RESUMO
Twelve regulated cell death programs have been described. We review in detail the basic biology of nine including death receptor-mediated apoptosis, death receptor-mediated necrosis (necroptosis), mitochondrial-mediated apoptosis, mitochondrial-mediated necrosis, autophagy-dependent cell death, ferroptosis, pyroptosis, parthanatos, and immunogenic cell death. This is followed by a dissection of the roles of these cell death programs in the major cardiac syndromes: myocardial infarction and heart failure. The most important conclusion relevant to heart disease is that regulated forms of cardiomyocyte death play important roles in both myocardial infarction with reperfusion (ischemia/reperfusion) and heart failure. While a role for apoptosis in ischemia/reperfusion cannot be excluded, regulated forms of necrosis, through both death receptor and mitochondrial pathways, are critical. Ferroptosis and parthanatos are also likely important in ischemia/reperfusion, although it is unclear if these entities are functioning as independent death programs or as amplification mechanisms for necrotic cell death. Pyroptosis may also contribute to ischemia/reperfusion injury, but potentially through effects in non-cardiomyocytes. Cardiomyocyte loss through apoptosis and necrosis is also an important component in the pathogenesis of heart failure and is mediated by both death receptor and mitochondrial signaling. Roles for immunogenic cell death in cardiac disease remain to be defined but merit study in this era of immune checkpoint cancer therapy. Biology-based approaches to inhibit cell death in the various cardiac syndromes are also discussed.
Assuntos
Morte Celular , Citotoxicidade Imunológica , Cardiopatias/patologia , Mitocôndrias Cardíacas/patologia , Miocárdio/patologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/metabolismo , Cardiopatias/imunologia , Cardiopatias/metabolismo , Cardiopatias/fisiopatologia , Humanos , Mitocôndrias Cardíacas/imunologia , Mitocôndrias Cardíacas/metabolismo , Miocárdio/imunologia , Miocárdio/metabolismo , Necrose , Piroptose , Transdução de SinaisRESUMO
Adrenergic modulation of voltage gated Ca2+ currents is a context specific process. In the heart Cav1.2 channels initiate excitation-contraction coupling. This requires protein kinase A (PKA) phosphorylation of the small GTPase Rad (Ras associated with diabetes) and involves direct phosphorylation of a1 subunit of the Cav1.2 at Ser1700. A contributing factor is the proximity of PKA to the channel through association with A-kinase anchoring proteins (AKAPs). Disruption of PKA anchoring by the disruptor peptide AKAP-IS prevents up-regulation of Cav1.2 currents in tsA-201 cells. Biochemical analyses demonstrate that Rad does not function as an A-kinase anchoring protein. Electrophysiological recording shows that channel mutants lacking phosphorylation sites (Cav1.2 STAA) lose responsivity to the second messenger cAMP. Measurements in cardiomyocytes isolated from Rad-/- mice show that adrenergic activation of Cav1.2 is attenuated but not completely abolished. Whole animal electrocardiography studies reveal that cardiac selective Rad knockout mice exhibited higher baseline left-ventricular ejection fraction (EF), greater fractional shortening (FS), and increased heart rate as compared to control animals. Yet, each parameter of cardiac function was slightly elevated when Rad-/- mice were treated with the adrenergic agonist isoproterenol. Thus, phosphorylation of Cav1.2 and dissociation of phospho-Rad from the channel are local cAMP responsive events that act in concert to enhance L-type calcium currents. This convergence of local PKA regulatory events at the cardiac L-type calcium channel may permit maximal ß-adrenergic influence on the fight-or-flight response.
RESUMO
Cell death is a crucial event underlying cardiac ischemic injury, pathological remodeling, and heart failure. Unlike apoptosis, necrosis had long been regarded as a passive and unregulated process. However, recent studies demonstrate that a significant subset of necrotic cell death is actively mediated through regulated pathways - a process known as "regulated necrosis". As a form of regulated necrosis, necroptosis is mediated by death receptors and executed through the activation of receptor interacting protein kinase 3 (RIPK3) and its downstream substrate mixed lineage kinase-like domain (MLKL). Recent studies have provided compelling evidence that necroptosis plays an important role in myocardial homeostasis, ischemic injury, pathological remodeling, and heart failure. Moreover, it has been shown that genetic and pharmacological manipulations of the necroptosis signaling pathway elicit cardioprotective effects. Important progress has also been made regarding the molecular mechanisms that regulate necroptotic cell death in vitro and in vivo. In this review, we discuss molecular and cellular mechanisms of necroptosis, potential crosstalk between necroptosis and other cell death pathways, functional implications of necroptosis in heart disease, and new therapeutic strategies that target necroptosis signaling.
