An Inducible and Vascular Smooth Muscle Cell-Specific Pink1 Knockout Induces Mitochondrial Energetic Dysfunction during Atherogenesis

Abstract

DNA damage and mitochondrial dysfunction are defining characteristics of aged vascular smooth muscle cells (VSMCs) found in atherosclerosis. Pink1 kinase regulates mitochondrial homeostasis and recycles dysfunctional organelles critical for maintaining energetic homeostasis. Here, we generated a new vascular-specific Pink1 knockout and assessed its effect on VSMC-dependent atherogenesis in vivo and VSMC energetic metabolism in vitro. A smooth muscle cell-specific and MHC-Cre-inducible flox'd Pink1^f/f kinase knockout was made on a ROSA26^+/0 and ApoE^-/- C57Blk6/J background. Mice were high fat fed for 10 weeks and vasculature assessed for physiological and pathogical changes. Mitochondrial respiratory activity was then assessed in wild-type and knockout animals vessels and isolated cells for their reliance on oxidative and glycolytic metabolism. During atherogenesis, we find that Pink1 knockout affects development of plaque quality rather than plaque quantity by decreasing VSMC and extracellular matrix components, collagen and elastin. Pink1 protein is important in the wild-type VSMC response to metabolic stress and induced a compensatory increase in hexokinase II, which catalyses the first irreversible step in glycolysis. Pink1 appears to play an important role in VSMC energetics during atherogenesis but may also provide insight into the understanding of mitochondrial energetics in other diseases where the regulation of energetic switching between oxidative and glycolytic metabolism is found to be important.

Keywords: DNA damage; atherosclerosis; glycolysis; mitochondrial dysfunction; oxidative phosphorylation.

Figure 1

Mice breeding scheme. (A) Original Pink1^f/f knockout out mice locus and (B) truncation site and original Southern blot by Glasl et al. [15]. (C) Flox’d Pink1^f/f mice were crossed with ApoE^−/− animals. Double homozygotes were then bred with Cre^+/0 Rosa^+/+ ApoE^−/− mice. (D) DNA genotyping for each allele (E) Pink excision pilot study used a Cre/ROSA beta galactosidase reporter after IP injection with tamoxifen in corn oil (n = 3). Cre-recombinase and X-gal staining was confirmed across entire sections of aortic rings (upper pair) of vessels and is counterstained with eosin (lower pair). (F) Whole abdominal aortic tissue were stained blue in presence of X-gal when Pink was excised and Cre was activated. (G) A Pink1-KO VSMC line was derived from Pink^f/f explant whole aortae and treatedwith hydroxytamoxifen (OHT) in culture to mediate Pink1 excision using a beta-galactosidase reporter (arrows) (×10) (scale bar 100 µm). (H) The atherosclerosis feeding protocol included littermate and sex-matched controls with groups fed up to 16 weeks of age to generate atherosclerotic lesions.

Figure 2

Pink1 mice body weight, serums and heart and blood pressures during high-fat feeding. (A) Body weights over experimental time course. (B) Blood serum values for glucose (C) cholesterol and (D) triglyceride across the high-fat feeding time course at T = 0, T = 5 and T = 10 weeks. (E) Mice heart rates @ T = 5 weeks. (F) Tail-cuff blood pressure values 5 weeks high-fat diet. Student t-test (n = 6) (ns—non significant), *** p ≤ 0.001. vs. control.

Figure 3

Pink1-KO ApoE^−/− mice have features of increased plaque vulnerability. Heart aortic root valves were sectioned at the coronary outflows and assessed for plaque quantity and quality. (A) Gross plaque structure and abundance of VSMC and macrophages, elastin andcollagens, hexII and Pink1 expression in plaque cap and IgG2A isotype controls (Olympus CellSenS) (scale 2 mm), control n = 9, Pink1-KO n = 15. (B) Representative whole aortae were taken from the aortic arch to the femoral bifurication and stained for total plaque burden with Oil red O, en face preparation (scale bar 500 µm). (C) Heart aortic valve (sinus) plaque cross-sectional area then quantified between groups. (D) Frequency of all discrete luminal surface aortic plaques were digitally extracted and plotted between control and Pink1-KO mice (Student t-test p ≥ 0.05, ns—not significant).

Figure 3

Pink1-KO ApoE^−/− mice have features of increased plaque vulnerability. Heart aortic root valves were sectioned at the coronary outflows and assessed for plaque quantity and quality. (A) Gross plaque structure and abundance of VSMC and macrophages, elastin andcollagens, hexII and Pink1 expression in plaque cap and IgG2A isotype controls (Olympus CellSenS) (scale 2 mm), control n = 9, Pink1-KO n = 15. (B) Representative whole aortae were taken from the aortic arch to the femoral bifurication and stained for total plaque burden with Oil red O, en face preparation (scale bar 500 µm). (C) Heart aortic valve (sinus) plaque cross-sectional area then quantified between groups. (D) Frequency of all discrete luminal surface aortic plaques were digitally extracted and plotted between control and Pink1-KO mice (Student t-test p ≥ 0.05, ns—not significant).

Figure 4

Pink1-KO ApoE^−/− mice have reduced VSMCs and extracellular plaque components. Quantification of plaque regions by immunohistochemistry for (A) VSMC (a-SMA+), (B) cap thickness, (C) extracellular collagen (sirius red), (D) elastin abundance (Verhoefff Van Gieson) stain, (E) macrophages (mac3+), (F) lipid void area, (G) necrotic area areas, and (H) hexokinase 2 abundance. Regions of stained plaque (n = 9–15). (ns—non significant), Student t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. control.

Figure 5

Pink1-KO ApoE^−/− mice have reduced respiratory capacity and ability to switch energetics to glycolysis compared to wild type. (A) Inhibition of mitochondrial respiration in Pink1+/+ wild-type VSMC retains a switching effect by (B) concomitant energetic switching by upregulation of glycolysis (ECAR). (C) In contrast, Pink1-KO cells have a decreased basal oxygen consumption rate (OCR) and sensitivity to inhibition of mitochondrial respiratory chain inhibitors; oligomycin (Arrow 1), uncoupler FCCP (Arrow 2) and rotenone and mxyothiazol (Arrow 4). (D) Loss of compensatory switch to glycolysis after oligomycin (Arrow 1) glucose bolus (Arrow 3) and 2-deoxy-glucose inhibitor 2-DG (Arrow 4) (n = 5). (E) Reduced mitochondrial oxygen consumption in Pink1 KO aortic tissue after high-fat diet both basally and specifically at complex I- and IV-dependent respiration. (F) Confocal images of mitochondrial VSMC α-SMA (green) (i–iv), mitochondrial abundance (red) (ii–v) with nuclear dapi (blue) co-localised combined channels to compare gross mitochondrial mass abundance between wild-type (Pink1+/+), control (upper) and Pink1 KO (lower) (n = 12) (scale 20 µm). One-way ANOVA with Dunnett’s post hoc test (n = 5), * p ≤ 0.05, *** p ≤ 0.001 vs. control.