We compared the RNA levels between old (24-month-old) and young (3-month-old) skeletal muscle using high-throughput RNA sequencing analysis to evaluate the gene expression changes. Based on the fold changes in gene expression, we selected the top 100 up-regulated genes and 100 down-regulated genes for further analysis (Fig. 1). The results showed that the genes involved in muscle functions and metabolism were among the largest transcriptional changes observed in aged animals.
Alterations in muscle sarcomere myosin gene expression: Up-regulation of embryonic and cardiac myosin genes in aging muscle
Transcriptome analysis revealed that some up-regulated genes were associated with muscle sarcomere structure (Additional file 1: Table S1). The embryonic form of myosins Myh3 (Myosin heavy chain 3), Myh7 (Myosin heavy chain 7), Myl4 (Myosin light chain 4), and Myl2 (Myosin light chain 2) as well as embryonic tropomyosin Tnnt2 (Troponin T2, cardiac type) and embryonic heart gene Ak147021 (Gm5532) were strongly up-regulated in 24-month-old muscle (Additional file 1: Table S1). Genes associated with type I muscle fibers, Tnnt1 (Troponin T1, slow skeletal type) and Atp2a2 (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2), were up-regulated in 24-month-old muscle (2.53- and 1.93- fold respectively), indicating a fast-to-slow muscle fiber transition during aging. Genes associated with both type I skeletal and cardiac muscles such as Myl2, Myl3 (Myosin light chain 3), Myoz2 (Myozenin 2), and Hspb7 (Heat shock protein family B (small) member 7) were up-regulated (2.79-, 2.19-, 1.65-, and 1.97-fold respectively). Additionally, sarcomere Myom3 (Myomesin3) encoding a structural sarcomeric protein was found to be up-regulate (2.16-fold) in 24-month-old muscle.
We confirmed cardiac-related genes, including Myh6, Tnnt2, Sln (Sarcolipin), and Npr3 (Natriuretic peptide receptor 3), that were elevated in the aged skeletal muscle compared to young muscle (Fig. 2a). Ingenuity pathway analysis (IPA) showed that up-regulated genes were involved in cardiogenesis (Fig. 2b), such as Gata4 (GATA binding protein 4), Tbx5 (T-box 5), Hand2 (Heart and neural crest derivatives expressed 2), and Myocd (Myocardin), and in cardiac muscle contraction such as Ryr2 (Ryanodine receptor 2), as well as their downstream genes, Tnnt2, Tnnc1 (Troponin C), Myh6, Myh7, Casq2 (Capsequestrin, cardiac muscle isoform), Hspb7, and Tnni1 (Troponin I1). The embryonic Myl4 gene, known to interact with other cardiac-specific factors, also had a 2.48-fold increase in mRNA expression in the 24-month-old muscle (Fig. 2b, Additional file 1: Table S1). We evaluated the protein expression pattern of the cardiac-specific myosin isoform Myh6 in the skeletal muscle of young and old mice. Both the IHC staining and Western blotting analysis showed that aged skeletal muscle had higher Myh6 expression compared to young muscle (Fig. 2c-d). More stress accumulation and less repair capacity resulted in increased degenerated myofibers and regenerating myofibers (centrally located nuclei) observable in aged skeletal muscle; interestingly, Myh6 seems to have higher expression in the degenerated myofibers as indicated in Fig. 2c. We also observed other genes involved in heart function to have age-associated changes. Pln (Phospholamban), which is expressed mainly in heart that inhibits sarcoplasmic reticulum Ca++ATPase pumps and regulates myocardiac contractility, was highly up-regulated (8.84-fold). Gene encoding the transcriptional repressor Hdac9 (Histone deacetylase 9), which acts as a negative feedback regulator of myocyte differentiation, muscle endplate reinnervation, and cardiac hypertrophy, was up-regulated (2.60-fold) in the aged muscle. Transcription factor Mef2c (Myocyte enhancer factor 2C), which activates the expression of downstream cardiac genes during aging and repair/regeneration, was also transcriptionally up-regulated (1.25-fold). In addition, several genes involved in calcium ion metabolism were also up-regulated. For example, the cardiac-specific Ryr2 gene for mediating the release of sarcoplasmic calcium ion, and Trpc3 (Transient receptor potential cation channel, subfamily C, member 3), involved in forming calcium ion channels in both skeletal and cardiac muscles were also up-regulated in aged skeletal muscles (4.69- and 2.73-fold respectively).
