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. 2017 Apr 11:7:46307.
doi: 10.1038/srep46307.

Upregulation of sestrins protect atriums against oxidative damage and fibrosis in human and experimental atrial fibrillation

Zengxiang Dong  1 , Chaolan Lin  1 , Yujiao Liu  2 , Hongbo Jin  3 , Hong Wu  1 , Zhenjun Li  1 , Liping Sun  4 , Lu Zhang  4 , Xi Hu  1 , Yingying Wei  1 , Chengcheng Wang  1 , Wei Han  1
Affiliations

Upregulation of sestrins protect atriums against oxidative damage and fibrosis in human and experimental atrial fibrillation

Zengxiang Dong et al. Sci Rep. .

Abstract

Atrial Fibrillation (AF) is common in the elderly and Sestrins (Sesns) have been suggested to prevent age-related pathologies. The aim of this study was to investigate the effects of Sesns in AF. Clinical data were collected and a small sample of atrial appendage and atrium was obtained from patients undergoing valve repairment. The expression of Sesn1, Sesn2, and Sesn3 was significantly higher in patients with permanent atrial fibrillation (PmAF) than that in sinus rhythm (SR), and further greater in the left atrium than the right in PmAF patients. Superoxide anion and malondialdehyde were enhanced and positively correlated to the protein expression of Sesn1/2/3. Reactive oxygen species (ROS) production and Ca2+ overload were significantly decreased and cell survival was enhanced by overexpression of Sesns 1/2/3 in cultured HL-1 cells. Conversely, knockdown of Sesn1/2/3 resulted in significantly increased ROS and Ca2+ overload. In addition, the overexpression of Sesn1/2 significantly reduced the proliferation of fibroblasts, as well as decreased the protein expression of collagen and fibronectin1 in angiotensin II-stimulated cardiac fibroblasts. Our study demonstrated for the first time that Sesns expression is significantly up-regulated in AF, which therefore may protect hearts against oxidative damage and atrial fibrosis.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. The expression of Sesns in atrium of patients and cells.
(A) The location of Sesns proteins expressed in atria detected by immunofluorescence. Sesns proteins stained with red fluorescence. Nuclei stained with blue fluorescence. Magnification: ×100. The location of Sesns proteins expressed in HL-1 cells and cardiac fibroblasts detected by immunofluorescence. Sesns proteins stained with green fluorescence. Nuclei stained with blue fluorescence. Magnification: ×400. (B) The expression of Sesns protein detected with western blot. Lane1: Molecular weight standards; Lane 2: Left atrium in SR group; Lane 3: Right atrium in SR group; Lane 4: Left atrium in PmAF group; Lane 5: Right atrium in PmAF group. Quantification of relative immunoreactivity for bands, SR group n = 23, PmAF group n = 19 *P < 0.05, **P < 0.01 compared with the SR group; #P < 0.05, ##P < 0.01 compared with the LA in the PmAF group. SR = Sinus rhythm; PmAF = Permanent AF; LA = left atrium, RA = right atrium.
Figure 2
Figure 2. Histopathological changes in atrium of patients.
Atrial collagen determined by Masson staining. Atrial collagen in the right and left atrium of patients with PmAF were significantly higher than in patients with SR. Quantification of atrial collagen is shown. SR group n = 23, PmAF group n = 19, **P < 0.01 compared with the SR group. SR = Sinus rhythm; PmAF = Permanent AF; LA = left atrium, RA = right atrium. Magnification: ×100.
Figure 3
Figure 3. Relationship between oxidative stress and Sesns expression in patients.
(A) The amount of O2·− in SR and PmAF. (B) The amount of malondialdehyde in SR and PmAF. The production of O2·−and MDA from LA and RA increased significantly in PmAF group compared with that in SR group. (C) The amount of O2·− positively correlated with the protein expression of Sesn1, Sesn2, and Sesn3. (D) A similar relationship was observed between MDA and Sesn1, Sesn2, and Sesn3. SR group n = 23, PmAF group n = 19, **P < 0.01 compared with the SR group. SR = Sinus rhythm; PmAF = Permanent AF; LA = left atrium, RA = right atrium.
Figure 4
Figure 4. Field pacing and overexpression and knockdown of Sesns in HL-1 cells.
(A) Left panel: YC-2-S stimulator and six-well plates equipped with electrode slices; Right panel: Low panel: Two paralleled platinum electrodes; Cells were stimulated in 600 times per minute. (B) The expression of Sesns protein detected with western blot. Representative lanes of immunoblotting. Lane1: Control; Lane 2: Pacing group; Lane 3: Overexpression of Sesns group; Lane 4: Knockdown of Sesns group. Quantification of relative immunoreactivity for bands, n = 6, **P < 0.01 compared with the control group.
Figure 5
Figure 5. Effects of Sesns on ROS generation in paced HL-1 cells.
(A) Flow cytometry was used to analyze the amount of ROS in paced HL-1 cells by transient transfection of Sesns siRNA and Sesns cDNA for 24 h. The transiently expressed Sesn1/2/3 siRNA increased the amount of ROS. The transiently expressed Sesn1/2/3 cDNA decreased the amount of ROS. (B) Quantification of relative ROS. n = 6, **P < 0.01 compared with the control group; ΔΔP < 0.01 compared with pacing group; ##P < 0.01 compared with the pacing + siRNA C group; &&P < 0.01 compared with the pacing + pcDNA3.1 group.
Figure 6
Figure 6. Effects of Sesns on Ca2+ in paced HL-1 cells.
(A) Flow cytometry was used to analyze the amount of Ca2+ in paced HL-1 cells by transient transfection of Sesns siRNA and Sesns cDNA for 24 h. The transiently expressed Sesn1/2/3 siRNA increased the amount of Ca2+. The transiently expressed Sesn1/2/3 cDNA decreased the amount of Ca2+. (B) Quantification of relative Ca2+. n = 6, **P < 0.01 compared with the control group; ##P < 0.01 compared with the pacing + siRNA C group; &P < 0.05, &&P < 0.01 compared with the pacing + pcDNA3.1 group.
Figure 7
Figure 7. Effects of Sesns on survival rate of paced HL-1 cells.
(A) The survival of HL-1 cells after transient transfection of Sesns siRNA and Sesns cDNA was monitored by flow cytometry. (B) Quantification of survival rate. Under control conditions, very small fluorescent variations were present in HL-1 cells. Pacing did not significantly change the survival rate of HL-1 cells. The silencing of Sesns by transient transfection of Sesn1/2/3 siRNA did not decrease the survival rate of paced HL-1 cells. The transiently expressed Sesn1/2/3 cDNA significantly enhanced the survival of paced HL-1 cells. n = 6, ΔΔP < 0.01 compared with pacing group; &&P < 0.01 compared with the pacing + pcDNA3.1 group.
Figure 8
Figure 8. Sesns prevent AngII-induced proliferation of cardiac fibroblasts and collagen synthesis.
(A) CCK8 method was used to analyze the proliferation of cardiac fibroblasts induced by AngII or/and transient transfection of Sesn1/2 cDNA. (B–D) mRNA levels of COLI, COLIII and FN1 in cardiac fibroblasts induced by AngII or/and transient transfection of Sesn1/2 cDNA were measured by RT-PCR. (E–H) Protein levels of COLI, COLIII and FN1 in cardiac fibroblasts induced by AngII or/and transient transfection of Sesn1/2 cDNA were measured by western blot. Data are expressed as mean ± SEM (n = 6). **P < 0.01 compared with control group; #P < 0.05 compared with Ang II group.
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