We previously published an aging gene signature from rats which surveyed one skeletal muscle, gastrocnemius, in addition to three other tissues: liver, kidney, and the hippocampus [7]. Here, we sought to survey rodent skeletal muscle more deeply, to ask more granular questions:

1)

Are there consistent mRNA expression changes between rats and mice—as one might expect, since they are both rodents?

2)

Are there differences in age-related gene expression in the rat versus the mouse, which might suggest one might be a “better” or more representative model for what happens in the human—if, for example, one of the rodent species underwent changes which are also found in the human?

3)

Are there any changes specific to the mouse or the rat muscle—i.e., not seen in the other species?

4)

Do the various muscles within a species demonstrate similar or different age-related gene expression changes?

5)

Might any of the age-related gene expression changes in mice or rats explain their loss of muscle mass, or the decline in the patency of the neuromuscular junction which accelerates age-related weakness, since the skeletal muscle becomes functionally denervated [25,26,27,28]?

In addition to studying multiple limb muscles, we added the diaphragm to this study, since this muscle is contracting constantly throughout life, to get an idea as to whether constant use distinguishes that muscle from any of the aging changes found in the limbs. We surveyed these gene expression changes in conjunction with age-related changes in skeletal muscle mass, which have previously been associated with declines in muscle function [6]; indeed, while some have debated whether muscle hypertrophy causes an improvement in muscle function, no one argues against the notion that decreases in muscle mass induce muscle weakness—since there are fewer sarcomeres to contract.

Before undertaking to answer these questions, we did some quality control work. First, we compared our data with publicly available gene expression datasets for rodent skeletal muscle aging from SarcoAtlas (https://sarcoatlas.scicore.unibas.ch) [29,30,31]). SarcoAtlas is a valuable resource that includes time course transcriptomic data from mice (N = 8–9 per group) and from rats (N = 9–10 per group). Specifically, data from Wistar rat gastrocnemius muscles in SarcoAtlas includes five ages, with 24-month-old rats being the oldest group [30, 32]. Eighty-eight percent of age-related upregulated genes and 81% of downregulated genes identified in the Wistar rat gastrocnemius are consistent with upregulated and downregulated genes, respectively, in our study (SD rat gastrocnemius).

The mouse aging time course in SarcoAltas includes samples from the gastrocnemius muscle of six different ages, ranging from 8 to 28 months [29]. Age-related genes from SarcoAtlas overlap with our data: 24% for upregulated genes and 8% for downregulated genes are consistent with our upregulated and downregulated genes, respectively. We note that SarcoAtlas defines age-related genes differently than us: they use principal component analyses to identify genes that align well with the age of the animal [29]. While it was reassuring to see consistency between our data and prior-published studies, we should note this current study is unique in that it combines multiple elements which allow for novel findings: the multiplicity of time points studied, the number of muscles analyzed, the N of animals in each group analyzed to better assure data accuracy, and the number of species analyzed—which uniquely allows for mouse versus rat comparisons. Also, we analyzed both, males and females in mice.

Having gone through the relevant literature comparisons, we were ready to address the questions asked. As to the question: Are there differences between age-related changes in rat muscle versus mouse muscle, the answer is clearly “yes”, and these differences are quite striking. The rat but not the mouse showed a decrease in mitochondrial gene expression as a result of age, a finding which has been reported in human sarcopenia [33]. Also, the rat showed a more dramatic increase in innate immune and inflammatory signaling, compared with the mouse, including an increase in interferon signaling, that may be a marker for DNA damage in old muscles [34,35,36]. We had previously reported upregulation of interferon response genes in the gastrocnemius and the non-muscle tissues (liver, kidney, and hippocampus) in the rat [7]. Age-related increase in the innate immune and inflammatory signaling was also seen in mouse skeletal muscles; however, this response was more modest compared with rat and was primarily localized to the fast limb muscles (gastrocnemius and tibias anterior). Age-related downregulation of the extracellular matrix genes was another commonality between mouse and rat muscles, albeit this downregulation was more prevalent in mouse muscles. At least in mice, downregulation of the ECM theme occurs in multiple tissues throughout the lifespan [37]. Extracellular matrix changes have been reported to occur during aging and sarcopenia, resulting in decreased muscle function [38]; the extracellular matrix in muscle is critical for anchoring the muscle fibers, and thus the sarcomere, to allow for muscle contraction [39,40,41].

As to the question as to whether one rodent model be superior over the other when it comes to modelling the human, both species may be useful; however, differences need to be taken into account as these will influence what therapeutic targets are being investigated.

