SIRT1 protects the heart from ER stress-induced cell death through eIF2α deacetylation

Abstract

Over the past decade, endoplasmic reticulum (ER) stress has emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases including heart failure. Cardiac therapy based on ER stress modulation is viewed as a promising avenue toward effective therapies for the diseased heart. Here, we tested whether sirtuin-1 (SIRT1), a NAD⁺-dependent deacetylase, participates in modulating ER stress response in the heart. Using cardiomyocytes and adult-inducible SIRT1 knockout mice, we demonstrate that SIRT1 inhibition or deficiency increases ER stress-induced cardiac injury, whereas activation of SIRT1 by the SIRT1-activating compound STAC-3 is protective. Analysis of the expression of markers of the three main branches of the unfolded protein response (i.e., PERK/eIF2α, ATF6 and IRE1) showed that SIRT1 protects cardiomyocytes from ER stress-induced apoptosis by attenuating PERK/eIF2α pathway activation. We also present evidence that SIRT1 physically interacts with and deacetylates eIF2α. Mass spectrometry analysis identified lysines K141 and K143 as the acetylation sites on eIF2α targeted by SIRT1. Furthermore, mutation of K143 to arginine to mimic eIF2α deacetylation confers protection against ER stress-induced apoptosis. Collectively, our findings indicate that eIF2α deacetylation on lysine K143 by SIRT1 is a novel regulatory mechanism for protecting cardiac cells from ER stress and suggest that activation of SIRT1 has potential as a therapeutic approach to protect the heart against ER stress-induced injury.

Figure 1

Knockout of SIRT1 sensitizes mice to ER stress-induced cardiac injury. (a) WT and SIRT1 iKO mice were injected i.p. with TN (2 mg/kg) or vehicle (150 mM dextrose) for 16 h and the levels of SIRT1 and ER stress markers GRP94 and GRP78 were analyzed by western blot in heart tissue. β-Actin was used as loading control. Relative expression of proteins (normalized to WT CTL) is presented in bar graphs. Error bars, S.E.M. (n=3). (b-g) Echocardiographic data were recorded 72 h after vehicle or TN treatment. (b) Representative images obtained by transthoracic echocardiography. (c) EF of WT and SIRT1 iKO mice 72 h after vehicle or TN treatment. (n=9). (d) Left ventricular internal dimension (diastole), LVIDd, in vehicle or TN-treated mice (n=9). (e) Left ventricular internal dimension (systole), LVIDs, in vehicle or TN-treated mice (n=9). (f) Total wall thickness (diastole), TWTd, in vehicle or TN-treated mice (n=9). (g) Total wall thickness (systole), TWTs, in vehicle or TN-treated mice (n=9). Results are presented as mean±S.E.M. *P<0.05, **P<0.01, ***P<0.005 versus respective control. ^$P<0.05, ^$$P<0.01, ^$$$P<0.005 versus WT TN. (h) WT and SIRT1 iKO mice were injected i.p. with TN (2 mg/kg) or with vehicle (150 mM dextrose) for 72 h and subjected to TUNEL assay. Representative micrographs showing apoptotic cardiomyocyte nuclei (brown) are presented (scale bar=40 μm). Quantification of apoptotic nuclei is showed in bar graph. Error bars, S.E.M. (n=6). (i) Freshly isolated ARVMs were left untreated or treated with 10 μg/ml TN for 24 h±10 μM EX527 or 1 μM STAC-3 pretreatment and cell viability was determined. Results presented in bar graph are expressed as mean±S.E.M. of percentages of dead cells (FDA-negative cells, n=6). **P<0.01, ***P<0.005 versus control. ^$P<0.05, ^$$P<0.01 versus TN alone

