Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress

Abstract

Chronic hypoxic stress induces epigenetic modifications mainly DNA methylation in cardiac fibroblasts, inactivating tumor suppressor genes (RASSF1A) and activating kinases (ERK1/2) leading to fibroblast proliferation and cardiac fibrosis. The Ras/ERK signaling pathway is an intracellular signal transduction critically involved in fibroblast proliferation. RASSF1A functions through its effect on downstream ERK1/2. The antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), decreases oxidative stress from chronic hypoxia, but its effects on these epigenetic changes have not been fully explored. To test our hypothesis, we used an in-vitro model: wild-type C57B6 male mice (WT) and transgenic males with an extra copy of human hEC-SOD (TG). The studied animals were housed in hypoxia (10% O2) for 21 days. The right ventricular tissue was studied for cardiac fibrosis markers using RT-PCR and Western blot analyses. Primary C57BL6 mouse cardiac fibroblast tissue culture was used to study the in-vitro model, the downstream effects of RASSF-1 expression and methylation, and its relation to ERK1/2. Our findings showed a significant increase in cardiac fibrosis markers: Collagen 1, alpha smooth muscle actin (ASMA), and SNAIL, in the WT hypoxic animals as compared to the TG hypoxic group (p < 0.05). The expression of DNA methylation enzymes (DNMT 1&3b) was significantly increased in the WT hypoxic mice as compared to the hypoxic TG mice (p < 0.001). RASSF1A expression was significantly lower and ERK1/2 was significantly higher in hypoxia WT compared to the hypoxic TG group (p < 0.05). Use of SiRNA to block RASSF1A gene expression in murine cardiac fibroblast tissue culture led to increased fibroblast proliferation (p < 0.05). Methylation of the RASSF1A promoter region was significantly reduced in the TG hypoxic group compared to the WT hypoxic group (0.59 vs. 0.75, respectively). Based on our findings, we can speculate that EC-SOD significantly attenuates RASSF1A gene methylation and can alleviate cardiac fibrosis induced by hypoxia.

The Applications and Mechanisms of Superoxide Dismutase in Medicine, Food, and Cosmetics

“…On the one hand, high expression of EC-SOD inhibited the increase in methylase activity and downregulated the expression of recombinant DNA methyltransferase 1(DNMT1) and DNMT-3b, thereby inhibiting the increase in HIF1α and biological oxidants. Another benefit of antioxidants was the chelation of hydrogen peroxide, which significantly reduced the activation of ERK1/2, both of which could counteract the oxidative stress associated with the pathogenesis of these cardiovascular diseases [ 62 ]. In addition, SOD3-deficient rats developed severe renal failure, arterial hypertension, and left ventricular hypertrophy at 21 weeks of age [ 62 ].…”

Spatiotemporal AMPKα2 deletion in mice induces cardiac dysfunction, fibrosis and cardiolipin remodeling associated with mitochondrial dysfunction in males only

“…Even though the link between AMPK and cardiac fibrosis has already been suggested since the activation of AMPK can suppress fibrosis induced by various stresses (for review see [16]), regulation of the fibrotic response by AMPKα2 is still poorly understood, on the contrary to the role of AMPKα1, the major AMPK isoform in cardiac fibroblasts, which has clearly been demonstrated to control the proliferation of cardiac fibroblasts and the development of fibrosis especially through the TGF-β1/p38 axis [15]. From another side, it cannot be excluded that fibrosis observed in males was linked to mitochondrial dysfunction since it has recently been shown that oxidative stress plays a key role in myocardial fibrosis development [46,47] and excessive ROS production is a well-known feature of altered mitochondria. Nevertheless, cardiac fibrosis in OVX group was not associated with clear alteration of electron transfer chain, rather suggesting that cardiac fibrosis was an earlier event than important mitochondrial perturbations in these mice and giving weight to the hypothesis of a link between AMPK and fibrosis.…”

Epigenetics Approaches toward Precision Medicine for Idiopathic Pulmonary Fibrosis: Focus on DNA Methylation

“…Chronic hypoxia induces oxidative stress. Recently, the administration of the antioxidant enzyme extracellular superoxide dismutase (EC-SOD) has been shown to alleviate the hypoxia-induced DNA methylation of the Ras association domain family 1 isoform A (RASSF1A) through the Ras/ERK pathway [ 116 ].…”