Deltorphin‐D Decreases H202 Induced Myocardial Cell Necrosis in Rat Myoblast Cell Line

Oxidative damage has been suggested to play a critical role in acute myocardial infarction (AMI) and other cardiovascular disease where prolonged ischemia occurs. During reperfusion reactive oxygen species such as H 2 O 2 and hydroxyl radicals which are derived from various sources are found in the myocardium. Lactic Acid Dehydrogenase (LDH) is a stable cytosolic enzyme released by cells when they are lysed. It has been used to measure cell mediated cytotoxicity and provides a sensitive marker of cell damage and necrosis as occurs in AMI. We used an H9C2 rat myoblast cell line to test the ischemic protective effect of the delta 2 specific opioid Deltorphin‐D. Cells were grown to confluency in 96 well plates in DMEM + 10% FBS and washed one time with assay buffer consisting of phenol red free DMEM + 2.5% FBS. Following the wash step, 50 μl Deltorphin‐D at concentrations of 0.1 to 10 mmol/l was added to the test cells. Cells were incubated at 37°C; 5% CO 2 for 30 min. Following preincubation 50 μl of H 2 O 2 was added giving a final H 2 O 2 concentration of 0.1 mm/L. The plate was incubated again at 37°C for 4 hr. Cell supernatants were assayed for LDH release using the Cytotox 96 Non‐Radioactive Cytotoxicity Assay from Promega, Madison, WI. Preincubation with Deltorphin‐D at all concentrations tested significantly decreased H 2 O 2 induced LDH release from 59 to 69% in H9C2 myoblasts compared to untreated controls. The mechanisms of ischemic protection provided by Deltorphin‐D and other delta 2 specific opioids may include blocking the formation of reactive oxygen species such as H 2 0 2 . Supported by ZymoGenetics, Inc., Seattle, WA