Darbepoetin-mediated cardioprotection after myocardial infarction involves multiple mechanisms independent of erythropoietin receptor-common beta-chain heteroreceptor

Abstract

Background and purpose: Darbepoetin, a long-acting erythropoietin derivative, attenuates cardiomyocyte apoptosis and improves short-term (3 days) cardiac function, but the mechanisms responsible are unknown. We investigated potential mechanisms by which darbepoetin exerts cardioprotection following myocardial infarction in mice and the significance of the erythropoietin receptor (EPOR)-common beta-chain (c-beta-chain) heteroreceptor.

Experimental approach: Mice underwent 60 min coronary occlusion followed by treatment with vehicle or a single dose of darbepoetin. Effects on gene expression, apoptosis and neutrophil accumulation in infarcted left ventricle were assessed 24 h later. Cardiac function, effects on vascularization and fibrosis were assessed 28 days later. The significance of EPOR-c-beta-chain heteroreceptor was examined 28 days after infarction using mice deficient in c-beta-chain.

Key results: Twenty-four hours after darbepoetin, mRNAs encoding haeme oxygenase-1 (HO-1), iNOS and brain natriuretic peptide (BNP) were markedly elevated only in infarcted regions, and the frequency of apoptotic cells attenuated. Inflammation was also attenuated with reductions in neutrophil numbers. Darbepoetin also elevated mRNAs encoding angiogenic factors: placental growth factor, monocyte chemoattractant protein-1 and interleukin-1beta. Twenty-eight days after treatment, CD31+ vessels in the infarct zone doubled and fibrosis reduced. Cardiac haemodynamics were improved. Darbepoetin also improved cardiac haemodynamics in c-beta-chain-deficient mice, increased HO-1 and iNOS expression and vessel numbers and attenuated fibrosis.

Conclusions and implications: Darbepoetin stimulates expression of haeme oxygenase, iNOS, BNP and angiogenic factors specifically in infarcted left ventricle that attenuates inflammation, apoptosis and fibrosis; elevate vessel numbers; and improve cardiac function. The EPOR-c-beta-chain heteroreceptor is not essential for these effects.

Figure 1

Darbepoetin, apoptosis and genes that regulate cell survival in infarct zone. (A) Apoptotic cells in the infarct zone 24 h after reperfusion determined using an ‘in situ Cell Death Detection Kit’ and direct cell counting, expressed as percentage of total cells in the infarct zone. (B) Total RNA isolated from infarct regions 24 h after reperfusion, and treatment with darbepoetin or vehicle was subjected to real-time PCR to quantify expression levels of genes that regulate responses to injury. Columns represent infarcted mice treated with vehicle (V) or darbepoetin (D; µg·kg⁻¹) or sham-operated mice (S) treated with darbepoetin. All results are means ± SEM of five mice in each group. *P < 0.05 from vehicle-treated mice.

Figure 2

Darbepoetin and neutrophil accumulation in infarcted myocardium. Top: neutrophil numbers in the infarct zone were determined by counting immunopositive neutrophils in the infarct zone 24 h after reperfusion. Results are mean ± SEM of five mice in each group. *P < 0.05 from infarct zone of vehicle-treated group. Bottom: photomicrographs of neutrophils accumulating in the infarct zone of mice treated with vehicle or darbepoetin (30 µg·kg⁻¹). Bar represents 100 µm.

Figure 3

Darbepoetin affects vessels in infarct zone and angiogenic genes. (A) Left: CD31-positive vessels in infarcted region of myocardium 28 days after treatment with vehicle or darbepoetin. Right: Cd31-positive vessels in the infarct zone of mice treated with vehicle or darbepoetin (30 µg·kg⁻¹). The bar represents 100 µm. (B) Expression of 28 angiogenic genes 24 h after myocardial infarction/sham operation and treatment with vehicle or darbepoetin. TNFSFR12a, tumour necrosis super family receptor 12a; CTGF, connective tissue growth factor; IL-10, interleukin-10; Nudt6, nudix (nucleoside diphosphate linked moiety X)-type motif 6; MMP9, matrix metalloproteinase-9; CSF-3, G-CSF; NRP1, neurophilin-1; Pofut 1, protein O-fucosyltransferase-1; PTN, pleiotrophin; CCL2, monocyte chemoattractant protein-1; Angpt14, angiopoietin-like-4; PTGS1, prostaglandin endoperoxide synthase-1; Sphk1, sphingosine kinase-1. All other genes follow standard accepted abbreviations. In (A), results are means ± SEM of five mice in each group. *P < 0.05 from sham-operated, darbepoetin-treated mice. In (B), results are means ± SEM of 10 mice in each group. *P < 0.05 from darbepoetin-treated, sham-operated mice; ⁺P < 0.05 from infarcted, vehicle-treated mice.

Figure 4

Darbepoetin and cardiac fibrosis. Fibrosis was assessed 28 days after ischaemia/sham operation and treatment with vehicle or darbepoetin. Top: cross-sections were stained with picrosirius red, and fibrosis expressed as % area stained with picrosirius red in each cross section. Results are means ± SEM from 10 mice in each group. *P < 0.05 from infarcted, vehicle-treated mice. Bottom: photomicrographs of cross sections of infarcted left ventricle stained with picrosirius red, demonstrating the extent of fibrosis in mice treated with vehicle or darbepoetin (30 µg·kg⁻¹).

Figure 5

Cardiac haemodynamics following ischaemic injury. LVSP, and LV + dp/dt and LV − dp/dt were measured in anaesthetized C57Bl6 mice using an intraventricular Miller catheter. Results are means ± SEM of 10 mice in each group. *P < 0.05 from infarcted, vehicle-treated mice.

Figure 7

Effects of darbepoetin on the expression of HO-1 and iNOS in infarcted left ventricle of c-β-chain-deficient mice 24 h after permanent coronary ligation, and fibrosis and number of vessels 28 days later. (A) iNOS expression in infarcted left ventricle of vehicle-treated mice. (B) iNOS expression in infarcted left ventricle of darbepoetin-treated mice; insert: non-immune IgG control. (C) HO-1 expression in infarcted left ventricle of vehicle-treated mice. (D) HO-1 expression in infarcted left ventricle of darbepoetin-treated mice; insert: non-immune IgG control. (E) Left ventricular fibrosis (F) vessels in border infarct zone of left ventricle. Results are means ± SEM of six to nine mice in each group. *P < 0.05 from vehicle-treated, infarcted mice.

Figure 6

Effects of darbepoetin on cardiac haemodynamics (LVSP, LV + dp/dt and LV − dp/dt) 28 days after subjecting C57Bl6 and c-β-chain-deficient mice to 1 h of ischaemia followed by reperfusion and also c-β-chain-deficient mice to permanent coronary ligation. Results are means ± SEM of six to nine mice in each group. *P < 0.05 from vehicle-treated, infarcted mice.