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Reperfusion injury is known to augment myocardial tissue injury following a heart attack partly due to the generation of reactive oxygen species (ROS) upon cardio‐angioplasty. Inhibition of protein kinase C beta II (PKCβII) during reperfusion with peptide inhibitor (N‐myr‐SLNPEWNET; PKCβ−) has been associated with decreased ROS release and leukocyte infiltration in rat hind‐limb and myocardial ischemia/reperfusion (I/R) studies, respectively. However, the role of activating PKCβII during the early reperfusion phase following myocardial ischemia is not known. It is thought that PKCβII activation augments tissue NADPH oxidase (NOX‐2) to enhance ROS release and exacerbate myocardial I/R (MI/R) injury. We were interested in studying the effects of myristoylated (myr‐) PKCβII peptide activator (N‐myr‐SVEIWD; myr‐PKCβ+) and myr‐PKCβ− in a rat MI/R model. Myristoylation of PKCβII peptides facilitate their entry into the cell in order to affect PKCβII activity by either augmenting or attenuating its translocation to cell membrane proteins, such as NOX‐2, via its selective receptor for activated C kinase domain. In this study, we hypothesize that myr‐PKCβ− will decrease infarct size and improve post‐reperfused cardiac function compared to non‐drug treated controls, whereas myr‐PKCβ+ treated hearts will not improve these parameters. Male Sprague‐Dawley rats (~300g) were anesthetized with an IP injection of pentobarbital (60mg/kg) and anticoagulated with 1000U of heparin. The heart was then removed and placed on the perfusion needle of the Langendorff heart apparatus and a pressure transducer was placed into the left ventricle to measure cardiac function. After establishing baseline for 15min, the heart was subjected to global MI(30min)/R(50min). Thereafter, 20μM of myr‐PKCβ + (n=6), 20 μM myr‐PKCβ− (n=6) or plasma without myr‐PKCβ+/− (control; n=8) was infused into the heart during the first 5min of reperfusion. Following reperfusion, all hearts were frozen at −20°C for 30min, sectioned into 2mm slices and incubated at 37°C in 1% triphenyltetrazolium chloride. The ratio of dead heart tissue (unstained) weight to total heart tissue weight was calculated for infarct size. Myr‐PKCβ− hearts significantly restored post‐reperfused left ventricular developed pressure by 68±11% of initial baseline values compared to control (32±5%, p<0.01) and myr‐PKCβ+ (49±8%, p<0.05) hearts. Additionally, myr‐PKCβ− significantly reduced infarct size to 16±3% compared to control (30±4%, p<0.05) hearts. Myr‐PKCβ+ hearts (infarct size of 26±4%) were not different from control hearts, suggesting that it would not be effective to attenuate MI/R injury. The data indicate that myr‐PKCβ− would be an effective treatment to reduce myocardial reperfusion injury when given to heart attack patients during cardio‐angioplasty.Support or Funding InformationThis research was supported by the Department of Bio‐Medical Sciences and the Division of Research at Philadelphia College of Osteopathic Medicine (PCOM) and by Young Therapeutics, LLC. 4170 City Avenue, Philadelphia, PA 19131This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Previous studies have shown that protein kinase C (PKC) alpha, beta II (βII) and zeta inhibition attenuates polymorphonuclear leukocyte (PMN) superoxide (SO) release, whereas PKC delta inhibition augments this response via inhibiting phosphorylation of PMN NADPH oxidase (NOX‐2). However, to date, there is no information regarding the role of PKCβII peptide activator on regulating PMN SO release. PKCβII activator and inhibitor peptides work by augmenting or attenuating PKCβII translocation after stimulation by N‐formyl‐L‐methionyl‐Lleucyl‐L‐phenylalanine (fMLP; a PMN chemotactic receptor agonist) or phorbol 12‐myristate 13‐acetate (PMA; a diacylglycerol mimetic). It is known that myristic acid (myr‐) conjugation is an effective means to facilitate intracellular delivery of the cargo sequence to regulate PKCβII activity. We hypothesize that myr‐PKCβII peptide‐activator (N‐myr‐SVEIWD; myr‐PKCβ+) would increase PMA/fMLP‐induced rat PMN SO release, whereas, myr‐PKCβII peptideinhibitor (N‐myr‐SLNPEWNET; myr‐PKCβ−) would attenuate this response compared to non‐drug treated controls or unconjugated native PKCβ+/− peptide sequences. PMNs were isolated via peritoneal lavage from male Sprague‐Dawley rats (~500g) and were incubated (5x106) for 15min at 37°C in the presence/absence of native/myr‐PKCβ+/− (20 mM; n=8[fMLP]) or SO dismutase (SOD;10mg/mL; n=8) as positive control. fMLP or PMA induced PMN SO release was measured spectrophotometrically at 550nm via reduction of ferricytochrome c for 90 sec (fMLP;1mM; n=19) or 360 sec (PMA;100nM; n=17). fMLP‐induced PMN SO release increased absorbance to 0.155±0.018 in non‐drug controls, 0.162±0.037 in myr‐PKCβ+, 0.136±0.025 in native PKCβ+, 0.070±0.012 in myr‐PKCβ− and 0.146±0.018 in native PKCβ− treated PMNs at 30 sec. Myr‐PKCβ− significantly decreased fMLP‐induced PMN SO release compared to nondrug controls, myr‐PKCβ+ and native PKC β+/− peptides (p<0.05). PMA‐induced PMN SO release increased absorbance to 0.41±0.039 in non‐drug controls, 0.517±0.047(n=7) in myr‐PKCβ+, 0.345±0.071(n=5) in native PKCβ+, 0.241±0.055(n=6) in myr‐PKCβ−, and 0.308±0.052(n=6) in native PKC β− at 360 sec. Myr‐PKCβ‐significantly attenuated PMA‐SO release compared to non‐drug controls and myr‐PKCβ+ (p<0.05). SOD‐treated samples showed >90% reduction of fMLP or PMA‐induced SO release and native PKCβ+/− was not different from non‐drug controls. Cell viability ranged between 94±2% and 98±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr‐PKCβ‐significantly attenuates fMLP/PMA‐induced SO release, whereas myr‐PKCβ+ only shows a trend to augment fMLP/PMA‐induced SO release. Additional studies will be conducted to determine if myr‐PKCβ+ will significantly augment PMN SO release.Support or Funding InformationThis research was supported by the Department of Bio‐Medical Sciences and the Division of Research at Philadelphia College of Osteopathic Medicine (PCOM) and by Young Therapeutics, LLC. 4170 City Avenue, Philadelphia, PA 19131This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.