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Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.

Noninvasive detection of nonalcoholic steatohepatitis (NASH), the progressive form of nonalcoholic fatty liver disease, promises to improve patient screening, accelerate drug trials, and reduce health care costs. On the basis of protease dysregulation of the biological pathways of fibrotic NASH, we developed the Glympse Bio Test System (GBTS) for multiplexed quantification of liver protease activity. GBTS-NASH comprises a mixture of 19 mass-barcoded PEGylated peptides that is administered intravenously and senses liver protease activity by releasing mass-barcoded reporters into urine for analysis by mass spectrometry. To identify a protease signature of NASH, transcriptomic analysis of 355 human liver biopsies identified a 13-protease panel that discriminated clinically relevant NASH ≥F2 fibrosis from F0-F1 with high classification accuracy across two independent patient datasets. We screened 159 candidate substrates to identify a panel of 19 peptides that exhibited high activity for our 13-protease panel. In the choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) mouse model, binary classifiers trained on urine samples discriminated fibrotic NASH from simple steatosis and healthy controls across a range of nondisease conditions and indicated disease regression upon diet change [area under receiver operating characteristics (AUROCs) > 0.97]. Using a hepatoprotective triple combination treatment (FXR agonist, ACC and ASK1 inhibitors) in a rat model of NASH, urinary classification distinguished F0-F1 from ≥F2 animals and indicated therapeutic response as early as 1 week on treatment (AUROCs >0.91). Our results support GBTS-NASH to diagnose fibrotic NASH via an infusion of peptides, monitor changes in disease severity, and indicate early treatment response.

Background: The farnesoid X receptor (FXR) influences hepatic metabolism, inflammation and liver fibrosis as key components of non-alcoholic steatohepatitis (NASH). We studied the effects of the non-steroidal FXR agonist cilofexor (formerly GS-9674) on portal pressure and fibrosis in experimental NASH., Methods: NASH was induced in Wistar rats using a choline-deficient high-fat diet plus intraperitoneal sodium nitrite injections. First, a dose-finding study was performed with 10 mg/kg and 30 mg/kg of cilofexor, focusing on histological readouts. Liver fibrosis was assessed by Picro-Sirius-Red, desmin staining and hepatic hydroxyproline content. Gene expression was determined by RT-PCR. In a subsequent hemodynamic study, rats received 30 mg/kg cilofexor with or without propranolol (25 mg/kg). Portal pressure, systemic hemodynamics and splanchnic blood flow were measured., Results: Cilofexor dose-dependently induced FXR target genes shp, cyp7a1 and fgf15 in hepatic and ileal tissues, paralleled by a dose-dependent reduction in liver fibrosis area (Picro-Sirius-Red) of -41% (10 mg/kg) and -69% (30 mg/kg), respectively. The 30 mg/kg cilofexor dose significantly reduced hepatic hydroxyproline content (-41%), expression of col1a1 (-37%) and pdgfr-β (-36%), as well as desmin area (-42%) in NASH rats. Importantly, cilofexor decreased portal pressure (11.9 ± 2.1 vs. 8.9 ± 2.2 mmHg; p = 0.020) without affecting splanchnic blood-flow or systemic hemodynamics. The addition of propranolol to cilofexor additionally reduced splanchnic inflow (-28%) but also mean arterial pressure (-25%) and heart rate (-37%)., Conclusion: The non-steroidal FXR agonist cilofexor decreased portal hypertension and reduced liver fibrosis in NASH rats. While cilofexor seems to primarily decrease sinusoidal resistance in cirrhotic portal hypertension, the combination with propranolol additionally reduced mesenteric hyperperfusion.

