Abnormal Skeletal Muscle Regeneration plus Mild Alterations in Mature Fiber Type Specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice.

Glycosylated α-dystroglycan provides an essential link between extracellular matrix proteins, like laminin, and the cellular cytoskeleton via the dystrophin-glycoprotein complex. In secondary dystroglycanopathy muscular dystrophy, glycosylation abnormalities disrupt a complex O-mannose glycan necessary for muscle structural integrity and signaling. Fktn-deficient dystroglycanopathy mice develop moderate to severe muscular dystrophy with skeletal muscle developmental and/or regeneration defects. To gain insight into the role of glycosylated α-dystroglycan in these processes, we performed muscle fiber typing in young (2, 4 and 8 week old) and regenerated muscle. In mice with Fktn disruption during skeletal muscle specification (Myf5/Fktn KO), newly regenerated fibers (embryonic myosin heavy chain positive) peaked at 4 weeks old, while total regenerated fibers (centrally nucleated) were highest at 8 weeks old in tibialis anterior (TA) and iliopsoas, indicating peak degeneration/regeneration activity around 4 weeks of age. In contrast, mature fiber type specification at 2, 4 and 8 weeks old was relatively unchanged. Fourteen days after necrotic toxin-induced injury, there was a divergence in muscle fiber types between Myf5/Fktn KO (skeletal-muscle specific) and whole animal knockout induced with tamoxifen post-development (Tam/Fktn KO) despite equivalent time after gene deletion. Notably, Tam/Fktn KO retained higher levels of embryonic myosin heavy chain expression after injury, suggesting a delay or abnormality in differentiation programs. In mature fiber type specification post-injury, there were significant interactions between genotype and toxin parameters for type 1, 2a, and 2x fibers, and a difference between Myf5/Fktn and Tam/Fktn study groups in type 2b fibers. These data suggest that functionally glycosylated α-dystroglycan has a unique role in muscle regeneration and may influence fiber type specification post-injury

Both presynaptic and postsynaptic components are present at neuromuscular junctions in 14 d regenerated muscle of Myf5/<i>Fktn</i> and Tam/<i>Fktn</i> KOs.

<p>(A) Proportion of NMJs positive for synaptophysin and BGTX in saline- or CTX-injected TA of Myf5/<i>Fktn</i> and Tam/<i>Fktn</i> KOs or grouped Myf5/Tam LCs. (B) Representative images showing localization of NMJ pre-synaptic (synaptophysin, red) and post-synaptic markers (BGTX, green) in Myf5/<i>Fktn</i> KO and Tam/<i>Fktn</i> KO and LC TAs injected with saline or CTX. Nuclei stained with DAPI (blue). Scale bar = 100μm. n = 7, LC; n = 4, Myf5 KO; n = 6 Tam KO.</p

Slow oxidative fibers are decreased in Myf5/<i>Fktn</i> KO and Tam/<i>Fktn</i> LC mice following CTX injection.

<p>(A) Quantification of slow type 1 oxidative fibers in TA muscle of saline- or CTX-injected Myf5/<i>Fktn</i> and Tam/<i>Fktn</i> LC or KO mice. *, p<0.05; **, p<0.01; ***, p < .001; two-way ANOVA with Bonferroni’s post-test (all genotypes combined) depicted on figures; two-way ANOVA per strain (Myf5/<i>Fktn</i> or Tam/<i>Fktn</i>) are also reported. (B) Whole tissue maps of CTX-injected TA muscle stained with anti-myosin heavy chain type 1 antibody (red), with sarcolemmal αDG core protein (green) and nuclear (blue) counterstains. Scale bar = 100 μm. n = 5, Myf5 LC; n = 7, Myf5 KO and Tam LC; n = 8, Tam KO.</p

Frequency of type 1 oxidative fibers decreases with age in iliopsoas and TA.

