Rapamycin reverses elevated mTORC1 signaling in lamin A/C-deficient mice, rescues cardiac and skeletal muscle function, and extends survival

Abstract

Mutations in LMNA, the gene that encodes A-type lamins, cause multiple diseases including dystrophies of the skeletal muscle and fat, dilated cardiomyopathy, and progeria-like syndromes (collectively termed laminopathies). Reduced A-type lamin function, however, is most commonly associated with skeletal muscle dystrophy and dilated cardiomyopathy rather than lipodystrophy or progeria. The mechanisms underlying these diseases are only beginning to be unraveled. We report that mice deficient in Lmna, which corresponds to the human gene LMNA, have enhanced mTORC1 (mammalian target of rapamycin complex 1) signaling specifically in tissues linked to pathology, namely, cardiac and skeletal muscle. Pharmacologic reversal of elevated mTORC1 signaling by rapamycin improves cardiac and skeletal muscle function and enhances survival in mice lacking A-type lamins. At the cellular level, rapamycin decreases the number of myocytes with abnormal desmin accumulation and decreases the amount of desmin in both muscle and cardiac tissue of Lmna(-/-) mice. In addition, inhibition of mTORC1 signaling with rapamycin improves defective autophagic-mediated degradation in Lmna(-/-) mice. Together, these findings point to aberrant mTORC1 signaling as a mechanistic component of laminopathies associated with reduced A-type lamin function and offer a potential therapeutic approach, namely, the use of rapamycin-related mTORC1 inhibitors.

Fig. 1

Signaling through the mTORC1 pathway is increased in Lmna^–/– mice. (A) Western blot analysis of Lmna^+/+ (n = 7) and Lmna^–/– (n = 8) mice heart tissue lysates. Phosphorylated mTOR^(S2448), S6 kinase^(T389), rpS6^(S235/S236), and 4E-BP1^(S65) are increased in Lmna^–/– mice compared to Lmna^+/+ mice. (B) Western blot analysis of Lmna^+/+ (n = 7) and Lmna^–/– (n = 8) mice skeletal muscle (quadriceps) tissue lysates. Phosphorylated rpS6^(S235/S236) and 4E-BP1^(S65) are increased in Lmna^–/– mice compared to Lmna^+/+ mice. However, phosphorylated S6 kinase^(T389) is not significantly increased. *P < 0.05; **P < 0.01.

Fig. 2

Treatment of Lmna^–/– mice with rapamycin improves heart and skeletal muscle function. (A) Representative echocardiograms of Lmna^+/+ and Lmna^–/– mice on a control or rapamycin diet. White arrow, LVEDD; red arrow, LVESD. (B) Normalized LVESD (P < 0.001), normalized LVEDD, fractional shortening, and MPI. Values are significantly different in Lmna^–/– mice fed control diet (n = 7) compared to Lmna^+/+ mice fed control diet (n = 4). Rapamycin in the diet significantly improved LVESD, fractional shortening, and MPI in Lmna^–/– mice (n = 7) compared to Lmna^–/– mice fed the control diet (n = 7). Black bars, control-fed; white bars, rapamycin-fed. IVRT, isovolemic relaxation time; IVCT, isovolemic contraction; LVET, LV ejection time. (C) Analysis of muscle function by rotarod test. There was a significant decrease in latency to fall and maximum speed reached in Lmna^–/– mice fed control diet (n = 5) compared to Lmna^+/+ mice fed control diet (n = 5). Dietary rapamycin significantly increased latency to fall and maximum speed reached in Lmna^–/– mice (n = 4) compared to Lmna^–/– mice fed the control diet (n = 5). Black bars, control-fed; white bars, rapamycinfed. *P < 0.05, **P < 0.01, Lmna^–/– control-fed versus Lmna^+/+ control-fed; ^#P < 0.05, ^##P < 0.01, Lmna^–/– rapamycin-fed versus Lmna^–/– control-fed.

Fig. 3

Treatment of Lmna^–/– mice with rapamycin increases survival. (A) Kaplan-Meier plot of Lmna^–/– mice (mixed 129Sv-C57BL/6J genetic background) fed control (n = 23) or diet that included encapsulated rapamycin (n = 23). Survival was significantly increased in Lmna^–/– mice fed dietary rapamycin (P = 0.0013). (B) Kaplan-Meier plot of Lmna^–/– mice (mixed 129Sv-C57BL/6J background) injected with vehicle (n = 11) or a higher dose of rapamycin (8 mg/kg) (n = 11). Survival was significantly increased in Lmna^–/– mice injected with rapamycin (P = 0.0002).

