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1.  Identification of εPKC targets during cardiac ischemic injury 
Background
Activation of ε protein kinase C (εPKC) protects hearts from ischemic injury. However, some of the mechanism(s) of εPKC mediated cardioprotection are still unclear. Identification of εPKC targets may aid to elucidate εPKC–mediated cardioprotective mechanisms. Previous studies, using a combination of εPKC transgenic mice and difference in gel electrophoresis (DIGE), identified a number of proteins involved in glucose metabolism, whose expression was modified by εPKC. These studies, were accompanied by metabolomic analysis, and suggested that increased glucose oxidation may be responsible for the cardioprotective effect of εPKC. However, whether these εPKC-mediated alterations were due to differences in protein expression or phosphorylation was not determined.
Methods and Results
Here, we used an εPKC-specific activator peptide, ψεRACK, in combination with phosphoproteomics to identify εPKC targets, and identified proteins whose phosphorylation was altered by selective activation of εPKC most of the identified proteins were mitochondrial proteins and analysis of the mitochondrial phosphoproteome, led to the identification of 55 spots, corresponding to 37 individual proteins, which were exclusively phosphorylated, in the presence of ψεRACK. The majority of the proteins identified were proteins involved in glucose and lipid metabolism, components of the respiratory chain as well as mitochondrial heat shock proteins.
Conclusion
In summary the protective effect of εPKC during ischemia involves phosphorylation of several mitochondrial proteins involved in glucose, lipid metabolism and oxidative phosphorylation. Regulation of these metabolic pathways by εPKC phosphorylation may lead to εPKC-mediated cardioprotection induced by ψεRACK.
PMCID: PMC3527096  PMID: 22453000
εPKC; ischemia; phosphorylation; mitochondria
2.  Ischaemic preconditioning improves proteasomal activity and increases the degradation of δPKC during reperfusion 
Cardiovascular Research  2009;85(2):385-394.
Aims
The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, δ and εPKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection.
Methods and results
Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished δPKC translocation by 3.8-fold and increased εPKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of δPKC decreased by 60 ± 2.7% in response to IPC, whereas the levels of εPKC did not significantly change. Prolonged ischaemia induced a 48 ± 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 ± 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of εPKC during IPC restored δPKC levels at the mitochondria while decreasing εPKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a δPKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol.
Conclusion
Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, δPKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, εPKC.
doi:10.1093/cvr/cvp334
PMCID: PMC2797452  PMID: 19820255
Cardioprotection; Ischaemia/reperfusion; Apoptosis; Proteasome; PKC; Ischaemic preconditioning
3.  εPKC phosphorylates the mitochondrial KATP+ channel during induction of ischemic preconditioning in the rat hippocampus 
Brain research  2007;1184:345-353.
Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (εPKC). A major goal in our laboratory is to define the cellular targets by which εPKC confers protection. We tested the hypothesis that εPKC targets the mitochondrial KATP+ channel ( mtKATP+) after IPC. Our results demonstrated a rapid translocation of εPKC to rat hippocampal mitochondria after IPC. Because in other tissues εPKC targets mtKATP+ channels, but its presence in brain mitochondria is controversial, we determined the presence of the KATP+ channel-specific subunits (Kir6.1 and Kir6.2) in mitochondria isolated from rat hippocampus. Next, we determined whether mtKATP+ channels play a role in the IPC induction. In hippocampal organotypic slice cultures, IPC and lethal ischemia were induced by oxygen-glucose deprivation. Subsequent cell death in the CA1 region was quantified using propidium iodide staining. Treatment with the KATP+ channel openers diazoxide or pinacidil 48 h prior to lethal ischemia protected hippocampal CA1 neurons, mimicking the induction of neuroprotection conferred by either IPC or εPKC agonist-induced preconditioning. Blockade of mtKATP+ channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or εPKC preconditioning. Both ischemic andεPKC agonist-mediated preconditioning resulted in phosphorylation of the mtKATP+ channel subunit Kir6.2. After IPC, selective inhibition of εPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of εPKC or its downstream effectors. Our results support the hypothesis that the brain mtKATP+ channel is an important target of IPC and the signal transduction pathways initiated by εPKC.
doi:10.1016/j.brainres.2007.09.073
PMCID: PMC2577914  PMID: 17988655
ischemic tolerance; diazoxide; protein kinase C; organotypic slice culture; cell death; signal transduction
4.  Dopamine and Ethanol Cause Translocation of εPKC Associated with εRACK: Cross-talk Between PKA and PKC Signaling Pathways 
Molecular pharmacology  2008;73(4):1105-1112.
Previously we found that neural responses to ethanol and the dopamine D2 receptor (D2) agonist NPA involve both epsilon protein kinase C (εPKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of εPKC and the relationship to PKA activation. In the present study, we used a new εPKC antibody, 14E6, that selectively recognizes active εPKC when not bound to its anchoring protein εRACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators, to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated εPKC and also induced translocation of both εPKC and its anchoring protein, εRACK to a new cytosolic site. The selective εPKC agonist, pseudo-εRACK, activated εPKC but did not cause translocation of the εPKC/εRACK complex to the cytosol. These data suggest a step-wise activation and translocation of εPKC following NPA or ethanol treatment where εPKC first translocates and binds to its RACK and subsequently the εPKC/εRACK complex translocates to a new subcellular site. Direct activation of PKA by Sp-cAMPS, PGE1 or the adenosine A2A receptor is sufficient to cause εPKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel cross-talk mechanism between εPKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Cross-talk between PKA and PKC may underlie some of the behaviors associated with alcoholism.
doi:10.1124/mol.107.042580
PMCID: PMC2692587  PMID: 18202306
5.  Post-Ischemic Activation of Protein Kinase C Epsilon Protects the Hippocampus from Cerebral Ischemic Injury via Alterations in Cerebral Blood Flow 
Neuroscience letters  2010;487(2):158-162.