<p>Oxidative type 1 fibers in (A) iliopsoas and (B) TA of 2, 4, and 8 wko Myf5/<i>Fktn</i>-deficient (KO) and control (LC) mice. *, p<0.05; **, p<0.01; ***, p<0.001, two-way ANOVA with Bonferroni’s post-test. Whole tissue (C) iliopsoas and (D) TA maps of sections stained with anti-myosin heavy chain type 1 antibody (red), with basement membrane perlecan or sarcolemmal αDG core protein (green) and nuclear (blue) counterstains. Scale bar = 100 μm. n = 4 for all 2 and 4 wko measurements (except Ilio LC, n = 3); n = 5 for all 8 wko measurements.</p

Minor delay in progression of glycolytic type 2x fiber switching in TA of Myf5/<i>Fktn</i> KO mice normalizes by 8 weeks.

<p>Glycolytic intermediate-twitch 2x fibers in Myf5/<i>Fktn</i>-deficient (KO) and control (LC) (A) iliopsoas and (B) TA muscles. *, p<0.05; **, p<0.01; ***, p<0.001, two-way ANOVA with Bonferroni’s post-test. Whole tissue (C) iliopsoas and (D) TA maps of sections stained with antibody detecting all myosin isoforms except type 2x (red), with basement membrane perlecan or sarcolemmal αDG core protein (green) and nuclear (blue) counterstains. Unstained (negative) fibers were counted to measure type 2x. Scale bar = 100μm. n = 4 for all 2 and 4 wko measurements (except Ilio 4wko LC, n = 3); n = 5 per 8 wko group.</p

Frequencies of type 2a oxidative and type 2b glycolytic fast-twitch fibers are unchanged in iliopsoas and TA between Myf5/<i>Fktn</i> KO and LC mice.

<p>(A) Oxidative type 2a fibers in iliopsoas (top) and TA (bottom) of 2, 4, and 8 wko Myf5/<i>Fktn</i>-deficient (KO) and control (LC) mice. (B) Glycolytic type 2b fibers in iliopsoas (top) and TA (bottom) of 2, 4, and 8 wko Myf5/<i>Fktn-</i>deficient (KO) and control (LC) mice. *, p<0.05; two-way ANOVA with Bonferroni’s post-test. n = 4 for all 2 and 4 wko experimental groups (except Ilio 4 wko LC, n = 3); n = 5 per 8 wko group.</p

Glycolytic type 2x, but not type 2b, fibers decrease following muscle regeneration.

<p>Glycolytic type 2b (A) or type 2x (B) fiber proportions in muscle-specific (Myf5) or whole-body (Tam) <i>Fktn</i> KO and LC muscle injected with saline or CTX. *, p<0.05; ***, p<0.001; two-way ANOVA with Bonferroni’s post-test (all genotypes combined) depicted on figures; two-way ANOVA per strain (Myf5/<i>Fktn</i> or Tam/<i>Fktn</i>) are also reported. (C) Whole tissue maps of TA muscle from Myf5/<i>Fktn</i> or Tam/<i>Fktn</i> KO and LC muscle injected with saline or CTX and stained with an antibody detecting all myosin heavy chain isoforms except type 2x. Unstained (negative) fibers were counted to measure type 2x. Scale bar = 100 μm. n = 5, Myf5 LC; n = 7, Myf5 KO and Tam LC; n = 8, Tam KO.</p

Fast oxidative fibers are increased in Tam/<i>Fktn</i> KO mice following CTX injection.

<p>(A) Quantification of fast type 2a oxidative fibers in TA muscle of saline- or CTX-injected Myf5/<i>Fktn</i> and Tam/<i>Fktn</i> LC or KO mice. *, p<0.05; **, p<0.01; ***, p < .001; two-way ANOVA with Bonferroni’s post-test (all genotypes combined) depicted on figures; two-way ANOVA per strain (Myf5/<i>Fktn</i> or Tam/<i>Fktn</i>) are also reported. (B) Whole tissue maps of CTX-injected TA muscle stained with anti-myosin heavy chain type 2a antibody (red), with sarcolemmal αDG core protein (green) and nuclear (blue) counterstains. Scale bar = 200 μm. n = 5, Myf5 LC; n = 7, Myf5 KO and Tam LC; n = 8, Tam KO.</p