Inhibition of mTORC1 signaling in aged rats counteracts the decline in muscle mass and reverses multiple parameters of muscle signaling associated with sarcopenia

There is a lack of pharmacological interventions available for sarcopenia, a progressive age-associated loss of muscle mass, leading to a decline in mobility and quality of life. We found mTORC1 (mammalian target of rapamycin complex 1), a well-established critical positive modulator of mass, to be hyperactivated in sarcopenic muscle. Furthermore, inhibition of the mTORC1 pathway counteracted sarcopenia as determined by observing an increase in muscle mass and fiber type cross sectional area, surprising because mTORC1 signaling has been shown to be required for muscle mass gains in some settings. Additionally, several genes related to senescence were downregulated, while gene expression indicators of neuromuscular junction denervation were diminished using a low dose of a rapalog. Therefore mTORC1 inhibition may delay the progression of sarcopenia by directly and indirectly modulating multiple age-associated pathways, implicating mTORC1 as a therapeutic target to treat sarcopenia.


Introduction 24
Skeletal muscle size is physiologically regulated by load and activity, and can decrease when 25 load is reduced. Muscle also atrophies, or decreases in size, in pathological conditions such as 26 cancer, immobilization and denervation (1). One setting where muscle mass and function are 27 diminished is old age. This loss of muscle is called sarcopenia, and it is associated with a 28 decrease in the ability to move, leading to morbidity and ultimately mortality (2); indeed, a 29 decrease in walking speed is one of the strongest predictors of mortality in humans, and this 30 finding is associated with sarcopenia (3, 4). In addition to frailty and sarcopenia, aging of course 31 affects every tissue system and greatly increases susceptibility to other serious diseases and co-32 morbidities, such as cancer, heart failure, chronic kidney disease, loss of vision, dementia and 33 Alzheimer's disease (1, 5, 6). 34 Experimental data strongly suggest the coordinated regulation of aging by distinct 35 molecular pathways (7); modulation of these pathways can counteract several age-related 36 diseases and co-morbidities, and prolong life (7-10). Of these signaling pathways, genetic or 37 pharmacological inhibition of the mammalian target of rapamycin (mTORC1) is thus far the 38 best-validated intervention to delay age-related pathophysiological changes (11). For instance, 39 the use of an mTORC1 inhibitor, rapamycin, even when administered at later stages in life, has 40 been shown to extend lifespan in mice (12-15). Pharmalogical agents related to rapamycin are 41 called "rapalogs". Use of a rapalog for aging-like indications has recently been translated to 42 human beings, where it was shown to improve responses to vaccinations in the elderly, 43 coincident with decreasing signs of immune-senescence (16). The low dose rapalog treatment 44 used in the human study was reverse-translated to rats, where it was shown that intervention late 45 in life could prevent signs of age-related kidney pathology (17). However, there has always been 46 concern about the potential effects of rapamycin and rapalogs on skeletal muscle. For example, 47 inhibition of the mTORC1 pathway was shown to entirely block responses to compensatory 48 hypertrophy in mice (18). This certainly gave the impression that activation of mTORC1 49 signaling was desireable for the maintenance of muscle mass. Most recently it was shown that 50 rapamcyin treatment inhibited muscle mass increase caused by myostatin loss (19). Thus it 51 seemed reasonable that inhibition of the pathway was not desireable in settings of muscle loss (1, 52 18, 20). 53 As to the pathway, Akt induces protein synthes in part by activation of mTORC1 54 signaling (18, 21). mTOR exists in the distinct complexes, mTORC1 and mTORC2. mTORC1 55 is characterized by the presence of RAPTOR (regulatory-associated protein of mTOR) (22), 56 while TORC2 binds to RICTOR (rapamycin-insensitive partner of mTOR) (23, 24). The 57 mTORC1 complex induces downstream signaling responsible for protein synthesis through 58 phosphorylation and activation of S6 Kinase 1 (S6K1), and via inhibition of 4E-BP1 (24, 25), 59 and is sensitive to inhibition by rapamycin and rapalogs. In addition to the anabolic function, Akt 60 also limits muscle protein degradation and atrophy by phosphorylating and thereby inhibiting the 61 FOXO (also known as Forkhead) family of transcription factors. Activation of FOXO3 is 62 sufficient to induce atrophy (26, 27); transgenic expression of FOXO1 also lead to an atrophic 63 phenotype (28, 29). Dephosphorylated FOXO1 and FOXO3 proteins translocate to the nucleus 64 where they induce transcription (30), upregulating the expression of the muscle-atrophy 65 associated E3 ligases, muscle RING finger 1 (MuRF1) and muscle atrophy F-box 66 (MAFbx)/Atrogin-1 (31-33). Both MuRF1 and MAFbx/Atrogin-1 are specifically upregulated in 67 atrophic conditions (34, 35), and target proteins that are critical for muscle structure and protein 68 synthesis for degradation, thereby inducing muscle loss (36-40). 69 mTORC1 inhibition has been widely suggested as a way to improve function in the 70 elderly in various tissues. However, its potential as a therapeutic intervention for the treatment of 71 sarcopenia has not been considered. Upon examination, we were surprised to learn that 72 mTORC1 signaling is upregulated rather than downregulated coincident with signs of sarcopenia 73 in rats, We therefore explored the effects of rapalog treatment in this setting. The results 74 demonstrate that the inhibition of mTORC1 is helpful in preventing pathological changes related 75 to sarcopenia.

