Lisa Maccatrozzo

Characterization of the myostatin gene in the gilthead seabream (Sparus aurata): sequence, genomic structure, and expression pattern.

Abstract: We report on the sequence and expression analysis of the myostatin gene (MSTN) in the gilthead seabream Sparus aurata. A 2189-bp transcript was isolated, encoding an open reading frame (385 amino acids) that showed 74% to 60% protein similarity with other vertebrate myostatins. Phylogenetic analysis of MSTN and other related genes confirmed the evolutionary relationships of the isolated sequence. The complete sequences of two introns were also determined. Intron-exon boundaries were conserved when compared with those of mammalian MSTN genes, whereas intron size was smaller. Reverse transcriptase polymerase chain reaction on total RNA extracted from different tissues and developmental stages revealed MSTN expression in the skeletal muscle, but also in other tissues. The observed expression profile differed from that in mammals, suggesting possible additional functions of myostatin in the teleost fish.

Molecular evidence for genetic subdivision of Antarctic krill (Euphausia superba Dana) populations

Antarctic krill (Euphausia superba Dana) is a key species in the Antarctic food web and occurs on a circum-continental scale. Population genetic structure of this species was investigated by sequence analysis of the ND1 mitochondrial gene in four population samples collected at different geographical localities around the Antarctic continent. Results indicate the existence of significant genetic differences between samples, and we suggest that oceanographic barriers could be sufficiently strong and temporally stable to restrict gene flow between distinct areas. Moreover, our data indicate that Antarctic krill is not at mutation–drift equilibrium and that the species possibly has a low effective population size as compared to the census size.

Canine adipose-derived-mesenchymal stem cells do not lose stem features after a long-term cryopreservation.

Adult stem cells are nowadays used for treating several pathologies. A putative stem cell population was found in the adipose tissue of mammals and canine adipose tissue-derived-mesenchymal stem cells (cA-MSC) have been shown to possess the capacity to differentiate into several lineages. The main goal of our research was to fully characterize cA-MSC and examine the effects of cryopreservation on their stemness features. Each sample of cA-MSC was analyzed immediately and then again after being frozen in liquid nitrogen for one year. After the cryopreservation period cells conserved their fibroblast-like morphology, alkaline phosphatase positivity and CD expression but showed a lower proliferation ratio and a lower telomerase activity in comparison with fresh cells. Finally, the cryopreservation protocol did not change the cA-MSC adipogenic, osteogenic and myogenic differentiative potential. Our data demonstrate that stored cA-MSC might represent a promising type of progenitor cell for autologous cellular-based therapies in veterinary medicine.

Fast fibres in a large animal: fibre types, contractile properties and myosin expression in pig skeletal muscles

SUMMARY Little is known about the influence of Myosin Heavy Chain (MHC) isoforms on the contractile properties of single muscle fibres in large animals. We have studied MHC isoform composition and contractile properties of single muscle fibres from the pig. Masseter, diaphragm, longissimus, semitendinosus, rectractor bulbi and rectus lateralis were sampled in female pigs (aged 6 months, mass 160 kg). RT-PCR, histochemistry, immunohistochemistry and gel electrophoresis were combined to identify and separate four MHC isoforms: MHC-slow and three fast MHC (2A, 2X, 2B). Maximum shortening velocity (Vo) and isometric tension (Po) were measured in single muscle fibres with known MHC isoform composition. Six groups of fibres (pure: slow, 2A, 2X and 2B, and hybrid: 2A-2X and 2X-2B) with large differences in Vo and Po were identified. Slow fibres had mean Vo=0.17±0.01 length s-1 and Po=25.1±3.3 mN mm-2. For fast fibres 2A, 2X and 2B, mean Vo values were 1.86±0.18, 2.55±0.19 and 4.06±0.33 length s-1 and mean Po values 74.93±8.36, 66.85±7.58 and 32.96±7.47 mN mm-2, respectively. An in vitro motility assay confirmed that Vo strictly reflected the functional properties of the myosin isoforms. We conclude that pig muscles express high proportions of fast MHC isoforms, including MHC-2B, and that Vo values are higher than expected on the basis of the scaling relationship between contractile parameters and body size.

A novel second myostatin gene is present in teleost fish

We report on the isolation and characterisation of the complete cDNA sequence encoding a novel bone morphogenetic protein‐like protein (sbMSTN‐b) in the teleost fish Sparus aurata. The encoded protein is 68% identical to S. aurata MSTN at the amino acid level, and homologues were also found in Umbrina cirrosa and Tetraodon nigroviridis. Phylogenetic analysis suggests that the MSTN‐b gene may be present in most, perhaps all, teleost fish species. RT‐PCR on different tissues/stages indicates that MSTN‐b is expressed almost exclusively in the central nervous system, starting from late larval stages. Quantitative analyses indicate an increase of sbMSTN‐b expression in the brain associated with metamorphosis, at the same time as completion of nervous system differentiation.

