Cholesteryl esters accumulate in the heart in a porcine model of ischemia and reperfusion

Abstract

Myocardial ischemia is associated with intracellular accumulation of lipids and increased depots of myocardial lipids are linked to decreased heart function. Despite investigations in cell culture and animal models, there is little data available on where in the heart the lipids accumulate after myocardial ischemia and which lipid species that accumulate. The aim of this study was to investigate derangements of lipid metabolism that are associated with myocardial ischemia in a porcine model of ischemia and reperfusion. The large pig heart enables the separation of the infarct area with irreversible injury from the area at risk with reversible injury and the unaffected control area. The surviving myocardium bordering the infarct is exposed to mild ischemia and is stressed, but remains viable. We found that cholesteryl esters accumulated in the infarct area as well as in the bordering myocardium. In addition, we found that expression of the low density lipoprotein receptor (LDLr) and the low density lipoprotein receptor-related protein 1 (LRP1) was up-regulated, suggesting that choleteryl ester uptake is mediated via these receptors. Furthermore, we found increased ceramide accumulation, inflammation and endoplasmatic reticulum (ER) stress in the infarcted area of the pig heart. In addition, we found increased levels of inflammation and ER stress in the myocardium bordering the infarct area. Our results indicate that lipid accumulation in the heart is one of the metabolic derangements remaining after ischemia, even in the myocardium bordering the infarct area. Normalizing lipid levels in the myocardium after ischemia would likely improve myocardial function and should therefore be considered as a target for treatment.

Figure 1. Increased accumulation of lipid droplets in the AAR and IA.

(A) Representative images of Oil red O stained cryosections. (B) Quantification of the total Oil red O stained area. Results are shown as mean ± SEM, **P<0.01 vs. control.

Figure 2. Increased levels of cholesteryl esters in the pig myocardium.

(A) Content of cholesteryl esters, (B) triglycerides and (C) free cholesterol, n = 7 per group. Results are shown as mean ± SEM, ***P<0.001 vs. control.

Figure 3. Increased expression of LDLr and LRP1.

(A) RT-QPCR analyses of mRNA expression of LDLr, VLDLr and SR-B1, n = 4 per group. (B) mRNA expression of VLDLr in HL-1 cells incubated in hypoxia for 0.5, 1, 6 and 24 h (n = 3). (C) Representative immunoblots of LDLr and LRP1. (D–E) Quantification of LDLr protein bands (D) and LRP1 protein bands (E) n = 6–7 per group. Results are shown as mean ± SEM, *P<0.05 vs. control, **P<0.01 vs. control, ***P<0.001 vs. control.

Figure 4. Increased levels of ceramides in ischemic myocardium.

Content of ceramide (A), sphingomyelin (B), phosphatidylcholine (C) and phosphatidylethanolamine (D), n = 7 per group. Results are shown as mean ± SEM, **P<0.01 vs. control, ***P<0.001 vs. control.

Figure 5. Increased expression of the cytokines IL-1β and IL-6 and ER stress markers in ischemic myocardium.

(A–B) RT-QPCR analyses of mRNA expression of IL-1β (A) and IL-6 (B), n = 6 per group. (C–D) RT-QPCR analyses of mRNA expression of CHOP (C) and ATF6B (D), n = 6 per group. (E) Representative immunoblots of Calnexin and GRP78. (F–G) Quantification of LDLr protein bands (F) and LRP1 protein bands (G) n = 7 per group. Results are shown as mean ± SEM, *P<0.05 vs. control, **P<0.01 vs. control, ***P<0.001 vs. control.