Abstract 18459: A Cell Permeable Protein Kinase C Beta II Peptide Inhibitor Mitigates the Generation of Reactive Oxygen Species in Rat ex-vivo and Porcine in-vivo Ischemia-Reperfusion Injury

Introduction: Restoration of blood flow following myocardial infarction is necessary to salvage ischemic tissue, but reperfusion is known to cause myocardial ischemia/reperfusion injury (MIR). Cytokine induced translocation of protein kinase C beta II (PKCβII) during reperfusion (R) stimulates the production of reactive oxygen species (ROS). Dual conjugation of PKCβII peptide inhibitor (PKCβII-) with myristic acid (myr) and trans-activator of transcription (Tat) (myr-Tat-CC-SLNPEWNET [myr-Tat-PKCβII-]) enhances intracellular delivery and reduces phorbol ester-induced neutrophil ROS. We hypothesize that 20ng/kg of myr-Tat-PKCβII- would exert cardioprotective effects in porcine MIR in-vivo by reducing stimulation of ROS.

Methods: Ex vivo: We tested 100nM - 10fmol myr-Tat-PKCβII- in ex-vivo rat MIR. Isolated hearts from anesthetized male SD rats underwent global I (30-min)/R(50-min). Myr-Tat-PKCβII- was given during first 5min of R. DP/dt max was measured via pressure transducer in the left ventricle. 1% TTC staining was used to determine infarct size. Data was analyzed via Fisher’s PLSD. In vivo: Regional I (1 hr)/R (3 hrs) was induced in male Yorkshire pigs. At R, myr-Tat-PKCβII- or scrambled control peptide (20ng/kg; ~100 pM blood volume [vol.] concentration [conc.]) was given by intra-arterial bolus. Ejection fraction (EF) was calculated. Infarct size was determined with Evans Blue dye and 1% TTC staining. Data was analyzed using Student’s t-test.

Results: Ex-vivo: Myr-Tat-PKCβII- (100nM - 100fmol; 10-14±3%; n=3-6) significantly reduced infarct size compared to controls (21±3%; n=21; p<0.05). DP/dt max was significantly improved (100pM-1pM; 961±274; n=5-6) compared to 100nM (163±77, n=3; p<0.05), 100fM (481±86, n=5; p<0.05), and 100pMscrambled myr-Tat-PKCβII- (386±86, n=4; p<0.05). In-vivo: Myr-Tat-PKCβII- 20ng/kg (~100pM blood vol. conc.) significantly restored EF to within 1.4±0.7% of baseline and reduced infarct size to (10.0±2.8%; n=4) compared to control (6.4±2.1%; 28.5±8.3%; n=6; p<0.05). Conclusions: Results suggest 20ng/kg (~100pM blood vol. conc.)) should continue to be used to test in porcine MIR in-vivo. Future studies include a 12-week porcine survival study to evaluate infarct size and cardiac function.