Lipin1 plays complementary roles in myofibre stability and regeneration in dystrophic muscles.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by dystrophin mutations, leading to the loss of sarcolemmal integrity, and resulting in progressive myofibre necrosis and impaired muscle function. Our previous studies suggest that lipin1 is important for skeletal muscle regeneration and myofibre integrity. Additionally, we discovered that mRNA expression levels of lipin1 were significantly reduced in skeletal muscle of DMD patients and the mdx mouse model. To understand the role of lipin1 in dystrophic muscle, we generated dystrophin/lipin1 double knockout (DKO) mice, and compared the limb muscle pathology and function of wild-type B10, muscle-specific lipin1 deficient (lipin1Myf5cKO ), mdx and DKO mice. We found that further knockout of lipin1 in dystrophic muscle exhibited a more severe phenotype characterized by increased necroptosis, fibrosis and exacerbated membrane damage in DKO compared to mdx mice. In barium chloride-induced muscle injury, both lipin1Myf5cKO and DKO showed prolonged regeneration at day 14 post-injection, suggesting that lipin1 is critical for muscle regeneration. In situ contractile function assays showed that lipin1 deficiency in dystrophic muscle led to reduced specific force production. Using a cell culture system, we found that lipin1 deficiency led to elevated expression levels of necroptotic markers and medium creatine kinase, which could be a result of sarcolemmal damage. Most importantly, restoration of lipin1 inhibited the elevation of necroptotic markers in differentiated primary lipin1-deficient myoblasts. Overall, our data suggests that lipin1 plays complementary roles in myofibre stability and muscle function in dystrophic muscles, and overexpression of lipin1 may serve as a potential therapeutic strategy for dystrophic muscles. KEY POINTS: We identified that lipin1 mRNA expression levels are significantly reduced in skeletal muscles of Duchenne muscular dystrophy patients and mdx mice. We found that further depletion of lipin1 in skeletal muscles of mdx mice induces more severe dystrophic phenotypes, including enhanced myofibre sarcolemma damage, muscle necroptosis, inflammation, fibrosis and reduced specific force production. Lipin1 deficiency leads to elevated expression levels of necroptotic markers, whereas restoration of lipin1 inhibits their expression. Our results suggest that lipin1 is functionally complementary to dystrophin in muscle membrane integrity and muscle regeneration.

Evolution of Transcriptional Repressors Impacts Caenorhabditis Vulval Development.

Comparative genomic sequence analysis has found that the genes for many chromatin-associated proteins are poorly conserved, but the biological consequences of these sequence changes are not understood. Here, we show that four genes identified for an Inappropriate Vulval cell Proliferation (ivp) phenotype in the nematode Caenorhabditis briggsae exhibit distinct functions and genetic interactions when compared with their orthologs in C. elegans. Specifically, we show that the four C. briggsae ivp genes encode the noncanonical histone HTZ-1/H2A.z and three nematode-specific proteins predicted to function in the nucleus. The mutants exhibit ectopic vulval precursor cell proliferation (the multivulva [Muv] phenotype) due to inappropriate expression of the lin-3/EGF gene, and RNAseq analysis suggests a broad role for these ivp genes in transcriptional repression. Importantly, although the C. briggsae phenotypes have parallels with those seen in the C. elegans synMuv system, except for the highly conserved HTZ-1/H2A.z, comparable mutations in C. elegans ivp orthologs do not exhibit synMuv gene interactions or phenotypes. These results demonstrate the evolutionary changes that can underlie conserved biological outputs and argue that proteins critical to repress inappropriate expression from the genome participate in a rapidly evolving functional landscape.