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Increased activation of the mTORC1 pathway with age 79 Given prior reports that mTORC1 inhibition was helpful to treat a variety of age-related 80 disorders, but also the data that mTORC1 activation is required for muscle hypertrophy, we 81 conducted a time course analysis of the mTORC1 pathway to get a full scope of how its activity 82 changes with age. In laboratory settings, Sprague Dawley rats have an average lifespan of up to 83 2.5 to 3 years (41). In our study, male rats ranging from 6-months to 27-months were used.

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Protein lysates from gastrocnemius muscles were probed for the downstream effector of 85 mTORC1, phosphorylated ribosomal protein S6 (rpS6), as a determinant of pathway activity.

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Basal (6 hours fasted) levels of phosphorylated rpS6 gradually increased as the rats aged, with a 87 substantial increase of about 10-fold in the oldest animals aged 27-months compared with 6-88 months. (Fig. 1A, B). The age-related increase in mTORC1 signaling coincided with a decrease 89 in muscle mass. At 21-months gastrocnemius muscle weights declined and progressively 90 atrophied at each later time point (Fig. 1C). Though muscle loss at this age is not a surprise, the 91 coincidence of this loss with mTORC1 activation was quite unexpected, given that it favors 92 muscle growth and hypertrophy.

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Skeletal muscle mass and quality is improved in sarcopenic rats treated with the rapalog 94 RAD001 95 Experimental evidence shows that the use of rapalogs as therapeutic agents is beneficial in 96 extending lifespan and counteracting age-related morbidities in humans and other evolutionarily 97 diverse species (reviewed in (42)). We sought to determine whether rapalog treatment could 98 counter the pathophysiological changes associated with sarcopenia. Aging rats display signs of 99 sarcopenia beginning at 18-months (43). In the present study, aged rats (22-months) were dosed 100 daily with either vehicle or 0.15mg/kg RAD001 for 6-weeks. This dose of RAD001 is equivalent 101 to a clinical dose of 0.5mg in humans, ensuring therapeutic relevance (16). Vehicle treated young 102 adult rats (7-months) served as a comparative baseline for aging effects. At the end of the 103 treatment, aged and young adult rats were 24-months and 9-months old respectively, and will be 104 referred to as such.

