Losartan restores skeletal muscle remodeling and protects against disuse atrophy in sarcopenia

Abstract

Sarcopenia, a critical loss of muscle mass and function because of the physiological process of aging, contributes to disability and mortality in older adults. It increases the incidence of pathologic fractures, causing prolonged periods of hospitalization and rehabilitation. The molecular mechanisms underlying sarcopenia are poorly understood, but recent evidence suggests that increased transforming growth factor-β (TGF-β) signaling contributes to impaired satellite cell function and muscle repair in aged skeletal muscle. We therefore evaluated whether antagonism of TGF-β signaling via losartan, an angiotensin II receptor antagonist commonly used to treat high blood pressure, had a beneficial impact on the muscle remodeling process of sarcopenic mice. We demonstrated that mice treated with losartan developed significantly less fibrosis and exhibited improved in vivo muscle function after cardiotoxin-induced injury. We found that losartan not only blunted the canonical TGF-β signaling cascade but also modulated the noncanonical TGF-β mitogen-activated protein kinase pathway. We next assessed whether losartan was able to combat disuse atrophy in aged mice that were subjected to hindlimb immobilization. We showed that immobilized mice treated with losartan were protected against loss of muscle mass. Unexpectedly, this protective mechanism was not mediated by TGF-β signaling but was due to an increased activation of the insulin-like growth factor 1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. Thus, blockade of the AT1 (angiotensin II type I) receptor improved muscle remodeling and protected against disuse atrophy by differentially regulating the TGF-β and IGF-1/Akt/mTOR signaling cascades, two pathways critical for skeletal muscle homeostasis. Thus, losartan, a Food and Drug Administration-approved drug, may prove to have clinical benefits to combat injury-related muscle remodeling and provide protection against disuse atrophy in humans with sarcopenia.

Fig. 1

Losartan improves muscle remodeling and in vivo function in sarcopenic mice. (A to C) Histological analyses of the tibialis anterior (TA) muscle after cardiotoxin (CT) injection of placebo-treated (left) and losartan-treated (right) 21-month-old C57BL/6 male mice. Hematoxylin-eosin (H&E) (A) shows evidence of early signs of regeneration indicated by open arrows and no phenotypic differences between the treatment groups. Developmental myosin immunofluorescence (B) confirms similar amounts of newly regenerating cells. H&E staining (C) at 19 days after CT reveals impaired regeneration in the placebo-treated animal evident by fibrosis (closed arrow). Scale bar, 200 μm. (D) The amount of fibrosis was quantified and expressed as a percentage of the damaged area. (E) In vivo function of the TA was assessed using the titanic/twitch ratio 19 days after injury. Data are means ± SEM (n = 4 to 7 animals). *P < 0.05; ***P < 0.001, unpaired t test.

Fig. 2

AT1 blockade modulates the canonical and noncanonical TGF-β pathways during muscle remodeling. (A and B) Western blot analyses of the TA using antibodies against the proteins indicated show the levels of the canonical and noncanonical TGF-β pathways in noninjected controls (N) and in the placebo (P) and losartan (L) groups at 4 days (A) and 19 days (B) after CT. Actin was used as a loading control. (C to F) Levels of p-p38 (C), pSmad2 (D), and pERK (E and F) were quantified using relative expression in arbitrary units (AUs). Data are means ± SEM (n = 4 animals). *P < 0.05, one-way ANOVA with Student-Newman-Keuls method.

Fig. 3

Modulation of TGF-β pathways affects expression of the myogenic regulatory pathway. (A and B) Western blot analyses of the myogenic regulatory pathway at 4 days (A) and 19 days (B) after CT in the TA of non-injected control (N), placebo-treated (P), and losartan-treated (L) mice. Actin was used as a loading control. (C and D) Relative expression was calculated for myogenin (C) and p21 (D) in arbitrary units (AUs). Data are means ± SEM (n = 4 animals). *P < 0.05, one-way ANOVA with Student-Newman-Keuls method.

Fig. 4

Losartan prevents disuse atrophy in sarcopenic mice. (A and B) H&E staining of the immobilized TA muscle of placebo-treated (left) and losartan-treated (right) animals does not show differences in the size of the individual myofibers (A) (scale bar, 100 μm) but reveal differences in the cross-sectional area (CSA) of the intact immobilized TA of the placebo group (B) (scale bar, 500 μm). There were also areas of fibrosis present (arrow) after immobilization only with placebo treatment (A). (C to F) Quantitative analyses were used to confirm histological findings by comparing the TAs from the nonimmobilized control (N), contralateral placebo (PC), immobilized placebo (PI), contralateral losartan (LC), and immobilized losartan (LI) limbs. (C) Comparisons were made between the TA weights of the contralateral and immobilized legs within the treatment groups (n = 5 to 6 animals). *P < 0.05, unpaired t test. (D to F) The evaluation of minimum Feret’s diameter (MFD), CSA, and myofiber count was made between the subgroups (n = 5 to 6 animals). **P < 0.01, one-way ANOVA with Student-Newman-Keuls method.

Fig. 5

Losartan does not inhibit canonical and noncanonical TGF-β pathways in immobilized aged mice. (A) Western blot analyses of the canonical and noncanonical TGF-β pathway in the nonimmobilized control (N) and immobilized placebo (P) and losartan (L) TAs. Actin was used as a loading control. (B) The levels of p-p38 were quantified using relative arbitrary units (AUs). Data are means ± SEM (n = 4 animals). *P < 0.05, one-way ANOVA with Student-Newman-Keuls method.

Fig. 6

AT1 blockade up-regulates the IGF-1/Akt/mTOR pathway during immobilization in sarcopenic muscle. (A to E) Analyses of the indicated proteins of the IGF-1/Akt/mTOR pathway (A) illustrate the effects of immobilization on the expression of pAkt (B), pFoxO3a (C), p-mTOR (D), and p4E-BP1 (E) in the TA of the nonimmobilized control (N) and immobilized placebo (P) and losartan (L) groups, expressed in relative arbitrary units (AUs). Actin was used as a loading control. Data are means ± SEM (n = 4 animals). *P < 0.05, one-way ANOVA with Student-Newman-Keuls method.