A Nonsense Variant in COL6A1 in Landseer Dogs with Muscular Dystrophy.

A novel canine muscular dystrophy in Landseer dogs was observed. We had access to five affected dogs from two litters. The clinical signs started at a few weeks of age and the severe progressive muscle weakness led to euthanasia between 5 and 15 months of age. The pedigrees of the affected dogs suggested a monogenic autosomal recessive inheritance of the trait. Linkage and homozygosity mapping indicated two potential genome segments for the causative variant on chromosomes 10 and 31 harboring a total of 4.8 Mb of DNA or 0.2% of the canine genome. Using the illumina sequencing technology we obtained a whole genome sequence from one affected Landseer. Variants were called with respect to the dog reference genome and compared to the genetic variants of 170 control dogs from other breeds. The affected Landseer dog was homozygous for a single private non-synonymous variant in the critical intervals, a nonsense variant in the COL6A1 gene (Chr31:39,303,964G>T; COL6A1:c.289G>T; p.E97*). Genotypes at this variant showed perfect concordance with the muscular dystrophy phenotype in all five cases and more than one thousand control dogs. Variants in the human COL6A1 gene cause Bethlem myopathy or Ullrich congenital muscular dystrophy. We therefore conclude that the identified canine COL6A1 variant is most likely causative for the observed muscular dystrophy in Landseer dogs. Based on the nature of the genetic variant in Landseer dogs and their severe clinical phenotype these dogs represent a model for human Ullrich congenital muscular dystrophy.

Dystrophic cardiomyopathy: role of TRPV2 channels in stretch-induced cell damage

Aims Duchenne muscular dystrophy (DMD), a degenerative pathology of skeletal muscle, also induces cardiac failure and arrhythmias due to a mutation leading to the lack of the protein dystrophin. In cardiac cells, the subsarcolemmal localization of dystrophin is thought to protect the membrane from mechanical stress. The absence of dystrophin results in an elevated stress-induced Ca2+ influx due to the inadequate functioning of several proteins, such as stretch-activated channels (SACs). Our aim was to investigate whether transient receptor potential vanilloid channels type 2 (TRPV2) form subunits of the dysregulated SACs in cardiac dystrophy. Methods and results We defined the role of TRPV2 channels in the abnormal Ca2+ influx of cardiomyocytes isolated from dystrophic mdx mice, an established animal model for DMD. In dystrophic cells, western blotting showed that TRPV2 was two-fold overexpressed. While normally localized intracellularly, in myocytes from mdx mice TRPV2 channels were translocated to the sarcolemma and were prominent along the T-tubules, as indicated by immunocytochemistry. Membrane localization was confirmed by biotinylation assays. Furthermore, in mdx myocytes pharmacological modulators suggested an abnormal activity of TRPV2, which has a unique pharmacological profile among TRP channels. Confocal imaging showed that these compounds protected the cells from stress-induced abnormal Ca2+ signals. The involvement of TRPV2 in these signals was confirmed by specific pore-blocking antibodies and by small-interfering RNA ablation of TRPV2. Conclusion Together, these results establish the involvement of TRPV2 in a stretch-activated calcium influx pathway in dystrophic cardiomyopathy, contributing to the defective cellular Ca2+ handling in this disease.

Elucidation of the gas-phase structure of a sugar-modified DNA analogue

Antisense oligonucleotides are medical agents for the treatment of genetic diseases that are designed to interact specifically with mRNA. This interaction either induces enzymatic degradation of the targeted RNA or modifies processing pathways, e.g. by inducing alternative splicing of the pre-mRNA. The latter mechanism applies to the treatment of Duchenne muscular dystrophy with a sugar-modified DNA analogue called tricyclo-DNA (tcDNA). In tcDNA the ribose sugar-moiety is extended to a three-membered ring system, which augments the binding affinity and the selectivity of the antisense oligonucleotide for its target. The advent of chemically modified nucleic acids for antisense therapy presents a challenge to diagnostic tools, which must be able to cope with a variety of structural analogues. Mass spectrometry meets this demand for non-enzyme based sequencing methods ideally, because the technique is largely unaffected by structural modifications of the analyte. Sequence coverage of a fully modified tcDNA 15mer can be obtained in a single tandem mass spectrometric experiment. Beyond sequencing experiments, tandem mass spectrometry was applied to elucidate the gas-phase structure and stability of tcDNA:DNA and tcDNA:RNA hybrid duplexes. Most remarkable is the formation of truncated duplexes upon collision-induced dissociation of these structures. Our data suggest that the cleavage site within the duplex is directed by the modified sugar-moiety. Moreover, the formation of truncated duplexes manifests the exceptional stability of the hybrid duplexes in the gas-phase. This stability arises from the modified sugar-moiety, which locks the tcDNA single strand into a conformation that is similar to RNA in A-form duplexes. The conformational particularity of tcDNA in the gas-phase was confirmed by ion mobility-mass spectrometry experiments on tcDNA, DNA, and RNA.

