Article

Pharmacological Inhibition of -Protein Kinase C Attenuates Cardiac Fibrosis and Dysfunction in Hypertension-Induced Heart Failure

July 2008
Hypertension 51(6):1565-9

July 2008
51(6):1565-9

DOI:10.1161/HYPERTENSIONAHA.107.109637

Source
PubMed

Authors:

Tomoyoshi Koyanagi

Lihan Sun

Xi'an Jiaotong-Liverpool University

Show all 5 authorsHide

Studies on genetically manipulated mice suggest a role for epsilon-protein kinase C (epsilonPKC) in cardiac hypertrophy and in heart failure. The potential clinical relevance of these findings was tested here using a pharmacological inhibitor of epsilonPKC activity during the progression to heart failure in hypertensive Dahl rats. Dahl rats, fed an 8% high-salt diet from the age of 6 weeks, exhibited compensatory cardiac hypertrophy by 11 weeks, followed by heart failure at approximately 17 weeks and death by the age of approximately 20 weeks (123+/-3 days). Sustained treatment between weeks 11 and 17 with the selective epsilonPKC inhibitor epsilonV1-2 or with an angiotensin II receptor blocker olmesartan prolonged animal survival by approximately 5 weeks (epsilonV1-2: 154+/-7 days; olmesartan: 149+/-5 days). These treatments resulted in improved fractional shortening (epsilonV1-2: 58+/-2%; olmesartan: 53+/-2%; saline: 41+/-6%) and decreased cardiac parenchymal fibrosis when measured at 17 weeks without lowering blood pressure at any time during the treatment. Combined treatment with epsilonV1-2, together with olmesartan, prolonged animal survival by 5 weeks (37 days) relative to olmesartan alone (from 160+/-5 to 197+/-14 days, respectively) and by approximately 11 weeks (74 days) on average relative to saline-treated animals, suggesting that the pathway inhibited by epsilonPKC inhibition is not identical to the olmesartan-induced effect. These data suggest that an epsilonPKC-selective inhibitor such as epsilonV1-2 may have a potential in augmenting current therapeutic strategies for the treatment of heart failure in humans.

Reviewing the Regulators of COL1A1

Article

Full-text available

Jun 2023
INT J MOL SCI

The collagen family contains 28 proteins, predominantly expressed in the extracellular matrix (ECM) and characterized by a triple-helix structure. Collagens undergo several maturation steps, including post-translational modifications (PTMs) and cross-linking. These proteins are associated with multiple diseases, the most pronounced of which are fibrosis and bone diseases. This review focuses on the most abundant ECM protein highly implicated in disease, type I collagen (collagen I), in particular on its predominant chain collagen type I alpha 1 (COLα1 (I)). An overview of the regulators of COLα1 (I) and COLα1 (I) interactors is presented. Manuscripts were retrieved searching PubMed, using specific keywords related to COLα1 (I). COL1A1 regulators at the epigenetic, transcriptional, post-transcriptional and post-translational levels include DNA Methyl Transferases (DNMTs), Tumour Growth Factor β (TGFβ), Terminal Nucleotidyltransferase 5A (TENT5A) and Bone Morphogenic Protein 1 (BMP1), respectively. COLα1 (I) interacts with a variety of cell receptors including integrinβ, Endo180 and Discoidin Domain Receptors (DDRs). Collectively, even though multiple factors have been identified in association to COLα1 (I) function, the implicated pathways frequently remain unclear, underscoring the need for a more spherical analysis considering all molecular levels simultaneously.

RENAL PKC EPSILON DEFICIENCY ATTENUATES ISCHEMIA REPERFUSION INJURY VIA IMPAIRED NEUTROPHIL INFILATRATION AND TNF ALPHA DEPENDENT APOPTOSIS INHIBITION

Article

May 2012

Phosphorylation and O-GlcNAcylation modulation of contractile proteins in heart failure

Article

Oct 2010

Emilie Dubois

Despite significant improvements in management of myocardial infarction (MI), left ventricular remodelling (LVR) remains a major complication and a strong predictor of both heart failure (HF) and death after MI. Although several variables, such as MI size, have been identified as risk factors, LVR remains difficult to predict in clinical practice. Better prediction could allow an individualized approach with more intense therapy and follow-up for such high-risk patients. The aim of my work is to identify molecular determinants of LVR to have a better understanding of physiopathological mechanisms of LVR. For that purpose, we studied post-translational modifications of contractile proteins in particular, phosphorylation and O-N-acetylglucosaminylation (O-GlcNAc). Then, we studied particularly troponin T (TnT) for which we could highlight a decrease of phosphorylation of serine 208 in LV and plasma of MI-rats. These results suggest that the level of circulating phosphorylated troponin T could be new biomarker of LVR and may help to predict the development of heart failure after MI. For this study, we worked in collaboration with INSERM unit U644 at Rouen using an experimental model of HF. MI was induced in rat by left coronary ligation and, the control rats undergoing the surgery without ligation. Initially, we performed differential phosphoproteomic study of LV in the late phase of the LVR (2 months post-MI). For this purpose, LV proteins were extracted and separated by two-dimensional electrophoresis. Gels were first stained by Pro-Q®Diamond (specific of phosphorylated proteins) and then by Sypro®Ruby (specific of total proteins). By bioinformatic analysis, we showed that 69 polypeptidic spots were modulated for their phosphorylation levels. We analyzed these spots by mass spectrometry and identified 30 proteins corresponding to 53 spots with modulationof phosphorylation. Among these proteins, we have chosen to study 6 contractile proteins: TnT, alpha-tropomyosin 1 (Tm-α1), desmin, αB-crystallin and myosin light chains 1 and 2 (MLC). For each described proteins, we have validated the modulation of phosphorylation and determined the aminoacid involved in the phosphorylation modulation using immunoprecipitation techniques with specific antibodies against the proteins and phospho-Tyrosine, -Threonine and -Serine antibodies confirming the screening performed by 2D-electrophoresis for the detection of phosphoproteins. We observed a significant decrease of phosphorylation on serine for Tm-α1, TnT and MLC-2 and on tyrosine residues for αB-crystallin as well as a significant increase in phosphorylation on tyrosine for MLC-1 and on serine residues for desmin, thus confirming the results obtained in two-dimensional electrophoresis. In order to complete analysis of the post-translational modifications, we studied the modifications of O-GlcNAc for each one of these proteins. We thus observed a significant decrease in O-GlcNAcylation of Tm-α1, αB-crystallin and desmin as well as an increase in O-GlcNAcylation of the MLC-3 and TnT. In addition, we have correlated these modulations of phosphorylation and O-GlcNAcylation levels with modulations of the activity of enzymes implied in these modulations. Indeed, by bioinformatic analysis of the TnT sequence and literature review, we highlighted that the protein kinase C and the protein phosphatase 2A could be implied in these modulations. We observed a decrease of protein kinase C epsilon isoform expression in the LV of MI- rats without modulation of protein phosphatase 2A activity. In addition, we showed an increase in the activity of O-GlcNAc transferase and a decreaseof O-GlcNAcase activity in LV of MI rats. [...]

Modifications post-traductionnelles des protéines contractiles cardiaques : nouveaux biomarqueurs du remodelage ventriculaire post-infarctus

Article

Emilie Dubois

Le remodelage ventriculaire gauche (RVG) est un processus complexe qui intervient après un infarctus du myocarde chez 30% des patients en dépit des meilleurs traitements connus actuellement. Le but de mon travail de thèse consistait à identifier les déterminants moléculaires du RVG dans le but de mieux en comprendre les mécanismes physiopathologiques. Pour cela, nous avons étudié les modifications post-traductionnelles des protéines contractiles du VG et en particulier, la phosphorylation et la O-N-acétylglucosaminylation (O-GlcNAc). Nous nous sommes ensuite particulièrement intéressés à la troponine T (TnT) pour laquelle nous avons ainsi pu mettre en évidence une diminution de la phosphorylation au niveau de la sérine 208 au niveau du VG et du plasma chez le rat, suggérant que cela pourrait être un marqueur du RVG post-infarctus. Pour cette étude, nous avons travaillé en collaboration avec l'unité INSERM U644 de Rouen sur un modèle expérimental d'insuffisance cardiaque. L'infarctus du myocarde est induit chez le rat par ligature de la branche descendante de l'artère coronaire gauche, les rats témoins subissant l'intervention mais sans ligature. Dans un premier temps, nous avons réalisé une étude globale du phosphoprotéome du VG en phase tardive du RVG (2 mois post-ligature). Pour cela, les protéines extraites du VG ont été séparées par électrophorèse bidimensionnelle puis colorées au Pro-Q®Diamond (spécifique des protéines phosphorylées) puis au Sypro®Ruby (spécifique des protéines totales). Par analyse bioinformatique, nous avons mis en évidences 69 spots polypeptidiques présentant des modulations de phosphorylation. Nous avons donc analysé ces spots par spectrométrie de masse et avons identifié 30 protéines correspondant à 53 spots polypeptidiques présentant des modulations de phosphorylation. Parmi ces protéines, nous avons choisi de nous concentrer et d'étudier 6 protéines contractiles : la TnT, l'alpha-tropomyosine 1 (α-Tm 1), la desmine, l'αB-crystalline et les chaînes légères de myosine 1 et 2 (MLC). Pour chacune de ces protéines, nous avons identifié le type d'acide aminé responsable de la phosphorylation et quantifié les modulations de phosphorylation dans le VG des rats insuffisants cardiaques (IC). De manière intéressante, nous avons observé que le VG des animaux IC présentait une diminution significative de la phosphorylation sur les résidus sérine pour l'α-Tm 1, la TnT et la MLC-2 et sur les résidus de tyrosine pour l'αB-crystalline ainsi qu'une augmentation significative de la phosphorylation sur les résidus tyrosine pour la MLC-1 et sur les résidus de sérines pour la desmine, confirmant ainsi les résultats obtenus en électrophorèse bidimensionnelle. Afin de compléter l'analyse des modifications post-traductionnelles, nous avons étudié les modifications de O-GlcNAc pour chacune de ces protéines. Nous avons ainsi observé une diminution significative de la O-GlcNAcylation de l'α-Tm 1, de la desmine et l'αB crystalline ainsi qu'une augmentation de la O-GlcNAcylation de la MLC-3 et de la TnT. Par ailleurs, nous avons pu corréler ces modulations de phosphorylation et de O-GlcNAcylation avec des modulations de l'activité des enzymes impliquées dans ces modulations. En effet, par analyse bioinformatique de la séquence de la TnT et par recherche bibliographique nous avons mis en évidence que la protéine kinase C et la protéine phosphatase 2A pourrait être impliquées dans ces modulations de phosphorylation. Nous avons alors mis en évidence une diminution de l'activité de la protéine kinase C epsilon dans le VG des rats IC mais sans variation de l'activité de la protéine phosphatase 2A. Par ailleurs, nous avons mis en évidence une augmentation de l'activité de la O-GlcNAc transférase et une diminution de l'activité de la O-GlcNAcase dans le VG des rats IC. Afin d'étudier les conséquences de ces modifications post-traductionnelles sur la fonction cardiaque, nous avons également utilisé un modèle de cœur isolé perfusé de rats par l'appareil Langendorff pour évaluer la fonction ventriculaire gauche en relation avec les modifications post-traductionnelles des protéines contractiles. La technique de cœur isolé perfusé nous a permis d'effectuer l'enregistrement des pressions du VG ainsi que des constantes de contractilité et de relaxation cardiaque. Nous avons étudié l'effet des modifications post-traductionnelles sur la fonction cardiaque par injection d'inhibiteurs spécifiques de l'isoforme ε de la protéine kinase C (peptides ε V1-2) afin de mimer une diminution de la phosphorylation et par injection d'inhibiteurs spécifiques de la O-GlcNAcase (PUGNAc) afin de mimer une augmentation de la O-GlcNAcylation. Nous avons ainsi évalué la fonction cardiaque dans des groupes d'animaux sains traités ou non par inhibiteurs mais également dans des groupes d'animaux IC traités ou non par inhibiteurs afin d'évaluer les effets bénéfiques ou délétères de ces traitements sur l'insuffisance cardiaque. L'étude globale du phosphoprotéome du VG nous a permis d'observer une diminution significative de la phosphorylation de la TnT sur la sérine 208 chez les rats IC. Nous avons donc synthétisé un peptide représentatif de cette séquence pour produire des anticorps dirigés spécifiquement contre la TnT phosphorylée en S208 (AC :P50753). Nous avons ainsi étudié la TnT phosphorylée en S208 ainsi que la forme O-GlcNAc de la TnT dans le plasma des rats IC. Nous avons mis en évidence à la fois une diminution de la TnT phosphorylée en S208 et une augmentation de la TnT O-GlcNAc dans le plasma des rats IC, suggérant que ces modifications pourraient être considérées comme marqueurs potentiels du RVG. Au vu de ces résultats, nous avons quantifié la TnT phosphorylée en S207 chez l'homme (AC : P45375) et la TnT O-GlcNAc dans deux populations indépendantes de patients coronariens présentant différents degrés de RVG (faible, intermédiaire et élevé). Nous avons ainsi observé une diminution significative de la TnT phosphorylée en S207 dans le plasma des patients présentant un RVG intermédiaire à élevé mais sans variation significative du niveau de TnT O-GlcNAc. Ces résultats suggèrent que la TnT phosphorylée en S207 pourrait être un biomarqueur du RVG. En conclusion, nous avons mis en évidence par analyse phosphoprotéomique globale des modulations du niveau de phosphorylation des protéines contractiles lors du RVG post-infarctus dans un modèle expérimental. Nous avons ensuite mis en évidence une balance phosphorylation / O-GlcNAc de la TnT en S208 dans le VG et dans le plasma des rats IC à 2 mois post-infarctus. Enfin, nous avons observé les mêmes modulations de phosphorylation chez l'homme et ainsi mis en évidence que la diminution de la phosphorylation de la TnT en S207 pourrait être un biomarqueur du RVG.

