Deficiency of emerin contributes differently to the pathogenesis of skeletal and cardiac muscles in LmnaH222P/H222P mutant mice

Abstract

Laminopathies are tissue-selective diseases that affect differently in organ systems. Mutations in nuclear envelopes, emerin (Emd) and lamin A/C (Lmna) genes, cause clinically indistinguishable myopathy called Emery-Dreifuss muscular dystrophy (EDMD) and limb-girdle muscular dystrophy. Several murine models for EDMD have been generated; however, emerin-null (Emd) mice do not show obvious skeletal and cardiac muscle phenotypes, and Lmna H222P/H222P mutant (H222P) mice show only a mild phenotype in skeletal muscle when they already have severe cardiomyopathy. Thus, the underlying molecular mechanism of muscle involvement due to nuclear abnormalities is still unclarified. We generated double mutant (Emd-/-/LmnaH222P/H222P; EH) mice to characterize dystrophic changes and to elucidate interactions between emerin and lamin A/C in skeletal and cardiac muscles. As H222P mice, EH mice grow normally and have breeding productivity. EH mice showed severer muscle involvement compared with that of H222P mice which was an independent of cardiac abnormality at 12 weeks of age. Nuclear abnormalities, reduced muscle fiber size and increased fibrosis were prominent in EH mice. Roles of emerin and lamin A/C in satellite cells function and regeneration of muscle fiber were also evaluated by cardiotoxin-induced muscle injury. Delayed increases in myog and myh3 expression were seen in both H222P and EH mice; however, the expression levels of those genes were similar with control and regenerated muscle fiber size was not different at day 7 after injury. These results indicate that EH mouse is a suitable model for studying skeletal muscle involvement, independent of cardiac function, in laminopathies and an interaction between emerin and lamin A/C in different tissues.

Fig 1. Growth curves and cardiac and skeletal muscle histology of mice at 30 weeks of age.

(A) Body weights of male mice at 6, 12, 18, 24, and 30 weeks of age. WT: grey dotted line (n = 17); Emd: grey line (n = 17), H222P: black dotted line (n = 13) and EH: black line (n = 17) mice. (B) Cardiac histology of mice at 30 weeks of age. (C) Skeletal muscle histology of H222P and EH mice at 30 weeks of age. quadriceps: QF; gastrocnemius: GC; soleus: SOL; paravertebral: PVM; abdomen: ABD; and diaphragm: DIA.

Fig 2. Cardiac muscle phenotypes of mice at 12 weeks of age.

(A) Evaluation of cardiac function by transthoracic echocardiographic analyses. Left ventricular ejection fractions (LVEFs) are shown (n = 8–9). (B) H&E staining of cryosections from cardiac muscle. (C) Immunostaining of laminin ɑ2 (green) and periostin (red) with DAPI (blue) from cardiac muscle. (D) Western blot analyses of periostin. The graph shows the quantification of periostin levels normalized to GAPDH (n = 3). (E) qPCR analyses of Tgfb2 (TGF β2), Postn (periostin), and Fn1 (fibronectin) mRNA levels in cardiac muscle (n = 4). (F) qPCR analyses of Nppa (natriuretic peptide A) and Nppb (natriuretic peptide B) mRNA levels in cardiac muscle (n = 4). In qPCR analyses, data were normalized by Gapdh mRNA and expressed as fold increases from WT mice. ***P<0.001 compared with WT mice.

Fig 3. Skeletal muscle functions of mice at 12 weeks of age.

(A) Average daily activity levels analyzed using a voluntary running wheel. Data were expressed as revolutions per day (n = 7). (B) Treadmill running to exhaustion. The average maximum speeds at which mice could not continue running are shown (n = 7). (C) Serum CK levels from sedentary and exercised mice (n = 7–8). **P<0.01 compared with WT mice; ##P<0.01 compared with Emd mice; $$$P<0.001 compared with H222P mice.

Fig 4. Skeletal muscle pathology of mice at 12 weeks of age.

(A) H&E staining of cryosections and (B) immunostaining of laminin ɑ2 (green) and periostin (red) with DAPI (blue) from soleus muscle. (C) Western blot analyses of periostin from soleus muscle. The graph shows quantification of periostin levels normalized to GAPDH (n = 4). (D) qPCR analyses of Tgfb2 (TGF β2), Postn (periostin), and Fn1 (fibronectin) mRNA levels in soleus muscle (n = 4). (E) Histogram of muscle fiber diameters in soleus muscle. Data are expressed as a percentage of total fibers. (F) The average muscle fiber size by measuring the minor axis of fibers (n = 3–4). (G) qPCR analyses of muscle atrophy-associated genes, Trim63 (MuRF-1), Gdf8 (myostatin) and Fbxo32 (atrogin-1) (n = 4). (H) The percentage of fibers with internal nuclei per total fibers (n = 4). (I) The percentage of embryonic myosin heavy chain (eMyHC)-positive regenerating fiber area per total muscle area (n = 4). (J) Immunostaining of laminin ɑ2 (green) and EBD (red) with DAPI (blue) from soleus muscle. Yellow arrows show the presence of EBD-positive necrotic myofibers. (K) qPCR analyses of muscle regeneration-related genes, Myh3 (eMyHC), Myog (myogenin), Pax7 (Pax7) and Myod1 (MyoD) (n = 4). In qPCR analyses, data were normalized by Gapdh mRNA and expressed as fold increases from WT mice. *P<0.05, **P<0.01, and ***P<0.001 compared with WT mice; #P<0.05, ##P<0.01, and ###P<0.001 compared with Emd mice; $P<0.05, $$P<0.01, and $$$P<0.001 compared with H222P mice.

Fig 5. Nuclear changes in cardiac and skeletal muscles.

(A and B) Western blot analyses of lamin A/C, emerin, LAP2α, and lamin B1 in (A) cardiac muscle and (B) skeletal muscle from WT, Emd, H222P, and EH mice. GAPDH was used as an internal control to ensure that equal sample volumes were applied. (C) Immunodetection of cardiac nuclei with abnormal shapes in EH mice. Lamin A/C (green) and either nesprin 1 (right, red) or LAP2α (left, red) were colocalized with DAPI-positive nuclei (blue). White arrows indicated elongated nuclei. (D) Immunodetection of nuclei with abnormal shapes in the soleus muscle from EH mice at 12 weeks of age. Bottom panels show that either nesprin 1 (upper, red) or LAP2α (lower, red) was mislocalized in lamin A/C (green) and DAPI-positive nuclei (blue). This mislocalization was also seen in lamin A/C and nesprin 1 double-stained cryosections in skeletal muscle from EH mice. (E) Electron microscopic observation of abnormal myonuclei from EH mice at 8 weeks of age. The irregular localization of highly condensed heterochromatin (left), enlarged and dysmorphic nuclei (middle left and middle right), and abnormal intranuclear vacuoles (right); scale bar = 2 μm.

Fig 6. Muscle regeneration from CTX-induced muscle injury.

(A) H&E staining of cryosections from the TA muscle of nontreated, and 3 days, 5 days, and 7 days after CTX injection (n = 4). (B) qPCR analyses of Myh3, Myh8, Myh1, Myh2, Myh4, Myog, Pax7, and Myod1 mRNA levels from the muscle of nontreated (CONT), and 3 days, 5 days, and 7 days after CTX injection. Data were normalized by Gapdh mRNA and expressed as fold increase of WT (CONT) mice. (C and D) The average regenerated muscle fiber size analyzed by measuring (C) the minor axis and (D) the area from the TA muscle of mice 7 days after CTX injection.