Pulmonary arterial hypertension, group 1 of the pulmonary hypertension disease family, involves pulmonary vascular remodelling, right ventricular dysfunction and cardiac failure. Oxidative stress, through activation of mitogen-activated protein kinases is implicated in these changes. Inhibition of apoptosis signal-regulating kinase 1, an apical mitogen-activated protein kinase, prevented pulmonary arterial hypertension developing in rodent models. Here, we investigate apoptosis signal-regulating kinase 1 in pulmonary arterial hypertension by examining the impact that its inhibition has on the molecular and cellular signalling in established disease. Apoptosis signal-regulating kinase 1 inhibition was investigated in in vivo pulmonary arterial hypertension and in vitro pulmonary hypertension models. In the in vivo model, male Sprague Dawley rats received a single subcutaneous injection of Sugen SU5416 (20 mg/kg) prior to two weeks of hypobaric hypoxia (380 mmHg) followed by three weeks normoxia (Sugen/hypoxic), then animals were either maintained for three weeks on control chow or one containing apoptosis signal-regulating kinase 1 inhibitor (100 mg/kg/day). Cardiovascular measurements were carried out. In the in vitro model, primary cultures of rat pulmonary artery fibroblasts and rat pulmonary artery smooth muscle cells were maintained in hypoxia (5% O 2 ) and investigated for proliferation, migration and molecular signalling in the presence or absence of apoptosis signal-regulating kinase 1 inhibitor. Sugen/hypoxic animals displayed significant pulmonary arterial hypertension compared to normoxic controls at eight weeks. Apoptosis signal-regulating kinase 1 inhibitor decreased right ventricular systolic pressure to control levels and reduced muscularised vessels in lung tissue. Apoptosis signal-regulating kinase 1 inhibition was found to prevent hypoxia-induced proliferation, migration and cytokine release in rat pulmonary artery fibroblasts and also prevented rat pulmonary artery fibroblast-induced rat pulmonary artery smooth muscle cell migration and proliferation. Apoptosis signal-regulating kinase 1 inhibition reversed pulmonary arterial hypertension in the Sugen/hypoxic rat model. These effects may be a result of intrinsic changes in the signalling of adventitial fibroblast., (© The Author(s) 2020.)

Iterative liver injury results in progressive fibrosis disrupting hepatic architecture, regeneration potential, and liver function. Hepatic stellate cells (HSCs) are a major source of pathological matrix during fibrosis and are thought to be a functionally homogeneous population. Here, we use single-cell RNA sequencing to deconvolve the hepatic mesenchyme in healthy and fibrotic mouse liver, revealing spatial zonation of HSCs across the hepatic lobule. Furthermore, we show that HSCs partition into topographically diametric lobule regions, designated portal vein-associated HSCs (PaHSCs) and central vein-associated HSCs (CaHSCs). Importantly we uncover functional zonation, identifying CaHSCs as the dominant pathogenic collagen-producing cells in a mouse model of centrilobular fibrosis. Finally, we identify LPAR1 as a therapeutic target on collagen-producing CaHSCs, demonstrating that blockade of LPAR1 inhibits liver fibrosis in a rodent NASH model. Taken together, our work illustrates the power of single-cell transcriptomics to resolve the key collagen-producing cells driving liver fibrosis with high precision., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Oxidative stress is an underlying component of acute and chronic kidney disease. Apoptosis signal-regulating kinase 1 (ASK1) is a widely expressed redox-sensitive serine threonine kinase that activates p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase kinases, and induces apoptotic, inflammatory, and fibrotic signaling in settings of oxidative stress. We describe the discovery and characterization of a potent and selective small-molecule inhibitor of ASK1, GS-444217, and demonstrate the therapeutic potential of ASK1 inhibition to reduce kidney injury and fibrosis. Activation of the ASK1 pathway in glomerular and tubular compartments was confirmed in renal biopsies from patients with diabetic kidney disease (DKD) and was decreased by GS-444217 in several rodent models of kidney injury and fibrosis that collectively represented the hallmarks of DKD pathology. Treatment with GS-444217 reduced progressive inflammation and fibrosis in the kidney and halted glomerular filtration rate decline. Combination of GS-444217 with enalapril, an angiotensin-converting enzyme inhibitor, led to a greater reduction in proteinuria and regression of glomerulosclerosis. These results identify ASK1 as an important target for renal disease and support the clinical development of an ASK1 inhibitor for the treatment of DKD.

