Preservation of skeletal muscle mass is achieved by maintaining a homeostatic balance between muscle regeneration, protein synthesis, and protein degradation. This balance is significantly perturbed during the physiological process of aging, leading to a loss of muscle mass and a decline in function over time. The decrease in the ability to regenerate after injury and the exaggerated atrophic response to disuse of sarcopenic muscles are two major clinical scenarios that contribute to morbidity and mortality in the aging population (19
Here, we demonstrate that the ability to repair skeletal muscle after injury is restored upon treatment with the AT1 receptor blocker losartan in sarcopenic mice. Furthermore, losartan treatment can prevent loss of muscle mass induced by hindlimb immobilization. Our data provide evidence that blockade of the AT1 receptor modulates multiple critical pathways associated with skeletal muscle homeostasis including the canonical and noncanonical TGF-β signaling cascades as well as the IGF-1/Akt/mTOR pathway.
TGF-β signaling, a known inhibitor of skeletal muscle regeneration and remodeling, promotes the formation of fibrotic tissue (7
). Previous studies showed that losartan inhibited canonical TGF-β signaling, thereby improving muscle regeneration and function in mouse models of MFS and DMD (20
). Indeed, administration of losartan after the induction of muscle laceration injuries in adult mice significantly decreased the formation of fibrosis (21
). Our results presented here shed further light into the mechanism of action of losartan in skeletal muscle. We demonstrate that blockade of the AT1 receptor during regeneration not only inhibits the canonical but also modulates the noncanonical TGF-β signaling cascade.
The TGF-β signaling pathway is one of the many pathways altered in skeletal muscle during the physiological process of aging. Specifically, increases in the canonical and noncanonical TGF-β pathways as well as alterations of Notch and WNT signaling pathways have been associated with an inability to activate satellite cells and repair injured muscle (3
). In the context of alterations of the canonical TGF-β signaling cascade, it has been suggested that an imbalance between TGF-β and Notch signaling increases the production of the cyclin-dependent kinase inhibitor p21 (3
). However, our data show an exaggerated increase of TGF-β signaling without an impaired muscle regeneration response or increase in p21 expression at early stages of the muscle repair process. In contrast, our results demonstrate that canonical TGF-β signaling remains increased during later stages of regeneration associated with a decrease of p21 expression. It is certainly possible that the different observations at early stages of muscle repair might be due to in vitro–performed experiments versus our in vivo mouse studies. However, our observations agree with previous evidence that an increase of canonical TGF-β signaling inhibits expression of p21 in the C2C12 murine muscle cell line (45
) and that p21 is indeed necessary for skeletal muscle differentiation and remodeling in response to injury in vivo (36
Our finding that losartan also modulates the noncanonical TGF-β signaling cascade is of particular interest because this pathway has previously been implicated in various stages of the muscle repair response. During the early stages of regeneration, we show injury-related expression of these proteins believed to be necessary for efficient regeneration. ERK1/2 is postulated to enhance myoblast proliferation during the acute stage of muscle repair; however, evidence suggests that its sustained expression may repress muscle-specific gene expression and myoblast differentiation (12
). Our data demonstrating an up-regulation of ERK1/2 in placebo-treated mice during the acute and later stages of regeneration further support this hypothesis. However, it is important to emphasize that the decrease in ERK signaling could be directly related to blockade of the AT1 receptor independent of TGF-β signaling (27
) (fig. S1
). In contrast to the biphasic expression of ERK, the expression of p38α is required throughout the process of muscle remodeling; it is critical for the exit of myoblasts from the cell cycle and the induction of muscle-specific genes necessary for myofiber recruitment and formation (13
). Our results show a delayed up-regulation of phosphorylated p38 in the placebo-treated mice at 19 days after CT as compared to the losartan-treated mice that have an increase at 4 days. Thus, we suggest that this delay in the expression of p38 contributes to the impaired muscle remodeling process observed in the placebo-treated mice.
