Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension
Pin-I Chen, … , Brian J. Feldman, Marlene Rabinovitch
Pin-I Chen, … , Brian J. Feldman, Marlene Rabinovitch
Published January 26, 2017
Citation Information: JCI Insight. 2017;2(2):e90427. https://doi.org/10.1172/jci.insight.90427.
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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 7

In PAECs under hypoxia, amphetamine impairs the transition from oxidative phosphorylation to glycolysis.

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In PAECs under hypoxia, amphetamine impairs the transition from oxidativ...
(A) Pulmonary artery endothelial cells (PAECs) were treated with vehicle (Veh), 0.5 mM amphetamine (AMPH), or AMPH with 10 μM sirtinol, and placed in hypoxia (Hx) for 24 hours with the stimuli. Extracellular acidification rates (ECARs) and oxygen consumption rates (OCRs) were measured using kits (Seahorse Bioscience). (B) Glycolysis, glycolytic reserve, and glycolytic capacity as well as baseline respiration, maximal respiration, and spare respiratory capacity. (C) Representative live-cell images of PAECs treated as in A, loaded with 100 nM MitoTracker Green FM to stain mitochondria, and 5 μM MitoSOX Red to detect mitochondrial superoxide. Scale bar: 10 μm. (D) Fluorescence intensities of MitoTracker and MitoSOX were quantified using ImageJ, and ROS production was expressed as the ratio of MitoSOX to MitoTracker fluorescence. (E) PAECs were treated as in A. Mitochondrial membrane potential was determined using the JC-1 dye assay. (F) PAECs were treated with 0.5 mM AMPH, or AMPH with 10 μM of antimycin A (AMA), and then cultured with the stimuli under normoxia (Nx) or Hx for 48 hours. Cell lysates were immunoblotted for γH2AX, cCasp3, β-actin, and H2AX (loading controls). (A and F) Line and dot plots represent mean ± SEM, n = 3 independent experiments. (B, D, and E) 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 3 to 4 replicates per experiment. **P < 0.005, ***P < 0.0005 vs. Nx+Veh; #P < 0.05, ##P < 0.005, ###P < 0.0005, ####P < 0.0001 vs. Hx+Veh; &P < 0.05, &&P < 0.005, &&&P < 0.0005, &&&&P < 0.0001 vs. Hx+AMPH; by 2-way ANOVA, Bonferroni’s post-test.