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

Cardiolipin Alterations during Obesity: Exploring Therapeutic Opportunities

Cardiolipin is a specific phospholipid of the mitochondrial inner membrane that participates in many aspects of its organization and function, hence promoting proper mitochondrial ATP production. Here, we review recent data that have investigated alterations of cardiolipin in different tissues in the context of obesity and the related metabolic syndrome. Data relating perturbations of cardiolipin content or composition are accumulating and suggest their involvement in mitochondrial dysfunction in tissues from obese patients. Conversely, cardiolipin modulation is a promising field of investigation in a search for strategies for obesity management. Several ways to restore cardiolipin content, composition or integrity are emerging and may contribute to the improvement of mitochondrial function in tissues facing excessive fat storage. Inversely, reduction of mitochondrial efficiency in a controlled way may increase energy expenditure and help fight against obesity and in this perspective, several options aim at targeting cardiolipin to achieve a mild reduction of mitochondrial coupling. Far from being just a victim of the deleterious consequences of obesity, cardiolipin may ultimately prove to be a possible weapon to fight against obesity in the future

Isolation and Phospholipid Enrichment of Muscle Mitochondria and Mitoplasts

International audienceThe efficient ATP production in mitochondria relies on the highly specific organization of its double membrane. Notably, the inner mitochondrial membrane (IMM) displays a massive surface extension through its folding into cristae, along which concentrate respiratory complexes and oligomer

Development and validation of a novel clinical scoring system for short‐term prediction of death in dogs with acute pancreatitis

Background: Acute pancreatitis (AP) is associated with a high death rate in dogs, but accurate predictors of early death are still lacking. Objectives: To develop a scoring system for prediction of short-term case fatality in dogs with AP. Animals: One hundred sixty-nine dogs with AP including 138 dogs in the training cohort and 31 dogs in the validation cohort. Methods: Multicenter, retrospective cohort study. Survival analysis was used to assess the associations with short-term death (within 30 days after admission). Independent predictors of death were identified by a stepwise selection method and used for the score calculation. Results: Death rate within 30 days after admission was 33% in the training cohort. Four independent risk factors for short-term death were identified in the training cohort: presence of systemic inflammatory response syndrome, coagulation disorders, increased creatinine and ionized hypocalcemia. Canine Acute Pancreatitis Severity (CAPS) score was developed to predict shortterm death, integrating these 4 factors in a weighted way. A simplified version of CAPS score (sCAPS) including respiratory rate instead of SIRS was also assessed. The area under the receiver-operating characteristic curve (AUC) of CAPS and sCAPS scores was 0.92 in the training cohort with an optimal cutoff of 11 (sensitivity, 89%; specificity, 90%) and 6 (sensitivity, 96%; specificity, 77%), respectively. CAPS and sCAPS score were validated in the validation cohort with respective AUC of 0.91 and 0.96. Conclusions and Clinical Importance: We propose 2 scoring systems that allow early and accurate prediction of short-term death in dogs with AP

Interactome Mapping of the Phosphatidylinositol 3-Kinase-Mammalian Target of Rapamycin Pathway Identifies Deformed Epidermal Autoregulatory Factor-1 as a New Glycogen Synthase Kinase-3 Interactor

The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors. Its perturbations are associated with cancer progression, type 2 diabetes, and neurological disorders. To better understand the mechanisms of action and regulation of this pathway, we initiated a large scale yeast two-hybrid screen for 33 components of the PI3K-mTOR pathway. Identification of 67 new interactions was followed by validation by co-affinity purification and exhaustive literature curation of existing information. We provide a nearly complete, functionally annotated interactome of 802 interactions for the PI3K-mTOR pathway. Our screen revealed a predominant place for glycogen synthase kinase-3 (GSK3) A and B and the AMP-activated protein kinase. In particular, we identified the deformed epidermal autoregulatory factor-1 (DEAF1) transcription factor as an interactor and in vitro substrate of GSK3A and GSK3B. Moreover, GSK3 inhibitors increased DEAF1 transcriptional activity on the 5-HT1A serotonin receptor promoter. We propose that DEAF1 may represent a therapeutic target of lithium and other GSK3 inhibitors used in bipolar disease and depression

