Proteomic and transcriptomic changes in hibernating grizzly bears reveal mechanisms that protect against muscle atrophy. Douaa A. Mugahid, Tutku G. Sengul, Xintian You, Yongbo Wang, Leif Steil, Nora Bergmann, Michael H. Radke, Andreas Ofenbauer, Manuela Gesell-Salazar, Andras Balogh, Stefan Kempa, Baris Tursun, Charles T. Robbins, Uwe Völker, Wei Chen, Lynne Nelson, Michael Gotthardt Supplement: Figures S1-S4 Tables S1-S4 1 Figures Supplement Figure 1, related to Figure 1 Fig. S1: Mapping changes in mRNA levels of insulin signaling. Pathway during hibernation (adapted from KEGG). Genes with decreased expression are depicted in blue, with increased expression in red. 2 Supplement Figure 2, related to Figure 3 Fig. S2: Changes in mRNA levels in aging skeletal muscle indicate alterations in metabolic regulation. (a) KEGG pathways with mRNAs regulated in aging female muscle. In red are the adjusted p-values per term. (b) Transcriptional changes in central glucose metabolism in aging muscle. 3 Supplement Figure 3, related to Figure 6 Fig. S3: Validation of the shRNA mediated knock-down in C2C12 cells. Protein expression was normalized to γ- tubulin. (a) Pdk4 protein and mRNA levels were reduced to <50% after shRNA treatment (n=3, mean± s.e.m., t- test, ***P < 0.001). (b) Serpinf1 protein and mRNA levels were reduced to <25% after shRNA treatment (n=3, mean± s.e.m., t-test, *P < 0.05, ***P < 0.001). (c) Over-expressing eGFP-tagged Pdk4 or Serpinf1 leads to a decrease in cell size (FSC-A) compared to eGFP controls (n=10000, mean± s.e.m., Bonferroni post-test, ***P < 0.001) 4 Supplement Figure 4, related to Figure 4 and S3 Fig. S4: Original gels for western blots indicating the cropped area. (a) Western Gels Figure 4g. (b) Western Gels Figure 4j. (c) Western Gels Figure S3a. (d) Western Gels Figure S3b. 5 Tables Table S1: Proteins quantified by Mass Spectrometry (separate pdf), Excel file available Table S2: Transcripts quantified by RNA seq (separate pdf), Excel file available Table S3: KEGG pathway enrichment analyses (separate pdf), Excel file available Table S4: Genes differentially regulated in hibernation relate to striated muscle growth. Gene OMIM Summary Human Relevance RASSF1 605082 Increased expression in patients with heart Heart failure, failure. Larger hearts in Rassf1 knockout cardiac mice after TAC-surgery to induce pressure hypertrophy overload.1 MYL3 16079 Essential myosin light chain. Mutations Cardiac have been associated with cardiac hypertrophy, hypertrophy and skeletal muscle disease.2,3 skeletal muscle disease CYC1 123980 Component of the electron transfer chain Skeletal myopathy, (ETC). A mutation in CYC1 reduces growth retardation protein stability in muscle of a patient with growth retardation.4 MYBPC1 160794 Myosin binding protein. Mutations are Distal associated with Distal arthrogryposis type I, arthrogryposis type characterized by reduced muscle mass.5 I, muscle atrophy TNNI3 191044 Associated with hypertrophic Hypertrophic cardiomyopathy.6 cardiomyopathy RNF11 612598 Involved in NFKB signaling.7,8 n.a. PPIA 123840 Encodes Cyclophilin A, which is