βIIPKC and εPKC isozymes as potential pharmacological targets in cardiac hypertrophy and heart failure

Article

Oct 2010
J MOL CELL CARDIOL

Cardiac hypertrophy is a complex adaptive response to mechanical and neurohumoral stimuli and under continual stressor, it contributes to maladaptive responses, heart failure and death. Protein kinase C (PKC) and several other kinases play a role in the maladaptative cardiac responses, including cardiomyocyte hypertrophy, myocardial fibrosis and inflammation. Identifying specific therapies that regulate these kinases is a major focus of current research. PKC, a family of serine/threonine kinases, has emerged as potential mediators of hypertrophic stimuli associated with neurohumoral hyperactivity in heart failure. In this review, we describe the role of PKC isozymes that is involved in cardiac hypertrophy and heart failure. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure".

PKCβII inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro-inflammatory responses

Article

Aug 2011
J CELL MOL MED

Protein kinase C βII (PKCβII) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted overexpression of PKCβII in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKCβII in HF development. Using a post-myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKCβII on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKCβII selective inhibitor (βIIV5-3 conjugated to TAT(47-57) carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT(47-57) carrier peptide alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKCβII inhibitor. Further, a 90% decrease in active TGFβ1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKCβII attenuates cardiac remodelling mediated by the TGF-SMAD signalling pathway. Therefore, sustained selective inhibition of PKCβII in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.

Role of Connexin 43 phosphorylation on Serine-368 by PKC in cardiac function and disease

Article

Full-text available

Jan 2023

Intercellular communication mediated by gap junction channels and hemichannels composed of Connexin 43 (Cx43) is vital for the propagation of electrical impulses through cardiomyocytes. The carboxyl terminal tail of Cx43 undergoes various post-translational modifications including phosphorylation of its Serine-368 (S368) residue. Protein Kinase C isozymes directly phosphorylate S368 to alter Cx43 function and stability through inducing conformational changes affecting channel permeability or promoting internalization and degradation to reduce intercellular communication between cardiomyocytes. Recent studies have implicated this PKC/Cx43-pS368 circuit in several cardiac-associated diseases. In this review, we describe the molecular and cellular basis of PKC-mediated Cx43 phosphorylation and discuss the implications of Cx43 S368 phosphorylation in the context of various cardiac diseases, such as cardiomyopathy, as well as the therapeutic potential of targeting this pathway.

Distinct Regulation of Cardiac Fibroblast Proliferation and Transdifferentiation by Classical and Novel Protein Kinase C Isoforms: Possible Implications for New Antifibrotic Therapies

Article

Nov 2020

Cardiac fibrosis is characterized by accumulation and activation of fibroblasts and excessive production of extracellular matrix, which results in myocardial stiffening and eventually leads to heart failure. While previous work suggests that protein kinase C (PKC) isoforms play a role in cardiac fibrosis and remodeling, the results are conflicting. Moreover, the potential of targeting PKC with pharmacological tools to inhibit pathological fibrosis has not been fully evaluated. Here we investigated the effects of selected PKC agonists and inhibitors on cardiac fibroblast (CF) phenotype, proliferation, and gene expression using primary adult mouse CFs, which spontaneously transdifferentiate into myofibroblasts in culture. A 48-h exposure to the potent PKC activator phorbol 12-myristate 13-acetate (PMA) at 10 nM concentration reduced the intensity of α-smooth muscle actin staining by 56% and periostin mRNA levels by 60% compared to control. The decreases were inhibited with the pan-PKC inhibitor Gö6983 and the inhibitor of classical PKC isoforms Gö6976, suggesting that classical PKCs regulate CF transdifferentiation. PMA also induced a 33% decrease in BrdU-positive CFs, which was inhibited with Gö6983 but not with Gö6976, indicating that novel PKC isoforms (nPKCs) regulate CF proliferation. Moreover, PMA downregulated the expression of collagen encoding genes Col1a1 and Col3a1 nPKC-dependently, showing that PKC activation attenuates matrix synthesis in CFs. The partial PKC agonist isophthalate derivative HMI-1b11 induced parallel changes in phenotype, cell cycle activity, and gene expression. In conclusion, our results reveal distinct PKC-dependent regulation of CF transdifferentiation and proliferation and suggest that PKC agonists exhibit potential as an antifibrotic treatment. Significance Statement Cardiac fibrosis is a pathological process that contributes to the development of heart failure. The molecular mechanisms regulating fibrosis in the heart are however not fully understood, which hinders the development of new therapies. Here, we demonstrate that classical and novel protein kinase C (PKC) isoforms distinctly regulate cardiac fibroblast transdifferentiation and proliferation, the two central processes in fibrosis. Our results indicate that pharmacological PKC activation may be a promising strategy to inhibit myocardial fibrosis.

Epub ahead of print Molecular mechanisms underlying gallic acid effects against cardiovascular diseases: An update review

Article

Full-text available

Aug 2019

Ghaidafeh Akbari

Objective: The prevalence of cardiovascular diseases (CVDs) is growing. CVDs are the major cause of mortality and have become one of the most important health challenges in developing countries. Gallic acid (GA) is a natural phytochemical which has been widely used against multiple conditions. The present review was designed to evaluate molecular mechanisms underlying the protective effects of this agent against CVDs. Materials and methods: Data discussed in this review were collected from the articles published in databases such as Science Direct, Scopus, PubMed, and Scientific Information Database between 1993 and 2018. Results: According to the experimental studies, GA has protective actions against CVDs through increasing antioxidant enzymes capacity, inhibition of lipid peroxidation and decreasing serum levels of cardiac marker enzymes, modulation of hemodynamic parameters, recovery of electrocardiogram aberrations, and preservation of histopathological changes. Conclusion: GA has potential cardioprotective action. Therefore, it has been suggested that this agent can be administered in underlying of CVDS.

Hypertrophy of human embryonic stem cell–derived cardiomyocytes supported by positive feedback between Ca2+ and diacylglycerol signals

Article

Full-text available

Aug 2019
PFLUG ARCH EUR J PHY

Human embryonic stem cell–derived cardiomyocytes develop pronounced hypertrophy in response to angiotensin-2, endothelin-1, and a selected mix of three fatty acids. All three of these responses are accompanied by increases in both basal cytoplasmic Ca²⁺ and diacylglycerol, quantified with the Ca²⁺ sensor Fluo-4 and a FRET-based diacylglycerol sensor expressed in these cardiomyocytes. The heart glycoside, ouabain (30 nM), and a recently developed inhibitor of diacylglycerol lipases, DO34 (1 μM), cause similar hypertrophy responses, and both responses are accompanied by equivalent increases of basal Ca²⁺ and diacylglycerol. These results together suggest that basal Ca²⁺ and diacylglycerol form a positive feedback signaling loop that promotes execution of cardiac growth programs in these human myocytes. Given that basal Ca²⁺ in myocytes depends strongly on the Na⁺ gradient, we also tested whether nanomolar ouabain concentrations might stimulate Na⁺/K⁺ pumps, as described by others, and thereby prevent hypertrophy. However, stimulatory effects of nanomolar ouabain (1.5 nM) were not verified on Na⁺/K⁺ pump currents in stem cell–derived myocytes, nor did nanomolar ouabain block hypertrophy induced by endothelin-1. Thus, low-dose ouabain is not a “protective” intervention under the conditions of these experiments in this human myocyte model. To summarize, the major aim of this study has been to characterize the progression of hypertrophy in human embryonic stem cell–derived cardiac myocytes in dependence on diacylglycerol and Na⁺ gradient changes, developing a case that positive feedback coupling between these mechanisms plays an important role in the initiation of hypertrophy programs.

Danhong Injection Reversed Cardiac Abnormality in Brain–Heart Syndrome via Local and Remote β-Adrenergic Receptor Signaling

Article

Full-text available

Jul 2018

Ischemic brain injury impacts cardiac dysfunction depending on the part of the brain affected, with a manifestation of irregular blood pressure, arrhythmia, and heart failure. Generally called brain–heart syndrome in traditional Chinese medicine, few mechanistic understanding and treatment options are available at present. We hypothesize that considering the established efficacy for both ischemic stroke and myocardial infarction (MI), Danhong injection (DHI), a multicomponent Chinese patent medicine, may have a dual pharmacological potential for treating the brain–heart syndrome caused by cerebral ischemic stroke through its multi-targeted mechanisms. We investigated the role of DHI in the setting of brain–heart syndrome and determined the mechanism by which it regulates this process. We induced Ischemia/Reperfusion in Wistar rats and administered intravenous dose of DHI twice daily for 14 days. We assessed the neurological state, infarct volume, CT scan, arterial blood pressure, heart rhythm, and the hemodynamics. We harvested the brain and heart tissues for immunohistochemistry and western blot analyses. Our data show that DHI exerts potent anti-stroke effects (infarct volume reduction: ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. vehicle. Neurological deficit correction: ∗p < 0.05 and ∗∗∗p < 0.001 vs. vehicle), and effectively reversed the abnormal arterial pressure (∗p < 0.05 vs. vehicle) and heart rhythm (∗∗p < 0.01 vs. vehicle). The phenotype of this brain–heart syndrome is strikingly similar to those of MI model. Quantitative assessment of hemodynamic in cardiac functionality revealed a positive uniformity in the PV-loop after administration with DHI and valsartan in the latter. Immunohistochemistry and western blot results showed the inhibitory effect of DHI on the β-adrenergic pathway as well as protein kinase C epsilon (PKCε) (∗∗p < 0.01 vs. model). Our data showed the underlying mechanisms of the brain–heart interaction and offer the first evidence that DHI targets the adrenergic pathway to modulate cardiac function in the setting of brain–heart syndrome. This study has made a novel discovery for proper application of the multi-target DHI and could serve as a therapeutic option in the setting of brain–heart syndrome.

Abnormal Downregulation of Caveolin-3 Mediates the Pro-Fibrotic Action of Microrna-22 in a Mouse Model of Myocardial Infarction

Article

Full-text available

Feb 2018

Background/aims: Cardiac fibrosis is an important cardiac remodeling event that can ultimately lead to the development of severe arrhythmia and heart failure. MicroRNAs (miRNAs) are involved in the pathogenesis of many cardiovascular diseases. Here, we aimed to investigate the effects of caveolin-3 (Cav3) on the pathogenesis of cardiac fibrosis and the underlying molecular mechanisms. Methods: Cav3 expression was decreased in cardiac fibrosis in vivo and in vitro model. To investigate the role of Cav3 in cardiac fibrosis, we transfected cardiac fibroblasts (CFs) with the siRNA of Cav3 and Cav3-overexpressing plasmid. The collagen content and proliferation of CFs were detected by qRT-PCR, western blot, MTT, and immunofluorescence. A luciferase reporter gene assay and gain/loss of function were used to detect the relationship between miR-22 and Cav3. Results: Cav3 depletion in CFs induced an increase in collagen content, cell proliferation, and phenotypic conversion of fibroblasts to myofibroblasts. Conversely, Cav3 overexpression in CFs was shown to inhibit angiotensin II-mediated excessive collagen deposition through protein kinase C (PKC)ε inactivation. Cav3 was experimentally confirmed as a direct target of miR-22, containing two seed binding sites in its 3'-untranslated region, and miR-22 was demonstrated to be significantly upregulated in the ischemic border zone in mice after myocardial infarction and in neonatal rat CFs pretreated with angiotensin II. miR-22 overexpression increased CFs proliferation, and collagen and α-smooth muscle actin levels in CFs, while the knockdown of endogenous miR-22 decreased CFs numbers. Conclusions: Our findings demonstrate that miR-22 accelerates cardiac fibrosis through the miR-22-Cav3-PKCε pathway, which, therefore, may represent a new therapeutic target for treatment of excessive fibrosis-associated cardiac diseases.

Effect of PKC inhibitor on experimental autoimmune myocarditis in Lewis rats

Article

Full-text available

Apr 2017

Myocarditis is a major cause of sudden, unexpected death in young people. However, it is still one of the most challenging diseases to treat in cardiology. In the present study, we showed that both expression level and activity of PKC-α were up-regulated in the rat heart of experimental autoimmune myocarditis (EAM). Intraperitoneal administration of PKC inhibitor (Ro-32-0432) at the end of the most severe inflammation period of EAM still significantly reduced the EAM induced expression of failure biomarkers. Furthermore, Ro-32-0432 reduced the ratio of Bax/Bcl-2 and suppressed the expression of cleaved caspase-3, both of which were increased in the heart of the EAM rats, suggesting an anti-apoptotic role of Ro-32-0432. Besides, Ro-32-0432 suppressed EAM-induced cardiac fibrosis and release of pro-inflammatory cytokines IL-1β and IL-17. These results suggest that inhibition of PKC may serve as a potential therapeutic strategy for the treatment of myocarditis.