Figure 2

SIRT1 protects cardiac cells from ER stress-induced apoptosis. (a) The percentage of dead cells (FDA-negative cells) was assessed by flow cytometry after treatment of H9c2 cells for 48 h with TG (5 μM) or TN (10 μg/ml) in the absence or presence of SIRT1 inhibitor EX527 (10 μM). (b-d) Same as (a) except that cells were stained with (b) PI to determine the percentage of necrosis/late apoptosis (PI high cells), (c) DiOC6(3) to estimate ΔΨm loss (DiOC6(3) low cells), (d) DEVD-NucView™ 488 to measure active caspase-3 (NucView 488-positive cells), (e) DAPI to analyze apoptotic nuclei. (f) H9c2 cells were transfected with SIRT1 or control siRNA and SIRT1 expression was assessed after 24 h by western blot. (g) H9c2 cells were transfected with SIRT1 or control siRNA for 24 h then treated for 48 h with TG or TN and cell death (FDA-negative cells) was measured by flow cytometry. Data in the bar graphs represent mean±S.E.M. (n=4). *P<0.05, **P<0.01, ***P<0.005 versus control. ^$P<0.05, ^$$$P<0.005 versus TG or TN alone

Figure 3

SIRT1 regulates UPR by modulating PERK/eIF2α pathway activation. (a–c) H9c2 cells were left untreated or treated with TG (5 μM) or TN (10 μg/ml) ±EX527 (10 μM) and the relative mRNA levels of UPR target genes were quantified by qPCR and expressed as fold change over untreated controls. Values represent mean±S.E.M. (n=6). **P<0.01, ***P<0.005 versus control. ^$P<0.05, ^$$P<0.01, ^$$$P<0.05 versus TG or TN alone. Members of the (a) ATF6 pathway, (b) IRE1 pathway and (c) PERK/eIF2α pathway. (d) Cells were left untreated or treated with TG (5 μM) or TN (10 μg/ml) for 15 min (p-PERK and PERK), 90 min (p-eIF2α and eIF2α) or 24 h (GADD34, ATF4 and CHOP) ±EX527 (10 μM). Representative western blots of the level of members of the PERK/eIF2α axis are presented. β-Actin was used as loading control. (e) WT and SIRT1 iKO mice were injected i.p. with TN (2 mg/kg) or with vehicle (150 mM dextrose) for 72 h, proteins from the left ventricle of the heart were analyzed. Representative western blot of the level of members of the PERK/eIF2α axis are presented. Bar graphs summarize changes in protein expression from three independent measurements. Data are represented as fold change over untreated WT mice. **P<0.01, ***P<0.005 versus WT CTL, ^$$$P<0.05 versus WT TN. Error bars, S.E.M.

Figure 4

Knockout of SIRT1 exacerbates PERK/eIF2α pathway activation and cardiac injury induced by ISO. (a) WT and SIRT1 iKO mice were injected subcutaneously with ISO (150 mg/kg) or vehicle (NaCl 0,9%) for 6 h and the levels of ER stress markers GRP94, GRP78, p-eIF2α, eIF2α and CHOP were analyzed by western blot in heart tissue. β-Actin was used as loading control. Relative expression of proteins (normalized to WT CTL) is presented in bar graphs. Error bars, S.E.M. (n=3). (b-g) Echocardiographic data were recorded 48 h after vehicle (NaCl 0,9%) or ISO treatment. (b) Representative images obtained by transthoracic echocardiography. (c) EF of WT and SIRT1 iKO mice after vehicle or ISO treatment. (n=5). (d) Left ventricular internal dimension (diastole), LVIDd, in vehicle or ISO-treated mice (n=5). (e) Left ventricular internal dimension (systole), LVIDs, in vehicle or ISO-treated mice (n= 5). (f) Total wall thickness (diastole), TWTd, in vehicle or ISO-treated mice (n=5). (g) Total wall thickness (systole), TWTs, in vehicle or ISO-treated mice (n=5). Results are presented as mean±S.E.M. **P<0.01, ***P<0.005 versus respective control. ^$P<0.05, ^$$P<0.01 versus WT ISO

