Mitochondrial Respiration Is Reduced in Atherosclerosis, Promoting Necrotic Core Formation and Reducing Relative Fibrous Cap Thickness

Abstract

Objective: Mitochondrial DNA (mtDNA) damage is present in murine and human atherosclerotic plaques. However, whether endogenous levels of mtDNA damage are sufficient to cause mitochondrial dysfunction and whether decreasing mtDNA damage and improving mitochondrial respiration affects plaque burden or composition are unclear. We examined mitochondrial respiration in human atherosclerotic plaques and whether augmenting mitochondrial respiration affects atherogenesis.

Approach and results: Human atherosclerotic plaques showed marked mitochondrial dysfunction, manifested as reduced mtDNA copy number and oxygen consumption rate in fibrous cap and core regions. Vascular smooth muscle cells derived from plaques showed impaired mitochondrial respiration, reduced complex I expression, and increased mitophagy, which was induced by oxidized low-density lipoprotein. Apolipoprotein E-deficient (ApoE^-/-) mice showed decreased mtDNA integrity and mitochondrial respiration, associated with increased mitochondrial reactive oxygen species. To determine whether alleviating mtDNA damage and increasing mitochondrial respiration affects atherogenesis, we studied ApoE^-/- mice overexpressing the mitochondrial helicase Twinkle (Tw⁺/ApoE^-/-). Tw⁺/ApoE^-/- mice showed increased mtDNA integrity, copy number, respiratory complex abundance, and respiration. Tw⁺/ApoE^-/- mice had decreased necrotic core and increased fibrous cap areas, and Tw⁺/ApoE^-/- bone marrow transplantation also reduced core areas. Twinkle increased vascular smooth muscle cell mtDNA integrity and respiration. Twinkle also promoted vascular smooth muscle cell proliferation and protected both vascular smooth muscle cells and macrophages from oxidative stress-induced apoptosis.

Conclusions: Endogenous mtDNA damage in mouse and human atherosclerosis is associated with significantly reduced mitochondrial respiration. Reducing mtDNA damage and increasing mitochondrial respiration decrease necrotic core and increase fibrous cap areas independently of changes in reactive oxygen species and may be a promising therapeutic strategy in atherosclerosis.

Keywords: atherosclerosis; mitochondria; reactive oxygen species; respiration; vascular smooth muscle.

Figure 1.

Human atherosclerosis shows reduced mitochondrial copy number and respiration. A, Mitochondrial copy number in normal human aorta and plaque (n=9). B and C, Seahorse profile for oxygen consumption rate (OCR) in human plaque segments from the media, shoulder, cap, and core with treatment with oligomycin (oligo), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and antimycin/rotenone (A/R; B) and respiratory reserve capacity (RRC; C; n=5–6). Representative Seahorse profile of human plaque vascular smooth muscle cells (VSMCs; blue) and normal aortic VSMCs (red; D) and OCR after FCCP (E) from n=5 to 7 cultures. F, Western blot and quantification of protein expression of mitochondrial protein complexes within the electron transport chain (complexes I–V) in normal human aortic or plaque VSMCs (n=3–5) relative to citrate synthase (CS). Data are mean±SEM. mtDNA indicates mitochondrial DNA.

Figure 2.

Mitophagy is increased by oxidized low-density lipoprotein (ox-LDL) and in plaque vascular smooth muscle cells (VSMCs). A, Confocal microscopic images of control normal human aortic or plaque VSMCs expressing mitochondrially targeted keima, either untreated or after treatment with 100 mg/mL ox-LDL or 5 mmol/L carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). Insets show high-power views of outlined areas. B and C, Red/green keima ratios for individual normal aortic VSMCs either untreated (C) or treated with 100 mg/mL ox-LDL for 4 to 24 h, 100 mg/mL native LDL for 24 h, or 5 mmol/L FCCP (B), or individual normal aortic VSMCs (C) or human plaque VSMCs either untreated or treated with ox-LDL or FCCP for 24 h (C). n≥20 images from 3 biological replicates. Western blots (D) and their quantification (E) for proteins involved in mitochondrial DNA synthesis and mitophagy in human aortic or plaque VSMCs, normalized to actin (n=3). PINK1 indicates PTEN-induced putative kinase 1; PolG, polymerase-γ; and TFAM, mitochondrial transcription factor A.

