RESUMEN
Since massive irreversible loss of cardiac myocytes occurs following myocardial injury, injection of human mesenchymal stem cells (hMSCs) has emerged as a promising therapeutic intervention. Despite the growing enthusiasm for this approach, the understanding of how hMSCs evoke cardiac improvement is ever more controversial. The present study critically tests hypothesis that hMSCs provide specific benefit directly to damaged ventricular myocytes. Cultures of neonatal mouse ventricular cardiac myocytes (nMCM) were subjected to two distinct acute stress protocols; incubations with either endotoxin, lipopolysaccharide (LPS) or toxic cytokine, IL-1ß. Myocyte injury was assessed in intracellular Ca(2+) signaling assays in fluo-3-loaded nMCMs that were imaged with high temporal resolution by fluorescent microscopy. Following LPS or IL-1ß treatment there was profound myocyte injury, manifest by chaotic [Ca(2+)](i) handling, quantified as a 3- to 5-fold increase in spontaneous [Ca(2+)](i) transients. Antibody neutralization experiments reveal such damage is mediated in part by interleukin-18 and not by tumor necrosis factor-α (TNF-α). Importantly, normal [Ca(2+)](i) signaling was preserved when cardiomyocytes were co-cultured with hMSCs. Since normal [Ca(2+)](i) handling was maintained in transwell cultures, where nMCMs and hMSCs were separated by a permeable membrane, a protective paracrine signaling cascade is operable. hMSCs provoke a genetic reprogramming of cardiomyocytes. LPS provokes release of TNFα from nMCMs which is blocked by hMSCs grown in co- or transwell cultures. Consistent with cytokine release, flow cytometry analyses reveal that hMSCs also block the LPS- and IL-1ß-dependent activation of cardiac transcription factor, NF-κB. Importantly, hMSC-conditioned medium restores normal Ca(2+) signaling in LPS- and IL-1ß-damaged nMCMs. These results reveal new evidence that hMSCs elicit protective and reparative effects on cardiac tissue through molecular reprogramming of the cardiac myocytes themselves. Thus these studies provide novel new insight into the cellular and molecular mechanisms that underlie the therapeutic benefit of hMSCs in the setting of heart failure. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".
Asunto(s)
Reprogramación Celular/fisiología , Ventrículos Cardíacos/citología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Adyuvantes Inmunológicos/farmacología , Animales , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Humanos , Interleucina-18/antagonistas & inhibidores , Interleucina-1beta/farmacología , Lipopolisacáridos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Modelos Biológicos , Miocitos Cardíacos/efectos de los fármacos , FN-kappa B/metabolismo , Comunicación Paracrina , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Although the function of protein kinase D1 (PKD) in cardiac cells has remained enigmatic, recent work has shown that PKD phosphorylates the nuclear regulators HDAC5/7 (histone deacetylase 5/7) and CREB, implicating this kinase in the development of dysfunction seen in heart failure. Additional studies have shown that PKD also phosphorylates multiple sarcomeric substrates to regulate myofilament function. Initial studies examined PKD through adenoviral vector expression of wild type PKD, constitutively active PKD (caPKD), or dominant negative PKD in cultured adult rat ventricular myocytes. Confocal immunofluorescent images of these cells reveal a predominant distribution of all PKD forms in a non-nuclear, Z-line localized, striated reticular pattern, suggesting the importance of PKD in Ca(2+) signaling in heart. Consistent with an established role of PKD in targeting cardiac troponin I (cTnI), caPKD expression led to a marked decrease in contractile myofilament Ca(2+) sensitivity with an unexpected electrical stimulus dependence to this response. This desensitization was accompanied by stimulus-dependent increases in cTnI phosphorylation in control and caPKD cells with a more pronounced effect in the latter. Electrical stimulation also provoked phosphorylation of regulatory site Ser(916) on PKD. The functional importance of this phospho-Ser(916) event is demonstrated in experiments with a phosphorylation-defective mutant, caPKD-S916A, which is functionally inactive and blocks stimulus-dependent increases in cTnI phosphorylation. Dominant negative PKD expression resulted in sensitization of the myofilaments to Ca(2+) and blocked stimulus-dependent increases in cTnI phosphorylation. Taken together, these data reveal that localized PKD may play a role as a dynamic regulator of Ca(2+) sensitivity of contraction in cardiac myocytes.