Rationale: Progression of pulmonary arterial hypertension (PAH) is associated with pathological remodeling of the pulmonary vasculature and the right ventricle (RV). Oxidative stress drives the remodeling process through activation of MAPKs (mitogen-activated protein kinases), which stimulate apoptosis, inflammation, and fibrosis., Objectives: We investigated whether pharmacological inhibition of the redox-sensitive apical MAPK, ASK1 (apoptosis signal-regulating kinase 1), can halt the progression of pulmonary vascular and RV remodeling., Methods: A selective, orally available ASK1 inhibitor, GS-444217, was administered to two preclinical rat models of PAH (monocrotaline and Sugen/hypoxia), a murine model of RV pressure overload induced by pulmonary artery banding, and cellular models., Measurements and Main Results: Oral administration of GS-444217 dose dependently reduced pulmonary arterial pressure and reduced RV hypertrophy in PAH models. The therapeutic efficacy of GS-444217 was associated with reduced ASK1 phosphorylation, reduced muscularization of the pulmonary arteries, and reduced fibrotic gene expression in the RV. Importantly, efficacy was observed when GS-444217 was administered to animals with established disease and also directly reduced cardiac fibrosis and improved cardiac function in a model of isolated RV pressure overload. In cellular models, GS-444217 reduced phosphorylation of p38 and JNK (c-Jun N-terminal kinase) induced by adenoviral overexpression of ASK1 in rat cardiomyocytes and reduced activation/migration of primary mouse cardiac fibroblasts and human pulmonary adventitial fibroblasts derived from patients with PAH., Conclusions: ASK1 inhibition reduced pathological remodeling of the pulmonary vasculature and the right ventricle and halted progression of pulmonary hypertension in rodent models. These preclinical data inform the first description of a causal role of ASK1 in PAH disease pathogenesis.

Background: Thioredoxin 2 (Trx2) is a key mitochondrial protein that regulates cellular redox and survival by suppressing mitochondrial reactive oxygen species generation and by inhibiting apoptosis stress kinase-1 (ASK1)-dependent apoptotic signaling. To date, the role of the mitochondrial Trx2 system in heart failure pathogenesis has not been investigated., Methods and Results: Western blot and histological analysis revealed that Trx2 protein expression levels were reduced in hearts from patients with dilated cardiomyopathy, with a concomitant increase in ASK1 phosphorylation/activity. Cardiac-specific Trx2 knockout mice develop spontaneous dilated cardiomyopathy at 1 month of age with increased heart size, reduced ventricular wall thickness, and a progressive decline in left ventricular contractile function, resulting in mortality due to heart failure by ≈4 months of age. The progressive decline in cardiac function observed in cardiac-specific Trx2 knockout mice was accompanied by the disruption of mitochondrial ultrastructure, mitochondrial membrane depolarization, increased mitochondrial reactive oxygen species generation, and reduced ATP production, correlating with increased ASK1 signaling and increased cardiomyocyte apoptosis. Chronic administration of a highly selective ASK1 inhibitor improved cardiac phenotype and reduced maladaptive left ventricular remodeling with significant reductions in oxidative stress, apoptosis, fibrosis, and cardiac failure. Cellular data from Trx2-deficient cardiomyocytes demonstrated that ASK1 inhibition reduced apoptosis and reduced mitochondrial reactive oxygen species generation., Conclusions: Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial reactive oxygen species production and ASK1-dependent apoptosis. Inhibition of ASK1 represents a promising therapeutic strategy for the treatment of dilated cardiomyopathy and heart failure., (© 2015 American Heart Association, Inc.)

Activation of protein kinase C∊ (PKC∊) confers protection against neuronal ischemia/reperfusion. Activation of PKC∊ leads to its translocation to multiple intracellular sites, so a mitochondria-selective PKC∊ activator was used to test the importance of mitochondrial activation to the neuroprotective effect of PKC∊. PKC∊ 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 mitochondria-selective activation of PKC∊ to protect primary brain cell cultures or mice subjected to ischemic stroke. Pretreatment with either general PKC∊ activator peptide, TAT-Ψ∊RACK, or mitochondrial-selective PKC∊ activator, TAT-Ψ∊HSP90, reduced cell death induced by simulated ischemia/reperfusion in neurons, astrocytes, and mixed neuronal cultures. The protective effects of both TAT-Ψ∊RACK and TAT-Ψ∊HSP90 were blocked by the PKC∊ antagonist ∊V1-2 , indicating that protection requires PKC∊ interaction with its anchoring protein, TAT-∊RACK. Further supporting a mitochondrial mechanism for PKC∊, neuroprotection by TAT-Ψ∊HSP90 was associated with a marked delay in mitochondrial membrane depolarization and significantly attenuated ROS generation during ischemia. Importantly, TAT-Ψ∊HSP90 reduced infarct size and reduced neurological deficit in C57/BL6 mice subjected to middle cerebral artery occlusion and 24 hr of reperfusion. Thus selective activation of mitochondrial PKC∊ 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., (Copyright © 2013 Wiley Periodicals, Inc.)

