Role of Sirt2 in Stress-induced Muscle Atrophy

Abstract

Skeletal muscle is one of the essential organs in our body, responsible for various functions such as locomotion, transport, protein storage, and thermoregulation. Under certain pathological conditions, there is a reduction in muscle mass known as muscle atrophy. Skeletal muscle atrophy occurs when there is an upsurge in protein degradation and a reduction in protein anabolism. Aging-associated reduction of muscle weight is known as sarcopenia. Individuals suffering from sarcopenia have severe degeneration, particularly in fast twitching (type II) muscle fiber.

Glucocorticoid signaling is one of the significant components influencing type II muscle fiber atrophy. Many post-translational modifications are known to alter the function of the glucocorticoid receptor. To enhance our knowledge of Glucocorticoid receptor-mediated muscle degeneration, we explored the role of SIRT2, a class III histone deacetylase member, in glucocorticoid receptor-mediated skeletal muscle degeneration. Various organs express SIRT2, including muscles. Nevertheless, the importance of SIRT2 in skeletal muscle atrophy remains poorly understood.

PART I: Development and validation of a glucocorticoid-based model to study muscle atrophy in primary myotubes

Traditional models utilize cell lines to study skeletal muscle atrophy in vitro. However, experiments performed in vitro using cell lines suffer from poor reproducibility and are often inconsistent with the results observed in vivo. On the other hand, primary myotube culture is a superior model for studying the skeletal muscle phenotype in a cell-autonomous manner. We standardized the isolation of primary myotubes from neonatal murine pups. We also standardized and validated the treatment of dexamethasone (a glucocorticoid receptor agonist) to induce muscle atrophy in primary myotubes. The results show that dexamethasone treatment can induce muscle atrophy in primary culture. Further, we observe an increase in atrogenes expression and a reduction in myotube diameter. Overall our data show that dexamethasone treatment is a simple, reliable, and efficient means to induce muscle atrophy in vitro.

PART II: Understanding the role of SIRT2 in stress-induced muscle atrophy

In the current study, we have focused on the role of SIRT2 (majorly a cytoplasmic protein) in muscle under dexamethasone-induced stress conditions. We characterized dexamethasone-mediated muscle atrophy in primary myotubes, SIRT2-deficient mice, and muscle-specific SIRT2-transgenic mice. Dexamethasone treatment decreases SIRT2 levels in primary myotube cultures, as well as in the skeletal muscles of mice. SIRT2 depletion aggravates stress-induced myotube atrophy, and consistent with this, SIRT2 overexpression protects against stress-induced myotube atrophy in vitro. SIRT2-deficient mice are more susceptible to stress-induced muscle atrophy than the wild-type control.

Increased expression of SIRT2 in cardiac-specific SIRT2-transgenic mice protects them from developing cardiac hypertrophy. However, the role of SIRT2 in skeletal muscles is currently poorly understood. To address this problem, we developed a novel animal model, a muscle-specific SIRT2-transgenic mouse, in the current study. Our data suggest that these mice have increased levels of SIRT2, specifically in skeletal muscle, and do not have any visible structural or functional defects. Further, upon corticosteroid (dexamethasone) treatment, these mice displayed resistance against reduced muscle weight and increased performance compared to controls. Muscle-specific transgenic mice also showed reduced expression of muscle atrophy markers.

Our results indicate that SIRT2 depletion leads to a decrease in the inhibitory phosphorylation of glucocorticoid receptors. Consistent with this, muscle-specific SIRT2-transgenic mice display increased phosphorylation of glucocorticoid receptors. Mechanistically, SIRT2 directly interacts with and deacetylates glucocorticoid receptors reducing its DNA binding affinity.

Overall, we observed that the SIRT2 protein level reduces upon treatment with glucocorticoid signaling activator Dexamethasone. Similarly, SIRT2-deficient mice were more susceptible to dexamethasone-mediated muscle degeneration. In comparison, Upon SIRT2 overexpression in primary myotubes, we found that the muscle fibers are protected against dexamethasone-mediated muscle degeneration. Similarly, muscle-specific SIRT2-transgenic mice were protected against stress-mediated muscle atrophy. We also determined two novel acetylation sites around the DNA binding domain of the Glucocorticoid receptor, critical for its DNA binding capability. We suspect that SIRT2 regulates these acetylation sites. Collectively, these findings suggest that SIRT2 plays a protective role against stress-induced muscle atrophy, possibly via alteration of glucocorticoid signaling.