Collagen VI deficiency reduces muscle pathology, but does not improve muscle function, in the γ-sarcoglycan-null mouse

Human Molecular Genetics, 2016, Vol. 25, No. 7 1357–1369 doi: 10.1093/hmg/ddw018 Advance Access Publication Date: 24 January 2016 Original Article ORIGINAL ARTICLE Collagen VI deficiency reduces muscle pathology, but does not improve muscle function, in the γ-sarcoglycan-null mouse 1 Howard Hughes Medical Institute, 2Department of Molecular Physiology and Biophysics, 3Department of Neurology and 4Department of Internal Medicine, The University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242, USA *To whom correspondence should be addressed at: Howard Hughes Medical Institute, The University of Iowa Roy J. and Lucille A. Carver College of Medicine, 4283 CBRB, 285 Newton Road, Iowa City, IA 52242, USA. Tel: +1 3193357867; Fax: +1 3193356957; Email: Abstract Muscular dystrophy is characterized by progressive skeletal muscle weakness and dystrophic muscle exhibits degeneration and regeneration of muscle cells, inflammation and fibrosis. Skeletal muscle fibrosis is an excessive deposition of components of the extracellular matrix including an accumulation of Collagen VI. We hypothesized that a reduction of Collagen VI in a muscular dystrophy model that presents with fibrosis would result in reduced muscle pathology and improved muscle function. To test this hypothesis, we crossed γ-sarcoglycan-null mice, a model of limb-girdle muscular dystrophy type 2C, with a Col6a2-deficient mouse model. We found that the resulting γ-sarcoglycan-null/Col6a2Δex5 mice indeed exhibit reduced muscle pathology compared with γ-sarcoglycan-null mice. Specifically, fewer muscle fibers are degenerating, fiber size varies less, Evans blue dye uptake is reduced and serum creatine kinase levels are lower. Surprisingly, in spite of this reduction in muscle pathology, muscle function is not significantly improved. In fact, grip strength and maximum isometric tetanic force are even lower in γ-sarcoglycan-null/Col6a2Δex5 mice than in γ-sarcoglycan-null mice. In conclusion, our results reveal that Collagen VI-mediated fibrosis contributes to skeletal muscle pathology in γ-sarcoglycan-null mice. Importantly, however, our data also demonstrate that a reduction in skeletal muscle pathology does not necessarily lead to an improvement of skeletal muscle function, and this should be considered in future translational studies. Introduction Limb-girdle muscular dystrophy type 2C (LGMD2C) manifests clinically as a progressive muscle weakness with calf hypertrophy, elevated serum creatine kinase (CK) levels and onset occurs during childhood (1). This form of muscular dystrophy is caused by autosomal recessive mutations in the γ-sarcoglycan gene (SGCG; OMIM #253700), which encodes the transmembrane protein γ-sarcoglycan (γ-SG) (2). Together with α-SG, β-SG, δ-SG and sarcospan, γ-SG forms the SG complex in cardiac and skeletal † Present address: Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands. Present address: Division of Cardiology, Department of Medicine, Kinki University Faculty of Medicine, Osaka, Japan. Received: October 12, 2015. Revised: December 24, 2015. Accepted: January 18, 2016 ‡ © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 1357 Jessica C. de Greef1,2,3,4,†, Rebecca Hamlyn1,2,3,4, Braden S. Jensen1,2,3,4, Raul O’Campo Landa1,2,3,4, Jennifer R. Levy1,2,3,4, Kazuhiro Kobuke1,2,3,4,‡ and Kevin P. Campbell1,2,3,4,* 1358 | Human Molecular Genetics, 2016, Vol. 25, No. 7 described over 15 years ago, is a Col6a1-deficient mouse that produces no α1(VI) mRNA and in which Collagen VI protein is not deposited in the ECM. Muscles of both heterozygous and homozygous mice feature fiber necrosis and phagocytosis, pronounced variation in fiber size, and an increased percentage of centrally nucleated fibers. However, aside from these histopathological changes, no obvious phenotype is detected, and as such the mouse model is regarded a good representation of the mild Bethlem myopathy (24). Many years later, two additional Collagen VI mouse models were generated, both targeting the Col6a3 gene. One of these (the Col6a3hm/hm mouse) expresses a very low level of a non-functional α3(VI) mRNA, which results in the absence of Collagen VI from the ECM. Again, the myopathic pathology of these mice is limited (25). The second Col6a3 mouse model lacks Exon 16 of the Col6a3 gene (Col6a3+/d16), mimicking the most common autosomal dominant mutation in Ullrich congenital muscular dystrophy patients. Mice heterozygous for this allele produce similar levels of the normal Col6a3 mRNA and an mRNA transcript with an in-frame deletion of 54 bp. The mutant α3(VI) protein exerts a dominant-negative effect on assembly of Collagen VI chains. The myopathic phenotype of these Col6a3+/d16 mice, and of Col6a3d16/d16 mice is again mild (26). To study the consequences of Collagen VI deposition in the ECM of skeletal muscle during the development of muscular dystrophy, we crossed a γ-SG-null mouse model with a mouse model carrying an in-frame deletion of Exon 5 of the Col6a2 gene (Col6a2Δex5 mice). Both mouse models were recently generated in our lab. We hypothesized that Collagen VI ablation in a muscular dystrophy mouse model that features a significant amount of fibrosis would ameliorate the muscle pathology, thereby restoring some muscle function. We found that the γ-SG-null/ Col6a2Δex5 mice indeed exhibited reduced muscle pathology, including reductions in the number of regenerating muscle fibers, variation in fiber size, Evans blue dye (EBD) uptake by skeletal muscles, and serum CK levels. However, muscle function was not improved, as assessed by measurements of grip strength and maximum isometric tetanic force. Results γ-SG-null mice exhibit severe muscle pathology including fibrosis To study the pathogenesis of LGMD2C and to further unravel the function of γ-SG, we developed a γ-SG-null mouse model in our laboratory. We generated this mouse model using a targeting vector that contains Exon 2 followed by a fragment of Intron 2, and then a neomycin resistance cassette flanked by two loxP sites (Supplementary Material, Fig. S1A). Loss of γ-SG was confirmed by immunofluorescence staining, as well as the absence of αSG, β-SG and δ-SG from the sarcolemma. Hematoxylin and eosin (H&E) staining showed that our γ-SG-null mice have severe muscle pathology similar to that commonly observed in patients with muscular dystrophy. It is characterized by a high percentage of centrally nucleated fibers and fiber size variation (Fig. 1A). In addition, high levels of serum CK levels were measured, both at baseline (Fig. 1B; P < 0.001) and 2 h after the mice (...truncated)