Protein Kinase C (PKC) is a family of serine/threonine-isozymes that are involved in many signaling events in normal and disease states. Previous studies from our lab have demonstrated that εPKC plays a pivotal role in neuroprotection induced by ischemic preconditioning. However, the role of εPKC during and after brain ischemia is not clearly defined. Therefore, in the present study, we tested the hypothesis that activation of εPKC during an ischemic event is neuroprotective. Furthermore, other studies have demonstrated that εPKC mediates cerebral ischemic tolerance in the rat brain by decreasing vascular tone. Thus, we also tested the effects of εPKC activation during ischemia on cerebral blood flow (CBF). We found that ψε-Receptors for activated C kinase (RACK), a εPKC-selective peptide activator, injected intravenously 30 minutes before induction of global cerebral ischemia conferred neuroprotection in the CA1 region of the rat hippocampus. Moreover, measurements of CBF before, during and after cerebral ischemia revealed a significant reduction in the reperfusion phase of rats pretreated with ψεRACK compared to Tat peptide (vehicle). Our results suggest that εPKC can protect the rat brain against ischemic damage by regulating CBF. Thus, εPKC may be one of the treatment modalities against ischemic injury.
doi:10.1016/j.neulet.2010.10.013
PMCID: PMC3004991  PMID: 20951185
Ischemia; epsilon Protein Kinase C; Cerebral Blood Flow; Neuroprotection
6.  Activation of aldehyde dehydrogenase 2 (ALDH2) confers cardioprotection in protein kinase C epsilon (PKCε) knockout mice 
Acute administration of ethanol can reduce cardiac ischemia/reperfusion injury. Previous studies demonstrated that the acute cytoprotective effect of ethanol on the myocardium is mediated by protein kinase C epsilon (PKCε). We recently identified aldehyde dehydrogenase 2 (ALDH2) as an PKCε substrate, whose activation is necessary and sufficient to confer cardioprotection in vivo. ALDH2 metabolizes cytotoxic reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), which accumulate during cardiac ischemia/reperfusion. Here, we used a combination of PKCε knockout mice and a direct activator of ALDH2, Alda-44, to further investigate the interplay between PKCε and ALDH2 in cardioprotection. We report that ethanol preconditioning requires PKCε, whereas direct activation of ALDH2 reduces infarct size in both wild type and PKCε knockout hearts. Our data suggest that ALDH2 is downstream of PKCε in ethanol preconditioning and that direct activation of ALDH2 can circumvent the requirement of PKCε to induce cytoprotection. We also report that in addition to ALDH2 activation, Alda-44 prevents 4-HNE induced inactivation of ALDH2 by reducing the formation of 4-HNE-ALDH2 protein adducts. Thus, Alda-44 promotes metabolism of cytotoxic reactive aldehydes that accumulate in ischemic myocardium. Taken together, our findings suggest that direct activation of ALDH2 may represent a method of harnessing the cardioprotective effect of ethanol without the side effects associated with alcohol consumption.
doi:10.1016/j.yjmcc.2009.10.030
PMCID: PMC2837767  PMID: 19913552
7.  Activation of εPKC Reduces Reperfusion Arrhythmias and Improves Recovery from Ischemia: Optical Mapping of Activation Patterns in the Isolated Guinea-pig Heart 
Introduction
Pervious biochemical and hemodymanic studies have highlighted the important role of εPKC in cardioprotection during ischemic preconditioning. However, little is known about the electrophysiological consequences of εPKC modulation in ischemic hearts. Membrane permeable peptide εPKC selective activator and inhibitor were used to investigate the role of εPKC modulation in reperfusion arrhythmias.
Methods
Protein transduction domain from HIV- TAT was used as a carrier for peptide delivery into intact Langendorff perfused guinea pig hearts. Action potentials were imaged and mapped (124 sites) using optical techniques and surface ECG was continuously recorded. Hearts were exposed to 30 min stabilization period, 15 min of no-flow ischemia, followed by 20 min reperfusion. Peptides (0.5 μM) were infused as follows: a) control (vehicle-TAT peptide; TAT-scrambled ψεRACK peptide); b) εPKC agonist (TAT-ψεRACK); c) εPKC antagonist (TAT-εV1).
Results
Hearts treated with εPKC agonist ψεRACK had reduced incidence of ventricular tachycardia (VT, 64%) and fibrillation (VF, 50%) compared to control (VT, 80%, p<0.05) and (VF, 70%, P<0.05). However, the highest incidence of VT (100%, P<0.05) and VF (80%) occurred in hearts treated with εPKC antagonist peptide εV1 compared to control and to εPKC agonist ψεRACK. Interestingly, at 20 min reperfusion, 100% of hearts treated with εPKC agonist ψεRACK exhibited complete recovery of action potentials compared to 40% (p<0.05) of hearts treated with εPKC antagonist peptide, εV1 and 65% (P<0.5) of hearts in control. At 20 min reperfusion, maps of action potential duration from εPKC agonist ψεRACK showed minimal dispersion (48.2±9 ms) compared to exacerbated dispersion (115.4±42 ms, P<0.05) in εPKC antagonist and control (67±20 ms, P<0.05). VT/VF and dispersion from hearts treated with scrambled agonist or antagonist peptides were similar to control.