secreted n.a. in response to increased ROS production. Knockout mice have smaller hearts in response to high ROS levels.9 PA2G4 602145 Knockouts are smaller with reduced n.a. circulating IGF-1.10 RORA 600825 Involved in circadian regulation and n.a. induces Bmal1 expression,11,12 which affects muscle function and size, increases PDK4 while reducing Pdha activity.13 ARID5B 608538 Decreased genomic methylation has been n.a. associated with reduced birth weight.14 NRG2 603818 Growth retardation in knockout mice.15 n.a. SERPINF1 172860 Enhances NFB signaling.16 n.a. 6 References 1. Oceandy, D. et al. Tumor suppressor Ras-association domain family 1 isoform A is a novel regulator of cardiac hypertrophy. Circulation 120, 607–616 (2009). 2. Poetter, K. et al. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle. Nat. Genet. 13, 63–69 (1996). 3. Olson, T. M., Karst, M. L., Whitby, F. G. & Driscoll, D. J. Myosin light chain mutation causes autosomal recessive cardiomyopathy with mid-cavitary hypertrophy and restrictive physiology. Circulation 105, 2337–2340 (2002). 4. Gaignard, P. et al. Mutations in CYC1, encoding cytochrome c1 subunit of respiratory chain complex III, cause insulin-responsive hyperglycemia. Am. J. Hum. Genet. 93, 384–389 (2013). 5. Gurnett, C. A. et al. Myosin binding protein C1: a novel gene for autosomal dominant distal arthrogryposis type 1. Hum. Mol. Genet. 19, 1165–1173 (2010). 6. Kimura, A. et al. Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat. Genet. 16, 379–382 (1997). 7. Shembade, N., Parvatiyar, K., Harhaj, N. S. & Harhaj, E. W. The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-kappaB signalling. EMBO J. 28, 513–522 (2009). 8. Maddirevula, S., Anuppalle, M., Huh, T.-L., Kim, S. H. & Rhee, M. Rnf11-like is a novel component of NF-κB signaling, governing the posterior patterning in the zebrafish embryos. Biochem. Biophys. Res. Commun. 422, 602–606 (2012). 9. Satoh, K. et al. Cyclophilin A promotes cardiac hypertrophy in apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 31, 1116–1123 (2011). 10. Zhang, Y. et al. Alterations in cell growth and signaling in ErbB3 binding protein-1 (Ebp1) deficient mice. BMC Cell Biol. 9, 69 (2008). 7 11. Akashi, M. & Takumi, T. The orphan nuclear receptor RORalpha regulates circadian transcription of the mammalian core-clock Bmal1. Nat. Struct. Mol. Biol. 12, 441–448 (2005). 12. Sato, T. K. et al. A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron 43, 527–537 (2004). 13. Dyar, K. A. et al. Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock. Mol Metab 3, 29–41 (2014). 14. Engel, S. M. et al. Neonatal genome-wide methylation patterns in relation to birth weight in the Norwegian Mother and Child Cohort. Am. J. Epidemiol. 179, 834–842 (2014). 15. Britto, J. M. et al. Generation and characterization of neuregulin-2-deficient mice. Mol. Cell. Biol. 24, 8221–8226 (2004). 