Purposes: We determined whether a small molecule inhibitor of apoptosis signal-regulating kinase 1 (ASK1-i) could reduce myocardial infarct size in a rat ischemia/reperfusion model., Methods and Results: Sprague-Dawley rats were randomized to 3 groups: ASK1-i infusion (n = 16), vehicle infusion (n = 16), or ischemic preconditioning (IPC; n = 15). Infusion of ASK1-i (10 mg/kg, iv) or vehicle commenced 45 minutes before myocardial ischemia. IPC consisted of 3 cycles of 3 minutes of coronary occlusion followed by 5 minutes of reperfusion immediately before index myocardial ischemia, which consisted of 30-minute left coronary occlusion followed by 180 minutes of reperfusion. Pathologic analysis revealed no significant difference in the ischemic risk size among the 3 groups. ASK1-I and IPC significantly reduced myocardial infarct size (27.7% ± 3.3%, 16.5% ± 3.4%, and 41.5% ± 4.8% in the ASK1-i group, the IPC group, and the vehicle group, respectively; P = 0.0002) and apoptosis (the percentage of apoptotic nuclei averaged 11.6% ± 1.0%, 10.2% ± 1.7%, and 17.7% ± 2.0% in the ASK1-i group, IPC group, and vehicle group, respectively, P = 0.0055)., Conclusions: A small molecule inhibitor of ASK1 was shown for the first time to reduce apoptosis and myocardial infarct size in a rat model of ischemia/reperfusion.

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.

Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na(+) channel 1.5 (Na(v)1.5), resulting in enhanced I(NaL). Conversely, an increase of I(NaL) would be expected to cause elevation of intracellular Ca(2+) and activation of CaMKII. However, a relationship between enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant (N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same association between CaMKII and Na(v)1.5 was also found in N(1325)S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction caused by excessive Na(+) influx.

Aims: Protein kinase C epsilon (PKCepsilon) is critical for cardiac protection from ischaemia and reperfusion (IR) injury. PKCepsilon substrates that mediate cytoprotection reside in the mitochondria. However, the mechanism enabling mitochondrial translocation and import of PKCepsilon 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 PKCepsilon., Methods and Results: Using an ex vivo perfused rat heart model of IR, we found that PKCepsilon translocates from the cytosol to the mitochondrial fraction following IR. Immunogold electron microscopy and mitochondrial fractionation demonstrated that following IR, mitochondrial PKCepsilon is localized within the mitochondria, on the inner mitochondrial membrane. Pharmacological inhibition of HSP90 prevented IR-induced interaction between PKCepsilon and the translocase of the outer membrane (Tom20), reduced mitochondrial import of PKCepsilon, and increased necrotic cell death by approximately 70%. Using a rational approach, we designed a 7-amino acid peptide activator of PKCepsilon, derived from an HSP90 homologous sequence located in the C2 domain of PKCepsilon (termed psiepsilonHSP90). Treatment with this peptide (conjugated to the cell permeating TAT protein-derived peptide, TAT(47-57)) increased PKCepsilon-HSP90 protein-protein interaction, enhanced mitochondrial translocation of PKCepsilon, increased phosphorylation and activity of an intra-mitochondrial PKCepsilon substrate, aldehyde dehydrogenase 2, and reduced cardiac injury in ex vivo and in vivo models of myocardial infarction., Conclusion: Our results suggest that HSP90-mediated mitochondrial import of PKCepsilon plays an important role in the protection of the myocardium from IR injury.

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 (PKCvarepsilon). We recently identified aldehyde dehydrogenase 2 (ALDH2) as a PKCvarepsilon 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 PKCvarepsilon knockout mice and a direct activator of ALDH2, Alda-44, to further investigate the interplay between PKCvarepsilon and ALDH2 in cardioprotection. We report that ethanol preconditioning requires PKCvarepsilon, whereas direct activation of ALDH2 reduces infarct size in both wild type and PKCvarepsilon knockout hearts. Our data suggest that ALDH2 is downstream of PKCvarepsilon in ethanol preconditioning and that direct activation of ALDH2 can circumvent the requirement of PKCvarepsilon 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., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

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 and the bioactivation of nitroglycerin to nitric oxide. In addition, chronic nitrate treatment results in ALDH2 inhibition and contributes to nitrate tolerance. Our laboratory 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 and found a strong inverse correlation between ALDH2 activity and infarct size. We also identified a small molecule ALDH2 activator 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.

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.