In conclusion
the results demonstrate that εPKC activation by ψεRACK peptide protects intact hearts from reperfusion arrhythmias and affords better recovery. On the other hand, inhibition of εPKC increased the incidence of arrhythmias and worsened recovery compared to controls. The results carry significant therapeutic implications for the treatment of acute ischemic heart disease by preconditioning-mimicking agents.
doi:10.1016/j.bbrc.2012.08.073
PMCID: PMC3459326  PMID: 22935420
cardiac electrophysiology; Protein Kinase C; reperfusion arrhythmia; optical mapping
8.  Aldehydic load and aldehyde dehydrogenase 2 profile during the progression of post-myocardial infarction cardiomyopathy: benefits of Alda-1 
Background/Objectives
We previously demonstrated that reducing cardiac aldehydic load by aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme responsible for metabolizing the major lipid peroxidation product, protects against acute ischemia/reperfusion injury and chronic heart failure. However, time-dependent changes in ALDH2 profile, aldehydic load and mitochondrial bioenergetics during progression of post-myocardial infarction (post-MI) cardiomyopathy is unknown and should be established to determine the optimal time window for drug treatment.
Methods
Here we characterized cardiac ALDH2 activity and expression, lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) adduct formation, glutathione pool and mitochondrial energy metabolism and H2O2 release during the 4 weeks after permanent left anterior descending (LAD) coronary artery occlusion in rats.
Results
We observed a sustained disruption of cardiac mitochondrial function during the progression of post-MI cardiomyopathy, characterized by >50% reduced mitochondrial respiratory control ratios and up to 2 fold increase in H2O2 release. Mitochondrial dysfunction was accompanied by accumulation of cardiac and circulating lipid peroxides and 4-HNE protein adducts and down-regulation of electron transport chain complexes I and V. Moreover, increased aldehydic load was associated with a 90% reduction in cardiac ALDH2 activity and increased glutathione pool. Further supporting an ALDH2 mechanism, sustained Alda-1 treatment (starting 24hrs after permanent LAD occlusion surgery) prevented aldehydic overload, mitochondrial dysfunction and improved ventricular function in post-MI cardiomyopathy rats.
Conclusion
Taken together, our findings demonstrate a disrupted mitochondrial metabolism along with an insufficient cardiac ALDH2-mediated aldehyde clearance during the progression of ventricular dysfunction, suggesting a potential therapeutic value of ALDH2 activators during the progression of post-myocardial infarction cardiomyopathy.
doi:10.1016/j.ijcard.2014.10.140
PMCID: PMC4405147  PMID: 25464432
myocardial infarction; 4-hydroxinonenal; oxidative stress; bioenergetics; aldehyde dehydrogenase 2
9.  Nociceptive-induced Myocardial Remote Conditioning Is Mediated By Neuronal Gamma Protein Kinase C 
Basic research in cardiology  2013;108(5):10.1007/s00395-013-0381-x.
Deciphering the remote conditioning molecular mechanism may provide targets to develop therapeutics that can broaden the clinical application. To further investigate this, we tested whether two protein kinase C isozymes, the ubiquitously expressed epsilon PKC (εPKC) and the neuronal specific gamma PKC (γPKC), mediate nociceptive-induced remote myocardial conditioning.
Male Sprague-Dawley rats were used for both in vivo and ex vivo myocardial ischemia-reperfusion protocols. For the in vivo studies, using a surgical abdominal incision for comparison, applying only to the abdomen either bradykinin or the εPKC activator (ψεRACK) reduced myocardial infarct size (45±1%, 44±2%, respectively, versus incision: 43±2%, and control: 63±2%, P < 0.001). Western blot showed only εPKC, and not γPKC, is highly expressed in the myocardium. However, applying a selective γPKC inhibitor (γV5-3) to the abdominal skin blocked remote protection by any of these strategies.
Using an ex vivo isolated heart model without an intact nervous system, only selective εPKC activation, unlike a selective classical PKC isozyme activator (activating α, β, βII and γ), reduced myocardial injury. Importantly, the classical PKC isozyme activator given to the abdomen in vivo (with an intact nervous system including γPKC) during myocardial ischemia reduced infarct size as effectively as an abdominal incision or ψεRACK (45±1% versus 45±2% and 47±1%, respectively). The classical PKC activator-induced protection was also blocked by spinal cord surgical transection.
These findings identified potential remote conditioning mimetics, with these strategies effective even during myocardial ischemia. A novel mechanism of nociceptive-induced remote conditioning, involving γPKC, was also identified.
doi:10.1007/s00395-013-0381-x
PMCID: PMC3856950  PMID: 23982492
infarct size; remote; incision; protein kinase C; gamma; epsilon
10.  Mitochondrial Aldehyde Dehydrogenase 2 Plays Protective Roles in Heart Failure After Myocardial Infarction via Suppression of the Cytosolic JNK/p53 Pathway in Mice 
Background
Increasing evidence suggests a critical role for mitochondrial aldehyde dehydrogenase 2 (ALDH2) in protection against cardiac injuries; however, the downstream cytosolic actions of this enzyme are largely undefined.
Methods and Results
Proteomic analysis identified a significant downregulation of mitochondrial ALDH2 in the heart of a rat heart failure model after myocardial infarction. The mechanistic insights underlying ALDH2 action were elucidated using murine models overexpressing ALDH2 or its mutant or with the ablation of the ALDH2 gene (ALDH2 knockout) and neonatal cardiomyocytes undergoing altered expression and activity of ALDH2. Left ventricle dilation and dysfunction and cardiomyocyte death after myocardial infarction were exacerbated in ALDH2‐knockout or ALDH2 mutant‐overexpressing mice but were significantly attenuated in ALDH2‐overexpressing mice. Using an anoxia model of cardiomyocytes with deficiency in ALDH2 activities, we observed prominent cardiomyocyte apoptosis and increased accumulation of the reactive aldehyde 4‐hydroxy‐2‐nonenal (4‐HNE). We subsequently examined the impacts of mitochondrial ALDH2 and 4‐HNE on the relevant cytosolic protective pathways. Our data documented 4‐HNE‐stimulated p53 upregulation via the phosphorylation of JNK, accompanying increased cardiomyocyte apoptosis that was attenuated by inhibition of p53. Importantly, elevation of 4‐HNE also triggered a reduction of the cytosolic HSP70, further corroborating cytosolic action of the 4‐HNE instigated by downregulation of mitochondrial ALDH2.