Changes in Z-band gene expression in aging muscle
The vertebrate striated muscle Z-band connects actin filaments of opposite polarity from adjacent sarcomeres and allows tension to be transmitted along a myofibril during contraction. Igfn1 (Immunoglobulin-like and fibronectin type III domain containing 1), Ky (Kyphoscoliosis peptidase), and Flnc (Filamin-C) are genes encoding the Z-band-associated proteins that provide structural support for skeletal muscle. All three genes as well as Des (Desmin) were up-regulated (1.63-, 1.35-, 1.50-, and 1.35-fold respectively) in 24-month-old muscle whereas the major component of sarcomere, Actn1 (alpha-actinin 1), was down-regulated (1.68-fold). The up-regulation of Igfn1 (1.63-fold) down-regulated protein synthesis by binding to the translation elongation factor eEF1A, and furthermore induced down-regulation of muscle protein biosynthesis in the 24-month-old muscle. Ky is specifically expressed in skeletal and cardiac muscles, and thus the up-regulation of Ky and Flnc may indicate an alteration in Z-band for force generation in the aged muscle. In summary, the expression changes of genes involved in muscle function and differentiation in aged muscle suggest an overall diminishment in muscle contractile function during aging.
Gene expression changes associated with the neuromuscular junction (NMJ)
Among the various factors involved in the development and function of the NMJ, we found that myotrophic and neurotrophic factor Igf1 (Insulin-like growth factor 1) was down-regulated (1.83-fold). Furthermore, several important genes associated with the NMJ, such as ApoE (Apolipoprotein E), Dok7 (Docking protein 7), Snta1 (Syntrophin alpha 1), and Aqp4 (Aquaporin 4), as well as genes coding several serpins, exhibited a significant decrease in gene expression in aging muscle (Additional file 1: Table S1). This indicates the dysfunction of specific aspects of neuromuscular synaptogenesis during aging.
In contrast to the down-regulation of the NMJ genes described above, two of the genes encoding the acetylcholine receptor (AChR) subunits Chrna1 (Cholinergic receptor nicotinic alpha 1 subunit) and Chrnb1 (Cholinergic receptor nicotinic beta 1 subunit) as well as the co-receptor of agrin Lrp4 (Low density lipoprotein receptor-related protein 4) were up-regulated (Additional file 1: Table S1, Fig. 2e). Lrp4 binds agrin released from motor neurons to regulate presynaptic differentiation at the NMJ. Lrp4 also binds with cholesterol carrier ApoE; hence, the up-regulation of Lrp4 may serve to compensate the down-regulation of ApoE for the survival of neurons. It is also possible that the observed increase of Chrna1, Chrnb1, and Lrp4 expressions reflect the change of muscle fiber types during aging as slow-twitch muscle fibers have wider NMJs and larger nerve terminal areas than the fast-twitch muscle fibers. We also observed strong up-regulation of the Mib1 (Mindbomb E3 ubiquitin protein ligase 1) gene (3.33-fold), which plays a critical function in reducing the number of presynaptic boutons and branches at the NMJ. Thus, the interconnected transcriptional changes of genes associated with the NMJ during aging suggests a decreased functioning of the NMJ and a compensatory mechanism for the adaptation of changing muscle fiber types.
Down-regulation of polyamine biosynthesis and amino acid metabolism: Implication for the loss of muscle mass during aging
Muscle hypertrophy and mass are positively correlated with polyamine accumulation. We observed down-regulation of Odc1 (Ornithine decarboxylase 1) and Amd1 (Adenosylmethionine decarboxylase 1), both encoding key enzymes involved in polyamine biosynthesis (2.65- and 1.97-fold respectively), in 24-month-old muscle. Further, Smox (spermine oxidase) and GR (glucocorticoid receptor) target Fkbp5 (FK506 binding protein 5), which help to maintain muscle mass, were also decreased (4.73- and 4.16-fold respectively). Furthermore, serine/threonine-protein kinase, encoded by Sgk1 (Serum/glucocorticoid regulated kinase 1), which is involved in the maintenance of muscle mass through the down-regulation of proteolysis and autophagy, was down-regulated (1.72-fold) in the aged muscle. These results are consistent with the loss of muscular mass of skeletal muscle fibers during aging. On the other hand, Forkhead transcription factors Foxo6 (forkhead box O6) and Foxo1 (forkhead box O1), which are involved in the maintenance of muscle mass, were down-regulated (2.00- and 2.27-fold respectively) in 24-month-old muscle. The measurement of myofiber diameter showed smaller myofibers in aged femoris rectus muscle compared with young one (Fig. 3a) Accordingly, decreased expression of those genes, which play critical functions on polyamine biosynthesis and amino acid metabolism, suggests the loss of muscle mass in aged mice.