Assuntos
Cardiopatias , Insuficiência Cardíaca , Apoptose , Humanos , Necroptose , Necrose , Proteínas Quinases/metabolismoRESUMO
Deubiquitinating enzymes (DUBs) appear to be a new class of regulators of cardiac homeostasis and disease. However, DUB-mediated signaling in the heart is not well understood. Herein we report a novel mechanism by which cylindromatosis (CYLD), a DUB mediates cardiac pathological remodeling and dysfunction. Cardiomyocyte-restricted (CR) overexpression of CYLD (CR-CYLD) did not cause gross health issues and hardly affected cardiac function up to age of one year in both female and male mice at physiological conditions. However, CR-CYLD overexpression exacerbated pressure overload (PO)-induced cardiac dysfunction associated with suppressed cardiac hypertrophy and increased myocardial apoptosis in mice independent of the gender. At the molecular level, CR-CYLD overexpression enhanced PO-induced increases in poly-ubiquitinated proteins marked by lysine (K)48-linked ubiquitin chains and autophagic vacuoles containing undegraded contents while suppressing autophagic flux. Augmentation of cardiac autophagy via CR-ATG7 overexpression protected against PO-induced cardiac pathological remodeling and dysfunction in both female and male mice. Intriguingly, CR-CYLD overexpression switched the CR-ATG7 overexpression-dependent cardiac protection into myocardial damage and dysfunction associated with increased accumulation of autophagic vacuoles containing undegraded contents in the heart. Genetic manipulation of Cyld in combination with pharmacological modulation of autophagic functional status revealed that CYLD suppressed autolysosomal degradation and promoted cell death in cardiomyocytes. In addition, Cyld gene gain- and/or loss-of-function approaches in vitro and in vivo demonstrated that CYLD mediated cardiomyocyte death associated with impaired reactivation of mechanistic target of rapamycin complex 1 (mTORC1) and upregulated Ras genes from rat brain 7 (Rab7), two key components for autolysosomal degradation. These results demonstrate that CYLD serves as a novel mediator of cardiac pathological remodeling and dysfunction by suppressing autolysosome efflux in cardiomyocytes. Mechanistically, it is most likely that CYLD suppresses autolysosome efflux via impairing mTORC1 reactivation and interrupting Rab7 release from autolysosomes in cardiomyocytes.
Assuntos
Cardiomiopatias/metabolismo , Enzima Desubiquitinante CYLD/metabolismo , Lisossomos/metabolismo , Miócitos Cardíacos/metabolismo , Pressão , Animais , Autofagia , Proteína 7 Relacionada à Autofagia/metabolismo , Encéfalo/metabolismo , Fibroblastos/metabolismo , Genes ras , Lisina/metabolismo , Lisossomos/ultraestrutura , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Transgênicos , Miocárdio/patologia , Miócitos Cardíacos/patologia , Ratos , Ubiquitinação , Regulação para Cima , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
BACKGROUND: Programmed cell death, including apoptosis, mitochondria-mediated necrosis, and necroptosis, is critically involved in ischemic cardiac injury, pathological cardiac remodeling, and heart failure progression. Whereas apoptosis and mitochondria-mediated necrosis signaling is well established, the regulatory mechanisms of necroptosis and its significance in the pathogenesis of heart failure remain elusive. METHODS: We examined the role of tumor necrosis factor receptor-associated factor 2 (Traf2) in regulating myocardial necroptosis and remodeling using genetic mouse models. We also performed molecular and cellular biology studies to elucidate the mechanisms by which Traf2 regulates necroptosis signaling. RESULTS: We identified a critical role for Traf2 in myocardial survival and homeostasis by suppressing necroptosis. Cardiac-specific deletion of Traf2 in mice triggered necroptotic cardiac cell death, pathological remodeling, and heart failure. Plasma tumor necrosis factor α level was significantly elevated in Traf2-deficient mice, and genetic ablation of TNFR1 largely abrogated pathological cardiac remodeling and dysfunction associated with Traf2 deletion. Mechanistically, Traf2 critically regulates receptor-interacting proteins 1 and 3 and mixed lineage kinase domain-like protein necroptotic signaling with the adaptor protein tumor necrosis factor receptor-associated protein with death domain as an upstream regulator and transforming growth factor ß-activated kinase 1 as a downstream effector. It is important to note that genetic deletion of RIP3 largely rescued the cardiac phenotype triggered by Traf2 deletion, validating a critical role of necroptosis in regulating pathological remodeling and heart failure propensity. CONCLUSIONS: These results identify an important Traf2-mediated, NFκB-independent, prosurvival pathway in the heart by suppressing necroptotic signaling, which may serve as a new therapeutic target for pathological remodeling and heart failure.
