Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., “MTMRs”). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder

RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes

Gene expression in a tissue-specific context depends on the combined efforts of epigenetic, transcriptional and post-transcriptional processes that lead to the production of specific proteins that are important determinants of cellular identity. Ribosomes are a central component of the protein biosynthesis machinery in cells; however, their regulatory roles in the translational control of gene expression in skeletal muscle remain to be defined. In a genetic screen to identify critical regulators of myogenesis, we identified a DEAD-Box RNA helicase, DDX27, that is required for skeletal muscle growth and regeneration. We demonstrate that DDX27 regulates ribosomal RNA (rRNA) maturation, and thereby the ribosome biogenesis and the translation of specific transcripts during myogenesis. These findings provide insight into the translational regulation of gene expression in myogenesis and suggest novel functions for ribosomes in regulating gene expression in skeletal muscles

MTM1-MTMR12 interactions in normal and disease states.

<p>Under normal conditions, MTM1 and MTMR12 interact in skeletal muscle and regulate skeletal muscle architecture and function. Loss of function mutations of <i>MTM1</i> (red cross) in skeletal muscle are associated with centronuclear myopathy and with a secondary reduction in MTMR12 levels. In centronuclear myopathy, disease causing missense mutations (red circle) that disrupt interactions between MTM1 and MTMR12 result in decreased stability of myotubularin causing myotubular myopathy associated with reduced levels of MTMR12. Loss of <i>MTMR12</i> in zebrafish and mammalian cells, results in decreased levels of myotubularin resulting in pathological changes similar to centronuclear myopathy.</p

Myotubularin and PtdIns3<i>P</i> alterations in <i>mtmr12</i> morphants.

<p>(A) Immunofluorescence of control and <i>mtmr12</i> knockdown fish showed significantly decreased myotubularin staining in <i>mtmr12</i> knockdown fish in images taken under identical conditions. Immunofluorescence detection of PtdIns3<i>P</i> showed apparent increases of this myotubularin substrate in <i>mtmr12</i> morphant embryos as compared to controls. (B) PtdIns3<i>P</i> levels are increased in <i>mtmr12</i>, <i>mtm1</i> and <i>mtm1-mtmr12</i> morphant zebrafish, *<i>P</i>≤0.05. Total lipids were extracted from zebrafish at 3 dpf and PtdIns3<i>P</i> levels were measured using a lipid-protein overlay enzyme-linked immunosorbent assay.</p

Abnormal histology of MTMR12-deficient zebrafish.

<p>(A)Toluidine blue stained longitudinal sections of skeletal muscle in control and morphant fish at 3 dpf. In comparison to the control fish, <i>mtmr12</i> morphants showed disorganized myofibers (arrowhead) with central nucleation (arrow), similar to histological changes observed in the skeletal muscle of <i>mtm1</i> morphant fish (arrow). Knockdown of both <i>mtm1</i> as well as <i>mtmr12</i> results in severe muscle disorganization greater than seen in <i>mtm1</i> or <i>mtmr12</i> alone morphants. (B) Centrally nucleated myofibers were quantified. Serial sections from 3–4 different embryos were analyzed and the relative number of centrally nucleated fibers in the middle somites (10–13) were counted. (C) Hematoxylin and Eosin staining of <i>mtmr12</i> morphant zebrafish at different time points. An increase in sarcomeric disorganization was observed at 3 dpf in comparison to 2 dpf in <i>mtmr12</i> morphants (arrow) (D) Centrally nucleated myofibers were quantified. Serial sections from 6 different embryos were analyzed and the relative number of centrally nucleated fibers in the middle somites (10–13) were counted. Scale bar = 10 µm.</p

Protein-protein interactions between myotubularin and MTMR12 proteins.

