Pretreatment with Naltrindole exhibits robust cardioprotection in an isolated rat heart model of ischemia-reperfusion injury

Previously, a novel three-amino acid peptide (tripeptide) that is structurally similar to other amino acid-based delta and kappa-opioid receptor agonists (the predominant opioid receptor subtypes in heart tissue) demonstrated a significant reduction in infarct size and improved cardiac function when administered during preconditioning in isolated rat hearts using the Langendorf model. The cardioprotective effects of tripeptide were blocked by naloxone (NX, broad-spectrum opioid antagonist) and nor-binaltorphine (BNI, kappa-opioid receptor antagonist), whereas naltrindole (NTI, delta-opioid receptor antagonist) seemed to augment the effects of tripeptide. To determine whether the cardioprotective effects of the combination were due to tripeptide or NTI, the effects of NTI and other opioid antagonists were evaluated individually in the same model. Therefore, the goal was to evaluate the effects of NTI, BNI, and NX independently. Hearts isolated from male Sprague-Dawley rats (~300g) were subjected to global ischemia (I, 30min)/reperfusion (R, 50min). NX (10 μM, n=6), BNI (5 μM, n=7), NTI (5 μM, n=8), or Krebs’ buffer control (control n=10) were given to the hearts 5 min prior to ischemia and during the first 5 min of reperfusion in a Langendorf model, perfused at a constant pressure of 80mmHg. Left ventricular (LV) functional indices were measured using an indwelling pressure transducer-tipped catheter. At the end of reperfusion (50min), hearts were frozen, sectioned (2 mm), and stained with 1% triphenyltetrazolium chloride (TTC). To determine infarct size, the weight of infarcted tissue was compared to total tissue at risk. All data were evaluated using ANOVA Student-Newman-Keuls post-hoc analysis. Control (untreated) hearts showed an elevated final LV end-diastolic pressure (LVEDP) of 60 ± 5 mmHg compared to an average initial baseline of 8 ± 1 mmHg (similar in all groups) and a mean infarct size of 36 ± 3% at 50 min post-reperfusion. NX and BNI demonstrated no significant cardioprotective effects compared to control with final LVEDP measures of 64 ± 8 mmHg and 61 ± 2 mmHg, respectively. Infarct size for NX (35 ± 5%) and BNI (36 ± 5%) treated hearts were similar to control hearts. By contrast, NTI significantly improved final LVEDP (17 ± 3mmHg) to near baseline values and reduced infarct size to 7 ± 2% compared to all groups (p\u3c 0.01). The results indicate that pretreatment with NTI can prevent I/R injury and restore post-reperfused heart function to near pre-ischemic levels. In future studies, we will evaluate the cardioprotective effects of NTI in an acute in vivo heart I/R model. In separate studies, we will attempt to determine the mechanism of action of NTI preconditioning, specifically whether cardioprotection is indeed mediated via delta receptor antagonism or via some other mechanism

Combinations of Host Biomarkers Predict Mortality among Ugandan Children with Severe Malaria: A Retrospective Case-Control Study