Figure 3.

Mitochondrial DNA (MtDNA) damage reduced mitochondrial respiration and increased ROS accumulation during murine atherosclerosis development. Six-wk-old chow-fed apolipoprotein E–deficient (ApoE^−/−) mice were compared with ApoE^−/− mice fat fed from 6 to 20 wk (n=4–7) and aortas examined for (A) mtDNA adducts and (B) mitochondrial complex or citrate synthase (CS) abundance. Representative Western blot (left) and quantification (right). Complex I- (C) and IV-supported respiration (D). E, Reactive oxygen species as measured by MitoP/B ratio in aortas. F, Representative Western blot (left) and quantification (right) of aortic manganese superoxide dismutase (MnSOD) abundance at 6 and 20 wk. G, mtDNA adducts in ApoE^−/− vascular smooth muscle cells isolated from 12- to 16-week-old mice with or without treatment with 100 μg/mL oxidized low-density lipoprotein (ox-LDL) for 24 h (n=8 biological replicates). Tub indicates tubulin.

Figure 4.

Twinkle increases mitochondrial respiration in aorta, vascular smooth muscle cells (VSMCs), and macrophages. A, Ex vivo respirometry for complex I-supported respiration in aortas from control apolipoprotein E–deficient (ApoE^−/−) or Tw⁺/ApoE^−/− mice (n=6–8) after 14-wk high-fat diet (HFD). Representative Seahorse profiles for oxygen consumption rate (OCR; B and D) or OCR after cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; C and E) in VSMCs (B and C) or macrophages (D and E) from control ApoE^−/− or Tw⁺/ApoE^−/− mice (n=6–9). After 14-wk HFD, reactive oxygen species were measured by MitoP/B ratio in aortas, hearts, and livers (F) or dichlorodihydrofluorescein diacetate (DCFDA) relative fluorescence units (RFU) in VSMCs (G) or macrophages (H) from control ApoE^−/− or Tw⁺/ApoE^−/− mice. Cultured VSMCs and macrophages were also treated with lipopolysaccharide (LPS) or tert-butyl hydroperoxide (t-BHP). n=4 to 5. Tw indicates Twinkle helicase.

Figure 5.

Twinkle mice show reduced necrotic core and increased fibrous cap areas. Histochemistry and immunohistochemistry of aortic root plaques of control apolipoprotein E–deficient (ApoE^−/−) and Tw⁺/ApoE^−/− mice after 14 wk of fat feeding. Sections were stained with Masson’s trichrome or antibodies to α-smooth muscle actin (α-SMA) or ki67 or underwent TUNEL. Scale bar: low power, 500 μm; high power, 100 μm. Tw indicates Twinkle helicase.

Figure 6.

Twinkle mouse plaques show reduced necrotic core and increased fibrous cap areas. Aortic root plaque % (A), necrotic core, and fibrous cap % areas (B) in control apolipoprotein E–deficient (ApoE^−/−) and Tw⁺/ApoE^−/− mice after fat feeding from 6 to 20 wk (n=11–18). Aortic root plaque % (C), necrotic core, and fibrous cap % areas (D) in ApoE^−/− mice transplanted with control ApoE^−/− or Tw⁺/ApoE^−/− bone marrow at 6 wk and fat feeding from 6 to 20 w (n=8–10). E, Cell number at day 0 and day 4 in vascular smooth muscle cells (VSMCs) derived from control ApoE^−/− and Tw⁺/ApoE^−/− mice (n=3). Apoptosis at baseline and after 50 μmol/L tert-butyl hydroperoxide (t-BHP) for 16 h in VSMCs (F) and macrophages (G) from control ApoE^−/− and Tw⁺/ApoE^−/− mice (n=4–5). BMT indicates bone marrow transplant; and Tw, Twinkle helicase.