ABSTRACT
Knock-out of the mouse RXRa gene was previously shown to result in a hypoplastic heart ventricular wall, histologically detectable in 12.5 dpc fetuses. We show here that a precocious ...differentiation can be detected as early as 8.5 dpc in ventricular cardiomyocytes of RXRα−/− mutants. This precocious differentiation, which is characterized by the presence of striated myofibrils, sarcoplasmic reticulum and intercalated disks, is found after 9.5 dpc in about 50% of RXRα−/− subepicardial myocytes. In contrast, wild-type subepicardial myocytes remain morphologically undifferentiated up to at least 16.5 dpc. A similar precocious differentiation was observed in 9.5 dpc subepicardial myocytes of several RXR0α−/− and RARα−/− mutants, as well as in vitamin A-deficient embryos. The proportion of differentiated subepicardial myocytes almost reached 100% in RXRα/RXRβ double null mutants, indicating a partial functional redundancy between RXRα and RXRβ. This differentiation defect was always paralleled by a decrease in the mitotic index. In addition, subepicardial myocytes of RXRα−/−, RXRα−/−1/RXRβ−/− or vitamin A deficient, but not of RXRβ−/− and RARα−/− embryos, were often flattened and more loosely connected to one another than those of WT embryos. Thus, retinoids are required at early stages of cardiac development to prevent differentiation, support cell proliferation and control the shape of ventricular myocytes, and both RXRs and RARs participate in the mediation of these functions.
Mammalian A-type proteins, ISCA1 and ISCA2, are evolutionarily conserved proteins involved in iron-sulfur cluster (Fe-S) biogenesis. Recently, it was shown that ISCA1 and ISCA2 form a heterocomplex ...that is implicated in the maturation of mitochondrial Fe
S
proteins. Here we report that mouse ISCA1 and ISCA2 are Fe
S
-containing proteins that combine all features of Fe-S carrier proteins. We use biochemical, spectroscopic and in vivo approaches to demonstrate that despite forming a complex, ISCA1 and ISCA2 establish discrete interactions with components of the late Fe-S machinery. Surprisingly, knockdown experiments in mouse skeletal muscle and in primary cultures of neurons suggest that ISCA1, but not ISCA2, is required for mitochondrial Fe
S
proteins biogenesis. Collectively, our data suggest that cellular processes with different requirements for ISCA1, ISCA2 and ISCA1-ISCA2 complex seem to exist.
The transcriptional coregulators PGC-1α and PGC-1β modulate the expression of numerous partially overlapping genes involved in mitochondrial biogenesis and energetic metabolism. The physiological ...role of PGC-1β is poorly understood in skeletal muscle, a tissue of high mitochondrial content to produce ATP levels required for sustained contractions. Here we determine the physiological role of PGC-1β in skeletal muscle using mice, in which PGC-1β is selectively ablated in skeletal myofibres at adulthood (PGC-1β(i)skm-/- mice). We show that myofibre myosin heavy chain composition and mitochondrial number, muscle strength and glucose homeostasis are unaffected in PGC-1β(i)skm-/- mice. However, decreased expression of genes controlling mitochondrial protein import, translational machinery and energy metabolism in PGC-1β(i)skm-/- muscles leads to mitochondrial structural and functional abnormalities, impaired muscle oxidative capacity and reduced exercise performance. Moreover, enhanced free-radical leak and reduced expression of the mitochondrial anti-oxidant enzyme Sod2 increase muscle oxidative stress. PGC-1β is therefore instrumental for skeletal muscles to cope with high energetic demands.
Nano-emulsions are very promising nano-carriers with high potential for loading lipophilic drugs. However, the surface of oil nano-droplets is a dynamic oil/water interface stabilized by surfactants, ...and its chemical modification to graft ligands is highly challenging. In this study we developed a new protocol for modification of the nano-droplets surface through a silica shell terminated by amine functions. It enabled preparation of nanocapsules of 65, 85 and 120 nm diameters with a surface coverage of
ca.
2 amino groups per nm
2
. The nanocapsule surface was then functionalized (41% efficiency) by a model fluorescent ligand (coumarin blue) with a carboxylic function. The evidence for the successful grafting was provided by spectrofluorometry, Förster resonance energy transfer, atomic force microscopy coupled with fluorescence imaging and fluorescence correlation spectroscopy. This simple protocol for surface functionalization of the liquid/liquid interface of lipid droplets may constitute a real advance regarding potential applications that need efficient decoration of droplets with ligands.
A new and simple method of modify and functionalize the liquid/liquid interface of nano-emulsion droplets.
Homozygous RAR beta mutants are growth-deficient, but are fertile and have a normal longevity. They display homeotic transformations and malformations of cervical vertebrae and a retrolenticular ...membrane. This latter abnormality arises from the persistence and hyperplasia of the primary vitreous body. In contrast, we found that abnormalities of cranial nerves IX and X which were previously proposed to be specific features of the RAR beta mutant phenotype (Luo et al., Mech. Dev. 53: 61-71, 1995) occur with the same low penetrance in wildtype littermates. Although the RAR beta protein is expressed at high levels in the striatum and interdigital mesenchyme, the brain and limbs of RAR beta mutants appear morphologically normal. RAR alpha/RAR beta double mutants display numerous visceral abnormalities, most of which are incompatible with post-natal life. The majority of these abnormalities was previously detected in RAR alpha/RAR beta2 mutants with the notable exceptions of agenesis of the stapedial (2nd aortic arch-derived) artery, thymic and spleen agenesis and abnormal inferior vena cava. RAR beta/RAR gamma double mutants show major ocular defects including a shortening of the ventral retina and pre-natal retinal dysplasia, both of which represent the only abnormalities of the fetal vitamin-A deficiency (VAD) syndrome not previously detected in RAR beta2/RAR gamma compound mutants. In addition, RAR beta is apparently functionally redundant with either RAR alpha or RAR gamma for the formation of a small subset of craniofacial skeletal elements, as well as for eyelid development and digit separation. We also provide evidence that, at least in some instances, this phenomenon of functional redundancy between RARs may be an artifactual consequence of gene knock-out.
