The authors have declared that no competing interests exist.
Conceived and designed the experiments: KCA SRZ. Performed the experiments: KCA SRZ. Analyzed the data: KCA SM SRZ. Contributed reagents/materials/analysis tools: KCA SRZ. Wrote the paper: KCA SRZ.
Levels of myostatin expression and physical activity have both been associated with transcriptome dysregulation and skeletal muscle hypertrophy. The transcriptome of triceps brachii muscles from male C57/BL6 mice corresponding to two genotypes (wild-type and myostatin-reduced) under two conditions (high and low physical activity) was characterized using RNA-Seq. Synergistic and antagonistic interaction and ortholog modes of action of myostatin genotype and activity level on genes and gene pathways in this skeletal muscle were uncovered; 1,836, 238, and 399 genes exhibited significant (FDR-adjusted P-value < 0.005) activity-by-genotype interaction, genotype and activity effects, respectively. The most common differentially expressed profiles were (i) inactive myostatin-reduced relative to active and inactive wild-type, (ii) inactive myostatin-reduced and active wild-type, and (iii) inactive myostatin-reduced and inactive wild-type. Several remarkable genes and gene pathways were identified. The expression profile of nascent polypeptide-associated complex alpha subunit (Naca) supports a synergistic interaction between activity level and myostatin genotype, while Gremlin 2 (Grem2) displayed an antagonistic interaction. Comparison between activity levels revealed expression changes in genes encoding for structural proteins important for muscle function (including troponin, tropomyosin and myoglobin) and for fatty acid metabolism (some linked to diabetes and obesity, DNA-repair, stem cell renewal, and various forms of cancer). Conversely, comparison between genotype groups revealed changes in genes associated with G1-to-S-phase transition of the cell cycle of myoblasts and the expression of Grem2 proteins that modulate the cleavage of the myostatin propeptide. A number of myostatin-feedback regulated gene products that are primarily regulatory were uncovered, including microRNA impacting central functions and Piezo proteins that make cationic current-controlling mechanosensitive ion channels. These important findings extend hypotheses of myostatin and physical activity master regulation of genes and gene pathways, impacting medical practices and therapies associated with muscle atrophy in humans and companion animal species and genome-enabled selection practices applied to food-production animal species.
Genetic and non-genetic conditions impact the molecular pathways and physiology of the skeletal muscle. The myostatin (Mstn) gene encodes a growth and differentiating factor and hormonal protein responsible for inhibition of muscle growth and proliferation in vertebrates. Myostatin negatively regulates muscle fiber number during skeletal muscle development [
Physical activity influences muscle fiber in manners akin to the effect of myostatin deficiency [
While targeted genetic and non-genetic studies have associated skeletal muscle hypertrophy to dysregulation of the IGF1-Akt-mTOR and myostatin-Smad2/3 signaling pathways, muscle atrophy has been associated to dysregulation of the autophagic-lysosomal and proteasomal pathways [
Few studies have evaluated the simultaneous effects of physical activity and myostatin genotype on the transcripts in the skeletal muscle of mice [
This study characterizes the complete transcriptome of triceps brachii muscles from C57/BL6 mice representing one of two genotype transcript levels (wild-type or myostatin typical and myostatin-reduced) and one of two physical activity levels (high and low) using massive parallel next-generation RNA sequencing. Synergistic, antagonistic and ortholog modes of action of the factors myostatin genotype and activity on genes and gene pathway profiles were investigated. This study is supported by: (a) mapping RNA sequencing reads to the mouse genome, identification of differentially expressed genes, and testing for differential expression among activity-genotype combination groups; (b) identification and interpretation of gene profiles revealing significant interaction between genotype and activity; (c) identification and interpretation of gene profiles revealing significant genotype (or activity) effect irrespective of activity (or genotype); and (d) functional analysis in support of the identification and interpretation of biological processes and pathways associated with genotype and activity levels. Our findings provide a basis to understand multifactorial gene regulation and dysregulation in triceps brachii and other skeletal muscles of mice.
Profiling information stems from an experiment comparing the transcriptome of a skeletal muscles, triceps brachii muscle of adult (6 months of age) male C57/BL6 mice. Difference in gene expression associated with two factors were studied. The factor termed genotype encompasses two levels: wild-type mice exhibiting baseline expression of the myostatin gene and myostatin-reduced mice exhibiting lower expression of the myostatin gene. The factor termed activity encompasses two levels: inactive and active. Four physical activity-by-genotype combination groups of mice were compared (n = 3/group): (1) active and myostatin-reduced, (2) inactive and wild-type (control genotype); (3) inactive and myostatin-reduced; and (4) active and wild-type. Prior to the trial, mice were housed in standard cages in groups of 2 or 3, given
Triceps brachii muscle transcriptome was studied using Illumina Genome Analyzer IIx (Illumina, Inc. San Diego, CA) producing 65-base long single-end reads. Data processing was performed using CASAVA software. The 65-base sequence reads were mapped to the mouse genome (mm9) using default settings and reads mapping to exons in the Refseq database (
The 65-base, single-end sequence reads from FastQ files were mapped to the mouse mm10 genome assembly accessed from the UCSC Genome Browser database (
Enrichment of functional categories and pathways among the differentially expressed genes was explored using the web service Database for Analysis, Validation, and Integrated Discovery (DAVID;
Beyond the identification of differentially expressed genes exhibiting significant physical activity-by-genotype interaction, this study aimed at uncovering the synergistic or antagonistic interplay between these factors. Six pairwise contrasts were used to profile the expression patterns: active wild-type vs inactive myostatin-reduced [AW-IM], active myostatin-reduced vs active wild-type [AM-AW], active myostatin-reduced vs inactive myostatin-reduced [AM-IM], inactive wild-type vs active wild-type [IW-AW], inactive wild-type vs active myostatin-reduced [IW-AM], and inactive wild-type vs inactive myostatin-reduced [IW-IM]. Among the genes that exhibited significant (FDR-adjusted P-value < 0.001) activity-by-genotype interaction, alternative profiles of over- and under-expression or non-significant (raw P-value < 0.00005 or FDR-adjusted P-value < 0.05) differential expression in each of the six contrasts between pairs of activity-genotype combinations were identified. The concept of synergism and antagonism has been used previously in studies on expression and regulation [
Considering that myostatin inhibition and physical activity are being explored as treatment options for muscle degeneration and other disorders, it is important to understand the impact of these factors at the gene co-regulation level. The RNA-Seq profile analyses revealed changes in the transcriptome of a skeletal muscle, the triceps brachii muscle, between C57/BL6 wild-type and myostatin-reduced mice under two physical activity conditions. First, the quality and quantity of the sequence reads was evaluated across samples. The average size of the RNA-Seq FastQ file was 1.3 G bases/sample. The average quality score Phred of the reads along the 65 positions across all samples was 30. The number of reads and quality scores along the reads were comparable across samples from all four activity-by-genotype groups. Likewise, the percentage of reads mapped to the mouse genome was similar across samples and was on average 84.6% (17,486,782 of 20,675,801 total reads mapped). Of these, 5,013,631 (28.7%) had multiple alignments (12,183 had >20 alignments).