Conclusions
Downregulation of ALDH2 in the mitochondria induced an elevation of 4‐HNE, leading to cardiomyocyte apoptosis by subsequent inhibition of HSP70, phosphorylation of JNK, and activation of p53. This chain of molecular events took place in both the mitochondria and the cytosol, contributing to the mechanism underlying heart failure.
doi:10.1161/JAHA.113.000779
PMCID: PMC4323818  PMID: 25237043
ALDH2; apoptosis; heart failure; myocardial infarction; p53
11.  Nicorandil attenuates carotid intimal hyperplasia after balloon catheter injury in diabetic rats 
Background
Diabetic patients suffer from undesired intimal hyperplasia after angioplasty. Nicorandil has a trend to reduce the rate of target lesion revascularization. However, whether nicorandil inhibits intimal hyperplasia and the possible mechanisms underlying it remain to be determined. We aimed at assessing the effect of nicorandil on intimal hyperplasia in diabetic rats.
Methods
After intraperitoneal injection of streptozotocin (STZ, 50 mg/kg), balloon injury model was established in carotid arteries of diabetic rats. Rats were randomized to vehicle, nicorandil (15 mg/kg/day) or 5-hydroxydecanoate (5-HD, 10 mg/kg/day), a mitochondrial ATP-sensitive potassium channel (mitoKATP channel)-selective antagonist. Perivascular delivery of εPKC siRNA was conducted to determine the role of εPKC pathway in intimal hyperplasia. In hyperglycemia environment (25 mM glucose), primary culture of vascular smooth muscle cells (VSMCs) were treated with nicorandil or 5-HD. Cell proliferation and cell migration were analyzed.
Results
Intimal hyperplasia significantly increased 14 days after balloon injury in diabetic rats (p < 0.01). Nicorandil inhibited intima development, reduced inflammation and prevented cell proliferation in balloon-injured arteries (p < 0.01). The protective effects of nicorandil were reversed by 5-HD (p < 0.05). εPKC was activated in balloon-injured arteries (p < 0.01). Nicorandil inhibited εPKC activation by opening mitoKATP channel. Perivascular delivery of εPKC siRNA inhibited intimal hyperplasia, inflammation and cell proliferation (p < 0.01). High glucose-induced VSMCs proliferation and migration were inhibited by nicorandil. εPKC activation induced by high glucose was also inhibited by nicorandil and that is partially reversed by 5-HD. εPKC knockdown prevented VSMCs proliferation and migration (p < 0.01).
Conclusions
Our study demonstrates that nicorandil inhibits intimal hyperplasia in balloon-injured arteries in diabetic rats. Nicorandil also prevents VSMCs proliferation and migration induced by high glucose. The beneficial effect of nicorandil is conducted via opening mitoKATP channel and inhibiting εPKC activation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12933-016-0377-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12933-016-0377-6
PMCID: PMC4826484  PMID: 27059601
Nicorandil; Intimal hyperplasia; Diabetes mellitus; ATP-sensitive potassium channel; Protein kinase C
12.  Ischemic Preconditioning Mediates Cyclooxygenase-2 Expression Via Nuclear Factor-Kappa B Activation in Mixed Cortical Neuronal Cultures 
Translational stroke research  2010;1(1):40-47.
Nuclear factor-kappaB (NF-κB) activation occurs following ischemic preconditioning (IPC) in brain. However, the upstream signaling messengers and down-stream targets of NF-κB required for induction of IPC remain undefined. In a previous study, we demonstrated that epsilon protein kinase c (εPKC) was a key mediator of IPC in brain. Activation of εPKC induced cyclooygenase-2 (COX-2) expression and conferred ischemic tolerance in the neuronal and hippocampal slice models. Here, we hypothesized that IPC-mediated COX-2 expression was mediated by NF-κB. We tested this hypothesis in mixed cortical neuron/astrocyte cell cultures. To simulate IPC or ischemia, cell cultures were exposed to 1 or 4 h of oxygen–glucose deprivation, respectively. Our results demonstrated translocation of p65 and p50 subunits of NF-κB into nucleus following IPC or εPKC activation. NF-κB inhibition with pyrrolidine dithiocarbamate (10 μM) abolished IPC or εPKC activator-mediated neuroprotection indicating that NF-κB activation was involved in ischemic tolerance. In parallel studies, inhibition of either εPKC or the extracellular signal-regulated kinase (ERK 1/2) pathway reduced IPC-induced NF-κB activation. Finally, inhibition of NF-κB blocked IPC-induced COX-2 expression. In conclusion, we demonstrated that IPC-signaling cascade comprises εPKC activation→ERK1/2 activation→NF-κB translocation to nucleus→COX-2 expression resulting in neuroprotection in mixed neuronal culture.
PMCID: PMC2893355  PMID: 20606709
Cerebral ischemia; Ischemic tolerance; Epsilon protein kinase C; Extracellular signal-regulated kinase (ERK1/2); Neuroprotection; Mixed cortical neuron/astrocyte cell cultures
13.  GABA synapses mediate neuroprotection after ischemic and εPKC preconditioning in rat hippocampal slice cultures 
Delayed neuroprotection against ischemic challenges is conferred by both ischemic preconditioning (IPC) and preconditioning by activation of the ε-isoform of protein kinase C (εPKC-PC). In vivo, ischemic preconditioning enhances GABA release and ameliorates glutamate release during lethal cerebral ischemia. We tested the hypothesis that IPC and εPKC-PC confer neuroprotection by GABA synapses in rat organotypic hippocampal slices. Ischemic preconditioning or εPKC-PC was induced with 15 mins oxygen-glucose deprivation (OGD) or ψεRACK, a selective εPKC activator; and test ischemia consisted of 40 mins OGD. At the time of peak neuroprotection (48 h after preconditioning), we recorded GABAA receptor-mediated miniature postsynaptic currents (GABA mPSCs) in vulnerable CA1 pyramidal neurons using whole-cell voltage clamp techniques. The frequency and amplitude of GABA mPSCs significantly increased 48 h after IPC. In contrast, εPKC-PC enhanced only the amplitude of GABA mPSCs with no effect on frequency. We next asked if neuroprotection depended on these changes in GABA synapses. Weak antagonism of the GABAA receptor with bicuculline (100 nmol/L) decreased the amplitude of GABA mPSCs by 20.9 ± 6.1%. When applied during test ischemia, 100 nmol/L bicuculline abolished neuroprotection conferred by either IPC or εPKC-PC. We conclude that neuroprotection conferred by preconditioning depends on functional modifications of GABA synapses.
