Peptides conjugated to myristic acid (myr) or trans activator of transcription (tat) have long been used to increase cell permeability via simple diffusion and endocytosis, respectively. Previously myr-PKCβII inhibitor (myr-PKCβII-) exerted cardioprotective effects in myocardial ischemia/reperfusion (I/R) and inhibited superoxide (SO) release from rat leukocytes by 40% (20 μM). By contrast, dual conjugated myr-tat-PKCβII inhibitor (myr-tat-PKCβII-) inhibited SO release by 90% at the same concentration. The purpose of this study was to determine the potency of myr-tat-PKCβII- compared to myr-PKCβII- in mitigating infarct size and restoring cardiac function in an ex-vivo rat model of myocardial I/R injury. Isolated hearts from male Sprague-Dawley rats (~300g) were subjected to global I(30-min)/R(50-min) . Left ventricular cardiac function was recorded using a pressure transducer. Treatments were infused during the first 5 min of R. After R, the hearts were sectioned (2 mm) from base to apex and stained with 1% triphenyltetrazolium chloride. Infarcted tissue was excised to determine infarct size (i.e. infarct tissue weight/ total tissue weight). Data were analyzed using ANOVA Fisher’s LSD analysis. Compared to untreated controls (23.2±3.1%, n=17), myr-tat-PKCβII- exerted a significant and similar decrease in infarct size that was observed from 100 nM (9.7±0.59%, n=3, p<0.05), 1 nM (10.0±2.3%, n=5, p<0.05), to 100 pM (12.6±2.3%, n=5, p<0.05); but not at 10 nM (13.8±3.6%, n=5). Cardiac function for all treatment groups did not significantly improve from control. However, at 100pM (1070±170 mmHg/s), +dP/dt max was significantly improved compared to all other treatment groups (p<0.05). The addition of myr to tat-conjugated peptides improved the cardioprotective effects ~200-fold compared to myr-PKCβII- and ~2000-fold compared to native peptide presumably by enhanced intracellular delivery of cargo. The ~50% reduction in infarct size suggests that myr-tat-PKCβII- is cardioprotective in I/R. Future studies will test our most effective dose determined in ex vivo hearts to restore post-reperfusion cardiac function combined with significant cardiac salvaging effect (reduced infarct size) in a porcine myocardial I/R model.
Ischemic heart disease remains the leading cause of death worldwide. Pharmacological agents that mimic the cardioprotective effects of ischemic preconditioning may have therapeutic potential as a secondary prevention strategy to resist infarction from subsequent cardiovascular events. Increased left ventricular end diastolic pressure (LVEDP) during ischemia, or ischemic peak pressure (IPP), is known to be correlated to infarct size. Recently our laboratory demonstrated that naltrindole (NTI), a selective delta opioid receptor antagonist, reduces IPP and infarct size when given prior to ischemia (preconditioning) in a Langendorff rat heart model. The purpose of this study was to examine the effects of NTI analogues naltriben (NTB, delta opioid receptor antagonist) and guanidinonaltrindole (GNTI, kappa opioid receptor antagonist) compared to NTI. Nor‐binaltrophine (BNI, kappa opioid receptor antagonist) and naloxone (NX, broad‐spectrum opioid receptor antagonist) were tested to evaluate cardioprotection by other opioid receptor antagonists. 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. LV cardiac function was measured using a pressure transducer. At the end of reperfusion, infarct size was assessed using 1% triphenyltetrazolium chloride staining and defined as infarcted tissue/total area at risk. Data were evaluated using ANOVA Student‐Neuman‐Keuls post‐hoc analysis. Control I/R hearts demonstrated an IPP of 39±3 mmHg compared to pre‐ischemic LVEDP of 9±1 mmHg at baseline (n=12, p<0.01), resulting in substantial infarct at the end of 45 min R (32±4%). NTI (n=7) and NTB (n=6) elicited cardiodepressive effects during preconditioning by reducing the maximal rate in the rise of LV pressure (dP/dt max) to 1581±379 mmHg/s and 929±243 mmHg/s, respectively, compared to control (2471±72 mmHg/s, p<0.01). IPP was reduced by NTI (18±3 mmHg/s) and NTB (15±3 mmHg/s) compared to all groups (p<0.05). Post‐reperfused dP/dt max and infarct size was most improved following NTI (1830±90 mmHg/s, 7±2%) and NTB (1846±140 mmHg/s, 7±2%) pretreatment, compared to control (777±142 mmHg/s, 32±4%, p<0.01). GNTI reduced infarct size (17 ± 4%, n=6, p<0.05), but did not exert a negative inotropic effect. Cardiac function and infarct size did not improve with BNI (n=7) or NX (n=6) pretreatment. These results suggest that NTI and analogues, GNTI and NTB, are cardioprotective against myocardial I/R injury. The negative inotropic effects of NTI and NTB were associated with ~75% reduction in infarct size compared to control. GNTI decreased infarct size by ~50% and these results suggest that NTI, NTB, and GNTI exert tissue‐salvaging effects independent of delta or kappa opioid receptor antagonism. In future studies, we will examine different ischemic time points to administer NTI and its analogues to determine optimal cardioprotection and investigate downstream effects on calcium handling.
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). In previous studies, cell‐permeable myristic acid conjugated PKCβII inhibitor (myr‐PKCβII‐) significantly attenuated PMN SO release induced by phorbol 12‐myristate 13‐acetate (PMA), a diacylglycerol mimetic. Myr‐PKCβII‐ was determined to be superior to unconjugated peptides and nontreated controls, suggesting enhanced intracellular delivery of cargo. We hypothesize that the simple diffusion of myr‐conjugation combined with the endocytotic mechanism of trans‐activator of transcription (Tat) would optimize the intracellular delivery of PKCβII‐ cargo compared to myr‐conjugation alone. In this study, we tested the concentration‐dependent effects of a dual myr‐Tat conjugated PKCβII‐ (myr‐Tat‐PKCβII‐; N‐myr‐Tat‐CC‐SLNPEWNET) on intracellular delivery compared to myr‐PKCβII‐, scrambled myr‐Tat‐PKCβII‐ (myr‐Tat‐PKCβII‐ scram), unconjugated PKCβII‐, and 0.5% dimethyl sulfoxide (DMSO) vehicle 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 PMA stimulation (100nM). The efficacy of intracellular drug 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‐PKCβII‐ 5μM (n=12, 0.392±0.04), 7.5μM (n=11, 0.397±0.05), 10μM (n=5, 0.211±0.05) and 20μM (n=5, 0.121±0.02) demonstrated a concentration‐dependent increase in intracellular delivery compared to DMSO vehicle control (n=84, 0.496±0.02, all p<0.05). Myr‐PKCβII‐ only significantly increased intracellular delivery at the 20μM concentration (n=27, 0.303±0.02, p<0.05) compared to DMSO vehicle control. Intracellular delivery of myr‐Tat‐PKCβII‐ 2μM (n=10, 0.436±0.06) and all concentrations of myr‐Tat‐PKCβII‐scram were not significantly different from DMSO vehicle controls. Results suggest that myr‐Tat dual conjugation is superior to myr‐conjugation alone at intracellular delivery of cell impermeant cargo. Future studies will investigate the concentration‐dependent effects of PKCβII‐ peptide conjugates on PMA‐induced PKCβII activity and translocation to membrane targets, such as NOX‐2, using immunocytochemistry and western blot analysis.
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