Journal: Medicine and Science in Sports and Exercise

Article Title: Sex-Specific Effect of CARM1 in Skeletal Muscle Adaptations to Exercise

doi: 10.1249/MSS.0000000000003333

Figure Lengend Snippet: Functional characterization of CARM1 skeletal mKO mice after chronic exercise. A. Daily VWR distance (km) of female (F) and male (M) WT mice and their age-matched CARM1 mKO littermates. B. Graphical summary of the average distance mice ran per day over the 8 wk they had access to a running wheel. Bars indicate group means with F and M values combined, whiskers represent SEMs, points illustrate individual results, and dashed lines denote sex-specific means (i.e., F, pink ; M, blue ). Statistical analysis was completed using an ANOVA and Tukey’s post hoc test. S, P < 0.05 main effect of sex; † P < 0.05 vs opposite sex same experimental group. n = 8–23. Graphical summaries of all-limb strength (C), all-limb fatigue (D), rotarod speed (E), rotarod time (F), as well as open-field test metrics, including distance traveled (G), average speed (H), ambulatory time (I), number of rearing events (L), and body mass (K) in F and M WT and mKO mice that were sedentary (SED) or VWR. Bars indicate group means with sexes combined, whiskers represent SEMs, points illustrate individual results, and dashed lines demonstrate sex-specific means. Statistical analysis was completed using an ANOVA and Tukey’s post hoc test. E, P < 0.05 main effect of exercise; G, P < 0.05 main effect of genotype; S, P < 0.05 main effect of sex; † P < 0.05 vs opposite sex within the same experimental group; * P < 0.05 vs WT SED. n = 3–14. Graphical summaries of TA (L), SOL (M), and GAST (N) muscle mass. Bars indicate group means with F and M values combined, whiskers represent SEMs, points illustrate individual results, and the dashed lines denote sex-specific means. Data are normalized to body mass and displayed relative to WT SED. Statistical analysis was completed using an ANOVA and Tukey’s post hoc test. E, P < 0.05 main effect of exercise; G, P < 0.05 main effect of genotype; S, P < 0.05 main effect of sex; E:G, P < 0.05 interaction effect between exercise and genotype; E:S P < 0.05 interaction effect between exercise and sex; † P < 0.05 vs opposite sex within the same experimental group; * P < 0.05 vs WT SED; # P < 0.05 vs WT VWR; % P < 0.05 vs mKO SED. n = 7–26.

Article Snippet: The following antibodies were used: CARM1 (1:5000; A300-421A; Bethyl Laboratories, Montgomery, TX), BRG1-associated factor 155 (BAF155) asymmetric dimethylation at arginine 1064 (mBAF155 Arg1064 , 1:1000; 94962; Cell Signaling, Danvers, MA), BAF155 (1:150; 11956S; Cell Signaling), mPABP1 Arg455/460 (1:1000; 3505S; Cell Signaling), polyadenylate-binding protein 1 (PABP1) asymmetric dimethylation at arginine 455/460 (mPABP1 Arg455/460 , 1:1000; 4992S; Cell Signaling), CARM1 substrate (1:1000; a kind gift from Dr. Mark Bedford, MD Anderson Cancer Center, University of Texas ( , , ), PRMT1 (1:5000; 07–404; EMD Millipore, Darmstadt, HE, Germany), PRMT5 (1:1000; 507–405; EMD Millipore), PRMT6 (1:5000; A300-929A; Bethyl laboratories), PRMT7 (1:100; Sc-376077; Santa Cruz, Santa Cruz, CA), MMA (1:1000; 8015; Cell Signaling), ADMA at glycine and arginine-rich sequences (1:200; 13522; Cell Signaling), SDMA (1:200; 13222; Cell Signaling), PGC-1α (1:1000; AB3242; EMD Millipore), BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3, 1:1000; 3769; Cell signaling), mitochondrial fission 1 protein (FIS1, 1:1000; 109516–1-AP; Proteintech, Rosemont, IL), optic atrophy-1 (OPA1, 1:1000; ab42364; Abcam, Cambridge, UK), total oxidative phosphorylation (OXPHOS, 1:1000; ab110413; Abcam), and citrate synthase (CS) (1:1000, ab96600, Abcam).