doi:10.1038/jcbfm.2008.126
PMCID: PMC2696173  PMID: 18957990
εPKC; inhibition; ischemia; ischemic tolerance; organotypic slice
14.  Protein kinase C epsilon activation delays neuronal depolarization during cardiac arrest in the euthermic arctic ground squirrel 
Journal of neurochemistry  2009;110(4):1170-1179.
During the pre-hibernation season, arctic ground squirrels (AGS) can tolerate 8 minutes of asphyxial cardiac arrest (CA) without detectable brain pathology. Better understanding of the mechanisms regulating innate ischemia tolerance in AGS has the potential to facilitate the development of novel, prophylactic agents to induce ischemic tolerance in patients at risk of stroke or cardiac arrest. We hypothesized that neuroprotection in AGS involves robust maintenance of ion homeostasis similar to anoxia-tolerant turtles. Ion homeostasis was assessed by monitoring ischemic depolarization (ID) in cerebral cortex during CA in vivo and during oxygen glucose deprivation in vitro in acutely prepared hippocampal slices. In both models, the onset of ID was significantly delayed in AGS compared to rats. The epsilon protein kinase C (εPKC) is a key mediator of neuroprotection and inhibits both Na+/K+-ATPase and voltage-gated sodium channels, primary mediators of the collapse of ion homeostasis during ischemia. The selective peptide inhibitor of εPKC (εV1–2) shortened the time to ID in brain slices from AGS but not in rats despite evidence that εV1–2 decreased activation of εPKC in brain slices from both rats and AGS. These results support the hypothesis that εPKC activation delays the collapse of ion homeostasis during ischemia in AGS.
doi:10.1111/j.1471-4159.2009.06196.x
PMCID: PMC2774829  PMID: 19493168
brain ischemia; heart arrest; tolerance; neuroprotection
15.  Aldehyde dehydrogenase 2 (ALDH2) rescues myocardial ischaemia/reperfusion injury: role of autophagy paradox and toxic aldehyde 
European Heart Journal  2010;32(8):1025-1038.
Aims
The present study was designed to examine the mechanism involved in mitochondrial aldehyde dehydrogenase (ALDH2)-induced cardioprotection against ischaemia/reperfusion (I/R) injury with a focus on autophagy.
Methods
Wild-type (WT), ALDH2 overexpression, and knockout (KO) mice (n = 4–6 for each index measured) were subjected to I/R, and myocardial function was assessed using echocardiographic, Langendroff, and edge-detection systems. Western blotting was used to evaluate AMP-dependent protein kinase (AMPK), Akt, autophagy, and the AMPK/Akt upstream signalling LKB1 and PTEN.
Results
ALDH2 overexpression and KO significantly attenuated and accentuated, respectively, infarct size, factional shortening, and recovery of post-ischaemic left ventricular function following I/R as well as hypoxia/reoxygenation-induced cardiomyocyte contractile dysfunction. Autophagy was induced during ischaemia and remained elevated during reperfusion. ALDH2 significantly promoted autophagy during ischaemia, which was accompanied by AMPK activation and mammalian target of rapamycin (mTOR) inhibition. On the contrary, ALDH2 overtly inhibited autophagy during reperfusion accompanied by the activation of Akt and mTOR. Inhibition and induction of autophagy mitigated ALDH2-induced protection against cell death in hypoxia and reoxygenation, respectively. In addition, levels of the endogenous toxic aldehyde 4-hydroxy-2-nonenal (4-HNE) were elevated by ischaemia and reperfusion, which was abrogated by ALDH2. Furthermore, ALDH2 ablated 4-HNE-induced cardiomyocyte dysfunction and protein damage, whereas 4-HNE directly decreased pan and phosphorylated LKB1 and PTEN expression.
Conclusion
Our data suggest a myocardial protective effect of ALDH2 against I/R injury possibly through detoxification of toxic aldehyde and a differential regulation of autophagy through AMPK- and Akt-mTOR signalling during ischaemia and reperfusion, respectively.
doi:10.1093/eurheartj/ehq253
PMCID: PMC3076664  PMID: 20705694
ALDH2; Myocardial ischaemia/reperfusion; Akt; AMPK; Autophagy; 4-HNE
16.  ALDH2 protects against stroke by clearing 4-HNE 
Cell Research  2013;23(7):915-930.
Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that metabolizes ethanol and toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE). Using an unbiased proteomic search, we identified ALDH2 deficiency in stroke-prone spontaneously hypertensive rats (SHR-SP) as compared with spontaneously hypertensive rats (SHR). We concluded the causative role of ALDH2 deficiency in neuronal injury as overexpression or activation of ALDH2 conferred neuroprotection by clearing 4-HNE in in vitro studies. Further, ALDH2-knockdown rats revealed the absence of neuroprotective effects of PKCε. Moderate ethanol administration that is known to exert protection against stroke was shown to enhance the detoxification of 4-HNE, and to protect against ischemic cerebral injury through the PKCε-ALDH2 pathway. In SHR-SP, serum 4-HNE level was persistently elevated and correlated inversely with the lifespan. The role of 4-HNE in stroke in humans was also suggested by persistent elevation of its plasma levels for at least 6 months after stroke. Lastly, we observed that 21 of 1 242 subjects followed for 8 years who developed stroke had higher initial plasma 4-HNE levels than those who did not develop stroke. These findings suggest that activation of the ALDH2 pathway may serve as a useful index in the identification of stroke-prone subjects, and the ALDH2 pathway may be a potential target of therapeutic intervention in stroke.
doi:10.1038/cr.2013.69
PMCID: PMC3698638  PMID: 23689279
ALDH2; 4-HNE; stroke; ethanol
17.  Differential Effects of Delta and Epsilon Protein Kinase C in Modulation of Postischemic Cerebral Blood Flow 
Cerebral ischemia causes cerebral blood flow (CBF) derangements resulting in neuronal damage by enhanced protein kinase C delta (δPKC) levels leading to hippocampal and cortical neuronal death after ischemia. Contrarily, activation of εPKC mediates ischemic tolerance by decreasing vascular tone providing neuroprotection. However, whether part of this protection is due to the role of differential isozymes of PKCs on CBF following cerebral ischemia remains poorly understood. Rats pretreated with a δPKC specific inhibitor (δV1-1, 0.5 mg/kg) exhibited attenuation of hyperemia and latent hypoperfusion characterized by vasoconstriction followed by vasodilation of microvessels after two-vessel occlusion plus hypotension. In an asphyxial cardiac arrest (ACA) model, rats treated with δ V1-1 (pre- and postischemia) exhibited improved perfusion after 24 h and less hippocampal CA1 and cortical neuronal death 7 days after ACA. On the contrary, εPKC-selective peptide activator, conferred neuroprotection in the CA1 region of the rat hippocampus 30 min before induction of global cerebral ischemia and decreased regional CBF during the reperfusion phase. These opposing effects of δ v. εPKC suggest a possible therapeutic potential by modulating CBF preventing neuronal damage after cerebral ischemia.
doi:10.1007/978-1-4614-1566-4_10
PMCID: PMC4086166  PMID: 22259083
18.  δPKC inhibition or εPKC activation repairs endothelial vascular dysfunction by regulating eNOS post-translational modification 
The balance between endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) and reactive oxygen species (ROS) production determines endothelial-mediated vascular homeostasis. Activation of protein kinase C (PKC) has been linked to imbalance of the eNOS/ROS system, which leads to endothelial dysfunction. We previously found that selective inhibition of delta PKC (δPKC) or selective activation of epsilon PKC (εPKC) reduces oxidative damage in the heart following myocardial infarction. In this study we determined the effect of these PKC isozymes in the survival of coronary endothelial cells (CVEC). We demonstrate here that serum deprivation of CVEC increased eNOS-mediated ROS levels, activated caspase-3, reduced Akt phosphorylation and cell number. Treatment with either the δPKC inhibitor, δV1-1, or the εPKC activator, ψεRACK, inhibited these effects, restoring cell survival through inhibition of eNOS activity. The decrease in eNOS activity coincided with specific de-phosphorylation of eNOS at Ser1179, and eNOS phosphorylation at Thr497 and Ser116. Furthermore, δV1-1 or ψεRACK induced physical association of eNOS with caveolin-1, an additional marker of eNOS inhibition, and restored Akt activation by inhibiting its nitration. Together our data demonstrate that 1) in endothelial dysfunction, ROS and reactive nitrogen species (RNS) formation result from uncontrolled eNOS activity mediated by activation of δPKC or inhibition of εPKC 2) inhibition of δPKC or activation of εePKC correct the perturbed phosphorylation state of eNOS, thus increasing cell survival. Since endothelial health ensures better tissue perfusion and oxygenation, treatment with a δPKC inhibitor and/or an εPKC activator in diseases of endothelial dysfunction should be considered.
doi:10.1016/j.yjmcc.2009.11.002
PMCID: PMC3760592  PMID: 19913548
19.  Sex differences in the phosphorylation of mitochondrial proteins result in reduced production of reactive oxygen species and cardioprotection in females 
Circulation research  2010;106(11):1681-1691.
Rationale
Although pre-menopausal females have a lower risk for cardiovascular disease, the mechanism(s) are poorly understood.
Objective
We tested the hypothesis that cardioprotection in females is mediated by altered mitochondrial protein levels and/or post-translational modifications.
Methods and Results
Using both an in vivo and an isolated heart model of ischemia and reperfusion (I/R), we found that females had less injury than males. Using proteomic methods we found that female hearts had increased phosphorylation and activity of aldehyde dehydrogenase-2 (ALDH2), an enzyme that detoxifies ROS generated aldehyde adducts, and that an activator of ALDH2 reduced I/R injury in males but had no significant effect in females. Wortmannin, an inhibitor of PI3K, blocked the protection and the increased phosphorylation of ALDH2 in females, but had no effect in males. Furthermore, we found an increase in phosphorylation of α-ketoglutarate dehydrogenase (αKGDH) in female hearts. αKGDH is a major source of ROS generation particularly with a high NADH/NAD ratio which occurs during I/R. We found decreased ROS generation in permeabilized female mitochondria given αKGDH substrates and NADH, suggesting that increased phosphorylation of αKGDH might reduce ROS generation by αKGDH. In support of this hypothesis, we found that PKC dependent phosphorylation of purified αKGDH reduced ROS generation. Additionally, myocytes from female hearts had less ROS generation following I/R than males and addition of wortmannin increased ROS generation in females to the same levels as in males.