Summary Myotubular myopathy (MTM) is a severe congenital muscle disease characterized by profound weakness, early respiratory failure and premature lethality. MTM is defined by muscle biopsy findings ...that include centralized nuclei and disorganization of perinuclear organelles. No treatments currently exist for MTM. We hypothesized that aberrant neuromuscular junction (NMJ) transmission is an important and potentially treatable aspect of the disease pathogenesis. We tested this hypothesis in two murine models of MTM. In both models we uncovered evidence of a disorder of NMJ transmission: fatigable weakness, improved strength with neostigmine, and electrodecrement with repetitive nerve stimulation. Histopathological analysis revealed abnormalities in the organization, appearance and size of individual NMJs, abnormalities that correlated with changes in acetylcholine receptor gene expression and subcellular localization. We additionally determined the ability of pyridostigmine, an acetylcholinesterase inhibitor, to ameliorate aspects of the behavioral phenotype related to NMJ dysfunction. Pyridostigmine treatment resulted in significant improvement in fatigable weakness and treadmill endurance. In all, these results describe a newly identified pathological abnormality in MTM, and uncover a potential disease-modifying therapy for this devastating disorder.
Skeletal muscle contraction is triggered by the excitation-contraction (E-C) coupling machinery residing at the triad, a membrane structure formed by the juxtaposition of T-tubules and sarcoplasmic ...reticulum (SR) cisternae. The formation and maintenance of this structure is key for muscle function but is not well characterized. We have investigated the mechanisms leading to X-linked myotubular myopathy (XLMTM), a severe congenital disorder due to loss of function mutations in the MTM1 gene, encoding myotubularin, a phosphoinositide phosphatase thought to have a role in plasma membrane homeostasis and endocytosis. Using a mouse model of the disease, we report that Mtm1 -deficient muscle fibers have a decreased number of triads and abnormal longitudinally oriented T-tubules. In addition, SR Ca 2+ release elicited by voltage-clamp depolarizations is strongly depressed in myotubularin-deficient muscle fibers, with myoplasmic Ca 2+ removal and SR Ca 2+ content essentially unaffected. At the molecular level, Mtm1 -deficient myofibers exhibit a 3-fold reduction in type 1 ryanodine receptor (RyR1) protein level. These data reveal a critical role of myotubularin in the proper organization and function of the E-C coupling machinery and strongly suggest that defective RyR1-mediated SR Ca 2+ release is responsible for the failure of muscle function in myotubular myopathy.
Retinoic acid (RA), the active vitamin A derivative, is an important developmental signaling molecule in vertebrates. In this study, we have assessed whether minimal numbers and/or specific ...distributions of RA-producing cells can support normal mouse embryonic development. Retinaldehyde dehydrogenase 2 (RALDH2) is the main RA-synthesizing enzyme acting during development. We have generated an embryonic stem (ES) cell line homozygous for an
Raldh2 gene disruption, and have analyzed chimeric embryos with various contributions of wild-type cells. Whereas embryos almost completely derived from
Raldh2
−/−
cells phenocopy the corresponding germline null mutants, the presence of even small numbers (<10%) of wild-type cells can rescue most of the morphogenetic defects, including embryonic turning and axial elongation, and left–right looping of the heart tube. No consistent bias in the distribution of wild-type cells was observed in the phenotypically rescued
Raldh2
−/−
chimeras. Analysis of an RA-sensitive transgene indicates that RA can diffuse from wild-type cells and elicit a widespread transcriptional response in
Raldh2-deficient cells. Our results show that few wild-type RA-producing cells, even when present in apparent random distributions, can support early morphogenesis of the mouse embryo. However, the
Raldh2
−/−
chimeric fetuses display lung abnormalities, persistent truncus arteriosus, and abnormal myocardial differentiation, showing that subsequent RA-dependent events cannot be fully rescued by the mosaic presence of wild-type cells.
ShcA is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation. We used the Cre-loxP technology and the ...smooth muscle protein-22 (Sm22) cre transgene, to ablate ShcA specifically in the cardiovascular system from early embryonic development. Conditionally mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart reveals aberrant intercalated z-disk and M-band structures, and misalignments of T-tubules with z-disk. We find that the neuregulin-1-ErbB3 signaling and the baroreceptor reflex response, which have been functionally associated, are both altered in the mutant mice. We further demonstrate that ShcA interacts with Cav-1 and the costameric protein plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Our studies demonstrate that ShcA modulates ErbB3/Neuregulin and Cav-1/dystrophin signaling, two crucial pathways for z-disk and costamere linkages in cardiomyocytes.
Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent ...occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.
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