Elevated MTORC1 signaling and impaired autophagy

Abstract

A-type lamins, generated from the LMNA gene by differential splicing, are type V intermediate filament proteins that polymerize to form part of the nuclear lamina, and are of considerable medical interest because missense mutations in LMNA give rise to a wide range of dystrophic and progeroid syndromes. Among these are dilated cardiomyopathy and two forms of muscular dystrophy (limb-girdle and Emery-Dreifuss), which are modeled in lmna^−/− mice and mice engineered to express human disease mutations. Our recent study demonstrates that cardiac and skeletal muscle pathology in lmna^−/− mice can be attributed to elevated MTORC1 signaling leading to impairment of autophagic flux. An accompanying paper from another laboratory shows similar impairments in mice engineered to express the LMNA H222P associated with dilated cardiomyopathy in humans and also in left ventricular tissue from human subjects. MTORC1 inhibition with rapalogs restores autophagic flux and improves cardiac function in both mouse models, and extends survival in the lmna^−/− mice. These findings elaborate a potential treatment option for dilated cardiomyopathy and muscular dystrophy associated with LMNA mutation and supplement growing evidence linking impaired autophagy to human disease.

Keywords: :

• dilated cardiomyopathy
• MTORC1 signaling
• autophagy
• nuclear lamina
• laminopathies
• muscular dystrophy
• aging

This article refers to:

While autophagy is clearly important for all cells, it is particularly essential for the maintenance of protein homeostasis in heart and skeletal muscle tissue, which require protein turnover to maintain their adaptability. For instance, mouse studies show that inhibition of autophagy results in cardiac dysfunction and the inability to successfully adapt to hemodynamic overload, while blockage of autophagy in skeletal muscle results in atrophy. In our study of mice lacking A-type lamins, which serve as a model for dilated cardiomyopathy (DCM) and skeletal muscle dystrophy, autophagy was found to be impaired as a result of aberrant MTORC1 pathway activity, and MTORC1 inhibition by rapamycin restores autophagy and improves cardiac function. An accompanying study led to similar findings in lmna^H222P/H222P mice, which express a human DCM disease variant. Here we compare findings from these studies and discuss the research questions and therapeutic possibilities that they portend.

Signaling through the MTORC1 Pathway is Elevated in lmna^−/− Mice

Compensatory growth is one mechanism used by heart and skeletal muscle tissue to offset inadequate function. MTORC1 pathway activation is important for this compensation, yet its hyperactivation is associated with cardiac hypertrophy in a variety of disease models. lmna^−/− and lmna^H222P/H222P mice develop DCM and serve as models for DCM with conduction system disease (CDM1A) associated with LMNA mutation. MTORC1 activity is inordinately high in both models, as determined by enhanced phosphorylation of MTOR, as well as downstream components of the pathway including RPS6KB/S6 kinase, RPS6 and/or EIF4EBP1 in heart tissue. Enhanced phosphorylation of RPS6 and EIF4EBP1 is also observed in skeletal muscle from lmna^−/− mice. Despite having increased MTORC1 signaling, lmna^−/− mice do not have enhanced protein translation and fail to exhibit compensatory hypertrophy in either heart or skeletal muscle. Instead, they develop DCM with ventricular wall thinning and muscular dystrophy with decreased myofiber cross-sectional area. This may be due in part to the mislocalization and aggregation of DES/desmin in the heart and skeletal muscle of lmna^−/− mice. DES, a cytoplasmic intermediate filament, forms a scaffold responsible for correctly positioning the contractile apparatus as well as organelles such as the mitochondria and nucleus within cardiac and skeletal muscle cells. Mutations in the Des gene result in DES aggregation, as well as cardiac and skeletal muscle dysfunction.

Autophagy is Upregulated in lmna^−/− Mice

MTORC1 not only regulates protein synthesis but also turnover of dysfunctional organelles and misfolded/aggregated proteins through inhibition of autophagy. In both lmna^−/− and lmna^H222P/H222P mice, molecular markers of autophagy are altered in a manner constant with reduced autophagic flux. We found that protein levels of LC3-I and -II, BECN1 and ATG7 are increased in heart and skeletal muscle of lmna^−/− mice. However, there were also indications that autophagic flux might be impaired. Despite apparent activation of autophagy, levels of SQSTM1/p62 are increased in the heart, suggesting a decrease in flux since SQSTM1 binds to proteins targeted for degradation and is itself degraded during autophagy. In addition, LAMP2A, a protein involved in chaperone-mediated autophagy, which has been shown to be activated when macroautophagy is blocked, is increased in heart and muscle tissue of lmna^−/− mice. In cardiac tissue of lmna^H222P/H222P mice, similar changes in levels of autophagy factors were found, including an increase in SQSTM1 levels consistent with impaired flux.

Rapamycin Reduces MTORC1-Mediated Inhibition of Autophagy in lmna^−/− Mice

Rapalogs, rapamycin and derivatives are specific and noncompetitive inhibitors of MTORC1 and are clinically approved in a range of disease indications. In both lmna^−/− and lmna^H222P/H222P mice, rapalog treatment reduces MTORC1 signaling, improves productive autophagy, restores cardiac function and, in the lmna^−/− case, extends survival. Specifically in heart tissue from rapamycin-treated lmna^−/− mice, LC3-I levels decrease, while LC3-II levels remain unchanged, indicating a shift to the formation of the lipidated form found in autophagosomes. In addition, there is a further increase in BECN1 and LAMP2A, while SQSTM1 levels decrease. The effects in skeletal muscle were less clear with regard to whether autophagy was enhanced. These findings suggest (1) that rapalogs may be effective agents in DCM associated with LMNA mutation and (2) that a full-scale assessment of autophagy in cardiac and skeletal muscle is warranted.

Conclusions

Elevated MTORC1 signaling is increasingly associated with pathology especially in, but not limited to, cardiac diseases. Perhaps most interestingly, elevated MTORC1 signaling is also associated with normal aging tissues in mice, including liver, hematopoietic stem cells and likely other tissues. Accordingly, rapamycin administered late in life slows aging and extends life span in mice. Impaired autophagy is also associated with aging in a range of model organisms. Together, these findings point to the importance of understanding links between MTORC1 signaling and autophagy in a range of diseases as well as in normal aging. With further study, we may find that interventions in the MTORC1 pathway can have an even broader range of therapeutic uses than are already in place.