DNA damage links mitochondrial dysfunction to atherosclerosis and the metabolic syndrome

Abstract

Rationale: DNA damage is present in both genomic and mitochondrial DNA in atherosclerosis. However, whether DNA damage itself promotes atherosclerosis, or is simply a byproduct of the risk factors that promote atherosclerosis, is unknown.

Objective: To examine the effect of DNA damage on atherosclerosis, we studied apolipoprotein (Apo)E(-/-) mice that were haploinsufficient for the protein kinase ATM (ataxia telangiectasia mutated), which coordinates DNA repair.

Methods and results: ATM(+/-)/ApoE(-/-) mice developed accelerated atherosclerosis and multiple features of the metabolic syndrome, including hypertension, hypercholesterolemia, obesity, steatohepatitis, and glucose intolerance. Transplantation with ATM(+/+) bone marrow attenuated atherosclerosis but not the metabolic syndrome. ATM(+/-) smooth muscle cells and macrophages showed increased nuclear DNA damage and defective DNA repair signaling, growth arrest, and apoptosis. Metabolomic screening of ATM(+/-)/ApoE(-/-) mouse tissues identified metabolic changes compatible with mitochondrial defects, with increased β-hydroxybutyrate but reduced lactate, reduced glucose, and alterations in multiple lipid species. ATM(+/-)/ApoE(-/-) mouse tissues showed an increased frequency of a mouse mitochondrial "common" deletion equivalent and reduced mitochondrial oxidative phosphorylation.

Conclusions: We propose that failure of DNA repair generates defects in cell proliferation, apoptosis, and mitochondrial dysfunction. This in turn leads to ketosis, hyperlipidemia, and increased fat storage, promoting atherosclerosis and the metabolic syndrome. Prevention of mitochondrial dysfunction may represent a novel target in cardiovascular disease.

Figure 1. ATM^+/−/ApoE^−/− mice have accelerated atherosclerosis.

A, Comparison of atherosclerotic lesions from ATM^+/+/ApoE^−/− (n=13) and ATM^+/−/ApoE^−/− mice (n=15). Descending aorta was stained for neutral lipid with oil red O (upper images). Scale bar is 1 mm. Lower images show aortic root section stained with H+E. Scale bars are 500 μm. B, Histogram of aortic root plaque area in ATM^+/+/ApoE^−/− and ATM^+/−/ApoE^−/− mice. C, Descending aorta (upper images) and aortic root sections (lower images) from ATM^+/+/ApoE^−/− (n=7) or ATM^+/−/ApoE^−/− mice (n=8), after ATM^+/+/ApoE^−/− BMT. D, Histogram of aortic root plaque area in transplanted mice.

Figure 2. ATM^+/−/ApoE^−/− mice show hyperlipidemia

A and B, Serum lipids in ATM^+/+/ApoE^−/− (black bars) (n=13) or ATM^+/−/ApoE^−/− (open bars) (n=15) mice fed either normal chow (A) or after 14 weeks of fat feeding (B). C and D, Serum lipids in ATM^+/+/ApoE^−/− or ATM^+/−/ApoE^−/− mice receiving ATM^+/+/ApoE^−/− BMT, fed either normal chow (n=7) (C) or after 14 weeks of fat feeding (n=8) (D).

Figure 3. ATM^+/−/ApoE^−/− mice show multiple features of the metabolic syndrome

A, Body weight for ATM^+/+/ApoE^−/− (n=10) and ATM^+/−/ApoE^−/− (n=6) mice after 14 weeks of high-fat feeding. B, Abdominal visceral fat pad from same experimental groups as in A. Scale bar is ≈4 cm. C, Representative (×20) images of white adipose tissue demonstrating adipocyte hypertrophy (upper images) and H and E-stained liver sections showing hepatic steatosis (lower images). Scale bar is 200 μm. D, Serum liver enzymes including alkaline phosphatase (AlkPhos), alanine transaminase (ALT), and aspartate transaminase (AST) in ATM^+/+/ApoE^−/− mice and ATM^+/−/ApoE^−/− mice. *P<0.001 (n=5). E, Fasted glucose tolerance test in ATM^+/+/ApoE^−/− mice and ATM^+/−/ApoE^−/− mice after 7 weeks of high-fat feeding (n=5). F, Insulin tolerance test in ATM^+/+/ApoE^−/− mice and ATM^+/−/ApoE^−/− mice after 7 weeks of high-fat feeding. All data are means; error bars represent SEMs. *P=<0.01 (n=5).

