Olaparib protects cardiomyocytes against oxidative stress and improves graft contractility during the early phase after heart transplantation in rats

Abstract

Background and purpose: Olaparib, rucaparib and niraparib, potent inhibitors of poly(ADP-ribose) polymerase (PARP) are approved as anti-cancer drugs in humans. Considering the previously demonstrated role of PARP in various forms of acute and chronic myocardial injury, we tested the effects of olaparib in in-vitro models of oxidative stress in cardiomyocytes, and in an in vivo model of cardiac transplantation.

Experimental approach: H9c2-embryonic rat heart-derived myoblasts pretreated with vehicle or olaparib (10μM) were challenged with either hydrogen peroxide (H₂ O₂ ) or with glucose oxidase (GOx, which generates H₂ O₂ in the tissue culture medium). Cell viability assays (MTT, lactate dehydrogenase) and Western blotting for PARP and its product, PAR was performed. Heterotopic heart transplantation was performed in Lewis rats; recipients were treated either with vehicle or olaparib (10 mg kg^-1 ). Left ventricular function of transplanted hearts was monitored via a Millar catheter. Multiple gene expression in the graft was measured by qPCR.

Key results: Olaparib blocked autoPARylation of PARP1 and attenuated the rapid onset of death in H9c2 cells, induced by H₂ O₂ , but did not affect cell death following chronic, prolonged oxidative stress induced by GOx. In rats, after transplantation, left ventricular systolic and diastolic function were improved by olaparib. In the transplanted hearts, olaparib also reduced gene expression for c-jun, caspase-12, catalase, and NADPH oxidase-2.

Conclusions and implications: Olaparib protected cardiomyocytes against oxidative stress and improved graft contractility in a rat model of heart transplantation. These findings raise the possibility of repurposing this clinically approved oncology drug, to be used in heart transplantation.

Linked articles: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Figure 1

Experimental protocol. Donor rats: haemodynamic measurements were assessed 1.5 h after a short intravenous infusion of either olaparib (10 mg·kg⁻¹) or the corresponding vehicle. Heart transplantation: a short intravenous infusion of either olaparib (10 mg·kg⁻¹) or the corresponding vehicle through the inferior vena cava was started immediately prior to releasing the aortic clamp and continued during the first 5 min of the reperfusion period. One and half hours after transplantation, left‐ventricular graft function was assessed, and tissue and blood samples were collected.

Figure 2

Olaparib protects heart myoblasts against oxidant‐induced damage. The viability of H9c2 cells treated with various concentration of olaparib (1–100 μM) alone or in combination with 300 μM or 1 mM H₂O₂ for 15 min (A) or 30 min (B) was assessed using MTT and LDH assays. Data shown are means ± SEM; *P < 0.05, significant protective effect of olaparib; n = 5 per group.

Figure 3

Olaparib protects cardiac myoblasts against acute, severe oxidative stress. (A) Experimental design. Reduction of MTT (B) and release of LDH (C) in H9c2 cells treated with 300 μM or 1 mM H₂O₂ in the absence or presence of 10 μM olaparib (olap). (D) Western blot analysis of PARylation , at 15 min after H₂O₂. ( E ) Western blot analysis of PARP1 expression, at 15min ‐24h, after H₂O₂. Data shown are means ± SEM. *P < 0.05, significant protective effect of olaparib; n = 5 per group.

Figure 4

Olaparib does not protect cardiac myoblasts against prolonged, low‐level oxidative stress. (A) Experimental design. Reduction of MTT (B) and release of LDH (C) in H9c2 cells treated with 0.05 U·mL⁻¹ GOx in the absence or presence of 10 μM olaparib (olap). (D) Western blot analysis of PARylation and PARP1 (* indicates a non‐specific band). Data shown are means ± SEM. n = 5 per group.

Figure 5

Olaparib does not protect cardiac myoblasts against prolonged, low‐level oxidative stress at later time points. (A) Experimental design. (B) Reduction of MTT and release of LDH in H9c2 cells treated with 0.05 U·mL⁻¹ GOx for 1 h followed by washout of GOx and incubated for additional 24 h in the absence or presence of 10 μM olaparib (olap). (C) Western blot analysis of PARylation and PARP1 (* indicates non‐specific band). Data shown are means ± SEM. n = 5 per group.

Figure 6

Olaparib improves left‐ventricular systolic and diastolic function of transplanted rat hearts. (A) Maximal slope of the systolic pressure increment (dP/dt_max)‐left‐ventricular volume, (B) maximal slope of the diastolic pressure decrement (dP/dt_min)‐left‐ventricular volume and (C) time constant of left‐ventricular pressure decay (Tau‐w). Data shown are means ± SEM. n = 6 rats per group. *P < 0.05, significantly different from control.

Figure 7

Olaparib affects gene expression in transplanted rat hearts. (A) c‐Jun, (B) bax, (C) caspase‐12, (D) caspase‐3, (E) SOD‐1, (F) GPX4, (G) catalase, (H) NOX2 and (I) NOX4. Data shown are means ± SEM. n = 6 rats per group. *P < 0.05, significantly different from control.

Figure 8

Olaparib treatment reduces neutrophil infiltration in the graft. (A) Representative photomicrographs of myeloperoxidase (MPO, magnification ×200; scale bar: 100 μm) and histological scores of (B) total number of myeloperoxidase‐expressing cells. Black arrows indicate myeloperoxidase positive cells (not all are marked). Data shown are as means ± SEM. n = 6 rats per group. *P < 0.05, significantly different from control.