CKIP-1 regulates mammalian and zebrafish myoblast fusion

Multinucleated muscle fibres arise by fusion of precursor cells called myoblasts. We previously showed that CKIP-1 ectopic expression in C2C12 myoblasts increased cell fusion. In this work, we report that CKIP-1 depletion drastically impairs C2C12 myoblast fusion in vitro and in vivo during zebrafish muscle development. Within developing fast-twich myotome, Ckip-1 localises at the periphery of fast precursor cells, closed to the plasma membrane. Unlike wild-type myoblasts that form spatially arrayed multinucleated fast myofibres, Ckip-1-deficient myoblasts show a drastic reduction in fusion capacity. A search for CKIP-1 binding partners identified the ARPC1 subunit of Arp2/3 actin nucleation complex essential for myoblast fusion. We demonstrate that CKIP-1, through binding to plasma membrane phosphoinositides via its PH domain, regulates cell morphology and lamellipodia formation by recruiting the Arp2/3 complex at the plasma membrane. These results establish CKIP-1 as a regulator of cortical actin that recruits the Arp2/3 complex at the plasma membrane essential for muscle precursor elongation and fusion

Differential physiological role of BIN1 isoforms in skeletal muscle development, function and regeneration

International audienceSkeletal muscle developmentand regeneration are tightly regulated processes. How the intracellular organization of muscle fibers is achieved during these steps is unclear. Here we focus on the cellular and physiological roles of amphiphysin 2(BIN1), a membrane remodeling protein mutated in both congenital and adult centronuclear myopathies(CNM),that is ubiquitously expressed and hasskeletal muscle-specific isoforms. We created and characterized constitutive, muscle-specific and inducible Bin1homozygous and heterozygous knockout mice targeting either ubiquitousor muscle-specific isoforms.Constitutive Bin1-deficient mice diedat birth from lack of feeding due to a skeletal muscle defect.T-tubules and other organelles weremisplaced and altered, supporting a general early role of BIN1 on intracellular organization in addition to membrane remodeling.Whereasrestricted deletion of Bin1in unchallenged adult muscles had no impact, the forced switch from the muscle-specificisoformsto the ubiquitousisoformsthrough deletion of the in-frame muscle–specific exon delayed muscle regeneration.Thus, BIN1 ubiquitous function is necessary for muscle development and function while its muscle-specific isoformsfine-tune muscle regenerationinadulthood, supporting that BIN1 centronuclear myopathy with congenital onset are due to developmental defects while later onsetmay be due to regeneration defects

A canine Arylsulfatase G (ARSG) mutation leading to a sulfatase deficiency is associated with neuronal ceroid lipofuscinosis

Neuronal ceroid lipofuscinoses (NCLs) represent the most common group of inherited progressive encephalopathies in children. They are characterized by progressive loss of vision, mental and motor deterioration, epileptic seizures, and premature death. Rare adult forms of NCL with late onset are known as Kufs’ disease. Loci underlying these adult forms remain unknown due to the small number of patients and genetic heterogeneity. Here we confirm that a late-onset form of NCL recessively segregates in US and French pedigrees of American Staffordshire Terrier (AST) dogs. Through combined association, linkage, and haplotype analyses, we mapped the disease locus to a single region of canine chromosome 9. We eventually identified a worldwide breed-specific variant in exon 2 of the Arylsulfatase G (ARSG) gene, which causes a p.R99H substitution in the vicinity of the catalytic domain of the enzyme. In transfected cells or leukocytes from affected dogs, the missense change leads to a 75% decrease in sulfatase activity, providing a functional confirmation that the variant might be the NCL-causing mutation. Our results uncover a protein involved in neuronal homeostasis, identify a family of candidate genes to be screened in patients with Kufs' disease, and suggest that a deficiency in sulfatase is part of the NCL pathogenesis