Mito-TEMPO protects cardiomyocytes from cigarette smoke medium-induced inflammation and apoptosis in vitro

[Speaker] Yingmin Liang:1
[Co-author] Mary S. M. Ip:1, Judith C. W. Mak:1,2
1:Medicine, The University of Hong Kong, Hong Kong, 2:Pharmacology & Pharmacy, The University of Hong Kong, Hong Kong

Background: Smoking has been regarded as the major risk factor for cardiovascular diseases (CVD). Reactive oxygen species (ROS) production has been implicated in the promoting inflammation and inducing apoptosis in pathogenesis of cigarette smoke (CS)-induced CVD. The mitochondria are important sources of ROS in the heart. Therefore, the therapeutic strategies specifically targeting mitochondrial ROS may have benefits. This study was aimed to investigate the effects of mitochondria-targeted antioxidant Mito-TEMPO on cigarette smoke medium (CSM)-exposed human AC16 cardiomyocytes in vitro.
Methods: The AC16 cell line was cultured in DMEM/F12 containing 12.5% fetal bovine serum, in a CO2 incubator at 37 Celsius. CSM was prepared by bubbling smoke from two cigarettes into 20ml serum-free medium, which was regarded as 100%. After serum starvation with 1% fetal bovine serum for 24h, cells were pretreated with Mito-TEMPO (1 micromolar) for 30 mins before 4% CSM was added and incubated for an additional 24h. Supernatant was collected for determination of interleukin (IL)-8 by ELISA. Cells were collected to perform MitoSox Red assay to determine mitochondrial superoxide by flow cytometry. Apoptotic cells were measured with Annexin V apoptosis detection kit. Cell lysates were prepared for Western blot analysis.
Results: Exposure of AC16 cells to CSM for 24h significantly induced mitochondrial superoxide production, which was prevented by Mito-TEMPO. Treatment with Mito-TEMPO inhibited CSM-induced IL-8 release and decreased cell apoptosis in cardiomyocytes. Mechanistic study revealed that the beneficial effect of Mito-TEMPO were associated with inhibition of NF-kappaB phosphorylation.
Conclusions: Inhibition of mitochondrial ROS by Mito-TEMPO reduced CSM-induced inflammation and apoptosis via NF-kappaB pathway in cardiomyocytes in vitro. Thus, mitochondria-targeted antioxidants may be an effective therapy for smoking-related cardiac complications.

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