FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1)

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1

Methanosarcina acetivorans C2A Topoisomerase IIIα, an Archaeal Enzyme with Promiscuity in Divalent Cation Dependence

Topoisomerases play a fundamental role in genome stability, DNA replication and repair. As a result, topoisomerases have served as therapeutic targets of interest in Eukarya and Bacteria, two of the three domains of life. Since members of Archaea, the third domain of life, have not been implicated in any diseased state to-date, there is a paucity of data on archaeal topoisomerases. Here we report Methanosarcina acetivorans TopoIIIα (MacTopoIIIα) as the first biochemically characterized mesophilic archaeal topoisomerase. Maximal activity for MacTopoIIIα was elicited at 30–35°C and 100 mM NaCl. As little as 10 fmol of the enzyme initiated DNA relaxation, and NaCl concentrations above 250 mM inhibited this activity. The present study also provides the first evidence that a type IA Topoisomerase has activity in the presence of all divalent cations tested (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+). Activity profiles were, however, specific to each metal. Known type I (ssDNA and camptothecin) and type II (etoposide, novobiocin and nalidixic acid) inhibitors with different mechanisms of action were used to demonstrate that MacTopoIIIα is a type IA topoisomerase. Alignment of MacTopoIIIα with characterized topoisomerases identified Y317 as the putative catalytic residue, and a Y317F mutation ablated DNA relaxation activity, demonstrating that Y317 is essential for catalysis. As the role of Domain V (C-terminal domain) is unclear, MacTopoIIIα was aligned with the canonical E. coli TopoI 67 kDa fragment in order to construct an N-terminal (1–586) and a C-terminal (587–752) fragment for analysis. Activity could neither be elicited from the fragments individually nor reconstituted from a mixture of the fragments, suggesting that native folding is impaired when the two fragments are expressed separately. Evidence that each of the split domains plays a role in Zn2+ binding of the enzyme is also provided

<i>FRG1</i> myoblasts exhibit a fusion defect that is rescued in <i>FRG1</i>/FHL1 myoblasts.

<p>Quantitative RT-PCR analysis of human FRG1 (A) and human FHL1 (B) mRNA in primary mouse myoblasts isolated from wild type mice, and two <i>FRG1</i> (1 and 2) and two <i>FRG1/FHL1</i> (1 and 2) mice. Data represent the mean +/- SEM of n = 3 independent experiments and was standardized to GAPDH and expressed relative to control wild type myoblasts. *p&lt;0.05. Representative images of primary myoblasts from wild type, <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice following 48 hours (C) or 96 hours (E) differentiation. Cultures were stained with the differentiation marker MHC (green) and DAPI for detection of the fusion index; the percentage of total nuclei localized within MHC-positive myotubes after 48 hours (D) and 96 hours (F) differentiation. Data represent the mean ± SEM from n = 3 independent experiments; wild type (n = 1); <i>FRG1</i> (n = 2); <i>FRG1</i>/<i>FHL1</i> (n = 2); ns not significant, **p&lt;0.005, ***p&lt;0.0001 determined by two-tailed Students T-test. Scale bars = 100μm.</p

FHL1 increases the proportion of muscle fibers with centralized nuclei in <i>FRG1</i> mice.

<p>Representative images of transverse sections of triceps (A) or quadriceps (B) muscles from 12-week-old <i>FRG1</i> and <i>FRG1/FHL1</i> mice stained with H&amp;E. Arrows indicate myofibers with centralized nuclei, an indicator of myoblast fusion <i>in vivo</i>. Scale bars = 100μm. For both triceps and quadriceps muscles the percentage of muscle fibers with centralized nuclei was quantified for wild type (n = 3), <i>FRG1</i> (n = 4) and <i>FRG1</i>/<i>FHL1</i> (n = 4) mice. The subset of these fibers containing multiple centralized nuclei was further quantified at 12 weeks of age. Data represent the mean ± SEM; ns not significant, *p&lt;0.05; **p&lt;0.005; ***p&lt;0.0005 determined by two-tailed Student’s T-test.</p

FHL1 does not alter satellite cell number or markers of satellite cell activation (MyoD) or differentiation (myogenin) in the triceps of <i>FRG1</i> mice.

<p>(A) Transverse muscle sections from the triceps of wild type, <i>FRG1</i> and <i>FRG1/FHL1</i> mice (aged 6- and 12-weeks) were co-stained with a satellite cell specific marker (pax7) and DAPI to detect nuclei. Arrows indicate pax7+ satellite cells. Boxed region indicates area shown in high magnification image inset. Scale bars = 100μm. The number of pax7+ satellite cells per 100 myofibers was counted for the triceps in mice aged (B) 6-weeks (<i>FRG1</i> n = 3 and <i>FRG1/FHL1</i> n = 3) and (C) 12-weeks (<i>FRG1</i> n = 4 and <i>FRG1/FHL1</i> n = 4). Quantitative RT-PCR analysis of pax7 (D- 6 weeks, E- 12 weeks) MyoD (F- 6 weeks, G- 12 weeks) and myogenin (H- 6 weeks, I- 12 weeks) mRNA in wild type, <i>FRG1</i> and <i>FRG1/FHL1</i> (n = 7 mice/genotype) triceps muscle. Data represent the mean ± SEM; ns not significant; *p&lt;0.05; **p&lt;0.001 determined by two-tailed Student’s T-test.</p