Myristoylated Protein Kinase C Epsilon Peptide Inhibitor Exerts Cardioprotective Effects in Rat and Porcine Myocardial Ischemia/Reperfusion: A Translational Research Study

Montgomery, Matthew; Adams, Jovan; Teng, Jane; Tekeleheymanot, Biruk; Ondrasik, Regina; Devine, Issachar; Perkins, Kerry-Anne; Chen, Qian; Barsotti, Robert; Young, Lindon H.

doi:10.17952/23aps.2013.178

Cited by 5 publications

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“…By comparison, we previously showed that 10 µM concentration significantly restored cardiac function (i.e. +dP/dt max ) at 10 min reperfusion and maintained this effect throughout the reperfusion period whether it was 45 or 90 min reperfusion [39]. The results for the current study clearly shows that the 10 µM concentration PKCε-can maintain its cardioprotective effect for an additional 45 min without significantly altering infarct size [i.e.…”

Section: Discussionsupporting

confidence: 66%

“…Interestingly, in a porcine acute intact regional I (1 hr)/R (3 hr) model, Montgomery M et al showed that Myr-PKCε-given IV at reperfusion to yield an estimated circulating concentration of 10 µM (0.8 mg/kg) restored post-reperfused cardiac function (i.e. ejection fraction) by 91±3% of initial baseline values and reduced infarct size to 13±0.3% [39]. In comparison, untreated controls only recovered to 69±3% of initial baseline and had infarct sizes of 34±4%.…”

Section: Discussionmentioning

confidence: 99%

“…Hearts were quickly isolated and attached to a Langendorff apparatus as previously described [39]. The I/R protocol consisted of a 15 min stabilization period, 30 min of global ischemia, and 90 min of reperfusion.…”

Section: Methodsmentioning

confidence: 99%

“…We previously found that Myr-PKCε-(10 μM) given at reperfusion elicited cardioprotective effects in an isolated perfused rat heart model subjected to I (30 min)/R (45 min) [39]. The aim of the present study is to determine the optimal concentration for the cardioprotective effects of Myr-PKCε-when the reperfusion period is doubled to 90 min.…”

Section: Introductionmentioning

confidence: 92%

“…Since BH 2 and BH 4 have equal affinity for the eNOS oxygenase-binding domain, an increase in the BH 2 to BH 4 ratio during reperfusion shifts eNOS towards the uncoupled state [34]. Hence, inhibiting PKCε during reperfusion will attenuate ROS mediated insults arising from uncoupled eNOS activity, the mK ATP channel, and leukocyte endothelial adhesion and penetration.We previously found that Myr-PKCε-(10 μM) given at reperfusion elicited cardioprotective effects in an isolated perfused rat heart model subjected to I (30 min)/R (45 min) [39]. The aim of the present study is to determine the optimal concentration for the cardioprotective effects of Myr-PKCε-when the reperfusion period is doubled to 90 min.…”

mentioning

confidence: 92%

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Protein Kinase C Epsilon PeptideInhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury

