Cell Metabolism, Volume 27 Supplemental Information Metabolic Maturation during Muscle Stem Cell Differentiation Is Achieved by mir-1/133a-mediated Inhibition of the Dlk1-Dio3 Mega Gene Cluster Stas Wüst, Stefan Dröse, Juliana Heidler, Ilka Wittig, Ina Klockner, Andras Franko, Erik Bonke, Stefan Günther, Ulrich Gärtner, Thomas Boettger, and Thomas Braun
Figure S1: Dlk1-Dio3 cluster mirnas are differentially expressed in freshly isolated MuSC and differentiated myotubes and induced by MEF2A Related to Figure 1. (A) RNA seq of males mice reveals strong and specific expression of Dlk1-Dio3 cluster genes in freshly isolated MuSCs (FI), which after massive reduction in proliferating MuSCs (P) are re-expressed at low levels in myotubes (M). (B-C) Affymetrix microarray-based GSEA (C2 gene sets) of proliferating versus freshly isolated MuSC (from male mice) (B); and myotubes versus freshly isolated MuSC (from male mice) (C). (D) GSEA (C3 gene sets) of myotubes versus freshly isolated MuSC. (E) RT-qPCR expression analysis of the Dlk1-Dio3 cluster lncrnas Rian and Mirg1 in freshly isolated MuSC and myotubes after 6 days in differentiation medium. (n=3/3 biological replicates (1 male, 2 females/1 male, 2 females); Mann- Whitney U test, one-tailed, *p=0.05) (F) Relative expression levels of mir-1 and mir-206 obtained by mirna seq in different tissues. (G-I) RT-qPCR expression analysis of Dlk1-Dio3 cluster components after forced expression of Mef2A (black bar) and Mef2A-dE4 (grey bars) in mouse embryonic fibroblasts (MEF) (G) C2C12 (H) and Sol8 (I) muscle cells (n=4/4/4 independent transfections; Mann- Whitney U test, one-tailed; *p<0.05).
Figure S2: mir-1/133a expression reduces MEF2a levels in vitro and maintains skeletal muscle mass in vivo Related to Figure 2. (A) Western blot analysis of MEF2A levels after transfection of mir-1, mir-133a or mir-1 and mir-133a into differentiating C2C12 cells. (RAL A serves as loading control; n=4/4 (males), Mann-Whitney U test; two-tailed; *p<0.05). (B) Scheme for conditional deletion of the mir-1-2/133a-1 gene. (C) RT-qPCR expression analysis of Mib1 (n=3/3/3 (males); Mann-Whitney U test, two-tailed) using primers located at the exons flanking the mirna cluster (mir-1/133a dko: mir-1-1/133a-2 -/- //mir-1-2/133a-1 fl/fl //Pax7-Cre + ) muscle compared to control muscle (Ctrl: mir-1-1/133a-2 +/+ //mir-1-2/133a-1 fl/fl //Pax7-Cre - ) and Pax7-Cre + control mice (Cre WT/tg = mir-1-1/133a-2 +/+ //mir-1-2/133a-1 wt/wt //Pax7-Cre + ) (D) Tibia length of mir-1/133a dko and ctrl mice at 12 weeks (n=7/8 (males); student s t-test; two-tailed).
