Potential mitochondrial dysfunction of skeletal muscles in mouse models of Osteogenesis imperfecta.

Victoria Gremminger1, Charlotte L Phillips1,2

1Department of Biochemistry, University of Missouri-Columbia2Department of Child Health, University of Missouri-Columbia

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder characterized by bone fragility, short stature, and muscle weakness[1]. Our lab studies two mouse models of OI, the +/G610C mouse modeling a moderate disease phenotype[2], and the oim/oim mouse modeling a severe OI phenotype[3]. Previously, we determined that oim/oim, but not +/G601C, mice exhibit severely reduced skeletal muscle contractile strength, suggesting a mutation specific inherent muscle pathology was likely [4]. To further evaluate the causes of this muscle weakness we evaluated citrate synthase activity, an indirect marker of mitochondrial content, in whole muscle homogenate and found increased activity in oim/oim mice relative to WT littermates despite poor muscle function[5]. This surprising discovery led us to hypothesize that oim/oim mitochondria were increased as a compensation mechanism due to poorly functioning mitochondria.

We chose to use transmission electron microscopy to evaluate mitochondria in both OI mouse models in order to assess mitochondrial number and note any gross morphological changes within the mitochondria. Since we knew muscle contractile generating force was decreased in oim/oim mice, we decided to evaluate the intermyofibrillar population of mitochondria. We used soleus muscle, an oxidative muscle, from four month old male mice. Soleus muscle was excised immediately following sacrifice and placed in glutaraldehyde fixative and processed for analysis by the Electron Microscopy Core. A sample size of three was evaluated for each of the three genotypes: WT, oim/oim, and +/G610C. Approximately ten images from each sample were acquired and used for assessing mitochondrial number; while ten additional images at a higher magnification were captured to assess any morphological changes.

We used classical stereological methods proposed by Gundersen et al to evaluate mitochondrial volume density (MVD), mitochondrial cross sectional area (CSA), and numerical density [6, 7]. Briefly, a grid with 144 grid points was laid over each image; MVD was calculated as a ratio of points intersecting mitochondria relative to the total 144 points. ImageJ (NIH) was used to determine the average mitochondrial CSA for each image, and the numerical density was calculated as MVD divided by CSA. In oim/oim soleus muscle, none of these parameters appeared different from their WT littermates (Figure 1), while in +/G610C mice, the CSA was increased compared to WT littermates (Figure 2). Higher magnification images were used to assess the morphology of mitochondria; no changes in gross morphology were seen in either +/G610C or oim/oim mice compared to their WT littermates.

Despite the fact that we did not see changes in mitochondria using TEM methodology, we have increasing evidence from a variety of different methods including respirometry, western blotting, and mtDNA copy number analysis that suggest mitochondrial dysfunction is present in oim/oim mice while we see milder mitochondrial alterations in +/G610C mice. Some limitations that may have prevented us from seeing these differences using EM include small sample size, muscle chosen for evaluation (oxidative vs. glycolytic), and the population of mitochondria chosen to study (intermyofibrillar vs. subsarcolemma). In the future, these parameters will need to be considered in order to minimize any limitations.

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Figure 1: Transmission electron microscopy in soleus muscle of 4 month old male mice. Intermyofibrillar mitochondria volume density, cross sectional area, and mitochondrial numerical density were assessed using classical stereological methods (Gundersen 1988) 10 representative images were selected per muscle for both WT (b) and oim/oim (c) groups. Analyses showed no differences between oim/oim and WT groups. In addition, high magnification images of WT (d) and oim/oim (e) failed to show morphological changes. n=3 for each genotype

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Figure 2: +/G610C mice exhibit increased mitochondrial cross sectional areas, while no changes in mitochondrial volume density or numerical density were observed for intermyofibrillar mitochondria in soleus muscle of 4 month old male mice. Images of WT (b) and +/G610C(c) used for quantification. High magnification images of WT (d) and +/G610C (e) did not show gross morphological changes. n=3 for each genotype; *p-value<0.05

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1. Forlino, A. and J.C. Marini, Osteogenesis imperfecta. The Lancet, 2016. 387(10028): p. 1657-1671.

2. Daley, E., et al., Variable bone fragility associated with an Amish COL1A2 variant and a knock-in mouse model. J Bone Miner Res, 2010. 25(2): p. 247-61.

3. Chipman SD, S.H., McBride DJ Jr.,Davisson MT, Marks SC Jr., Shuldiner AR, Wenstrup RJ, Rowe DW, Shapiro JR, Defective proa2(I) collagen synthesis in a recessive mutation in mice: A model of human osteogenesis imperfecta. Proc. Nati. Acad. Sci USA, 1993. 90: p. 1701-1705.

4. Gentry, B.A., et al., Skeletal muscle weakness in osteogenesis imperfecta mice. Matrix Biol, 2010. 29(7): p. 638-44.

5. Jeong, Y., et al., Soluble activin receptor type IIB decoy receptor differentially impacts murine osteogenesis imperfecta muscle function. Muscle Nerve, 2017.

6. Gundersen, H.J.G.B., T.F.; Korbo, L.; Marcussen, N.; Moller, A.; Nielsen, K.; Nyengaard, J.R.; Pakkenber, B.; Srensen, F.B.; Vesterby, A.; West, M.J. , Some new, simple and efficient sterological methods and their use in pathological research and diagnosis. APMIS, 1988. 96: p. 379-394.

7. Toledo, F.G., S. Watkins, and D.E. Kelley, Changes induced by physical activity and weight loss in the morphology of intermyofibrillar mitochondria in obese men and women. J Clin Endocrinol Metab, 2006. 91(8): p. 3224-7.

Figure 2: +/G610C mice exhibit increased mitochondrial cross sectional areas, while no changes in mitochondrial volume density or numerical density were observed for intermyofibrillar mitochondria in soleus muscle of 4 month old male mice. Images of WT (b) and +/G610C(c) used for quantification. High magnification images of WT (d) and +/G610C (e) did not show gross morphological changes. n=3 for each genotype; *p-value<0.05