Overall, 1,836 genes exhibited significant (FDR-adjusted P-value < 0.005) activity-by-genotype interaction.
Gene Name |
Log2Fold |
FDR-adjusted P-Value | |||||
---|---|---|---|---|---|---|---|
AM-AW |
AM-IM |
AW-IM |
IW-IM |
IW-AW |
IW-AM |
||
Dusp18 | -0.78 | -0.73 | -1.51 | -0.64 | -0.87 | -0.09 | 1.55E-13 |
Per1 | 1.22 | 0.40 | 1.62 | 0.82 | 0.79 | -0.42 | 1.55E-13 |
Atp1b2 | 0.62 | 0.34 | 0.96 | 0.31 | 0.65 | 0.023 | 1.55E-13 |
Tnnc1 | -0.89 | -4.14 | -5.03 | -3.67 | -1.36 | -0.47 | 1.55E-13 |
Zmynd17 | -1.25 | 0.47 | -0.78 | -1.63 | 0.85 | 2.09 | 1.55E-13 |
Myh7 | -0.72 | -4.87 | -5.58 | -4.37 | -1.20 | -0.49 | 1.55E-13 |
Tpm3 | -0.75 | -2.22 | -2.96 | -1.76 | -1.20 | -0.46 | 1.55E-13 |
Myl2 | -1.15 | -3.74 | -4.89 | -3.49 | -1.39 | -0.25 | 1.55E-13 |
Atp2a2 | -0.89 | -2.15 | -3.05 | -2.31 | -0.74 | 0.16 | 1.55E-13 |
Tnnt1 | -0.92 | -4.07 | -4.99 | -3.66 | -1.33 | -0.41 | 1.55E-13 |
Csrp3 | -0.91 | -1.92 | -2.83 | -2.01 | -0.82 | 0.09 | 1.55E-13 |
Fxyd6 | -0.76 | -2.05 | -2.81 | -1.96 | -0.85 | -0.08 | 1.55E-13 |
Myoz2 | -0.59 | -2.43 | -3.03 | -2.12 | -0.91 | -0.31 | 6.84E-13 |
Myl3 | -0.62 | -3.15 | -3.77 | -2.76 | -1.01 | -0.39 | 6.84E-13 |
Naca | 0.87 | 0.03 | 0.91 | 0.43 | 0.48 | -0.39 | 1.89E-12 |
Ak3 | 0.55 | -1.53 | -0.99 | 0.41 | -1.40 | -1.95 | 1.89E-12 |
Cyp1a1 | 2.04283 | -0.84607 | 1.19676 | 0.267273 | 0.929775 | -1.11319 | 3.16E-12 |
Gnb2l1 | 0.739797 | -0.36408 | 0.375364 | -0.32355 | 0.699348 | -0.04009 | 8.56E-12 |
Wnk2 | 1.10552 | 0.056718 | 1.16275 | 0.760786 | 0.402344 | -0.70346 | 2.45E-11 |
Ncor2 | 0.556591 | -0.14625 | 0.41082 | -0.02854 | 0.439626 | -0.11714 | 6.96E-11 |
Acta2 | 0.972604 | -0.62395 | 0.348594 | -0.39031 | 0.738874 | -0.23374 | 2.01E-10 |
Dhcr24 | 0.771441 | -0.05624 | 0.715401 | -0.12054 | 0.83638 | 0.064728 | 3.20E-10 |
Dusp23 | 0.468437 | 0.024808 | 0.493519 | 0.026371 | 0.467286 | -0.00141 | 1.01E-09 |
Ramp1 | -0.66605 | -0.05061 | -0.71666 | -0.92948 | 0.213069 | 0.87899 | 1.07E-09 |
Atrnl1 | -0.67904 | -0.04955 | -0.72883 | -0.49233 | -0.2365 | 0.442585 | 1.32E-09 |
* Genes exhibiting significant synergistic and antagonist activity-by-genotype interaction effects are displayed in italics. Ddha1, Lancl1, Fos, Tmem1, and Pmepa1 follow a synergistic pattern; Sln, Grem2, Mettl2, and Pak1 follow an antagonistic pattern.
1. When considering two values, A and B, Log2Fold Change = Log2 (B/A). For example, Log2Fold of the contrast AW-IM = log2(IM/AW).