Figure S3: Inactivation of mir-1/133a in skeletal muscle increases expression of components of the Dlk1-Dio3 cluster Related to Figure 2 and Figure 3. (A) List of Dlk1-Dio3 cluster mirs upregulated in mir-1/133a dko compared to ctrl TA muscles (Affymetrix microarray analysis n = 3/3 (males); log 2 scale; students t-test, two-tailed, significant differences marked in red; p<0.05). (B) RTqPCR expression analysis of Meg3, Mirg and Rian relative to Rplp1 using RNA isolated from TA muscles of ctrl and mir-1/133a dko mice (n=3/3 (males); Mann-Whitney U test; one-tailed; *p=0.05). (C) RT-qPCR expression analysis of Dlk1, Rtl1 and Dio3 relative to Rplp1 using RNA isolated from TA muscles of ctrl and mir-1/133a dko mice (n=3/3 (males); Mann-Whitney U test; one-tailed, not significant). (D) Cross sections of soleus and EDL muscles from ctrl and mir-1/133a dko mice (females) immunostained for slow muscle myosin. Scale bar: 30 µm. (E) Quantification of slow muscle myosin positive muscle fibers in 12 weeks old control and mir-1/133a dko mice (> 350 fibres/animal were counted; n=3/5 (males); Mann-Whitney U test; two-tailed, not significant) (F) Quantification of fibers with central nuclei in male control and dko mice at 12 weeks (>350, 400 and 1500 fibers/animal were counted for soleus, EDL and TA muscles respectively; n=3/5 (1 male, 2 females/2 males, 3 females); Mann-Whitney U test; one-tailed; *p<0.05). (G) Statistical analysis of oxygraph measurements of isolated EDL fibers for CI (complex I), CII (complex II) and L (leakage respiration). Mean rates and standard error are shown (n=4/4 (males); Mann-Whitney U test; twotailed, *p<0.05).
Figure S4: Loss of mir-1/133a in the skeletal muscle lineage disrupts the mitochondrial network but does not affect MuSC numbers and differentiation Related to Figure 4. (A) Immunofluorescence-based assessment of the number of Pax7 + -MuSC isolated from muscles of mir- 1/133a dko and ctrl mice 3d or 6d after induction of differentiation (n=3/3 (females), Mann-Whitney U test, two-tailed.). (B) Immunofluorescence-based assessment of the number of myogenin + -MuSC from mir-1/133a dko and ctrl muscles 3d and 6d after induction of differentiation (n=3/3 (females); Mann-Whitney U test; two-tailed). (C) RT-qPCR expression analysis of Meg3, Rian and Mirg in differentiating MuSC isolated from mir-1/133a dko and ctrl mice 6d after induction of differentiation (n=4/4 (females), Mann-Whitney U test; two-tailed; *p<0.05). (D) Immunofluorescence staining (MF20 antibody, green) of differentiating MuSC (from female mice), 3d and 6d after induction of differentiation. Nuclei were stained with DAPI (blue). Scale bar: 50 µm. (E) SDH staining of differentiating MuSC from female ctrl and mir-1/133a dko mice 3d and 6d after induction of differentiation The arrowhead points to mitochondrial aggregates in mir-1/133a dko-muscs. Scale bar: 25 µm. (F) SDH staining of WT female MuSCs transfected with mirnas mir-485, -493, and 543 or control mir 6 days after induction of differentiation. The arrowhead points to mitochondrial aggregates in transfected MuSCs. Scale bar: 25 µm. (G) RT-qPCR expression analysis of Opa1 and Mfn1 in WT MuSCs transfected with mirnas mir-485, -493, and 543 or control mir six days after induction of differentiation (n=3/3 (females); Mann-Whitney U test; one-tailed; *p=0.05).