Asunto(s)
Calcio/metabolismo , Regulación Enzimológica de la Expresión Génica , Miocardio/enzimología , Proteína Quinasa C/fisiología , Animales , Sitios de Unión , Señalización del Calcio , Ventrículos Cardíacos/citología , Masculino , Células Musculares/citología , Contracción Muscular , Fosforilación , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
LDL is the most abundant cholesterol transport vehicle in plasma and a major prognostic indicator of atherosclerosis. Hepatic LDL receptors limit circulating LDL levels, since cholesterol internalized by the liver can be excreted. As such, mechanisms regulating LDL receptor expression in liver cells are appealing targets for cholesterol-lowering therapeutic strategies. Activation of HepG2 cells with phorbol esters enhances LDL receptor mRNA levels through transcriptional and posttranscriptional mechanisms. Here, we show that 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced stabilization of receptor mRNA requires the activity of protein kinase C and is accompanied by activation of the major mitogen activated protein kinase pathways. Inhibitor studies demonstrated that receptor mRNA stabilization is independent of the extracellular signal-regulated kinase or p38(MAPK), but requires activation of the c-Jun N-terminal kinase (JNK). An essential role for JNK in stabilizing receptor mRNA was further confirmed through small interfering RNA (siRNA) experiments and by activating JNK through two protein kinase C-independent mechanisms. Finally, prolonged JNK activation increased steady-state levels of receptor mRNA and protein, and significantly enhanced cellular LDL-binding activity. These data suggest that JNK may play an important role in posttranscriptional control of LDL receptor expression, thus constituting a novel mechanism to enhance plasma LDL clearance by liver cells.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de LDL/genética , Línea Celular , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Hígado/citología , Hígado/metabolismo , MAP Quinasa Quinasa 7/genética , MAP Quinasa Quinasa 7/metabolismo , Sistema de Señalización de MAP Quinasas , Modelos Biológicos , Mutación , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Interferente Pequeño/genética , Transducción de Señal , Transducción Genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Defects of kinase-phosphatase signaling in cardiac myocytes contribute to human heart disease. The activity of one phosphatase, PP2A, is governed by B targeting subunits, including B56gamma1, expressed in heart cells. As the role of PP2A/B56gamma1 on the heart function remains largely unknown, this study sought to identify protein partners through unbiased, affinity purification-based proteomics combined with the functional validation. The results reveal multiple interactors that are localized in strategic cardiac sites to participate in Ca2+ homeostasis and gene expression, exemplified by the Ca pump, SERCA2a, and the splicing factor ASF/SF2. These results are corroborated by confocal imaging where adenovirally overexpressed B56gamma1 is found in z-line/t-tubule region and nuclear speckles. Importantly, overexpression of B56gamma1 in cultured myocytes dramatically impairs cell contractility. These results provide a global view of B56gamma1-regulated local signaling and heart function.
Asunto(s)
Miocardio/metabolismo , Fosfoproteínas Fosfatasas/química , Proteómica/métodos , Empalme Alternativo , Cromatografía Liquida , Humanos , Espectrometría de Masas , Microscopía Confocal , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Proteína Fosfatasa 2 , Estructura Terciaria de Proteína , Proteínas de Unión al ARN , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Factores de Empalme Serina-Arginina , Transducción de SeñalRESUMEN
Protein phosphatase 2A (PP2A) is a multifunctional protein phosphatase with critical roles in excitable cell signaling. In the heart, PP2A function is linked with modulation of beta-adrenergic signaling and has been suggested to regulate key ion channels and transporters including Na/Ca exchanger, ryanodine receptor, inositol 1,4,5-trisphosphate receptor, and Na/K ATPase. Although many of the functional roles and molecular targets for PP2A in heart are known, little is established regarding the cellular pathways that localize specific PP2A isoform activities to subcellular sites. We report that the PP2A regulatory subunit B56alpha is an in vivo binding partner for ankyrin-B, an adapter protein required for normal subcellular localization of the Na/Ca exchanger, Na/K ATPase, and inositol 1,4,5-trisphosphate receptor. Ankyrin-B and B56alpha are colocalized and coimmunoprecipitate in primary cardiomyocytes. Using multiple strategies, we identified the structural requirements on B56alpha for ankyrin-B association as a 13 residue motif in the B56alpha COOH terminus not present in other B56 family polypeptides. Finally, we report that reduced ankyrin-B expression in primary ankyrin-B(+/-) cardiomyocytes results in disorganized distribution of B56alpha that can be rescued by exogenous expression of ankyrin-B. These new data implicate ankyrin-B as a critical targeting component for PP2A in heart and identify a new class of signaling proteins targeted by ankyrin polypeptides.