Assuntos
Apoptose/fisiologia , Miócitos Cardíacos/metabolismo , Fator 2 Associado a Receptor de TNF/deficiência , Remodelação Ventricular/fisiologia , Animais , Animais Recém-Nascidos , Cardiotônicos/metabolismo , Morte Celular/fisiologia , Células Cultivadas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/patologia , Necrose/patologia , Necrose/prevenção & controle , Ratos , Ratos Sprague-Dawley , Fator 2 Associado a Receptor de TNF/genéticaRESUMO
Microtubules (MTs) have many roles in ventricular myocytes, including structural stability, morphological integrity, and protein trafficking. However, despite their functional importance, dynamic MTs had never been visualized in living adult myocytes. Using adeno-associated viral vectors expressing the MT-associated protein plus end binding protein 3 (EB3) tagged with EGFP, we were able to perform live imaging and thus capture and quantify MT dynamics in ventricular myocytes in real time under physiological conditions. Super-resolution nanoscopy revealed that EB1 associated in puncta along the length of MTs in ventricular myocytes. The vast (~80%) majority of MTs grew perpendicular to T-tubules at a rate of 0.06µm∗s(-1) and growth was preferentially (82%) confined to a single sarcomere. Microtubule catastrophe rate was lower near the Z-line than M-line. Hydrogen peroxide increased the rate of catastrophe of MTs ~7-fold, suggesting that oxidative stress destabilizes these structures in ventricular myocytes. We also quantified MT dynamics after myocardial infarction (MI), a pathological condition associated with increased production of reactive oxygen species (ROS). Our data indicate that the catastrophe rate of MTs increases following MI. This contributed to decreased transient outward K(+) currents by decreasing the surface expression of Kv4.2 and Kv4.3 channels after MI. On the basis of these data, we conclude that, under physiological conditions, MT growth is directionally biased and that increased ROS production during MI disrupts MT dynamics, decreasing K(+) channel trafficking.
Assuntos
Ventrículos do Coração/metabolismo , Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Animais , Camundongos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Ligação Proteica , Transporte Proteico , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND: Programmed necrosis (necroptosis) plays an important role in development, tissue homeostasis, and disease pathogenesis. The molecular mechanisms that regulate necroptosis in the heart and its physiological relevance in myocardial remodeling and heart failure remain largely unknown. METHODS AND RESULTS: Here, we identified an obligate function for TAK1 (transforming growth factor ß-activated kinase 1, gene name Map3k7) in regulating necroptotic myocyte death, myocardial remodeling, and heart failure propensity. Cardiac-specific ablation of Map3k7 in mice induced spontaneous apoptosis and necroptosis that led to adverse remodeling and heart failure, and these effects were abolished by ablation of tumor necrosis factor receptor-1. Mechanistically, TAK1 functions as a molecular switch in tumor necrosis factor receptor-1 signaling by regulating the formation of 2 cell death complexes, RIP 1 (receptor-interacting protein 1)-FADD (Fas-associated protein with death domain)-caspase 8 and RIP1-RIP3, a process that is dependent on FADD and caspase 8 as scaffolding molecules. Importantly, inhibition of RIP1 or RIP3 largely blocked necroptotic cell death, adverse remodeling, and heart failure in TAK1-deficient mice. CONCLUSIONS: These results indicate that TAK1 functions as a key survival factor in the heart by directly antagonizing necroptosis, which is critical for the maintenance of myocardial homeostasis and the prevention of adverse myocardial remodeling.