Down-regulation of carbohydrate, amino acid metabolism, and mitochondrial gene expression in aging skeletal muscle: Implication in loss of strength in aging muscle
Transcriptome analysis revealed the expression of several metabolic genes that were strongly down-regulated in the 24-month-old muscle in comparison with the 3-month-old muscle (Additional file 1: Table S2). Skeletal muscle is a highly energy-dependent tissue. Our data revealed a reduced expression of genes involved in glucose metabolism in aged muscle (Additional file 1: Table S2). First, genes involved in glycogen biosynthesis and utilization were down-regulated in the skeletal muscle of 24-month-old mice as compared with 3-month-old mice. For example, the Ppp1r3c (Protein phosphatase 1 regulatory subunit 3C) gene, which encodes the regulatory subunit of protein phosphatase-1 that is involved in the activation of glycogen synthase, was strongly down-regulated (3.67-fold).
Several enzymes involved in gluconeogenesis were also down-regulated in aged muscle. Pck1 (Phosphoenolpyruvate carboxykinase 1), which encodes cytosolic phosphoenolpyruvate carboxykinase and serves as the key control element in gluconeogenesis, was strongly down-regulated (3.54-fold). Gpt2 (Glutamic pyruvate transaminase (alanine aminotransferase) 2), which encodes mitochondrial alanine transaminase and participates in gluconeogenesis, was also down-regulated (2.06-fold). In addition, Pgk1 (Phosphoglycerate kinase 1), which encodes phosphoglycerate kinase that participates in glycolysis, was down-regulated (1.48-fold). Slc16a3 (Solute carrier family 16 member 3), which encodes MCT4 (Monocarboxylate transporter 4) lactate transporter involved in anaerobic glycolysis energy production of fast-twitch muscle fibers, was down-regulated (1.82-fold). On the other hand, the glycolysis inhibitor Rrad (Ras related glycolysis inhibitor and calcium channel regulator), which inhibits glucose uptake, was up-regulated (3.59-fold) in 24-month-old skeletal muscle. The down-regulation of genes that function in glycogen metabolism and glycolysis suggests that the aging muscle will not be able to sustain muscle contractile activities.
Besides carbohydrates, amino acids oxidation can serve as an energy source for muscle by converting amino acid into glucose. We found that several enzymes involved in amino acid catabolism and the urea cycle were down-regulated or silenced in aging muscle (Additional file 1: Table S2). There seems to be no alternative source of energy to compensate for the lack of energy in the muscle cells of aging animals; however, more research on the metabolic state of skeletal muscle is necessary to determine this.
The mitochondrial translational activator encoded by Mss51 (Mitochondrial Translational Activator, as known as Zmynd17) is a fast-twitch fiber gene and was strongly up-regulated (4.66-fold) in aged muscle. Nr1d1 (Nuclear receptor subfamily, group D, member 1), which is a transcription repressor that coordinates circadian rhythms with metabolism was up-regulated 3.5-fold in 24-month-old skeletal muscle. This transcription regulator has been shown to regulate skeletal muscle mitochondria biogenesis and autophagic regulation. Glul (Glutamate-Ammonia Ligase), which encodes mitochondrial glutamine synthetase, was under-expressed (2.71-fold). This enzyme is involved in the regulation of glycogen synthesis via the up-regulation of glycogen synthase. Slc25a25 (Solute Carrier Family 25 Member 25), which encodes ATP-Mg2+/Pi inner mitochondrial membrane solute transporter and participates in muscle ATP production, was also strongly down-regulated (4.99-fold) in 24-month-old muscle. In addition, we also observed strong down-regulation of the Fkbp5 gene (4.16-fold), which regulates metabolism during stress. These results suggest that aging muscle has reduced ATP production and mitochondria activity, and particularly, aging muscles are less well-adapted to stress.