PRKCE gene encoding protein kinase C-epsilon—Dual roles at sarcomeres and mitochondria in cardiomyocytes

Article

Sep 2016

Protein kinase C-epsilon (PKCε) is an isoform of a large PKC family of enzymes that has a variety of functions in different cell types. Here we discuss two major roles of PKCε in cardiac muscle cells; specifically, its role in regulating cardiac muscle contraction via targeting the sarcomeric proteins, as well as modulating cardiac cell energy production and metabolism by targeting cardiac mitochondria. The importance of PKCε action is described within the context of intracellular localization, as substrate selectivity and specificity is achieved through spatiotemporal targeting of PKCε. Accordingly, the role of PKCε in regulating myocardial function in physiological and pathological states has been documented in both cardioprotection and cardiac hypertrophy.

Multifunctional roles of PKCδ: Opportunities for targeted therapy in human disease

Article

May 2016

The serine-threonine protein kinase, Protein Kinase C-δ (PKCδ), is emerging as a bi-functional regulator of cell death and proliferation. Studies in PKCδ -/- mice have confirmed a pro-apoptotic role for this kinase in response to DNA damage, and a tumor promoter role in some oncogenic contexts. In non-transformed cells, inhibition of PKCδ suppresses release of cytochrome c and caspase activation, indicating a function upstream of apoptotic pathways. Data from PKCδ -/- mice demonstrates a role for PKCδ in the execution of DNA damage-induced and physiologic apoptosis. This has led to the important finding that inhibitors of PKCδ can be used therapeutically to reduce irradiation and chemotherapy-induced toxicity. In contrast, PKCδ is a tumor promoter in mouse models of mammary gland and lung cancer, and increased PKCδ expression is a negative prognostic indicator in Her2 + and other subtypes of human breast cancer. Understanding how these distinct functions of PKCδ are regulated is critical for the design of therapeutics to target this pathway. This review will discuss what is currently known about biological roles of PKCδ and prospects for targeting PKCδ in human disease.

Protein Kinase C Epsilon Contributes to NADPH Oxidase Activation in a Pre-Eclampsia Lymphoblast Cell Model

Article

Full-text available

Dec 2013

Pre-eclampsia is a pregnancy-specific disorder characterised by hypertension and proteinuria, which in severe cases results in multi-system disturbances. The maternal syndrome is associated with a pro-inflammatory state, consisting of leukocyte activation, which is thought to contribute to the widespread endothelial dysfunction. We previously showed increased activation of NADPH oxidase in pre-eclampsia, in both neutrophils and B-lymphoblast cell lines (B-LCLs). In this study, the mechanism by which NADPH oxidase activity is increased in pre-eclampsia was further investigated. NADPH oxidase activity was found to be increased in phorbol-12-myristate-13-acetate (PMA) stimulated B-LCLs isolated from women with pre-eclampsia. This correlated with an increase in protein kinase C (PKC) substrate phosphorylation, p47-phox phosphorylation (a regulatory component of NADPH oxidase) and p47-phox directed-kinase activity. Using ion exchange and hydroxyapatite chromatography we identified a major peak of PMA regulated p47-phox kinase activity. Chromatography fractions were probed for PKC isoforms. We found the major peak of p47-phox kinase activity could not be separated from the elution profile of PKC epsilon. Using a peptide inhibitor of PKC epsilon, PMA-induced reactive oxygen species (ROS) production could be reduced to that of a normal B-LCL. These data suggest a pro-inflammatory role for PKC epsilon in the pathogenesis of pre-eclampsia.

Expression of Ser Phosphorylated PKC Epsilon in Experimental Crescentic Glomerulonephritis: An Immunohistochemical Study

Article

Full-text available

Apr 2014

PKCε, a DAG-dependent, Ca2+- independent kinase attenuates extent of fibrosis following tissue injury, suppresses apoptosis and promotes cell quiescence. In crescentic glomerulonephritis (CGN), glomerular epithelial cells (GEC) contribute to fibro-cellular crescent formation while they also transdifferentiate to a mesenchymal phenotype. The aim of this study was to assess PKCε expression in CGN. Using an antibody against PKC-ε phosphorylated at Ser729, we assessed its localization in rat model of immune-mediated rapidly progressive CGN. In glomeruli of control animals, pPKCε was undetectable. In animals with CGN, pPKCε was expressed exclusively in glomerular epithelial cells (GEC) and in GEC comprising fibrocellular crescents that had acquired a myofibroblast-type phenotype. In non-immune GEC injury induced by puromycin aminonucleoside and resulting in proteinuria of similar magnitude as in CGN, pPKCε expression was absent. There was constitutive pPKCε expression in distal convoluted tubules, collecting ducts and thick segments of Henley's loops in both control and experimental animals. We propose that pPKCε expression occurring in GEC and in fibrocellular crescentic lesions in CGN may facilitate PKCε dependent pathologic processes.

Calponin1 inhibits dilated cardiomyopathy development in mice through the εPKC pathway

Article

Feb 2014

Calponin1 (CNN1) is involved in the regulation of smooth muscle contraction in physiological situation and it also expresses abnormally in a variety of pathological situations. We found that the expression of CNN1 decreased significantly in the heart tissue of a cTnT(R141W) transgenic dilated cardiomyopathy (DCM) mouse model and an adriamycin (ADR)-induced DCM mouse model, suggesting that CNN1 is involved in the pathogenesis of DCM. However, the role of CNN1 on cardiac function, especially on pathogenesis of DCM, has not been clarified. In this study, we tested whether rescued expression of CNN1 could prevent the development of DCM and investigated its possible mechanisms. The DCM phenotypes were significantly improved with the transgenic expression of CNN1 in the cTnT(R141W)×CNN1 double transgenic (DTG) mice, which was demonstrated by the survival, cardiac geometry and function analyses, as well as microstructural and ultrastructural observations based on echocardiography and histology examination. The expression of CNN1 could also resist the cardiac geometry breakage and dysfunction in the ADR-induced DCM mice model. Meanwhile, the epsilon isoform of protein kinase C (εPKC) activator and inhibitor could reverse the activation of εPKC/ERK/mTOR pathway and DCM phenotypes in the cTnT(R141W) and cTnT(R141W)×CNN1 double transgenic (DTG) mice. εPKC/ERK/mTOR pathway activation induced by the rescued expression of CNN1 contributed to the improvement of cardiac dysfunction and pathological changes observed in the DTG mice. CNN1 could be a therapeutic target to prevent the development of DCM and heart failure (HF).

Expression and Regulation of Osteopontin in the Diabetic Heart

Article

Jan 2011

Abigail Goodman

Diabetes leads to several alterations in cardiac structure, one of which is fibrosis of the ventricular myocardium. Myocardial fibrosis is a common underlying factor in most cardiac pathologies. Osteopontin (OPN) is a small phospho-protein that has been implicated in fibrotic tissue remodeling. In the heart, the expression of OPN protein is increased after acute and chronic pathologies. Upregulation of OPN coincides with a transition to heart failure and a direct role for OPN in the progression of diabetic cardiomyopathy to heart failure has been reported. The overall objective of this project is to determine if OPN is upregulated in the heart in a model of type 2 diabetes and if OPN is increased in cardiac cells in response to high glucose. The hypotheses of the project are that OPN expression is increased in the type 2 diabetic heart, cardiac cells contribute to the increased OPN expression in the heart, and high glucose increases OPN expression in cardiac cells. Our proposed pathway of high glucose induced OPN expression is mediated by Angiotensin II (Ang II) and protein kinase C (PKC). In this study I use a type 2 diabetic rat model to determine if OPN expression is increased in the LV. Isolated neonatal rat ventricular myocytes and fibroblasts were used to determine upregulation of high glucose in cardiac cells and to elucidate the regulation of OPN expression in response to high glucose by Ang II and PKC. My results show that OPN expression is increased in the LV of a model of type 2 diabetes. Further I determined that both myocytes and fibroblasts increase OPN expression in response to high glucose. Inhibition of Ang II receptors and production decreased OPN expression in response to high glucose. To determine PKC regulation of OPN expression general and classical PKC inhibitors were used, both of which inhibited increased OPN expression in response to high glucose. A role for PKC in OPN expression was determined by overexpressing constitutively active and dominant negative recombinant PKC proteins. These results provide a better understanding of the signal transduction pathways leading to the cardiac dysfunction seen in diabetic patients.

Novel Drp1 inhibitor diminishes aberrant mitochondrial fission and neurotoxicity

Article

Full-text available

Dec 2012
J CELL SCI

Excessive mitochondrial fission is associated with the pathology of a number of neurodegenerative diseases. Therefore, inhibitors of aberrant mitochondrial fission could provide important research tools as well as potential leads for drug development. Using a rational approach, we designed a novel and selective peptide inhibitor, P110, of excessive mitochondrial fission. P110 inhibits Drp1 enzyme activity and blocks Drp1/Fis1 interaction in vitro and in cultured neurons whereas it has no effect on the interaction between Drp1 and other mitochondrial adaptors, as demonstrated by co-immunoprecipitation. Further, using a model of Parkinson's disease (PD) in culture, we demonstrated that P110 is neuroprotective by inhibiting mitochondrial fragmentation and ROS production and subsequently improving mitochondrial membrane potential and mitochondrial integrity. P110 increased neuronal cell viability by reducing apoptosis and autophagic cell death, and reduced neurite loss of primary dopaminergic neurons in this PD cell culture model. We also found that P110 treatment appears to have minimal effects on mitochondrial fission and cell viability under basal conditions. Finally, P110 required the presence of Drp1 to inhibit mitochondrial fission under oxidative stress conditions. Together, our findings suggest that P110, as a selective peptide inhibitor of Drp1, might be useful for treatment of diseases in which excessive mitochondrial fission and mitochondrial dysfunction occur.

Rosuvastatin-attenuated heart failure in aged spontaneously hypertensive rats via PKCα/β2 signal pathway

Article

Sep 2012
J CELL MOL MED

There are controversies concerning the capacity of Rosuvastatin to attenuate heart failure in end-stage hypertension. The aim of the study was to show whether the Rosuvastatin might be effective or not for the heart failure treatment. Twenty-one spontaneously hypertensive rats (SHRs) aged 52 weeks with heart failure were randomly divided into three groups: two receiving Rosuvastatin at 20mg.kg(-1) .d(-1) and 40mg.kg(-1) .d(-1) , respectively, and the third, placebo for comparison with 7 Wistar-Kyoto rats (WKYs) as controls. After an 8-week treatment, the systolic blood pressure (SBP) and echocardiographic features were evaluated; mRNA level of B-type natriuretic peptide (BNP) and plasma NT-proBNP concentration, measured; the heart tissues, observed under electron microscope (EM); myocardial sarcoplasmic reticulum Ca(2+) pump (SERCA-2) activity and mitochondria cytochrome C oxidase (CCO) activity, measured; the expression of SERCA-2a, phospholamban (PLB), ryanodine receptor2 (RyR2), sodium-calcium exchanger1 (NCX1), Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase inhibitor-1 (PPI-1), detected by western blot and RT-qPCR; and the total and phosphorylation of protein kinase Cα/β (PKCα/β), measured. Aged SHRs with heart failure was characterized by significantly decreased left ventricular ejection fraction and left ventricular fraction shortening, enhanced left ventricular end diastolic diameter and LV Volume, accompanied by increased plasma NT-proBNP and elevated BNP gene expression. Damaged myofibrils, vacuolated mitochondria and swollen sarcoplasmic reticulum were observed by EM. Myocardium mitochondria CCO and SERCA-2 activity decreased. The expressions of PLB and NCX1 increased significantly with up-regulation of PPI-1 and down-regulation of CaMKII, while that of RyR2 decreased. Rosuvastatin was found to ameliorate the heart failure in aged SHRs and to improve changes of SERCA-2a, PLB, RyR2, NCX1, CaMKII and PPI-1; PKCα/β2 signal pathway, to be suppressed; the protective effect of Rosuvastatin, to be dose-dependent. In conclusion, the heart failure of aged SHRs that was developed during the end-stage of hypertension could be ameliorated by Rosuvastatin. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundationfor Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Biphasic Time Course of the Changes in Aldosterone Biosynthesis Under High-Salt Conditions in Dahl Salt-Sensitive Rats

Article

Full-text available

Mar 2012
ARTERIOSCL THROM VAS

The comorbidity of excess salt and elevated plasma aldosterone has deleterious effects in cardiovascular disease. We evaluated the mechanisms behind the paradoxical increase in aldosterone biosynthesis in relation to dietary intake of salt. Dahl salt-sensitive (Dahl-S) and salt-resistant (Dahl-R) rats were fed a high-salt diet, and plasma and tissue levels of aldosterone in the adrenal gland and heart were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry. In Dahl-S rats, we found that the delayed and paradoxical increase in aldosterone biosynthesis after the initial and appropriate response to high salt. The late rise in aldosterone biosynthesis was accompanied by upregulation of CYP11B2 expression in the zona glomerulosa and increased adrenal angiotensin II levels and renin-angiotensin system components. It preceded the appearance of left ventricular systolic dysfunction and renal insufficiency. Blockade of angiotensin AT(1) receptors reversed the paradoxical increase in aldosterone biosynthesis. In contrast, Dahl-R rats maintained the initial suppression of aldosterone biosynthesis. Aldosterone levels in the heart closely paralleled those in the plasma and adrenal gland and disappeared after bilateral adrenalectomy. Chronic salt overload in Dahl-S rats stimulates aberrant aldosterone production via activation of the local renin-angiotensin system in the adrenal gland, thereby creating the comorbidity of excess salt and elevated plasma aldosterone.