2. AW-IM refers to the active wild-type vs. inactive myostatin-reduced contrast group
3. AM-AW refers to the active myostatin-reduced vs. active wild-type contrast group
4. AM-IM refers to the active myostatin-reduced vs. inactive myostatin-reduced contrast group
5. IW-AW refers to the inactive wild-type vs. active wild-type contrast group
6. IW-AM refers to the inactive wild-type vs. active myostatin-reduced contrast group
7. IW-IM refers to the jnactive wild-type vs. inactive myostatin-reduced contrast group
*Expanded gene names, listed in alphabetical order: Acta2 = actin, alpha 2, smooth muscle, aorta; Ak3 = adenylate kinase 3; Atp1b2 = ATPase, Na+/K+ transporting, beta 2 polypeptide; Atp2a2 = ATPase, Ca++ transporting, cardiac muscle, slow twitch 2; Atrnl1 = Attractin-Like 1; Csrp3 = cysteine and glycine-rich protein 3; Cyp1a1 = Cytochrome P450, Family 1, Subfamily A, Polypeptide 1; Ddah1 = dimethylarginine dimethylaminohydrolase 1; Dhcr24 = 24-Dehydrocholesterol Reductase; Dusp18 = dual specificity phosphatase 18; Dusp23 = Dual Specificity Phosphatase 23; Fos = FBJ Murine Osteosarcoma Viral Oncogene Homolog; Fxyd6 = FXYD domain-containing ion transport regulator 6; Gnb2l1 = Guanine Nucleotide Binding Protein (G Protein), Beta Polypeptide 2-Like; Grem2 = Gremlin 2, DAN Family BMP Antagonist; Lancl1 = LanC Lantibiotic Synthetase Component C-Like 1; Mettl21e = methyltransferase like 21E; Myh7 = myosin, heavy chain 7, cardiac muscle, beta; Myl2 = myosin, light polypeptide 2, regulatory, cardiac, slow; Myl3 = myosin, light polypeptide 3; Myoz2 = myozenin 2; Naca = nascent polypeptide-associated complex alpha polypeptide; Ncor2 = Nuclear Receptor Corepressor 2; Pak1 = p21 protein (Cdc42/Rac)-activated kinase 1
Per1 = period circadian clock 1; Pmepa1 = Prostate Transmembrane Protein, Androgen Induced 1; Ramp1 = Receptor (G Protein-Coupled) Activity Modifying Protein 1; Sln = Sarcolipin; Tmem100 = Transmembrane Protein 100; Tnnc1 = troponin C, cardiac/slow skeletal; Tnnt1 = troponin T1, skeletal, slow; Tpm3 = tropomyosin 3, gamma; Wnk2 = WNK Lysine Deficient Protein Kinase 2; Zmynd17 = zinc finger, MYND-type containing 17
Among active mice, the genotype difference was associated with the fewest number of differentially expressed genes among all pairwise contrasts. Likewise, among wild-type mice, activity level was associated with the second lowest number of differentially expressed genes among all pairwise contrasts. In contrast, changes in activity level elicited more differentially expressed genes in myostatin-reduced mice than in wild-type mice.
Gene Name |
Log2(Fold) |
||
---|---|---|---|
IW-AW2 | IW-AM3 | IW-IM4 | |
M6prbp1 | -0.55 | -0.92 | 0.47 |
Pak1 | -0.60 | -1.16 | 1.07 |
Casq2 | -0.87 | -1.25 | -0.75 |
Fos | 0.79 | 1.57 | -1.53 |
Dhrs4 | -0.63 | -0.64 | -0.60 |
Gm5514 | -1.44 | -1.21 | -1.52 |
Ddah1 | 0.59 | 2.23 | -2.15 |
Fabp3 | -0.75 | -0.63 | -0.93 |
Got1 | -0.62 | -0.55 | -0.44 |
2310076L09Rik | -0.87 | -0.72 | -1.04 |
Mafb | -0.78 | -1.07 | 0.58 |
EG225594 | -1.64 | -1.55 | 2.22 |
4832428D23Rik | -1.20 | -3.91 | 4.48 |
BDH1 | -1.83 | -1.47 | -2.24 |
ZMYND11 | 0.85 | 2.10 | -1.63 |
Gck | 1.17 | 1.48 | -0.70 |
Esrrb | -1.26 | -1.28 | -1.28 |
Acaa2 | -0.59 | -0.53 | -0.69 |
Ankrd2 | -1.18 | -0.92 | -1.59 |
Actn2 | -0.62 | -0.78 | -0.87 |
Egr1 | 1.56 | 1.65 | -1.48 |
Myom3 | -1.13 | -1.08 | -1.93 |
9830123M21Rik | 0.69 | 2.52 | -0.49 |
Rn45s | -0.80 | -1.94 | 1.16 |
NNT | -0.79 | -0.72 | -0.70 |
Dgat2 | -0.83 | -0.70 | -0.97 |
H19 | -0.68 | -0.89 | 0.43 |
Tbc1d1 | 0.72 | 0.98 | -0.91 |
IL15 | -1.05 | -1.39 | 1.06 |
Myh2 | -1.08 | -0.85 | -1.78 |
COL22A1 | 0.63 | 0.74 | -0.48 |
ORF63 | 0.85 | 0.93 | -0.75 |
IDH2 | -0.85 | -0.67 | -0.92 |
M6prbp1 | -0.55 | -0.92 | 0.47 |
1. When considering two values, A and B, Log2Fold Change = Log2 (B/A). For example, Log2Fold of the contrast AW-IM = log2(IM/AW).
