Understanding the Role of SIRT2 in Cardiac Hypertrophy, Cell Death and Glucose Homeostasis

Abstract

Over the last century the major causes of human deaths and ailments has shifted from acute infectious diseases to chronic aging related disorders like cancer and type-2 diabetes. The aging process is a universal property of most organisms, accompanied by a subtle, progressive, and often irreversible decline of physiological and reproductive functions resulting in an increased vulnerability to environmental challenge and a growing risk of disease and death. In molecular terms, it can be understood as a decline of the homeostatic mechanisms that ensure the function of cells, tissues, organs, and organ systems. However, caloric restriction (CR), i.e. reduced intake of food has been shown to ameliorate the effects of aging to some extent. One of the ways through CR slows down aging is by activating a group of enzymes known as Sirtuins, are a family of NAD+ dependant Class III histone deacetylases, which were first characterized in yeast. In S. cerevisiae, overexpression of Sir2, the founding member of the family, was shown to extend the replicative lifespan, while its depletion decreased lifespan. In mammals, there are seven orthologues of Sir2 (SIRT1-7) with a conserved catalytic domain. They differ in their enzymatic activity, subcellular localisation and therefore their binding partners and target molecules. SIRT1, 6 and 7 are localized in nucleus, SIRT3, 4 and 5 are mitochondrial sirtuins. SIRT2, which is mostly cytoplasmic but translocate to the nucleus during G2/M transition phase in cell cycle, has a NAD+-dependent deacetylase activity along with Mono-ADP-Ribosyl transferase activity. SIRT2 regulates microtubule dynamics of the cell by acetylation at K40 residue of tubulin. Although, generally deemed to be a tumour suppressor as it regulates cell cycle progression, it was observed to be upregulated in some cancers. One of the major targets of SIRT2 are FoxO family of transcription factors, which are involved in regulation of processes as varied as cell death, adipocyte differentiation and autophagy. By deacetylating them, SIRT2 can regulate their activity, stability and sub-cellular localization. However, the role of SIRT2 in cardiac diseases, oxidative stress response and regulation of insulin resistance is unknown. In this work, we studied the role of SIRT2 in cardiac hypertrophy, cell death and glucose homeostasis. PART 1: Role of SIRT2 in cardiac hypertrophy To find out if SIRT2 plays a role in cardiac hypertrophy, we treated mice with Isoproterenol, a well-accepted model of pathological hypertrophy. We found SIRT2 protein levels to be significantly downregulated in Isoproterenol-treated mice hearts as compared to the controls. In vitro experiments with primary cardiomyocytes yielded the same results. SIRT2 knockout mice spontaneously developed age dependent cardiac hypertrophy and fibrosis. We found that SIRT2 binds to and deacetylates the transcription factor NFATc2. NFATc2 activation is a well-known modulator of cardiac fetal-programme and cardiac hypertrophy. SIRT2 deficiency leads to hyperacetylation of NFATc2, while acetylation enhances nuclear localization of NFATc2. Further, we have demonstrated that GSK3 activity is markedly reduced in Sirt2 knockout mice, a hallmark of hypertrophic hearts. It has been shown that cardiac specific GSK3 overexpression ameliorates cardiac hypertrophy. We found GSK3 activity increased by SIRT2 mediated deacetylation of Lys246 and Lys183 of GSK3α and GSK3β respectively. Interestingly, reduced GSK activity in SIRT2-deficient mice was independent of inhibitory phosphorylation at Ser9. Moreover, GSK3 is required for the anti-hypertrophic function of SIRT2. PART 2: Role of SIRT2 in regulating oxidative stress-induced cell death Our body is continually exposed to variety of exogenous or endogenous stresses. Chronic stresses can disrupt nearly every system in our body and can eventually lead to serious life-threatening illnesses such as heart attacks, kidney disease and cancer. Sirtuins are known to regulate cell death. SIRT1 increases cell survival, by modulating activity of p53 and FOXO3 deacetylation mediated degradation. SIRT3 decreases cell death by improving mitochondrial function. However, role of SIRT2 in cell death is not well understood. In this study we found that SIRT2-depleted cells are resistant to oxidative stress and show enhanced survival. Similarly, SIRT2-KO mice showed enhanced resistance to acetaminophen-induced hepatocyte cell death. Mechanistically, acetylation of JNK at K153 by p300 acetyl transferase reduced JNK phosphorylation and activity, whereas deacetylation of JNK by SIRT2 promoted its phosphorylation as well its activity. Our molecular simulation and biological assays demonstrated that acetylation of JNK at K153 impairs ATP binding, hence reduces its activity. Our results indicate that deacetylation of JNK by SIRT2 promotes oxidative stress-induced cell death. PART 3: Understanding the role of SIRT2 in glucose homeostasis Glucose is one of the major sources of energy for human body, hence regulation of glucose is tightly regulated. Skeletal muscles play a key role in glucose homeostasis. Skeletal muscles comprise around 40-50% of the total body mass in humans and around 80% of glucose in the body is utilized by skeletal muscles. Impaired glucose uptake by muscle cells leads to insulin resistance. SIRT2 improves glucose uptake in hepatocytes through deacetylation of glucokinase regulatory protein (GKRP) or SIRT2 mediated phosphoenolpyruvate carboxy kinase (PEPCK) deacetylation, thus modulating gluconeogenesis in liver. However, role of SIRT2 in glucose homeostasis in muscle tissue is not well studied. Upon induction of high fat diet (HFD) mediated insulin resistance, SIRT2 levels were increased in skeletal muscle of mice. We found SIRT2-KO mice to be highly resistant to HFD induced insulin resistance as revealed by markedly higher glucose clearance rate. Mechanistically, SIRT2 mediated deacetylation of IRS1 was found to promote the inhibitory phosphorylation of IRS1 at Ser307 through activate JNK, which is associated with impaired insulin signalling. We found that SIRT2 overexpression reduced the insulin-stimulated membrane localization of GLUT4 transporter. Our results further suggest that inhibition of SIRT2 reverses the palmitate-induced reduction in cellular glucose uptake and promotes glucose uptake in insulin resistant myotubes and therefore ameliorates the effects of insulin resistance. In the present work, we have elucidated the role of SIRT2 cardiac myocytes, hepatocytes and skeletal myotubes. We believe that modulation of SIRT2 activity could be a potential therapeutic strategy to treat diseases related to aging