ET-1 from endothelial cells is required for complete angiotensin II-induced cardiac fibrosis and hypertrophy

Article

Full-text available

Feb 2012

Hypertensive patients develop cardiac hypertrophy and fibrosis with increased stiffness, contractile deficit and altered perfusion. Angiotensin II (AngII) is an important factor in the promotion of this pathology. The effects of AngII are partly mediated by endothelin-1 (ET-1) and transforming growth factor-β. The exact feature of these pathways and the intercellular communications involved remain unclear. In this study, we explored the role of endothelial cell-derived ET-1 in the development of AngII-induced cardiac fibrosis and hypertrophy. We used mice with vascular endothelial cell specific ET-1 deficiency (VEETKO) and their wild type littermates (WT). Mice were infused for one week with AngII (3.2mg/kg/day, n=12) or vehicle (0.15mol/L NaCl and 1mmol/L acetic acid, n=5), using subcutaneous mini-pumps. Hearts were stained with hematoxylin-eosin and masson's trichrome for histology. Cardiac gene expression and protein abundance were measured by Northern Blot, real time PCR and Western Blot. AngII-induced cardiac hypertrophy, interstitial and perivascular fibrosis were less pronounced in VEETKO mice compared to WT. Blood pressure increased similarly in both genotypes. Expression of connective tissue growth factor, tumor growth factor-β, collagen I and III in response to AngII required endothelial ET-1. Endothelial ET-1 was also necessary to the elevation in protein kinase C δ abundance and ERK1/2 activation. AngII-induced elevation in PKCε abundance was however ET-1 independent. This study underscores the significance of ET-1 from the vasculature in the process of AngII-induced cardiac hypertrophy and fibrosis, independently from blood pressure. Endothelial ET-1 represents therefore a possible pharmacological target.

Therapeutic Potential for Protein Kinase C Inhibitor in Vascular Restenosis

Article

Full-text available

Jun 2011
J CARDIOVASC PHARM T

Vascular restenosis, an overreaction of biological response to injury, is initialized by thrombosis and inflammation. This response is characterized by increased smooth muscle cell migration and proliferation. Available pharmacological treatments include anticoagulants, antiplatelet agents, immunosuppressants, and antiproliferation agents. Protein kinase C (PKC), a large family of serine/threonine kinases, has been shown to participate in various pathological stages of restenosis. Consequently, PKC inhibitors are expected to exert a wide range of pharmacological activities therapeutically beneficial for restenosis. In this review, the roles of PKC isozymes in platelets, leukocytes, endothelial cells, and smooth muscle cells are discussed, with emphasis given to smooth muscle cells. We will describe cellular and animal studies assessing prevention of restenosis with PKC inhibitors, particularly targeting -α, -β, -δ, and -ζ isozymes. The delivery strategy, efficacy, and safety of such PKC regulators will also be discussed.

Highly Specific Modulators of Protein Kinase C Localization: Applications to Heart Failure

Article

Jun 2010
Drug Discov Today Dis Mech

Heart failure (HF) in which the blood supply does not match the body's needs, affects 10% of the population over 65 years old. The protein kinase C (PKC) family of kinases has a key role in normal and disease states. Here we discuss the role of PKC in HF and focus on the use of specific PKC regulators to identify the mechanism leading to this Pathology and potential leads for therapeutics.

Sustained Inhibition of Protein Kinase C Inhibits Vascular Restenosis After Balloon Injury and Stenting

Article

Sep 2010
CIRCULATION

ε protein kinase C (εPKC) is involved in vascular smooth muscle cell (VSMC) activation, but little is known about its function in vascular pathology. We aimed at assessing the role of εPKC in the development of restenosis. Rat models of aortic balloon injury with or without subsequent stenting were used. Rats were treated with the selective ψεPKC activator ε receptor for activated protein kinase C (ψεRACK), the selective εPKC inhibitor εV1-2, or saline. Both down-stream cascades of the platelet-derived growth factor receptor via extracellular signal-regulated kinase and Akt, respectively, were evaluated in vivo and in VSMC cultures. Intimal hyperplasia with luminal obliteration developed in saline-treated balloon-injured rat aortas (20.3±8.0%), and ψεRACK significantly promoted neointima development (32.4±4.9%, P=0.033), whereas εV1-2 significantly inhibited luminal narrowing (9.2±4.3%, P=0.039). εPKC inhibition led to significantly reduced VSMC extracellular signal-regulated kinase phosphorylation in vivo, whereas Akt phosphorylation was not markedly affected. Neointimal proliferation in vivo and platelet-derived growth factor-induced VSMC proliferation/migration in vitro were significantly inhibited by εV1-2. The inhibition of the platelet-derived growth factor pathway was mediated by inhibiting down-stream extracellular signal-regulated kinase and Akt phosphorylation. In vitro, εV1-2 showed inhibitory properties on endothelial cell proliferation, but that did not prevent reendothelialization in vivo. εV1-2 showed proapoptotic effects on VSMC in vitro. After stent implantation, luminal restenosis (quantified by optical coherence tomography imaging) was significantly reduced with εV1-2 (8.0±2.0%) compared with saline (20.2±9.8%, P=0.028). εPKC seems to be centrally involved in the development of neointimal hyperplasia. We suggest that εPKC inhibition may be mediated via inhibition of extracellular signal-regulated kinase and Akt activation. εPKC modulation may become a new therapeutic target against vascular restenosis.

PARP inhibition delays transition of hypertensive cardiopathy to heart failure in spontaneously hypertensive rats

Article

Full-text available

Jun 2009
CARDIOVASC RES

Oxidative stress followed by abnormal signalling can play a critical role in the development of long-term, high blood pressure-induced cardiac remodelling in heart failure (HF). Since oxidative stress-induced poly(ADP-ribose)polymerase (PARP) activation and cell death have been observed in several experimental models, we investigated the possibility that inhibition of nuclear PARP improves cardiac performance and delays transition from hypertensive cardiopathy to HF in a spontaneously hypertensive rat (SHR) model of HF. SHRs were divided into two groups: one received no treatment (SHR-C) and the other (SHR-L) received 5 mg/kg/day L-2286 (PARP-inhibitor) orally for 46 weeks. A third group was a normotensive age-matched control group (CFY) and a fourth was a normotensive age-matched group receiving L-2286 treatment 5 mg/kg/day (CFY+L). At the beginning of the study, systolic function was similar in both CFY and SHR groups. In the SHR-C group at the end of the study, eccentric hypertrophy with poor left ventricular (LV) systolic function was observed, while PARP inhibitor treatment preserved systolic LV function. Due to these favourable changes, the survival rate of SHRs was significantly improved (P < 0.01) by the administration of the PARP inhibitor (L-2286). The PARP inhibitor used did not affect the elevated blood pressure of SHR rats, but moderated the level of plasma-BNP (P < 0.01) and favourably influenced all the measured gravimetric parameters (P < 0.05) and the extent of myocardial fibrosis (P < 0.05). The inhibition of PARP increased the phosporylation of Akt-1/GSK-3beta (P < 0.01), ERK 1/2 (P < 0.01), and PKC epsilon (P < 0.01), and decreased the phosphorylation of JNK (P < 0.05), p-38 MAPK (P < 0.01), PKC pan betaII and PKC zeta/lambda (P < 0.01), and PKC alpha/betaII and delta (P < 0.05). These data demonstrate that chronic inhibition of PARP induces long-term favourable changes in the most important signalling pathways related to oxidative stress. PARP inhibition also prevents remodelling, preserves systolic function, and delays transition of hypertensive cardiopathy to HF in SHRs.

Diabetes mellitus accelerates left ventricular diastolic dysfunction through activation of the renin–angiotensin system in hypertensive rats

Article

May 2009
Hypertens Res

Diabetes mellitus (DM) is a major risk factor for heart failure, independent of coronary artery disease or hypertension (HT). Therefore, our study was designed to examine the mechanisms of DM-induced left ventricular (LV) diastolic dysfunction. In this study, we made five different 10-week treatment groups of Dahl salt-sensitive rats as follows: Control; a low-salt (0.5% NaCl) diet, HT; a high-salt (5% NaCl) diet, DM; a low-salt diet with streptozotocin (STZ) injection (30 mg kg(-1) i.p.), HT+DM; a high-salt diet with STZ injection, and the Olmesartan group; a high-salt diet with STZ treated with an angiotensin receptor blocker, olmesartan (1 mg kg(-1) day(-1)). Cardiac diastolic dysfunction with a preserved systolic function was noted in the HT group, and was most prominently noted in the HT+DM group, characterized by enhanced cardiac fibrosis, whereas the extent of HT and myocardial hypertrophy was comparable between the two groups. Myocardial expressions of collagen III, transforming growth factor-beta2, angiotensin-converting enzyme (ACE), angiotensin II type-1 receptor and myocardial oxidative stress (evaluated by 4-hydroxy-2-nonenal-modified protein) were mostly enhanced in the HT+DM group. Importantly, there was a positive correlation between the extent of diastolic dysfunction and that of myocardial ACE expression. All these cardiac abnormalities induced by DM and HT were ameliorated in the olmesartan group. These results indicate that DM accelerates diastolic dysfunction in hypertensive heart disease through activation of the renin-angiotensin system, with subsequent inflammatory and oxidative stresses and myocardial fibrosis, suggesting that an inhibition of the system is effective for the treatment of diastolic dysfunction in this combined disorder.

12-O-tetradecanoylphorbol-13-acetate Reduces Activation of Hepatic Stellate Cells by Inhibiting the Hippo Pathway Transcriptional Coactivator YAP

Article

Full-text available

Dec 2022

Although protein kinase C (PKC) regulates various biological activities, including cell proliferation, differentiation, migration, tissue remodeling, gene expression, and cell death, the antifibrotic effect of PKC in myofibroblasts is not fully understood. We investigated whether 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, reduced the activation of hepatic stellate cells (HSCs) and explored the involvement of the Hippo pathway transcriptional coactivator YAP. We analyzed the effect of TPA on the proliferation and expression of α-smooth muscle actin (SMA) in the LX-2 HSC line. We also analyzed the phosphorylation of the Hippo pathway molecules YAP and LATS1 and investigated YAP nuclear translocation. We examined whether Gö 6983, a pan-PKC inhibitor, restored the TPA-inhibited activities of HSCs. Administration of TPA decreased the growth rate of LX-2 cells and inhibited the expression of α-SMA and collagen type I alpha 1 (COL1A1). In addition, TPA induced phosphorylation of PKCδ, LATS1, and YAP and inhibited the nuclear translocation of YAP compared with the control. These TPA-induced phenomena were mostly ameliorated by Gö 6983. Our results indicate that PKCδ exerts an antifibrotic effect by inhibiting the Hippo pathway in HSCs. Therefore, PKCδ and YAP can be used as therapeutic targets for the treatment of fibrotic diseases.

Protective effects of dioscin against isoproterenol-induced cardiac hypertrophy via adjusting PKCε/ERK-mediated oxidative stress

Article

Jun 2021
EUR J PHARMACOL

Cardiac hypertrophy (CH) plays a central role in cardiac remodeling and is an independent risk factor for cardiac events. It is imperative to find drugs with protective effect on CH. Dioscin, one natural product, shows various pharmacological activities, and PKCepsilon (PKCε) plays an important role in the physiological hypertrophic responses. Thus, we aimed to investigate the possible protective effect of dioscin on CH through PKCε. In the present study, the isoproterenol (ISO)-induced H9C2 cells and primary cardiomyocytes models, and the ISO-induced rat model were established, and the pharmacodynamics and mechanism of dioscin were investigated. In vitro results prompted that, dioscin significantly improved ISO-induced cardiomyocyte hypertrophy, decreased the levels of cell size, protein content of single cell, reactive oxygen species, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), beta-myosin heavy chain (β-MHC). Moreover, in vivo, changes in histopathological of the animals caused by ISO are improved by dioscin. And dioscin decreased the index of CH and the levels of CK, MDA, LDH, and increased the levels of GSH, SOD and GSH-Px. Mechanism research showed that dioscin inhibited the expression levels of PKCε, and affected the expression levels of p-MEK, p-ERK, Nrf2, Keap1 and HO-1 to inhibit oxidative stress. In addition, the results of ISO-induced CH in PKCε siRNA transfected H9C2 cells and C57BL/6 mice further showed that the protective effect of dioscin on CH, which was mediated by inhibition of PKCε/ERK signal pathway. In summary, dioscin can effectively inhibit CH by regulating PKCε-mediated oxidative stress, which should be considered as one potent candidate for new drug research and development to treat CH in the future.

