Rapid effects of 17beta-estradiol on TRPV5 epithelial Ca2+ channels in rat renal cells.

The renal distal tubules and collecting ducts play a key role in the control of electrolyte and fluid homeostasis. The discovery of highly calcium selective channels, Transient Receptor Potential Vanilloid 5 (TRPV5) of the TRP superfamily, has clarified the nature of the calcium entry channels. It has been proposed that this channel mediates the critical Ca(2+) entry step in transcellular Ca(2+) re-absorption in the kidney. The regulation of transmembrane Ca(2+) flux through TRPV5 is of particular importance for whole body calcium homeostasis.In this study, we provide evidence that the TRPV5 channel is present in rat cortical collecting duct (RCCD(2)) cells at mRNA and protein levels. We demonstrate that 17beta-estradiol (E(2)) is involved in the regulation of Ca(2+) influx in these cells via the epithelial Ca(2+) channels TRPV5. By combining whole-cell patch-clamp and Ca(2+)-imaging techniques, we have characterized the electrophysiological properties of the TRPV5 channel and showed that treatment with 20-50nM E(2) rapidly (\u3c5min) induced a transient increase in inward whole-cell currents and intracellular Ca(2+) via TRPV5 channels. This rise was significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV5.These data demonstrate for the first time, a novel rapid modulation of endogenously expressed TRPV5 channels by E(2) in kidney cells. Furthermore, the results suggest calcitropic effects of E(2). The results are discussed in relation to present concepts of non-genomic actions of E(2) in Ca(2+) homeostasis

Progressive Structural Defects in Canine Centronuclear Myopathy Indicate a Role for HACD1 in Maintaining Skeletal Muscle Membrane Systems

Mutations in HACD1/PTPLA cause recessive congenital myopathies in humans and dogs. Hydroxyacyl-coA dehydratases are required for elongation of very long chain fatty acids, and HACD1 has a role in early myogenesis, but the functions of this striated muscle-specific enzyme in more differentiated skeletal muscle remain unknown. Canine HACD1 deficiency is histopathologically classified as a centronuclear myopathy (CNM). We investigated the hypothesis that muscle from HACD1-deficient dogs has membrane abnormalities in common with CNMs with different genetic causes. We found progressive changes in tubuloreticular and sarcolemmal membranes and mislocalized triads and mitochondria in skeletal muscle from animals deficient in HACD1. Furthermore, comparable membranous abnormalities in cultured HACD1-deficient myotubes provide additional evidence that these defects are a primary consequence of altered HACD1 expression. Our novel findings, including T-tubule dilatation and disorganization, associated with defects in this additional CNM-associated gene provide a definitive pathophysiologic link with these disorders, confirm that dogs deficient in HACD1 are relevant models, and strengthen the evidence for a unifying pathogenesis in CNMs via defective membrane trafficking and excitation-contraction coupling in muscle. These results build on previous work by determining further functional roles of HACD1 in muscle and provide new insight into the pathology and pathogenetic mechanisms of HACD1 CNM. Consequently, alterations in membrane properties associated with HACD1 mutations should be investigated in humans with related phenotypes

A dog model for centronuclear myopathy (CNM) carrying the most common DNM2 mutation

International audienceMutations in DNM2 cause autosomal dominant centronuclear myopathy (ADCNM), a rare disease characterized by skeletal muscle weakness and structural anomalies of the myofibres including nuclear centralization and mitochondrial mispositioning. Following the clinical report of a Border Collie male with exercise intolerance and histopathological hallmarks of CNM on the muscle biopsy, we identified the c.1393C>T (R465W) mutation in DNM2, corresponding to the most common ADCNM mutation in humans. In order to establish a large animal model for longitudinal and preclinical studies on the muscle disorder, we collected sperm samples from the Border Collie male and generated a dog cohort for subsequent clinical, genetic, and histological investigations. Four of the five offspring carried the DNM2 mutation and showed muscle atrophy and a mildly impaired gait. Morphological examinations of transverse muscle sections revealed CNM-typical fibres with centralized nuclei and remodelling of the mitochondrial network. Overall, the DNM2-CNM dog represents a faithful animal model for the human disorder, allows the investigation of ADCNM disease progression, and constitutes a valuable complementary tool to validate innovative therapies established in mice

A COLQ Missense Mutation in Sphynx and Devon Rex Cats with Congenital Myasthenic Syndrome

International audienceAn autosomal recessive neuromuscular disorder characterized by skeletal muscle weakness, fatigability and variable electromyographic or muscular histopathological features has been described in the two related Sphynx and Devon Rex cat breeds (Felis catus). Collection of data from two affected Sphynx cats and their relatives pointed out a single disease candidate region on feline chromosome C2, identified following a genome-wide SNP-based homozygosity mapping strategy. In that region, we further identified COLQ (collagen-like tail subunit of asymmetric acetylcholinesterase) as a good candidate gene, since COLQ mutations were identified in affected humans and dogs with endplate acetylcholinesterase deficiency leading to a synaptic form of congenital myasthenic syndrome (CMS). A homozygous c.1190G\textgreaterA missense variant located in exon 15 of COLQ, leading to a C397Y substitution, was identified in the two affected cats. C397 is a highly-conserved residue from the C-terminal domain of the protein; its mutation was previously shown to produce CMS in humans, and here we confirmed in an affected Sphynx cat that it induces a loss of acetylcholinesterase clustering at the neuromuscular junction. Segregation of the c.1190G\textgreaterA variant was 100% consistent with the autosomal recessive mode of inheritance of the disorder in our cat pedigree; in addition, an affected, unrelated Devon Rex cat recruited thereafter was also homozygous for the variant. Genotyping of a panel of 333 cats from 14 breeds failed to identify a single carrier in non-Sphynx and non-Devon Rex cats. Finally, the percentage of healthy carriers in a European subpanel of 81 genotyped Sphynx cats was estimated to be low (3.7%) and 14 control Devon Rex cats were genotyped as wild-type individuals. Altogether, these results strongly support that the neuromuscular disorder reported in Sphynx and Devon Rex breeds is a CMS caused by a unique c.1190G\textgreaterA missense mutation, presumably transmitted through a founder effect, which strictly and slightly disseminated in these two breeds. The presently available DNA test will help owners avoid matings at risk

Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice

International audienceSyncytins are envelope genes from endogenous retroviruses, " captured " for a role in pla-centation. They mediate cell-cell fusion, resulting in the formation of a syncytium (the syncy-tiotrophoblast) at the fetomaternal interface. These genes have been found in all placental mammals in which they have been searched for. Cell-cell fusion is also pivotal for muscle fiber formation and repair, where the myotubes are formed from the fusion of mononucle-ated myoblasts into large multinucleated structures. Here we show, taking advantage of mice knocked out for syncytins, that these captured genes contribute to myoblast fusion, with a >20% reduction in muscle mass, mean muscle fiber area and number of nuclei per fiber in knocked out mice for one of the two murine syncytin genes. Remarkably, this reduction is only observed in males, which subsequently show muscle quantitative traits more similar to those of females. In addition, we show that syncytins also contribute to muscle repair after cardiotoxin-induced injury, with again a male-specific effect on the rate and extent of regeneration. Finally, ex vivo experiments carried out on murine myoblasts demonstrate the direct involvement of syncytins in fusion, with a >40% reduction in fusion index upon addition of siRNA against both syncytins. Importantly, similar effects are observed with primary myoblasts from sheep, dog and human, with a 20–40% reduction upon addition of siRNA against the corresponding syncytins. Altogether, these results show a direct contribution of the fusogenic syncytins to myogenesis, with a demonstrated male-dependence of the effect in mice, suggesting that these captured genes could be responsible for the muscle sexual dimorphism observed in placental mammals

In Vivo Myoblasts Tracking Using the Sodium Iodide Symporter Gene Expression in Dogs

Stem cell-based therapies are a promising approach for the treatment of degenerative muscular diseases; however, clinical trials have shown inconclusive and even disappointing results so far. Noninvasive cell monitoring by medicine imaging could improve the understanding of the survival and biodistribution of cells following injection. In this study, we assessed the canine sodium iodide symporter (cNIS) reporter gene as an imaging tool to track by single-photon emission computed tomography (SPECT/CT) transduced canine myoblasts after intramuscular (IM) administrations in dogs. cNIS-expressing cells kept their myogenic capacities and showed strong 99 mTc-pertechnetate (99 mTcO4-) uptake efficiency both in vitro and in vivo. cNIS expression allowed visualization of cells by SPECT/CT along time: 4 h, 48 h, 7 days, and 30 days after IM injection; biopsies collected 30 days post administration showed myofiber's membranes expressing cNIS. This study demonstrates that NIS can be used as a reporter to track cells in vivo in the skeletal muscle of large animals. Our results set a proof of concept of the benefits NIS-tracking tool may bring to the already challenging cell-based therapies arena in myopathies and pave the way to a more efficient translation to the clinical setting from more accurate pre-clinical results.status: publishe

Genotypes for chromosome C2 candidate region.

<p>SNP genotypes for each cat were manually inspected in Excel to identify homozygous regions shared by the two affected cats. Only one region from chromosome C2 spanning from position 137108027 bp to position 140984522 bp (according to the updated felCat5 SNP manifest for the Illumina Feline 63k SNP genotyping array, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137019#pone.0137019.ref020" target="_blank">20</a>]) was consistent with the highly-probable heterozygous status of the sire and dam, the non-homozygously mutated status of the proband’s healthy littermate and the inferred heterozygous status of the paternal grandmother. This region encompassed 3.9 Mb. Homozygosity for the allele shared by the affected sib-pair is shown in light green. Heterozygosity or homozygosity for the opposite allele is shown in yellow. Missing genotypes are noticed 0. Chr: chromosome. bp: base pairs.</p

Histological and histochemical features of the disease.

<p>Cryosections (6 μm) of cervical muscle from a four-month-old affected Sphynx kitten (A-C) and from her healthy littermate (D-F). Haematoxylin-eosin staining (A, D) showed a wide range of fibre size with more rounded fibres surrounded by a thicker endomysium in the affected kitten (A) compared to the healthy control (D). No internal nuclei were present on both sections. Myosin adenosine triphosphatase isoforms (ATPase activity at pH 9.4, B and E) showed a slight increase in pale type-1 fibres in the affected kitten (B) compared to the healthy control (E) but no fibre-type aggregation. Motor end-plates were labelled with alpha-bungarotoxin (for acetylcholine receptors AchR; C and F insets; green) and esterase activity (for AchE; C, F; brown stain). Muscle nuclei were stained with Dapi (C and F insets; blue). Sections from the healthy littermate (F) showed a perfect colocalization of compacted AchR (green) and AchE (brown). In contrast, sections from the affected cat showed a faint, abnormally dispersed AchE staining in myofibres with normal clusters of AchR (C).</p

Congenital neuromuscular disorder in a Sphynx kitten.

<p>Picture of the four-month-old Sphynx female kitten presented at the Neurology clinics located at the Alfort School of Veterinary Medicine campus, in Maisons-Alfort, France. The kitten displayed a peculiar gait while walking, with ventroflexion of her neck. Note the marked dorsal protrusion of scapulae. No significant muscle atrophy was noticed.</p

In Vivo Myoblasts Tracking Using the Sodium Iodide Symporter Gene Expression in Dogs

International audienc