<p>(A) GST pull down of MTM1-GST recombinant protein with MTMR12 showed a direct interaction between the two proteins (above). Coomassie blue stained gel (below) showing GST and MTM1-GST protein used in the pull down (B) Co-IP experiments from Cos-1 transfected cells with MTM1 and MTMR12-B10 constructs showing the interaction of the two proteins in the cellular context <i>in-vivo</i> (C) MTMR12 co-immunoprecipitates with myotubularin (using 1G1 monoclonal antibody) in mouse muscle lysates revealed with anti-MTMR12 polyclonal antibody (upper panel) and with the 2827 polyclonal anti-myotubularin (bottom panel). (D) Confocal microscopic immunofluorescence studies of longitudinal frozen sections of skeletal muscle from mouse tibialis anterior muscle with sarcomeric markers. Individual immunostaining with 2827 anti-myotubularin or anti-MTMR12 showed similar striated localization in skeletal muscle (top panel). Double-immunostaining with both proteins showed a co-localization of myotubularin and MTMR12 to Triads and partial co-localization with the ryanodine receptor, RyR1, a sarcoplasmic reticulum marker, but not with α-actinin, a Z-line marker. Scale bar = 10 µm.</p

Myotubularin-MTMR12 interactions in XLMTM.

<p>(A) Schematic diagram of different domains of myotubularin protein displaying representative pathogenic mutations found in XLMTM patients or an artificial inactivating mutation C375S* (GRAM, N terminal lipid or protein interacting domain; RID, putative membrane targeting motif; PTP/DSP, phosphatase domain; SID, protein-protein interacting domain; CC, coiled-coil domain; PDZB, PDZ binding site). (B) Wild-type or mutant MTM1-B10 fusion proteins with indicated missense mutations and wild-type MTMR12-GFP proteins were overexpressed in Cos1 cells. Immunoprecipitation of protein extracts with anti-B10 tag antibody showed that mutations on GRAM or RID domains disrupt the interactions between MTM1 and MTMR12. (C) Western blotting of XLMTM patient myotubes showed that mutants that decrease the stability of myotubularin protein also results in a reduction of MTMR12 levels. Histograms depict the western quantification for panels (B) and (C). Asterisks indicate statistically significant differences from measurements of wild type controls, P≤0.05.</p

Loss of protein stability in the absence of myotubularin-MTMR12 interactions.

<p>(A) Knockdown of <i>mtmr12</i> resulted in a strong decrease of myotubularin protein in <i>mtmr12</i> morphant zebrafish at 3 dpf by western blotting. Western blot analysis was done on three independently injected clutches (n in each clutch = 50–75). The histogram at right shows normalized amounts of myotubularin in control and <i>mtmr12</i> morphant zerbrafish, *<i>P</i>≤0.01. (B) siRNA-mediated knockdown of <i>Mtmr12</i> in C2C12 myoblasts leads to decreased protein levels of MTM1. Histograms showed western blot quantification of MTM1 and MTMR12 with reference to α-actinin as a loading control. Data represent mean of 3 independent experiments, * P<0.05. (C) <i>Mtmr12</i> siRNA treated myoblasts were differentiated into myotubes and tested for protein expression of MTM1, MTMR12, desmin and myogenin. <i>Mtmr12</i> knockdown in myotubes leads to decreased protein levels of MTM1 and increased amounts of the intermediate filament protein desmin, but do not affect myogenin levels. α-Actinin was used as the loading control (histograms). Data represent mean of 3 independent experiments, * P<0.05. (D) Labeling of α-actinin and desmin in C2C12 myoblast and myotubes treated with <i>Mtmr12</i> siRNA or scramble control siRNA. <i>Mtmr12</i> knockdown cells showed abnormal accumulation of desmin in both stages (arrow). (E) Quantification of myotubes at 2, 4, 6 and 9 days of differentiation showed no significant differences between siRNA-<i>Mtmr12</i> cells and scramble siRNA. Data were obtained from 2 independent experiments (* P<0.05) and minimum of 100 cells per condition were counted. (F) <i>Mtm1</i> knockout mice exhibited highly reduced levels of MTMR12 protein in skeletal muscle at pre-symptomatic (2 weeks) as well as symptomatic stages (5 weeks). The histogram on right shows normalized amounts of MTMR12 in control and <i>Mtm1KO Mice</i>, *P≤0.05. (G) siRNA-based <i>Mtm1</i> knockdown in C2C12 cells led to no reduction in MTMR12 protein as seen by western blot analysis. β-actin/GAPDH were used as loading controls in western blots. The histogram at right shows normalized amounts of MTMR12 in control and <i>Mtm1</i> knockdown cell line, *P≤0.05.</p