PKCε stimulation of TRPV1 orchestrates carotid body responses to asthmakines

Article

Full-text available

Dec 2020
J PHYSIOL-LONDON

Key points We have previously shown that carotid body stimulation by lysophosphatidic acid elicits a reflex stimulation of vagal efferent activity sufficient to cause bronchoconstriction in asthmatic rats. Here, we show that pathophysiological concentrations of asthma‐associated prototypical Th2 cytokines also stimulate the carotid bodies. Stimulation of the carotid bodies by these asthmakines involves a PKCε–transient receptor potential vanilloid 1 (TRPV1) signalling mechanism likely dependent on TRPV1 S502 and T704 phosphorylation sites. As the carotid bodies’ oxygen sensitivity is independent of PKCε–TRPV1 signalling, systemic blockade of PKCε may provide a novel therapeutic target to reduce allergen‐induced asthmatic bronchoconstriction. Consistent with the therapeutic potential of blocking the PKCε–TRPV1 pathway, systemic delivery of a PKCε‐blocking peptide suppresses asthmatic respiratory distress in response to allergen and reduces airway hyperresponsiveness to bradykinin. Abstract The autonomic nervous system orchestrates organ‐specific, systemic and behavioural responses to inflammation. Recently, we demonstrated a vital role for lysophosphatidic acid in stimulating the primary autonomic oxygen chemoreceptors, the carotid bodies, in parasympathetic‐mediated asthmatic airway hyperresponsiveness. However, the cacophony of stimulatory factors and cellular mechanisms of carotid body activation are unknown. Therefore, we set out to determine the intracellular signalling involved in carotid body‐mediated sensing of asthmatic blood‐borne inflammatory mediators. We employed a range of in vitro and rat in situ preparations, site‐directed mutagenesis, patch‐clamp, nerve recordings and pharmacological inhibition to assess cellular signalling. We show that the carotid bodies are also sensitive to asthma‐associated prototypical Th2 cytokines which elicit sensory nerve excitation. This provides additional asthmatic ligands contributing to the previously established reflex arc resulting in efferent vagal activity and asthmatic bronchoconstriction. This novel sensing role for the carotid body is mediated by a PKCε‐dependent stimulation of transient receptor potential vanilloid 1 (TRPV1), likely via TRPV1 phosphorylation at sites T704 and S502. Importantly, carotid body oxygen sensing was unaffected by blocking either PKCε or TRPV1. Further, we demonstrate that systemic PKCε blockade reduces asthmatic respiratory distress in response to allergen and airway hyperresponsiveness. These discoveries support an inflammation‐dependent, oxygen‐independent function for the carotid body and suggest that targeting PKCε provides a novel therapeutic option to abate allergic airway disease without altering life‐saving autonomic hypoxic reflexes.

Targeting Mitochondrial Fission-Fusion Imbalance in Heart Failure

Article

Full-text available

Dec 2020

Purpose of the Review This review focuses on the central role of mitochondrial fission-fusion imbalance in heart failure. We also discuss the development of pharmacological strategies capable of re-establishing mitochondrial dynamics in heart failure. Recent Findings Heart failure is a degenerative disease and a major cause of morbidity and mortality worldwide. Loss of mitochondrial fission-fusion balance and consequent impaired cardiac bioenergetics are hallmarks of heart failure. Therefore, the identification of maladaptive molecular signatures that contribute to impaired mitochondrial dynamics and bioenergetics, such as activation of protein kinase C βII, becomes a valuable platform and strategy to generate novel, and more effective, pharmacological tools to treat heart failure. Summary Here, we discuss critical post-translational modifications (phosphorylation) of mitochondrial dynamics-related proteins in failing hearts. We also highlight some druggable protein-protein interactions that control mitochondrial dynamics as potential targets to treat heart failure.

Apelin attenuates TGF-β1-induced epithelial to mesenchymal transition via activation of PKC-ε in human renal tubular epithelial cells

Article

Aug 2017

Epithelial to mesenchymal transition (EMT), a process whereby fully differentiated epithelial cells transition to a mesenchymal phenotype, has been implicated in the pathogenesis of renal fibrosis. Apelin, a bioactive peptide, has recently been recognized to protect against renal profibrotic activity, but the underlying mechanism has not yet been elucidated. In this study, we investigated the regulation of EMT in the presence of apelin-13 in vitro. Expression of the mesenchymal marker alpha-smooth muscle actin (α-SMA) and the epithelial marker E-cadherin was examined by immunofluorescence and western blotting in transforming growth factor beta 1 (TGF-β1)-stimulated human proximal tubular epithelial cells. Expression of extracellular matrix, fibronectin and collagen-I was examined by quantitative real-time PCR and ELISA. F13A, an antagonist of the apelin receptor APJ, and small interfering RNA targeting protein kinase C epsilon (PKC-ε) were used to explore the relevant signaling pathways. Apelin attenuated TGF-β1-induced EMT, and inhibited the EMT-associated increase in α-SMA, loss of E-cadherin, and secretion of extracellular matrix. Moreover, apelin activated PKC-ε in tubular epithelial cells, which in turn decreased phospho-Smad2/3 levels and increased Smad-7 levels. APJ inhibition or PKC-ε deletion diminished apelin-induced modulation of Smad signaling and suppression of tubular EMT. Our findings identify a novel PKC-ε-dependent mechanism in which apelin suppresses TGF-β1-mediated activation of Smad signaling pathways and thereby inhibits tubular EMT. These results suggest that apelin may be a new agent that can suppress renal fibrosis and retard chronic kidney disease progression.

MicroRNA-101b attenuates cardiomyocyte hypertrophy by inhibiting PKCε signaling

Article

Dec 2016
FEBS LETT

Previously, a surgical regression model identified microRNA-101b (miR-101b) as a potential inhibitor of cardiac hypertrophy. Here, we investigated the anti-hypertrophic mechanism of miR-101b using neonatal rat ventricular myocytes. miR-101b markedly suppressed agonist-induced cardiac hypertrophy as shown by cell size and fetal gene expression. By systems biology approaches, we identified protein kinase C epsilon (PKCε) as the major target of miR-101b. Our results from qRT-PCR, western blot, and luciferase reporter assays confirm that PKCε is a direct target of miR-101b. In addition, we found that effectors downstream of PKCε (p-AKT, p-ERK1/2, p-NFAT and p-GSK3β) are also affected by miR-101b. Our study reveals a novel inhibitory mechanism for miR-101b as a negative regulator of cardiac hypertrophy. This article is protected by copyright. All rights reserved.

Gallic acid prevents isoproterenol-induced cardiac hypertrophy and fibrosis through regulation of JNK2 signaling and Smad3 binding activity

Article

Full-text available

Oct 2016

Gallic acid, a type of phenolic acid, has been shown to have beneficial effects in inflammation, vascular calcification, and metabolic diseases. The present study was aimed at determining the effect and regulatory mechanism of gallic acid in cardiac hypertrophy and fibrosis. Cardiac hypertrophy was induced by isoproterenol (ISP) in mice and primary neonatal cardiomyocytes. Gallic acid pretreatment attenuated concentric cardiac hypertrophy. It downregulated the expression of atrial natriuretic peptide, brain natriuretic peptide, and beta-myosin heavy chain in vivo and in vitro. Moreover, it prevented interstitial collagen deposition and expression of fibrosis-associated genes. Upregulation of collagen type I by Smad3 overexpression was observed in cardiac myoblast H9c2 cells but not in cardiac fibroblasts. Gallic acid reduced the DNA binding activity of phosphorylated Smad3 in Smad binding sites of collagen type I promoter in rat cardiac fibroblasts. Furthermore, it decreased the ISP-induced phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) protein in mice. JNK2 overexpression reduced collagen type I and Smad3 expression as well as GATA4 expression in H9c2 cells and cardiac fibroblasts. Gallic acid might be a novel therapeutic agent for the prevention of cardiac hypertrophy and fibrosis by regulating the JNK2 and Smad3 signaling pathway.

Protein Kinase C as Regulator of Vascular Smooth Muscle Function and Potential Target in Vascular Disorders

Chapter

Jan 2016

Vascular smooth muscle (VSM) plays an important role in maintaining vascular tone. In addition to Ca²⁺-dependent myosin light chain (MLC) phosphorylation, protein kinase C (PKC) is a major regulator of VSM function. PKC is a family of conventional Ca²⁺-dependent α, β, and γ, novel Ca²⁺-independent δ, e(open), θ, and η, and atypical ξ, and ι/λ isoforms. Inactive PKC is mainly cytosolic, and upon activation it undergoes phosphorylation, maturation, and translocation to the surface membrane, the nucleus, endoplasmic reticulum, and other cell organelles; a process facilitated by scaffold proteins such as RACKs. Activated PKC phosphorylates different substrates including ion channels, pumps, and nuclear proteins. PKC also phosphorylates CPI-17 leading to inhibition of MLC phosphatase, increased MLC phosphorylation, and enhanced VSM contraction. PKC could also initiate a cascade of protein kinases leading to phosphorylation of the actin-binding proteins calponin and caldesmon, increased actin-myosin interaction, and VSM contraction. Increased PKC activity has been associated with vascular disorders including ischemia-reperfusion injury, coronary artery disease, hypertension, and diabetic vasculopathy. PKC inhibitors could test the role of PKC in different systems and could reduce PKC hyperactivity in vascular disorders. First-generation PKC inhibitors such as staurosporine and chelerythrine are not very specific. Isoform-specific PKC inhibitors such as ruboxistaurin have been tested in clinical trials. Target delivery of PKC pseudosubstrate inhibitory peptides and PKC siRNA may be useful in localized vascular disease. Further studies of PKC and its role in VSM should help design isoform-specific PKC modulators that are experimentally potent and clinically safe to target PKC in vascular disease.

Selective phosphorylation inhibitor of δPKC-PDK protein-protein inter-actions; application for myocardial injury in vivo

Article

May 2016

Protein kinases regulate numerous cellular processes, including cell growth, metabolism and cell death. Because the primary sequence and the three-dimensional structure of many kinases are highly similar, the development of selective inhibitors for only one kinase is challenging. Furthermore, many protein kinases are pleotropic, mediating diverse and sometimes even opposing functions by phosphorylating multiple protein substrates. Here, we set up to develop an in-hibitor of a selective protein kinase phosphorylation of only one of its substrates. Focusing on the pleotropic delta pro-tein kinase C (δPKC), we used a rational approach to identify a distal docking site on δPKC for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned that an inhibitor of PDK's docking should selectively inhibit the phosphorylation of only PDK without affecting phosphorylation of the other δPKC substrates. Our approach identified a selective inhibitor of PDK docking to δPKC with an in vitro Kd of ~50 nM and reducing cardiac injury IC50 of ~5 nM. This inhibitor, which did not affect the phosphorylation of other δPKC substrates even at 1 μM, demonstrated that PDK phosphorylation alone is critical for δPKC-mediated injury by heart attack. The approach we describe is likely applicable for the identification of other substrate-specific kinase inhibitors.

Olmesartan ameliorates pressure overload-induced cardiac remodeling through inhibition of TAK1/p38 signaling in mice

Article

Jan 2016

Aims: Many studies have demonstrated the potent effects of ARB administration on heart failure. However, the mechanism of the potent effects of ARB on cardiac remodeling is less well understood. We investigated the role of Olmesartan on the fibrosis and hypertrophy in mouse heart. Materials and methods: We employed TAC surgery, a mouse model of chronic cardiac failure. All the mice were separated into three groups: the sham group, TAC group and TAC plus Olmesartan group (given Olmesartan treatment after TAC). We analyzed left ventricle remodeling, and function by echocardiography or pathology. We further detected the level of marker genes involved in fibrosis and hypertrophy and in cultured neonatal rat cardiac fibroblasts and myocytes infected by constitutively active TAK1 and p38MAPK. After TAC, all the mice developed hypertrophy, worse cardiac function and malignant remodeling in left ventricle. Key findings: Olmesartan improved heart remodeling and function without changing pressure of blood. Moreover, Olmesartan reduced the level of transforming growth factor β activated kinase-1 (TAK1) and phospho-p38 MAPK. In neonatal rat cardiac fibroblast cells and cardiomyocytes, Olmesartan also decreased TAK1 and p38 MAPK activation triggered by TGFβ1 or AngII. The inhibitory effect of Olmesartan was abrogated by overexpression of constitutively active TAK1 and p38 MAPK by adenovirus system. Significance: Our results suggest Olmesartan improves heart remodeling and function induced by pressure overload. P38 MAPK inactivation attenuated by olmesartan via inhibition of TAK1 pathway plays an important role in the process.