Assuntos
Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , MAP Quinase Quinase Quinases/fisiologia , Transdução de Sinais/fisiologia , Remodelação Ventricular/fisiologia , Animais , Apoptose/fisiologia , Caspase 8/fisiologia , Linhagem Celular , Modelos Animais de Doenças , Proteína de Domínio de Morte Associada a Fas/fisiologia , Insuficiência Cardíaca/mortalidade , Homeostase/fisiologia , MAP Quinase Quinase Quinases/deficiência , MAP Quinase Quinase Quinases/genética , Camundongos , Camundongos Knockout , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Necrose/fisiopatologia , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologiaRESUMO
RATIONALE: Both nuclear factors of activated T cells (NFAT) and nuclear factor-κB (NFκB) are Rel homology domain (RHD)-containing transcription factors whose independent activities are critically involved in regulating cardiac hypertrophy and failure. OBJECTIVE: To determine the potential functional interaction between NFAT and NFκB signaling pathways in cardiomyocytes and its role in cardiac hypertrophy and remodeling. METHODS AND RESULTS: We identified a novel transcriptional regulatory mechanism whereby NFκB and NFAT directly interact and synergistically promote transcriptional activation in cardiomyocytes. We show that the p65 subunit of NFκB coimmunoprecipitates with NFAT in cardiomyocytes, and this interaction maps to the RHD within p65. Overexpression of the p65-RHD disrupts the association between endogenous p65 and NFATc1, leading to reduced transcriptional activity. Overexpression of IκB kinase ß (IKKß) or p65-RHD causes nuclear translocation of NFATc1, and expression of a constitutively nuclear NFATc1-SA mutant similarly facilitated p65 nuclear translocation. Combined overexpression of p65 and NFATc1 promotes synergistic activation of NFAT transcriptional activity in cardiomyocytes, whereas inhibition of NFκB with IκBαM or dominant negative IKKß reduces NFAT activity. Importantly, agonist-induced NFAT activation is reduced in p65 null mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs. In vivo, cardiac-specific deletion of p65 using a Cre-loxP system causes a ≈50% reduction in NFAT activity in luciferase reporter mice. Moreover, ablation of p65 in the mouse heart decreases the hypertrophic response after pressure overload stimulation, reduces the degree of pathological remodeling, and preserves contractile function. CONCLUSIONS: Our results suggest a direct interaction between NFAT and NFκB that effectively integrates 2 disparate signaling pathways in promoting cardiac hypertrophy and ventricular remodeling.
Assuntos
Cardiomegalia/metabolismo , Miocárdio/metabolismo , Fatores de Transcrição NFATC/metabolismo , Fator de Transcrição RelA/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Animais Recém-Nascidos , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Cardiomegalia/prevenção & controle , Células Cultivadas , Modelos Animais de Doenças , Ecocardiografia Doppler , Regulação da Expressão Gênica , Genes Reporter , Quinase I-kappa B/metabolismo , Imunoprecipitação , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Contração Miocárdica , Miocárdio/patologia , Fatores de Transcrição NFATC/genética , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fator de Transcrição RelA/deficiência , Fator de Transcrição RelA/genética , Ativação TranscricionalRESUMO
The slip ammonia from selective catalytic reduction (SCR) of NOx in coal-fired flue gas can result in deterioration of the utilities or even the environmental issues. To achieve selective catalytic oxidation (SCO) of slip ammonia, Ru-modified Ce-Zr solid solution catalysts were prepared and evaluated under various conditions. It was found that the Ru/Ce(0.6)Zr(0.4)O2(polyvinylpyrrolidone (PVP)) catalyst displayed significant catalytic activity and the slip ammonia was almost completely removed with the coexistence of NOx and SO2. Interestingly, the effect of SO2 on NH3 oxidation was bifacial, and the N2 selectivity of the resulting products was as high as 100% in the presence of SO2 and NH3. The mechanism of the SCO of NH3 over Ru/Ce(0.6)Zr(0.4)O2(PVP) was studied using various techniques, and the results showed that NH3 oxidation follows an internal SCR (iSCR) mechanism. The adsorbed ammonia was first activated and reacted with lattice oxygen atoms to form an -HNO intermediate. Then, the -HNO mainly reacted with atomic oxygen from O2 to form NO. Meanwhile, the formed NO interacted with -NH2 to N2 with N2O as the byproduct, but the presence of SO2 can effectively inhibit the production of N2O.
