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

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.

Observational studies have indicated that SAS is associated with a high risk for serious hypertension [19]. IH-induced cardiomyocyte damage occurs due to the increases in intracellular reactive oxygen species during reoxygenation after hypoxia [20][21][22][23]. Moreover, IH may cause lipid peroxidation [24], protein oxidation, DNA damage [25], and attenuation of antioxidant enzyme capacity, reducing the number of cardiomyocytes and 2. Results 2.1. The Gene Expression Levels of Cd38, Ryr2, and Fkbp12.6 in Cardiomyocytes Were Decreased by IH We exposed mouse P19.CL6 and rat H9c2 cardiomyocytes to normoxia or IH for 24 h. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we measured the mRNA levels of Cd38 (which encodes ADP-ribosyl cyclase/cyclic ADP-ribose [cADPR] hydrolase, EC 3.2.2.6), Ryr2, and Fkbp12.6 (a cADPR receptor [Fkbp1b]) in mouse P19.CL6 and rat H9c2 cells. As shown in Figures 1 and 2, IH significantly decreased the mRNA levels of Cd38, Ryr2, and Fkbp12.6 in mouse P19.CL6 and rat H9c2 cardiomyocytes, respectively. 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).  6 in rat H9c2 cardiomyocytes. Rat H9c2 cells were treated with normoxia or IH for 24 h. The mRNA levels were measured by realtime 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.
Furthermore, we measured the Cd38, Ryr2, and Fkbp12.6 protein levels in the H9c2 cells using Western blot analyses, and the results showed that the protein levels were significantly decreased by IH (p = 0.0205, p = 0.0241, and p = 0.0078, respectively) ( Figure  3). 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).  Rat H9c2 cells were treated with normoxia or IH for 24 h. The mRNA levels were measured by realtime 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.
Furthermore, we measured the Cd38, Ryr2, and Fkbp12.6 protein levels in the H9c2 cells using Western blot analyses, and the results showed that the protein levels were significantly decreased by IH (p = 0.0205, p = 0.0241, and p = 0.0078, respectively) ( Figure  3). 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.
Furthermore, we measured the Cd38, Ryr2, and Fkbp12.6 protein levels in the H9c2 cells using Western blot analyses, and the results showed that the protein levels were significantly decreased by IH (p = 0.0205, p = 0.0241, and p = 0.0078, respectively) ( Figure 3).  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.

The Promoter Activities of Cd38, Ryr2, and Fkbp12.6 Were Not Decreased by IH
To determine whether the IH-induced decreases in the Cd38, Ryr2, and Fkbp12.6 mRNA levels were caused by the inactivation of transcription, we fused a 3456 bp fragment containing 3187 bp of the human CD38 promoter, a 1260 bp fragment containing 1250 bp of the human RYR2 promoter, and a 1805 bp fragment containing 1696 bp of the human FKBP12.6 promoter to the luciferase gene of pGL4.17. The reporter constructs were transfected into H9c2 cardiomyocytes. After IH stimulation, the promoter activities of CD38, RYR2, and FKBP12.6 were not decreased by IH in H9c2 cardiomyocytes (p = 0.9630, p = 0.2735, and p = 0.3563, respectively) ( Figure 4), suggesting that the gene expression levels of Cd38, Ryr2, and Fkbp12.6 in response to IH were not regulated by transcription. , 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 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.

The Promoter Activities of Cd38, Ryr2, and Fkbp12.6 Were Not Decreased by IH
To determine whether the IH-induced decreases in the Cd38, Ryr2, and Fkbp12.6 mRNA levels were caused by the inactivation of transcription, we fused a 3456 bp fragment containing 3187 bp of the human CD38 promoter, a 1260 bp fragment containing 1250 bp of the human RYR2 promoter, and a 1805 bp fragment containing 1696 bp of the human FKBP12.6 promoter to the luciferase gene of pGL4.17. The reporter constructs were transfected into H9c2 cardiomyocytes. After IH stimulation, the promoter activities of CD38, RYR2, and FKBP12.6 were not decreased by IH in H9c2 cardiomyocytes (p = 0.9630, p = 0.2735, and p = 0.3563, respectively) ( Figure 4), suggesting that the gene expression levels of Cd38, Ryr2, and Fkbp12.6 in response to IH were not regulated by transcription. 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.