5. IW-AW refers to the inactive wild-type vs. active wild-type contrast group
6. IW-AM refers to the inactive wild-type vs. active myostatin-reduced contrast group
7. IW-IM refers to the jnactive wild-type vs. inactive myostatin-reduced contrast group
*Expanded gene names, listed in alphabetical order: 2310076L09Rik = RIKEN cDNA 2310076L09 gene; 4832428D23Rik = RIKEN cDNA 4832428D23 gene; 9830123M21Rik = RIKEN cDNA 9830123M21 gene; Acaa2 = acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase); Actn2 = actinin alpha 2; Ankrd2 = ankyrin repeat domain 2 (stretch responsive muscle); BDH1 = 3-hydroxybutyrate dehydrogenase, type 1; Casq2 = calsequestrin 2; COL22A1 = collagen, type XXII, alpha 1; Ddah1 = dimethylarginine dimethylaminohydrolase 1; Dgat2 = diacylglycerol O-acyltransferase 2; Dhrs4 = dehydrogenase/reductase (SDR family) member 4; EG225594 = predicted gene 4841; Egr1 = early growth response 1; Esrrb = estrogen related receptor, beta; Fabp3 = fatty acid binding protein 3, muscle and heart; similar to mammary-derived growth inhibitor; Fos = FBJ osteosarcoma oncogene; Gck = glucokinase;Gm5514 = lactate dehydrogenase B; predicted gene 5514; Got1 = similar to Aspartate aminotransferase, cytoplasmic (Transaminase A) (Glutamate oxaloacetate transaminase 1); glutamate oxaloacetate transaminase 1, soluble; H19 = H19 fetal liver mRNA; IDH2 = Isocitrate Dehydrogenase 2 (NADP+), Mitochondrial; IL15 = interleukin 15; M6prbp1 = mannose-6-phosphate receptor binding protein 1; Mafb = v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (avian); Myh2 = myosin, heavy polypeptide 2, skeletal muscle, adult, myosin, heavy polypeptide 1, skeletal muscle, adult; Myom3 = myomesin family, member 3; Nnt = nicotinamide nucleotide transhydrogenase; ORF63 = open reading frame 63; Pak1 = p21 protein (Cdc42/Rac)-activated kinase 1; Rn45s = RNA, 45S Pre-Ribosomal 5; Tbc1d1 = TBC1 domain family, member 1; similar to TBC1 domain family member 1; ZMYND11 = zinc finger, MYND domain containing 11
Notably, some functional categories enriched among the genes differentially expressed in the IW-IM contrast (Table D in
Other genes that exhibited a significant interaction did not reach high significance in particular contrasts; however, the integration of consistent borderline significant contrasts resulted in a significant overall interaction effect (
The interaction pattern of cysteine and glycine-rich protein 3 (CSRP3) and Myozenin 2 (Myoz2) were characterized by highest expression in inactive myostatin-reduced mice relative to all other activity level-genotype groups. The consistent interaction patterns of Myosin, light polypeptide 2 (Myl2) and myosin, light polypeptide 3 (Myl3) can be summarized by over-expression in inactive myostatin-reduced mice relative to all other activity level-genotype groups. The pathways of these Myosin genes could result in this consistent profile. Myosin light chains (Myls) modulate muscle contraction and may be involved in myogenesis or muscle regeneration [
Profile |
Number of Genes |
|||||
---|---|---|---|---|---|---|
AM-AW | AM-IM | AW-IM | IW-IM | IW-AW | IW-AM | |
0 | 0 | 0 | 0 | 0 | 0 | 1509 |
0 | 0 | +1 | 0 | 0 | 0 | 183 |
0 | 0 | -1 | 0 | 0 | 0 | 146 |
0 | -1 | -1 | 0 | 0 | 0 | 142 |
0 | -1 | 0 | 0 | 0 | 0 | 86 |
0 | +1 | 0 | 0 | 0 | 0 | 83 |
0 | 0 | +1 | +1 | 0 | 0 | 71 |
0 | +1 | +1 | 0 | 0 | 0 | 68 |
0 | -1 | -1 | -1 | 0 | 0 | 67 |
0 | +1 | +1 | +1 | 0 | 0 | 54 |
1. The six contrast groups are ordered as follows- active wild-type vs. inactive myostatin-reduced, active myostatin-reduced vs. active wild-type, active myostatin-reduced vs. jnactive myostatin-reduced, inactive wild-type vs. active wild-type, inactive wild-type vs. active myostatin-reduced, and jnactive wild-type vs. inactive myostatin-reduced. Each number of the 6 in the Profile “code” refers to each contrast in order (the first number in the Profile “code” denotes significance level for the first group, active wild-type vs. inactive myostatin-reduced, the second number denotes significance level for the second group, etc.)
2. Unlisted profiles include < 50 genes
Tables
Category | Term | Number of Genes | P-Value | FDR-adjusted P-value |
---|---|---|---|---|
Score = 16.28 | ||||
KEGG PATHWAY | mmu00190:Oxidative phosphorylation | 31 | 6.69E-33 | 6.84E-30 |
KEGG PATHWAY | mmu05012:Parkinson’s disease | 30 | 6.01E-31 | 6.14E-28 |
GOTERM BP FAT | GO:0022900~electron transport chain | 24 | 1.58E-27 | 2.39E-24 |
KEGG PATHWAY | mmu05010:Alzheimer’s disease | 30 | 9.82E-27 | 1.00E-23 |
KEGG PATHWAY | mmu05016:Huntington’s disease | 30 | 1.16E-26 | 1.19E-23 |
GOTERM BP FAT | GO:0006091~generation of precursor metabolites and energy | 29 | 2.84E-25 | 4.30E-22 |
GOTERM BP FAT | GO:0055114~oxidation reduction | 32 | 3.95E-17 | 5.99E-14 |
GOTERM MF FAT | GO:0015078~hydrogen ion transmembrane transporter activity | 12 | 1.27E-11 | 1.65E-08 |
GOTERM MF FAT | GO:0015077~monovalent inorganic cation transmembrane transporter activity | 12 | 2.47E-11 | 3.20E-08 |