Assuntos
Poluentes Atmosféricos/química , Amônia/química , Rubídio/química , Adsorção , Catálise , Cério/química , Óxido Nítrico/química , Oxirredução , Óxidos/química , Oxigênio/química , Povidona , Espectroscopia de Infravermelho com Transformada de Fourier , Dióxido de Enxofre/química , Zircônio/químicaRESUMO
The cardiac calcium channel CaV1.2 conducts L-type calcium currents that initiate excitation-contraction coupling and serves as a crucial mediator of ß-adrenergic regulation of the heart. We evaluated the inotropic response of mice with mutations in C-terminal phosphoregulatory sites under physiological levels of ß-adrenergic stimulation in vivo, and we assessed the impact of combining mutations of C-terminal phosphoregulatory sites with chronic pressure-overload stress. Mice with Ser1700Ala (S1700A), Ser1700Ala/Thr1704Ala (STAA), and Ser1928Ala (S1928A) mutations had impaired baseline regulation of ventricular contractility and exhibited decreased inotropic response to low doses of ß-adrenergic agonist. In contrast, treatment with supraphysiogical doses of agonist revealed substantial inotropic reserve that compensated for these deficits. Hypertrophy and heart failure in response to transverse aortic constriction (TAC) were exacerbated in S1700A, STAA, and S1928A mice whose ß-adrenergic regulation of CaV1.2 channels was blunted. These findings further elucidate the role of phosphorylation of CaV1.2 at regulatory sites in the C-terminal domain for maintaining normal cardiac homeostasis, responding to physiological levels of ß-adrenergic stimulation in the fight-or-flight response, and adapting to pressure-overload stress.
RESUMO
Oxidative stress has been shown to induce cell death in a wide range of human diseases including cardiac ischemia/reperfusion injury, drug induced cardiotoxicity, and heart failure. However, the mechanism of cell death induced by oxidative stress remains incompletely understood. Here we provide new evidence that oxidative stress primarily induces ferroptosis, but not apoptosis, necroptosis, or mitochondria-mediated necrosis, in cardiomyocytes. Intriguingly, oxidative stress induced by organic oxidants such as tert-butyl hydroperoxide (tBHP) and cumene hydroperoxide (CHP), but not hydrogen peroxide (H2O2), promoted glutathione depletion and glutathione peroxidase 4 (GPX4) degradation in cardiomyocytes, leading to increased lipid peroxidation. Moreover, elevated oxidative stress is also linked to labile iron overload through downregulation of the transcription suppressor BTB and CNC homology 1 (Bach1), upregulation of heme oxygenase 1 (HO-1) expression, and enhanced iron release via heme degradation. Strikingly, oxidative stress also promoted HO-1 translocation to mitochondria, leading to mitochondrial iron overload and lipid reactive oxygen species (ROS) accumulation. Targeted inhibition of mitochondrial iron overload or ROS accumulation, by overexpressing mitochondrial ferritin (FTMT) or mitochondrial catalase (mCAT), respectively, markedly inhibited oxidative stress-induced ferroptosis. The levels of mitochondrial iron and lipid peroxides were also markedly increased in cardiomyocytes subjected to simulated ischemia and reperfusion (sI/R) or the chemotherapeutic agent doxorubicin (DOX). Overexpressing FTMT or mCAT effectively prevented cardiomyocyte death induced by sI/R or DOX. Taken together, oxidative stress induced by organic oxidants but not H2O2 primarily triggers ferroptotic cell death in cardiomyocyte through GPX4 and Bach1/HO-1 dependent mechanisms. Our results also reveal mitochondrial iron overload via HO-1 mitochondrial translocation as a key mechanism as well as a potential molecular target for oxidative stress-induced ferroptosis in cardiomyocytes.
Assuntos
Sobrecarga de Ferro , Estresse Oxidativo , Humanos , Espécies Reativas de Oxigênio , Morte Celular , Ferro , Miócitos CardíacosRESUMO
High levels of circulating catecholamines cause cardiac injury, pathological remodeling, and heart failure, but the underlying mechanisms remain elusive. Here we provide both in vitro and in vivo evidence that excessive ß-adrenergic stimulation induces ferroptosis in cardiomyocytes, revealing a novel mechanism for catecholamine-induced cardiotoxicity and remodeling. We found that isoproterenol, a synthetic catecholamine, promoted glutathione depletion and glutathione peroxidase 4 (GPX4) degradation in cardiomyocytes, leading to GPX4 inactivation and enhanced lipid peroxidation. Isoproterenol also promoted heme oxygenase 1 (HO-1) expression by downregulating the transcription suppressor BTB and CNC homology 1 (Bach1), leading to increased labile iron accumulation through heme degradation. Moreover, isoproterenol markedly induced the accumulation of free iron and lipid reactive oxygen species (ROS) in the mitochondria, while targeted inhibition of iron overload and ROS accumulation within mitochondria effectively inhibited ferroptosis in cardiomyocytes. Importantly, isoproterenol administration markedly induced ferroptosis in the myocardium in vivo, associated with elevated non-heme iron accumulation driven by HO-1 upregulation. Strikingly, blockade of ferroptosis with ferrostatin-1 or inhibition of HO-1 activity with zinc protoporphyrin (ZnPP) effectively alleviated cardiac necrosis, pathological remodeling, and heart failure induced by isoproterenol administration. Taken together, our results reveal that catecholamine stimulation primarily induces ferroptotic cell death in cardiomyocyte through GPX4 and Bach1-HO-1 dependent signaling pathways. Targeting ferroptosis may represent a novel therapeutic strategy for catecholamine overload-induced myocardial injury and heart failure.