Techniques: Functional Assay

Journal: Medicine and Science in Sports and Exercise

Article Title: Sex-Specific Effect of CARM1 in Skeletal Muscle Adaptations to Exercise

doi: 10.1249/MSS.0000000000003333

Figure Lengend Snippet: Effect of long-term exercise on CARM1 activity in skeletal muscle. A. Representative Western blots of CARM1 (short and long exposure), BRG1-associated factor 155 (BAF155) asymmetric dimethylation at arginine 1064 (mBAF155 Arg1064 ), total BAF155 (top band, indicated by arrow), polyadenylate-binding protein 1 (PABP1) asymmetric dimethylation at arginine 455/460 (mPABP1 Arg455/460 ), total PABP1, and arginine methylated CARM1 substrates in TA muscles from F and M WT and mKO SED and VWR animals. The ponceau stain demonstrates equal loading while approximate molecular weights (MW, in kDa) are to the right of each image. Dashed lines denote where images were cropped to rearrange nonadjacent lanes from the same immunoblot. Graphical summaries of CARM1 (B), mBAF155 Arg1064 (C), BAF155 (D), BAF155 methylation status (E), calculated as the levels of mBAF155 Arg1064 relative to BAF155 content, mPABP1 Arg455/460 (F), PABP1 (G), PABP1 methylation status (H), and arginine methylated CARM1 substrates (I). Bars indicate group means with sexes combined, whiskers represent SEMs, points illustrate individual results, and dashed lines demonstrate sex-specific means. Data are expressed relative to WT SED. Statistical analysis was completed using an ANOVA and Tukey’s post hoc test. E, P < 0.05 main effect of exercise; G, P < 0.05 main effect of genotype; S, P < 0.05 main effect of sex; E:G, P < 0.05 interaction effect between exercise and genotype; * P < 0.05 vs WT SED; # P < 0.05 vs WT VWR. n = 8–26.

Article Snippet: The following antibodies were used: CARM1 (1:5000; A300-421A; Bethyl Laboratories, Montgomery, TX), BRG1-associated factor 155 (BAF155) asymmetric dimethylation at arginine 1064 (mBAF155 Arg1064 , 1:1000; 94962; Cell Signaling, Danvers, MA), BAF155 (1:150; 11956S; Cell Signaling), mPABP1 Arg455/460 (1:1000; 3505S; Cell Signaling), polyadenylate-binding protein 1 (PABP1) asymmetric dimethylation at arginine 455/460 (mPABP1 Arg455/460 , 1:1000; 4992S; Cell Signaling), CARM1 substrate (1:1000; a kind gift from Dr. Mark Bedford, MD Anderson Cancer Center, University of Texas ( , , ), PRMT1 (1:5000; 07–404; EMD Millipore, Darmstadt, HE, Germany), PRMT5 (1:1000; 507–405; EMD Millipore), PRMT6 (1:5000; A300-929A; Bethyl laboratories), PRMT7 (1:100; Sc-376077; Santa Cruz, Santa Cruz, CA), MMA (1:1000; 8015; Cell Signaling), ADMA at glycine and arginine-rich sequences (1:200; 13522; Cell Signaling), SDMA (1:200; 13222; Cell Signaling), PGC-1α (1:1000; AB3242; EMD Millipore), BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3, 1:1000; 3769; Cell signaling), mitochondrial fission 1 protein (FIS1, 1:1000; 109516–1-AP; Proteintech, Rosemont, IL), optic atrophy-1 (OPA1, 1:1000; ab42364; Abcam, Cambridge, UK), total oxidative phosphorylation (OXPHOS, 1:1000; ab110413; Abcam), and citrate synthase (CS) (1:1000, ab96600, Abcam).

Techniques: Activity Assay, Western Blot, Binding Assay, Methylation, Muscles, Staining

Journal: Medicine and Science in Sports and Exercise

Article Title: Sex-Specific Effect of CARM1 in Skeletal Muscle Adaptations to Exercise

doi: 10.1249/MSS.0000000000003333

Figure Lengend Snippet: PRMT expression and function in CARM1 mKO mice. A. Typical Western blots for PRMT1, PRMT5, PRMT6, PRMT7, as well as MMA-, ADMA-, and SDMA-marked proteins in TA muscles from F and M WT and mKO SED and VWR animals. The ponceau stain demonstrates equal loading while approximate molecular weight (MW, kDa) are to the right of each image. The dashed lines illustrate where representative immunoblots were cropped allowing rearrangement of lanes. Graphical summaries of PRMT1 (B), PRMT5 (C), PRMT6 (D), PRMT7 (E), MMA (F), ADMA (G), and SDMA (H) in TA muscles of mice from all experimental groups. Bars indicate group means with sexes combined, whiskers represent SEMs, points illustrate individual results, and dashed lines demonstrate sex-specific means. Data are expressed relative to WT SED. Statistical analysis was completed using an ANOVA and Tukey’s post hoc test. G, P < 0.05 main effect of genotype; S, P < 0.05 main effect of sex; E:S, P < 0.05 interaction effect between exercise and sex; * P < 0.05 vs WT SED. n = 8–26.