GOTERM MF FAT | GO:0022890~inorganic cation transmembrane transporter activity | 12 | 1.55E-09 | 2.01E-06 |
KEGG PATHWAY | mmu04260:Cardiac muscle contraction | 10 | 2.24E-07 | 2.29E-04 |
GOTERM MF FAT | GO:0015002~heme-copper terminal oxidase activity | 6 | 5.10E-07 | 6.61E-04 |
GOTERM MF FAT | GO:0016675~oxidoreductase activity, acting on heme group of donors | 6 | 5.10E-07 | 6.61E-04 |
GOTERM MF FAT | GO:0016676~oxidoreductase activity, acting on heme group of donors, oxygen as acceptor | 6 | 5.10E-07 | 6.61E-04 |
GOTERM MF FAT | GO:0004129~cytochrome-c oxidase activity | 6 | 5.10E-07 | 6.61E-04 |
Category | Term | Number of Genes | P-Value | FDR-adjusted P-value |
---|---|---|---|---|
Score = 5.07 | ||||
GOTERM BP FAT | GO:0006091~generation of precursor metabolites and energy | 24 | 1.74E-10 | 2.86E-07 |
GOTERM BP FAT | GO:0022900~electron transport chain | 15 | 8.68E-09 | 1.43E-05 |
GOTERM BP FAT | GO:0055114~oxidation reduction | 32 | 6.91E-07 | 1.1E-03 |
GOTERM BP FAT | GO:0045333~cellular respiration | 9 | 7.84E-06 | 0.01 |
GOTERM BP FAT | GO:0015980~energy derivation by oxidation of organic compounds | 11 | 7.88E-06 | 0.01 |
GOTERM BP FAT | GO:0022904~respiratory electron transport chain | 6 | 1.10E-04 | 0.18 |
GOTERM BP FAT | GO:0006119~oxidative phosphorylation | 7 | 4.14E-04 | 0.68 |
Score = 4.18 | ||||
GOTERM BP FAT | GO:0006091~generation of precursor metabolites and energy | 24 | 1.74E-10 | 2.86E-07 |
KEGG PATHWAY | mmu00190:Oxidative phosphorylation | 16 | 7.12E-09 | 8.25E-06 |
GOTERM BP FAT | GO:0022900~electron transport chain | 15 | 8.68E-09 | 1.43E-05 |
KEGG PATHWAY | mmu05016:Huntington’s disease | 18 | 1.82E-08 | 2.11E-05 |
KEGG PATHWAY | mmu05012:Parkinson’s disease | 15 | 7.73E-08 | 8.96E-05 |
KEGG PATHWAY | mmu05010:Alzheimer’s disease | 16 | 6.59E-07 | 7.63E-04 |
GOTERM BP FAT | GO:0006119~oxidative phosphorylation | 7 | 4.14E-04 | 0.68 |
Category | Term | Number of Genes | P-Value | FDR-adjusted P-value |
---|---|---|---|---|
Score = 2.43 | ||||
GOTERM BP FAT | GO:0001525~angiogenesis | 8 | 3.00E-04 | 0.48 |
Gene under-expression in active wild-type and active myostatin-reduced relative to inactive myostatin-reduced is a clear example of activity-by-genotype interaction because the combination of inactivity and myostatin-reduced genotypes was associated with higher gene expression than activity (regardless of genotype). Among genes sharing the first profile (under-expression in active wild-type and active myostatin-reduced relative to inactive myostatin-reduced) one highly enriched functional cluster (enrichment score = 16.28) was identified (
Among the profile cluster characterized by genes under- or over-expressed in the AW-IM contrast and not differentially expressed in all other contrasts (
Among the profile characterized by genes under- or over-expressed in the AM-IM contrast and not differentially expressed in all other contrasts (
Gene |
Log2 (Myostatin-reduced/Wild-type) | FDR-adjusted P-value |
---|---|---|
ERCC2 | 4.19 | 2.5E-03 |
DGCR8 | 4.09 | 2.5E-03 |
METTL21E | 3.48 | 2.5E-03 |
GSPT1 | 2.38 | 2.5E-03 |
ACTC1 | 2.29 | 2.5E-03 |
GREM2 | 1.91 | 2.5E-03 |
SLN | 1.89 | 2.5E-03 |
CDH4 | 1.88 | 2.5E-03 |
F830016B08RIK | 1.87 | 2.5E-03 |
KATNAL2 | 1.74 | 2.5E-03 |
IL12A | 1.47 | 2.5E-03 |
MYBPH | 1.34 | 2.5E-03 |
VASH2 | 1.30 | 2.5E-03 |
*Expanded gene names, listed in alphabetical order: ACTC1 = actin, alpha, cardiac muscle 1; CDH4 = cadherin 4, type 1, R-cadherin; DGCR8 = DGCR8 microprocessor complex subunit; ERCC2 = Excision Repair Cross-Complementing Rodent Repair Deficiency, Complementation Group 2; F830016B08RIK = RIKEN cDNA F830016B08 gene; GREM2 = gremlin 2, DAN family BMP antagonist; GSPT1 = G1 to S phase transition 1; IL12A = interleukin 12A (natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1, p35); KATNAL2 = katanin p60 subunit A-like 2; METTL21E = methyltransferase like 21E; MYBPH = myosin binding protein H; SLN = sarcolipin; VASH2 = vasohibin 2
Clusters of functional categories enriched (enrichment score > 3.0) among the genes differentially expressed between genotypes (raw P-value < 0.00005 comparable to FDR-adjusted P-value < 0.005) are listed in
Category | Term | Number of Genes | P-Value | FDR-adjusted P-value |
---|---|---|---|---|
Score = 3.59 | ||||
KEGG PATHWAY | mmu05410:Hypertrophic cardiomyopathy (HCM) | 10 | 7.69E-05 | 0.09 |
KEGG PATHWAY | mmu04260:Cardiac muscle contraction | 9 | 2.65E-04 | 0.31 |
KEGG PATHWAY | mmu05414:Dilated cardiomyopathy | 9 | 8.16E-04 | 0.96 |
Score = 3.43 | ||||
GO BP FAT | GO:0007167~enzyme linked receptor protein signaling pathway | 20 | 7.05E-06 | 0.01 |
GO BP | GO:0007179~transforming growth factor beta receptor signaling pathway | 7 | 5.38E-04 | 0.89 |
1 False Discovery Rate adjusted P-value. Only terms with FDR-adjusted P-value < 0.99 or with more than 5 genes are listed
Gene |
Log2(Active/Inactive) | FDR-adjusted P-value |
---|---|---|
ERCC2 | 3.95 | 1.54E-03 |
BDH1 | 2.38 | 1.54E-03 |
GM1078 | 2.28 | 1.54E-03 |
BC048679 | 2.28 | 1.54E-03 |
LRRC52 | 1.92 | 1.54E-03 |
