Antiarrhythmogenic Effect of Reconstituted High-Density Lipoprotein Against Ischemia/Reperfusion in Rats
Satoshi Imaizumi, MD*,
Shin-ichiro Miura, MD, PhD*,*,
Kazuto Nakamura, MD, PhD , ,
Yoshihiro Kiya, MD*,
Yoshinari Uehara, MD, PhD*,
Bo Zhang, PhD*,
Yoshino Matsuo, PhD*,
Hidenori Urata, MD, PhD ,
Munehito Ideishi, MD, PhD*,
Kerry-Anne Rye, PhD||,
Masataka Sata, MD, PhD, and
Keijiro Saku, MD, PhD, FACC*
* Department of Cardiology, Fukuoka University School of Medicine, Fukuoka, Japan
Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan
Department of Advanced Clinical Science and Therapeutics, University of Tokyo Graduate School of Medicine, Tokyo, Japan
Department of Cardiovascular Diseases, Fukuoka University Chikushi Hospital, Chikushino, Japan
|| Lipid Research Group, Heart Research Institute, Camperdown, Sydney, New South Wales, Australia.
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Figure 1 Experimental Protocol
Rats underwent 5 min of occlusion of the left coronary artery followed by 3 min of reperfusion. The control group was infused with phosphate-buffered saline (PBS) 10 min before occlusion. In the reconstituted high-density lipoprotein (rHDL), apolipoproteinA-I (A-I), and 1-palmitoyl-2-oleoyl-phosphatidyl-choline (POPC) groups, rHDL (6 mg/kg as A-I), A-I (6 mg/kg), or POPC (15 mg/kg) were infused instead of PBS. The L-NAME+rHDL, PD98059+rHDL, and wortmannin+rHDL groups were additionally injected with N-nitro-L-arginine methyl ester hydrochloride (L-NAME) (10 mg/kg), PD98059 (4 mg/kg), or wortmannin (16 µg/kg), respectively, 5 min before the infusion of rHDL. The L-NAME, PD98059, and wortmannin groups were identical to the L-NAME+rHDL, PD98059+rHDL, and wortmannin+rHDL groups except that rHDL was not administered. The animals were killed immediately after reperfusion for the collection of heart and blood samples. ECG = electrocardiogram; iv = intravenously.
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Figure 2 Lipoprotein Profiles
Lipoprotein profiles as determined by capillary isotachophoresis (top panels) and lipid profiles as analyzed by agarose gel electrophoresis and differential staining (bottom panels) in plasma from Wistar rats before rHDL injection (A), and 5 min (B), 10 min (C), and 30 min (D) after rHDL injection. Peak 1, fast-migrating HDL (fHDL); peaks 2 and 3, intermediate-migrating HDL (iHDL); peaks 4, slow-migrating HDL (sHDL). Abbreviations as in Figure 1.
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Figure 3 Effects of rHDL on Reperfusion-Induced Arrhythmia
(A) Distribution of ventricular arrhythmias during reperfusion in the control group, rHDL group, and L-NAME+rHDL group. The orange cells are the times during which the rat was in ventricular fibrillation (VF); the yellow cells show periods of ventricular tachycardia (VT); and the gray cells are periods during which neither VF nor VT was evident. (B and C) Duration (in seconds) of VT (B) and VF (C) during reperfusion. *p < 0.05 for the rHDL group versus the control group. Abbreviations as in Figure 1.
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Figure 4 Plasma NO Levels
Plasma nitric oxide (NO) levels in rats: (A) NO3, (B) NO2, and (C) NOx (NO3+NO2). Blood samples were drawn by cardiac puncture at the end of reperfusion, and plasma NO3 and NO2 levels were evaluated. The NO3, NO2, and NOx levels (%) are shown relative to the control. The control sample was defined as 100% NO production, and the percent increase or decrease in NO production relative to the control was calculated for each sample. The asterisk *p < 0.05 for the rHDL group versus the rHDL+L-NAME group. **p < 0.01 for the rHDL group versus the control group. ***p < 0.05 for the rHDL group versus the control group. Abbreviations as in Figure 1.
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Figure 5 Localization and Expression Levels of eNOS
(A) Immunohistochemical staining for endothelial nitric oxide synthase (eNOS) and phospho-eNOS (p-eNOS) from rat hearts after reperfusion in the control and rHDL groups. Representative sections are shown (magnification x800). (B) The rHDL activated p-eNOS after 10 min in human coronary artery endothelial cells. Representative pictures are shown. The graph shows the ratio of p-eNOS to total eNOS relative to 1.0 for no treatment. *Significant increase (p < 0.05) versus no treatment. Abbreviations as in Figure 1.
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Figure 6 Expression Levels of Akt, ERK, and eNOS
(A) The rHDL activated p-Akt, p-ERK, and p-eNOS after 10 min of incubation in human coronary artery endothelial cells. (B) The rHDL-induced extracellular-signal-regulated kinase (ERK) activation was blocked by wartmannin and PD98059, but not L-NAME. The rHDL (5 µg/ml)-induced Akt activation was blocked by wartmannin (100 nmol/l), but not by PD98059 (3 µmol/l) or L-NAME (1 µmol/l). (C) The scavenger receptor class B, type I (SR-BI), adenosine triphosphate-binding cassette transporter (ABC) A1, or ABCG1 cells were transfected into ldlA7 cells. Forty-eight hours after transfection, the cells were cultured under serum-free conditions for an additional 24 h. The rHDL (5 µg/ml) was added to the medium for 10 min, and then the cells were lysed and analyzed. Representative pictures are shown (A to C). The graph shows the ratio of phospho- to total eNOS, Akt, and ERK in each sample relative to 1.0 for no treatment. *Significant increase (p < 0.05) versus no treatment. Abbreviations as in Figure 1.
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