a large mammal (the pig) in order to ascertain whether distinct expression changes of this factor might be linked to the fiber-type composition of the muscle examined and/or to specific developmental stages. To assess the expression pattern of mstn in relation to myogenic proliferative (Pax7 and MyoD) and differentiative (myogenin) markers, we evaluated muscles with different myosin heavy-chain compositions sampled during pre- and post-natal development and on myogenic cells isolated from the same muscles. Skeletal muscles showed higher levels of mRNA for mstn and all other genes examined during fetal development than after birth. The wide distribution of mstn was also confirmed by immunohistochemistry experiments supporting evidence for cytoplasmic staining in early fetal periods as well as the localization in type 1 fibers at the end of the gestation period. Extraocular muscles, in contrast, did not exhibit decreasing mRNA levels for mstn or other genes even in adult samples and expressed higher levels of both mstn mRNA and protein compared with skeletal muscles. Experiments carried out on myogenic cells showed that mstn mRNA levels decreased when myoblasts entered the differentiation program and that cells isolated at early post-natal stages maintained a high level of Pax7 expression. Our results showed that mstn had a specific expression pattern whose variations depended on the muscle type examined, thus supporting the hypothesis that at birth, porcine myogenic cells continue to be influenced by hyperplastic/proliferative mechanisms.

Glial cell line-derived neurotrophic factor expression in the retina of adult zebrafish (Danio rerio).

The glial cell line-derived neurotrophic factor (GDNF) is a well-known growth factor acting on many neuronal populations of central, peripheral and autonomous nervous system. This factor was also previously detected in the retina of developing rat and chicken while no data are available for the zebrafish. In this study transcripts of GDNF mRNA were observed in adult retina extracts by RT-PCR. The presence of the GDNF protein was confirmed by SDS-PAGE and Western blotting analysis in adult retina homogenates. In situ hybridization and immunohistochemical experiments demonstrated that GDNF mRNA and protein localized in the photoreceptors, in the outer nuclear layer, in the inner plexiform layer and in the ganglion cell layer. These results showed that the expression of GDNF is not probably restricted during development but it might be involved in the physiology of adult zebrafish retina.

Fiber types in canine muscles: myosin isoform expression and functional characterization.

This study was aimed to achieve a definitive and unambiguous identification of fiber types in canine skeletal muscles and of myosin isoforms that are expressed therein. Correspondence of canine myosin isoforms with orthologs in other species as assessed by base sequence comparison was the basis for primer preparation and for expression analysis with RT-PCR. Expression was confirmed at protein level with histochemistry, immunohistochemistry, and SDS-PAGE combined together and showed that limb and trunk muscles of the dog express myosin heavy chain (MHC) type 1, 2A, and 2X isoforms and the so-called "type 2dog" fibers express the MHC-2X isoform. MHC-2A was found to be the most abundant isoform in the trunk and limb muscle. MHC-2X was expressed in most but not all muscles and more frequently in hybrid 2A-2X fibers than in pure 2X fibers. MHC-2B was restricted to specialized extraocular and laryngeal muscles, although 2B mRNA, but not 2B protein, was occasionally detected in the semimembranosus muscle. Isometric tension (P(o)) and maximum shortening velocity (V(o)) were measured in single fibers classified on the basis of their MHC isoform composition. Purified myosin isoforms were extracted from single muscle fibers and characterized by the speed (V(f)) of actin filament sliding on myosin in an in vitro motility assay. A close proportionality between V(o) and V(f) indicated that the diversity in V(o) was due to the different myosin isoform composition. V(o) increased progressively in the order 1/slow < 2A < 2X < 2B, thus confirming the identification of the myosin isoforms and providing their first functional characterization of canine muscle fibers.