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To determine if we were able to ameliorate age-related muscle loss with rapalog 106 treatment, we measured the wet weights of the tibialis anterior (TA), plantaris, gastrocnemius, 107 and soleus muscles. Consistent with previous data, all muscles, except for the soleus muscle 108 from 24-month old vehicle treated rats had considerably reduced mass compared to 9-month old 109 rats ( Fig. 2A). RAD001 treatment did not lead to further atrophy in any of these muscles. On the 110 contrary, rapalog appeared to be protective for aged animals, and reduced extensive muscle mass 111 loss. Plantaris and TA muscles showed a surprising increase in mass, with the TA muscle being 112 significantly increased compared to vehicle treated animals ( Fig. 2A). Our data provide strong 113 evidence that when administered to sarcopenic rats, low dose rapalog treatment is not detrimental 114 to muscle mass. Rather, it allows for animals to maintain or gain muscle.

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Changes in muscle mass often reflect morphological alterations in tissue. We performed 116 histological analysis on H&E stained plantaris muscle cross-sections. Tissue from 9-month old 117 rats had normal morphology, typical of healthy muscle (Fig. 2B). In contrast, we detected several 118 indicators of distressed muscle in aged animals that received only vehicle. A high proportion of 119 fibers had a smaller cross-sectional area, a phenotype associated with muscle atrophy (Fig. 2D, 120 E). Moreover, about 23% of myofibers from vehicle treated 24-month old muscles presented 121 with central nuclei, indicative of prior degeneration and ongoing regeneration (Fig. 2B, C). There 122 was a striking reduction in the number of myofibers with central nuclei in 24-month old plantaris 123 muscles treated with RAD001 compared with aged matched muscles treated with vehicle ( Fig.   124 2B, C). In addition, consistent with the observed trend of increased plantaris muscle mass in 125 RAD001 treated rats, the average myofiber cross-sectional area tended to increase (Fig. 2D) -the 126 most obvious change was a reduced frequency of very small, mis-shaped atrophic myofibers 127 (Fig. 2E). Taken together, these data show that a low dose rapalog treatment for 6 weeks can 128 counteract age-related morpho-pathological changes in sarcopenic skeletal muscle -especially 129 signs of degeneration requiring regeneration, as measured by the presence of central nuclei.

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Chronic activation of the mTORC1 pathway by muscle-specific deletion of Tsc1, a 131 negative regulator of mTORC1, has been shown to cause a late-onset myopathy, with muscle 132 atrophy in young adult mice (44). Inhibition of mTORC1 activity using rapamycin was able to 133 reverse the observed pathological changes and normalize muscle mass in these animals (44).

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Despite evidence of similarly sustained mTORC1 signaling in aged muscle, its inhibition has not  Fig. S1). These data confirm that the relatively low dose of the rapalog used in the present 141 study was sufficient to inhibit mTORC1 signaling in aged skeletal muscle. 142 mTORC1 inhibition reverses molecular changes associated with sarcopenia 143 We previously reported on age-related gene expression changes that help to demonstrate the 144 molecular pathogenesis of sarcopenia (43). These data revealed the transcriptional upregulation 145 of several pathways, including pathways related to innate inflammation and senescence, cellular 146 processes modulated by mTORC1. Because RAD001-treated animals displayed a remarkable 147 sparing of muscle morho-pathology and mass with mTORC1 inhibition, we sought to determine 148 the molecular changes that could account for this.

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The E3 ligase MuRF1 is an important regulator of atrophy (1, 34). MuRF1 gene 150 expression was analyzed in young and old muscles treated with vehicle, and in old muscles  The onset of senescence with age is associated with the inability to efficiently repair and 165 recover muscle, a contributing factor to the progressive decline in muscle mass in sarcopenia.

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Cell cycle proteins Cdkn1a (p21) and Cdkn2a (p16) are known cellular senescence markers that Gadd45a, known to be markers of functional denervation (43), were determined by RT-qPCR. In 180 agreement with our previous observations, all of these genes were significantly upregulated in 181 muscles from vehicle treated 24-month old animals compared to 9-month old controls (Fig. 5).

