Prompt reperfusion is vital to resuscitating ischemic myocardium. However, cardiomyocyte death still occurs due to ischemia-reperfusion (I/R) injury, which is mediated in part by oxidative stress. Major sources of reactive oxygen species (ROS) during I/R are NADPH oxidase (NOX-2) and mitochondria, which are principally activated by protein kinase C βII (PKCβII). Previously, myristic acid (Myr) and trans-activator of transcription (Tat) conjugated PKCβII inhibitor (Myr-Tat-PKCβII-; N-Myr-Tat-CC-SLNPEWNET) exhibited cardioprotective effects in ex vivo rat hearts. In this study, we hypothesize that Myr-Tat-PKCβII- will mitigate cardiac injury in an in vivo porcine myocardial I/R model compared to scrambled peptide controls. Male Yorkshire pigs (38-50kg) underwent regional I(1hr)/R(3hrs) through balloon-assisted occlusion of the second diagonal branch of the left anterior descending coronary artery (LAD) supplying ~40% of the anterior portion of the myocardium. During reperfusion (balloon deflation), a bolus of Myr-Tat-PKCβII- or scrambled control was infused into the LAD. Cardiac function was evaluated as the relative change in ejection fraction (EF) at the end of 3hr reperfusion compared to baseline. Serial measurements of serum creatine phosphokinase (CPK), troponin I, and myoglobin were evaluated to assess cardiac injury. Post-reperfused hearts were stained with Evans Blue dye to identify the area at risk (AR) and 1% triphenyltetrazolium chloride to demarcate the area of necrosis (AN). Infarct size (AN/AR), EF, and cardiac injury markers were analyzed via Student’s t-test. Myr-Tat-PKCβII- preserved EF with a relative change of 1.2±2.8% compared to 8.9±2.2% in control hearts (p<0.05) from mean baseline EF (61.4±0.5%). Myr-Tat-PKCβII- significantly decreased myoglobin levels at 1hr reperfusion (135±132ng/mL, n=4) compared to scrambled control (1022±346ng/mL, n=3 p<0.05). Myr-Tat-PKCβII- reduced infarct size to 13.5±3.9% (n=4) compared to scrambled control hearts (27.5±7.9%, n=6). CPK and troponin I levels were comparable in both groups. These results suggest Myr-Tat-PKCβII- mitigates cardiac injury when given at reperfusion onset. Future studies will examine Myr-Tat-PKCβII- in an 8 week in-vivo, porcine MI survival study.

Polymorphonuclear leukocyte (PMN) superoxide (SO) production by NADPH oxidase (NOX‐2) activation contributes to myocardial ischemia‐reperfusion (I/R) injury. Protein kinase C beta‐II (PKC βII) is a principal mediator of NOX‐2 activation via phosphorylation of NOX‐2 cytosolic protein p47phox. Phorbol 12‐myristate 13‐acetate (PMA) is a known broad‐spectrum PKC agonist that induces PMN SO release. In prior studies, selective PKCβII inhibition with myristoylated PKCβII peptide inhibitor (N‐myr‐SLNPEWNET; myr‐PKCβII‐) attenuated PMA‐induced PMN SO release and myocardial I/R injury in a dose‐dependent manner. However, the role of myristoylation mediating the inhibitory effects of myr‐PKCβII‐ on PMN SO release needs to be determined. Therefore, we aim to confirm the role of PKCβII by using myristoylated PKCβII peptide activator (N‐myr‐SVEIWD; myr‐PKCβII+) and myr‐PKCβII‐ that influence PKCβII translocation. Whereas, myr‐scrambled PKCβII‐ (N‐myr‐WNPESLNTE; myr‐PKCβII‐scram) is a control for myristoylation. We hypothesize that myr‐PKCβII+ should augment, myr‐PKCβII‐should attenuate, and myr‐PKCβII‐scram should have no effect on PMA‐induced PMN SO release compared to non‐treated and unconjugated peptide controls. PMNs (5×106) isolated from male Sprague‐Dawley rats (~400g) were incubated for 15 min at 37°C in the presence/absence of SO dismutase (SOD; 10μg/mL, positive control), unconjugated PKCβII+/− (20 μM), myr‐PKCβII+/− (20 