Assuntos
Ferroptose , Insuficiência Cardíaca , Humanos , Ferroptose/genética , Espécies Reativas de Oxigênio/metabolismo , Cardiotoxicidade , Catecolaminas/farmacologia , Isoproterenol/farmacologia , Ferro/metabolismoRESUMO
Mutations in TGF-ß-activated kinase 1 binding protein 2 (TAB2) have been implicated in the pathogenesis of dilated cardiomyopathy and/or congenital heart disease in humans, but the underlying mechanisms are currently unknown. Here, we identified an indispensable role for TAB2 in regulating myocardial homeostasis and remodeling by suppressing receptor-interacting protein kinase 1 (RIPK1) activation and RIPK1-dependent apoptosis and necroptosis. Cardiomyocyte-specific deletion of Tab2 in mice triggered dilated cardiomyopathy with massive apoptotic and necroptotic cell death. Moreover, Tab2-deficient mice were also predisposed to myocardial injury and adverse remodeling after pathological stress. In cardiomyocytes, deletion of TAB2 but not its close homolog TAB3 promoted TNF-α-induced apoptosis and necroptosis, which was rescued by forced activation of TAK1 or inhibition of RIPK1 kinase activity. Mechanistically, TAB2 critically mediates RIPK1 phosphorylation at Ser321 via a TAK1-dependent mechanism, which prevents RIPK1 kinase activation and the formation of RIPK1-FADD-caspase-8 apoptotic complex or RIPK1-RIPK3 necroptotic complex. Strikingly, genetic inactivation of RIPK1 with Ripk1-K45A knockin effectively rescued cardiac remodeling and dysfunction in Tab2-deficient mice. Together, these data demonstrated that TAB2 is a key regulator of myocardial homeostasis and remodeling by suppressing RIPK1-dependent apoptosis and necroptosis. Our results also suggest that targeting RIPK1-mediated cell death signaling may represent a promising therapeutic strategy for TAB2 deficiency-induced dilated cardiomyopathy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Apoptose , Cardiomiopatia Dilatada , Necroptose , Proteína Serina-Treonina Quinases de Interação com Receptores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Camundongos , Camundongos Knockout , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Heart failure afflicts ~5 million people and causes ~300,000 deaths a year in the United States alone. Heart failure is defined as a deficiency in the ability of the heart to pump sufficient blood in response to systemic demands, which results in fatigue, dyspnea, and/or edema. Identifying new therapeutic targets is a major focus of current research in the field. We and others have identified critical roles for protein kinase C (PKC) family members in programming aspects of heart failure pathogenesis. More specifically, mechanistic data have emerged over the past 6-7 years that directly implicate PKCα, a conventional PKC family member, as a nodal regulator of heart failure propensity. Indeed, deletion of the PKCα gene in mice, or its inhibition in rodents with drugs or a dominant negative mutant and/or inhibitory peptide, has shown dramatic protective effects that antagonize the development of heart failure. This review will weigh all the evidence implicating PKCα as a novel therapeutic target to consider for the treatment of heart failure. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure."
Assuntos
Insuficiência Cardíaca/tratamento farmacológico , Terapia de Alvo Molecular , Proteína Quinase C-alfa/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica , Insuficiência Cardíaca/enzimologia , Humanos , Camundongos , Camundongos Knockout , Contração Miocárdica , Miocárdio/enzimologia , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/genética , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent alpha-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCalpha and PKCbeta, whereas the novel isoform, PKC, negatively regulates these events. PKCdelta also negatively regulates thrombus formation but not alpha-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCalpha or PKCbeta showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKC. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.
