EP1 activation inhibits doxorubicin-cardiomyocyte ferroptosis via Nrf2

Abstract

Chemotherapeutic agents, such as doxorubicin (DOX), may cause cardiomyopathy, even life-threatening arrhythmias in cancer patients. Ferroptosis-an iron-dependent oxidative form of programmed necrosis, plays a pivotal role in DOX-induced cardiomyopathy (DIC). Prostaglandins (PGs) are bioactive signaling molecules that profoundly modulate cardiac performance in both physiologic and pathologic conditions. Here, we found that PGE₂ production and its E-prostanoid 1 receptor (EP1) expression were upregulated in erastin (a ferroptosis inducer) or DOX-treated cardiomyocytes. EP1 inhibition markedly aggravated erastin or DOX-induced cardiomyocyte ferroptosis, whereas EP1 activation exerted opposite effect. Genetic depletion of EP1 in cardiomyocytes worsens DOX-induced cardiac injury in mice, which was efficiently rescued by the ferroptosis inhibitor Ferrostatin-1 (Fer-1). Mechanistically, EP1 activation protected cardiomyocytes from DOX-induced ferroptosis by promoting nuclear factor erythroid 2-related factor 2 (Nrf2)-driven anti-oxidative gene expression, such as glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11). EP1 was coupled with G_αq to elicit intracellular Ca²⁺ flux and activate the PKC/Nrf2 cascade in ferroptotic cardiomyocytes. EP1 activation also prevents DOX-induced ferroptosis in human cardiomyocytes. Thus, PGE₂/EP1 axis protects cardiomyocytes from DOX-induced ferroptosis by activating PKC/Nrf2 signaling and activation of EP1 may represent an attractive strategy for DIC prevention and treatment.

Keywords: Cardiomyocyte ferroptosis; DOX-induced cardiomyopathy; EP1; Nrf2.

Fig. 1

EP1 expression is upregulated in cardiomyocyte in response to erastin or DOX treatment A, B. Effect of Fer-1 (2 μM, ferroptosis inhibitor), DFO (100 μM, iron chelator), Z-VAD (10 μM, apoptosis inhibitor), Nec-1S (10 μM, necroptosis inhibitor), Trolox (100 μM, antioxidant) or Baf-A1 (1 nM, autophagy inhibitor) on living H9C2 cells treated with DOX (2 μM) (A) or erastin (3 μM) (B). *P < 0.05 vs vehicle; A, n = 5; B, n = 8. C. The PG profiles of H9C2 cells challenged by erastin or DOX. H9C2 cells were treated with erastin or DOX; and arachidonic acid (30 μM) was added into culture after washing for additional 30 min, culture supernatants were collected for PG measurement. *P < 0.05 vs control; n = 6. D. Relative mRNA levels of various prostaglandin receptors in H9C2 cells in response to erastin. *P < 0.05 vs control; n = 6. E. Effect of various prostaglandin receptors agonist (BW245C (10 μM), DP1 agonist; DK-PGD₂ (1 μM), DP2 agonist; 17-PT-PGE₂ (2.5 μM), EP1 agonist; Butaprost (5 μM), EP2 agonist; Sulprostone (1 μM), EP3 agonist; CAY10684 (1 μM), EP4 agonist; Latanoprost (2 μM), FP agonist; Cicaprost (1 μM), IP agonist; U46619 (1 μM), TP agonist) on living H9C2 cells treated with erastin. *P < 0.05 vs vehicle; n = 8. F. Time-dependent EP1 expression in H9C2 cells in response to erastin. *P < 0.05 vs 0 h; n = 6. G. Dose-dependent EP1 expression in H9C2 cells in response to DOX. *P < 0.05 vs 0 nM; n = 4. H. Western blot analysis of HA-EP1 expression in heart tissue from EP1-N-HA mice treated with DOX. I. Quantification of the HA-EP1 expression in H. *P < 0.05 vs vehicle; n = 4.

Fig. 2

Activation of EP1 inhibits DOX-induced ferroptosis in H9C2 cells A. Representative H&E images of H9C2 cells treated with 17-PT-PGE₂ or SC-51322 in the presence of erastin. Scale bars: 20 μm B. Effect of 17-PT-PGE₂ or SC-51322 on living H9C2 cells treated with different concentrations of erastin. *P < 0.05 vs DMSO; n = 4–6. C. Representative flow cytometric plots showing intracellular ROS levels of H9C2 cells treated with 17-PT-PGE₂ or SC-51322 in the presence of erastin. D. Quantification of ROS production in (C). *P < 0.05 vs indicated; n = 5. E. MDA levels in H9C2 cells treated with 17-PT-PGE₂ or SC-51322 in the presence of erastin. *P < 0.05 vs indicated; n = 4. F. Western blot analysis of GPX4 expression in H9C2 cells treated with 17-PT-PGE₂ or SC-51322 in the presence of erastin. G. Quantification of relative protein expression levels of GPX4 in (F). *P < 0.05 as indicated; n = 4. H. Effect of 17-PT-PGE₂ or SC-51322 of living H9C2 cells in the presence of DOX. *P < 0.05 vs DMSO; n = 8. I. MDA levels in H9C2 cells treated with 17-PT-PGE₂ and SC-51322 in the presence of DOX. *P < 0.05 vs indicated; n = 4–5. J. Western blot analysis of GPX4 expression in H9C2 cells treated with 17-PT-PGE₂ or SC-51322 in the presence of DOX. K. Quantification of relative protein expression levels of GPX4 in (J) *P < 0.05 as indicated; n = 4.

