Arteriosclerosis, Thrombosis, and Vascular Biology Mitochondrial Ca 2+ Uptake Drives Endothelial Injury By Radiation Therapy

BACKGROUND: Radiation therapy strongly increases the risk of atherosclerotic vascular disease, such as carotid stenosis. Radiation induces DNA damage, in particular in mitochondria, but the upstream and downstream signaling events are poorly understood. The objective of this study was to define such mechanisms. METHODS: Endothelial-specific MCU (mitochondrial Ca 2+ uniporter) knockout and C57Bl6/J mice with or without a preinfusion of a mitoTEMPO (mitochondrial reactive oxygen species [ROS] scavenger) were exposed to a single dose of cranial irradiation. 24, and 240 hours postirradiation, vascular reactivity, endothelial function, and mitochondrial integrity were assessed ex vivo and in vitro. RESULTS: In cultured human endothelial cells, irradiation with 4 Gy increased cytosolic Ca 2+ transients and the mitochondrial Ca 2+ concentration ([Ca 2+ ] mt ) and activated MCU. These outcomes correlated with increases in mitochondrial ROS ( mt ROS), loss of NO production, and sustained damage to mitochondrial but not nuclear DNA. Moreover, irradiation impaired activity of the ETC (electron transport chain) and the transcription of ETC subunits encoded by mitochondrial DNA ( mt DNA). Knockdown or pharmacological inhibition of MCU blocked irradiation-induced mt ROS production, mt DNA damage, loss of NO production, and impairment of ETC activity. Similarly, the pretreatment with mitoTEMPO, a scavenger of mt ROS, reduced irradiation-induced Ca 2+ entry, and preserved both the integrity of the mt DNA and the production of NO, suggesting a feed-forward loop involving [Ca 2+ ] m and mt ROS. Enhancement of DNA repair in mitochondria, but not in the nucleus, was sufficient to block prolonged mt ROS elevations and maintain NO production. Consistent with the findings from cultured cells, in C57BL/6J mice, head and neck irradiation decreased endothelium-dependent vasodilation, and mt DNA integrity in the carotid artery after irradiation. These effects were prevented by endothelial knockout of MCU or infusion with mitoTEMPO. CONCLUSIONS: Irradiation-induced damage to mt DNA is driven by MCU-dependent Ca 2+ influx and the generation of mt ROS. Such damage leads to reduced transcription of mitochondrial genes and activity of the ETC, promoting sustained mt ROS production that induces endothelial dysfunction. Our findings suggest that targeting MCU and mt ROS might be sufficient to mitigate irradiation-induced vascular disease. GRAPHIC ABSTRACT: A graphic abstract is available for this article. high prevalence of atherosclerotic vascular nm, and its emission was recorded at 535 nm. Recordings were performed every 5 s for at least 10 minutes. Real-time Pericam fluorescence ratios were recorded after PDGF (platelet-derived growth factor) was added (20 ng/ mL) and were quantified using ImageJ. Peak amplitude (R) was calculated by subtracting the baseline fluorescence ratio from the peak fluorescence ratio. The AUC was determined by subtracting the AUC value obtained in the absence of agonist over the same recoding period. Summary data represent the average difference in basal and peak mitochondrial [Ca 2+ ] ([Ca 2+ ] mt ). Kruskal-Wallis test and Dunn post hoc test were used for data sets where normal distribution could not be assumed. Two-tailed unpaired Student t test and 1-way ANOVA, followed by Tukey multiple comparison test, were used for data sets with normal distribution. Two-way ANOVA followed by Tukey multiple comparison test was used for grouped data sets. A P <0.05 was considered significant.