Genetic Reduction of DNA Damage Repair Protein ATM Kinase Attenuates Endothelium‐Dependent Dilation and NO Bioavailability via Enhanced Superoxide in Old but not Young Mice

Abstract

Advanced age is associated with accumulated DNA damage and disruption of selective DNA damage repair pathways which together can accelerate the aging phenotype. In the vasculature, aging is characterized by impaired endothelium‐dependent dilation (EDD) and reduced nitric oxide (NO) bioavailability. Activation of ataxia telangiectasia mutated (ATM) kinase, a key mediator of the response to double‐stranded DNA (dsDNA) breaks, leads to upregulation of its effectors p53 and p21. Importantly, expression of these proteins is negatively correlated to EDD and has been demonstrated to be increased in the endothelium with aging. While these findings suggest that dsDNA breaks impair EDD, it is unknown if alterations in ATM per se modulate EDD. We hypothesized that genetic deletion of ATM would impair endothelial function in young mice (4–6 mo, n=7–9/group) leading to a vascular phenotype that phenocopies aging. In young mice, EDD in response to acetylcholine (Ach) in isolated mesenteric arteries was similar between young ATM +/+ and ATM −/− mice in the absence or presence of the NO synthase inhibitor, L‐NAME (both p>0.05) (Fig 1). Endothelium‐independent dilation (EID) to sodium nitroprusside (SNP) was also not different between young ATM +/+ and ATM −/− mice (p>0.05). Thus, contrary to our hypothesis, EDD and NO were preserved despite ATM deletion, suggesting that an insufficient accumulation of DNA damage may have occurred in young mice to impair endothelial function. We next hypothesized that an additional genomic stressor, such as aging, may be necessary to uncover the effects of ATM deficiency on endothelial function. However, ATM −/− mice die at ~6 mo due to malignant thymic lymphomas, therefore, to explore the effects of an age‐related accumulation of DNA damage on endothelial function, we assessed Ach‐mediated EDD in mesenteric arteries of aged (18–19 mo) ATM +/+ and ATM +/− mice (n=5–13). While aging alone (4–6 vs. 18–19 mo, ATM +/+) only tended to reduce EDD (p=0.1), EDD was significantly blunted in old ATM +/− compared to age‐matched ATM +/+ (p<0.01) (Fig 2A). This impairment in EDD resulted from decreased NO bioavailability, evidenced by a ~35% reduction in NO‐dependent vasodilation in aged ATM +/− compared to ATM +/+ mice (p<0.01) (Fig 2B). EID to SNP did not differ between groups (p>0.05). To determine if increases in reactive oxygen species may underlie blunted EDD/NO in aged ATM +/− mice, EDD was assessed in the presence of the superoxide scavenger, TEMPOL. TEMPOL restored EDD in aged ATM +/− mice (p<0.05) to levels not different from young mice (p>0.05). Here, we provide novel evidence that although ATM deficiency alone is insufficient to impair EDD in young mice, in the face of a known genomic stressor like aging, ATM deficiency leads to impaired EDD and reduced NO via increased superoxide. These findings support the concept that accumulation of DNA damage and genomic instability with advanced age impairs endothelial function.Support or Funding InformationFunding Information: Funded in part by awards from the NIH, R01 AG048366, R01 AG050238, R01 AG060395, K99 AT010017, T32 5T32HL139451‐02, and by US Department of Veterans Affairs I01BX004492ATM kinase deletion does not impair endothelium dependent dilation (EDD) or nitric oxide (NO) bioavailability in mesenteric arteries of young mice.EDD was assessed in response to increasing concentrations of acetylcholine (Ach) in isolated mesenteric arteries from young (4–6 mo) mice lacking ATM (ATM −/−) or wildtype (ATM +/+) littermate controls. To determine the contribution of NO to dilation, Ach responses were repeated in the presence of the NO synthase inhibitor, L‐NAME. †, difference after L‐NAME. p<0.05. Data are means ± SEM.Figure 1ATM kinase deficiency impairs endothelium dependent dilation (EDD) and nitric oxide (NO) bioavailability in mesenteric arteries of aged mice.(A) EDD was assessed in response to increasing concentrations of acetylcholine (Ach) in isolated mesenteric arteries from aged (18–19 mo) mice heterozygous for ATM (ATM +/−) and wildtype (ATM +/+) controls. Ach dose‐responses were repeated in the presence of the NO synthase inhibitor, L‐NAME. (B) NO‐dependent vasodilation, i.e. the difference in maximal dilation to Ach in the absence or presence of L‐NAME, was calculated. *, difference from ATM+/+. †, difference after L‐NAME. p<0.05. Data are means ± SEM.Figure 2