Figure 4. ATM^+/− cells demonstrate genomic instability, abnormal cell proliferation, and apoptosis

A, Micronuclei in macrophages and VSMCs from ATM^+/+/ApoE^−/− and ATM^+/−/ApoE^−/− mice (n=3). B, Quantification of DNA comet tails lengths by grade (inset) in ATM^+/+/ApoE^−/− and ATM^+/−/ApoE^−/− VSMCs. Cells were incubated for 1 hour with 10 μmol/L t-BHP and comet tails estimated 1 hour later (scale bar=20 μm), n=3. C and D, Percentage of ATM^+/+/ApoE^−/− and ATM^+/−/ApoE^−/− VSMCs undergoing proliferation (C) or apoptosis in ATM^+/+/ApoE^−/− and ATM^+/−/ApoE^−/− VSMCs and macrophages (D) assessed by time-lapse videomicroscopy over 24 hours, both basally and after exposure to 10 μmol/L t-BHP (n=3). E, Immunocytochemistry of DNA damage foci expression of phospho-ATM (red) and γ-H2AX (green) in ATM^+/+/ApoE^−/− and ATM^+/−/ApoE^−/− VSMCs after 10 μmol/L t-BHP for 1 hour and 1-hour recovery. Nuclei are counterstained with DAPI (blue). Scale bar is 20 μm. F, Western blot of ATM^+/+/ApoE^−/− and ATM^+/−/ApoE^−/− VSMCs basally (0 hour), after 1 hour of treatment with 10 μmol/L t-BHP (1 hour), and after 1 hour of recovery (2 hours) or ATM^+/+/ApoE^−/− and ATM^+/−/ApoE^−/− macrophages after 1 hour of treatment with 10 μmol/L t-BHP and 1 hour of recovery.

Figure 5. ATM^+/− mice show a defect in oxidative phosphorylation on metabolomic screening

A, High-resolution 500 MHz ¹H-NMR spectra of liver extracts from ATM^+/+/ApoE^−/− and ATM^+/−/ApoE^−/− mice. Top, β-Hydroxybutyrate, lactate, and alanine peaks are labeled. Bottom, Multiple peaks show the glucose region in NMR spectra. B, β-Hydroxybutyrate concentrations in liver, pancreas, and plasma of ATM^+/−/ApoE^−/− mice. Data are shown as relative changes in ATM^+/+/ApoE^−/− compared with ATM^+/+/ApoE^−/− mice (means±SEM). *P<0.05, **P<0.01. C, Major lipid changes in the liver and pancreas. Data are shown as relative changes in ATM^+/+/ApoE^−/− compared with ATM^+/+/ApoE^−/− mice (means±SEM). *P<0.05, **P<0.01, ***P<0.001. Black bars indicate ATM^+/+/ApoE^−/−; unfilled bars, ATM^+/−/ApoE^−/−.

Figure 6. ATM^+/− mitochondria have increased ROS production and MtDNA damage

A, ROS production assayed using the ROS-sensitive fluorochrome DCFDA in ATM^+/+ and ATM^+/− VSMCs. B, MtDNA adducts determined by semiquantitative PCR. C, Degree of heteroplasmy in tissues from ATM^+/−/ApoE^−/− and ATM^+/+/ApoE^−/− mice after 14 weeks of high-fat feeding. D, Fold increase in mitochondrial common mutation in tissues from ATM^+/−/ApoE^−/− relative to ATM^+/+/ApoE^−/− mice. BAT indicates brown adipose tissue; WAT, white adipose tissue. E, Quantitative fluorescent Western blot of liver mitochondrial respiratory complex proteins relative to the nuclear-encoded mitochondrial protein MnSOD. F, Liver mitochondrial complex I respiratory activity normalized to the nuclear-encoded mitochondrial protein citrate synthase.

Figure 7

Proposed model of increased atherosclerosis and metabolic syndrome in ATM^+/−/ApoE^−/− mice.