The Promoter Activities of Cd38, Ryr2, and Fkbp12.6 Were Not Decreased by IH
To determine whether the IH-induced decreases in the Cd38, Ryr2, and Fkbp12.6 mRNA levels were caused by the inactivation of transcription, we fused a 3456 bp fragment containing 3187 bp of the human CD38 promoter, a 1260 bp fragment containing 1250 bp of the human RYR2 promoter, and a 1805 bp fragment containing 1696 bp of the human FKBP12.6 promoter to the luciferase gene of pGL4.17. The reporter constructs were transfected into H9c2 cardiomyocytes. After IH stimulation, the promoter activities of CD38, RYR2, and FKBP12.6 were not decreased by IH in H9c2 cardiomyocytes (p = 0.9630, p = 0.2735, and p = 0.3563, respectively) ( Figure 4), suggesting that the gene expression levels of Cd38, Ryr2, and Fkbp12.6 in response to IH were not regulated by transcription. , 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 , 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.

The Pten Level Was Significantly Increased by IH
Wu et al. recently reported that overexpression of Pten suppresses the expression of CD38 in airway smooth muscle cells [51]. Therefore, it is possible that the IH-induced downregulation of the Cd38, Ryr2, and Fkbp12.6 is possibly caused by the upregulation of Pten. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes to normoxia or IH and determined the Pten mRNA levels using real-time RT-PCR. We found that the Pten mRNA levels in both H9c2 and P19.CL6 cells were significantly increased in response to IH exposure ( Figure 5).
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.

The Pten Level Was Significantly Increased by IH
Wu et al. recently reported that overexpression of Pten suppresses the expression of CD38 in airway smooth muscle cells [51]. Therefore, it is possible that the IH-induced downregulation of the Cd38, Ryr2, and Fkbp12.6 is possibly caused by the upregulation of Pten. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes to normoxia or IH and determined the Pten mRNA levels using real-time RT-PCR. We found that the Pten mRNA levels in both H9c2 and P19.CL6 cells were significantly increased in response to IH exposure ( Figure 5). 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).
We further measured the Pten protein levels in H9c2 cells using Western blot analysis, and we confirmed that the level of Pten was significantly increased by IH ( Figure  6: p = 0.0417). 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. 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).
We further measured the Pten protein levels in H9c2 cells using Western blot analysis, and we confirmed that the level of Pten was significantly increased by IH ( Figure 6: 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.

The Pten Level Was Significantly Increased by IH
Wu et al. recently reported that overexpression of Pten suppresses the expression of CD38 in airway smooth muscle cells [51]. Therefore, it is possible that the IH-induced downregulation of the Cd38, Ryr2, and Fkbp12.6 is possibly caused by the upregulation of Pten. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes to normoxia or IH and determined the Pten mRNA levels using real-time RT-PCR. We found that the Pten mRNA levels in both H9c2 and P19.CL6 cells were significantly increased in response to IH exposure ( Figure 5). We further measured the Pten protein levels in H9c2 cells using Western blot analysis, and we confirmed that the level of Pten was significantly increased by IH ( Figure  6: p = 0.0417). 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 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. The mechanism of Cd38, Ryr2, and Fkbp12.6 expression in cardiomyocytes was investigated by knocking down the Pten gene by means of RNA interference in rat H9c2 cells. The expression levels of Cd38, Ryr2, and Fkbp12.6 were significantly decreased by IH in the presence of scrambled RNA. By contrast, the introduction of siPten inhibited the IH-induced decreases in the mRNA levels of Cd38, Ryr2, and Fkbp12.6 in H9c2 cells (Figure 7). These results indicated that the observed decreases in the Cd38, Ryr2, and Fkbp12.6 levels in response to IH were caused by Pten expression. The mechanism of Cd38, Ryr2, and Fkbp12.6 expression in cardiomyocytes was investigated by knocking down the Pten gene by means of RNA interference in rat H9c2 cells. The expression levels of Cd38, Ryr2, and Fkbp12.6 were significantly decreased by IH in the presence of scrambled RNA. By contrast, the introduction of siPten inhibited the IHinduced decreases in the mRNA levels of Cd38, Ryr2, and Fkbp12.6 in H9c2 cells ( Figure  7). These results indicated that the observed decreases in the Cd38, Ryr2, and Fkbp12.6 levels in response to IH were caused by Pten expression. 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.