Asunto(s)
Ancirinas/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Miocitos Cardíacos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Monoéster Fosfórico Hidrolasas/química , Fracciones Subcelulares/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Proteína Fosfatasa 2 , Ratas , Ratas Sprague-Dawley , Especificidad por SustratoRESUMEN
Loss-of-function mutations in the inward rectifier potassium channel, Kir2.1, cause Andersen-Tawil syndrome (ATS-1), an inherited disorder of periodic paralysis and ventricular arrhythmias. Here, we explore the mechanism by which a specific ATS-1 mutation (V302M) alters channel function. Val-302 is located in the G-loop, a structure that is believed to form a flexible barrier for potassium permeation at the apex of the cytoplasmic pore. Consistent with a role in stabilizing the G-loop in an open conformation, we found the V302M mutation specifically renders the channel unable to conduct potassium without altering subunit assembly or attenuating cell surface expression. As predicted by the position of the Val-302 side chain in the crystal structure, amino acid substitution analysis revealed that channel activity and phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity are profoundly sensitive to alterations in the size, shape, and hydrophobicity of side chains at the Val-302 position. The observations establish that the Val-302 side chain is a critical determinant of potassium conduction through the G-loop. Based on our functional studies and the cytoplasmic domain crystal structure, we suggest that Val-302 may influence PIP2 gating indirectly by translating PIP2 binding to conformational changes in the G-loop pore.
Asunto(s)
Síndrome de Andersen/metabolismo , Sistema de Conducción Cardíaco/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Potasio/metabolismo , Sustitución de Aminoácidos , Síndrome de Andersen/genética , Animales , Línea Celular , Permeabilidad de la Membrana Celular/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Transporte Iónico/genética , Metionina/genética , Metionina/metabolismo , Mutación Missense , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Valina/química , Valina/genética , Valina/metabolismo , Xenopus laevisRESUMEN
The Nuclear Factor-kappaB (NF-kappaB) signaling pathway has been linked to several pathologic processes in the myocardium including cardiomyocyte proinflammatory cytokine release, ischemia/reperfusion injury, hypertrophy and apoptosis. However, very little is known about the intracellular mechanisms that govern NF-kappaB activity in the myocardial cells. Recent advances in our understanding of the regulation of NF-kappaB signaling in non-myocyte systems suggest that the activity of the NF-kappaB pathway is tightly regulated by a diversity of stress-activated signaling intermediates through direct post-translational modification of various components of the NF-kappaB pathway. In this review, we will focus on these recent revelations and their implications not only in cardiac pathologies, but in the development of new therapeutic strategies to manage heart disease.
Asunto(s)
Cardiopatías/metabolismo , Miocardio/metabolismo , FN-kappa B/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Animales , Citosol/metabolismo , Regulación de la Expresión Génica , Corazón , Humanos , Proteínas I-kappa B/metabolismo , Modelos Biológicos , FN-kappa B/fisiología , Fosforilación , Transducción de Señal , Transcripción GenéticaRESUMEN
A central feature of heart disease is a molecular remodeling of signaling pathways in cardiac myocytes. This study focused on novel molecular elements of MAPK-mediated alterations in the pattern of gene expression of the protein phosphatase 2A (PP2A). In an established model of sustained JNK activation, a 70% decrease in expression of the targeting subunit of PP2A, B56alpha, was observed in either neonatal or adult cardiomyocytes. This loss in protein abundance was accompanied by a decrease of 69% in B56alpha mRNA steady-state levels. Given that the 3'-untranslated region of this transcript contains adenylate-uridylate-rich elements known to regulate mRNA degradation, experiments explored the notion that instability of B56alpha mRNA accounts for the response. mRNA time-course analyses with real-time PCR methods showed that B56alpha transcript was transformed from a stable (no significant decay over 1 h) to a labile form that rapidly degraded within minutes. These results were supported by complementary experiments that revealed that the RNA-binding protein AUF1, known to destabilize target mRNA, was increased fourfold in JNK-activated cells. A variety of other stress-related stimuli, such as p38 MAPK activation and phorbol ester, upregulated AUF1 expression in cultured cardiac cells as well. In addition, gel mobility shift assays demonstrated that p37AUF1 binds with nanomolar affinity to segments of the B56alpha 3'-untranslated region. Thus these studies provide new evidence that signaling-induced mRNA instability is an important mechanism that underlies the changes in the pattern of gene expression evoked by stress-activated pathways in cardiac cells.