Synthetic Thyroid Hormone and Thyroid Hormone Analogues for Treatment of Heart Failure

Article

Jan 2009

Heart failure (HF) should be seen in a unique scenario of altered systemic homeostasis, in which heart dysfunction, peripheral organ dysfunction, and derangement of the neuroendocrine and immune systems represent chronic crosstalking between stress stimuli, with continuous activation of the stress response. The thyroid hormone (TH) system is profoundly involved in cardiovascular and systemic homeostasis. In HF, the most frequent alteration of TH metabolism is a low-triiodothyronine state, which may participate directly in progression of HF. Initial results have shown that TH replacement therapy in patients with HF improves cardiac performance, hemodynamic and exercise performance. It also induces deactivation of the neuroendocrine profile, as a result of the significant reductions in vasoconstrictor/sodium-retaining norepinephrine and aldosterone. It in the plasma levels of their counterpart, N-terminal pro B-type natriuretic peptide (NT-proBNP). Depending on the pathophysiology of the HF, two strategies of TH replacement therapy have been suggested: (1) the cardiosystemic strategy, which involves administration of synthetic T4 or T3, and (2) the cardioselective one, using TH analogues, in particular 3,5-diiodothyropropionic acid (DITPA). The rationale of these two approaches is based on the pathophysiology of HF progression, which is linked to progressive impairment of systolic-diastolic cardiac function, but also to systemic disturbance, which frequently progresses independently of deteriorating cardiac function.

Renal PKC- deficiency attenuates acute kidney injury and ischemic allograft injury via TNF- -dependent inhibition of apoptosis and inflammation

Article

Full-text available

Jul 2014

Acute kidney injury (AKI) increases the risk of morbidity and mortality after major surgery and transplantation. We investigated the effect of protein kinase C (PKC) epsilon deficiency on AKI and ischemic allograft damage after kidney transplantation (ktx). PKC epsilon deficient and wild type (WT) control mice were subjected to 35 minutes of renal pedicle clamping to induced AKI. PKC epsilon deficiency was associated with a marked improvement in survival and an attenuated loss of kidney function. Furthermore, functional MRI studies revealed better renal perfusion in PKC epsilon deficient than WT mice one day after IRI. Acute tubular necrosis and neutrophil infiltration were markedly reduced in PKC epsilon deficient mice. To determine whether this resistance to IRI resulted from changes in local renal cells or infiltrating leukocytes, we studied a life supporting renal transplant model of ischemic graft injury. We transplanted kidneys from H2b PKC epsilon deficient mice (129/SV) and their corresponding WT littermates into MHC-incompatible H2d recipients (BALB/c) and induced ischemic graft injury by prolonged ischemia time. Recipients of WT allografts developed severe renal failure and died within 10 days of transplantation. Recipients of PKC epsilon deficient allografts had better renal function and survival. They had less generation of reactive oxygen species and up-regulation of pro-inflammatory proteins (i.e. ICAM-1, i-NOS, TNF-alpha) and showed less tubular epithelial cell apoptosis and inflammation in their allografts. These data suggest that local renal PKC epsilon expression mediates pro-apoptotic and pro-inflammatory signaling and that an inhibitor of PKC epsilon signaling could be used to prevent hypoxia-induced AKI.

Protein kinase C, an elusive therapeutic target?

Article

Nov 2012
NAT REV DRUG DISCOV

Protein kinase C (PKC) has been a tantalizing target for drug discovery ever since it was first identified as the receptor for the tumour promoter phorbol ester in 1982. Although initial therapeutic efforts focused on cancer, additional indications - including diabetic complications, heart failure, myocardial infarction, pain and bipolar disorder - were targeted as researchers developed a better understanding of the roles of eight conventional and novel PKC isozymes in health and disease. Unfortunately, both academic and pharmaceutical efforts have yet to result in the approval of a single new drug that specifically targets PKC. Why does PKC remain an elusive drug target? This Review provides a short account of some of the efforts, challenges and opportunities in developing PKC modulators to address unmet clinical needs.

Up-regulation of myocardial connexin-43 in spontaneously hypertensive rats fed red palm oil is most likely implicated in its anti-arrhythmic effects

Article

Aug 2012
CAN J PHYSIOL PHARM

The purpose of this study was to test our hypothesis that red palm oil (RPO) intake may affect abnormalities of myocardial connexin-43 (Cx43) and protein kinase Cε (PKCε) signaling, and consequently the propensity of the spontaneously hypertensive rat heart (SHR) heart to arrhythmias. SHR and Wistar-Kyoto (WKY) rats fed a standard rat chow plus red palm oil (200 µL/day) for 5 weeks were compared with untreated rats. Cytosolic but not particulate PKCε expression as well as Cx43-mRNA, total Cx43 proteins, and its phoshorylated forms were increased, and disordered localization of Cx43 was attenuated in the left ventricle of RPO-fed SHR compared with untreated rats. These alterations were associated with suppression of early post-ischemic-reperfusion-related ventricular tachycardia and electrically inducible ventricular fibrillation. However, the treatment dose of RPO caused down-regulation of myocardial Cx43, but did not alter its cell membrane distribution or overall PKCε expression in WKY rats. It was, however, associated with poor arrhythmia protection, suggesting overdosing. Results indicate that SHR benefit from RPO intake, particularly because of its apparent anti-arrhythmic effects. This protection can be, in part, attributed to the preservation of cell-to-cell communication via up-regulation of myocardial Cx43, but not with PKCε activation.

Regulation of PKC by Protein–Protein Interactions in Cancer

Chapter

Apr 2010

Protein kinase C (PKC) was first identified in 1977 by Nishizuka’s group as a proteolytically activated protein kinase. It was subsequently found that the enzyme is activated by calcium and anionic phospholipids. Unsaturated diacylglycerol (DAG) was then found to be an essential activator of PKC, linking PKC activation to tyrosine kinase- or G-protein-coupled receptor-mediated inositol phospholipid hydrolysis. In addition to phospholipids, DAG, and calcium (depending on the isozyme), PKC isozymes are also regulated by protein–protein interactions. In a variety of experimental models, PKC isozymes have been found to mediate different and often opposing roles in tumor growth, and their activities are further regulated by these multiple intra- and intermolecular protein–protein interactions. In clinical samples, the levels of protein or activities of PKCs are dysregulated when compared to normal tissue of the same origin and correlate with poor prognosis. PKC regulates multiple aspects of tumorigenesis, including cell proliferation, angiogenesis, metastasis, and apoptosis, making it a major regulator in the transformation to malignant phenotype. This review focuses on the current understanding of PKC regulation by protein–protein interactions as it relates to cancer. We summarize known roles for each domain of PKC and discuss intramolecular interactions that regulate the activation state of the enzyme, as well as intermolecular interactions that determine the specificity of the signaling of each PKC isozyme. We also demonstrate how identification of the molecular sites of specific protein–protein interactions within PKC and between PKC and other proteins has led to the design of effective isozyme-selective activators and inhibitors of PKC and discuss how these pharmacological tools can assist in determining the role of specific PKC isozymes in tumorigenesis. KeywordsAngiogenesis-Cancer-Metastasis-Protein kinase C-Protein–protein interaction

Molecular Signaling Mechanisms of Myocardial Stretch: Implications for Heart Disease

Chapter

Jan 2010

With every heartbeat, myocardial cells are subjected to substantial mechanical stretch. Stretch is a potent stimulus for growth, differentiation, migration, remodeling and gene expression. Mechanical load is a major cause of cardiac hypertrophy. Since the initial observation of stretch-induced growth, our understanding of this complex field has been steadily growing, but remains incomplete. The mechanisms by which myocardial cells convert mechanical stimuli into biochemical signals that result in physiologic and pathological changes remain to be completely understood. Integrins, caveolae and focal adhesions have been shown to have important mechanosensing roles in cardiac myocytes. Downstream effectors activated by mechanosensors include guanine-nucleotide binding proteins (G-proteins), mitogen-activated protein (MAP) kinases, Janus-associated kinase/signal transducers and activators of transcription (JAK/Stat), protein kinase C (PKC) and protein kinase B/Akt pathways. Multiple levels of crosstalk exist between these pathways. Early studies have implicated most of these pathways in cardiac injury and growth response, however, more recent advancements in the development of kinase-specific inhibitors and genetically-engineered animal models have revealed significant new insights. Recent studies suggest that acute mechanical stretch activates protective pathways including c-jun N-terminal kinase (JNK) and Akt as a tolerance response, rather than injury-related signaling cascades such as p38 MAP kinase. However, chronic stretch/mechanical load creates an imbalance that favors the injury related pathway by an unknown mechanism in the myocardium. The following chapter provides an overview of the fundamental processes of stretch-activated mechano-signaling in myocardial cells, and recent advances in our understanding of this increasingly important field.

Regulation of cardiac excitability by protein kinase C isozymes

Article

Full-text available

Jan 2012

Cardiac excitability and electrical activity are determined by the sum of individual ion channels, gap junctions and exchanger activities. Electrophysiological remodeling during heart disease involves changes in membrane properties of cardiomyocytes and is related to higher prevalence of arrhythmia-associated morbidity and mortality. Pharmacological and genetic manipulation of cardiac cells as well as animal models of cardiovascular diseases are used to identity changes in electrophysiological properties and the molecular mechanisms associated with the disease. Protein kinase C (PKC) and several other kinases play a pivotal role in cardiac electrophysiological remodeling. Therefore, identifying specific therapies that regulate these kinases is the main focus of current research. PKC, a family of serine/threonine kinases, has been implicated as potential signaling nodes associated with biochemical and biophysical stress in cardiovascular diseases. In this review, we describe the role of PKC isozymes that are involved in cardiac excitability and discuss both genetic and pharmacological tools that were used, their attributes and limitations. Selective and effective pharmacological interventions to normalize cardiac electrical activities and correct cardiac arrhythmias will be of great clinical benefit.

ALDH2 Activator Inhibits Increased Myocardial Infarction Injury by Nitroglycerin Tolerance

Article

Nov 2011
Sci Transl Med

Nitroglycerin, which treats impaired cardiac function through vasodilation as it is converted to nitric oxide, is used worldwide for patients with various ischemic and congestive cardiac diseases, including angina pectoris. Nevertheless, after continuous treatment, the benefits of nitroglycerin are limited by the development of tolerance to the drug. Nitroglycerin tolerance is a result of inactivation of aldehyde dehydrogenase 2 (ALDH2), an enzyme essential for cardioprotection in animals subjected to myocardial infarction. Here, we tested the hypothesis that the tolerance that develops as a result of sustained nitroglycerin treatment increases cardiac injury by subsequent myocardial infarction. In a rat model of myocardial infarction, 16 hours of prior, sustained nitroglycerin treatment resulted in infarcts that were twice as large as those in untreated control animals and in diminished cardiac function at 3 days and 2 weeks after the myocardial infarction. We also sought to identify a potential treatment to protect against this increased cardiac damage. Nitroglycerin inhibited ALDH2 activity in vitro, an effect that was blocked by Alda-1, an activator of ALDH2. Co-administration of Alda-1 with the nitroglycerin prevented the nitroglycerin-induced increase in cardiac dysfunction after myocardial infarction in rats, at least in part by enhancing metabolism of reactive aldehyde adducts that impair normal protein functions. If our animal studies showing that nitroglycerin tolerance increases cardiac injury upon ischemic insult are corroborated in humans, activators of ALDH2 such as Alda-1 may help to protect patients with myocardial infarction from this nitroglycerin-induced increase in cardiac injury while maintaining the cardiac benefits of the increased nitric oxide concentrations produced by nitroglycerin.

The function of protein kinase C in myocardial fibrosis

Article

Dec 2010

Myocardial fibrosis is the common results of the development of a variety of heart diseases which leads to extracellular matrix protein metabolic disorders and causes cardiac remodeling owing to cardiac fibroblasts proliferation, eventually results in malignant arrhythmia, heart failure, and even the occurrence of sudden cardiac death. Effective inhibition of myocardial remodeling could prevent the occurrence of sudden death. To know the protein kinase C (PKC) effective mechanism of regulation on myocardial fibrosis, a new therapeutic target for reversing myocardial remodeling might be provided.

Matrix metalloproteinases modulated by protein kinase Cε mediate resistin-induced migration of human coronary artery smooth muscle cells

Article

Apr 2011

Emerging evidence showed that resistin induces vascular smooth muscle cell (VSMC) migration, a critical step in initiating vascular restenosis. Adhesion molecule expression and cytoskeletal rearrangement have been observed in this progress. Given that matrix metalloproteinases (MMPs) also regulate cell migration, we hypothesized that MMPs may mediate resistin-induced VSMC migration. Human VSMCs were treated with recombinant human resistin at physiologic (10 ng/mL) and pathologic (40 ng/mL) concentrations for 24 hours. Cell migration was determined by the Boyden chamber assay. MMP and tissue inhibitor metalloproteinase (TIMP) mRNA and protein levels were measured with real-time PCR and ELISA. MMP enzymatic activity was measured by zymography. In another experiment, neutralizing antibodies against MMP-2 and MMP-9 were coincubated with resistin in cultured VSMCs. The regulation of MMP by protein kinase C (PKC) was determined by εV1-2, a selective PKCε inhibitor. Resistin-induced smooth muscle cell (SMC) migration was confirmed by the Boyden chamber assay. Forty nanograms/milliliter resistin increased SMC migration by 3.7 fold. Additionally, resistin stimulated MMP-2 and -MMP9 mRNA and protein expressions. In contrast, the TIMP-1 and TIMP-2 mRNA levels were inhibited by resistin. Neutralizing antibodies against MMP-2 and MMP-9 effectively reversed VSMC migration. Furthermore, resistin activated PKCε, but selective PKCε inhibitor suppressed resistin-induced MMP expression, activity, and cell migration. Our study confirmed that resistin increased vascular smooth muscle cell migration in vitro. In terms of mechanism, resistin-stimulated cell migration was associated with increased MMP expression, which was dependent on PKCε activation.

