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.

Ischemia‐reperfusion (I/R) injury mediated by excessive reactive oxygen species (ROS) is a well‐known phenomenon causing paradoxical myocardial damage after cardio‐angioplasty, coronary bypass, or organ transplantation following ischemic injury. Protein kinase C beta II isoform (PKCβII) inhibition using a cell‐permeable myristic acid (myr‐) conjugated PKCβII peptide inhibitor (N‐myr‐SLNPEWNET; myr‐PKCβII−) given at reperfusion significantly attenuated ROS release in previous animal I/R studies. However, prior studies did not explore the possibility that myristic acid conjugation itself contributes to the attenuation of I/R injury. We hypothesize that myristic acid conjugation is not responsible for attenuation of ROS‐induced I/R damage and that myr‐PKCβII− will reduce infarct size and improve post‐reperfused cardiac function compared to scrambled myr‐PKCβII− peptide (N‐myr‐WNPESLNTE; myr‐PKCβII‐scram), myr‐PKCβII activator peptide (N‐myr‐SVEIWD; myr‐PKCβII+), and plasma controls. Hearts isolated from male Sprague‐Dawley rats (~300g) were subjected to 30 min of global ischemia and myr‐PKCβII− (20μM), myr‐PKCβII+ (20μM), myr‐PKCβII‐scram (20μM), or plasma (control) was given at initial reperfusion during the first five minutes. Thereafter, Krebs’ buffer was reperfused into hearts at a constant pressure (80 mmHg) throughout the remainder of reperfusion (50 min). Left ventricular (LV) cardiac function indices were measured using a pressure transducer, and infarct size of frozen post‐reperfused hearts was determined using 1% triphenyltetrazolium chloride staining comparing infarcted tissue vs. total tissue weight. Data were evaluated using ANOVA with Bonferroni‐Dunn post‐hoc analysis. Myr‐PKCβII‐significantly improved both post‐reperfused cardiac relaxation function indices compared to all groups (p<0.05). LV end diastolic pressure (LVEDP; mmHg) and the maximal rate of decline of LVEDP (mmHg/s) at 50 min post‐reperfusion significantly improved with myr‐PKCβII− (41±5 and 1088±84; n=16) compared to plasma‐control (61±4 and 731±95; n=14), myr‐PKCβII+ (58±4 and 716±84; n=13), or myr‐PKCβII‐scram (68±1 and 451±78; n=8) hearts. Additionally, myr‐PKCβII− significantly reduced infarct size (%) to 13±2 compared to either plasma‐control (24±4) or myr‐PKCβII‐scram (24±2; both p<0.05), whereas myr‐PKCβII+ (21±3) did not differ significantly from myr‐PKCβII‐scram or plasma‐control. Results suggest that myr‐conjugation is not responsible for the cardioprotective effects observed with myr‐PKCβII− in I/R injury. Myr‐PKCβII− may be an effective therapeutic to improve clinical outcomes after coronary bypass, cardio‐angioplasty, or organ transplantation. 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 research license is supported by Young Therapeutics, LLC. lindonyo@pcom.edu.

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.