LDHB | 1.88 | 1.54E-03 |
TNNC1 | 1.86 | 1.54E-03 |
EGLN3 | 1.82 | 1.54E-03 |
MYL2 | 1.82 | 1.54E-03 |
TNNT1 | 1.78 | 1.54E-03 |
MYH7 | 1.78 | 1.54E-03 |
MYOM3 | 1.76 | 1.54E-03 |
ESRRB | 1.74 | 1.54E-03 |
SLC26A10 | 1.65 | 1.54E-03 |
FHL2 | 1.65 | 1.54E-03 |
ANKRD2 | 1.63 | 1.54E-03 |
MYH2 | 1.61 | 1.54E-03 |
TM6SF1 | 1.59 | 1.54E-03 |
IQSEC2 | 1.57 | 1.54E-03 |
VAV2 | 1.53 | 1.54E-03 |
TPM3 | 1.51 | 1.54E-03 |
*Expanded gene names, listed in alphabetical order:ANKRD2 = ankyrin repeat domain 2 (stretch responsive muscle); BC048679 = cDNA sequence BC048679; BDH1 = 3-hydroxybutyrate dehydrogenase, type 1; EGLN3 = egl-9 family hypoxia-inducible factor 3; ERCC2 = excision repair cross-complementing rodent repair deficiency, complementation group 2; ESRRB = estrogen-related receptor beta; FHL2 = four and a half LIM domains 2; GM1078 = SH3 domain binding kinase family, member 3; IQSEC2 = IQ motif and Sec7 domain 2; LDHB = lactate dehydrogenase B; LRRC52 = leucine rich repeat containing 52; MYH2 = myosin, heavy chain 2, skeletal muscle, adult; MYH7 = myosin, heavy chain 7, cardiac muscle, beta; MYL2 = myosin, light chain 2, regulatory, cardiac, slow; MYOM3 = myomesin 3; SLC26A10 = solute carrier family 26, member 10; TM6SF1 = transmembrane 6 superfamily member 1; TNNC1 = troponin C type 1 (slow); TNNT1 = troponin T type 1 (skeletal, slow); TPM3 = tropomyosin 3; VAV2 = vav 2 guanine nucleotide exchange factor
Clusters of functional categories enriched (enrichment score > 3.0) among the genes differentially expressed between activity levels (raw P-value < 0.00005 or FDR-adjusted P-value < 0.005) are listed in
Category | Term | Number of Genes | P-Value | FDR-adjusted P-value |
---|---|---|---|---|
Score = 19.79 | ||||
KEGG PATHWAY | mmu00190:Oxidative phosphorylation | 51 | 5.29E-29 | 6.39E-26 |
KEGG PATHWAY | mmu05012:Parkinson’s disease | 50 | 2.05E-27 | 2.47E-24 |
GOTERM BP FAT | GO:0006091~generation of precursor metabolites and energy | 65 | 2.35E-27 | 4.12E-24 |
KEGG PATHWAY | mmu05010:Alzheimer’s disease | 55 | 7.43E-25 | 8.98E-22 |
KEGG PATHWAY | mmu05016:Huntington’s disease | 53 | 5.65E-23 | 6.82E-20 |
GOTERM BP FAT | GO:0022900~electron transport chain | 39 | 4.30E-22 | 7.54E-19 |
GOTERM MF FAT | GO:0015078~hydrogen ion transmembrane transporter activity | 23 | 4.07E-11 | 6.28E-08 |
GOTERM MF FAT | GO:0015077~monovalent inorganic cation transmembrane transporter activity | 23 | 1.44E-10 | 2.22E-07 |
GOTERM MF FAT | GO:0022890~inorganic cation transmembrane transporter activity | 26 | 2.46E-09 | 3.79E-06 |
Score = 8.42 | ||||
KEGG PATHWAY | mmu04260:Cardiac muscle contraction | 28 | 1.13E-14 | 1.35E-11 |
GOTERM MF FAT | GO:0015078~hydrogen ion transmembrane transporter activity | 23 | 4.07E-11 | 6.28E-08 |
GOTERM MF FAT | GO:0015077~monovalent inorganic cation transmembrane transporter activity | 23 | 1.44E-10 | 2.22E-07 |
GOTERM MF FAT | GO:0022890~inorganic cation transmembrane transporter activity | 26 | 2.46E-09 | 3.79E-06 |
GOTERM MF FAT | GO:0016675~oxidoreductase activity, acting on heme group of donors | 10 | 7.32E-07 | 1.13E-03 |
GOTERM MF FAT | GO:0016676~oxidoreductase activity, acting on heme group of donors, oxygen as acceptor | 10 | 7.32E-07 | 1.13E-03 |
GOTERM MF FAT | GO:0015002~heme-copper terminal oxidase activity | 10 | 7.32E-07 | 1.13E-03 |
GOTERM MF FAT | GO:0004129~cytochrome-c oxidase activity | 10 | 7.32E-07 | 1.13E-03 |
Both genotype and activity level were associated with significant changes in gene expression, irrespective of the remainder factor indicating main effects. The more extreme fold change estimates observed in the genotype relative to the activity contrasts (based on the top differentially expressed genes) indicate that the genotypes considered in this study have a higher impact on gene expression than the physical activity levels evaluated (Tables
Consideration of the number of differentially expressed genes across pairwise contrasts alone uncovered insightful interaction patterns that are cornerstone for more complex patterns across contrasts. Among all pairwise contrasts, myostatin-related genotype differences within the active group (contrast AM-AW) were associated with the fewest number of differentially expressed genes (86 genes), suggesting that activity may be picking up and modulating or compensating some expression regulated by myostatin. The second lowest number of differentially expressed genes (119 genes) was related to differences in activity level within wild-type mice (contrast IW-AW), indicating that activity alone was associated with more limited changes in gene expression than those observed in the combination of activity and myostatin reduction. Finally, considering the fact that activity level elicited more differentially expressed genes in myostatin-reduced than in wild-type mice along with the finding that the greatest number of differentially expressed genes is in the AW-IM contrast confirms the hypothesized synergistic impact of physical activity and the silencing of myostatin on gene expression.