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, using an unbiased proteomic search, we 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 before 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 wild-type or mutant ALDH2 who are subjected to cardiac ischemia, such as during coronary bypass surgery.

PKC (protein kinase C) isoenzymes are related protein kinases, involved in many signalling events in normal state and in disease. Basic research into identifying the molecular basis of PKC selectivity led to simple strategies to identify selective competitive inhibitor peptides and allosteric agonist peptides of individual PKC isoenzymes. The strategies and rationale used to identify these peptide regulators of protein-protein interaction may be applicable to other signalling events. Importantly, the PKC-regulating peptides proved to be useful pharmacological tools and may serve as drugs or drug leads for a variety of human diseases.

Mitochondria mediate diverse cellular functions including energy generation and ROS (reactive oxygen species) production and contribute to signal transduction. Mitochondria are also key regulators of cell viability and play a central role in necrotic and apoptotic cell death pathways induced by cardiac ischaemia/reperfusion injury. PKC (protein kinase C) epsilon plays a critical role in cardioprotective signalling pathways that protect the heart from ischaemia/reperfusion. Emerging evidence suggests that the cardioprotective target of PKCepsilon resides at the mitochondria. Proposed mitochondrial targets of PKCepsilon include mitoK(ATP) (mitochondrial ATP-sensitive K(+) channel), components of the MPTP (mitochondrial permeability transition pore) and components of the electron transport chain. This review highlights mitochondrial targets of PKCepsilon and their possible role in cardioprotective signalling in the setting of ischaemia/reperfusion injury.

Current treatment for acute myocardial infarction (AMI) is aimed at limiting the duration of ischemia by either mechanical (balloon catheters) or enzymatic (thrombolytics) means to disrupt the occlusion. While these treatments are effective in limiting the duration of ischemia, no therapeutic treatment is currently available to prevent ischemic injury and to reduce reperfusion injury, which occurs after these interventions. The development of rationally designed PKC isozyme-selective regulator peptides has permitted investigation into the role of specific PKC isozymes in ischemia-reperfusion (IR) injury. Based on these studies, it is now evident that epsilon and deltaPKC have distinct temporal and opposing roles in regulating myocardial damage induced by IR. Activation of epsilonPKC before ischemia protects the heart by mimicking preconditioning, whereas inhibition of deltaPKC during reperfusion protects the heart from reperfusion-induced damage. These cardioprotective effects have been observed in isolated cardiomyocytes, isolated perfused hearts and in vivo in all species tested including mouse, rat and pig and may provide the basis for future therapeutic agents. Having established the efficacy of PKC isozyme-specific regulators in reducing IR injury, the next challenge is to outline the molecular mechanisms regulated by delta and epsilonPKC isozymes that result in enhanced tolerance to IR. In this review, we discuss progress that has been made in establishing cytoprotective mechanisms, which arise as a consequence of epsilonPKC activation or deltaPKC inhibition, and how they may lead to protection in the setting of myocardial ischemia reperfusion.

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-3beta (GSK-3beta), and cardioprotection. We conclude that PDK1 mediates preconditioning in the heart by regulating activating PKB-GSK-3beta to regulate mitochondrial but not sarcolemmal membrane potential. 3'Phosphoinositide-dependent kinase-1 (PDK1) is essential for ischemic preconditioning of the myocardium.

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 AMPKalpha1/alpha2 under normoxic, ischemic, and anoxic conditions. In the heart lacking cardiac muscle LKB1, the basal activity of AMPKalpha2 was vastly reduced and not increased by ischemia or anoxia. Phosphorylation of AMPKalpha2 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 AMPKalpha2, significant basal activity of AMPKalpha1 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 AMPKalpha1, 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 AMPKalpha2 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 AMPKalpha1 in cardiac muscle.

Cardiac sarcolemmal ATP-sensitive K+ (K(ATP)) channels, composed of Kir6.2 and SUR2A subunits, are regulated by intracellular ATP and they couple the metabolic status of the cell with the membrane excitability. On the basis of previous studies, we have suggested that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) may be a part of the sarcolemmal K(ATP)-channel protein complex. A polypeptide of approximately 42 kDa was immunoprecipitated with an anti-SUR2A antibody from guinea-pig cardiac membrane fraction and identified as GAPDH. Immunoprecipitation/western blotting analysis with anti-Kir6.2, anti-SUR2A and anti-GAPDH antibodies showed that GAPDH is a part of the sarcolemmal K(ATP)-channel protein complex in vivo. Further studies with immunoprecipitation/western blotting and the membrane yeast two-hybrid system showed that GAPDH associates physically with the Kir6.2 but not the SUR2A subunit. Patch-clamp electrophysiology showed that GAPDH regulates K(ATP)-channel activity irrespective of high intracellular ATP, by producing 1,3-bisphosphoglycerate, a K(ATP)-channel opener. These results suggest that GAPDH is an integral part of the sarcolemmal K(ATP)-channel protein complex, where it couples glycolysis with the K(ATP)-channel activity.

