Poster Title Here Mitochondrial Deficiency in Primary Muscle Cells from Mdx Mice Robert Shine, Peter Dwyer, Lyndsay Olson, Jennifer Truong, Matthew Goddeeris, Effie Tozzo, Eric Bell Mitobridge, Inc. Cambridge, MA 02138

Abstract Results Results

Duchenne muscular dystrophy (DMD) is a recessive, fatal X-linked disease that is characterized M ito c h o n d ria l c o n trib u te d A T P is re d u c e d Mdx myoblasts have decreased expression by progressive wasting due to a loss of function in dystrophin, a protein that is of OXPHOS complexes

part of a complex that bridges the cytoskeleton and extracellular matrix. The mdx mouse, an in m d x m y o b la s ts a n d m y o tu b e s T

animal model for DMD, has a point mutation in the dystrophin that results in a loss of 1 .5 2.0

W l function. This study uses primary muscle satellite cell derived myoblasts and myotubes to W T

a WT m determine differences in mitochondrial biology between the mdx mice and wild type (WT) control s M D X

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mice. Compared to cells isolated from WT mice, mdx cells have reductions in mitochondrial 1 .0 **** f

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bioenergetics. Moreover, mdx cells have reduced levels of mitochondria which may partially *** e T

g 1.0 explain the reduction in bioenergetics. Interestingly, the mitochondrial phenotype is apparent ****

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Materials and Analysis a

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B 0 B 0 D WT and mdx myoblast isolation and culture: Quadricep and gastrocnemius muscles from a C

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single mouse were pooled and subjected to a mechanical/collagenase digestion. Isolated g g U li li myoblasts were cultured on Matrigel-coated cultureware in DMEM/F-12 with 10% FBS and O O Complex I II III IV V 20ng/mL bFGF. Myoblast homogeneity was determined by a co-staining for vimentin and M y o b la s t M y o tu b e desmin (data not shown). Figure 6: RNA from WT or mdx myoblasts was isolated using a NucleoSpin RNA kit (Macherey-Nagle) and converted to cDNA Myotube Differentiation: Confluent myoblasts were changed to DMEM + 2%FBS + - Figure 3: Cells were treated for 24h. The ATP levels were determined using the Cell TiterGlo assay (Promega). Significant with the High Capacity cDNA Reverse Transcriptase Kit (Thermo). Gene expression was then determined using the SmartChip difference between WT and mdx was determined by Mann-Whitney Test or unpaired T-Test; **p<0.01, ***p<0.001, ****p<0.0001, Real-Time PCR system (Wafergen) and analyzed with Qbase. Significant difference between WT and mdx were determined by selenium-transferrin for 5 days with the assay performed Day 6. myoblast n=40, myotube n=8. unpaired T-test; *p<0.05, n=9. Mitochondrial Characterization Assays: Procedural information is contained in figure legends. Statistical Analysis: Myoblast data generated from 3 pairs of WT/mdx mice were combined for D iffe re n tia l e x p re s s io n in s e le c te d g e n e s analysis. Myotube data is from 1 pair of WT/mdx mice. Data was graphed as boxplots and o f in te re s t to m ito c h o n d ria l fu n c tio n

statistical significance determined using GraphPad Prism. Comparisons were performed between T Palmitate oxidation is reduced 3

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M 1.5 Figure 7: RNA from WT or mdx myoblasts was isolated using a NucleoSpin RNA kit (Macherey-Nagle) and converted to cDNA

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/ WT MDX with the High Capacity cDNA Reverse Transcriptase Kit (Thermo). Gene expression was then determined using the SmartChip

0 1.0 Real-Time PCR system (Wafergen) and analyzed with Qbase. Significant difference between WT and mdx were determined by 9 unpaired T-test; *p<0.05, n=9.

5 Figure 4: WT and mdx myoblasts were starved for 18h in low media with carnitine. KHB plus BSA or BSA-palmitate was

X 0.5 added and oxygen consumption was analyzed using a Seahorse Metabolic Analyzer. The palmitate response was determined by

E calculating a ratio of the FCCP-induced BSA-palmitate respiration/FCCP-induced BSA respiration. Significant difference between WT and mdx determined by unpaired T-test; ****p<0.0001, n=45 (WT)/43 (mdx). 0.0 Conclusions and Future Directions WT MDX WT MDX Myoblast Myotube It has been previously demonstrated that mdx muscle has mitochondrial-related deficits including decreased spare respiratory capacity1, complex 1 mediated ATP production2, and mitochondrial Figure 1: Membrane potential was determined with JC-10 membrane potential assay (Abcam). 3.3μM FCCP was used to ablate 3 membrane potential as a negative control. Statistical significance between WT and mdx was determined by unpaired T-test; M d x m y o b la s ts e x h ib it re d u c e d biomass . However, it was unclear when these deficits manifest in the muscle and what the ****p<0.0001, myoblast n=32, myotube n=8. underlying defect(s) in mitochondrial biology might be.

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M d x m y o b la s ts d e m o n s tra te 1 .5 We have demonstrated that the mdx-derived muscle cells exhibit significant mitochondrial defects W

and that these defects are apparent before myogenesis has completed. These defects appear to re d u c e d N A D + le v e ls

m have a direct impact on cell physiology, as we observe reduced cellular ATP and inefficient fatty

5 o acid utilization. The functional deficit in mitochondria could be due to reduced mitochondrial

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0 .0 References

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o F **** F W T M D X W T M D X 0 1Schuh RA, et al. Am J Physiol Regul Integr Comp Physiol. 302(6):R712-9. 2012. l 6 C o p y N u m b e r C itr a te S y n th a s e 2 o N 6 Rybalka E, et al. PLoS One. 9(12):e115763. 2014. tr M 8 3 n N K Figure 5: Mitochondrial number was determined two ways. For copy number, gDNA was extracted and analyzed by Godin R, et al. J. Physiol. 590(21):5487-502. 2012. o F C determining the ratio of 16s(mito)/HK2(nuclear). was determined by using protein lysate in the Citrate Synthase Assay Kit (Sigma). Significant difference between WT and mdx copy number was determined with unpaired T-test; Figure 2: Myoblasts were treated for 24h with 1mM NMN or 10nM FK866. The myoblasts were then analyzed using the NAD- **p<0.025. Significant difference between WT and mdx CS was determined by Mann-Whitney test; ****p<0.0001, copy number Glo Assay (Promega). Significant difference between WT and mdx was determined by Mann-Whitney Test; ****p<0.0001, n=44. n=5 (one pair of mice), citrate synthase n=40.