Muscle cell differentiation (GO:0042692) and muscle organ development (GO:0007517) were two BP terms enriched among the genes differentially expressed in the IW-IM and AM-AW contrasts. These categories are consistent with the known role of myostatin on cell differentiation and proliferation in triceps. Multiple studies have confirmed the direct impact of myostatin on these muscles. Specifically, myostatin-deficient mice have significantly larger tricep muscles than wild-type mice [
Enrichment of hypertrophic and dilated cardiomyopathy KEGG pathways (mmu05410 and mmu05414, respectively) was observed among genes differentially expressed in the IW-IM and AM-AW contrasts. Although hypertrophic cardiomyopathy is characterized by an hypertrophied heart muscle while tricep samples were used in this study, our results suggest that the expression of genes in similar biological processes are altered by myostatin genotype regardless of activity level. Our result is consistent with a previous report that a hypertrophic cardiomyopathy mutation is expressed in the messenger RNA of skeletal as well as cardiac muscle [
Finally, enrichment of the vasculature development BP (GO:0001944) among the genes differentially expressed in the AM-IM and IW-IM contrasts suggests that activity level within myostatin-reduced mice and myostatin genotype within inactive mice have comparable impact on the expression of genes in the vascular development pathway. While inactivity may have counteracted the effect of myostatin reduction in the former contrast, myostatin reduction may have counteracted the effect of inactivity in the latter contrast. Our results offer support at the gene expression level to claims that the processes that regulate blood vessel development can also enable the adult to adapt to changes in tissues that can be elicited by activity or pathologies [
CSRP3 and Myoz2 shared the same interaction pattern of highest expression in inactive myostatin-reduced relative to all other activity level genotype groups. The parallel expression profiles of these two genes detected in the present study is in agreement with previous reports. The expression of CSRP3 and Myoz2 is high in skeletal muscles [
Clusters of expression profiles among genes exhibiting significant activity-by-genotype interaction were identified as well. Among genes sharing the first profile (under-expression in active wild-type and active myostatin-reduced relative to inactive myostatin-reduced and similar expression levels across all other activity-genotype groups), KEGG pathways for several inflammation-associated neurodegenerative conditions including Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease were enriched. Our results are in agreement with reports that myostatin causes sporadic inclusion body myositis (sIBM), a muscle-wasting disease that has pathogenesis similar to that of Alzheimer’s and Parkinson’s diseases [
Additionally, oxidative phosphorylation, electron transport chain, energy generation, and mitochondrial ATP synthesis GO terms were enriched among the profile characterized by genes under- or over-expressed in the AW-IM contrast and not differentially expressed in all other contrasts. These findings are consistent with the electron transport chain, or the flow of electrons resulting from NADH and FADH2 oxidation, that establishes an electrochemical gradient vital in powering ATP synthesis in oxidative phosphorylation, the final stage of aerobic cell respiration. Myostatin reduction, although not affecting phosphorylated compound concentrations and intracellular pH at rest, causes up to a 206% increase in ATP cost of contraction as well as limiting the shift toward oxidative metabolism during muscle activity [
Examples of synergistic or antagonistic mode of action of genotype and activity factors on gene expression are listed in
The identification of significant interactions enabled the detection of synergistic effects between genotype and activity. For genes Naca, Dusp23, and Dhcr24, the difference in expression between myostatin genotype groups was more extreme than between activity groups (Table A in
Among the genes differentially expressed (FDR-adjusted P-Value < 0.005 and log2(fold change) > |1.3|) between myostatin-reduced and wild-type mice (
Among the rest of the genes differentially expressed between genotype groups, four provided remarkable insight into myostatin’s effects on the gene networks of muscle development. The protein coded by the Piezo-type mechanosensitive ion channel component 1 (Piezo1) allows cells to react to physical stimuli. Mechanosensitive ion channels play a key role in the physiology of smooth muscle [
Many of the differentially expressed genes between active and inactive mice, unsurprisingly, are associated with the biological processes of contractile response of muscles to activity. A notable finding is that Ercc2 was differentially expressed between genotype groups and between activity groups as well, yet this gene did not exhibit a significant activity-by-genotype interaction effect. A similar molecular mechanism is speculated for both comparisons. Among other genes differentially expressed between activity groups, Tnnt1 [
Novel associations between differentially expressed genes and activity level were also identified in this study. Many of these genes have indirect links to muscle function and activity, but the actual mechanism uncovered is unique and unexpected. The differentially expressed gene 3-hydroxybutyrate dehydrogenase (Bdh1) encodes an enzyme involved in the interconversion of acetoacetate and (R)-3-hydroxybutyrate, essential for fatty acid catabolism. Also, Bdh1 mRNA is found in all forms of muscle [
The enrichment of GO biological process terms related to vasculature development (angiogenesis, blood vessel development, vasculature development, blood vessel morphogenesis, and tube development) among the genes differentially expressed in the AM-IM and IW-IM contrasts suggests that the combination of activity and myostatin-reduced genotype has comparable impact to the combination of inactivity and wild-type typical myostatin genotype on the expression of genes in the vascular development pathway. A link between vascular development and muscle development is expected based on the logical physiological association of the two organ systems. Vasculature is modified in order to meet the metabolic requirements of tissue cells in response to changes in metabolic rate; oxygen is a major control element of this adaptation, as hypoxia initiates various signals which in turn lead to an increase in vessel growth [
The study of the impact of physical activity and myostatin level on gene expression in the triceps brachii muscles of C57/BL6 mice uncovered novel and confirmed known associations at the gene and gene network levels. Novel and significant interaction effects were observed for some genes (e.g. Naca, Grem2) including synergistic effects (e.g. Naca, Dhcr24) and antagonistic effects (e.g. Mettl21e, Cyp1a1, Mpz). Functional analysis of genes presenting significant interaction effects uncovered novel (e.g. angiogenesis) and expected (e.g. oxidative phosphorylation, electron transport chain) enriched pathways and biological processes.