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 approximately 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.

We employed Cre/loxP technology to generate mPDK1(-/-) mice, which lack PDK1 in cardiac muscle. Insulin did not activate PKB and S6K, nor did it stimulate 6-phosphofructo-2-kinase and production of fructose 2,6-bisphosphate, in the hearts of mPDK1(-/-) mice, consistent with PDK1 mediating these processes. All mPDK1(-/-) mice died suddenly between 5 and 11 weeks of age. The mPDK1(-/-) animals had thinner ventricular walls, enlarged atria and right ventricles. Moreover, mPDK1(-/-) muscle mass was markedly reduced due to a reduction in cardiomyocyte volume rather than cardiomyocyte cell number, and markers of heart failure were elevated. These results suggested mPDK1(-/-) mice died of heart failure, a conclusion supported by echocardiographic analysis. By employing a single-cell assay we found that cardiomyocytes from mPDK1(-/-) mice are markedly more sensitive to hypoxia. These results establish that the PDK1 signalling network plays an important role in regulating cardiac viability and preventing heart failure. They also suggest that a deficiency of the PDK1 pathway might contribute to development of cardiac disease in humans.

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+ (K(ATP)) 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 K(ATP) channel regulatory subunit, but not Kir6.2, a K(ATP) 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)-1alpha and dominant negative HIF-1beta 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 K(ATP) 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-1alpha-independent signaling cascade leading to an increase in SUR2A protein, a higher density of K(ATP) channels, and a cellular phenotype more resistant to acute metabolic stress.

ATP-sensitive K(+) (K(ATP)) channels in the heart are normally closed by high intracellular ATP, but are activated during ischemia to promote cellular survival. These channels are heteromultimers composed of Kir6.2 subunit, an inwardly rectifying K(+) channel core, and SUR2A, a regulatory subunit implicated in ligand-dependent regulation of channel gating. Here, we have shown that the muscle form (M-LDH), but not heart form (H-LDH), of lactate dehydrogenase is directly physically associated with the sarcolemmal K(ATP) channel by interacting with the Kir6.2 subunit via its N-terminus and with the SUR2A subunit via its C-terminus. The species of LDH bound to the channel regulated the channel activity despite millimolar concentration of intracellular ATP. The presence of M-LDH in the channel protein complex was required for opening of K(ATP) channels during ischemia and ischemia-resistant cellular phenotype. We conclude that M-LDH is an integral part of the sarcolemmal K(ATP) channel protein complex in vivo, where, by virtue of its catalytic activity, it couples the metabolic status of the cell with the K(ATP) channels activity that is essential for cell protection against ischemia.

Objectives: The main objective of the present study was to establish whether 17beta-estradiol (E2) regulates expression of cardiac adenosine triphosphate-sensitive potassium (K(ATP)) channel., Background: Based on our previous studies that demonstrate gender-specific differences in sarcolemmal K(ATP) channels, we have hypothesized that the main estrogen, E2, may regulate expression of cardiac K(ATP) channels., Methods: Reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for Kir6.2 and sulfonylurea receptor 2A (SUR2A) subunits was performed on total ribonucleic acid (RNA) from rat embryonic heart-derived H9c2 cells. Immunoprecipitation and Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was done on membrane fraction of H9c2 cells. Whole cell electrophysiology and digital epifluorescent Ca(2+) imaging were performed on living H9c2 cells. All experiments were done in cells incubated 24 h with or without 100 nM E2., Results: The RT-PCR revealed higher levels of SUR2A, but not Kir6.2, messenger RNA (mRNA) in E2-treated, relative to untreated, cells. Increase of the level of only the SUR2A subunit could change the number of sarcolemmal K(ATP) channels only if the Kir6.2 is in excess over SUR2A. Indeed, RT-PCR analysis demonstrated considerably lower levels of SUR2A mRNA compared with Kir6.2 mRNA. Significantly higher levels of both Kir6.2 and SUR2A protein subunits were found in the membrane fraction of E2-treated cells compared with untreated ones, and the density of current evoked by pinacidil (100 microM), a K(ATP) channel opener, was significantly higher in E2-treated compared with untreated cells. To test the effect of E2 on cellular response to hypoxia-reoxygenation, we have measured on-line, intracellular concentration of Ca(2+) in H9c2 cells exposed to hypoxia-reoxygenation. Intracellular Ca(2+) loading induced by hypoxia-reoxygenation was significantly decreased by treatment with E2. This E2-mediated protection was inhibited by HMR 1098 (30 microM), but not by 5-hydroxydecanoate (50 microM)., Conclusions: In conclusion, this study has demonstrated that E2 increases levels of SUR2A subunit, stimulates K(ATP) channel formation and protects cardiac cells from hypoxiareoxygenation.