Fig. 4

Signaling through the mTORC1 pathway in heart and muscle of Lmna^–/– mice is reduced by rapamycin treatment but is dependent on dosage. (A) Phosphorylated mTOR^(S2448), S6 kinase^(T389), and rpS6^(S235/S236) were decreased in hearts from Lmna^–/– mice fed the rapamycin diet (R) (n = 6) compared to Lmna^–/– mice fed the control diet (C) (n = 6). Phosphorylation of 4E-BP1^(S65) was not significantly reduced. (B) Phosphorylated mTOR^(S2448), S6 kinase^(T389), rpS6^(S235/S236), and 4E-BP1^(S65) were not significantly reduced in muscle from Lmna^–/– mice fed the rapamycin diet (R) (n = 6) compared to Lmna^–/– mice fed the control diet (C) (n = 5). (C) Immunohistochemistry of heart tissue sections from Lmna^–/– mice fed the rapamycin diet (n = 3), which showed a significant decrease in the percentage of cardiomyocytes with accumulation of phosphorylated rpS6 compared to Lmna^–/– mice fed the control diet (n = 3). (D and E) Phosphorylated rpS6^(S235/S236) was decreased in heart and muscle from Lmna^–/– mice injected with rapamycin (R) (8 mg/kg) (n = 4) compared to vehicle-injected Lmna^–/– mice (C) (n = 4). Panel (D) is heart and panel (E) is muscle. *P < 0.05; **P < 0.01.

Fig. 5

Rapamycin reduces abnormal desmin accumulation in Lmna^–/– mice. (A) Desmin protein levels in heart and skeletal muscle of rapamycin-treated Lmna^–/– mice (n = 6) compared to control-fed Lmna^–/– mice (n = 6) as measured by Western blot analysis. (B) Accumulation of desmin in the myocytes of Lmna^–/– mice compared to control Lmna^+/– mice by immunohistochemistry of muscle sections. The percentage of cells with accumulation of desmin in the myocytes of Lmna^–/– mice fed rapamycin (n = 4) was significantly reduced compared to Lmna^–/– mice fed the control diet (n = 4). DAPI, 4′,6-diamidino-2-phenylindole. (C) Accumulation of desmin in the cardiomyocytes of Lmna^–/– mice compared to control Lmna^+/– mice by immunohistochemistry of heart sections. The percentage of cells with accumulation of desmin in the cardiomyocytes of Lmna^–/– mice fed rapamycin (n = 4) was not different from that in Lmna^–/– mice fed the control diet (n = 4). *P < 0.05.

Fig. 6

Autophagy markers are increased in heart and skeletal muscle tissue of Lmna^–/– mice. (A) LC3-I, LC3-II, Atg7, beclin 1, and phospho-ULK1 (P-ULK1) in heart. Western blot analysis of heart tissue lysates shows a significant increase in levels of LC3-I, LC3-II, Atg7, and beclin 1 protein in Lmna^–/– mice compared to Lmna^+/+ mice. Phospho-ULK1 levels are not significantly different. For LC3 blot: Lmna^+/+ mice (n = 7) (black bars); Lmna^–/– mice (n = 8) (white bars). For all other blots: Lmna^+/+ mice (n = 5) and Lmna^–/– mice (n = 4). (B) p62 and Lamp2a in heart. Western blot analysis of heart tissue shows a significant increase in p62 protein in Lmna^–/– mice (n = 6) compared to Lmna^+/+ mice (n = 7). Lamp2a protein levels are also significantly different in Lmna^–/– mice (n = 4) compared to Lmna^+/+ mice (n = 5). (C) LC3-I, LC3-II, Atg7, beclin 1, and phospho-ULK1 in muscle. Western blot analysis of muscle tissue lysates shows a significant increase in LC3-I, LC3-II, Atg7, and beclin 1 protein levels in Lmna^–/– mice compared to Lmna^+/+ mice. Phospho-ULK1 levels are also significantly different. For the LC3 blot: Lmna^+/+ mice (n = 7) (black bars); Lmna^–/– mice (n = 6) (white bars). For all other blots, Lmna^+/+ mice (n = 7) and Lmna^–/– mice (n = 6). (D) p62 and Lamp2a in muscle. Western blot analysis of muscle tissue shows a significant increase in Lamp2a protein levels in Lmna^–/– mice (n = 6) compared to Lmna^+/+ mice (n = 7) but not in p62 protein levels. *P < 0.05; **P < 0.01.

Fig. 7

Rapamycin enhances autophagy-mediated degradation in hearts from Lmna^–/– mice. (A) Effect of rapamycin on autophagy markers in heart. Western blot analysis of heart tissue shows a significant decrease in LC3-I (n = 6), p62 (n = 6), and beclin 1 (n = 6) protein levels [but no change in LC3-II (n = 6) protein levels] in Lmna^–/– mice treated with rapamycin compared to control Lmna^–/– mice (n = 5 to 6). Atg7, Lamp2a, and phospho-ULK1 are not significantly changed in Lmna^–/– mice treated with rapamycin (n = 6) compared to control-treated Lmna^–/– mice (n = 5). (B) Effect of rapamycin on autophagy markers in muscle. Western blot analysis of muscle tissue shows no significant change in LC3-I, LC3-II, Atg7, and phospho-ULK1 protein levels in Lmna^–/– mice (n = 6) treated with rapamycin compared to control-treated Lmna^–/– mice (n = 5). There was a significant increase in p62, beclin 1, and Lamp2a protein levels in Lmna^–/– mice treated with rapamycin (n = 6) compared to control Lmna^–/– mice (n = 5). *P < 0.05; **P < 0.01.