It is interesting that all the Mup (Major urinary protein) genes were silenced in the aged muscle (Additional file 1: Table S2). The major function of Mup is chemical communication but recent studies have shown that one of the members, Mup1, is involved in the regulation of glucose and lipid metabolism and energy expenditure.
Changes in expression of fatty acid metabolism during aging
Another energy source for muscle contraction is fatty acids. Transcriptome analysis also revealed changes in the lipid metabolism (Additional file 1: Table S2). The Lpin1 (Lipin 1) gene, which encodes phosphatidic acid phosphohydrolase was found to be down-regulated (1.53-fold) in 24-month-old muscle. Fabp1 (Fatty acid-binding protein 1) and ApoB (Apolipoprotein B), which are involved in the transport of long chain fatty acid and fat molecules respectively, were found silenced in 24-month-old muscle (Additional file 1: Table S2). This result is consistent with the accumulation of adipose-like tissues in the 24-month-old muscle (Fig. 3b-f). Several apolipoprotein genes, such as Apoc3 (Apolipoprotein C3), Apoa5 (Apolipoprotein A5) and Apoc2/Apoc4 (Apolipoprotein C2/ Apolipoprotein C4), were also silenced in the aged muscle.
Aside from adipose-like tissues infiltration, we also found severe degenerated myofibers accompanied by collagen accumulation (Fig. 3g-k) in the rectus femoris muscles of old mice. Based on the information from National Toxicology Program of NIH (https://ntp.niehs.nih.gov/nnl/musculoskeletal/skel_musc/degen/skeletal-muscle-degeneration_508.pdf), degenerated myofibers look either pale or dark; additionally histological observation includes various changes, such as cell swelling, vacuolation, loss of striation, fragmentation, and rupture of fibers. In aged skeletal muscle, we observed pale and swollen myofibers, ruptured fibers, as well as regenerated myofibers, centrally positioned nuclei in the myofibers (Fig. 3l-n). In Fig. 3f, k, n, we used blinded test to quantify degenerated myofibers, including pale, swelling, and ruptured fibers, meanwhile we also quantified myofibers that had centrally located nuclei that indicated myofibers under regeneration. Adipose tissues observed in skeletal muscle were measured using SPOT software to show the fat infiltration proportion. Subsequently, we analyzed the ultrastructure of rectus femoris muscle from young and aged mice. Transmission electron microscopy showed that myofilaments in young skeletal muscle were orderly and closely arranged (Fig. 4a); however, loose and twisted arrangements and enlarged sarcoplasmic reticulum (SR) were observed in aged muscle (Fig. 4b). The ultrastructure of aged skeletal muscle also showed enlarged multivesicular structure, which seems to be the late endosome or multivescular body (MVB) as well as apparent lipofuscin granules (Fig. 4c). Aged mice have a diminished capacity for the regeneration of skeletal muscle and displacement of differentiated adipose tissues, resulting in the degeneration and loss of skeletal myofibers.
Changes in immune genes during aging
The switch from fast-to-slow muscle fiber was accompanied with a decrease in immune cell population and increase in cardiac vessel markers. In aged skeletal muscle, we found that specific genes related to macrophage, T lymphocytes, B lymphocytes, and complement system were silenced or strongly down-regulated (Additional file 1: Table S3) and vessel endothelial markers were up-regulated (Additional file 1: Table S4). For example, genes encoding the B-cell specific surface molecules Cd19 (Cluster of differentiation 19), Cd79a (Cluster of differentiation 79a) and Cd22 (Cluster of differentiation 22) as well as Btla (B and T lymphocyte associated) and Cxcr5 (Chemokine (C-X-C motif) receptor 5) of T cells and Cd209b (Cluster of differentiation 209b) of dendritic cells were silenced or strongly down-regulated in 24-month-old muscle (Additional file 1: Table S3). Angiogenesis and vasculogenesis in skeletal muscle have been shown to be modulated by monocytes and macrophages [24,25,26,27]. The decline of endothelial gene expression could lead to out-of-control blood pressure. Taken together, the aged skeletal muscle cells and their environment have reduced immune functions compared with young individuals. The repair/regeneration of muscle injury is compromised with the decline of immune function, which further accelerates the aging process.