Rescue of <i>mtmr12</i> morphant phenotypes by <i>MTM1</i>.

<p>The ability of human <i>MTM1</i> or <i>MTMR12</i> transcripts to rescue abnormalities seen in morphant zebrafish was classified in to phenotypic index of five groups: Normal, mild, moderate, severe and dead, described in the table depending on body length, birefringence and ultrastructure of skeletal muscle. (A) Polarized light microscopy of 3 dpf live embryos showed that birefringence of <i>mtmr12</i> morphant embryos increased significantly upon overexpression of human <i>MTM1</i> mRNA. (B) Overexpression of human <i>MTMR12</i> mRNA in <i>mtm1</i> morphant fish resulted in a mild rescue of skeletal muscle defects as seen by birefringence of zebrafish embryos. (C) Overexpression of human <i>MTM1</i> mRNA in <i>mtm1-mtmr12</i> morphant fish resulted in a moderate rescue of skeletal muscle defects as seen by birefringence of zebrafish embryos. (D) Electron microscopy showed normal skeletal muscle structure of <i>mtmr12</i> and <i>mtm1-mtmr12</i> morphant fish rescued with <i>MTM1</i> mRNA but displayed disorganized triads in <i>mtm1</i> morphants that were rescued with <i>MTMR12</i> mRNA. (E) Quantification of the body length and disorganized triads in morphant and rescued fish. Body length was measured in 10–15 embryos in each group. Total number of triads were counted in at least 15 myofibers within each embryo (n = 5 embryos). P≤0.05.</p

Skeletal muscle abnormalities in <i>ddx27</i> mutant zebrafish.

<p>(A) Microscopic visualization of control and mutant larval zebrafish (<i>osoi</i>) at 5 days post fertilization (dpf). Mutant fish display leaner muscles (left panel) and exhibit highly reduced birefringence in comparison to control (right panel). Mutant fish also exhibit pericardial edema (arrow) (B) Genetic mapping of <i>osoi</i> mutant by initial bulk segregant analysis identified linkage on chromosome 6. Fine mapping of chromosome 6 resolved flanking markers z41548 and z14467, with a candidate genome region containing six candidate genes that were sequenced by Sanger sequencing (C) Overexpression of human <i>DDX27</i> mRNA results in a significant decrease in mutant zebrafish phenotype (D) Whole-mount Immunofluorescence was performed on control and <i>ddx27</i> mutant larvae (Z-stack confocal image, 4dpf) (scale bar: 50μm) (E) Immunofluorescence on newly isolated (Day 0) and cultured (Day1 and 3) EDL myofibers from wild-type mice (scale bar: 10μm). (F) Western blot showing relative expression of Ddx27 and myogenic markers (MyoD, MyoG and MF20) in proliferating C2C12 myoblasts in growth media (50% confluence) or in differentiation media for 3 days (D0-3). GAPDH was used as the control. (G) Schematic diagram of nucleolus depicting nucleolar domains. Eukaryotic nucleolus has tripartite architecture: Fibrillar center (FC); Dense fibrillar component (DFC) and granular compartment (GC). Immunofluorescence of human myoblasts with DDX27 and nucleolar markers labeling each compartment of nucleolus (scale bar: 2μm).</p