3-Deaza-cADPR Attenuated the IH-Induced Decreases in the Cd38, Ryr2, and Fkbp12.6 Levels
CD38 was originally found to be a surface antigen/marker of B lymphocytes, monocytes, and natural killer cells [57]. In 1993, three research groups independently found cADPR synthesizing activity in Cd38 [58][59][60]. cADPR binds to Fkbp12.6 to dissociate Fkbp12.6 from the Ryr2 complex and induces Ca 2+ release from the Ryr2 intracellular Ca 2+ channel [38,61]. To confirm the correlation between the expression levels of Cd38, Ryr2, and Fkbp12.6 and the Cd38-cADPR-mediated signaling pathway, we added 3-deaza-cADPR, a cell-permeable cADPR agonist [62,63], into H9c2 cell culture medium, and then we subjected the cells to normoxia or IH for 24 h. Following IH stimulation, the mRNA levels of Cd38, Ryr2, and Fkbp12.6 were measured. The results showed that the IH-induced decreases in the mRNA levels of Cd38, Ryr2, and Fkbp12. 6 were attenuated in the presence of 3-deaza-cADPR (Figure 8), indicating that the observed reduction in the mRNA levels in response to IH was induced by the inhibition of the Cd38-cADPR-mediated signaling pathway. 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.

3-Deaza-cADPR Attenuated the IH-Induced Decreases in the Cd38, Ryr2, and Fkbp12.6 Levels
CD38 was originally found to be a surface antigen/marker of B lymphocytes, monocytes, and natural killer cells [57]. In 1993, three research groups independently found cADPR synthesizing activity in Cd38 [58][59][60]. cADPR binds to Fkbp12.6 to dissociate Fkbp12.6 from the Ryr2 complex and induces Ca 2+ release from the Ryr2 intracellular Ca 2+ channel [38,61]. To confirm the correlation between the expression levels of Cd38, Ryr2, and Fkbp12.6 and the Cd38-cADPR-mediated signaling pathway, we added 3-deaza-cADPR, a cell-permeable cADPR agonist [62,63], into H9c2 cell culture medium, and then we subjected the cells to normoxia or IH for 24 h. Following IH stimulation, the mRNA levels of Cd38, Ryr2, and Fkbp12.6 were measured. The results showed that the IH-induced decreases in the mRNA levels of Cd38, Ryr2, and Fkbp12.6 were attenuated in the presence of 3-deaza-cADPR (Figure 8), indicating that the observed reduction in the mRNA levels in response to IH was induced by the inhibition of the Cd38-cADPR-mediated signaling pathway. 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.