Background: Severe malaria is a leading cause of childhood mortality in Africa. However, at presentation, it is difficult to predict which children with severe malaria are at greatest risk of death. Dysregulated host inflammatory responses and endothelial activation play central roles in severe malaria pathogenesis. We hypothesized that biomarkers of these processes would accurately predict outcome among children with severe malaria. Methodology/Findings: Plasma was obtained from children with uncomplicated malaria (n = 53), cerebral malaria (n = 44) and severe malarial anemia (n = 59) at time of presentation to hospital in Kampala, Uganda. Levels of angiopoietin-2, von Willebrand Factor (vWF), vWF propeptide, soluble P-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), soluble endoglin, soluble FMS-like tyrosine kinase-1 (Flt-1), soluble Tie-2, C-reactive protein, procalcitonin, 10 kDa interferon gamma-induced protein (IP-10), and soluble triggering receptor expressed on myeloid cells-1 (TREM-1) were determined by ELISA. Receiver operating characteristic (ROC) curve analysis was used to assess predictive accuracy of individual biomarkers. Six biomarkers (angiopoietin-2, soluble ICAM-1, soluble Flt-1, procalcitonin, IP-10, soluble TREM-1) discriminated well between children who survived severe malaria infection and those who subsequently died (area under ROC curve>0.7). Combinational approaches were applied in an attempt to improve accuracy. A biomarker score was developed based on dichotomization and summation of the six biomarkers, resulting in 95.7% (95% CI: 78.1-99.9) sensitivity and 88.8% (79.7-94.7) specificity for predicting death. Similar predictive accuracy was achieved with models comprised of 3 biomarkers. Classification tree analysis generated a 3-marker model with 100% sensitivity and 92.5% specificity (cross-validated misclassification rate: 15.4%, standard error 4.9%). Conclusions: We identified novel host biomarkers of pediatric severe and fatal malaria (soluble TREM-1 and soluble Flt-1) and generated simple biomarker combinations that accurately predicted death in an African pediatric population. While requiring validation in further studies, these results suggest the utility of combinatorial biomarker strategies as prognostic tests for severe malaria

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-

Myristic Acid-Trans-Activator of Transcription Dual Conjugation Enhances Intracellular delivery of protein kinase C beta II peptide inhibitor for concentration-dependent attenuation of superoxide release in isolated rat polymorphonuclear leukocytes

Protein kinase C beta II (PKCβII) activation promotes polymorphonuclear (PMN) superoxide (SO) production by phosphorylating serine and threonine amino acid residues on NADPH oxidase (NOX-2). Previously, myristic acid conjugated (or myristoylated) PKCβII inhibitor (myr-PKCβII-) significantly attenuated PMN SO release when dual conjugated to myristic acid and Trans-activator of transcription (myr-Tat-PKCβII-; N-myr-Tat-CC-SLNPEWNET) compared to myr-conjugation alone. However, the optimal concentration of myr-Tat-PKCβII- has yet to be determined. We hypothesized that myr-Tat conjugation would enhance the intracellular delivery of PKCβII- cargo and attenuate SO release in a concentration-dependent manner while retaining greater cell viability at lower concentrations. This study tested the concentration-dependent effects of myr-Tat-PKCβII- on SO release and cell viability compared to myr-Tat-PKCβII scrambled (myr-Tat-PKCβII- scram; N-myr-Tat-CC-WNPESLNTE), unconjugated PKCβII-, and untreated control group. Rat PMNs were incubated for 15 min at 37°C with either unconjugated PKCβII- (20μM), myr-Tat-PKCβII- (2μM, 5μM, 7.5μM, 10μM, and 20μM), or myr-Tat-PKCβII-scram (2μM, 5μM, 7.5μM, 10μM, and 20μM). PMN SO release was calculated by the change in absorbance at 550 nm over 390 sec via ferricytochrome c reduction after phorbol-12-myristate-13 acetate (PMA) stimulation (100nM). Intracellular delivery was evaluated by the magnitude of PMA-induced PMN SO release attenuation with the PKCβII- cargo. Data were analyzed with ANOVA Fisher’s PLSD post-hoc analysis. Myr-Tat toxicity occurs at concentrations higher than 2 μM. Myr-Tat PKCβII- significantly decreased SO release compared to control while retaining similar cell viability at 5 μM (n=15, 0.384±0.03) and 7.5μM (n=11, 0.391±0.05) concentrations. Myr-Tat PKCβII-scram 5μM (n=7, 0.44±0.07) and 7.5μM (n=5, 0.409±0.11) were not different from control. Results suggest that Myr-Tat-PKCβII- 5μM significantly attenuates PMA-induced PMA SO release while retaining cell viability at \u3e 80%. Future studies will assess the effects of myr- or myr-Tat-PKCβII- peptides on PKCβII- translocation activity

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