Gas-phase fragmentation and conformation of the sugar-modified antisense oligonucleotide tcDNA

Tricyclo-DNA (tcDNA) is a sugar- and backbone-modified analogue of DNA that is currently tested as antisense oligonucleotide for the treatment of Duchenne muscular dystrophy. The name tricyclo-DNA is derived from the modified sugar-moiety: the deoxyribose is extended to a three-membered ring system. This modification is designed to limit the flexibility of the structure, thus giving rise to entropically stabilized hybrid duplexes formed between tcDNA and complementary DNA or RNA oligonucleotides. While the structural modifications increase the biostability of the therapeutic agent, they also render the oligonucleotide inaccessible to enzyme-based sequencing methods. Tandem mass spectrometry constitutes an alternative sequencing technique for partially and fully modified oligonucleotides. For reliable sequencing, the fragmentation mechanism of the structure in question must be understood. Therefore, the presented work evaluates the effect of the modified sugar-moiety on the gas-phase dissociation of single stranded tcDNA. Moreover, our experiments reflect the exceptional gas-phase stability of hybrid duplexes that is most noticeable in the formation of truncated duplex ions upon collision-induced dissociation. The stability of the duplex arises from the modified sugar-moiety, as the rigid structure of the tcDNA single strand minimizes the change of the entropy for the annealing. Moreover, the tc-modification gives rise to extended conformations of the nucleic acids in the gas-phase, which was studied by ion mobility spectrometry-mass spectrometry.

Central and peripheral defects in motor units of the diaphragm of spinal muscular atrophy mice.

Spinal muscular atrophy (SMA) is characterized by motoneuron loss and muscle weakness. However, the structural and functional deficits that lead to the impairment of the neuromuscular system remain poorly defined. By electron microscopy, we previously found that neuromuscular junctions (NMJs) and muscle fibres of the diaphragm are among the earliest affected structures in the severe mouse SMA model. Because of certain anatomical features, i.e. its thinness and its innervation from the cervical segments of the spinal cord, the diaphragm is particularly suitable to characterize both central and peripheral events. Here we show by immunohistochemistry that, at postnatal day 3, the cervical motoneurons of SMA mice receive less stimulatory synaptic inputs. Moreover, their mitochondria become less elongated which might represent an early stage of degeneration. The NMJs of the diaphragm of SMA mice show a loss of synaptic vesicles and active zones. Moreover, the partly innervated endplates lack S100 positive perisynaptic Schwann cells (PSCs). We also demonstrate the feasibility of comparing the proteomic composition between diaphragm regions enriched and poor in NMJs. By this approach we have identified two proteins that are significantly upregulated only in the NMJ-specific regions of SMA mice. These are apoptosis inducing factor 1 (AIFM1), a mitochondrial flavoprotein that initiates apoptosis in a caspase-independent pathway, and four and a half Lim domain protein 1 (FHL1), a regulator of skeletal muscle mass that has been implicated in several myopathies.

STUDIES INTO THE MOLECULAR MECHANISMS REGULATING MUSCULAR ATROPHY RESULTING FROM HINDLIMB IMMOBILIZATION IN THE RAT (PROTEIN, SYNTHESIS, RNA)

The purpose of the work performed in this dissertation was to examine some of the possible regulatory mechanisms involved in the initiation of muscular atrophy during periods of decreased muscle utilization resulting from hindlimb immobilization in the rat. A 37% decrease in the rate of total muscle protein synthesis which has been observed to occur in the first 6 h of immobilization contributes significantly to the observed loss of protein during immobilization.^ The rates of cytochrome c and actin synthesis were determined in adult rat red vastus lateralis and gastrocnemius muscles, respectively, by the constant infusion and incorporation of ('3)H-tyrosine into protein. The fractional synthesis rates of both actin and cytochrome c were significantly decreased (P < 0.05) in the 6th h of hindlimb immobilization.^ RHA was extracted from adult rat gastrocnemius muscle by modification of the phenol: chloroform: SDS extraction procedures commonly used for preparation of RNA for hybridization analysis from other mammalian tissues. RNA content of rat gastrocnemius muscle, as determined by this method of extraction and its subsequent quantification by UV absorbance and orcinol assay, was significantly greater than the RNA content previously determined for adult rat gastrocnemius by other commonly employed methods.^ RNA extracted by this method from gastrocnemius muscles of control and 6h immobilized rats was subjected to "dot blot" hybridization to ('32)P-labelled probe from plasmid p749, containing a cDNA sequence complementary to (alpha)-actin mRNA and from rat skeletal muscle. (alpha)-Actin specific mRNA content as estimated by this procedure is not significantly decreased in rat gastrocnemius following 6h or hindlimb immobilization. However, (alpha)-actin specific mRNA content is significantly decreased (P < 0.05) in adult rat gastrocnemius (alpha)-actin specific mRNA is not decreased in adult rat gastrocnemius muscle following 6h of immobilization, a time when actin synthesis is significantly decreased, it is concluded that a change in (alpha)-actin specific mRNA content is not the initiating event responsible for the early decrease in actin synthesis observed in the 6th h of immobilization. ^