Discussion
In this study, we demonstrated that IH exposure induced the reduction in Cd38, Ryr2, and Fkbp12.6 mRNA levels in cardiomyocytes. We further studied the mechanisms by which IH downregulates the mRNA levels of Cd38, Ryr2, and Fkbp12.6, and we speculated that Pten-mediated downregulation is involved in the process. We then knocked down Pten, and the IH-induced downregulation of Cd38, Ryr2, and Fkbp12.6 was not observed in siPten-transfected cardiomyocytes, suggesting that the dysfunction of the Cd38-cADPR signal system in cardiomyocytes is induced by IH via the overexpression of Pten. To verify this possibility, we added 3-deaza-cADPR, a cell-permeable cADPR agonist, into the cardiomyocyte culture medium and found that the IH-induced decreases in Cd38, Ryr2, and Fkbp12.6 were attenuated, indicating that IH indeed reduces Cd38 expression, Cd38 activity (cADPR synthesizing ADP-ribosyl cyclase activity), and cADPR concentration in cardiomyocytes. The reduced cADPR concentration in turn decreased the recipients of cADPR, Ryr2, and Fkbp12.6 in cardiomyocytes.
SAS patients frequently suffer from CVDs such as hypertension, coronary disease, and heart failure [10][11][12]. The prevalence of SAS in patients with heart failure ranges from 15% to 59%, and the mortality rate among patients with severe SAS is significantly high [27][28][29][30]. We recently reported that IH increases renin expression in juxtaglomerular cells [46], as well as the expression of dopamine β-hydroxylase and phenylethanolamine Nmethyltransferase in catecholamine-synthesizing neuroblastoma cells [64], causing SAS patients to become hypertensive. However, how cardiac dysfunction develops in SAS patients remains elusive. 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.

Discussion
In this study, we demonstrated that IH exposure induced the reduction in Cd38, Ryr2, and Fkbp12.6 mRNA levels in cardiomyocytes. We further studied the mechanisms by which IH downregulates the mRNA levels of Cd38, Ryr2, and Fkbp12.6, and we speculated that Pten-mediated downregulation is involved in the process. We then knocked down Pten, and the IH-induced downregulation of Cd38, Ryr2, and Fkbp12.6 was not observed in siPten-transfected cardiomyocytes, suggesting that the dysfunction of the Cd38-cADPR signal system in cardiomyocytes is induced by IH via the overexpression of Pten. To verify this possibility, we added 3-deaza-cADPR, a cell-permeable cADPR agonist, into the cardiomyocyte culture medium and found that the IH-induced decreases in Cd38, Ryr2, and Fkbp12.6 were attenuated, indicating that IH indeed reduces Cd38 expression, Cd38 activity (cADPR synthesizing ADP-ribosyl cyclase activity), and cADPR concentration in cardiomyocytes. The reduced cADPR concentration in turn decreased the recipients of cADPR, Ryr2, and Fkbp12.6 in cardiomyocytes.
The Cd38-cADPR signaling pathway has been reported to antagonize cardiomyocyte differentiation of mouse embryonic stem cells [70]. Meanwhile, cADPR activates Ryr2 [47] in cardiomyocytes, Cd38 is expressed in heart [71], isoproterenol increases ADP-ribosyl cyclase activity of Cd38 in ventricular muscle [72], cADPR levels are decreased in rat myocardial ischemia [73], male Cd38 knockout mice have shown cardiac hypertrophy [49], Ryr2 knockout mice have died on embryonic day 10 with morphological abnormalities in their heart tube [74], altered stoichiometry of FKBP12.6 relative to that of RYR2 has been reported to a cause abnormal Ca 2+ leak through RYR2 in heart failure [48], and Fkbp12.6 knockout male mice have shown cardiac hypertrophy [55], indicating that the Cd38-cADPR signaling pathway is indispensable in cardiomyocyte functioning.
Pten acts as a phosphatase that dephosphorylates phosphatidylinositol 3,4,5-triphosphate (PIP 3 ). Pten specifically catalyzes the dephosphorylation of the 3 phosphate of the inositol ring in PIP 3 , producing a biphosphate product (phosphatidylinositol 4,5-bisphosphate). This dephosphorylation is important because it results in the inhibition of the Akt signaling pathway, which plays a key role in multiple signal transduction pathways [75]. Pten is involved in the regulation of cellular processes, including cell survival, proliferation, and migration, and it participates in diverse physiological and pathological processes [75]. Overexpression of Pten suppresses the expression of Cd38 in airway smooth muscle cells [54]. PTEN expression is increased by RYR2 knockdown, whereas increased RYR2 expression inhibits PTEN expression in pancreatic cancer cells [76]. In adult mice, cardiac-specific Pten knockout preserves heart function, decreases scar size, and promotes cardiomyocyte proliferation after myocardial infarction stress [77]. Recently, Ashikawa et al. have reported that intraperitoneal injection of a Pten inhibitor, bisperoxovanadium-pic, ameliorated left ventricular inflammation, fibrosis, and diastolic dysfunction in DS/Obese (DahlS.Z-lepr fa /Lepr fa ) rats [78]. In the present study, IH reduced the expression of the components of the Cd38-cADPR signal system in cardiomyocytes via Pten overexpression; therefore, Pten inhibitors can preserve cardiac cell functions and may serve as new drugs for cardiomyocyte functioning.
This study demonstrated that the gene expression levels of Cd38, Ryr2, and Fkbp12.6 decreased via the upregulation of Pten in IH-treated cardiomyocytes. It is suggested that, in SAS patients, downregulation of Cd38, Ryr2, and Fkbp12.6 may decrease the function of the Cd38-cADPR signaling in cardiomyocytes, leading to the failure of cardiac functions.

