Myristic Acid Conjugation Itself is Not Responsible for Robust Cardioprotective Effects Observed with Protein Kinase C Beta II Peptide Inhibitor in Ischemia‐Reperfusion Injury

Abstract

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 InformationThis 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.