Overall, we found that the rat has a wider range of age-related transcriptional changes. Modeling muscle aging in the mouse may leave out some of the age-related changes that have been found to be relevant to the human sarcopenia, e.g., decline in signatures of mitochondrial function and oxidative phosphorylation [33]. We note that the lack of reduced mitochondrial function in our mouse muscle data is consistent with the findings of Ham et al. [30], while Borsch et al. [29] reported reduced mitochondrial function in the muscles of mouse, rat, and human. These discrepancies may reflect a difference in how the gene pathway enrichment tests were performed: Ham et al. 2020 and our study used Fisher’s exact test while Borsch et al. 2021 used GESA [29]. Thus, changes in mitochondrial function in mouse muscles might be relatively subtle and subjected to interpretation depending on the method used in analysis. Indeed, when we analyzed the rodent time course data from SarcoAltas, we found more genes related to mitochondrial function are reduced in rat muscles compared with mouse muscles.

In addition, it should be noted that our oldest groups of male and female mice were 27 months. Other laboratories have found more pronounced age-related muscle wasting and more robust molecular changes in mice if they are allowed to age even more, resulting in “geriatric mice” [30, 42,43,44]. We find it challenging to age C57Bl6J mice (purchased from the Jackson laboratory, USA) beyond 27 months, since there is a high attrition rate (> 50%) and many surviving mice develop various types of malignancies (tumors). With the high tumor burden, it is challenging to differentiate whether skeletal muscles are responding to natural aging or cachectic environment.

Substantial differences in the sarcopenic phenotype between mice and rats are intriguing. We found that lower limb muscles in rats are more impacted by aging compared to the same muscles in mice. One possibility for more pronounced sarcopenia in the lower limbs of rats compared with mice, is more severe age-related deterioration of muscle innervation. Even though myofiber denervation occurs in both mice [45, 46] and rats [32], more severe loss of innervation may be a significant contributor to the more severe sarcopenia seen in rats. To this end, we observed a more robust dysregulation of the NMJ stability markers in the lower limb muscles of rats, compared with mice, pointing to more severe functional denervation of myofibers in rats.

Interestingly, the diaphragm muscle was relatively spared, and we saw milder changes in the aging transcriptome in the rat diaphragm compared with the lower limb muscle. Similar to our findings, Pannérec et al. reported more severe sarcopenia in the lower limb muscles of old Wistar rats, compared with the triceps muscle [32]. These differences were attributed to more severe age-associated changes in the lumbar-region of the spinal cord that innervates lower limbs, compared with the cervical region that innervates the diaphragm [32]. In rats, diaphragm motor units are innervated by phrenic motoneurons located at C3–C5 levels of the cervical spinal cord [47]. Thus, it is possible that similar to sparing of the triceps muscle in old rats [32], the diaphragm muscle is also spared due to relatively robust motoneurons in the upper region of the spinal cord.

Deterioration of NMJs and myofiber denervation is also a feature of human sarcopenia [25, 27, 48]. Moreover, loss of motoneuron numbers in old human spinal cords may be a contributing factor for severe cases of sarcopenia encountered in geriatric population [25].

Thus, the presence of a more severe denervation-driven phenotype in rats, combined with the significant downregulation of mitochondrial function, should be taken into account when choosing the mouse or the rat to model human sarcopenia. The rat is likely to be a more useful model to discover targets related to loss of innervation and mitochondrial disfunction in settings of sarcopenia.

As to the key question about whether there are consistent changes, which might point to evolutionary conserved mechanisms of aging, the main alteration which is consistent is the increase in innate immune and inflammatory signaling, with a particular focus on interferon signaling. Interestingly, there was a striking overlap between the age-related pathways identified in our study in both species with the pathways upregulated in cultured senescent cells [49]. For instance, pathways enriched by genes responsive to interferon alpha and gamma, TNF-alpha, and other pro-inflammatory cytokines that are induced in old rodent muscles, are also induced in senescent cells [49]. Increase in interferon responsive genes and pro-inflammatory pathways may suggest accumulation of senescent cells and DNA damage events in old rodent muscles. DNA damage itself is capable of inducing interferon response genes and driving innate immune response [34, 35]. Age-related increase in innate immune response may also be due to the change in heterochromatin which causes the exposure and activation of repetitive elements, including retrotransposons that has previously been observed [36]. Since we conducted bulk tissue transcriptomic analyses, we did not determine cell types that contribute to innate immune and inflammatory signaling in aging rodent skeletal muscles. Further studies aimed at identification of RNA and protein localization in tissue sections are warranted to reveal these cell types.