μM), or myr‐PKCβII‐scram (20 μM). SO release was evaluated by the absorbance change (at 550 nm) via ferricytochrome c reduction after PMA stimulation (100 nM) for 390 sec. Data were analyzed by ANOVA using Bonferroni‐Dunn post‐hoc analysis. Myr‐PKCβII‐ significantly attenuated PMA‐induced PMN SO release (0.29±0.02; n=36; p<0.05) when compared to myr‐PKCβII+ (0.42±0.03; n=29), myr‐PKCβII‐scram (0.53±0.05; n=10), and non‐treated controls (0.41±0.02; n=55). Unconjugated PKCβII+ (0.41±0.04; n=16) and PKCβII‐(0.40±0.04; n=28) were similar to non‐treated controls. SOD (n=8) significantly reduced SO release by 94±7% compared to all groups (p<0.01). Cell viability determined by 0.2% trypan blue exclusion was similar in all groups, 94±2%. Unexpectedly, myr‐PKCβII‐scram rather than myr‐PKCβII+ PMNs exhibited the highest PMA‐induced PMN SO release but was not significantly different from untreated controls. Additional experiments will determine whether myr‐PKCβII‐scram significantly enhances SO release. Results suggest myr‐ conjugation improved myr‐PKCβII‐ delivery compared to unconjugated PKCβII‐ but does not contribute to the inhibitory effects of PMA‐induced PMN SO release. Therefore, myr‐PKCβII‐ may be an effective therapeutic intervention to limit inflammation‐induced I/R injury. Support or Funding Information This research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine. Current license is supported by Young Therapeutics, LLC.

Protein kinase C beta II (PKCβII) activates polymorphonuclear leukocyte (PMN) superoxide (SO) production via NADPH oxidase (NOX-2) phosphorylation to exacerbate myocardial ischemia/reperfusion (I/R) injury. In prior studies, myristoylation (myr) of PKCβII peptide inhibitor (N-myr-SLNPEWNET; myr-PKCβII-), which disrupts PKCβII translocation/phosphorylation of NOX-2, was shown to dose-dependently attenuate PMN SO release induced by phorbol 12-myristate 13-acetate (PMA), a broad-spectrum PKC agonist. However, the role of myr on the inhibitory effects of myr-PKCβII- has yet to be elucidated. We hypothesized that myr-PKCβII peptide activator (N-myr-SVEIWD; myr-PKCβII+) would augment, myr-PKCβII- would attenuate, and scrambled myr-PKCβII- (N-myr-WNPESLNTE; myr-PKCβII-scram), a control for myr, would not affect PMA-induced PMN SO release compared to unconjugated peptides and nontreated controls. Rat PMNs (5х10 6 ) were incubated for 15 min at 37 o C in the presence/absence of SO dismutase (SOD; 10 μg/mL), unconjugated PKCβII+/-, myr-PKCβII+/-, or myr-PKCβII-scram (all 20 μM). SO release was measured by the change in absorbance at 550 nm via ferricytochrome c reduction after PMA (100 nM) stimulation for 390 sec. Data were analyzed by ANOVA using Student-Newman-Keuls post hoc analysis. Myr-PKCβII- significantly attenuated SO release (0.30±0.02; n=27; p<0.05) compared to nontreated controls (0.46±0.01; n=73), myr-PKCβII+ (0.46±0.03; n=25), unconjugated PKCβII+ (0.43±0.04; n=15), PKCβII- (0.43±0.02; n=22) and myr-PKCβII-scram (0.65±0.04; n=22). SOD (n=8), which rapidly converts SO to H 2 O 2 , significantly reduced absorbance by 94±7%, indicating that absorbance increased mainly due to PMA stimulation. Cell viability (trypan blue exclusion) was similar in all groups (94±2%). Unexpectedly, myr-PKCβII-scram significantly stimulated the highest increase in absorbance compared to all groups (p<0.01). Future studies will determine whether myr-PKCβII-scram augments absorbance by a different mechanism. Results suggest that myr improves myr-PKCβII- delivery compared to unconjugated PKCβII- but does not affect inhibition of PMA-induced PMN SO release. Myr-PKCβII- may thus effectively limit inflammation-induced I/R injury.

