Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes

Abstract

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes.

Keywords: C/EBP-homologous protein (CHOP); Cardiomyocytes; Endoplasmic reticulum-Stress; Inositol requiring kinase 1a (IRE1a); Sigma 1 receptor (Sigmar1); spliced-X-box binding protein 1 (XBP1s).

Figure 1. Sigmar1 knockdown increases ER-stress induced CHOP expression in cardiomyocytes

(A) Western blot showing time- and dose-dependent changes in tunicamycin-induced CHOP expression in cardiomyocytes. (B) Western blot showing siRNA knockdown of Sigmar1 in cardiomyocytes. (C,D) Western blot showing siRNA knockdown of Sigmar1 in cardiomyocytes increased ER-stress induced CHOP expression in NRCs. Bars represent mean ± S.E.M.; n=3 experiments.

Figure 2. Sigmar1 knockdown increases ER-stress-induced nuclear accumulation of CHOP in cardiomyocytes

Confocal microscopic observation of ER-stress-induced CHOP (green) nuclear localization following Sigmar1 knockdown in cardiomyocytes. Cardiomyocytes are counterstained with α-cardiac actin antibody (red) and nuclei with DAPI (blue). n=3 experiments.

Figure 3. Effect of Sigmar1 knockdown on ER-stress induced cellular damage in cardiomyocytes

Tunicamycin-induced LDH release in NRCs. Bars represent mean ± S.E.M.; ***, P<0.001 compared with control group by Tukey’s post hoc test; n=3 experiments.

Figure 4. Sigmar1 overexpression decreases ER-stress-induced CHOP expression in cardiomyocytes

(A) Western blot showing adenovirus-mediated overexpression of Sigmar1 in cardiomyocytes. Representative Western blot (B) and densitometric quantitation (C) showing Sigmar1 overexpression decreased ER-stress-induced CHOP expression in NRCs. Bars represent mean ± S.E.M.; n=3 experiments.

Figure 5. Sigmar1 overexpression decreases ER-stress-induced nuclear accumulation of CHOP in cardiomyocytes

Confocal microscopic observation of ER-stress induced CHOP (green) nuclear localization following Sigmar1 overexpression in cardiomyocytes. Cardiomyocytes are counterstained with α-cardiac actin antibody (red) and nuclei with DAPI (blue); n=3 experiments.

Figure 6. Effect of Sigmar1 overexpression on ER-stress-induced cellular damage in cardiomyocytes

Tunicamycin induced LDH release in NRCs. Bars represent mean ± S.E.M.; n=3 experiments. Abbreviation: NS, not significant.

Figure 7. Effect of Sigmar1 expression on ER-stress response pathways

(A) Effect of adenoviral-mediated Sigmar1 overexpression on tunicamycin-induced changes in expression of proteins involved in the ER-stress response pathway and (B) densitometric quantitation of p-IRE1α. (C) Effect of Sigmar1 knockdown by siRNA transfection on tunicamycin-induced changes in the expression of adaptive ER-stress proteins and (D) densitometric quantitation of p-IRE1α. Bars represent mean ± S.E.M.; n=3 experiments. *, P<0.05 and **, P<0.01 compared with control group; ^#, P<0.05 compared with control tunicamycin treatment group by Tukey’s post hoc test. (E) Co-immunoprecipitation (Co-IP) studies showing IRE1α interaction with Sigmar1 in cardiomyocytes. Whole cell extracts from Sigmar1 adenoviral overexpressed (Sigmar1 Ade) or control infected (β-Gal) cardiomyocytes were immunoprecipitated with anti-IRE1α antibody. The abundance of immunoprecipitated Sigmar1 was detected by Western blotting using anti-Sigmar1 antibody.

Figure 8. Effect of Sigmar1 overexpression on XBP1s expression in NRCs

(A) Sigmar1 overexpression increases the expression of XBP1s in tunicamycin treated NRCs. Bars represent mean ± S.E.M.; n=3 experiments. *, P<0.05 and **, P<0.01 compared with control group by Tukey’s post hoc test. (B) Confocal microscopic observation of the changes in XBP1s (green) nuclear localization by Sigmar1 overexpression in tunicamycin treated cardiomyocytes (cardiac troponin I: red; DAPI: blue).

Figure 9. Effect of Sigmar1 siRNA knockdown on XBP1s expression in NRCs

(A) Sigmar1 knockdown by siRNA transfection decreases the expression of XBP1s in tunicamycin-treated NRCs. Bars represent mean ± S.E.M.; n=3 experiments. *, P<0.05 compared with control group by Tukey’s post hoc test. (B) Confocal microscopic observation of the changes in XBP1s (green) nuclear localization by Sigmar1-siRNA knockdown in tunicamycin-treated cardiomyocytes (cardiac troponin I: red; DAPI: blue).