Downregulation of the Cd38-Cyclic ADP-Ribose Signaling in Cardiomyocytes by Intermittent Hypoxia via Pten Upregulation

Abstract

Sleep apnea syndrome (SAS) is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia, IH), and it is a risk factor for cardiovascular disease (CVD) and insulin resistance/type 2 diabetes. However, the mechanisms linking IH stress and CVD remain elusive. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes to experimental IH or normoxia for 24 h to analyze the mRNA expression of the components of Cd38-cyclic ADP-ribose (cADPR) signaling. We found that the mRNA levels of cluster of differentiation 38 (Cd38), type 2 ryanodine receptor (Ryr2), and FK506-binding protein 12.6 (Fkbp12.6) in H9c2 and P19.CL6 cardiomyocytes were significantly decreased by IH, whereas the promoter activities of these genes were not decreased. By contrast, the expression of phosphatase and tensin homolog deleted from chromosome 10 (Pten) was upregulated in IH-treated cells. The small interfering RNA for Pten (siPten) and a non-specific control RNA were introduced into the H9c2 cells. The IH-induced downregulation of Cd38, Ryr2, and Fkbp12.6 was abolished by the introduction of the siPten, but not by the control RNA. These results indicate that IH stress upregulated the Pten in cardiomyocytes, resulting in the decreased mRNA levels of Cd38, Ryr2, and Fkbp12.6, leading to the inhibition of cardiomyocyte functions in SAS patients.

Keywords: Cd38; Fkbp12.6; Pten; Ryr2; cardiomyocytes; intermittent hypoxia; sleep apnea syndrome.

Figure 1

The mRNA levels of mouse (A) Cd38, (B) Ryr2, and (C) Fkbp12.6 in mouse P19.CL6 cardiomyocytes. Cardiomyocytic-differentiated mouse P19.CL6 cells were treated with normoxia or IH for 24 h. The mRNA levels were measured by real-time RT-PCR and normalized by rat insulinoma gene (Rig)/ribosomal protein S15 (RpS15) as an internal standard. The mRNA level exposed to normoxia was set to 1.0. Data are expressed as the mean ± SE of the samples (n = 4 to 6). Statistical analyses were performed using Student’s t-test. IH significantly decreased the mRNA levels of Cd38, Ryr2, and Fkbp12.6 in mouse P19.CL6 cells. In addition, correlation analyses revealed that the correlation coefficient(s) between Cd38 vs. Ryr2, Cd38 vs. Fkbp12.6, and Ryr2 vs. Fkbp12.6 were 0.618, 0.912, and 0.566, respectively, indicating that there are positive correlation(s).

Figure 2

The mRNA levels of rat (A) Cd38, (B) Ryr2, and (C) Fkbp12.6 in rat H9c2 cardiomyocytes. Rat H9c2 cells were treated with normoxia or IH for 24 h. The mRNA levels were measured by real-time RT-PCR and normalized by Rig/RpS15 as an internal standard. The mRNA level exposed to normoxia was set to 1.0. Data are expressed as the mean ± SE of the samples (n = 4 to 6). Statistical analyses were performed using Student’s t-test. IH significantly decreased the mRNA levels of Cd38, Ryr2, and Fkbp12.6 in rat H9c2 cells.

Figure 3

Relative protein expression levels of (A) Cd38, (B) Ryr2, and (C) Fkbp12.6 in rat H9c2 cardiomyocytes subjected to IH. The Cd38, Ryr2, and Fkbp12.6 band densities were quantified through image analysis and then normalized to β-actin, as measured in the same blot. Each bar represents the mean of six independent measurements. The relative expression levels of the Cd38, Ryr2, and Fkbp12.6 are arbitrarily presented. The protein level exposed to normoxia was set to 1.0. The results are expressed as the mean ± SE in arbitrary units. A representative immunoblot with the apparent molecular weight is shown in the upper panel.

Figure 4

Luciferase assays of the promoter activities of (A) CD38, (B) RYR2, and (C) FKBP12.6 in H9c2 cardiomyocytes. Reporter plasmids, prepared by inserting the promoter fragments of human CD38 (−3187~+269), human RYR2 (−1250~+10), and human FKBP12.6 (−1696~+109) upstream of a firefly luciferase reporter gene in pGL4.17 vector, were transfected into rat H9c2 cells. After the cells were exposed either to IH or to normoxia for 24 h, the cells were lysed and the promoter activities of CD38, RYR2, and FKBP12.6 were measured. The promoter activity was normalized for variations in transfection efficiency, with β-galactosidase activity as an internal standard and the promoter activity of cells exposed to normoxia was set to 1.0. Data are presented as the mean ± SE of the samples (n = 5 to 6) and were analyzed using Student’s t-test.

Figure 5

The mRNA levels of Pten in rat H9c2 (left) and mouse P19.CL6 (right) cardiomyocytes. Rat H9c2 and cardiomyocytic-differentiated mouse P19.CL6 cells were treated with normoxia or IH for 24 h. The mRNA levels were measured by real-time RT-PCR and normalized by Rig/RpS15 as an internal standard. The mRNA level exposed to normoxia was set to 1.0. Data are expressed as mean ± SE of the samples (n = 4 to 6). Statistical analyses were performed using Student’s t-test. IH significantly increased the mRNA levels of Pten in rat H9c2 and mouse P19.CL6 cardiomyocytes. Correlation analysis in P19.CL6 cells revealed that the correlation coefficient(s) between Cd38 vs. Pten and Fkbp12.6 vs. Pten were −0.221 and −0.362, respectively, indicating that there are negative correlation(s).

Figure 6

Relative protein expression of Pten in rat H9c2 myocytes subjected to IH. The Pten band densities were quantified through image analysis and then normalized to β-actin, as measured in the same blot. The protein level exposed to normoxia was set to 1.0. Each bar represents the mean value of six independent experiments (n = 6). The relative expression of the Pten is arbitrarily presented. The results are expressed as mean ± SE in arbitrary units. A representative immunoblot with the apparent molecular weight is also shown in the right panel.

Figure 7

Effects of siPten on the IH-induced gene expression of Cd38, Ryr2, and Fkbp12.6. SiPten and scrambled RNA (control) were transfected into H9c2 cardiomyocytes, which in turn were subjected to IH or normoxia for 24 h. The mRNA levels of the Cd38, Ryr2, and Fkbp12.6 were measured via real-time RT-PCR, with Rig/RpS15 as the endogenous control. The mRNA level exposed to normoxia was set to 1.0. Data are expressed as the mean ± SE of six independent experiments (n = 6). Student’s t-test was used in statistical analyses.

Figure 8

The mRNA levels of Cd38, Ryr2, and Fkbp12.6 in the presence or absence of 3-deaza-cADPR. No differences in Ryr2 and Fkbp12.6 mRNA levels were observed under normoxia and under IH in the presence of 3-deaza-cADPR (0.6979-fold decrease in 3-deaza-cADPR (+) (p = 0.5260) and 1.076-fold increase in 3-deaza-cADPR (+) (p = 0.5501), respectively). By contrast, the Cd38 mRNA level under IH increased by 1.214-fold relative to that under normoxia in the presence of 3-deaza-cADPR (p = 0.0211). Although the mRNA levels of Cd38, Ryr2, and Fkbp12.6 decreased in response to IH in the absence of 3-deaza-cADPR (3-deaza-cADPR (−) controls), this trend was not observed following the addition of the 3-deaza-cADPR (3-deaza-cADPR (+)). The mRNA level exposed to normoxia was set to 1.0. Data are expressed as the mean ± SE of six independent experiments (n = 6). Student’s t-test was employed in statistical analyses.