Article Snippet: The following antibodies were used: CARM1 (1:5000; A300-421A; Bethyl Laboratories, Montgomery, TX), BRG1-associated factor 155 (BAF155) asymmetric dimethylation at arginine 1064 (mBAF155 Arg1064 , 1:1000; 94962; Cell Signaling, Danvers, MA), BAF155 (1:150; 11956S; Cell Signaling), mPABP1 Arg455/460 (1:1000; 3505S; Cell Signaling), polyadenylate-binding protein 1 (PABP1) asymmetric dimethylation at arginine 455/460 (mPABP1 Arg455/460 , 1:1000; 4992S; Cell Signaling), CARM1 substrate (1:1000; a kind gift from Dr. Mark Bedford, MD Anderson Cancer Center, University of Texas ( , , ), PRMT1 (1:5000; 07–404; EMD Millipore, Darmstadt, HE, Germany), PRMT5 (1:1000; 507–405; EMD Millipore), PRMT6 (1:5000; A300-929A; Bethyl laboratories), PRMT7 (1:100; Sc-376077; Santa Cruz, Santa Cruz, CA), MMA (1:1000; 8015; Cell Signaling), ADMA at glycine and arginine-rich sequences (1:200; 13522; Cell Signaling), SDMA (1:200; 13222; Cell Signaling), PGC-1α (1:1000; AB3242; EMD Millipore), BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3, 1:1000; 3769; Cell signaling), mitochondrial fission 1 protein (FIS1, 1:1000; 109516–1-AP; Proteintech, Rosemont, IL), optic atrophy-1 (OPA1, 1:1000; ab42364; Abcam, Cambridge, UK), total oxidative phosphorylation (OXPHOS, 1:1000; ab110413; Abcam), and citrate synthase (CS) (1:1000, ab96600, Abcam).

Techniques: Expressing, Western Blot, Muscles, Staining, Molecular Weight

Journal: Medicine and Science in Sports and Exercise

Article Title: Sex-Specific Effect of CARM1 in Skeletal Muscle Adaptations to Exercise

doi: 10.1249/MSS.0000000000003333

Figure Lengend Snippet: Mitochondria-related protein expression in CARM1 mKO mice. (A) Typical Western blots of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), mitochondrial fission 1 protein (FIS1), optic atrophy-1 (OPA1), representative subunits of oxidative phosphorylation (OXPHOS) complexes I (CI)-CV, and citrate synthase (CS) in TA muscles from F and M WT and mKO SED and VWR mice. Ponceau stains demonstrate equal loading while approximate molecular weight (MW, kDa) are to the right of each image. Dashed lines denote where images were cropped to rearrange nonadjacent lanes from the same immunoblot. Graphical summaries of PGC-1α (B), BNIP3 (C), FIS1 (D), OPA1 (E), OXPHOS (F), and CS protein (G) contents. Bars indicate group means with sexes combined, whiskers represent SEMs, points illustrate individual results, and dashed lines demonstrate sex-specific means. Data are expressed relative to WT SED. Statistical analysis was completed using an ANOVA and Tukey’s post hoc test. G, P < 0.05 main effect of genotype; E:S, P < 0.05 interaction effect between exercise and sex; G:S, P < 0.05 interaction effect between genotype and sex. * P < 0.05 vs WT SED; # P < 0.05 vs WT VWR. n = 8–25. ( H ) CS activity in the GAST muscles of mice from all experimental groups. Bars indicate group means with sexes combined, whiskers represent SEMs, points illustrate individual results, and dashed lines demonstrate sex-specific means. Data are expressed relative to WT SED. Statistical analysis was completed using an ANOVA and Tukey’s post hoc test. n = 7–8.