Expression of eight distinct MHC isoforms in bovine striated muscles: evidence for MHC-2B presence only in extraocular muscles

SUMMARY This study aimed to analyse the expression of myosin heavy chain (MHC) isoforms in bovine muscles, with particular attention to the MHC-2B gene. Diaphragm, longissimus dorsi, masseter, several laryngeal muscles and two extraocular muscles (rectus lateralis and retractor bulbi) were sampled in adult male Bos taurus (age 18-24 months, mass 400-500 kg) and analysed by RT-PCR, gel electrophoresis and immunohistochemistry. Transcripts and proteins corresponding to eight MHC isoforms were identified: MHC-α and MHC-β/slow (or MHC-1), two developmental isoforms (MHC-embryonic and MHC-neonatal), three adult fast isoforms (MHC-2A, MHC-2X and MHC-2B) and the extraocular isoform MHC-Eo. All eight MHC isoforms were found to be co-expressed in extrinsic eye muscles, retractor bulbi and rectus lateralis, four (β/slow, 2A, 2X, neonatal) in laryngeal muscles, three (β/slow, 2A and 2X) in trunk and limb muscles and two (β/slow and α) in masseter. The expression of MHC-2B and MHC-Eo was restricted to extraocular muscles. Developmental MHC isoforms (neonatal and embryonic) were only found in specialized muscles in the larynx and in the eye. MHC-α was only found in extraocular and masseter muscle. Single fibres dissected from masseter, diaphragm and longissimus were classified into five groups (expressing, respectively, β/slow, α, slow and 2A, 2A and 2X) on the basis of MHC isoform electrophoretical separation, and their contractile properties [maximum shortening velocity (v0) and isometric tension (P0)] were determined. v0 increased progressively from slow to fast 2A and fast 2X, whereas hybrid 1-2A fibres and fibres containing MHC-α were intermediate between slow and fast 2A.

Successful recellularization of human tendon scaffolds using adipose-derived mesenchymal stem cells and collagen gel.

The major goal of regenerative medicine is to determine experimental techniques that take maximal advantage of reparative processes that occur naturally in the animal body. Injection of mesenchymal stem cells into the core of a damaged tendon represents such an approach. Decellularization of native tendons as potential targets and seeding protocols are currently under investigation. The aim of our study was to manufacture a recellularized biocompatible scaffold from cadaveric tissue for use in total or partial tendon injuries. Results showed that it was possible to introduce proliferating cells into the core of a decellularized tendon to treat the scaffold with a collagen gel. The method was effective in maintaining scaffold extracellular matrix and for expressing collagen type I and cartilage oligomeric matrix protein by injecting mesenchymal stem cells. Copyright

Characterization of the myostatin gene and a linked microsatellite marker in shi drum (Umbrina cirrosa, Sciaenidae)

Abstract We report the characterisation of the gene coding for myostatin (or growth differentiation factor 8, GDF8) in the shi drum, Umbrina cirrosa . Because of its relevant role in muscle growth regulation, GDF8 is an important candidate locus for improvement of animal production. RT-PCR and RACE protocols were used to obtain the full-length GDF8 cDNA sequence. The complete cDNA was 2086-bp long, containing an open reading frame of 376 amino acids. Similarity searches on protein databases give highest values with zebrafish GDF8 (291 identical residues over 376 compared amino acid positions), and other vertebrate GDF8s. This evidence suggests that the putative protein isolated is the GDF8 homologue in the shi drum. At the genomic level, the position and complete sequence was determined for two introns. While intron–exon boundaries were conserved compared to mammalian GDF8 genes, both introns were considerably smaller, of 342 and 735 bp, respectively. In the 3′ noncoding region of the cDNA, a microsatellite repeat (AC 29 ) was present. This repeat region was examined at the genomic level, and its size polymorphism assessed in a preliminary way, revealing four distinct alleles. This microsatellite locus might represent a useful polymorphic marker for studying phenotype/genotype association. We also examined the pattern of expression of sdGDF8 , which is wider in the shi drum compared to other vertebrates, suggesting possible additional functions of myostatin in the teleost fish. These results should be taken into account if null mutants for GDF8 in this species are to be produced, as they might have a more severe phenotype than observed in mammals.

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.

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.