Real-Time RT-PCR
Total RNA was isolated from H9c2 and P19.CL6 cells using an RNeasy Plus Cell Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesized from total RNA as a template using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), as previously described [40,46,64,80,81]. Real-time PCR was performed using an SYBR ® Fast qPCR Kit (KAPA Biosystems, Boston, MA, USA) and a Thermal Cycler Dice Real Time System (Takara Bio, Kusatsu, Japan). All the PCR primers were synthesized by Nihon Gene Research Laboratories, Inc. (NGRL; Sendai, Japan); the primer sequences for each primer set are shown in Table 1. PCR was performed with an initial step of 3 min at 95 • C, followed by 40 cycles of 3 s at 95 • C and 20 s at 60 • C for Rig/RpS15, Cd38, Ryr2, Fkbp12.6, and Pten. The mRNA expression levels were normalized to the mRNA level of Rig/RpS15, as previously described [40,46,64,80,81].

RNA Interference
The siRNA directed against rat Pten was synthesized by NGRL. The sense sequence of the siRNA for the rat Pten was 5 -GGAACAAUAUUGAUGAUGUtt-3 (corresponding to the residues 140-158 of NM_031606.1). Silencer ® Select scrambled siRNA was purchased from Ambion and was used as a control. The transfection of the siRNA into H9c2 cells was performed using Lipofectamine ® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The cells were each transfected with 5 pmol of each siRNA in a 24-well culture dish, as previously described [40,46,80,81,84].

Addition of 3-Deaza-cADPR
H9c2 cells were adjusted at 2 × 10 5 cells/mL and the 0.5 mL cell suspension was seeded into each well of a 24-well plate. After incubation at 37 • C overnight, the medium was replaced with fresh medium with or without 3-deaza-cADPR (Sigma; finally adjusted to 10 nM). The cells were further incubated at 37 • C in an IH or normoxia condition for 24 h. Cellular RNA preparation and real-time RT-PCR were performed as described in Section 4.2.

Data Analysis
The results are expressed as mean ± SE. Statistical significance was determined using Student's t-test performed in GraphPad Prism software (GraphPad Software, La Jolla, CA, USA).

Acknowledgments:
We are indebted to Satoshi Ueno (Nara Medical University) for encouragement. Ribosomal protein S15 Pten

Conflicts of
Phosphatase and tensin homolog deleted from chromosome 10 Rig/RpS15 Rat insulinoma gene/Ribosomal protein S15 RT-PCR Reverse transcriptase-polymerase chain reaction SAS Sleep apnea syndrome siRNA Small interfering RNA RyR Ryanodine receptor