Activation of PKCε confers protection against neuronal ischemia/reperfusion. Since activation of PKCε leads to its translocation to multiple intracellular sites, a mitochondrial-selective PKCε activator was used to test the importance of mitochondrial activation to the neuroprotective effect of PKCε. PKCε can regulate key cytoprotective mitochondrial functions including electron transport chain activity, reactive oxygen species (ROS) generation, mitochondrial permeability transition, and detoxification of reactive aldehydes. We tested the ability of mitochondrial selective activation of PKCε to protect primary brain cell cultures or mice subjected to ischemic stroke. Pre-treatment with either general PKCε activator peptide, ψεRACK, or mitochondrial-selective PKCε activator, ψεHSP90, reduced cell death induced by simulated ischemia/reperfusion in neurons, astrocytes, and mixed neuronal cultures. The protective effects of both ψεRACK and ψεHSP90 were blocked by the PKCε antagonist, εV1–2, indicating protection requires PKCε interaction with its anchoring protein, εRACK. Further supporting a mitochondrial mechanism for PKCε, neuroprotection by ψεHSP90 was associated with a marked delay in mitochondrial membrane depolarization and significantly attenuated ROS generation during ischemia. Importantly, ψεHSP90 reduced infarct size and reduced neurological deficit in C57/BL6 mice subjected to middle cerebral artery occlusion and 24 hours of reperfusion. Thus selective activation of mitochondrial PKCε preserves mitochondrial function in vitro and improves outcome in vivo, suggesting potential therapeutic value clinically when brain ischemia is anticipated, including neurosurgery and cardiac surgery.
mitochondria; astrocytes; acute stroke; cell culture; animal models
Ning, S., Budas, G. R., Churchill, E. N., Chen, C., Knox, S. J. and Mochly-Rosen, D. Mitigation of Radiation-Induced Dermatitis by Activation of Aldehyde Dehydrogenase 2 Using Topical Alda-1 in Mice.
Radiation-induced dermatitis is a debilitating clinical problem in cancer patients undergoing cancer radiation therapy. It is also a possible outcome of exposure to high levels of radiation due to accident or hostile activity. We report that activation of aldehyde dehydrogenase 2 (ALDH2) enzymatic activity using the allosteric agonist, Alda-1, significantly reduced 4-hydroxynonenal adducts accumulation, delayed the onset of radiation dermatitis and substantially reduced symptoms in a clinically-relevant model of radiation-induced dermatitis. Importantly, Alda-1 did not radioprotect tumors in mice. Rather, it increased the sensitivity of the tumors to radiation therapy. This is the first report of reactive aldehydes playing a role in the intrinsic radiosensitivity of normal and tumor tissues. Our findings suggest that ALDH2 represents a novel target for the treatment of radiation dermatitis without reducing the benefit of radiotherapy.
The physiological effects of ethanol are dependent upon the amount and duration of consumption. Chronic excessive consumption can lead to diseases such as liver cirrhosis, and cardiac arrhythmias, while chronic moderate consumption can have therapeutic effects on the cardiovascular system. Recently, it has also been observed that acute administration of ethanol to animals prior to an ischemic event provides significant protection to the heart. This review focuses on the different modalities of chronic vs. acute ethanol consumption and discusses recent evidence for a protective effect of acute ethanol exposure and the possible use of ethanol as a therapeutic agent.
PKC; ethanol; ischemic preconditioning; ischemia; reperfusion; cardiac protection
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.
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.
Protein kinase C epsilon (PKCε) is critical for cardiac protection from ischaemia and reperfusion (IR) injury. PKCε substrates that mediate cytoprotection reside in the mitochondria. However, the mechanism enabling mitochondrial translocation and import of PKCε to enable phosphorylation of these substrates is not known. Heat shock protein 90 (HSP90) is a cytoprotective protein chaperone that participates in mitochondrial import of a number of proteins. Here, we investigated the role of HSP90 in mitochondrial import of PKCε.
Methods and results
Using an ex vivo perfused rat heart model of IR, we found that PKCε translocates from the cytosol to the mitochondrial fraction following IR. Immunogold electron microscopy and mitochondrial fractionation demonstrated that following IR, mitochondrial PKCε is localized within the mitochondria, on the inner mitochondrial membrane. Pharmacological inhibition of HSP90 prevented IR-induced interaction between PKCε and the translocase of the outer membrane (Tom20), reduced mitochondrial import of PKCε, and increased necrotic cell death by ∼70%. Using a rational approach, we designed a 7-amino acid peptide activator of PKCε, derived from an HSP90 homologous sequence located in the C2 domain of PKCε (termed ψεHSP90). Treatment with this peptide (conjugated to the cell permeating TAT protein-derived peptide, TAT47–57) increased PKCε–HSP90 protein–protein interaction, enhanced mitochondrial translocation of PKCε, increased phosphorylation and activity of an intra-mitochondrial PKCε substrate, aldehyde dehydrogenase 2, and reduced cardiac injury in ex vivo and in vivo models of myocardial infarction.