Fig. 3

EP1 activation reduces DOX-induced ferroptosis in cardiomyocytes through evoking Nrf2 activity A. Effect of GPX4 inhibitor RSL3 on erastin-induced ferroptosis of H9C2 cells after17-PT-PGE₂ treatment. *P < 0.05 vs indicated; n = 8. B. Western blot analysis of p-Nrf2 and Nrf2 expression in H9C2 cells treated with 17-PT-PGE₂ or SC-51322 in the presence of erastin. C. Quantification of relative protein expression levels in (B). *P < 0.05 as indicated; n = 4. D. Western blot analysis of p-Nrf2 and Nrf2 expression in H9C2 cells treated with 17-PT-PGE₂ or SC-51322 in the presence of DOX. E. Quantification of relative protein expression levels in (D). *P < 0.05 as indicated; n = 4. F. Western blot analysis of nuclear p-Nrf2 and Nrf2 expression in H9C2 cells treated with17-PT-PGE₂ or SC-51322 in the presence of erastin. G. Quantification of relative protein expression levels in (F). *P < 0.05 as indicated; n = 4. H. Western blot analysis of nuclear p-Nrf2 and Nrf2 expression in H9C2 cells treated with 17-PT-PGE₂ or SC-51322 in the presence of DOX. I. Quantification of relative protein expression levels in (H). *P < 0.05 as indicated; n = 4. J, K. Effect of ML-385 (5 μM) on cell viability (J) and intracellular ROS production (K) in H9C2 cells treated with 17-PT-PGE₂ in the presence of erastin. *P < 0.05 as indicated; (J), n = 8; (K), n = 6. L. Western blot analysis of effect of ML-385 on the expression of GPX4 and SLC7A11 in H9C2 cells treated with 17-PT-PGE₂ in the presence of erastin. M. Quantification of relative protein expression levels in (L). *P < 0.05 as indicated; n = 4. N. Western blot analysis of effect of ML-385 on the expression of GPX4 and SLC7A11 in H9C2 cells treated with 17-PT-PGE₂ in the presence of DOX. O. Quantification of relative protein expression levels in (N). *P < 0.05 as indicated; n = 4.

Fig. 4

Blockage of PKC abolishes EP1-mediated anti-ferroptosis effect on DOX-treated cardiomyocytes through suppressing Nrf2 A. Western blot analysis of effect of staurosporine (20 nM) on the expression of p-Nrf2 and Nrf2 in H9C2 cells treated with 17-PT-PGE₂ in the presence of erastin. B. Quantification of relative protein expression levels in (A). *P < 0.05 as indicated; n = 4. C, D. Effect of staurosporine on the cell viability (C) and intracellular ROS production (D) in H9C2 cells treated with 17-PT-PGE₂ in the presence of erastin. *P < 0.05 as indicated; (C), n = 8; (D), n = 7. E. Western blot analysis of effect of staurosporine on the expression of p-Nrf2, Nrf2, GPX4 and SLC7A11 in H9C2 cells treated with17-PT-PGE₂ in the presence of erastin. F. Quantification of relative protein expression levels in (E). *P < 0.05 as indicated; n = 4. G. Western blot analysis of effect of staurosporine on the expression of p-Nrf2, Nrf2, GPX4 and SLC7A11 in H9C2 cells treated with 17-PT-PGE₂ in the presence of DOX. H. Quantification of relative protein expression levels in (G). *P < 0.05 as indicated; n = 4.