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Miocitos Cardíacos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/metabolismo , Animales , Animales Recién Nacidos , Secuencia de Bases , Células Cultivadas , Activación Enzimática/fisiología , Regulación de la Expresión Génica/fisiología , Ribonucleoproteína Nuclear Heterogénea D0 , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , MAP Quinasa Quinasa 7/genética , MAP Quinasa Quinasa 7/metabolismo , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/genética , Unión Proteica , Proteína Fosfatasa 2 , ARN Mensajero/genética , Ratas , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Emerging evidence suggests that both human stem cells and mature stromal cells can play an important role in the development and growth of human malignancies. In contrast to these tumor-promoting properties, we observed that in an in vivo model of Kaposi's sarcoma (KS), intravenously (i.v.) injected human mesenchymal stem cells (MSCs) home to sites of tumorigenesis and potently inhibit tumor growth. We further show that human MSCs can inhibit the in vitro activation of the Akt protein kinase within some but not all tumor and primary cell lines. The inhibition of Akt activity requires the MSCs to make direct cell-cell contact and can be inhibited by a neutralizing antibody against E-cadherin. We further demonstrate that in vivo, Akt activation within KS cells is potently down-regulated in areas adjacent to MSC infiltration. Finally, the in vivo tumor-suppressive effects of MSCs correlates with their ability to inhibit target cell Akt activity, and KS tumors engineered to express a constitutively activated Akt construct are no longer sensitive to i.v. MSC administration. These results suggest that in contrast to other stem cells or normal stromal cells, MSCs possess intrinsic antineoplastic properties and that this stem cell population might be of particular utility for treating those human malignancies characterized by dysregulated Akt.
Asunto(s)
Efecto Injerto vs Tumor/inmunología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/inmunología , Sarcoma de Kaposi/inmunología , Animales , Modelos Animales de Enfermedad , Activación Enzimática/inmunología , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Proteína Oncogénica v-akt/inmunología , Sarcoma de Kaposi/patología , Sarcoma de Kaposi/terapia , Células del Estroma/inmunología , Células del Estroma/trasplante , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
TNF-alpha is recognized as a significant contributor to myocardial dysfunction. Although several studies suggest that members of the NF-kappaB family of transcription factors are essential regulators of myocardial TNF-alpha gene expression, recent developments in our understanding of the modulation of NF-kappaB activity through posttranslational modification of NF-kappaB subunits suggest that the present view of NF-kappaB-dependent cytokine expression in heart is incomplete. Therefore, the goal of the present study was to examine the role of p65 subunit phosphorylation in the regulation of TNF-alpha production in cultured neonatal ventricular myocytes. Bacterial LPS-induced TNF-alpha production is accompanied by a 12-fold increase in phosphorylation of p65 at Ser536, a modification associated with enhancement of p65 transactivation potential. Pharmacological inhibition of IKK-beta reduced LPS-induced TNF-alpha production 38-fold, TNF-alpha mRNA levels 6-fold, and IkappaB-alpha phosphorylation 5-fold and degraded IkappaB-alpha 2-fold and p65 phosphorylation 6-fold. Overexpression of dominant-negative p65 reduced TNF-alpha production 3.5-fold, whereas overexpression of dominant-negative IKK-beta reduced LPS-induced TNF-alpha production 2-fold and p65 phosphorylation 2-fold. Overexpression of dominant-negative IKK-alpha had no effect on p65 phosphorylation or TNF-alpha production, revealing that IKK-beta, not IKK-alpha, plays a central role in regulation of p65 phosphorylation at Ser536 and TNF-alpha production in heart. Finally, we demonstrated, using a chromatin immunoprecipitation assay, that LPS stimulates recruitment of Ser536-phosphorylated p65 to the TNF-alpha gene promoter in cardiac myocytes. Taken together, these data provide compelling evidence for the role of NF-kappaB signaling in TNF-alpha gene expression in heart and highlight the importance of this proinflammatory gene-regulatory pathway as a potential therapeutic target in the management of cytokine-induced myocardial dysfunction.