Figure S5: Loss of mir-1/133a and up-regulation of Dlk1-Dio3 cluster mirnas do not repress the mtor pathway in skeletal muscle cells Related to Figure 4. (A, B) GO-term enrichment analysis of predicted mir-1 (A) and mir-133a (B) target genes using microarray data obtained from control and mir-1/133a dko TA muscles (n=3/3 (males)). (C) Microarray based analysis (Affymetrix) of target genes and pathways described to be regulated by mir-1/133a or involved in mitochondrial activity (n=3/3 (males), Mann-Whitney U test; *p=0.05) (D) Depiction of a potential mir-1 target site within the 3 UTR of Slc25a23 mrna (microrna.org, release 2010-11-01, mirsvr score: -0.1156, PhastCons score: 0.5265). (E) RT-qPCR expression analysis of Slc25a23 mrna in dko and control TA muscles (n=3/3 (males); Mann-Whitney U test; one-tailed; *p=0.05). (F, G) Western blot analysis of target genes and pathways described to be regulated by mir-1/133a (n=3/3, Mann-Whitney U test, not significant). (H) Comparative analysis of changes in the expression of mtor pathway-components in mir-1/133a dko and Mef2A-tg muscles using transcriptome (Affymetrix microarrays) and proteome data (quantitative mass spectrometry), (Affy: n=3/3 (males) (dko/ctrl), n=3/4 (males) (Mef2A-tg/ctrl), Students t-test; Proteome: n=6/6 (males) (mir-1/133a dko/ctrl); n=4/4 (males) (Mef2A-tg/ctrl), Mann-Whitney U test, one-tailed; significant [p<0.05] differences marked in red). (I) Quantification of centralized nuclei (n=3/3 (males); 230 fibers per animal were counted). (J) Quantification of mitochondrial structure by TEM. (Mitochondria counted per animal: ctrl: 52, 62, 22; dko: 55, 66, 63; Mef2A-tg: 44, 40, 65; n=3/3/3 (males); *p=0.05; Mann- Whitney U test; one-tailed). (K) Statistical analysis of oxygraph measurements of isolated EDL fibers for CI (complex I), CII (complex II) and L (leakage respiration). Mean rates and standard deviations are shown (n=5/4 (males); Mann-Whitney U test, two-tailed; **p<0.01,). (L) Western blot analysis of 4E-BP1 (Ser65) and p70 S5K (Thr389) phosphorylation in ctrl, mir-1/133a dko and Mef2A-tg TA muscles using RAL A as loading control (n=5/4/4 (males); Mann-Whitney U test; one-tailed; *p<0.05, **p<0.01). (M) Western blot analysis of AKT phosphorylation in ctrl, mir-1/133a dko and Mef2A-tg TA muscles using RAL A as loading control (n=5/4/4 (males); Mann-Whitney U test).
Figure S6: Loss of mir-1/133a but not increased expression of Mef2A augments Dynamin-2 expression levels -Related to Figure 5. (A-B) Western blot analysis of DNM2 levels in mir-1/133a dko and ctrl TA muscles (A) and in Mef2A-tg and ctrl TA muscles (B), (n=3/3 (males); Mann- Whitney U test; one-tailed; *p=0.05). (C) Comparative analysis of changes in the expression of mitochondrial coded proteins in mir-1/133a dko and Mef2A-tg muscles using transcriptome (Affymetrix microarrays) and proteome data (quantitative mass spectrometry), (Affy mir-1/133a dko: n=3/3 (males), Mef2A-tg: n=3/4 (males), student s t-test; Proteome: mir-1/133a dko n=6/6, Mef2A-tg n=4/4 (males), Mann-Whitney U test, one-tailed). (D, E) Western blot analysis of cytochrome c oxidase (COX1) and ND1 (NADH dehydrogenase) levels in ctrl, mir-1/133a dko and Mef2A-tg TA muscles (n=5/4/4 (males); Mann-Whitney U test; one-tailed; *p<0.05, **p<0.01).
Figure S7: Forced expression of Mef2A increases expression of Dlk1-Dio3 cluster components and disturbs the mitochondrial network in differentiating MuSC -Related to Figure 7. (A, B) Western blot analysis of MEF2A levels in isolated control and Mef2A-tg MuSCs 6d after induction of differentiation (n=4/4 (males); *p<0.05; student s t-test; two-tailed; *p<0.05). (C) Succinate dehydrogenase (SDH) staining of isolated control and Mef2A-tg MuSCs (from male mice) 6d after induction of differentiation. Scale bar 25µm (D) RT-qPCR expression analysis of the Dlk1-Dio3 cluster lncrnas Meg3, Rian and Mirg in isolated control and Mef2A-tg MuSCs 6d after induction of differentiation (n=4/4 (males); Mann-Whitney U test; two-tailed; *p<0.05).