The opening of sarcolemmal K(ATP) channels is considered to be an important endogenous cardioprotective mechanism. On the other hand, age-dependent changes in the myocardial susceptibility to ischemia and hypoxia have been observed in different species, including humans. Here, we have hypothesized that aging might be associated with the changes in sarcolemmal K(ATP) channels. Therefore, the main objective of the present study was to establish whether aging changes expression of cardiac sarcolemmal ATP-sensitive K+ (K(ATP)) channels. RT-PCR using primers specific for K(ATP) channel subunits, Kir6.2, Kir6.1 and SUR2A subunits was performed using total RNA from guinea-pig ventricular tissue. Whole cell electrophysiology was done on isolated guinea-pig ventricular cardiomyocytes. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Tissue and cells were harvested from young and old, male and female guinea-pigs. RT-PCR analysis did not reveal significant age-related changes in levels of Kir6.1 or Kir6.2 mRNAs. However, levels of SUR2A were significantly lower in old than in young females. Such age-differences were not observed with cardiac tissue from male animals. In both old and young males, pinacidil (100 microM) induced outward currents. The difference between current density of pinacidil-sensitive component in females, but not males, was statistically significant. Western blotting analysis revealed higher levels of Kir6.2 and SUR2A proteins in cardiac membrane fraction from young than old females. The present study demonstrates that in females, but not males, aging is associated with decrease in number of cardiac K(ATP) channels which is due to decrease in levels of the SUR2A subunit.

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 approximately 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.

Objectives: The main objective of this study was to establish whether gender regulates expression and/or properties of cardiac ATP-sensitive K(+) (K(ATP)) channels., Background: Recently, evidence has been provided that differing cardiac responses in males and females to metabolic stress may result from gender-specific difference(s) in the efficiency of endogenous cardioprotective mechanism(s) such as K(ATP) channels., Methods: A reverse transcription polymerase chain reaction (RT-PCR) using primers specific for Kir6.2, Kir6.1 and SUR2A subunits was performed on total RNA from guinea pig ventricular tissue. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Whole-cell, single-channel electrophysiology and digital epifluorescent Ca(2+) imaging were performed on isolated guinea pig ventricular cardiomyocytes., Results: The RT-PCR revealed higher levels of SUR2A, but not Kir6.1 and Kir6.2, messenger RNA in female tissue relative to male tissue, while much higher levels of both Kir6.2 and SUR2A proteins in cardiac membrane fraction in female tissue compared with male tissue were found. In both male and female tissue, pinacidil (100 microM), a K(ATP) channel opener, induced outward whole-cell currents. The current density of the pinacidil-sensitive component was significantly higher in female tissue than it was in male tissue, while no differences in single K(ATP) channel properties between genders were observed. Ischemia-reperfusion challenge induced significant intracellular Ca(2+) loading in male, but not female, cardiomyocytes. To test the hypothesis that SUR2A expression is the limiting factor in K(ATP) channel formation, we took different volumes of Kir6.2 and SUR2A complementary DNA (cDNA) from the same cDNA pool and subjected them to PCR. In order to obtain a band having 50% of the maximal intensity, a volume of SUR2a cDNA approximately 20 times the volume of Kir6.2 cDNA was required., Conclusions: This study has demonstrated that female tissue expresses higher levels of functional cardiac K(ATP) channels than male tissue due to the higher expression of the SUR2A subunit, which has an impact on cardiac response to ischemia-reperfusion challenge.