Asunto(s)
Lipopolisacáridos/farmacología , Miocitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Animales Recién Nacidos , Western Blotting , Células Cultivadas , Cromatina/metabolismo , Citocinas/biosíntesis , Inmunoprecipitación , Ratones , Mutagénesis Sitio-Dirigida/efectos de los fármacos , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Fosforilación , Transducción de Señal/efectos de los fármacos , Quinasa de Factor Nuclear kappa BRESUMEN
Protein phosphatase 2A (PP2A) is widely distributed in heart tissues, yet its precise cellular functions are poorly understood. This study is based on the notion that PP2A action is governed by interactions of the core enzyme with B targeting/regulatory subunits. The subcellular localizations of two B subunits, B56alpha and B56gamma1, were assessed using adenovirus-driven expression of epitope-tagged (hemagglutinin, HA) in cultured neonatal and adult rat ventricular myocytes. Confocal imaging revealed that HA-B56alpha was excluded from the nucleus and decorated striated structures, whereas HA-B56gamma1 was principally found in the nucleus. Precise immunolabeling studies showed that B56gamma1 was concentrated in intranuclear structures known as nuclear speckles, macromolecular structures that accumulate transcription and splicing factors. Western blot analyses revealed that overexpression of either B subunit had no effect on the levels of other PP2A subunits in cultured neonatal cardiac cells. However, overexpression of only B56gamma1 increased whole cell PP2A activity by 40% when measured in cell extracts. Finally, B56gamma1 did not alter global gene expression or expression of hypertrophic gene markers such as alpha-skeletal actin. However, morphometric analyses of confocal images revealed that B56gamma1 alters the dynamic assembly/disassembly process of nuclear speckles in heart cells. These studies provide new insight into mechanisms of PP2A targeting in the subnuclear architecture in cardiomyocytes and into the role of this phosphatase in nuclear signaling.
Asunto(s)
Núcleo Celular/enzimología , Miocitos Cardíacos/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Núcleo Celular/ultraestructura , Células Cultivadas , Técnicas de Transferencia de Gen , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Miocitos Cardíacos/ultraestructura , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 2 , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
BACKGROUND: Heme oxygenase-1 (HO-1) is a stress protein and the rate-limiting enzyme in heme degradation. We sought to examine the notion that protein kinases and phosphatases through phosphorylation and dephosphorylation modulate the HO-1 expression in cardiomyocytes under hypoxic conditions. METHODS AND RESULTS: Exposure of neonatal rat cardiomyocytes to hypoxia markedly induced the HO-1 expression, as assessed by Northern blot, Western blot, and transfection assay. The hypoxia-induced HO-1 expression was blocked by the kinase inhibitors staurosporine and SB202190 in a dose-dependent manner. Hypoxia decreased the activity of phosphatase-1 (PP-1). To examine the effect of PP-1 inhibition on HO-1 expression we used the phosphatase inhibitor okadaic acid (OA) and an antisense vector. OA treatment or overexpression of the antisense PP-1 transcript markedly induced HO-1 expression. Furthermore, transfection assay using HO-1 promoter constructs revealed the involvement of the nuclear factor kB (NF-kB) and Activator protein-1 (AP-1) in the hypoxia-induced activation of the HO-1 gene. The HO-1 promoter activity was modulated by OA under normoxic conditions or staurosporine under hypoxia. CONCLUSIONS: Our results suggest that activation of protein kinases and downregulation of PP-1 activity contribute to the hypoxia-induced HO-1 gene expression and that the proximal HO-1 promoter region containing NF-kB and AP-1 binding sites is likely to play a role in the transcriptional activation of the HO-1 gene in cardiomyocytes in response to hypoxic stress.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/metabolismo , Hipoxia/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Regulación hacia Abajo/fisiología , Expresión Génica , Hemo-Oxigenasa 1 , FN-kappa B/fisiología , Fosforilación , Regiones Promotoras Genéticas , Ratas , Ratas Wistar , Transducción de Señal/fisiología , Factor de Transcripción AP-1/fisiología , TransfecciónRESUMEN
