Dual conjugation of protein kinase C epsilon peptide inhibitor with myristic acid and trans-activator of transcription mitigates myocardial ischemia-reperfusion injury in an in vivo porcine model

Abstract Protein Kinase C epsilon (PKCε) signaling is known to mediate superoxide production from mitochondrial and uncoupled endothelial nitric oxide synthase sources in myocardial ischemia-reperfusion (I/R) injury. Previously, PKCε peptide inhibitor conjugated with myristic acid and trans-activator of transcription (N-Myr-Tat-CC-EAVSLKPT [PKCε-]; Myr-Tat-PKCε-) exhibited profound reduction in infarct size compared to Myr-PKCε- or Tat-PKCε- in ex vivo rat hearts♰. This study aims to evaluate the effects of Myr-Tat-PKCε- in porcine myocardial I/R in vivo compared to a scrambled control peptide. Male Yorkshire castrated pigs (38-54kg) were subjected to regional I(1hr)/R(3hrs) via catheter-balloon in the left anterior descending coronary artery (LAD) at the location of the second LAD branch. Myr-Tat-PKCε- or Myr-Tat-PKCε-scrambled control peptide (N-Myr-Tat-CC-LSETKPAV [PKCε-scram]; Myr-Tat-PKCε-scram) bolus (0.2 mg/kg) was administered into the LAD at reperfusion. Echocardiography was used to determine ejection fraction (EF). Following reperfusion, hearts were excised and stained. The area at risk (AR) and area of necrosis (AN) were identified with 1% Evans Blue dye and 1% triphenyltetrazolium chloride respectively. Infarct size (AN/AR) and EF were analyzed with unpaired Student’s t-test. Myr-Tat-PKCε-scram exhibited a reduced final EF compared to baseline (551 vs 621%, n=3). Myr-Tat-PKCε- significantly increased final EF back to baseline (591 vs 591%, n=5; p\u3c0.05). Myr-Tat-PKCε- exhibited a reduction in infarct size (102%, n=4; p\u3c0.01) compared to Myr-Tat-PKCε-scram (297%, n=3). Results suggest that Myr-Tat-PKCε- mitigates myocardial I/R injury when administered during reperfusion. Future studies will test the effects of Myr-Tat-PKCε- in an 8-week porcine myocardial I/R survival study to determine its therapeutic potential for heart attack patients

Protein Kinase C Beta II Inhibitor combined with Conjugated Myristic Acid and Trans-Activator of Transcription achieves cardiovascular protection in porcine myocardial ischemia/reperfusion model

Introduction While timely reperfusion is critical during resuscitation of ischemic myocardium, oxidative stress still leads to ischemia/reperfusion (I/R) injury and ultimately, cardiomyocyte death. The major targets for mitigating oxidative stress are NADPH oxidase (NOX-2) and mitochondria, which are both activated by Protein Kinase C Beta II (PKCβII). In previous studies with an ex-vivo rat heart I/R model, Myristic Acid (Myr) and Trans-Activator Transcription (Tat) conjugated PKCβII inhibitor (Myr-Tat-PKCβII-; N-Myr-Tat-CC-SLNPEWNET) showed cardioprotective effects and a decrease in infarct area. In this study, we investigated the cardioprotective effects of Myr-Tat-PKCβII- in comparison with a scrambled peptide control. Methods Regional I(1 hour)/R(3 hours) was induced in Male Yorkshire pigs (38-50kg) using balloon-assisted occlusion of the second diagonal branch of the Left Anterior Descending Artery (LAD) which is responsible for supplying 40% of the anterior myocardium. At the end of ischemia, the balloon was deflated to allow for reperfusion and the LAD was treated immediately with a bolus of either Myr-Tat-PKCβII- or scrambled peptide. Cardiac function was measured by monitoring changes in ejection fraction (EF). Cardiac injury was assessed through routine measurements of serum creatine phosphokinase (CPK), troponin I, and myoglobin. After I/R, the hearts were stained with Evans Blue dye to identify the area at risk (AR) and 1% triphenyltetrazolium chloride to determine the area of necrosis (AN). Infarct size was then quantified (AN/AR) and was analyzed via Student’s t-test, along with EF and cardiac marker measurements. Results Analysis showed that Myr-Tat-PKCβII- significantly restored EF to within 1.40.7% of baseline compared to controls which only restored EF to within 6.42.1% (p\u3c0.05) of baseline. Myr-Tat-PKCβII- showed a significant decrease in serum myoglobin levels at 1 hr of reperfusion (135132 ng/mL, n=4) compared to scrambled control (1022346 ng/mL, n=3 p\u3c0.05). Myr-Tat-PKCβII- reduced infarct size to 10.0±2.8%; n=4; compared to scrambled control hearts (28.5±8.3%; n=6; *p\u3c0.05). CPK and Troponin I levels were comparable in both groups. These results suggest that Myr-Tat-PKCβII- can help prevent cardiac injury when given immediately after an ischemic event. Discussion Data from ex-vivo rat heart I/R model, coupled with data from this in-vivo porcine I/R model indicate the efficacy of Myr-Tat-PKCβII- in preventing oxidative stress-induced cardiac injury. These findings suggest that Myr-Tat-PKCβII- could be useful in the clinical setting when administered immediately after cardiac resuscitation following an ischemic event. Future studies include the treatment of human umbilical vein endothelial cells with Myr-Tat-PKCβII- prior to hypoxia and at the beginning of reperfusion conditions, followed by cell viability assays in comparison with the untreated control. This additional data can help determine the optimal dose to use in an 8 week survival study using the same porcine myocardial I/R protocol for Myr-Tat-PKCβII-

Infarct-sparing effects of naltrindole and analogues through inhibition of myocardial hypercontracture during ischemia/reperfusion injury

Coronary artery disease remains the leading cause of death worldwide. Reduction in infarct size may be a potential preventive strategy for cardiovascular complications. Increased left ventricular end-diastolic pressure (LVEDP) or Ischemic Peak Pressure (IPP) during ischemia, is associated with infarct size. Previously, we showed that naltrindole (NTI, selective delta opioid receptor antagonist), reduced LVEDP and infarct size in ex vivo rat hearts. This study examined the effects of NTI and analogues naltriben (NTB, delta opioid receptor antagonist) and guanidinonaltrindole (GNTI, kappa opioid receptor antagonist) (all 5 μM) compared to control hearts. Isolated hearts from male Sprague-Dawley rats (~300g) were subjected to 30-min global ischemia (I)/45-min reperfusion (R) with treatments infused for 5 min before I and during the first 5 min of R. After R, 1% triphenyltetrazolium chloride staining assessed infarct size. Data were evaluated using ANOVA Student-Neuman-Keuls post-hoc analysis. NTI (n=8) and NTB (n=6) elicited a transient decrease in the maximal rate in the rise of LV pressure (dP/dt max) to 1581±379 mmHg/s and 929±243 mmHg/s, respectively when given prior to I compared to control (2471±72 mmHg/s, n=12, p These results suggest that NTI, NTB and GNTI are cardioprotective against myocardial I/R injury. The negative inotropic effects of NTI and NTB were associated with ~75% reduction and GNTI with ~50% reduction in infarct size compared to control. These results suggest that NTI and analogues exert tissue-salvaging effects. Future studies will examine the cardioprotective effects of NTI and analogues given at different ischemic times