Angiotensin I-Converting Enzyme Inhibitors Are Allosteric Enhancers of Kinin B1 and B2 Receptor Function

Article

Feb 2010
Hypertension

The beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors go beyond the inhibition of ACE to decrease angiotensin (Ang) II or increase kinin levels. ACE inhibitors also affect kinin B1 and B2 receptor (B1R and B2R) signaling, which may underlie some of their therapeutic usefulness. They can indirectly potentiate the actions of bradykinin (BK) and ACE-resistant BK analogs on B2Rs to elevate arachidonic acid and NO release in laboratory experiments. Studies indicate that ACE inhibitors and some Ang metabolites increase B2R functions as allosteric enhancers by inducing a conformational change in ACE. This is transmitted to B2Rs via heterodimerization with ACE on the plasma membrane of cells. ACE inhibitors are also agonists of the B1R, at a Zn-binding sequence on the second extracellular loop that differs from the orthosteric binding site of the des-Arg-kinin peptide ligands. Thus, ACE inhibitors act as direct allosteric B1R agonists. When ACE inhibitors enhance B2R and B1R signaling, they augment NO production. Enhancement of B2R signaling activates endothelial NO synthase, yielding a short burst of NO; activation of B1Rs results in a prolonged high output of NO by inducible NO synthase. These actions, outside inhibiting peptide hydrolysis, may contribute to the pleiotropic therapeutic effects of ACE inhibitors in various cardiovascular disorders.

Effects of Olmesartan on Apelin/APJ and Akt/Endothelial Nitric Oxide Synthase Pathway in Dahl Rats With End-stage Heart Failure

Article

Nov 2009
J CARDIOVASC PHARM

Apelin and its cognate G protein-coupled receptor APJ constitute a signaling pathway with a positive inotropic effect on cardiac function, and the apelin/APJ pathway seems to have opposing physiological role to the renin-angiotensin system. We investigated whether angiotensin II receptor blocker olmesartan could improve cardiac function associated with apelin/APJ and Akt/endothelial nitric oxide synthase (eNOS) pathway in Dahl salt-sensitive hypertensive (DS) rats with end-stage heart failure using NOS inhibitor L-N(G)-nitroarginine methyl ester (L-NAME). High salt-loaded DS rats were treated with (1) vehicle, (2) olmesartan, and (3) olmesartan plus L-NAME for 7 weeks. Decreased end-systolic elastance and percent fractional shortening in failing rats were significantly ameliorated by olmesartan. Increased atherosclerosis and vascular remodeling and fibrosis factors such as procollagen type I and III and fibronectin expression in DS rats were inhibited by olmesartan. Downregulation of apelin and APJ expression and phosphorylation of Akt and eNOS in failing rats were significantly increased by olmesartan. In addition,administration of L-NAME completely abrogated the olmesartan-mediated improvement of cardiac function and remodeling, and apelin/APJ expression and Akt/eNOS phosphorylation. These findings suggest that olmesartan may improve cardiac dysfunction and remodeling associated with apelin/APJ and Akt/eNOS pathway in DS rats with end-stage heart failure.

Tissue Inhibitors of Metalloproteinases: Evolution, Structure and Function

Article

Full-text available

Mar 2000
Biochim Biophys Acta Protein Struct Mol Enzymol

The matrix metalloproteinases (MMPs) play a key role in the normal physiology of connective tissue during development, morphogenesis and wound healing, but their unregulated activity has been implicated in numerous disease processes including arthritis, tumor cell metastasis and atherosclerosis. An important mechanism for the regulation of the activity of MMPs is via binding to a family of homologous proteins referred to as the tissue inhibitors of metalloproteinases (TIMP-1 to TIMP-4). The two-domain TIMPs are of relatively small size, yet have been found to exhibit several biochemical and physiological/biological functions, including inhibition of active MMPs, proMMP activation, cell growth promotion, matrix binding, inhibition of angiogenesis and the induction of apoptosis. Mutations in TIMP-3 are the cause of Sorsby’s fundus dystrophy in humans, a disease that results in early onset macular degeneration. This review highlights the evolution of TIMPs, the recently elucidated high-resolution structures of TIMPs and their complexes with metalloproteinases, and the results of mutational and other studies of structure–function relationships that have enhanced our understanding of the mechanism and specificity of the inhibition of MMPs by TIMPs. Several intriguing questions, such as the basis of the multiple biological functions of TIMPs, the kinetics of TIMP–MMP interactions and the differences in binding in some TIMP–metalloproteinase pairs are discussed which, though not fully resolved, serve to illustrate the kind of issues that are important for a full understanding of the interactions between families of molecules.

Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and varepsilon PKC

Article

Full-text available

Oct 2001

Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported. Here, deltaPKC-selective activator and inhibitor peptides were designed rationally, based on molecular modeling and structural homology analyses. Together with previously identified activator and inhibitor peptides of epsilonPKC, deltaPKC peptides were used to identify cardiac functions of these isozymes. In isolated cardiomyocytes, perfused hearts, and transgenic mice, deltaPKC and epsilonPKC had opposing actions on protection from ischemia-induced damage. Specifically, activation of epsilonPKC caused cardioprotection whereas activation of deltaPKC increased damage induced by ischemia in vitro and in vivo. In contrast, deltaPKC and epsilonPKC caused identical nonpathological cardiac hypertrophy; activation of either isozyme caused nonpathological hypertrophy of the heart. These results demonstrate that two related PKC isozymes have both parallel and opposing effects in the heart, indicating the danger in the use of therapeutics with nonselective isozyme inhibitors and activators. Moreover, reduction in cardiac damage caused by ischemia by perfusion of selective regulator peptides of PKC through the coronary arteries constitutes a major step toward developing a therapeutic agent for acute cardiac ischemia.

Salt and hypertension. Lessons from animal models that relate to human hypertension

Article

Feb 1991

L Tobian

A high NaCl diet can raise blood pressure in both susceptible people and in susceptible animals, and the mechanisms are probably quite similar for both humans and animals. The possibly harmful effects of a high NaCl diet are not unexpected since both prehistoric man and mammals evolved in a low NaCl world. Evolutionary forces molded mammals to adapt well to a low sodium intake; the modern high NaCl intake goes "against the grain" of this adaptation. The high NaCl diet can cause premature mortality by raising blood pressure in susceptible people. We have new evidence that in a hypertensive setting, a high NaCl diet can increase mortality even though it does not cause a further rise of blood pressure. Multiple small cerebral infarcts are a partial cause of this excess mortality. Recent evidence also indicates that a high potassium diet reduces the rise of blood pressure caused by a high NaCl diet, whereas a low normal potassium intake encourages an NaCl-induced rise of blood pressure. It is the combination of kidneys that tends to retain NaCl together with a high NaCl intake that produces a rise in blood pressure. This combination tends to cause NaCl retention, which can trigger a rise in blood pressure in susceptible humans and animals. Such a rise in blood pressure can augment renal NaCl excretion and regain the previous NaCl balance. In the Dahl salt-sensitive (DS) rat, there are several renal abnormalities that would tend to encourage sodium retention. By analogy, renal "abnormalities" are probably present in people susceptible to hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased Protein-Kinase-C and isozyme redistribution in pressure-overload cardiac-hypetrophy in the rat

Article

Dec 1994

Protein kinase C (PKC) activity and isozyme distribution were evaluated during development of pressure-overload-induced cardiac hypertrophy. Three-week-old rats were loosely banded on the ascending aorta (left ventricular hypertrophy [LVH] group). Two weeks later, when left ventricular mass was 50% greater than in the sham-operated control group and cardiac mass was still rapidly increasing beyond that of normal growth, PKC activity and [3H]phorbol 12,13-dibutyrate (PDBu) binding capacity were determined. In LVH, PKC activity was 119 +/- 14%, 158 +/- 17%, and 152 +/- 9% of the control value in cytosol, membrane, and nuclear-cytoskeletal fractions, respectively (n = 9 or 10). [3H]PDBu binding assay revealed increased PKC concentration in LVH cytosolic (control, 0.51 +/- 0.06 pmol/L per milligram; LVH, 0.78 +/- 0.09 pmol/L per milligram; n = 5; P < .05) and membrane fractions (control, 1.33 +/- 0.15; LVH, 2.32 +/- 0.39; n = 5; P < .05). Scatchard analysis indicated no difference in Kd values between control and LVH groups. Immunoblot analysis using PKC isoform-specific antibodies showed that both Ca(2+)-dependent (alpha and beta) and Ca(2+)-independent (delta, epsilon, and zeta) isoforms were present in the left ventricle. Compared with the control value, there was increased concentration in the membrane and nuclear-cytoskeletal fractions for beta 1,2 and epsilon and in the cytosol for beta 1,2. PKC-delta could be detected only in the nuclear-cytoskeletal fraction and was not changed in LVH. PKC-alpha and -zeta were present in all three fractions but were not altered in LVH. These data indicate that PKC activity and concentration increase during development of LVH induced by pressure overload. The increased PKC isozymes were mainly limited to PKC-beta 1,2 and PKC-epsilon, and the increase was present mainly in the membrane and nuclear-cytoskeletal fractions.

Molecular signaling mechanisms controlling growth and function of cardiac fibroblasts

Article

Nov 1995

Cardiac fibroblasts appear to be important in producing and maintaining the extracellular matrix (ECM) of the heart. The abnormal proliferation of cardiac fibroblasts and deposition of the ECM protein, collagen, associated with hypertension and myocardial infarction, may adversely affect the performance of the heart. Several groups of factors affect collagen gene expression and/or growth of cardiac fibroblasts. Angiotensin II, aldosterone and endothelins play a central role in the remodeling of the ECM in hypertension, and decrease collagenase activity and/or increase collagen synthesis in cultured cells. Regulatory peptides that are generally elevated at sites of injury, such as TGF-beta 1 and PDGF, increase collagen synthesis and/or stimulate mitogenesis. Mechanical stretch enhances collagen expression and cell proliferation, responses which could in part be due to integrin activation. Cytokines may stimulate or inhibit cell growth, the latter through prostaglandin formation. Angiotensin II is a principal determinant in vivo of cardiac fibroplasia and synthesis of the ECM proteins, collagen and fibronectin. Cardiac fibroblasts possess G-protein-coupled AT1 receptors for angiotensin II that couple to activation of multiple signalling pathways, including: phospholipase C-beta, with the subsequent release of Ca2+ from intracellular stores and activation of protein kinase C, mitogen-activated protein kinases, tyrosine kinases, phospholipase D, phosphatidic acid formation, and the STAT family of transcription factors. Cardiac fibroblasts respond to angiotensin II with hyperplastic/hypertrophic growth, and increased expression of collagen, fibronectin, and integrins. The mechanisms by which the AT1 receptor activates multiple signalling pathways are not known, although the receptor might interact at some level with both integrins and cytokine receptors. Different signalling pathways of the AT1 receptor may subserve different cellular responses, such as mitogenesis, ECM synthesis, or an inflammatory/stress response. Crosstalk among the signalling pathways of the AT1 receptor, and those of G-protein, cytokine, and growth-factor receptors, may determine the ultimate response of the cell.

Sustained in vivo cardiac protection by a rationally designed peptide that causes ?? protein kinase C translocation

Article

Nov 1999

Brief periods of cardiac ischemia trigger protection from subsequent prolonged ischemia (preconditioning). epsilon Protein kinase C (epsilonPKC) has been suggested to mediate preconditioning. Here, we describe an epsilonPKC-selective agonist octapeptide, psiepsilon receptor for activated C-kinase (psiepsilonRACK), derived from an epsilonPKC sequence homologous to its anchoring protein, epsilonRACK. Introduction of psiepsilonRACK into isolated cardiomyocytes, or its postnatal expression as a transgene in mouse hearts, increased epsilonPKC translocation and caused cardio-protection from ischemia without any deleterious effects. Our data demonstrate that epsilonPKC activation is required for protection from ischemic insult and suggest that small molecules that mimic this epsilonPKC agonist octapeptide provide a powerful therapeutic approach to protect hearts at risk for ischemia.

Excessive activation of matrix metalloproteinases coincides with left ventricular remodeling during transition from hypertrophy to heart failure in hypertensive rats

Article

May 2002
J AM COLL CARDIOL

We sought to elucidate how the local activation of matrix metalloproteinases (MMPs) is balanced by that of the endogenous tissue inhibitors of MMP (TIMPs) during left ventricular (LV) remodeling. Although it is known that the extracellular matrix (ECM) must be altered during LV remodeling, its local regulation has not been fully elucidated. In Dahl salt-sensitive rats with hypertension, in which a stage of concentric, compensated left ventricular hypertrophy (LVH) at 11 weeks is followed by a distinct stage of congestive heart failure (CHF) with LV enlargement and dysfunction at 17 weeks, we determined protein and messenger ribonucleic acid (mRNA) levels of LV myocardial TIMP-2 and -4 and MMP-2, as well as their concomitant activities. No changes were found at the LVH stage. However, during the transition to CHF, TIMP-2 and -4 activities, protein and mRNA levels were all sharply increased. At the same time, the MMP-2 mRNA and protein levels and activities, as determined by gelatin zymography, as well as by an antibody capture assay, showed a substantial increase during the transition to CHF. The net MMP activities were closely related to increases in LV diameter (r = 0.763) and to systolic wall stress (r = 0.858) in vivo. Both TIMPs and MMP-2 remained inactive during hypertrophy, per se; they were activated during the transition to CHF. At this time, the activation of MMP-2 surpassed that of TIMPs, possibly resulting in ECM breakdown and progression of LV enlargement.