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Interestingly, treatment of aged animals with RAD001 reduced the transcriptional upregulation 183 of these denervation-associated gene markers in relation to their vehicle treated age-matched 184 counterparts (Fig. 5). These data suggest that suppressing the mTORC1 pathway in aged animals 185 could be protective against age-associated denervation. Age-associated diseases comprise many of the most serious conditions afflicting human beings: 189 sarcopenia and frailty, cancer, heart disease, Alzheimer's disease, and chronic kidney disease.

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The mTORC1 inhibitor rapamycin and its analogs (rapalogs) have been shown to extend lifespan 191 (12-15) and delay many of these age-related conditions (9-11). These findings have even been 192 extended to human beings, where a rapalog reversed immune-senescence and increased 193 responses to vaccines that normally decline with age in the elderly (16). One area which caused 194 some concern when it came to giving mTORC1 inhibitors to aged subjects was skeletal muscle, 195 since mTORC1 activation mediates protein synthesis (51) and mTORC1 inhibition blocks load-196 dependent hypertrophy (52). However, when we examined mTORC1 signaling in skeletal 197 muscles in rats, at ages where sarcopenia occurs (43), we were surprised to see that signaling was 198 increased rather than decreased -there was an age-related increase in the phosphorylation of 199 rpS6, a readout of mTORC1 activity. Coincident with elevated mTORC1 signaling, there was a 200 progressive decrease in skeletal muscle mass. These findings at least established that activation 201 of mTORC1 was coincident with atrophy, and therefore was not sufficient to prevent muscle loss 202 in sarcopenic conditions. We therefore asked whether counter-regulating this age-associated 203 increase in mTORC1 signaling may perhaps be beneficial for skeletal muscle, and thus we 204 treated aged rats for six weeks with a rapalog, RAD001, at a clinically relevant low dose (we 205 reverse-translated the low dose that had been used in a human study) (16). Treatment with a 206 similar low dose of the rapalog RAD001, although with a distinct dosing regimen (intermittent 207 dosing) had recently been shown to delay age-related changes in the kidney (17). 208 We were surprised to see that skeletal muscle mass increased rather than decreased as a 209 result of mTORC1 inhibition. This was not due to adverse events such as edema; muscles -210 particularly the TA, showed increased mass, and examination of individual myofibers showed a 211 trend towards increased cross-sectional area; very small atrophic fibers that are found with age 212 were in particular absent with rapalog treatment. With age, there is a dramatic increase in fibers 213 with central nuclei -a sign of muscle undergoing degeneration followed by regeneration.

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Treatment with the rapalog for six weeks decreased the number of myofibers with central nuclei 215 by almost half, which is a marker that there was less functional degeneration, requiring 216 subsequent regeneration. In line with this, there were also signs that functional denervation 217 occured with age; this impression was bolstered molecularly by the demonstration that gene 218 markers associated with denervation, including MuSK and several of the acetyl choline receptor 219 genes, were increased with age -consistent with a prior report (43). These denervation markers 220 were each counter-regulated by the rapalog, indicating that rapalog treatment prevented 221 functional denervation, providing an additional mechanism for preservation of muscle mass.

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Rapalog treatment decreased mTORC1 signaling detected by de-phosphorylation of 223 S6K1 and its downstream target rpS6. Coincident with this, mRNA levels of the putative 224 atrophy marker MuRF1 were significantly reduced. In addition to MuRF1, the metallothionein 225 MT1 was downregulated by mTORC1 inhibition. We had previously shown that this is a high-226 fidelity marker of atrophy, and knocking out the MT genes in mice causes muscle hypertrophy 227 (45). This finding too is consistent with the increase in mass observed in the present study, and 228 provides further mechanistic rationale. As for the senescence markers p16 and p21, they were 229 elevated in aged muscle when compared to young muscle, and reversed towards the "younger   Anti-rabbit and anti-mouse IgG HRP-conjugated secondary antibodies were also from Cell 282 Signaling Technologies. Densitometric analysis was performed using Fiji 1.51n software.   The authors thank Danuta Lubicka for formulating RAD001, as well as study support associates 336 and the veterinary team for maintaining aged rats, and assistance in animal experimentation.