Conclusion
These data suggest that post-translational modifications can modify ROS handling and play an important role in female cardioprotection.
doi:10.1161/CIRCRESAHA.109.213645
PMCID: PMC3127199  PMID: 20413785
gender difference; cardioprotection; mitochondria; proteomics; aldehyde dehydrogenase
20.  Ischemic Preconditioning Targets the Respiration of Synaptic Mitochondria via Protein Kinase Cε 
In the brain, ischemic preconditioning (IPC) diminishes mitochondrial dysfunction after ischemia and confers neuroprotection. Activation of ε protein kinase C (εPKC) has been proposed to be a key neuroprotective pathway during IPC. We tested the hypothesis that IPC increases the levels of εPKC in synaptosomes from rat hippocampus, resulting in improved synaptic mitochondrial respiration. Preconditioning significantly increased the level of hippocampal synaptosomal εPKC to 152% of sham-operated animals at 2 d of reperfusion, the time of peak neuroprotection. We tested the effect of εPKC activation on hippocampal synaptic mitochondrial respiration 2 d after preconditioning. Treatment with the specific εPKC activating peptide, tat-ψεRACK (tat-ψε-receptor for activated C kinase), increased the rate of oxygen consumption in the presence of substrates for complexes I, II, and IV to 157, 153, and 131% of control (tat peptide alone). In parallel, we found that εPKC activation in synaptosomes from preconditioned animals resulted in altered levels of phosphorylated mitochondrial respiratory chain proteins: increased serine and tyrosine phosphorylation of 18 kDa subunit of complex I, decreased serine phosphorylation of FeS protein in complex III, increased threonine phosphorylation of COX IV (cytochrome oxidase IV), increased mitochondrial membrane potential, and decreased H2O2 production. In brief, ischemic preconditioning promoted significant increases in the level of synaptosomal εPKC. Activation of εPKC increased synaptosomal mitochondrial respiration and phosphorylation of mitochondrial respiratory chain proteins. We propose that, at 48 h of reperfusion after ischemic preconditioning, εPKC is poised at synaptic mitochondria to respond to ischemia either by direct phosphorylation or activation of the εPKC signaling pathway.
doi:10.1523/JNEUROSCI.5471-07.2008
PMCID: PMC2678917  PMID: 18417696
cerebral ischemia; phosphorylation; electron transport chain; neuroprotection; cell death; hippocampus
21.  Aldehyde dehydrogenase 2 in cardiac protection: a new therapeutic target? 
Trends in cardiovascular medicine  2009;19(5):158-164.
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is emerging as a key enzyme involved in cytoprotection in the heart. ALDH2 mediates both the detoxification of reactive aldehydes such as acetaldehyde and 4-hydroxy-2-nonenal (4-HNE) and the bioactivation of nitroglycerin (GTN) to nitric oxide (NO). In addition, chronic nitrate treatment results in ALDH2 inhibition and contributes to nitrate tolerance. Our lab recently identified ALDH2 to be a key mediator of endogenous cytoprotection. We reported that ALDH2 is phosphorylated and activated by the survival kinase protein kinase C epsilon (PKCε) and found a strong inverse correlation between ALDH2 activity and infarct size. We also identified a small molecule ALDH2 activator (Alda-1) which reduces myocardial infarct size induced by ischemia/reperfusion in vivo. In this review, we discuss evidence that ALDH2 is a key mediator of endogenous survival signaling in the heart, suggest possible cardioprotective mechanisms mediated by ALDH2, and discuss potential clinical implications of these findings.
doi:10.1016/j.tcm.2009.09.003
PMCID: PMC2856486  PMID: 20005475
22.  Mast cells and εPKC: A role in cardiac remodeling in hypertension-induced heart failure 
Heart failure (HF) is a chronic syndrome in which pathological cardiac remodeling is an integral part of the disease and mast cell (MC) degranulation-derived mediators have been suggested to play a role in its progression. Protein kinase C (PKC) signaling is a key event in the signal transduction pathway of MC degranulation. We recently found that inhibition of εPKC slows down the progression of hypertension-induced HF in salt-sensitive Dahl rats fed a high-salt diet. We therefore determined whether εPKC inhibition affects MC degranulation in this model. Six week-old male Dahl rats were fed with a high-salt diet to induce systemic hypertension, which resulted in concentric left ventricular hypertrophy at the age of 11 weeks, followed by myocardial dilatation and HF at the age of 17 weeks. We administered εV1-2 an εPKC-selective inhibitor peptide (3 mg/Kg/day), δV1-1, a δPKC-selective inhibitor peptide (3 mg/Kg/day), TAT (negative control; at equimolar concentration; 1.6 mg/Kg/day) or olmesartan (angiotensin receptor blocker [ARB] as a positive control; 3mg/Kg/day) between 11 weeks and 17 weeks. Treatment with εV1-2 attenuated cardiac MC degranulation without affecting MC density, myocardial fibrosis, microvessel patency, vascular thickening and cardiac inflammation in comparison to TAT- or δV1-1-treatment. Treatment with ARB also attenuated MC degranulation and cardiac remodeling, but to a lesser extent when compared to εV1-2. Finally, εV1-2 treatment inhibited MC degranulation in isolated peritoneal MCs. Together, our data suggest that εPKC inhibition attenuates pathological remodeling in hypertension-induced HF, at least in part, by preventing cardiac MC degranulation.