16. Yabe, T., Sanagi, T., Schwartz, J. P. & Yamada, H. Pigment epithelium-derived factor induces pro-inflammatory genes in neonatal astrocytes through activation of NF-kappa B and CREB. Glia 50, 223–234 (2005). Supplementary table S1: Proteins quantified by Mass Spectrometry Primary.Protein.Name Sybmol Protein.Description SA_Active A_Active C2_Active C1_Active SA_Hibernation A_Hibernation C2_Hibernation C1_Hibernation p‐value Activity p‐value Age p‐value Interaction PRDX3_HUMAN PRDX3 Thioredoxin‐dependent peroxide reductase, mitochondrial OS=Homo sapiens GN=PRDX3 PE=1 SV=3 161482.2 117631.2 167560.9 149663.9 70567.58 89352.1 88619.39 96890.27 8.27E‐03 2.85E‐01 8.20E‐01 FKBP3_HUMAN FKBP3 Peptidyl‐prolyl cis‐trans isomerase FKBP3 OS=Homo sapiens GN=FKBP3 PE=1 SV=1 22601.64 24929.97 20030.69 18531.02 28560.94 25989.35 25795.3 22983.8 2.16E‐02 3.52E‐02 5.34E‐01 NDUS2_HUMAN NDUFS2 NADH dehydrogenase [ubiquinone] iron‐sulfur protein 2, mitochondrial OS=Homo sapiens GN=NDUFS2 PE=1 SV 41785.93 43633.75 31596.21 34064.82 23385.2 19089.09 41156.4 36979.53 1.06E‐02 7.77E‐02 1.19E‐03 G6PI_HUMAN GPI Glucose‐6‐phosphate isomerase OS=Homo sapiens GN=GPI PE=1 SV=4 540308 334053 216598.8 231263.7 534012.75 657969.12 440073.97 383743.84 4.87E‐02 3.26E‐02 8.25E‐01 ODPB_HUMAN PDHB Pyruvate dehydrogenase E1 component subunit beta, mitochondrial OS=Homo sapiens GN=PDHB PE=1 SV=3 882959.9 831176.5 708099.3 884739 648777.12 592426.31 610186.25 574431.69 1.08E‐02 4.15E‐01 7.58E‐01 LEG1_HUMAN LGALS1 Galectin‐1 OS=Homo sapiens GN=LGALS1 PE=1 SV=2 112953.4 130414.6 147095.7 143857.1 204433.64 199484.25 209957.11 172125.33 3.93E‐03 5.74E‐01 1.74E‐01 CAZA2_HUMAN CAPZA2 F‐actin‐capping protein subunit alpha‐2 OS=Homo sapiens GN=CAPZA2 PE=1 SV=3 130210.4 135367.3 133938.3 112673.2 102768.62 98811.41 91443.65 94306 5.09E‐03 1.96E‐01 8.96E‐01 CATA_HUMAN CAT Catalase OS=Homo sapiens GN=CAT PE=1 SV=3 23430.05 16023.12 28397.99 32902.75 45158.78 60153.01 50919.24 73116.64 1.03E‐02 2.23E‐01 9.17E‐01 SCG2_HUMAN SCG2 Secretogranin‐2 OS=Homo sapiens GN=SCG2 PE=1 SV=2 10295.79 6845.84 9899.9 10008.21 64792.81 95403.58 18074.76 4606.31 1.23E‐02 1.60E‐02 1.40E‐02 NDRG2_HUMAN NDRG2 Protein NDRG2 OS=Homo sapiens GN=NDRG2 PE=1 SV=2 99327.66 145680 56727.93 64999.53 310046.12 198522.81 245779.12 228472.66 7.29E‐03 2.67E‐01 5.07E‐01 IMA4_HUMAN KPNA4 Importin subunit alpha‐4 OS=Homo sapiens GN=KPNA4 PE=1 SV=1 3192.11 7044.49 3246.44 2809.08 10685.01 10039.32 6720.41 9596.3 1.31E‐02 1.53E‐01 9.65E‐01 PHS_HUMAN PCBD1 Pterin‐4‐alpha‐carbinolamine dehydratase OS=Homo sapiens GN=PCBD1 PE=1 SV=2 16859.67 13996.25 15622.82 10919.19 51930.26 25551.42 35456.54 30619.6 3.46E‐02 5.97E‐01 8.08E‐01 CH10_HUMAN HSPE1 10 kDa heat shock protein, mitochondrial OS=Homo sapiens GN=HSPE1 PE=1 SV=2 58697.41 33825.93 92876.98 79033.93 29859.24 29427.82 40000.32 34480.71 1.08E‐02 3.10E‐02 9.12E‐02 PCSK1_HUMAN PCSK1N ProSAAS OS=Homo sapiens GN=PCSK1N PE=1 SV=1 36593.58 25465.44 21479.35 17178.29 57192.74 55946.88 22005.87 23914.88 8.63E‐03 1.72E‐03 2.26E‐02 IDH3G_HUMAN IDH3G Isocitrate dehydrogenase [NAD] subunit gamma, mitochondrial OS=Homo sapiens GN=IDH3G PE=1 SV=1 130471.7 129124.2 93678.37 125692.4 86674.2 80367.52 95099.37 103780.52 2.87E‐02 8.16E‐01 9.99E‐02 CISY_HUMAN CS Citrate synthase, mitochondrial OS=Homo sapiens GN=CS PE=1 SV=2 559682.4 521152.8 386764.9