Assuntos
Plaquetas/enzimologia , Colágeno/farmacologia , Proteína Quinase C/metabolismo , Trombose/sangue , Trombose/enzimologia , Animais , Anticoagulantes/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/deficiência , Isoenzimas/metabolismo , Camundongos , Ativação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/deficiência , Trombose/induzido quimicamente , Trombose/fisiopatologiaRESUMO
RATIONALE: Apoptosis signal-regulating kinase (ASK)1 is a central upstream kinase in the greater mitogen-activated protein kinase cascade that mediates growth and death decisions in cardiac myocytes in response to diverse pathological stimuli. OBJECTIVE: However, the role that ASK1 plays in regulating the cardiac hypertrophic response in vivo remains controversial. METHODS AND RESULTS: Here, we generated mice with cardiac-specific and inducible overexpression of ASK1 in the heart to assess its gain-of-function effect. ASK1 transgenic mice exhibited no induction of cardiac hypertrophy or pathology at 3 and 12 months of age, and these mice showed an identical hypertrophic response to controls following 2 weeks of pressure-overload stimulation or isoproterenol infusion. Although ASK1 overexpression did not alter the cardiac hypertrophic response, it promoted cardiomyopathy and greater TUNEL following pressure-overload stimulation and myocardial infarction. Indeed, ASK1 transgenic mice showed a greater than 2-fold increase in ischemia reperfusion-induced injury to the heart compared with controls. Examination of downstream signaling showed a prominent activation of mitogen-activated protein kinase kinase 4/6 and c-Jun NH(2)-terminal kinase (JNK)1/2 (but not p38 or extracellular signal-regulated kinases [ERKs]), inhibition of calcineurin-NFAT (nuclear factor of activated T cells), and induction of Bax in the hearts of ASK1 transgenic mice following 1 and 8 weeks of pressure-overload stimulation. Mechanistically, cardiomyopathy associated with ASK1 overexpression after 8 weeks of pressure overload was significantly reduced in the calcineurin Abeta-null (CnAbeta(-/-)) background. CONCLUSIONS: These results indicate that ASK1 does not directly regulate the cardiac hypertrophic response in vivo, but it does alter cell death and propensity to cardiomyopathy, in part, through a calcineurin-dependent mechanism.
Assuntos
Cardiomiopatias/patologia , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 5/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Animais , Apoptose/fisiologia , Calcineurina/metabolismo , Cálcio/metabolismo , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Marcação In Situ das Extremidades Cortadas , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Transgênicos , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Protein kinase (PK)Calpha, PKCbeta, and PKCgamma comprise the conventional PKC isoform subfamily, which is thought to regulate cardiac disease responsiveness. Indeed, mice lacking the gene for PKCalpha show enhanced cardiac contractility and reduced susceptibility to heart failure. Recent data also suggest that inhibition of conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 enhances heart function and antagonizes failure, although the isoform responsible for these effects is unknown. Here, we investigated mice lacking PKCalpha, PKCbeta, and PKCgamma for effects on cardiac contractility and heart failure susceptibility. PKCalpha(-/-) mice, but not PKCbetagamma(-/-) mice, showed increased cardiac contractility, myocyte cellular contractility, Ca(2+) transients, and sarcoplasmic reticulum Ca(2+) load. PKCalpha(-/-) mice were less susceptible to heart failure following long-term pressure-overload stimulation or 4 weeks after myocardial infarction injury, whereas PKCbetagamma(-/-) mice showed more severe failure. Infusion of ruboxistaurin (LY333531), an orally available PKCalpha/beta/gamma inhibitor, increased cardiac contractility in wild-type and PKCbetagamma(-/-) mice, but not in PKCalpha(-/-) mice. More importantly, ruboxistaurin prevented death in wild-type mice throughout 10 weeks of pressure-overload stimulation, reduced ventricular dilation, enhanced ventricular performance, reduced fibrosis, and reduced pulmonary edema comparable to or better than metoprolol treatment. Ruboxistaurin was also administered to PKCbetagamma(-/-) mice subjected to pressure overload, resulting in less death and heart failure, implicating PKCalpha as the primary target of this drug in mitigating heart disease. As an aside, PKCalphabetagamma triple-null mice showed no defect in cardiac hypertrophy following pressure-overload stimulation. In conclusion, PKCalpha functions distinctly from PKCbeta and PKCgamma in regulating cardiac contractility and heart failure, and broad-acting PKC inhibitors such as ruboxistaurin could represent a novel therapeutic approach in treating human heart failure.
