Leiomodin-3-deficient mice display nemaline myopathy with fast-myofiber atrophy

Abstract

Nemaline myopathy (NM) is one of the most common forms of congenital myopathy, and affects either fast myofibers, slow myofibers, or both. However, an animal model for congenital myopathy with fast-myofiber-specific atrophy is not available. Furthermore, mutations in the leiomodin-3 (LMOD3) gene have recently been identified in a group of individuals with NM. However, it is not clear how loss of LMOD3 leads to NM. Here, we report a mouse mutant in which the piggyBac (PB) transposon is inserted into the Lmod3 gene and disrupts its expression. Lmod3(PB/PB) mice show severe muscle weakness and postnatal growth retardation. Electron microscopy and immunofluorescence studies of the mutant skeletal muscles revealed the presence of nemaline bodies, a hallmark of NM, and disorganized sarcomeric structures. Interestingly, Lmod3 deficiency caused muscle atrophy specific to the fast fibers. Together, our results show that Lmod3 is required in the fast fibers for sarcomere integrity, and this study offers the first NM mouse model with muscle atrophy that is specific to fast fibers. This model could be a valuable resource for interrogating myopathy pathogenesis and developing therapeutics for NM as well as other pathophysiological conditions with preferential atrophy of fast fibers, including cancer cachexia and sarcopenia.

Keywords: Fast-myofiber atrophy; Leiomodin-3; Mouse; Nemaline myopathy.

Fig. 1.

Disruption of Lmod3 expression causes growth retardation in Lmod3^PB/PB mice. (A) Schematic representation of the genomic region of the Lmod3 gene and the position of PB insertion. Black box: exon. White box: untranslated region (UTR). Arrows: primers for quantitative RT-PCR. (B) Quantitative RT-PCR analysis of Lmod3 mRNA from TA muscles of 5-week-old mice with indicated genotypes. n=5∼7. (C) Western blotting of 5-week-old mouse TA muscles with antibodies against Lmod3 and β-tubulin (as loading control). (D) Growth curves of male Lmod3^+/+ mice (n=5), Lmod3^PB/+ mice (n=11) and Lmod3^PB/PB mice (n=6). (E) A picture of three 1-week-old male littermates with genotype labeled. ^, # or * indicates P<0.05; ^^, ## or ** indicates P<0.01; ### or *** indicates P<0.001.

Fig. 2.

Lmod3^PB/PB mice show muscle weakness due to atrophy of myofibers. (A) A picture of unskinned hindlimbs of three 4-week-old male littermates with genotype labeled. (B) Lean mass and fat mass of 5-week-old Lmod3^PB/PB males (black bar, n=10) and Lmod3^+/+ males (white bar, n=5) measured by EchoMRI. ** indicates P<0.01. (C) Forelimb grip strength of 4-week-old Lmod3^PB/PB males (black bar, n=4) and Lmod3^+/+ males (white bar, n=5). (D-G) H&E staining of TA muscles from a 5-week-old Lmod3^PB/PB male (F,G) and a wild-type male littermate control (D,E). Scale bar: 50 µm. Yellow arrows: atrophic muscle fibers with internalized nuclei.

Fig. 3.

Disorganized sarcomere structure in Lmod3^PB/PB muscles. Longitudinal sections of 4-week-old TA muscles from Lmod3^PB/PB or Lmod3^PB/+ mice stained with phalloidin (A,E), α-actinin (B,F), DAPI (C,G) or merged (D,H). Scale bar: 20 µm. Inlets show magnification of marked regions.

Fig. 4.

Disorganized Z-line and nemaline bodies in Lmod3^PB/PB muscles. (A,B) Modified Gomori trichrome staining on 4-week-old TA muscles from Lmod3^PB/+ and Lmod3^PB/PB mice. Scale bar: 50 µm. White arrows: nemaline bodies. (C-F) Electron microscopy images of EDL muscles from 6-week-old Lmod3^PB/+ (C) and Lmod3^PB/PB (D-F) mice. Scale bar: 1 µm. Yellow arrow: Z-line streaming. Red arrowheads: nemaline bodies.

Fig. 5.

Disruption of Lmod3 causes atrophy specifically to fast myofibers. (A-R) Cross-sections of 4-week-old TA muscles from Lmod3^PB/+ and Lmod3^PB/PB mice stained with the antibodies indicated. Scale bar: 200 µm. (S-U) Size distribution of cross-sectional area (CSA) of type-IIb (S), type-I (T) and type-IIa (U) myofibers in TA muscles from 4-week-old Lmod3^PB/+ and Lmod3^PB/PB mice (n=4). Panels S-U show P<0.001 in Kolmogorov–Smirnov test. (V) Relative ratio between the number of myofibers of a specific fiber type to the number of total myofibers in TA muscles from Lmod3^PB/+ and Lmod3^PB/PB mice (n=4). *P<0.05.