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 4

Amphetamine-induced SIRT1 stabilization under hypoxia exaggerates caspase-3 activation and DNA damage (see also Supplemental Figure 3).

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Amphetamine-induced SIRT1 stabilization under hypoxia exaggerates caspas...
Pulmonary artery endothelial cells (PAECs) were treated daily with 0.5 mM amphetamine (AMPH) for 3 days and then cultured with vehicle (Veh) or AMPH under normoxia (Nx) or hypoxia (Hx) for (A) 48 hours or (B) 8 hours. Cells were (A) immunoblotted for acetylated histone 3 (H3K9Ac), histone 4 (H4K8Ac), and β-actin (for normalization) or (B) extracted for RNA and then screened for histone deacetylase (HDAC) expression via quantitative real-time PCR (qRT-PCR). qRT-PCR results are displayed as a heatmap with blue to yellow indicating low to high levels relative to Veh for each condition. (C) PAECs were treated as described in A, with or without sirtinol (10 μM) during the treatment. Cell lysates were immunoblotted for γH2AX, cleaved caspase-3 (cCasp3), and β-actin (loading control). (D) PAECs were transfected with 20 nM control (siCtl) or SIRT1 (siSIRT1) siRNA. Twenty-four hours after transfection, cells were treated as in A and analyzed for SIRT1, p-Akt, γH2AX, cCasp3, and β-actin (loading control). Dot plots represent mean ± SEM, n = 3–4. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001 vs. (siCtl or Vec)+Nx+Veh; ##P < 0.005, ####P < 0.0001 vs. (siCtl or Vec)+Hx+Veh; $$P < 0.005 vs. siSIRT1+Hx+Veh; &P < 0.05, &&&&P < 0.0005 vs. (siCtl or Vec)+Hx+AMPH; by 2-way ANOVA, Bonferroni’s post-test.