Article Snippet: The following antibodies were used: CARM1 (1:5000; A300-421A; Bethyl Laboratories, Montgomery, TX), BRG1-associated factor 155 (BAF155) asymmetric dimethylation at arginine 1064 (mBAF155 Arg1064 , 1:1000; 94962; Cell Signaling, Danvers, MA), BAF155 (1:150; 11956S; Cell Signaling), mPABP1 Arg455/460 (1:1000; 3505S; Cell Signaling), polyadenylate-binding protein 1 (PABP1) asymmetric dimethylation at arginine 455/460 (mPABP1 Arg455/460 , 1:1000; 4992S; Cell Signaling), CARM1 substrate (1:1000; a kind gift from Dr. Mark Bedford, MD Anderson Cancer Center, University of Texas ( , , ), PRMT1 (1:5000; 07–404; EMD Millipore, Darmstadt, HE, Germany), PRMT5 (1:1000; 507–405; EMD Millipore), PRMT6 (1:5000; A300-929A; Bethyl laboratories), PRMT7 (1:100; Sc-376077; Santa Cruz, Santa Cruz, CA), MMA (1:1000; 8015; Cell Signaling), ADMA at glycine and arginine-rich sequences (1:200; 13522; Cell Signaling), SDMA (1:200; 13222; Cell Signaling), PGC-1α (1:1000; AB3242; EMD Millipore), BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3, 1:1000; 3769; Cell signaling), mitochondrial fission 1 protein (FIS1, 1:1000; 109516–1-AP; Proteintech, Rosemont, IL), optic atrophy-1 (OPA1, 1:1000; ab42364; Abcam, Cambridge, UK), total oxidative phosphorylation (OXPHOS, 1:1000; ab110413; Abcam), and citrate synthase (CS) (1:1000, ab96600, Abcam).

Techniques: Expressing, Western Blot, Muscles, Molecular Weight, Activity Assay

Journal: Medicine and Science in Sports and Exercise

Article Title: Sex-Specific Effect of CARM1 in Skeletal Muscle Adaptations to Exercise

doi: 10.1249/MSS.0000000000003333

Figure Lengend Snippet: Effect of exercise on skeletal muscle mitochondrial morphology in F and M CARM1 mKO mice. A. Representative transmission electron micrograph (TEM) images of TA muscles from F mice from each experimental group at ×15,000 (first column), ×30,000 (second column), or ×75,000 zoom (third and fourth columns). The third column displays NM, whereas the fourth column depicts grossly distorted, ABM. The white boxes in the ×15,000 column correspond to the images shown in the ×30,000 column. Scale bars are indicated. B. Typical TEM of TA muscles from M mice from each experimental group. Scale bars are indicated. Graphical summaries of SS (C) and IMF (D) mitochondrial size (μM 2 ), SS (E), and IMF (F) mitochondrial fractional area (%), and SS (G) and IMF (H) mitochondria classified as abnormal (%). Bars indicate group means with sexes combined, whiskers represent SEMs, points illustrate individual results, and dashed lines demonstrate sex-specific means. Statistical analysis was completed using an ANOVA and Tukey’s post hoc test. E, P < 0.05 main effect of exercise; G, P < 0.05 main effect of genotype; E:G, P < 0.05 interaction effect between exercise and genotype; E:S, P < 0.05 interaction effect between exercise and sex; E:G:S, P < 0.05 interaction effect between exercise, genotype and sex. * P < 0.05 vs WT SED; # P < 0.05 vs WT VWR; % P < 0.05 vs mKO SED. n = 19–71.

Article Snippet: The following antibodies were used: CARM1 (1:5000; A300-421A; Bethyl Laboratories, Montgomery, TX), BRG1-associated factor 155 (BAF155) asymmetric dimethylation at arginine 1064 (mBAF155 Arg1064 , 1:1000; 94962; Cell Signaling, Danvers, MA), BAF155 (1:150; 11956S; Cell Signaling), mPABP1 Arg455/460 (1:1000; 3505S; Cell Signaling), polyadenylate-binding protein 1 (PABP1) asymmetric dimethylation at arginine 455/460 (mPABP1 Arg455/460 , 1:1000; 4992S; Cell Signaling), CARM1 substrate (1:1000; a kind gift from Dr. Mark Bedford, MD Anderson Cancer Center, University of Texas ( , , ), PRMT1 (1:5000; 07–404; EMD Millipore, Darmstadt, HE, Germany), PRMT5 (1:1000; 507–405; EMD Millipore), PRMT6 (1:5000; A300-929A; Bethyl laboratories), PRMT7 (1:100; Sc-376077; Santa Cruz, Santa Cruz, CA), MMA (1:1000; 8015; Cell Signaling), ADMA at glycine and arginine-rich sequences (1:200; 13522; Cell Signaling), SDMA (1:200; 13222; Cell Signaling), PGC-1α (1:1000; AB3242; EMD Millipore), BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3, 1:1000; 3769; Cell signaling), mitochondrial fission 1 protein (FIS1, 1:1000; 109516–1-AP; Proteintech, Rosemont, IL), optic atrophy-1 (OPA1, 1:1000; ab42364; Abcam, Cambridge, UK), total oxidative phosphorylation (OXPHOS, 1:1000; ab110413; Abcam), and citrate synthase (CS) (1:1000, ab96600, Abcam).

Techniques: Transmission Assay, Muscles