Reduction of myocardial ischaemia-reperfusion injury by inactivating oxidized phospholipids

Abstract

Aims: Myocardial ischaemia followed by reperfusion (IR) causes an oxidative burst resulting in cellular dysfunction. Little is known about the impact of oxidative stress on cardiomyocyte lipids and their role in cardiac cell death. Our goal was to identify oxidized phosphatidylcholine-containing phospholipids (OxPL) generated during IR, and to determine their impact on cell viability and myocardial infarct size.

Methods and results: OxPL were quantitated in isolated rat cardiomyocytes using mass spectrophotometry following 24 h of IR. Cardiomyocyte cell death was quantitated following exogenously added OxPL and in the absence or presence of E06, a 'natural' murine monoclonal antibody that binds to the PC headgroup of OxPL. The impact of OxPL on mitochondria in cardiomyocytes was also determined using cell fractionation and Bnip expression. Transgenic Ldlr-/- mice, overexpressing a single-chain variable fragment of E06 (Ldlr-/--E06-scFv-Tg) were used to assess the effect of inactivating endogenously generated OxPL in vivo on myocardial infarct size. Following IR in vitro, isolated rat cardiomyocytes showed a significant increase in the specific OxPLs PONPC, POVPC, PAzPC, and PGPC (P < 0.05 to P < 0.001 for all). Exogenously added OxPLs resulted in significant death of rat cardiomyocytes, an effect inhibited by E06 (percent cell death with added POVPC was 22.6 ± 4.14% and with PONPC was 25.3 ± 3.4% compared to 8.0 ± 1.6% and 6.4 ± 1.0%, respectively, with the addition of E06, P < 0.05 for both). IR increased mitochondrial content of OxPL in rat cardiomyocytes and also increased expression of Bcl-2 death protein 3 (Bnip3), which was inhibited in presence of E06. Notably cardiomyocytes with Bnip3 knock-down were protected against cytotoxic effects of OxPL. In mice exposed to myocardial IR in vivo, compared to Ldlr-/- mice, Ldlr-/--E06-scFv-Tg mice had significantly smaller myocardial infarct size normalized to area at risk (72.4 ± 21.9% vs. 47.7 ± 17.6%, P = 0.023).

Conclusions: OxPL are generated within cardiomyocytes during IR and have detrimental effects on cardiomyocyte viability. Inactivation of OxPL in vivo results in a reduction of infarct size.

Figure 1

Identification of fragmented OxPL molecules in NNCM during IR. (A–E) Single MRM ion chromatograms of fragmented OxPL identified in NNCM during IR. Cells were incubated under IR conditions are described in the Methods section. (F) Presence of most abundant OxPL in cultured NNCM after IR compared to control. The most abundant compound was PONPC, which had a significant increase after exposure to IR. n = 4 separate cultures, each done in triplicate (*P < 0.05, ANOVA).

Figure 2

Changes in OxPL levels during rat coronary ligation IR injury. (A) TTC staining of cross section of rat myocardium in control and after 1 h of ischaemia with 24 h of reperfusion. (B) Significant increase in OxPL levels in rat myocardium after 24 h of IR injury, n = 4. Levels of the different OxPL (POVC, PGPC, PONPC, KOdiAPC, and PAzPC) have been each compared in controls vs. IR via the Student’s t-test, *P < 0.05).

Figure 3

Cell viability of NNCM exposed to increasing concentrations of aldehyde and carboxylic acid based OxPL compared to cell viability after treatment with non-oxidized control PSPC. (A) Images of rat NNCM stained using the vital dyes, calcein-AM (green—live), and ethidium homodimer-1 (red—dead), exposed to the indicated concentrations of the control phospholipid PSPC, and the OxPL POVPC, PAzPC, and PONPC for 4 h at 37°C. (B) Cell viability measured as percent cell death after incubations with control PSPC and indicated OxPL (n = 4, separate cultures, each done in triplicate) (*P < 0.05, ^#P < 0.01, vs. PSPC at same concentration, ANOVA).

Figure 4

Cellular and mitochondrial fragmented OxPC in cardiomyocytes during IR injury. (A) Significant increase in PONPC and POVPC in mitochondria in cells undergoing IR compared to control cardiomyocytes. Level of POVPC and PONPC in control vs. IR *P < 0.05, Student’s t-test) (n = 4). (B) There is specific enrichment of POVPC and PONC in mitochondria undergoing IR when compared to cellular membrane. *P < 0.05, Student’s t-test (n = 4).

Figure 5

Mitochondrial permeability of NNMC in presence of OxPL. (A) Representative images of NNCM stained with calcein-AM and CoCl₂ with exposure to increasing concentrations of POVPC and PONPC to determine mitochondrial permeability as compared to non-oxidized control PSPC. (B) Mitochondrial permeability of NNCM to increasing concentrations of PONPC and POVPC is depicted as the fold change over the fluorescence of non-oxidized control PSPC treated cardiomyocytes stained with calcein-AM and CoCl₂ at equal concentrations OxPL’s significantly decreased fluorescence compared to PSPC if *P < 0.05, ^#P < 0.01, (n = 4) ANOVA.

Figure 6

Attenuation of OxPL mediated cell death by E06. Representative images of NNCM treated with POVPC and PONPC, and in presence of E06. NNCM stained using the vital dyes, calcein-AM (green—live) and ethidium homodimer-1 (red—dead), co-treated with 5 μM of POVPC and PONPC and 10 μg/mL of OxLDL-specific E06 antibody for 2 h at 37°C (A). There was significant inhibition of OxPL-induced cell death at 5 μM concentration (B). (n = 4 separate cultures, each done in triplicate) (*P < 0.05 when compare to POVPC and PONPC without E06) ANOVA.

Figure 7

OxPL induce cell death through a Bnip3 pathway. (A) Representation of mRNA expression in neonatal cardiomyocytes when exposed to PONPC and POVPC in both the presence and absence of E06. (PSPC vs. POVPC, PSPC vs. PONPC ^#P < 0.05, Student’s t-test), (POVPC vs. POVPC + E06, *P < 0.05, Student’s t-test) (PONPC vs. PONPC + E06, *P < 0.05, Student’s t-test) (n = 4). (B) Cell viability of NNCM exposed to POVPC in wild-type and Bnip3KD cardiomyocytes. (C) Percent cell death in NNCM exposed to POVPC in wild-type and Bnip3KD cardiomyocytes (n = 3) (WT POVPC vs. Bnip KD POVPC, ^##P < 0.05, Student’s t-test).

Figure 8

Mice expressing scFV-E06 have reduced infarct size following myocardial IR. (A) Representative myocardial TTC staining of LDLR^−/− (n = 15) and scFvE06/LDLR^−/− (n = 14) groups 7 days after 60 min of ischaemia. White indicates infarct; red, viable myocardium; and non-blue, AAR. (B) There were no significant difference in AAR normalized by LV mass distal to the ligation for ischaemia (AAR/LV). Compared to controls, E06-scFv/Ldlr^−/− mice had 65.9% smaller IA/AAR (P = 0.0023) (C) and 58.5% smaller IA/LV (P = 0.0025) ANOVA. TTC, 2, 3, 5-triphenyltetrazolium chloride; AAR, area at risk; LV, left ventricle; IA, infarct area.