The role of cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG) in necrotic and apoptotic pathways of many cell types is well established; however its role in the ischemic preconditioning (IPC) of cardiomyocytes is not clearly defined. In the current study, we assessed the hypothesis that PKG protects against cell death following ischemidreperfusion injury in myocytes subjected to IPC. Freshly isolated adult rat ventricular myocytes were subjected to IPC by incubating in ischemic buffer for 30 minutes (min) followed by incubation in normal medium for 30 min. Prolonged simulated ischemia (SI) was created by incubating myocytes in the ischemic buffer for 90 min and reoxygenation (RO) for 120 min in the normal medium. Necrosis was determined by trypan blue exclusion and apoptosis was assessed by TUNEL assay. IPC reduced necrosis as shown by significant decrease in trypan blue positive cells as compared to virgin non-preconditioned myocytes subjected to SI and RO alone (p<.01). Similarly, the number of TUNEL positive myocytes following SI and 18 hrs of RO were significantly reduced in the IPC group. Treatment with PKG inhibitor, KT5832 (2pM) completely abolished the protection against necrosis by IPC. However, KT5832 failed to abolish the protective affect of IPC against apoptosis. Furthermore, myocytes infected with an adenoviral construct of PKG-la (1 x 1 o4 particles/cell) significantly reduced the number of trypan blue and TUNEL positive cells. These results suggest that the PKG signaling pathway plays an essential role in the preconditioning of myocytes against necrosis following SI / RO injury. Furthermore, while the overexpression of PKG protects myocytes against necrosis, as well as apoptosis, IPC may not induce a sufficient level of PKG during 18 hours of RO to induce protection against apoptosis.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-2441 |
| Date | 01 January 2005 |
| Creators | Caligtan, Marc J. |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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