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 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.

Body, ambient and felt temperature: An attempt to resolve a human and mice dilemma.

Mice thermoneutral zone lies at temperatures much higher than expected when considering the geographical extension of the species. Growing evidence shows that mouse-dependent thermogenesis experimentation needs to cope with temperatures below those at which the animals are most comfortable. The associated physiological changes interfere with experimental results, thereby highlighting the apparently trivial subject of room-temperature. Working at above 25 °C is difficult for researchers and animal care technicians. Herein, we explore alternative solutions related to living habits of wild mice that could improve translation of research on mice to humans. Standard murine environments are often colder than those in laboratory facilities and their behavior is mainly characterized by a gregarious, nesting and exploratory way of life. Optimization of their thermal environment can thus also be achieved by avoiding individual housing and providing high-quality nesting material and devices that would allow locomotor activity, hence muscle thermogenesis. These options have additional relevance in terms of animal welfare. When precise monitoring of the temperature is required, temperature-controlled cabinets can be used for the duration of the experiments. During the manipulation of mice, a heated laminar flow hood or tray could create an optimized microenvironment. The specification of temperature-related data in publications should contain information on the translatability of the described mouse models to humans. Furthermore, publications should describe the premises of the laboratory in relation to housing possibilities and murine behavior.

Acupuncture has potential in managing axial stiffness in steeplechase racehorses: a blinded prospective randomized preliminary study.

OBJECTIVE: Evaluate the short-term effects of acupuncture on the dynamic manifestations of axial stiffness in steeplechase racehorses. ANIMALS: 12 steeplechase racehorses presenting signs of axial stiffness during training. METHODS: Horses were randomly assigned to either an acupuncture treatment by an experienced certified acupuncturist (n = 6) or no treatment as negative controls (6). The horses' locomotion was evaluated during training before treatment (D0) and 7 (D7) and 14 (D14) days after by their rider and trainer through a questionnaire. Additionally, the improvement of their dorsal flexibility 2 days after treatment was evaluated subjectively at the trot, free jumping at the canter was evaluated by expert clinicians, and free jumping at the trot was evaluated objectively via inertial measurement units. RESULTS: Significantly more horses were improved on D7 and D14 in the acupuncture group (6/6) compared with the control group (1/5; P =.01) according to the scores set by the trainer and riders. Subjective evaluation of the dorsal flexibility also revealed a significant improvement (P = .04) for horses receiving the acupuncture treatment (median improvement score, 0.50 [reference range, 0.5 to 0.9]) compared with control horses (-0.25 [reference range, -0.5 to 0]). CLINICAL RELEVANCE: Acupuncture may be an interesting nondoping strategy to improve clinical signs of axial stiffness and performance on steeplechase racehorses.

Hacd2 deficiency in mice leads to an early and lethal mitochondrial disease.

OBJECTIVE: Mitochondria fuel most animal cells with ATP, ensuring proper energetic metabolism of organs. Early and extensive mitochondrial dysfunction often leads to severe disorders through multiorgan failure. Hacd2 gene encodes an enzyme involved in very long chain fatty acid (C ≥ 18) synthesis, yet its roles in vivo remain poorly understood. Since mitochondria function relies on specific properties of their membranes conferred by a particular phospholipid composition, we investigated if Hacd2 gene participates to mitochondrial integrity. METHODS: We generated two mouse models, the first one leading to a partial knockdown of Hacd2 expression and the second one, to a complete knockout of Hacd2 expression. We performed an in-depth analysis of the associated phenotypes, from whole organism to molecular scale. RESULTS: Thanks to these models, we show that Hacd2 displays an early and broad expression, and that its deficiency in mice is lethal. Specifically, partial knockdown of Hacd2 expression leads to death within one to four weeks after birth, from a sudden growth arrest followed by cachexia and lethargy. The total knockout of Hacd2 is even more severe, characterized by embryonic lethality around E9.5 following developmental arrest and pronounced cardiovascular malformations. In-depth mechanistic analysis revealed that Hacd2 deficiency causes altered mitochondrial efficiency and ultrastructure, as well as accumulation of oxidized cardiolipin. CONCLUSIONS: Altogether, these data indicate that the Hacd2 gene is essential for energetic metabolism during embryonic and postnatal development, acting through the control of proper mitochondrial organization and function.

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.

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.

The 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 oligomers of the ATP synthase. Evidence has accumulated to highlight the importance of a specific phospholipid composition of the IMM to support mitochondrial oxidative phosphorylation. Contribution of specific phospholipids to mitochondrial ATP production is classically studied by modulating the activity of enzymes involved in their synthesis, but the interconnection of phospholipid synthesis pathways often impedes the determination of the precise role of each phospholipid. Here, we describe a protocol to specifically enrich mitochondrial membranes with cardiolipin or phosphatidylcholine, as well as a fluorescence-based method to quantify phospholipid enrichment. This method, based on the fusion of lipid vesicles with isolated mitochondria, may further allow a precise evaluation of phospholipid contribution to mitochondrial functions.

Cardiolipin content controls mitochondrial coupling and energetic efficiency in muscle.

