Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension
Pin-I Chen, Aiqin Cao, Kazuya Miyagawa, Nancy F. Tojais, Jan K. Hennigs, Caiyun G. Li, Nathaly M. Sweeney, Audrey S. Inglis, Lingli Wang, Dan Li, Matthew Ye, Brian J. Feldman, Marlene Rabinovitch
Pin-I Chen, Aiqin Cao, Kazuya Miyagawa, Nancy F. Tojais, Jan K. Hennigs, Caiyun G. Li, Nathaly M. Sweeney, Audrey S. Inglis, Lingli Wang, Dan Li, Matthew Ye, Brian J. Feldman, Marlene Rabinovitch
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Research Article Cell biology Vascular biology

Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension

Abstract

Amphetamine (AMPH) or methamphetamine (METH) abuse can cause oxidative damage and is a risk factor for diseases including pulmonary arterial hypertension (PAH). Pulmonary artery endothelial cells (PAECs) from AMPH-associated-PAH patients show DNA damage as judged by γH2AX foci and DNA comet tails. We therefore hypothesized that AMPH induces DNA damage and vascular pathology by interfering with normal adaptation to an environmental perturbation causing oxidative stress. Consistent with this, we found that AMPH alone does not cause DNA damage in normoxic PAECs, but greatly amplifies DNA damage in hypoxic PAECs. The mechanism involves AMPH activation of protein phosphatase 2A, which potentiates inhibition of Akt. This increases sirtuin 1, causing deacetylation and degradation of HIF1α, thereby impairing its transcriptional activity, resulting in a reduction in pyruvate dehydrogenase kinase 1 and impaired cytochrome c oxidase 4 isoform switch. Mitochondrial oxidative phosphorylation is inappropriately enhanced and, as a result of impaired electron transport and mitochondrial ROS increase, caspase-3 is activated and DNA damage is induced. In mice given binge doses of METH followed by hypoxia, HIF1α is suppressed and pulmonary artery DNA damage foci are associated with worse pulmonary vascular remodeling. Thus, chronic AMPH/METH can induce DNA damage associated with vascular disease by subverting the adaptive responses to oxidative stress.

Authors

Pin-I Chen, Aiqin Cao, Kazuya Miyagawa, Nancy F. Tojais, Jan K. Hennigs, Caiyun G. Li, Nathaly M. Sweeney, Audrey S. Inglis, Lingli Wang, Dan Li, Matthew Ye, Brian J. Feldman, Marlene Rabinovitch

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Figure 6

Stabilization of SIRT1 by amphetamine deacetylates and destabilizes HIF1α, impairing its transcriptional activity.

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Stabilization of SIRT1 by amphetamine deacetylates and destabilizes HIF1...
(A) Pulmonary artery endothelial cells (PAECs) were treated daily with vehicle (Veh) or the indicated dose of amphetamine (AMPH) for 3 days and then continued with vehicle or AMPH under normoxia (Nx) or hypoxia (Hx) for 48 hours. Cell lysates were immunoblotted for HIF1α and β-actin (for normalization). (B) PAECs were treated daily with 0.5 mM AMPH or AMPH with sirtinol (10 μM) for 3 days, and then cultured with the same stimuli under Nx or Hx for 24 hours. Cell lysates were immunoprecipitated with anti–acetyl-lysine antibody or IgG as negative control. Immunoprecipitates were probed for HIF1α to evaluate the levels of acetylated HIF1α. Whole-cell lysates were immunoblotted to determine the levels of total HIF1α and β-actin (loading control). A representative experiment is shown; 3 independent experiments were conducted with similar results. (C) PAECs were transfected with control or SIRT1 siRNAs, treated as in A and analyzed for HIF1α levels. (D) Cignal HIF firefly luciferase reporter and Renilla luciferase plasmids were cotransfected with control or SIRT1 siRNAs into PAECs. Twenty-four hours after transfection, cells were treated with 0.5 mM AMPH as in A and assessed for HIF activity, shown as the ratio of Firefly- to Renilla-luciferase-catalyzed light emission. (E and F) PAECs were treated as in B but with only 8 hours of Nx/Hx; RNA was extracted, and PDK1 and COX4I1 mRNA evaluated by qRT-PCR. (G) PAECs transfected and treated as in C were analyzed for PDK1 and COX4I1 protein levels. In C and G, β-actin was probed as a loading control, and SIRT1 to verify SIRT1 knockdown efficiency. (A, C, and G) Dot plots represent mean ± SEM, n = 3–4. (D, E, and F) Box-and-whisker plots represent values within the interquartile range (boxes) and the minimum to maximum (whiskers). The line within the box shows the median. n = 3 independent experiments with 2 to 3 replicates per experiment. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001 vs. Nx+Veh or siCtl+Nx+Veh; #P < 0.05, ##P < 0.005, ###P < 0.0005 vs. Hx+Veh or siCtl+Hx+Veh; &P < 0.05 vs. Hx+AMPH or siCtl+Hx+AMPH; by 2-way ANOVA, Bonferroni’s post-test.