Efectos fisiológicos sobre la aptitud muscular y aeróbica de un programa de ejercicio en gimnasio

El objetivo del presente trabajo es estudiar los efectos que produce un programa de ejercicio de fuerza resistencia, sobre el VO2MAX., en una población que concurre a un gimnasio de la ciudad de La Plata. La intervención se inicia realizando evaluaciones que permitieran estimar el VO2 máx y el 1MR de algunos ejercicios para valorar de fuerza. Esta investigación es de carácter exploratorio y descriptivo, teniendo en cuenta cumple con las características de una muestra no probabilística, por su forma de selección y su población de estudio. Dentro de las variables estudiadas se cuenta con, una variable dependiente que es el consumo de Oxigeno máximo (VO2 máx), que es la central del estudio, y el plan de entrenamiento que la variable independiente, estas dos variables son de carácter cuantitativo. El desarrollo de esta investigación demuestra que la intervención realizada durante estos dos meses, modifico la variable central analizada, (el VO2máx.), lo cual se comprueba al comparar los resultados del proceso con la primera evaluación, dichos valores fueron mejorados en distintos porcentajes. la intervención se realiza solo en el área de fuerza, y se recomienda a la muestra que continúe con su vida como lo venía haciendo para influir lo menos posible en los resultados

Efectos de un programa de flexibilidad en el desarrollo de la fuerza muscular en jugadoras de fútbol femenil

Se analizó el impacto de un programa de entrenamiento de flexibilidad sobre el desarrollo de la fuerza muscular en 16 jugadores de futbol con edad de 19.032.7 años. Se entrenó durante 30 días y 5 veces por semanas, donde el grupo "A" realizó entrenamiento de flexibilidad, mientras que "B" el entrenamiento regular. Se midió la flexibilidad, 1RM, salto vertical, peso, talla, circunferencia de pantorrilla y muslo. Los resultados muestran valores para A y B respectivamente, donde el IGF fue de 91.01 18.3 y 111.93 23.5; 78.22 29, y 79.03 29.1. La circunferencia femoral, 48.04 3.6 cms y 49.54 3.4 cms.; 47.56 4.9 y 47.89 5.2. Circunferencia de pantorrilla, 33.83 2.7 cm y 35.21 2.4 cm; 33.83 2 y 33.73 2.8. Fuerza 48.13 7.8 Kg. y 53.38 8.2 Kg.; 52.63 8.6 Kg. y 53.39 9.1 Kg. Potencia anaeróbica, 34.13 2.9 cm. y 36.63 1.7 cm; 38.25 4.7 y 37.06 3.4. Como conclusión se tiene que el uso la flexibilidad impacta de forma positiva en el IGF y por tanto en el desarrollo favorable muscular de jugadoras de fútbol.

Efectos fisiológicos sobre la aptitud muscular y aeróbica de un programa de ejercicio en gimnasio

El objetivo del presente trabajo es estudiar los efectos que produce un programa de ejercicio de fuerza resistencia, sobre el VO2MAX., en una población que concurre a un gimnasio de la ciudad de La Plata. La intervención se inicia realizando evaluaciones que permitieran estimar el VO2 máx y el 1MR de algunos ejercicios para valorar de fuerza. Esta investigación es de carácter exploratorio y descriptivo, teniendo en cuenta cumple con las características de una muestra no probabilística, por su forma de selección y su población de estudio. Dentro de las variables estudiadas se cuenta con, una variable dependiente que es el consumo de Oxigeno máximo (VO2 máx), que es la central del estudio, y el plan de entrenamiento que la variable independiente, estas dos variables son de carácter cuantitativo. El desarrollo de esta investigación demuestra que la intervención realizada durante estos dos meses, modifico la variable central analizada, (el VO2máx.), lo cual se comprueba al comparar los resultados del proceso con la primera evaluación, dichos valores fueron mejorados en distintos porcentajes. la intervención se realiza solo en el área de fuerza, y se recomienda a la muestra que continúe con su vida como lo venía haciendo para influir lo menos posible en los resultados