Our results suggest that HSP90-mediated mitochondrial import of PKCε plays an important role in the protection of the myocardium from IR injury.
Protein kinase C epsilon; Mitochondria; Protein–protein interaction; Ischaemia reperfusion; Heat shock protein 90
There is substantial interest in the development of drugs that limit the extent of ischemia-induced cardiac damage caused by myocardial infarction or by certain surgical procedures. Here an unbiased proteomic search identified mitochondrial aldehyde dehydrogenase 2 (ALDH2) as an enzyme whose activation correlates with reduced ischemic heart damage in rodent models. A high-throughput screen yielded a small-molecule activator of ALDH2 (Alda-1) that, when administered to rats prior to an ischemic event, reduced infarct size by 60%, most likely through its inhibitory effect on the formation of cytotoxic aldehydes. In vitro, Alda-1 was a particularly effective activator of ALDH2*2, an inactive mutant form of the enzyme that is found in 40% of East Asian populations. Thus, pharmacologic enhancement of ALDH2 activity may be useful for patients with wildtype or mutant ALDH2 subjected to cardiac ischemia, such as during coronary bypass surgery. (140/140 words)
Cardiac sarcolemmal ATP-sensitive K+ (KATP) channels, composed of Kir6.2 and SUR2A subunits, couple the metabolic status of cells with the membrane excitability. Based on previous functional studies, we have hypothesized that creatine kinase (CK) may be a part of the sarcolemmal KATP channel protein complex. The inside-out and whole cell patch clamp electrophysiology applied on guinea pig cardiomyocytes showed that substrates of CK regulate KATP channels activity. Following immunoprecipitation of guinea-pig cardiac membrane fraction with the anti-SUR2 antibody, Coomassie blue staining revealed, besides Kir6.2 and SUR2A, a polypeptide at ∼48 kDa. Western blotting analysis confirmed the nature of putative Kir6.2 and SUR2A, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis identified p48 kDa as a muscle form of CK. In addition, the CK activity was found in the anti-SUR2A immunoprecipitate and the cross reactivity between an anti-CK antibody and the anti-SUR2A immunoprecipitate was observed as well as vice verse. Further results obtained at the level of recombinant channel subunits demonstrated that CK is directly physically associated with the SUR2A, but not the Kir6.2, subunit. All together, these results suggest that the CK is associated with SUR2A subunit in vivo, which is an integral part of the sarcolemmal KATP channel protein complex.
heart; KATP channels; SUR2A; Kir6.2
Chronic exposure to lower oxygen tension may increase cellular resistance to different types of acute metabolic stress. Here, we show that 24-h-long exposure to slightly decreased oxygen tension (partial pressure of oxygen (PO2) of 100 mm Hg instead of normal 144 mm Hg) confers resistance against acute hypoxia/reoxygenation-induced Ca2+ loading in heart-derived H9c2 cells. The number of ATP-sensitive K+ (KATP) channels were increased in cells exposed to PO2 = 100 mm Hg relative to cells exposed to PO2 = 144 mm Hg. This was due to an increase in transcription of SUR2A, a KATP channel regulatory subunit, but not Kir6.2, a KATP channel pore-forming subunit. PO2 = 100 mm Hg also increased the SUR2 gene promoter activity. Experiments with cells overexpressing wild type of hypoxia-inducible factor (HIF)-1α and dominant negative HIF-1β suggested that the HIF-1-signaling pathway did not participate in observed PO2-mediated regulation of SUR2A expression. On the other hand, NADH inhibited the effect of PO2 = 100 mm Hg but not the effect of PO2 = 20 mm Hg. LY 294002 and PD 184 352 prevented PO2-mediated regulation of KATP channels, whereas rapamycin was without any effect. HMR 1098 inhibited the cytoprotective effect of PO2 = 100 mm Hg, and a decrease of PO2 from 144 to 100 mm Hg did not change the expression of any other gene, including those involved in stress and hypoxic response, as revealed by Affymetrix high density oligonucleotide arrays. We conclude that slight hypoxia activates HIF-1α-independent signaling cascade leading to an increase in SUR2A protein, a higher density of KATP channels, and a cellular phenotype more resistant to acute metabolic stress.
