Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for cardiovascular disease (CVD) and insulin resistance. However, the mechanisms linking IH stress and CVD remain elusive. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes either to sustained hypoxia (1% O2 [SH]), 70 cycles/24 h of IH (5 min SH/10 min normoxia [21% O2]), mimicking SAS patients, or normoxia for 24 h, analyzed mRNA expression and found that IH significantly increased the mRNA levels of regenerating gene (Reg) IV and hepatocyte growth factor (Hgf). Reg IV and Hgf in IH-treated cell medium were also increased. In order to analyze the IH-induced expression mechanisms of Reg IV and Hgf genes, we prepared reporter plasmids containing Reg IV and Hgf upstream of luciferase reporter gene, and transfected them into P19.CL6 cardiomyocytes. The promoter activities of the genes were not increased by IH. Target mRNA search of microRNA (miR)s revealed that both the mRNAs have a potential target sequence for miR-499. The miR-499 level of IH-treated cells was significantly decreased than that of normoxia-treated cells. To clarify the role of miR-499, miR-499 mimic and non-specific control RNA (miR-499 mimic NC) were introduced into P19.CL6 cells, and the IH-induced up-regulation of Reg IV and Hgf was abolished by introduction of miR-499 mimic. We also measured apoptosis of IH/SH-stimulated P19.CL6 cells by TUNEL method and found that SH but not IH increased apoptosis and that Reg IV and Hgf in cultured medium significantly reduced. The addition of Reg IV and/or Hgf in the culture medium attenuated the SH-induced apoptosis. These results indicate that IH stress increased levels of Reg IV and Hgf mRNA via the inhibition of the miR-499-mediated mRNA degradation, leading to protect cardiomyocytes against hypoxia -induced apoptosis.


S. Takasawa: None. A. Itaya-hironaka: None. A. Yamauchi: None. M. Makino: None. S. Sakuramoto-tsuchida: None. T. Uchiyama: None. Y. Takeda: None. Y. Kyotani: None. H. Ota: None.

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