One might ask about mechanism: how are these genes perturbed? We found that alterations in several transcription factors could explain many of the changes observed: e.g., Runx3, Cebpa, Ikzf1, Nef2, Stat3, Esrra, and Nfyb. We did not directly query these factors in this study. We merely performed RT-qPCR amplification of selected transcription factors in all examined muscles of mice (Fig. S12) and rats (Fig. S13). These data showed that while a few transcription factors are regulated at the gene expression level, expression of others is not impacted by age (Figs. S12 and S13). Ideally, protein dynamics for these factors need to be determined. Indeed, once one does that, the next question would be how those factors might be activated, or whether the changes in transcription are epigenetically induced, making particular genes more accessible to these transcription factors. These questions will require extensive additional research and are thus left for future studies.

As to the question, “are various muscles different, similar or the same, within a species, when it comes to age-related changes?” we did see fairly similar changes across muscle types, but one could not say the changes were “the same”. For example, the diaphragm, which is constantly contracting throughout life, in order for the animal to breathe, showed the least number of age-related gene changes. This is consistent with the notion that exercise is helpful to avoid the effects of age. The changes which were seen in the diaphragm were upregulation of several inflammation pathways, including cytokine signaling and “innate immunity”, again suggestive of the idea that the muscle is undergoing a loss of heterochromatin, resulting in DNA damage [36].

Having a gene expression time course from rodents is indispensable to studying aging, but is difficult to prepare due to the cost in time and resources. Previous collections of rodent gene expression time courses made tradeoffs among the coverages of time points, tissues, sexes, and species, as well as the sample sizes for each condition [29, 30, 37]. Some of these limitations are now overcome by our data set.

After conducting this unbiased study, we next went back to the data to enquire about particular genes which have been previously shown to be upregulated in settings of skeletal muscle atrophy [21,22,23,24] (Fig. 5). We divided the figure into two sections, Fig. 5A surveys three select genes that increase coincident with denervation: Chrnd, MuSK, and Ncam1. Figure 5B surveys five genes that are associated with skeletal muscle atrophy [21]: Fboxo32, more commonly known as MAFbx or Atrogin-1; Trim63, more commonly known as MuRF1; Hdac4; Mt1, also known as Metallothionein 1—the gene is called Mt1A in the rat; Mt2 also known as Metallothionein 2 the gene is called Mt2A in the rat. The data show that markers of muscle atrophy are much more pronounced in the rat than in the mouse—there we saw a statistically significant upregulation of MuRF1, Hdac4, Mt1/Mt1A, and MT2/MT2A in the rat. None of these genes showed age-related transcriptional regulation in the mouse. Interestingly, MAFbx/Atrogin1/FbxO32 was not upregulated during sarcopenia in the mouse or the rat, distinguishing sarcopenia from more acute settings of atrophy. We had similar findings when we surveyed rat sarcopenia proteomically in the past [6], thus repeating the finding that MAFbx is not upregulated during sarcopenia in the rat, even though there is a significant loss of the skeletal muscle. As for the denervation marker genes, MuSK and acetylcholine receptor delta subunit (Chrnd) and Ncam1 are all significantly upregulated in the rat but not the mouse, indicating that the rat is functionally denervated. Prior studies do show that mice too experience NMJ changes with age [44, 46, 50], but this study shows that molecularly, there are much clearer signs of functional denervation in the rat than the mouse with age.

Sarcopenia is clearly distinct from simple acute muscle atrophy. We find signs of functional denervation, loss of mitochondrial gene expression, and significant increases in inflammation in the rat model, where the onset of age-related decline in muscle mass was evident at 21 months and further progressed by 27 months. In the mouse, age-related decline in muscle mass was less dramatic compared with the rat, and there were much fewer signs of age-related gene alterations. Although, similar to rat, mouse also displayed age-related increases in the immune and inflammatory signaling and downregulation of the ECM-related genes.

After comparing mice to rats in order to find conserved differences throughout evolution, and taking into account the prior reports on geriatric mice, one is left with the strong impression that sarcopenia is primarily a disease of inflammation, with perhaps consequent inputs due to changes in the extracellular matrix and loss of mitochondrial activity, and with an additional component contributed by the motoneuron, resulting in functional denervation—all contributing to and finally resulting in weakness, frailty, and then death. It remains to be seen if any of these processes can be pharmacologically perturbed so as to preserve muscle function in old age—we and others have already shown one instance where this was found, by using a rapalog to block some of the age-related inflammatory changes seen [51, 52]—this treatment also improved aged muscle phenotypes [51, 52]. This work needs to be followed up, asking if any of the sarcopenia-associated pathways can be counter-regulated, as this was done with pharmacological inhibition of mTORC1 signaling [30, 53], so as to continue to discover a robust set of interventions that might keep the elderly strong, healthy, and active.