Cardioprotective effects of Cu(II)ATSM in human vascular smooth muscle cells and cardiomyocytes mediated by Nrf2 and DJ-1

Abstract

Cu^(II)ATSM was developed as a hypoxia sensitive positron emission tomography agent. Recent reports have highlighted the neuroprotective properties of Cu^(II)ATSM, yet there are no reports that it confers cardioprotection. We demonstrate that Cu^(II)ATSM activates the redox-sensitive transcription factor Nrf2 in human coronary artery smooth muscle cells (HCASMC) and cardiac myocytes (HCM), leading to upregulation of antioxidant defense enzymes. Oral delivery of Cu^(II)ATSM in mice induced expression of the Nrf2-regulated enzymes in the heart and aorta. In HCASMC, Cu^(II)ATSM increased expression of the Nrf2 stabilizer DJ-1, and knockdown of Nrf2 or DJ-1 attenuated Cu^(II)ATSM-mediated heme oxygenase-1 and NADPH quinone oxidoreductase-1 induction. Pre-treatment of HCASMC with Cu^(II)ATSM protected against the pro-oxidant effects of angiotensin II (Ang II) by attenuating superoxide generation, apoptosis, proliferation and increases in intracellular calcium. Notably, Cu^(II)ATSM-mediated protection against Ang II-induced HCASMC apoptosis was diminished by Nrf2 knockdown. Acute treatment with Cu^(II)ATSM enhanced the association of DJ-1 with superoxide dismutase-1 (SOD1), paralleled by significant increases in intracellular Cu^(II) levels and SOD1 activity. We describe a novel mechanism by which Cu^(II)ATSM induces Nrf2-regulated antioxidant enzymes and protects against Ang II-mediated HCASMC dysfunction via activation of the Nrf2/DJ-1 axis. Cu^(II)ATSM may provide a therapeutic strategy for cardioprotection via upregulation of antioxidant defenses.

Figure 1

Cu^(II) ATSM induces nuclear translocation of Nrf2 and antioxidant protein expression in HCASMC. HCASMC were treated with Cu^(II)ATSM (0.1, 0.5, 1 and 10 µM) for 12 h and expression of (A) HO-1 and (B) GCLM assessed by immunoblotting with densitometric analysis relative to α-tubulin. Data denote mean ± S.E.M., n = 5, *P < 0.05, **P < 0.001 vs vehicle (one-way ANOVA and Bonferroni post hoc analysis). (C) HCASMC were treated with Cu^(II)ATSM (0.1, 0.5, 1 and 10 µM, 30 min) and phosphorylation of Nrf2 at serine 40 assessed by immunoblotting with densitometric analysis relative to total Nrf2. Data denote mean ± S.E.M., n = 4, *P < 0.05, **P < 0.001 vs vehicle (one-way ANOVA and Bonferroni post hoc analysis). (D) HCASMC were treated with Cu^(II)ATSM (1 µM, 4 h) and Nrf2 localisation assessed by fluorescence microscopy (scale bar = 5 µm). Induction of (E) HO-1 and (F) NQO1 protein in response to Cu^(II)ATSM (1 µM, 12 h) was assessed following transient transfection of cells with scramble (Scr) si-RNA or Nrf2 siRNA. Data denote mean ± S.E.M., n = 4, *P < 0.05, ***P < 0.001 (two-way ANOVA and Bonferroni post hoc analysis).

Figure 2

Cu^(II)ATSM induces Nrf2 target antioxidant protein expression in HCM in vitro. HCM were treated with Cu^(II)ATSM (1 µM, 12 h) and expression of (A) HO-1, (B) GCLM and (C) NQO1 assessed by immunoblotting with densitometric analysis relative to α-tubulin. Data denote mean ± S.E.M., n = 4, *P < 0.05 vs vehicle (Student’s t-test). (D) Induction of HO-1 in HCM in response to Cu^(II)ATSM (1 µM, 12 h) following transient transfection of cells with scramble (Scr) si-RNA or Nrf2 si-RNA. Data denote mean ± S.E.M., n = 4, *P < 0.05, **P < 0.01 (two-way ANOVA and Bonferroni post hoc analysis).

Figure 3

Oral delivery of Cu^(II)ATSM induces Nrf2 target antioxidant protein expression in murine heart and aorta. Heart (A) and aortic (B) tissue homogenates from C57/BL6 male mice administered Cu^(II)ATSM by oral gavage (30 mg/kg) were immunoblotted for HO-1, Prx1, GCLM and NQO1. Data denotes mean ± S.E.M., n = 5 animals per group, *P < 0.05 and **P < 0.01 vs vehicle (Student’s t-test).