Among the genes exhibiting significant main genotype effect, known (e.g. Sln, Grem2) and novel (e.g. Piezo1, Ercc2, Gspt1) associations were detected. Functional analysis of genes presenting significant genotype effect uncovered novel (e.g. dilated and hypertrophic cardiomyopathy) and expected (e.g. muscle cell differentiation, muscle organ development) enriched pathways and biological processes. Likewise, among the genes exhibiting significant main activity effect, known (e.g. MB, Tpm and novel (e.g. Bdh1, Esrrβ) associations were detected. Functional analysis of genes presenting significant activity effect uncovered novel (e.g. Alzheimer’s, Parkinson’s and Huntington’s disease) and expected (e.g. oxidative phosphorylation, cardiac muscle contraction) enriched pathways and biological processes. While several genes and functional categories enriched among the differentially expressed genes uncovered in this study were consistent with previous reports, the identity and profile of the genes exhibiting the most extreme interaction and main genotype and activity effects opened new avenues of inquiry on the role of specific genes in skeletal muscle development and the effects of myostatin and physical activity on muscle function. The present study centered on the comparison of four genotype-activity groups based on transcriptome information from a specific skeletal muscle type, mouse strain, gender, and age. Consideration of additional muscle types, genotypes, activities, ages, and genders would help identify additional synergistic and antagonistic relationships between these factors.
The findings from the present study could have medical implications on preventive practices and therapies associated with muscle atrophy in humans and companion animal species and genome-enabled selection practices applied to food-production animal species. The study of changes in gene expression in response to myostatin gene expression level in skeletal muscle tissue involved genes that code for a number of proteins that are feedback regulated by the myostatin molecule. The functions of the genes exhibiting differential expression between genotype groups are primarily regulatory. This functional category includes microRNA and Piezo proteins that make the list of the top 10 differentially expressed genes, side-by-side with Grem2 proteins that modulate the metalloprotein BMP-mediated cleavage of the myostatin propeptide. The role of genes regulated by microRNAs was unanticipated, especially because these genes seem to impact central functions such as the G1 to S phase transition of the cell cycle of myoblasts. The role of genes coding for Piezo proteins that make mechanosensitive ion channels, which in turn regulate cationic currents in the cells, was also remarkable and unanticipated, especially because of the consistent profile of the genes in this family. The study of changes in gene expression patterns in response to activity level revealed enrichment of genes that code structural proteins important for muscle function, including troponin, tropomyosin and myoglobin proteins. Activity was also associated with differential expression of genes important for fatty acid metabolism, some linked to type II diabetes and obesity and others to DNA-repair capacity, stem cell renewal, and various forms of cancer.
Our results provide evidence supporting the role of myostatin as a master regulator and the hypothesis that physical activity affect the expression of genes associated with homeostatic balance between storage of fat and muscle growth. Down-regulation of myostatin expression enables muscle growth at full expense of storage of fat, a condition that is hardwired at the regulatory level (e.g. through antagonists of metalloenzymes responsible for the myostatin activation). During activity, the changes in gene expression associated with balance between storage of fat and growth appears more instantaneous and subtle. This balance involves the regulation of metabolic pathways of fatty acid synthesis and does not impinge on oxidative phosphorylation pathways. The master regulatory functions of myostatin identified in this study should now be explored at the biochemical level to identify details of the regulatory networks, especially because of their potential to assist in the development of muscular disorders.
Table A: Differentially expressed genes (FDR-adjusted P-value <.01) across activity-genotype contrasts.
Table B: Enriched (enrichment score > 3) clusters of Gene Ontology (GO) biological process (BP), molecular function (MF) Functional Annotation Tool (FAT) categories, and KEGG pathways among differentially expressed genes (FDR-adjusted P-value < 0.01) in the active myostatin-reduced vs jnactive myostatin-reduced contrast group.
Table C: Enriched (enrichment score > 2.0) clusters of Gene Ontology (GO) biological process (BP), molecular function (MF) Functional Annotation Tool (FAT) categories, and KEGG pathways among differentially expressed genes (FDR-adjusted P-value < 0.01) in the inactive wild-type vs active wild-type contrast group.
Table D: Enriched (enrichment score > 3) clusters of Gene Ontology (GO) biological process (BP), molecular function (MF) Functional Annotation Tool (FAT) categories, and KEGG pathways among differentially expressed genes (FDR-adjusted P-value < 0.01) in the inactive wild-type vs inactive myostatin-reduced contrast group.
Table E: Enriched (enrichment score > 3) clusters of Gene Ontology (GO) biological process (BP), molecular function (MF) Functional Annotation Tool (FAT) categories, and KEGG pathways among the genes differentially expressed between active and inactive mice (FDR-adjusted P-value < 0.05.
Table F: Enriched (enrichment score > 3) clusters of Gene Ontology (GO) biological process (BP), molecular function (MF) Functional Annotation Tool (FAT) categories, and KEGG pathways among differentially expressed genes (FDR-adjusted P-value < 0.01) in the active wild-type vs inactive myostatin-reduced contrast group.
Table G: Genes differentially expressed (FDR-adjusted P-value < 0.01) between myostatin-reduced and wild-type mice in triceps brachii muscle.
Table H: Enriched (enrichment score > 3) clusters of Gene Ontology (GO) biological process (BP), molecular function (MF) Functional Annotation Tool (FAT) categories, and KEGG pathways among differentially expressed genes (FDR-adjusted P-value < 0.005) between triceps brachii muscle of myostatin-reduced and wild-type mice.
Table I: Genes differentially expressed (FDR-adjusted P-value < 0.01) between active and sedentary mice in triceps brachii muscle.
Table J: Enriched (enrichment score > 2) clusters of Gene Ontology (GO) biological process (BP), molecular function (MF) Functional Annotation Tool (FAT) categories, and KEGG pathways among differentially expressed genes (FDR-adjusted P-value < 0.005) between triceps brachii muscle of active and sedentary mice.
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