doi:10.1016/j.yjmcc.2008.08.009
PMCID: PMC2657602  PMID: 18804478
Mast cell degranulation; protein kinase C; PKC-selective inhibitor peptide; cardiac remodeling; heart failure
23.  Preconditioning mediated by sublethal oxygen–glucose deprivation-induced cyclooxygenase-2 expression via the signal transducers and activators of transcription 3 phosphorylation 
The signal transducers and activators of transcription (STATs) were found to be essential for cardioprotection. However, their role in preconditioning (PC) neuroprotection remains undefined. Previously, our studies showed that PC mediated a signaling cascade that involves activation of epsilon protein kinase C (εPKC), extracellular signal-regulated kinase (ERK1/2), and cyclooxygenase-2 (COX-2) pathways. However, the intermediate pathway by which ERK1/2 activates COX-2 was not defined. In this study, we investigated whether the PC-induced signaling pathway requires phosphorylation of STAT isoforms for COX-2 expression. To mimic PC or lethal ischemia, mixed cortical neuron/astrocyte cell cultures were subjected to 1 and/or 4 h of oxygen–glucose deprivation (OGD), respectively. The results indicated serine phosphorylation of STAT3 after PC or εPKC activation. Inhibition of either εPKC or ERK1/2 activation abolished PC-induced serine phosphorylation of STAT3. Additionally, inhibition of STAT3 prevented PC-induced COX-2 expression and neuroprotection against OGD. Therefore, our findings suggest that PC signaling cascade involves STAT3 activation after εPKC and ERK1/2 activation. Finally, we show that STAT3 activation mediates COX-2 expression and ischemic tolerance.
doi:10.1038/jcbfm.2008.26
PMCID: PMC2645802  PMID: 18398416
cerebral ischemia; extracellular signal-regulated kinase (ERK1/2); ischemic tolerance; neuroprotection; phosphorylation; protein kinase C
24.  Impaired Cardiac SIRT1 Activity by Carbonyl Stress Contributes to Aging-Related Ischemic Intolerance 
PLoS ONE  2013;8(9):e74050.
Reactive aldehydes can initiate protein oxidative damage which may contribute to heart senescence. Sirtuin 1 (SIRT1) is considered to be a potential interventional target for I/R injury management in the elderly. We hypothesized that aldehyde mediated carbonyl stress increases susceptibility of aged hearts to ischemia/reperfusion (I/R) injury, and elucidate the underlying mechanisms with a focus on SIRT1. Male C57BL/6 young (4-6 mo) and aged (22-24 mo) mice were subjected to myocardial I/R. Cardiac aldehyde dehydrogenase (ALDH2), SIRT1 activity and protein carbonyls were assessed. Our data revealed that aged heart exhibited increased endogenous aldehyde/carbonyl stress due to impaired ALDH2 activity concomitant with blunted SIRT1 activity (P<0.05). Exogenous toxic aldehydes (4-HNE) exposure in isolated cardiomyocyte verified that aldehyde-induced carbonyl modification on SIRT1 impaired SIRT1 activity leading to worse hypoxia/reoxygenation (H/R) injury, which could all be rescued by Alda-1 (ALDH2 activator) (all P<0.05). However, SIRT1 inhibitor blocked the protective effect of Alda-1 on H/R cardiomyocyte. Interestingly, myocardial I/R leads to higher carbonylation but lower activity of SIRT1 in aged hearts than that seen in young hearts (P<0.05). The application of Alda-1 significantly reduced the carbonylation on SIRT1 and markedly improved the tolerance to in vivo I/R injury in aged hearts, but failed to protect Sirt1+/− knockout mice against myocardial I/R injury. This was verified by Alda-1 treatment improved postischemic contractile function recovery in ex vivo perfused aged but not in Sirt1+/− hearts. Thus, aldehyde/carbonyl stress is accelerated in aging heart. These results provide a new insight that impaired cardiac SIRT1 activity by carbonyl stress plays a critical role in the increased susceptibility of aged heart to I/R injury. ALDH2 activation can restore this aging-related myocardial ischemic intolerance.
doi:10.1371/journal.pone.0074050
PMCID: PMC3769351  PMID: 24040162
25.  AMP-Dependent Kinase and Autophagic Flux are Involved in Aldehyde Dehydrogenase 2-Offered Protection against Cardiac Toxicity of Ethanol 
Free radical biology & medicine  2011;51(9):1736-1748.
Mitochondrial aldehyde dehydrogenase-2 (ALDH2) alleviates ethanol toxicity although the precise mechanism is unclear. This study was designed to evaluate the effect of ALDH2 on ethanol-induced myocardial damage with a focus on autophagy. Wild-type FVB and transgenic mice overexpressing ALDH2 were challenged with ethanol (3 g/kg/d, i.p.) for 3 days and cardiac mechanical function was assessed using the echocardiographic and IonOptix systems. Western blot analysis was used to evaluate essential autophagy markers, Akt and AMPK and their downstream signaling mTOR. Ethanol challenge altered cardiac geometry and function evidenced by enlarged ventricular end systolic and diastolic diameters, decreased cell shortening and intracellular Ca2+ rise, prolonged relengthening and intracellular Ca2+ decay, as well as reduced SERCA Ca2+ uptake, the effects of which were mitigated by ALDH2. Ethanol challenge facilitated myocardial autophagy as evidenced by enhanced expression of Beclin, ATG7 and LC3B II, as well as mTOR dephosphorylation, which was alleviated by ALDH2. Ethanol challenge-induced cardiac defect and apoptosis were reversed by the ALDH-2 agonist Alda-1, the autophagy inhibitor 3-MA, and the AMPK inhibitor compound C whereas the autophagy inducer rapamycin and the AMPK activator AICAR mimicked or exacerbated ethanol-induced cell injury. Ethanol promoted or suppressed phosphorylation of AMPK and Akt, respectively, in FVB but not ALDH2 murine hearts. Moreover, AICAR nullified Alda-1-induced protection against ethanol-triggered autophagic and functional changes. Ethanol increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by Alda-1 and 3-MA. Lysosomal inhibition using bafilomycin A1, E64D and pepstatin A obliterated Alda-1- but not ethanol-induced responses in GFP-LC3 puncta. Our results suggested that ALDH2 protects against ethanol toxicity through altered Akt and AMPK signaling and regulation of autophagic flux.
doi:10.1016/j.freeradbiomed.2011.08.002
PMCID: PMC3188331  PMID: 21871561
Ethanol; ALDH2; myocardial dysfunction; autophagy; autophagy flux; Akt; AMPK

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