Brief periods of ischemia and reperfusion that precede sustained ischemia lead to a reduction in myocardial infarct size. This phenomenon, known as ischemic preconditioning, is mediated by signaling pathway(s) that are yet to be fully defined. 3′-phosphoinositide-dependent kinase-1 (PDK1) has been implicated in numerous cellular processes. However, the involvement of PDK1 in preconditioning has yet to be elucidated. Studying PDK1 is not as straightforward as it is for the majority of kinases, due to the lack of a specific inhibitor of PDK1. Therefore, we have taken advantage of PDK1 hypomorphic mutant mice with reduced expression of PDK1 to study the role of PDK1 in preconditioning. Whole heart and single cell models of preconditioning demonstrated that the hearts and cardiac cells from PDK1 hypomorphic mice could not be preconditioned. The cardioprotective effect of PDK1 was not related to the effect that preconditioning has on sarcolemmal membrane action potential as revealed by di-8-ANEPPS, a sarcolemmal-potential sensitive dye, and laser confocal microscopy. In contrast, experiments with JC-1, a mitochondrial membrane potential-sensitive dye, has demonstrated that intact PDK1 levels were required for preconditioning-mediated regulation of mitochondrial membrane potential. Western blotting combined with functional experiments have shown that intact PDK1 levels were required for preconditioning-induced phosphorylation of protein kinase B (PKB), glycogen synthase kinase-3β (GSK-3β), and cardioprotection. We conclude that PDK1 mediates preconditioning in the heart by regulating activating PKB-GSK-3β to regulate mitochondrial but not sarcolemmal membrane potential. 3′phosphoinositide-dependent kinase-1 (PDK1) is essential for ischemic preconditioning of the myocardium.
GSK-3; hypoxia; ischemia; mitochondria; cardioprotection
The opening of sarcolemmal and mitochondrial ATP-sensitive K+ (KATP) channels in the heart is believed to mediate ischemic preconditioning, a phenomenon whereby brief periods of ischemia/reperfusion protect the heart against myocardial infarction. Here, we have applied digital epifluorescent microscopy, immunoprecipitation and Western blotting, perforated patch clamp electrophysiology, and immunofluorescence/laser confocal microscopy to examine the involvement of KATP channels in cardioprotection afforded by preconditioning. We have shown that adult, stimulated-to-beat, guinea-pig cardiomyocytes survived in sustained hypoxia for ∼17 min. An episode of 5-min-long hypoxia/5-min-long reoxygenation before sustained hypoxia dramatically increased the duration of cellular survival. Experiments with different antagonists of KATP channels, applied at different times during the experimental protocol, suggested that the opening of sarcolemmal KATP channels at the beginning of sustained hypoxia mediate preconditioning. This conclusion was supported by perforated patch clamp experiments that revealed activation of sarcolemmal KATP channels by preconditioning. Immunoprecipitation and Western blotting as well as immunofluorescence and laser confocal microscopy showed that the preconditioning is associated with the increase in KATP channel proteins in sarcolemma. Inhibition of trafficking of KATP channel subunits prevented preconditioning without affecting sensitivity of cardiomyocytes to hypoxia in the absence of preconditioning. We conclude that the preconditioning is mediated by the activation and trafficking of sarcolemmal KATP channels.
ischemia; heart; Kir6.2; SUR2A; cardioprotection
Recent studies indicate that the LKB1 is a key regulator of the AMP-activated protein kinase (AMPK), which plays a crucial role in protecting cardiac muscle from damage during ischemia. We have employed mice that lack LKB1 in cardiac and skeletal muscle and studied how this affected the activity of cardiac AMPKα1/α2 under normoxic, ischemic, and anoxic conditions. In the heart lacking cardiac muscle LKB1, the basal activity of AMPKα2 was vastly reduced and not increased by ischemia or anoxia. Phosphorylation of AMPKα2 at the site of LKB1 phosphorylation (Thr172) or phosphorylation of acetyl-CoA carboxylase-2, a downstream substrate of AMPK, was ablated in ischemic heart lacking cardiac LKB1. Ischemia was found to increase the ADP-to-ATP (ADP/ATP) and AMP-to-ATP ratios (AMP/ATP) to a greater extent in LKB1-deficient cardiac muscle than in LKB1-expressing muscle. In contrast to AMPKα2, significant basal activity of AMPKα1 was observed in the lysates from the hearts lacking cardiac muscle LKB1, as well as in cardiomyocytes that had been isolated from these hearts. In the heart lacking cardiac LKB1, ischemia or anoxia induced a marked activation and phosphorylation of AMPKα1, to a level that was only moderately lower than observed in LKB1-expressing heart. Echocardiographic and morphological analysis of the cardiac LKB1-deficient hearts indicated that these hearts were not overtly dysfunctional, despite possessing a reduced weight and enlarged atria. These findings indicate that LKB1 plays a crucial role in regulating AMPKα2 activation and acetyl-CoA carboxylase-2 phosphorylation and also regulating cellular energy levels in response to ischemia. They also provide genetic evidence that an alternative upstream kinase can activate AMPKα1 in cardiac muscle.
cellular energy metabolism; hypoxia; cardiovascular physiology; AMP-activated protein kinase