Figure 4

Cu^(II)ATSM mediated induction of HO-1 expression in HCASMC is dependent on DJ-1. Induction of (A) DJ-1, (B) HO-1 and (C) NQO1 by Cu^(II)ATSM (1 µM, 12 h) was assessed in HCASMC transiently transfected with scramble (Scr) si-RNA or DJ-1 si-RNA, and expression determined by immunoblotting relative to α-tubulin. Data denote mean ± S.E.M., n = 4, P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA and Bonferroni post hoc analysis).

Figure 5

Cu^(II)ATSM pre-treatment protects against Ang II-mediated superoxide generation, apoptosis, proliferation and intracellular Ca²⁺ elevation. (A) HCASMC were pre-treated with Cu^(II)ATSM (1 µM, 12 h) and then with angiotensin II (Ang II, 200 nM, 4 h). Superoxide generation was assessed using L-012 enhanced chemiluminescence measured over 10 min. (B) HCASMC were treated with 200 nM Ang II for 4 h, prior to superoxide measurement in the presence of Cu^(II)ATSM (1 µM). (C) Mitochondrial superoxide generation was determined using MitoSOX red in HCASMC following treatment with Ang II (200 nM, 4 h) and subsequent treatment with Cu^(II)ATSM (1 µM, 30 min). Data denote mean ± S.E.M., n = 4–6, ^#P < 0.01 vs control, ^‡P < 0.01 compared to Cu^(II)ATSM treatment, (one-way ANOVA and Bonferroni post hoc analysis), (D) Annexin V fluorescence was used to assess Ang II (200 nM, 12 h) induced apoptosis in HCASMC following Nrf2 or DJ-1 siRNA knockdown and pre-treatment with Cu^(II)ATSM (1 µM, 12 h). Data denote mean ± S.E.M., n = 4, ***P < 0.001 vs control, ^‡P < 0.001 vs cells treated with Ang II in the absence of Cu^(II)ATSM, ^###P < 0.001 vs cells treated with Cu^(II)ATSM only (one-way ANOVA and Bonferroni post hoc analysis). (E) HCASMC proliferation was assessed following pre-treatment with Cu^(II)ATSM (1 µM, 12 h) prior to Ang II (200 nM, 72 h). (F) Ang II (200 nM, 30 min) mediated changes in intracellular Ca²⁺ (Fura-2AM fluorescence) in HCASMC pretreated with Cu^(II)ATSM (1 µM, 12 h). Data denote mean ± S.E.M., n = 4, ***P < 0.001 compared to control, ^#P < 0.001 vs Ang II (one-way ANOVA and Bonferroni post hoc analysis).

Figure 6

Cu^(II)ATSM increases association of DJ-1 with SOD1, intracellular Cu^(II) levels and SOD activity in HCASMC. (A) HCASMC were treated with Cu^(II)ATSM (1 µM, 1 h), immunoprecipitated for DJ-1 and immunobloted for DJ-1 and SOD1 (representative of n = 4 donors). Intracellular levels of Cu^(II) were assessed in HCAMSC treated with Cu^(II)ATSM (1 µM, 30 min) using either ICP-MS (B) or Phen Green SK fluorescence (C). Data denote mean ± S.E.M., n = 4, **P < 0.01 vs Veh, (Students’ t-test). (D) HCASMC were treated with Cu^(II)ATSM (0.1, 0.5, 1 and 10 µM, 15 min) and phosphorylation of ERK 1/2 detected by immunoblotting and analysed by densitometry relative to α-tubulin. Data denote mean ± S.E.M., n = 4, *P < 0.05, **P < 0.001 vs vehicle (one-way ANOVA and Bonferroni post hoc analysis). (E) HCASMC were treated with Cu^(II)ATSM (1 µM, 30 min) and SOD1 activity assessed. Data denote mean ± S.E.M., n = 4, *P < 0.05 vs Veh, (Students’ t-test). (F) Annexin V fluorescence to assess Ang II (200 nM, 12 h) induced apoptosis in HCASMC following SOD1 knockdown by siRNA and pre-treatment with Cu^(II)ATSM (1 µM, 12 h). Data denote mean ± S.E.M., n = 4, ***P < 0.001 vs si-Scr control, ^#P < 0.001 vs si-SOD1 control, ^‡P < 0.001 vs si-Scr cells treated with Ang II in the absence of Cu^(II)ATSM, ^†P < 0.001 vs si-SOD1 cells treated with Ang II in the absence of Cu^(II)ATSM (one-way ANOVA and Bonferroni post hoc analysis).