Protective role of retinoid X receptor in H9c2 cardiomyocytes from hypoxia/reoxygenation injury in rats

Abstract

BACKGROUND: Retinoid X receptor (RXR) plays a central role in the regulation of intracellular receptor signaling pathways. The activation of RXR has protective effect on H 2 O 2 -induced apoptosis of H9c2 ventricular cells in rats. But the protective effect and mechanism of activating RXR in cardiomyocytes against hypoxia/reoxygenation (H/R)-induced oxidative iniury are still unclear. METHODS:The model of H/R injury was established through hypoxia for 2 hours and reoxygenation for 4 hours in H9c2 cardiomyocytes of rats. 9-cis-retinoic acid (9-cis RA) was obtained as an RXR agonist, and HX531 as an RXR antagonist. Cultured cardiomyocytes were randomly divided into four groups: sham group, H/R group, H/R+9-cis RA -pretreated group (100 nmol/L 9-cis RA), and H/R+9-cis RA+HX531-pretreated group (2.5 μmol/L HX531). The cell viability was measured by MTT, apoptosis rate of cardiomyocytes by flow cytometry analysis, and mitochondrial membrane potential (ΔΨm) by JC-1 fluorescent probe, and protein expressions of Bcl-2, Bax and cleaved caspase-9 with Western blotting. All measurement data were expressed as mean±standard deviation, and analyzed using one-way ANOVA and the Dunnett test. Differences were considered signifi cant when P was <0.05. RESULTS:Pretreatment with RXR agonist enhanced cell viability, reduced apoptosis ratio, and stabled ΔΨm. Dot blotting experiments showed that under H/R stress conditions, Bcl-2 protein level decreased, while Bax and cleaved caspase-9 were increased. 9-cis RA administration before H/R stress prevented these effects, but the protective effects of activating RXR on cardiomyocytes against H/R induced oxidative injury were abolished when pretreated with RXR pan-antagonist HX531. CONCLUSION:The activation of RXR has protective effects against H/R injury in H9c2 cardiomyocytes of rats through attenuating signaling pathway of mitochondria apoptosis.

DNA Methylation Changes Are Associated With an Incremental Ascent to High Altitude

“…The RXRa pathway was altered by hypobaric hypoxia exposure in the rat brain (Sethy et al, 2011). RXRs play a protective role in H9c2 cardiomyocytes from hypoxia/reoxygenation–induced oxidative injury in rats (Shan et al, 2014). We found decreased methylation of the CpG island located in the promoter region of RXRa , which may be associated with increased expression of RXRa .…”

Oxidative stress does not play a primary role in the toxicity induced with clinical doses of doxorubicin in myocardial H9c2 cells

“…Moreover, while the administration of all-trans-Retinoic acid during the differentiation forces the cells to acquire a cardiac muscle phenotype [ 20 ], the cell population still remains heterogeneous that makes fastidious any quantitative analyses as each phenotype requires to be discriminated. To overcome this issue, we opted for a short-term differentiation [ 29 ]. Cells were differentiated up to two days prior to analyses to be performed.…”

“…As the adult heart tissue contains mostly cardiac cells that lost their proliferative activity, i.e., differentiated cardiomyocytes, we induced the cell differentiation to be in line with the in vivo conditions. Therefore, cells were deprived of serum by diminishing the FBS to 1 % [ 20 ] to arrest their proliferation prior to the experiments to be performed in accordance with [ 29 ]. 24 h later, cells were treated with doxorubicin (DOX; SigmaAldrich) up to 24 h prior analyses.…”

“…We selected the H9c2 cell line as in vitro cardiac cell model. However, to stay in compliance with the differentiated state of the cardiomyocytes in vivo, we performed experiments on differentiated H9c2 cells as previously reported [ 29 ]. We assessed several functional and morphological features to highlight the cell death mechanisms linked to DOX cardiotoxicity.…”

Retinoid X Receptor Antagonists

“…Huang et al used 12 as a tool to show that activation of RXR has a protective effect against hypoxia-reoxygenation disorder in H9c2 cardiomyocytes [ 92 ]. Franklin and colleagues revealed that phagocytosis and remyelination of myelin debris accompanying aging progressed upon activation of RXR using 12 [ 93 ].…”