Figure 5

SIRT1 physically interacts with and deacetylates eIF2α. (a) Immunoprecipitation of acetylated proteins from H9c2 lysate followed by immunoblotting with the indicated antibodies. Input, input fraction; Output, supernatant after immunoprecipitation; IP, immunoprecipitate; IgG: negative control. (b) eIF2α was immunoprecipitated from H9c2 lysate and its level of acetylation was analyzed by immunoblotting with anti-acetyl-lysine antibody. (c) eIF2α was immunoprecipitated from control or H9c2 cells treated with EX527 (10 μM, 4 h) and its level of acetylation was determined by immunoblotting with anti-acetyl-lysine antibody. The ratio of acetylated versus total eIF2α is presented relative to control. (d) The level of acetylation of eIF2α was determined from immunoprecipitates of left ventricle extracts of WT or SIRT1 iKO mice. The ratio of acetylated versus total eIF2α is presented relative to WT mice. (e) The physical interaction between endogenous SIRT1 and eIF2α was demonstrated by co-immunoprecipitation. SIRT1 was precipitated from H9c2 lysate with anti-SIRT1 antibody and blotted with anti-eIF2α antibody, and vice versa. Negative controls: IgGr, rabbit IgG, IgGm, mouse IgG. (f) eIF2α was immunoprecipitated from control or H9c2 cells treated with TN (10 μg/ml, 4 h) and its level of acetylation was determined by immunoblotting with anti-acetyl-lysine antibody. Ratios of acetylated versus total eIF2α are presented relative to control. (g) H9c2 cells were treated with TN (10 μg/ml, 4 h) ±EX527 (10 μM), eIF2α was immunoprecipitated and its levels of acetylation and phosphorylation were analyzed. Ratios of acetylated and phosphorylated versus total eIF2α are presented relative to TN alone. (h) WT and SIRT1 iKO mice were injected with TN (2 mg/kg), eIF2α was immunoprecipitated from left ventricle and its levels of acetylation and phosphorylation were analyzed. Ratios of acetylated or phosphorylated versus total eIF2α are presented relative to WT mice

Figure 6

Lysines K141 and K143 are the acetylation sites on eIF2α targeted by SIRT1. (a) NanoLC-MS/MS spectrum of acetylated eIF2α peptides containing lysine K141. (b) NanoLC-MS/MS spectrum of acetylated eIF2α peptides containing lysine K143. (c) Relative acetylated K141 or K143 peptide count in control or EX527 (10 μM) treated cells. (d) Sequence alignment of the region surrounding K141 and K143 residues of eIF2α

Figure 7

Acetylation of eIF2α on lysine K143 regulates ER stress-induced cell death. (a–c) Wild-type and mutant eIF2α expression plasmids were transfected into H9c2 cells for 24 h, ER stress was induced or not by TG (5 μM) or TN (10 μg/ml) and cell death was analyzed in transfected cells by flow cytometry after 48 h by FDA assay. For acetylation-defective mutants, either lysine 141 (K141R) or 143 (K143R) or both residues (K141R/K143R) were replaced with arginine. For eIF2α phosphorylation-defective mutant, serine 52 (S52A) was replace by alanine. (a) Expression levels of the Flag-tagged eIF2α mutants. β-Actin was used as loading control. (b) Effect of wild-type and mutant eIF2α expression on cell death. (c and d) Effect of wild-type and mutant eIF2α on (c) TG-induced cell death (FDA-negative cells), (d) TN-induced cell death (FDA-negative cells). Results are expressed as mean±S.E.M. (n=6). *P<0.05, versus WT eIF2α. (e) Model of SIRT1 protection against ER stress-induced injury in cardiac cells. Upon ER stress, eIF2α is acetylated on K141/K143 residues leading to induction of cardiac injury through cardiomyocyte apoptosis. The deacetylation of eIF2α by SIRT1 protects cardiomyocytes from ER stress-induced apoptosis and heart dysfunction