Mechanisms of altered skeletal muscle action potentials in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) patients suffer from progressive and debilitating motor dysfunction for which only palliative treatment is currently available. Previously, we discovered reduced skeletal muscle Cl- channel (ClC-1) and inwardly rectifying K+ channel (Kir) currents in R6/2 HD transgenic mice. To further investigate the role of ClC-1 and Kir currents in HD skeletal muscle pathology, we measured the effect of reduced ClC-1 and Kir currents on action potential (AP) repetitive firing in R6/2 mice using a two-electrode current clamp. We found that R6/2 APs had a significantly lower peak amplitude, depolarized maximum repolarization, and prolonged decay time compared with wild type (WT). Of these differences, only the maximum repolarization was accounted for by the reduction in ClC-1 and Kir currents, indicating the presence of additional ion channel defects. We found that both KV1.5 and KV3.4 mRNA levels were significantly reduced in R6/2 skeletal muscle compared with WT, which explains the prolonged decay time of R6/2 APs. Overall, we found that APs in WT and R6/2 muscle significantly and progressively change during activity to maintain peak amplitude despite buildup of Na+ channel inactivation. Even with this resilience, the persistently reduced peak amplitude of R6/2 APs is expected to result in earlier fatigue and may help explain the motor impersistence experienced by HD patients. This work lays the foundation to link electrical changes to force generation defects in R6/2 HD mice and to examine the regulatory events controlling APs in WT muscle.

Lipin1 is required for skeletal muscle development by regulating MEF2c and MyoD expression.

KEY POINTS: Lipin1 is critical for skeletal muscle development. Lipin1 regulates MyoD and myocyte-specific enhancer factor 2C (MEF2c) expression via the protein kinase C (PKC)/histone deacetylase 5-mediated pathway. Inhibition of PKCµ activity suppresses myoblast differentiation by inhibiting MyoD and MEF2c expression. ABSTRACT: Our previous characterization of global lipin1-deficient (fld) mice demonstrated that lipin1 played a novel role in skeletal muscle (SM) regeneration. The present study using cell type-specific Myf5-cre;Lipin1fl/fl conditional knockout mice (Lipin1Myf5cKO ) shows that lipin1 is a major determinant of SM development. Lipin1 deficiency induced reduced muscle mass and myopathy. Our results from lipin1-deficient myoblasts suggested that lipin1 regulates myoblast differentiation via the protein kinase Cµ (PKCµ)/histone deacetylase 5 (HDAC5)/myocyte-specific enhancer factor 2C (MEF2c):MyoD-mediated pathway. Lipin1 deficiency leads to the suppression of PKC isoform activities, as well as inhibition of the downstream target of PKCµ, class II deacetylase HDAC5 nuclear export, and, consequently, inhibition of MEF2c and MyoD expression in the SM of lipin1Myf5cKO mice. Restoration of diacylglycerol-mediated signalling in lipin1 deficient myoblasts by phorbol 12-myristate 13-acetate transiently activated PKC and HDAC5, and upregulated MEF2c expression. Our findings provide insights into the signalling circuitry that regulates SM development, and have important implications for developing intervention aimed at treating muscular dystrophy.

Lipin-1 regulates Bnip3-mediated mitophagy in glycolytic muscle.

Autophagy of mitochondria (mitophagy) is essential for maintaining muscle mass and healthy skeletal muscle. Patients with heritable phosphatidic acid phosphatase lipin-1-null mutations present with severe rhabdomyolysis and muscle atrophy in glycolytic muscle fibers, which are accompanied with mitochondrial aggregates and reduced mitochondrial cytochrome c oxidase activity. However, the underlying mechanisms leading to muscle atrophy as a result of lipin-1 deficiency are still not clear. In this study, we found that lipin-1 deficiency in mice is associated with a marked accumulation of abnormal mitochondria and autophagic vacuoles in glycolytic muscle fibers. Our studies using lipin-1-deficient myoblasts suggest that lipin-1 participates in B-cell leukemia (BCL)-2 adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3)-regulated mitophagy by interacting with microtubule-associated protein 1A/1B-light chain (LC)3, which is an important step in the recruitment of mitochondria to nascent autophagosomes. The requirement of lipin-1 for Bnip3-mediated mitophagy was further verified in vivo in lipin-1-deficient green fluorescent protein-LC3 transgenic mice (lipin-1-/--GFP-LC3). Finally, we showed that lipin-1 deficiency in mice resulted in defective mitochondrial adaptation to starvation-induced metabolic stress and impaired contractile muscle force in glycolytic muscle fibers. In summary, our study suggests that deregulated mitophagy arising from lipin-1 deficiency is associated with impaired muscle function and may contribute to muscle rhabdomyolysis in humans.-Alshudukhi, A. A., Zhu, J., Huang, D., Jama, A., Smith, J. D., Wang, Q. J., Esser, K. A., Ren, H. Lipin-1 regulates Bnip3-mediated mitophagy in glycolytic muscle.