Unbalanced energy partitioning participates in the rise of obesity, a major public health concern in many countries. Increasing basal energy expenditure has been proposed as a strategy to fight obesity yet raises efficiency and safety concerns. Here, we show that mice deficient for a muscle-specific enzyme of very-long-chain fatty acid synthesis display increased basal energy expenditure and protection against high-fat diet-induced obesity. Mechanistically, muscle-specific modulation of the very-long-chain fatty acid pathway was associated with a reduced content of the inner mitochondrial membrane phospholipid cardiolipin and a blunted coupling efficiency between the respiratory chain and adenosine 5'-triphosphate (ATP) synthase, which was restored by cardiolipin enrichment. Our study reveals that selective increase of lipid oxidative capacities in skeletal muscle, through the cardiolipin-dependent lowering of mitochondrial ATP production, provides an effective option against obesity at the whole-body level.

Differential physiological roles for BIN1 isoforms in skeletal muscle development, function and regeneration.

Skeletal muscle development and 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 has skeletal muscle-specific isoforms. We created and characterized constitutive muscle-specific and inducible Bin1 homozygous and heterozygous knockout mice targeting either ubiquitous or muscle-specific isoforms. Constitutive Bin1-deficient mice died at birth from lack of feeding due to a skeletal muscle defect. T-tubules and other organelles were misplaced and altered, supporting a general early role for BIN1 in intracellular organization, in addition to membrane remodeling. Although restricted deletion of Bin1 in unchallenged adult muscles had no impact, the forced switch from the muscle-specific isoforms to the ubiquitous isoforms through deletion of the in-frame muscle-specific exon delayed muscle regeneration. Thus, ubiquitous BIN1 function is necessary for muscle development and function, whereas its muscle-specific isoforms fine tune muscle regeneration in adulthood, supporting that BIN1 CNM with congenital onset are due to developmental defects, whereas later onset may be due to regeneration defects.

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 short-term 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.

Targeted Lipidomic Analysis of Myoblasts by GC-MS and LC-MS/MS.

Lipids represent ∼10% of the cell dry mass and play essential roles in membrane composition and physical properties, energy storage, and signaling pathways. In the developing or the regenerating skeletal muscle, modifications in the content or the flipping between leaflets of membrane lipid components can modulate the fusion capacity of myoblasts, thus constituting one of the regulatory mechanisms underlying myofiber growth. Recently, few genes controlling these qualitative and quantitative modifications have started to be unraveled. The precise functional characterization of these genes requires both qualitative and quantitative evaluations of a global lipid profile. Here, we describe a lipidomic protocol using mass spectrometry, allowing assessing the content of fatty acids, glycerophospholipids, and cholesterol in the routinely used C2C12 mouse myoblast cell line, or in primary cultures of mouse myoblasts.

HACD1, a regulator of membrane composition and fluidity, promotes myoblast fusion and skeletal muscle growth.

The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies, yet the underlying cellular and molecular mechanisms remain elusive. In this study, we investigate the role of HACD1/PTPLA, which is involved in the elongation of the very long chain fatty acids, in muscle fibre formation. In humans and dogs, HACD1 deficiency leads to a congenital myopathy with fibre size disproportion associated with a generalized muscle weakness. Through analysis of HACD1-deficient Labradors, Hacd1-knockout mice, and Hacd1-deficient myoblasts, we provide evidence that HACD1 promotes myoblast fusion during muscle development and regeneration. We further demonstrate that in normal differentiating myoblasts, expression of the catalytically active HACD1 isoform, which is encoded by a muscle-enriched splice variant, yields decreased lysophosphatidylcholine content, a potent inhibitor of myoblast fusion, and increased concentrations of ≥ C18 and monounsaturated fatty acids of phospholipids. These lipid modifications correlate with a reduction in plasma membrane rigidity. In conclusion, we propose that fusion impairment constitutes a novel, non-exclusive pathological mechanism operating in congenital myopathies and reveal that HACD1 is a key regulator of a lipid-dependent muscle fibre growth mechanism.

Phosphorylation of NBR1 by GSK3 modulates protein aggregation.

The autophagy receptor NBR1 (neighbor of BRCA1 gene 1) binds UB/ubiquitin and the autophagosome-conjugated MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) proteins, thereby ensuring ubiquitinated protein degradation. Numerous neurodegenerative and neuromuscular diseases are associated with inappropriate aggregation of ubiquitinated proteins and GSK3 (glycogen synthase kinase 3) activity is involved in several of these proteinopathies. Here we show that NBR1 is a substrate of GSK3. NBR1 phosphorylation by GSK3 at Thr586 prevents the aggregation of ubiquitinated proteins and their selective autophagic degradation. Indeed, NBR1 phosphorylation decreases protein aggregation induced by puromycin or by the DES/desmin N342D mutant found in desminopathy patients and stabilizes ubiquitinated proteins. Importantly, decrease of protein aggregates is due to an inhibition of their formation and not to their autophagic degradation as confirmed by data on Atg7 knockout mice. The relevance of NBR1 phosphorylation in human pathology was investigated. Analysis of muscle biopsies of sporadic inclusion body myositis (sIBM) patients revealed a strong decrease of NBR1 phosphorylation in muscles of sIBM patients that directly correlated with the severity of protein aggregation. We propose that phosphorylation of NBR1 by GSK3 modulates the formation of protein aggregates and that this regulation mechanism is defective in a human muscle proteinopathy.