Assuntos
Insuficiência Cardíaca/prevenção & controle , Indóis/farmacologia , Maleimidas/farmacologia , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Miocárdio/enzimologia , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Cardiomegalia/enzimologia , Cardiomegalia/etiologia , Cardiomegalia/fisiopatologia , Cardiomegalia/prevenção & controle , Modelos Animais de Doenças , Fibrose , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Metoprolol/farmacologia , Camundongos , Camundongos Knockout , Infarto do Miocárdio/complicações , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Proteína Quinase C beta , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/deficiência , Proteína Quinase C-alfa/genética , Edema Pulmonar/enzimologia , Edema Pulmonar/etiologia , Edema Pulmonar/fisiopatologia , Edema Pulmonar/prevenção & controle , Fatores de TempoRESUMO
RATIONALE: Pathological cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca(2+) needed to maintain cardiac function when systolic wall stress is increased. Hypertrophic Ca(2+) binds to calmodulin (CaM) and activates the phosphatase calcineurin (Cn) and CaM kinase (CaMK)II. Cn dephosphorylates cytoplasmic NFAT (nuclear factor of activated T cells), inducing its translocation to the nucleus where it activates antiapoptotic and hypertrophic target genes. Cytoplasmic CaMKII regulates Ca(2+) handling proteins but whether or not it is directly involved in hypertrophic and survival signaling is not known. OBJECTIVE: This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn. METHODS AND RESULTS: Green fluorescent protein-tagged NFATc3 was used to determine the cellular location of NFAT in cultured neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes. Constitutively active (CaMKII-CA) or dominant negative (CaMKII-DN) mutants of cytoplasmic targeted CaMKII(deltac) were used to activate and inhibit cytoplasmic CaMKII activity. In NRVM CaMKII-DN (48.5+/-3%, P<0.01 versus control) increased, whereas CaMKII-CA decreased (5.9+/-1%, P<0.01 versus control) NFAT nuclear translocation (Control: 12.3+/-1%). Cn inhibitors were used to show that these effects were caused by modulation of Cn activity. Increasing Ca(2+) increased Cn-dependent NFAT translocation (to 71.7+/-7%, P<0.01) and CaMKII-CA reduced this effect (to 17.6+/-4%). CaMKII-CA increased TUNEL and caspase-3 activity (P<0.05). CaMKII directly phosphorylated Cn at Ser197 in CaMKII-CA infected NRVMs and in hypertrophied feline hearts. CONCLUSION: These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn.
Assuntos
Calcineurina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomegalia/metabolismo , Núcleo Celular/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição NFATC/metabolismo , Transporte Ativo do Núcleo Celular/genética , Animais , Calcineurina/genética , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Calmodulina/genética , Calmodulina/metabolismo , Cardiomegalia/genética , Cardiomegalia/patologia , Caspase 3/genética , Caspase 3/metabolismo , Gatos , Núcleo Celular/genética , Citoplasma/genética , Citoplasma/metabolismo , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Células K562 , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutação , Miócitos Cardíacos/patologia , Fatores de Transcrição NFATC/genética , Fosforilação/genética , Ratos , Ratos Sprague-DawleyRESUMO
In this study, we identified an unexpected pro-cell death role for NFκB in mediating oxidative stress-induced necrosis, and provide new mechanistic evidence that NFκB, in cooperation with HDAC3, negatively regulates Nrf2-ARE anti-oxidative signaling through transcriptional silencing. We showed that genetic inactivation of NFκB-p65 inhibited, whereas activation of NFκB promoted, oxidative stress-induced cell death and HMGB1 release, a biomarker of necrosis. Moreover, NFκB-luciferase activity was elevated in cardiomyocytes after simulated ischemia/reperfusion (sI/R) or doxorubicin (DOX) treatment, and inhibition of NFκB with Ad-p65-shRNA or Ad-IκBαM diminished sI/R- and DOX-induced cell death and HMGB1 release. Importantly, NFκB negatively regulated Nrf2-ARE activity and the expression of antioxidant proteins. Mechanistically, co-immunoprecipitation revealed that p65 was required for Nrf2-HDAC3 interaction and transcriptional silencing of Nrf2-ARE activity. Further, the ability of HDAC3 to repress Nrf2-ARE activity was lost in p65 deficient cells. Pharmacologic inhibition of HADCs or NFκB with trichostatin A (TSA) or BMS-345541, respectively, increased Nrf2-ARE activity and promoted cell survival after sI/R. In vivo, NFκB transcriptional activity in the mouse heart was significantly elevated after ischemia/reperfusion (I/R) injury, which was abolished by cardiomyocyte-specific deletion of p65 using p65fl/flNkx2.5-Cre mice. Moreover, genetic ablation of p65 in the mouse heart attenuated myocardial infarct size after acute I/R injury and improved cardiac remodeling and functional recovery after chronic myocardial infarction. Thus, our results identified NFκB as a key regulator of oxidative stress-induced necrosis by suppressing the Nrf2-ARE antioxidant pathway through an HDAC3-dependent mechanism. This study also revealed a new pathogenic role of NFκB in cardiac ischemic injury and pathological remodeling.