Previously, naltrindole (NTI; selective delta opioid receptor antagonist) was shown to improve post-reperfused cardiac function and reduced infarct size when given prior to ischemia (I)/ reperfusion (R) in ex-vivo rat hearts. Conversely, naloxone (NX, broad-spectrum opioid antagonist) and nor-binaltrophine (BNI, selective kappa receptor antagonist) were similar to control hearts. In this study, the effects of NTI derivatives naltriben (NTB, delta receptor antagonist) and guanidonaltrindole (GNTI, kappa receptor antagonist) were compared to NTI, BNI, and NX. Isolated hearts from male SD rats (300g) were subjected to global I(30min)/R(45min). Treatments were given 5 min before I (preconditioning) and during the first 5 min of R. Left ventricular (LV) cardiac function was measured using a pressure transducer. At the end of reperfusion, infarcted heart tissue was compared to total tissue weight. Data were evaluated using ANOVA. As shown in Table 1, NTI, NTB, and GNTI significantly improved post-reperfused cardiac function and reduced infarct size compared to control hearts. NTI and NTB elicited direct effects on cardiac function when given during preconditioning in contrast to all other study groups and were the most robust at reducing infarct size and restoring post reperfusion cardiac function. The negative inotropic effects of NTI and NTB were correlated with a decrease in the rise of ischemic pressure. GNTI also elicited significant improvement in post-reperfused cardiac function and reduction of infarct size compared to BNI which suggests a separate cardioprotective mechanism that this NTI derivative may exert in contrast to kappa opioid receptor inhibition. Results suggest that NTI and derivatives, GNTI and NTB, are cardioprotective against I/R injury resulting in reduced ischemic peak pressure (NTI/NTB) and infarct size. In future studies, we will examine the mechanism of the protective effects of NTI and derivatives in hearts subjected to I/R injury.

Reactive oxygen species (ROS) induced ischemia-reperfusion (I/R) injury is a phenomenon causing paradoxical myocardial damage after cardio-angioplasty, coronary bypass and organ transplantation. Previous studies show that a cell-permeable myristic acid (myr-) conjugated PKCβII peptide inhibitor given at reperfusion prevents PKCβII translocation (N - myr-SLNPEWNET; myr-PKCβII-) and significantly attenuates ROS mediated I/R injury. We included a scrambled myr-PKCβII- (N-myr-WNPESLNTE; myr-PKCβII-scram) to examine the effects of myr separately. We hypothesize that myr-PKCβII- will improve and myr-PKCβII activator peptide (N-myr-SVEIWD; myr-PKCβII+) will exacerbate infarct size and post-reperfused cardiac function compared to myr-PKCβII-scram and untreated controls. Hearts isolated from male Sprague-Dawley rats (~300g) were perfused with Krebs’ buffer at a constant pressure of 80mmHg and subjected to 30 min of global ischemia and 50 min reperfusion. Myr-PKCβII-, myr-PKCβII+, myr-PKCβII-scram (all 20μM), or untreated control were given during the first five minutes of reperfusion. Left ventricular (LV) dP/dt max and dP/dt min (mmHg/s) were measured using a pressure transducer, and infarct size was determined using 1% triphenyltetrazolium chloride staining comparing infarcted tissue vs. total tissue weight. Data were evaluated using ANOVA with Student-Newman-Keuls post-hoc analysis. Myr-PKCβII- (n=17) significantly improved LV dP/dt max and dP/dt min to 1535±107 and 1063±83 at 50 min post-reperfusion compared to untreated control (815±107 and 722±89; n=15); myr-PKCβII-scram (513±78 and 433±66; n=12), and myr-PKCβII+ (860±118 and 694±81; n=14) (all p<0.05). Myr-PKCβII- significantly reduced infarct size (%) to 13±2 compared to untreated control (24±4); myr-PKCβII-scram (22±2), and myr-PKCβII+ (21±3) (all p<0.05). Unexpectedly, myr-PKCβII-scram significantly depressed post-reperfused LV dP/dt max and LV dP/dt min compared to untreated control and other treated groups (p<0.05). Results suggest that myr-PKCβII- exerted significant cardioprotective effects compared to untreated controls, myr- PKCβII+ and myr-PKCβII-scram and would improve clinical outcomes after cardio-angioplasty or organ transplantation.