Efectos de un programa de flexibilidad en el desarrollo de la fuerza muscular en jugadoras de fútbol femenil

Se analizó el impacto de un programa de entrenamiento de flexibilidad sobre el desarrollo de la fuerza muscular en 16 jugadores de futbol con edad de 19.032.7 años. Se entrenó durante 30 días y 5 veces por semanas, donde el grupo "A" realizó entrenamiento de flexibilidad, mientras que "B" el entrenamiento regular. Se midió la flexibilidad, 1RM, salto vertical, peso, talla, circunferencia de pantorrilla y muslo. Los resultados muestran valores para A y B respectivamente, donde el IGF fue de 91.01 18.3 y 111.93 23.5; 78.22 29, y 79.03 29.1. La circunferencia femoral, 48.04 3.6 cms y 49.54 3.4 cms.; 47.56 4.9 y 47.89 5.2. Circunferencia de pantorrilla, 33.83 2.7 cm y 35.21 2.4 cm; 33.83 2 y 33.73 2.8. Fuerza 48.13 7.8 Kg. y 53.38 8.2 Kg.; 52.63 8.6 Kg. y 53.39 9.1 Kg. Potencia anaeróbica, 34.13 2.9 cm. y 36.63 1.7 cm; 38.25 4.7 y 37.06 3.4. Como conclusión se tiene que el uso la flexibilidad impacta de forma positiva en el IGF y por tanto en el desarrollo favorable muscular de jugadoras de fútbol.

Efectos de un programa de flexibilidad en el desarrollo de la fuerza muscular en jugadoras de fútbol femenil

Se analizó el impacto de un programa de entrenamiento de flexibilidad sobre el desarrollo de la fuerza muscular en 16 jugadores de futbol con edad de 19.032.7 años. Se entrenó durante 30 días y 5 veces por semanas, donde el grupo "A" realizó entrenamiento de flexibilidad, mientras que "B" el entrenamiento regular. Se midió la flexibilidad, 1RM, salto vertical, peso, talla, circunferencia de pantorrilla y muslo. Los resultados muestran valores para A y B respectivamente, donde el IGF fue de 91.01 18.3 y 111.93 23.5; 78.22 29, y 79.03 29.1. La circunferencia femoral, 48.04 3.6 cms y 49.54 3.4 cms.; 47.56 4.9 y 47.89 5.2. Circunferencia de pantorrilla, 33.83 2.7 cm y 35.21 2.4 cm; 33.83 2 y 33.73 2.8. Fuerza 48.13 7.8 Kg. y 53.38 8.2 Kg.; 52.63 8.6 Kg. y 53.39 9.1 Kg. Potencia anaeróbica, 34.13 2.9 cm. y 36.63 1.7 cm; 38.25 4.7 y 37.06 3.4. Como conclusión se tiene que el uso la flexibilidad impacta de forma positiva en el IGF y por tanto en el desarrollo favorable muscular de jugadoras de fútbol.

Gene Expression Profiling in Limb-Girdle Muscular Dystrophy 2A

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in calpain 3 (CAPN3). Calpain 3 plays different roles in muscular cells, but little is known about its functions or in vivo substrates. The aim of this study was to identify the genes showing an altered expression in LGMD2A patients and the possible pathways they are implicated in. Ten muscle samples from LGMD2A patients with in which molecular diagnosis was ascertained were investigated using array technology to analyze gene expression profiling as compared to ten normal muscle samples. Upregulated genes were mostly those related to extracellular matrix (different collagens), cell adhesion (fibronectin), muscle development (myosins and melusin) and signal transduction. It is therefore suggested that different proteins located or participating in the costameric region are implicated in processes regulated by calpain 3 during skeletal muscle development. Genes participating in the ubiquitin proteasome degradation pathway were found to be deregulated in LGMD2A patients, suggesting that regulation of this pathway may be under the control of calpain 3 activity. As frizzled-related protein (FRZB) is upregulated in LGMD2A muscle samples, it could be hypothesized that β-catenin regulation is also altered at the Wnt signaling pathway, leading to an incorrect myogenesis. Conversely, expression of most transcription factor genes was downregulated (MYC, FOS and EGR1). Finally, the upregulation of IL-32 and immunoglobulin genes may induce the eosinophil chemoattraction explaining the inflammatory findings observed in presymptomatic stages. The obtained results try to shed some light on identification of novel therapeutic targets for limb-girdle muscular dystrophies