Fig. 5

EP1 activation protects cardiomyocytes from DOX-induced ferroptosis by mobilizing Ca²⁺ to activate PKC/Nrf2 signaling A. Effect of FR900359 (1 μM) or PTX (10 μM) on the cell viability of H9C2 cells treated with 17-PT-PGE₂ in the presence of erastin. *P < 0.05 as indicated; n = 8. B. Representative fluorescence images showing the intracellular Ca²⁺ spark in neonatal rat cardiomyocytes treated with 17-PT-PGE₂. Scale bars: 10 μm C. The intracellular Ca²⁺ flux in neonatal rat cardiomyocytes treated with 17-PT-PGE₂. The arrow indicates the injection of 17-PT-PGE₂. D. PKC kinase activity in H9C2 cells after 17-PT-PGE₂ treatment. *P < 0.05 as indicated; n = 5–6. E, F. The effect of U73122 (5 μM) on the cell viability (E) and intracellular ROS production (F) in H9C2 cells treated with 17-PT-PGE₂ in the presence of erastin. *P < 0.05 as indicated; E, n = 8; F, n = 5–6. G. Western blot analysis of effect of U73122 on the expression of p-Nrf2, Nrf2, GPX4 and SLC7A11 in H9C2 cells treated with 17-PT-PGE₂ in the presence of erastin. H. Quantification of relative protein expression levels in (G). *P < 0.05 as indicated; n = 4. I. Western blot analysis of effect of U73122 on the expression of p-Nrf2, Nrf2, GPX4 and SLC7A11 in H9C2 cells treated with 17-PT-PGE₂ in the presence of DOX. J. Quantification of relative protein expression levels in (I). *P < 0.05 as indicated; n = 4. K. Schematic illustration of EP1-mediated myocardial protection in DOX-induced cardiomyocytes ferroptosis through Ca²⁺/PKC/Nrf2 signaling pathway.

Fig. 6

EP1 deletion in cardiomyocytes exacerbates DOX-induced cardiac injury in mice through promoting cardiac ferroptosis A. Schematic diagram of the treatment of Fer-1 in DOX-challenged mice. B, C. Quantitative analysis of cardiac EF (B) and FS (C) in EP1^f/f and EP1^f/fMHC^Cre mice treated with DOX in the presence or absence of Fer-1. *P < 0.05 vs indicated; n = 8. D. The heart/body weight ratio of EP1^f/f and EP1^f/fMHC^Cre mice treated with DOX in the presence or absence of Fer-1. *P < 0.05 vs indicated; n = 8. E. Representative images of H&E and Sirius red staining of heart form EP1^f/f and EP1^f/fMHC^Cre mice treated with DOX in the presence or absence of Fer-1. H&E Scale bars: 1 mm; Sirius red Scale bars: 50 μm F. Quantification of collagen content in (E). *P < 0.05 vs indicated; n = 8. G, I. The gene expressions of Anp (G), Bnp (H), Myh7 (I) in the cardiac tissue from EP1^f/f and EP1^f/fMHC^Cre mice treated with DOX in the presence or absence of Fer-1. *P < 0.05 vs indicated; (G), n = 8; (H), n = 5–8; (I), n = 5–8. J, K. MDA levels in the serum (J) and cardiac tissue (K) from EP1^f/f and EP1^f/fMHC^Cre mice treated with DOX in the presence or absence of Fer-1. *P < 0.05 vs indicated; (J), n = 6; (K), n = 5–8. L. Western blot analysis of p-Nrf2, Nrf2, GPX4 and SLC7A11 expression in cardiac tissues from DOX-treated EP1^f/f and EP1^f/fMHC^Cre mice. M-P. Quantification of protein expression of p-Nrf2 (M), Nrf2 (N), GPX4 (O), SLC7A11 (P) in (L). *P < 0.05 as indicated; n = 6. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

EP1 activation prevents DOX-induced ferroptosis in human cardiomyocytes A. Cell viability of AC16 cells treated with 17-PT-PGE₂ (2.5 μM) or SC-51322 (10 μM) in the presence of erastin (5 μM). *P < 0.05 vs indicated; n = 8. B. Representative flow cytometric plots showing the intracellular ROS levels in AC16 cells treated with 17-PT-PGE₂ or SC-51322 in the presence of erastin. C. Quantification of ROS production in (B). *P < 0.05 vs indicated; n = 5–8. D. Western blot analysis of p-Nrf2 and total Nrf2 expression in AC16 cells treated with 17-PT-PGE₂ or SC-51322in the presence of erastin. E. Quantification of relative protein expression levels in (D). *P < 0.05 as indicated; n = 4. F. Cell viability of AC16 cells treated with 17-PT-PGE₂ or SC-51322 in the presence of DOX. *P < 0.05 vs indicated; n = 8. G. Representative flow cytometric plots showing the intracellular ROS levels in AC16 cells treated with 17-PT-PGE₂ or SC-51322 in the presence of DOX. H. Quantification of ROS production in (G). *P < 0.05 vs indicated; n = 6. I. Western blot analysis of p-Nrf2 and total Nrf2 expression in AC16 cells treated with 17-PT-PGE₂ or SC-51322 in the presence of DOX. J. Quantification of relative protein expression levels in (I). *P < 0.05 as indicated; n = 4.