Evidence suggests that the canonical and noncanonical TGF-β pathway regulates members of the MRF family (46
). These factors include MyoD, Myf5, myogenin, and MRF4. Additional key players during myogenesis are Pax7, which is expressed during satellite cell activation, and p21, which permits irreversible withdrawal of satellite cells from the cell cycle, a critical and necessary step for the differentiation and maturation of muscle fibers (36
). Our observations of an increase in Pax7 and MyoD at 4 and 19 days after CT injection in placebo-treated animals suggest that aged mice fail to transition from a state of satellite cell proliferation toward muscle differentiation and fusion (47
). It is likely that losartan-induced blunting of the canonical and noncanonical TGF-β signaling pathways permits muscle remodeling by improving the physiological environment of satellite cells, which is critical for satellite cell function and their ability to regenerate and repopulate myofibers (33
Additionally, we investigated disuse atrophy, which poses a frequent problem for individuals of all ages, but is particularly challenging for older adults. When skeletal muscle is subjected to immobilization for a period of time, muscle atrophy occurs (1
). This atrophic response is a completely reversible process in the younger population (1
); however, as a result of the physiological process of aging, animal models and humans are known to exhibit an exaggerated atrophy in response to disuse and an inability to rebuild muscle mass after immobilization (19
). Studies performed in human subjects reported a 30% loss of skeletal muscle mass after only 2 weeks of immobilization in older men as compared to a loss of less than 2% in young men, and only 2.5% of the loss muscle repopulated (43
). Our data suggest that the decrease in muscle mass of aged rodents and humans subjected to immobilization is in fact due to a loss of muscle fibers rather than actual atrophy of myofibers generally observed in the young (39
).This provides a mechanistic explanation for the exaggerated response to disuse and the inability to recover with aging. Furthermore, the ability to prevent this loss of muscle fibers with losartan provides a rationale to explore this drug as a potential therapeutic option for disuse atrophy in older adults.
We did not observe significant alterations in the canonical or non-canonical TGF-β signaling pathways in our placebo- or losartan-treated immobilized animals with the exception of p38. Previous studies have shown immobilization-induced alterations in these pathways. Specifically, an increase in the MAP kinase pathway has been suggested to contribute to the loss of muscle mass during disuse atrophy (17
). The levels of p38 expression in the losartan-treated immobilized TA were significantly reduced, supporting the notion that when p38 is up-regulated during immobilization, it induces atrophy (39
). Because our analyses were performed after 21 days of immobilization, it is possible that transient alterations of these pathways may have occurred at an earlier time point. Because altered TGF-β signaling did not appear to play a major role in conferring protection against disuse atrophy in this immobilization model, we performed analyses of the IGF-1/Akt/mTOR pathway, which is a critical mediator of skeletal muscle proteolysis and synthesis and has been shown to be modulated by losartan treatment in skeletal muscle (40
). Phosphorylated Akt phosphorylates and activates mTOR signaling, thereby causing an increase of protein synthesis. In addition, Akt phosphorylates and inactivates the transcription factor FoxO3a, preventing muscle protein degradation. The IGF-1/Akt/mTOR pathway and the inactivated form of FoxO3a are down-regulated during various challenges, causing muscle atrophy (17
). Our analyses of placebo-treated, immobilized TA muscle of aged mice revealed the expected decrease of members of the IGF-1/Akt/mTOR signaling cascade pathway. In contrast, losartan treatment prevented down-regulation of the expression profile of this pathway and resulted in an up-regulation of mTOR activation, suggesting that increased protein synthesis and inhibition of protein degradation may contribute to protection against disuse atrophy in sarcopenia.
Our results indicate that the blockade of the AT1 receptor has beneficial effects on skeletal muscle remodeling in response to injury and conferring protection against disuse atrophy in sarcopenia by modulating the TGF-β and IGF-1/Akt/mTOR signaling cascades. Previous studies in young rats have shown that angiotensin II is necessary for a hypertrophic response elicited by muscle overload and that the effect may be partly mediated by the AT1 receptor (50
). Together, these results suggest that there are age-related differences in response to AT1 receptor blockers in skeletal muscle.
With the number of individuals older than 60 years doubling over the next 40 years, sarcopenia is a major public health problem (51
). Additionally, normal muscle mass and strength are required to perform daily activities. Skeletal muscle injuries and disuse atrophy are clinical scenarios that increase morbidity and rehabilitation time of the aging population and represent additional challenges for geriatric healthcare providers. Our observations show that losartan can effectively improve skeletal muscle regeneration and preserve mass in physiological challenging conditions using a sarcopenic mouse model. Notably, losartan is a Food and Drug Administration–approved drug that is well tolerated in all age groups, with rare events of low blood pressure reported as a side effect in the elderly population (52
). In our studies, losartan was administered before the induction of either injury or immobilization; thus, future clinical trials should consider administering losartan during the early stages of muscle injury and/or immobilization. In summary, these preclinical studies provide the basis for new therapeutic strategies in patients with sarcopenia.