Differing roles for sur-2/MED23 in C. elegans and C. briggsae vulval development.

Normal vulval development in the nematode Caenorhabditis briggsae is identical to that in the related Caenorhabditis elegans. However, several experiments suggest that there are differences between the two species with respect to the contribution of EGF/Ras signaling. To investigate these differences genetically, we have characterized a C. briggsae mutant strain that phenocopies the effect observed when C. briggsae animals are treated with U0126, an inhibitor of the EGF pathway component MEK. We identify that the gene affected in the mutant strain is Cbr-sur-2, which encodes a MED23 mediator complex protein that acts downstream of EGF signaling in C. elegans and other organisms, such as mammals. When Cbr-sur-2 and Cel-sur-2 mutants are compared, we find that the production of additional vulval cells from P5.p and P7.p in C. elegans is dependent on proper development of P6.p, while C. briggsae does not have a similar requirement. Combined chemical and genetic interference with the EGF pathway completely eliminates vulval development in C. elegans but not in C. briggsae. Our results provide genetic evidence for the differing requirements for EGF signaling in the two species.

Automated Image Analysis Pipeline Development to Monitor Disease Progression in Muscular Dystrophy Using Cell Profiler.

Muscular dystrophies are inherited disorders that are characterized by progressive muscle degeneration. These disorders are caused by mutations in the genes encoding structural elements within the muscle, which leads to increased vulnerability to mechanical stress and sarcolemma damage. Although myofibers have the capacity to regenerate, the newly formed myofibers still harbor genetic mutation, which induces continuous cycles of muscle fiber death and regeneration. This repeated cycling is accompanied by an inflammatory response which eventually provokes excessive fibrotic deposition. The histopathological changes in skeletal muscle tissue are central to the disease pathogenesis. Analysis of muscle histopathology is the gold standard for monitoring muscle health and disease progression. However, manual, or semi-manual quantification methods, are not only immensely tedious but can be subjective. Here, we present four image analysis pipelines built in CellProfiler which enable users without a background in computer vision or programming to quantitatively analyze biological images. These image analysis pipelines are designed to quantify skeletal muscle histopathological staining for membrane damage, the abundance and size distribution of regenerating muscle fibers, inflammation via quantification of CD68+ M1 macrophages, and collagen deposition. Additionally, we discuss methods to address common errors associated with the quantification of microscopy images. These automated tools can not only improve workflow efficiency but can provide a better understanding of the histopathological progression of muscular dystrophy.

Loss of membrane integrity drives myofiber death in lipin1-deficient skeletal muscle.

Mutations in lipin1 are suggested to be a common cause of massive rhabdomyolysis episodes in children; however, the molecular mechanisms involved in the regulation of myofiber death caused by the absence of lipin1 are not fully understood. Loss of membrane integrity is considered as an effective inducer of cell death in muscular dystrophy. In this study, we utilized a mouse line with selective homozygous lipin1 deficiency in the skeletal muscle (Lipin1Myf5cKO ) to determine the role of compromised membrane integrity in the myofiber death in lipin1-deficient muscles. We found that Lipin1Myf5cKO muscles had significantly elevated proapoptotic factors (Bax, Bak, and cleaved caspase-9) and necroptotic proteins such as RIPK1, RIPK3, and MLKL compared with WT mice. Moreover, Lipin1Myf5cKO muscle had significantly higher membrane disruptions, as evidenced by increased IgG staining and elevated uptake of Evans Blue Dye (EBD) and increased serum creatine kinase activity in Lipin1Myf5cKO muscle fibers. EBD-positive fibers were strongly colocalized with apoptotic or necroptotic myofibers, suggesting an association between compromised plasma membrane integrity and cell death pathways. We further show that the absence of lipin1 leads to a significant decrease in the absolute and specific muscle force (normalized to muscle mass). Our work indicates that apoptosis and necroptosis are associated with a loss of membrane integrity in Lipin1Myf5cKO muscle and that myofiber death and dysfunction may cause a decrease in contractile force.