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.

Upregulation of Reg IV and Hgf mRNAs by Intermittent Hypoxia via Downregulation of microRNA-499 in Cardiomyocytes

“…The cells were kept at 37 °C, 5% CO 2 , and 95% humidity, and the medium was changed every day. Cells were exposed to either normoxia (21% O 2 , 5% CO 2 , and balanced N 2 ), intermittent hypoxia (IH: 70 cycles of 5 min sustained hypoxia [1% O 2 , 5% CO 2 , and balanced N 2 ] and 10 min normoxia), or sustained hypoxia (1% O 2 , 5% CO 2 , and balanced N 2 ) using a custom-designed, computer-controlled incubation chamber attached to an external O 2 -CO 2 -N 2 computer-driven controller (O 2 programmable control, 9200EX, Wakenbtech CO., Ltd., Kyoto, Japan), as described [ 3 , 101 , 102 , 103 , 104 , 105 , 106 , 107 ]. These conditions are similar to the conditions reported in patients with severe degrees of SAS: in severe cases of SAS, patients are repeatedly exposed to severe hypoxemia followed by mild hypoxemia or normoxia (i.e., IH).…”

“…Total RNA was isolated using an RNeasy plus mini kit (Qiagen, Hilden, Germany) from H9c2 and P19.CL6 cells, 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 described [ 73 , 74 , 75 , 79 , 82 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 ]. Real-time PCR was performed using SYBR ® Fast qPCR kit (KAPA Biosystems, Boston, MA, USA) and a Thermal Cycler Dice Real Time System (Takara Bio, Kusatsu, Japan).…”

“…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 rat insulinoma gene (Rig)/ribosomal protein S15 ( RpS15 ), Il-6, Il-8 , Il-17A , Il-18 , Il-33 , Tgfβ1 , Ccl2, Cxcl12, Tnfα , Vegf-A , Flt-1 , Flk-1 , Cd38 , Reg I , Reg II , PAP II/Reg IIIα , PAP I/Reg IIIβ , PAP III/Reg IIIγ , Reg IIIδ , Reg IV , Extl3 ( Reg receptor ), Hgf , and c-Met ( Hgf receptor ). The mRNA expression levels were normalized to the mRNA level of Rig/RpS15 , as described [ 75 , 79 , 85 , 104 , 105 , 106 , 107 ]. For miR, total RNA, including miRNA, was isolated from P19.CL6 cells using the miRNeasy mini kit (Qiagen) according to the manufacturer’s instructions.…”

“…Reporter plasmids were prepared by inserting the promoter fragments of mouse Reg IV (−2008–+29) and rat Hgf (−1336–+59) upstream of a firefly luciferase reporter gene in the pGL4.17[ luc2 /Neo] vector (Promega, Madison, WI) and pGL3-Basic (Promega) [ 36 ], respectively. The reporter plasmids were transfected into mouse P19.CL6 cells differentiated into cardiomyocytes using Lipofectamine ® 3000 (Invitrogen), as described [ 36 , 73 , 74 , 75 , 79 , 82 , 102 , 105 , 106 , 107 , 108 , 109 ]. The cells were exposed to either 70 cycles/24 h of IH, mimicking the cardiomyocytes of SAS patients, or normoxia for 24 h. After the cells were exposed to IH, they were lysed, and promoter activities were measured.…”

The Impact of Intermittent Hypoxia on Metabolism and Cognition

“…In contrast, the expression of phosphatase and tensin homolog deleted from chromosome 10 (Pten) was upregulated by IH stimulation. The introduction of siRNA into Pten abolished the IH-induced reduction of Cd38, Ryr2, and Fkbp12.6 [ 113 ]. We treated H9c2 rat cardiomyocytes with 3-deaza-cADPR, a cell-permeable cADPR agonist [ 114 ], and introduced it 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 (ADP-ribosyl cyclase activity for cADPR synthesis), and cADPR concentration in cardiomyocytes.…”