2017

J Cardiobiol

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Background: The generation of reactive oxygen species (ROS) during myocardial ischemia (I)/reperfusion (R) contributes to postreperfusion cardiac injury. The increase in ROS is attributed in part to the uncoupling of endothelial nitric oxide synthase (eNOS) that produces ROS instead of nitric oxide (NO) and mitochondrial derived ROS which in part is derived from opening of mitochondrial ATPsensitive K + channels (mK ATP ). Both the activity of uncoupled eNOS and the opening of mK ATP channels in mitochondria are stimulated by protein kinase C epsilon (PKCε) that is activated during reperfusion. We hypothesize that a cell permeable PKCε peptide inhibitor, (N-myristic acid-EAVSLKPT, Myr-PKCε-) given at reperfusion will improve postreperfused cardiac function and attenuate infarct size compared to untreated isolated perfused rat hearts subjected to ischemic reperfusion injury. Methods:Male Sprague-Dawley rats (275-325 g) were anesthetized with sodium pentobarbital (60 mg/kg) and anticoagulated with heparin 1000 units given intraperitoneally. The hearts were excised and attached to a Langendorff perfusion system. All hearts were subjected to 30 min of global ischemia, followed by 90 min of reperfusion. Myr-PKCε-was dissolved in Krebs' buffer and infused during the first 10 min of reperfusion at final concentrations of 5, 10 and 20 µM.Results: Myr-PKCε-treated hearts (10 and 20 μM) exhibited significant improvement in post-reperfused cardiac function at 90 min compared to untreated controls. The maximal rate of left ventricular developed pressure generation (+dP/dt max ) of Myr-PKCε-treated hearts showed significant recovery to 56±4% (10 μM, p<0.05, n=8) and 50±3%; (20 μM, p<0.05, n=8) of initial baseline values. By contrast, the +dP/ dt max of both untreated control hearts (n=9) and those treated with the lowest concentration of Myr-PKCε-(5 μM, n=8) recovered to only 30±4% and 33±4% of initial baseline values, respectively. Interestingly, all Myr-PKCε-treated hearts (5-20 μM) showed significant reduction in infarct size to 28±2% compared to untreated control hearts, which averaged 38±3% (p<0.05, n=9). Conclusion:The results suggest that Myr-PKCε-given at reperfusion effectively reduces infarct size, improves cardiac function and is a putative treatment that could aid in clinical myocardial infarction/ organ transplantation patient recovery. [3][4][5][6][7]. The necessity for effective pharmacological intervention still exists, despite numerous basic studies firmly establishing that I/R injury can be delayed or even reduced by the introduction of cardioprotective agents or strategies prior to a prolonged ischemic insult (preconditioning) or at the start of reperfusion [1,8]. Such interventions have been shown to reduce infarct size, attenuate the frequency and severity of reperfusion-induced arrhythmias, and prevent endothelial dysfunction [9][10][11][12][13].Preconditioning of the myocardium can be induced by several rounds of brief ischemia (i.e. less than 4 min) followed by several rounds of brief reperfus...

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Section: Discussionsupporting

confidence: 66%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 92%

mentioning

confidence: 92%

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Protein Kinase C Epsilon PeptideInhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury

2017

J Cardiobiol

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Modulation of Nitric Oxide Release in Human Umbilical Vein Endothelial Cells by Myristolated‐PKC Epsilon Activator/Inhibitor Peptides

et al. 2018

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Ischemia/reperfusion (I/R) injury plays a critical role in endothelial dysfunction as a consequence of limited nitric oxide (NO) bioavailability and excess generation of reactive oxygen species (ROS) generated by endothelial nitric oxide synthase (eNOS) during reperfusion. Activated protein kinase C epsilon (PKCɛ) binds to a specific receptor for activated C kinase (RACK‐1), facilitating its translocation from the cytosol to the cell membrane for phosphorylation of targets, such as eNOS, at serine‐1177 to augment activity. Previous in vitro and in vivo animal studies have shown that PKCɛ activation stimulates eNOS activity, increasing NO release; whereas PKCɛ inhibition using a peptide inhibitor that interferes with its interaction with RACK‐1 results in decreased NO release. However, the modulation of PKCɛ‐mediated eNOS activity has yet to be demonstrated in human cells. In this study, we aim to demonstrate enhancement and attenuation of NO release in human umbilical vein endothelial cells (HUVECs) using cell‐permeable, myristic‐acid conjugated PKCɛ activator (Myr‐PKCɛ+), Myr‐HDAPIGYD (MW=1097 g/mol), and inhibitor (Myr‐PKCɛ−), Myr‐EAVSLKPT (MW=1054 g/mol), peptides respectively. Single‐donor HUVECs at passages 3–4 were grown to confluence in 6‐well (35 mm) plates. NO release was measured in real time using a calibrated NO electrode following the administration of 10 μM Myr‐PKCɛ+ or 10 μM Myr‐PKCɛ− treatments in the absence or presence of 10 μM acetylcholine (Ach) stimulation of NO release. Basal NO release (375±175 pM) was determined by measuring the difference between wells with and without cells (n=4). Myr‐PKCɛ+ increased NO levels by 36±13 pM (p<0.01, n=5) and Myr‐PKCɛ− decreased NO levels by 41±55 pM, p<0.01, n=5) significantly compared to baseline in the absence of Ach stimulation. In the presence of 10 μM Ach only, NO release significantly increased by 61±15 pM above baseline (p<0.01, n=5). In Ach‐stimulated cells, there was an additional significant increase in NO release by 70±23 pM (p<0.01, n=5) when treated with Myr‐PKCɛ+ compared to baseline, although there was no significance compared to the initial Ach‐stimulated NO release. Following initial 10 μM Ach administration, Myr‐PKCɛ− significantly reduced NO release by 48±16 pM, (p<0.01, n=5) compared to baseline. These results suggest Myr‐PKCɛ+ presumably increases NO release via activation of eNOS, whereas Myr‐PKCɛ− attenuates eNOS activity by inhibiting its phosphorylation in HUVECs; thus, eNOS modulation by these peptides demonstrate comparable effects across species. Elucidating the role of eNOS modulation is important for developing therapeutic interventions in the context of human organ I/R injury, in which uncoupled eNOS produces ROS instead of NO. Future studies will evaluate the effects of Myr‐PKCɛ+ and Myr‐PKCɛ− on NO release in HUVECs during reperfusion following prolonged ischemia compared to non‐ischemic controls.Support or Funding InformationThis study was supported by the Center for Chronic Disorders of Aging, the Division of Research and the Department of Biomedical Sciences at Philadelphia College of Osteopathic Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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Protein Kinase C Epsilon Peptide Inhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury

et al. 2017

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During myocardial ischemia/reperfusion (I/R), the generation of reactive oxygen species (ROS) contributes to post‐reperfusion cardiac injury and contractile dysfunction. Activation of protein kinase C epsilon (PKC ɛ) during I/R has been shown to increase ROS release, in part, by its stimulation of increased uncoupled endothelial nitric oxide synthase activity. We hypothesize that using a cell permeable PKC ɛ peptide inhibitor (PKC ɛ‐) (N‐myr‐EAVSLKPT, MW=1054 g/mol, 10μM or 20μM) will improve post‐reperfused cardiac function and attenuate infarct size compared to untreated controls in isolated perfused rat hearts subjected to I(30 min)/R(90 min). Male Sprague‐Dawley rats (275–325 g) were anesthetized with sodium pentobarbital (60 mg/kg) and anticoagulated with heparin 1000 units IP. PKC ɛ‐was dissolved in Krebs' buffer and infused during the first 10 min of reperfusion. PKC ɛ‐treated hearts exhibited significant improvement in post‐reperfused cardiac function at 90 min in the maximal rate of left ventricular developed pressure (+dP/dtmax): 56±5%; n=6 (10μM) and 46±3%; n=4 (20μM) compared to untreated controls (n=6) which only recovered to 32±5% of baseline values for +dP/dtmax respectively (p<0.05). Furthermore, PKC ɛ‐treated hearts showed significant reduction in infarct size of 27±2% (10μM) and 28 ±2% (20μM) compared to untreated control I/R hearts, 40±3% (p<0.05). The results suggest that PKC ɛ‐is effective in improving cardiac function and reducing infarct size and is a putative treatment that could aid in clinical myocardial infarction/organ transplantation patient recovery.Support or Funding InformationThis study was supported by the Center for Chronic Disorders of Aging, the Division of Research and the Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.

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Myristoylated Protein Kinase C Epsilon Peptide Inhibitor Exerts Cardioprotective Effects in Rat and Porcine Myocardial Ischemia/Reperfusion: A Translational Research Study

Cited by 5 publications

References 0 publications

Protein Kinase C Epsilon PeptideInhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury

Protein Kinase C Epsilon PeptideInhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury

Modulation of Nitric Oxide Release in Human Umbilical Vein Endothelial Cells by Myristolated‐PKC Epsilon Activator/Inhibitor Peptides

Protein Kinase C Epsilon Peptide Inhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury

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