Tissue Angiotensin II During Progression or Ventricular Hypertrophy to Heart Failure in Hypertensive Rats; Differential Effects on PKCε and PKCβ

Article

Nov 2002

The protein kinase C (PKC) family has been implicated as second messengers in mechanosensitive modulation of cardiac hypertrophy. However, little information is available on the role of expression and activation of specific cardiac PKC isozymes during development of left ventricular hypertrophy (LVH) and failure (LVF). Dahl salt-sensitive rats fed an 8% salt diet developed systemic hypertension and concentric LVH at 11 weeks of age that is followed by left ventricle (LV) dilatation and global hypokinesis at 17 weeks. Among several PKC isozymes expressed in the LV myocardium, only PKC epsilon showed a 94% increase at the LVH stage. At the LVF stage, however, PKC epsilon returned to the control level, whereas PKC beta I and beta II increased by 158% and 155%, respectively. Hearts were studied at each stage using the Langendorff set-up, and a LV balloon was inflated to achieve an equivalent diastolic wall stress. Following mechanical stretch, PKC epsilon was significantly activated in LVH myocardium in which tissue angiotensin II levels were increased by 59%. Pre-treatment with valsartan, an AT(1)-receptor blocker, abolished the stretch-mediated PKC epsilon activation. Mechanical stretch no longer induced PKC epsilon activation in LVF. Chronic administration of valsartan blunted the progression of LVF and inhibited the increase in PKC beta. Mechanosensitive PKC epsilon activation is augmented and therefore may contribute to the development of compensatory hypertrophy. This effect was dependent on activation of tissue angiotensin II. However, this compensatory mechanism becomes inactive in LVF, where PKC beta may participate in the progression to cardiac dysfunction and LV remodeling.

Opposing effects of delta- and zeta-protein kinase C isozymes on cardiac fibroblast proliferation: use of isozyme-selective inhibitors

Article

Sep 2003

Neonatal primary cardiac fibroblasts in defined medium continue to proliferate. Here, we show that phorbol ester inhibited and transforming growth factor-beta1 (TGFbeta1) stimulated this fibroblast proliferation. Cardiac fibroblasts contain six protein kinase C (PKC) isozymes: alpha-, delta-, epsilon-, betaI-, betaII-, and zeta-PKC. To evaluate the effect of different PKC isozymes on the proliferation of these cells, we used isozyme-selective PKC inhibitors. Inhibition of endogenous delta-PKC with deltaV1-1, an isozyme-selective translocation inhibitor, resulted in increased basal thymidine incorporation by 58 +/- 12% of control cells, but did not affect TGFbeta1-induced cell growth. Inhibition of endogenous zeta-PKC in neonatal rat cardiac fibroblasts with zeta-pseudosubstrate, a selective inhibitor for the atypical PKC isozymes, revealed an opposite effect; this inhibitor reduced basal growth to 45 +/- 11% and TGFbeta1-induced growth to 61 +/- 10%. Other isozyme-specific inhibitors used in this study did not alter basal or TGFbeta1-stimulated fibroblast growth. Taken together, our data provide evidence that delta-PKC inhibits and zeta-PKC stimulates proliferation of neonatal rat cardiac fibroblasts.

Protein kinase C isoform-selective signals that lead to cardiac hypertrophy and the progression of heart failure

Article

Oct 2003

Protein kinase C isoforms comprise a family of structurally related serine/threonine kinases that are activated by second messenger molecules formed via receptor-dependent activation of phospholipase C. Cardiomyocytes co-express multiple protein kinase C isoforms which play key roles in a spectrum of adaptive and maladaptive cardiac responses. This chapter focuses on the structural features, modes of activation, and distinct cellular actions of individual PKC isoforms in the heart. Particular emphasis is placed on progress that comes from studies in molecular models of PKC isoform overexpression or gene deletion in mice. Recent studies that distinguish the functional properties of novel PKC isoforms (PKC(delta) and PKC(epsilon)) from each other, and from the actions of the conventional PKC isoforms, and suggest that these proteins may play a particularly significant role in pathways leading to cardiac growth and/or cardioprotection also are considered.

Suppression of graft coronary artery disease by a brief treatment with a selective epsilon PKC activator and a delta PKC inhibitor in murine cardiac allografts

Article

Oct 2004
CIRCULATION

Inhibiting delta protein kinase C (deltaPKC) during reperfusion and activating epsilon PKC (epsilonPKC) before ischemia each limits cardiac ischemic injury. Here, we examined whether limiting ischemia-reperfusion injury inhibits graft coronary artery disease (GCAD) and improves murine cardiac allografting. Hearts of FVB mice (H-2q) were transplanted into C57BL/6 mice (H-2b). epsilonPKC activator (psiepsilonRACK) was injected intraperitoneally (20 nmol) into donor mice 20 minutes before procurement. Hearts were then perfused with psiepsilonRACK (1.5 nmol) through the inferior vena cava (IVC) and subsequently submerged in psiepsilonRACK (0.5 micromol/L) for 20 minutes at 4 degrees C. Before reperfusion, the peritoneal cavity of recipients was irrigated with deltaPKC inhibitor (deltaV1-1, 300 nmol); control animals were treated with normal saline. The total ischemic time to the organ was 50 minutes. Two hours after transplantation, production of inflammatory cytokines and adhesion molecules, cardiomyocyte apoptosis, and caspase-3 and caspase-9 (but not caspase-8) activities were significantly reduced in the PKC regulator-treated group. Fas ligand levels (but not Fas) were also significantly reduced in this group. Importantly, GCAD indices, production of inflammatory cytokines, and adhesion molecules were significantly decreased and cardiac allograft function was significantly better as measured up to 30 days after transplantation. An epsilonPKC activator and a deltaPKC inhibitor together reduced GCAD. Clinically, these PKC isozyme regulators may be useful for organ preservation and prevention of ischemia-reperfusion injury and graft coronary artery disease in cardiac transplantation.

Cardioprotection by -Protein Kinase C Activation From Ischemia: Continuous Delivery and Antiarrhythmic Effect of an -Protein Kinase C-Activating Peptide

Article

Feb 2005
CIRCULATION

We previously showed that a selective activator peptide of epsilon-protein kinase C (PKC), psi(epsilon)RACK, conferred cardioprotection against ischemia-reperfusion when delivered ex vivo before the ischemic event. Here, we tested whether in vivo continuous systemic delivery of psi(epsilon)RACK confers sustained cardioprotection against ischemia-reperfusion in isolated mouse hearts and whether psi(epsilon)RACK treatment reduces infarct size or lethal arrhythmias in porcine hearts in vivo. After psi(epsilon)RACK was systemically administered in mice either acutely or continuously, hearts were subjected to ischemia-reperfusion in an isolated perfused model. Whereas psi(epsilon)RACK-induced cardioprotection lasted 1 hour after a single intraperitoneal injection, continuous treatment with psi(epsilon)RACK induced a sustained preconditioned state during the 10 days of delivery. There was no desensitization to the therapeutic effect, no downregulation of epsilonPKC, and no adverse effects after sustained psi(epsilon)RACK delivery. Porcine hearts were subjected to ischemia-reperfusion in vivo, and psi(epsilon)RACK was administered by intracoronary injection during the first 10 minutes of ischemia. psi(epsilon)RACK treatment reduced infarct size (34+/-2% versus 14+/-1%, control versus psi(epsilon)RACK) and resulted in fewer cases of ventricular fibrillation during ischemia-reperfusion (87.5% versus 50%, control versus psi(epsilon)RACK). The epsilonPKC activator psi(epsilon)RACK induced cardioprotection both in vivo and ex vivo, reduced the incidence of lethal arrhythmia during ischemia-reperfusion, and did not cause desensitization or downregulation of epsilonPKC after sustained delivery. Thus, psi(epsilon)RACK may be useful for patients with ischemic heart disease. In addition, the psi(epsilon)RACK peptide should be a useful pharmacological agent for animal studies in which systemic and sustained modulation of epsilonPKC in vivo is needed.

Increased Collagen Deposition and Diastolic Dysfunction but Preserved Myocardial Hypertrophy After Pressure Overload in Mice Lacking PKC

Article

Apr 2005
CIRC RES

Overexpression and activation of protein kinase C-epsilon (PKCepsilon) results in myocardial hypertrophy. However, these observations do not establish that PKCepsilon is required for the development of myocardial hypertrophy. Thus, we subjected PKCepsilon-knockout (KO) mice to a hypertrophic stimulus by transverse aortic constriction (TAC). KO mice show normal cardiac morphology and function. TAC caused similar cardiac hypertrophy in KO and wild-type (WT) mice. However, KO mice developed more interstitial fibrosis and showed enhanced expression of collagen Ialpha1 and collagen III after TAC associated with diastolic dysfunction, as assessed by tissue Doppler echocardiography (Ea/Aa after TAC: WT 2.1+/-0.3 versus KO 1.0+/-0.2; P<0.05). To explore underlying mechanisms, we analyzed the left ventricular (LV) expression pattern of additional PKC isoforms (ie, PKCalpha, PKCbeta, and PKCdelta). After TAC, expression and activation of PKCdelta protein was increased in KO LVs. Moreover, KO LVs displayed enhanced activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), whereas p42/p44-MAPK activation was attenuated. Under stretch, cultured KO fibroblasts showed a 2-fold increased collagen Ialpha1 (col Ialpha1) expression, which was prevented by PKCdelta inhibitor rottlerin or by p38 MAPK inhibitor SB 203580. In conclusion, PKCepsilon is not required for the development of a pressure overload-induced myocardial hypertrophy. Lack of PKCepsilon results in upregulation of PKCdelta and promotes activation of p38 MAPK and JNK, which appears to compensate for cardiac hypertrophy, but in turn, is associated with increased collagen deposition and impaired diastolic function.

The cardiac fibroblast: Therapeutic target in myocardial remodeling and failure

Article

Feb 2005

Cardiac fibroblasts play a central role in the maintenance of extracellular matrix in the normal heart and as mediators of inflammatory and fibrotic myocardial remodeling in the injured and failing heart. In this review, we evaluate the cardiac fibroblast as a therapeutic target in heart disease. Unique features of cardiac fibroblast cell biology are discussed in relation to normal and pathophysiological cardiac function. The contribution of cardiac fibrosis as an independent risk factor in the outcome of heart failure is considered. Candidate drug therapies that derive benefit from actions on cardiac fibroblasts are summarized, including inhibitors of angiotensin-aldosterone systems, endothelin receptor antagonists, statins, anticytokine therapies, matrix metalloproteinase inhibitors, and novel antifibrotic/anti-inflammatory agents. These findings point the way to future challenges in cardiac fibroblast biology and pharmacotherapy.

Pharmacological inhibition of epsilon PKC suppresses chronic inflammation in murine cardiac transplantation model

Article

Nov 2007

Epsilon protein kinase C (epsilonPKC) plays pivotal roles in myocardial infarction and in heart failure. Although cardiac transplantation is a well-established therapy for severe heart failure, allograft rejection and host inflammatory responses limit graft function and reduce life expectancy. Here we determined whether sustained epsilonPKC inhibition beginning 3 days after transplantation suppress allograft rejection and improve cardiac transplantation using a murine heterotopic transplantation model. Hearts of FVB mice (H-2(q)) were transplanted into C57BL/6 mice (H-2(b)). Delivery of the epsilonPKC inhibitor, TAT(47-57)-epsilonV1-2 (epsilonV1-2, n=9, 20 mg/kg/day), or the carrier control peptide, TAT(47-57) (TAT, n=8), by osmotic pump began 3 days after transplantation and continued for the remaining 4 weeks. epsilonV1-2 treatment significantly improved the beating score throughout the treatment. Infiltration of macrophages and T cells into the cardiac grafts was significantly reduced and parenchymal fibrosis was decreased in animals treated with epsilonV1-2 as compared with control treatment. Finally, the rise in pro-fibrotic cytokine, TGF-beta and monocyte recruiting chemokine MCP-1 levels was almost abolished by epsilonV1-2 treatment, whereas the rise in PDGF-BB level was unaffected. These data suggest that epsilonPKC activity contributes to the chronic immune response in cardiac allograft and that an epsilonPKC-selective inhibitor, such as epsilonV1-2, could augment current therapeutic strategies to suppress inflammation and prolong graft survival in humans.

Pharmacological Inhibition of -Protein Kinase C Attenuates Cardiac Fibrosis and Dysfunction in Hypertension-Induced Heart Failure

Abstract

No full-text available

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