Role of Sirtuin 2 in the development of tissue fibrosis

Abstract

Wound healing is a crucial physiological process required for the healthy lifespan of an organism. Dysregulation in the process of wound healing leads to the production of excess extracellular matrix leading to fibrosis. Ageing is associated with increased cell death and decreased regeneration in various organs. This results in fibrosis in multiple organs in aging organisms, accompanied by changes in organ structure and loss in organ function. Aging is a universal process and multiorgan fibrosis is a common factor in organ failure due to age, ultimately leading to mortality. Sirtuins are a family of deacetylases that have been implicated in the regulation of aging in various organisms. Although, role of some of the sirtuins have been studied in fibrosis in an organ dependent manner, not much is understood about the specific role of SIRT2 in multi-organ fibrosis. The present work is focused on developing a simple and efficient in vitro model system to study fibrosis and using this model system along with in vivo animal models to study the role of SIRT2 in multi-organ fibrosis. Development of an in vitro model system to study fibrosis One of the key components of a healthy heart is cardiac fibroblasts. In vitro studies on cardiac fibrosis require access to cultured cardiac fibroblasts. The procedures used currently for cultivating cardiac fibroblasts are laborious and call for specialized chemicals and equipment. The isolation of primary cardiac fibroblasts is generally a byproduct of culturing primary cardiomyocytes, as is evident by analyzing a few of the recently published and widely cited papers. Very few protocols have been specially designed for the culture of primary cardiac fibroblasts. The protocol described in the study helps in easily and effectively isolating enriched cardiac fibroblasts with good viability of the cells. The yield and purity of the cultured cardiac fibroblasts are influenced by several factors, including the quality of the reagents used for the culture, the makeup of the digestion mixture employed, the conditions maintained throughout the digestion of the heart tissue, and the age of the pups utilized for culture. The current study outlines a comprehensive and streamlined procedure for isolating and cultivating primary cardiac fibroblasts from neonatal murine pups. Using treatment with transforming growth factor (TGF)β–1, we show that the cultured fibroblasts can be transdifferentiated into myofibroblasts, simulating the alterations that occur in fibroblasts during cardiac fibrosis in vivo. Studies on the numerous facets of cardiac fibrosis, inflammation, fibroblast proliferation, and growth can be done using these cultured primary cardiac fibroblast cells. Investigating the role of SIRT2 in the development of tissue fibrosis Fibrosis is an aging-associated disorder and fibrotic diseases are a major cause of multi-organ failure with age. Multiple studies indicate that the TGF-β/SMAD signaling pathway is the major regulator of fibrosis. However, endogenous regulators of the TGF-β/SMAD signaling are not well understood. In our study, we identify SIRT2 as a critical regulator of TGF- β/SMAD signaling, the transdifferentiation of fibroblasts to myofibroblasts, and resulting fibrosis. Using in vivo and in vitro model systems, we demonstrate that SIRT2 deficiency spontaneously transforms fibroblasts to myofibroblasts accompanied by increased expression of α-SMA, FN1, and Col3a1 in multiple organs including heart, liver, kidney, and muscles. On the other hand, overexpression of SIRT2 attenuates the induction of fibrosis by preventing fibroblast transdifferentiation. Through RNA-seq analysis, we identify TGF-β/SMAD signaling as a downstream target of SIRT2. We observe an increase in the activation of the TGF-β/SMAD pathway in vivo, in the organs of SIRT2-/- mice including heart and liver, and in vitro in SIRT2 deficient primary fibroblasts. Mechanistically, we show that SIRT2 binds to and deacetylates SMAD3 to regulate its transcriptional activity. Using mass-spectrometric analysis, we identify the residues Lys29 and Lys44 in the DNA binding domain of SMAD3 as the targets of SIRT2 for deacetylation. Interestingly, inhibition of TGF-β/SMAD signaling rescues fibrosis in SIRT2-depleted fibroblasts. Remarkably, we observe that the levels and activity of SIRT2 are downregulated in aged mice, accompanied by increased fibrosis and hyperactive TGF-β/SMAD signaling. Similarly, SIRT2 levels and activity are downregulated in failing human heart samples along with increased fibrosis. Overall, our study indicates that SIRT2 plays a protective role against multi-organ fibrosis, and therefore may be a potential therapeutic target for the treatment of fibrosis-related disorders