Cardiomyocyte Hypoxia-Reoxygenation Model

The HR injury model was mimicked in vitro by 45 min of hypoxia and 6 h of reoxygenation. The primary cardiomyocytes were isolated from WT and CK2α^CKO mice according to our previous study [44 (link)]. To inhibit the mitophagy, siRNA against FUNDC1 was transfected to cardiomyocytes isolated from WT mice. To observe the autophagic flux, Bafilomycin-A1 (0.5 μM, Selleck Chemicals) was used 12 h before treatment. Details on MTT assay, TUNEL staining and caspase3/9 activities are described in the supplemental methods. The siRNAs specific against the expression of CK2α and FUNDC1 or control siRNAs were purchased Santa Cruz Biotechnology. The siRNA transfection was based on our previous study [45 (link)], and the transfection efficiency was confirmed by western blots.

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Zhou H., Zhu P., Wang J., Zhu H., Ren J, & Chen Y. (2018). Pathogenesis of cardiac ischemia reperfusion injury is associated with CK2α-disturbed mitochondrial homeostasis via suppression of FUNDC1-related mitophagy. Cell Death and Differentiation, 25(6), 1080-1093.

Publication 2018

Bafilomycin-A1 control siRNAs

Assay Autophagic Bafilomycin a1 Cardiomyocytes Caspase3 Ck2α Hypoxia Inhibit Injury Mice Mitophagy Sirna Transfection Tunel Western blots

Corresponding Organization : University of Wyoming

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Protocol cited in 7 other protocols

Variable analysis

independent variables

Hypoxia duration (45 min)
Reoxygenation duration (6 h)
Transfection of siRNA against FUNDC1 in cardiomyocytes isolated from WT mice

dependent variables

Cell viability (MTT assay)
Apoptosis (TUNEL staining, caspase 3/9 activities)

control variables

Cardiomyocytes isolated from WT and CK2α^CKO mice
Treatment with Bafilomycin-A1 (0.5 μM, 12 h before) to observe autophagic flux

controls

Positive control: Cardiomyocytes isolated from WT mice
Negative control: Cardiomyocytes isolated from CK2α^CKO mice

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