Studies have suggested that integration of kinase and phosphatase activities maintains the steady-state L-type Ca(2+) current in ventricular myocytes, a balance disrupted in failing hearts. As we have recently reported that the PP1/PP2A inhibitor calyculin A evokes pronounced increases in L-type I(Ca), the goal of this study was to identify the counteracting kinase and phosphatase that determine 'basal'I(Ca) in isolated mouse ventricular myocytes. Whole-cell voltage-clamp studies, with filling solutions containing 10 mm EGTA, revealed that calyculin A (100 nm) increased I(Ca) at test potentials between -42 and +49 mV (44% at 0 mV) from a holding potential of -80 mV. It also shifted the V(0.5) (membrane potential at half-maximal) of both activation (from -17 to -25 mV) and steady-state inactivation (from -32 to -37 mV) in the hyperpolarizing direction. The broad-spectrum protein kinase inhibitor, staurosporine (300 nm), was without effect on I(Ca) when added after calyculin A. However, by itself, staurosporine decreased I(Ca) throughout the voltage range examined (50% at 0 mV) and blocked the response to calyculin A, indicating that the phosphatase inhibitor's effects depend upon an opposing kinase activity. The PKA inhibitors Rp-cAMPs (100 microm in the pipette) and H89 (1 microm) failed to reduce basal I(Ca) or to block the calyculin A-evoked increase in I(Ca). Likewise, calyculin A was still active with 10 mm intracellular BAPTA or when Ba(2+) was used as the charge carrier. These data eliminate roles for protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMKII) as counteracting kinases. However, the protein kinase C (PKC) inhibitors Ro 31-8220 (1 microm) and Gö 6976 (200 nm) decreased steady-state I(Ca) and blunted the effect of calyculin A. PP2A is not involved in this regulation as intracellular applications of 10-100 nm okadaic acid or 500 nm fostriecin failed to increase I(Ca). However, PP1 is important, as dialysis with 2 microm okadaic acid or 500 nm inhibitor-2 mimicked the increases in I(Ca) seen with calyculin A. These in situ studies identify constitutive activity of PP1 and the counteracting activity of certain isoforms of PKC, in pathways distinct from receptor-mediated signalling cascades, as regulatory components that determine the steady-state level of cardiac L-type I(Ca).
Asunto(s)
Canales de Calcio Tipo L/fisiología , AMP Cíclico/análogos & derivados , Ácido Egtácico/análogos & derivados , Miocitos Cardíacos/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Proteína Quinasa C/fisiología , Animales , Fenómenos Biofísicos , Biofisica , Tampones (Química) , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Ácido Egtácico/farmacología , Conductividad Eléctrica , Inhibidores Enzimáticos/farmacología , Homeostasis , Isoenzimas/fisiología , Masculino , Toxinas Marinas , Ratones , Ratones Endogámicos , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteína Fosfatasa 1 , Estaurosporina/farmacología , Tionucleótidos/farmacologíaRESUMEN
Mutations in ion channels involved in the generation and termination of action potentials constitute a family of molecular defects that underlie fatal cardiac arrhythmias in inherited long-QT syndrome. We report here that a loss-of-function (E1425G) mutation in ankyrin-B (also known as ankyrin 2), a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans. Mice heterozygous for a null mutation in ankyrin-B are haploinsufficient and display arrhythmia similar to humans. Mutation of ankyrin-B results in disruption in the cellular organization of the sodium pump, the sodium/calcium exchanger, and inositol-1,4,5-trisphosphate receptors (all ankyrin-B-binding proteins), which reduces the targeting of these proteins to the transverse tubules as well as reducing overall protein level. Ankyrin-B mutation also leads to altered Ca2+ signalling in adult cardiomyocytes that results in extrasystoles, and provides a rationale for the arrhythmia. Thus, we identify a new mechanism for cardiac arrhythmia due to abnormal coordination of multiple functionally related ion channels and transporters.
Asunto(s)
Ancirinas/genética , Muerte Súbita Cardíaca/etiología , Síndrome de QT Prolongado/genética , Mutación/genética , Potenciales de Acción , Animales , Ancirinas/fisiología , Bradicardia/complicaciones , Bradicardia/genética , Bradicardia/metabolismo , Bradicardia/fisiopatología , Canales de Calcio/metabolismo , Señalización del Calcio , Electrocardiografía , Femenino , Corazón/fisiopatología , Frecuencia Cardíaca , Heterocigoto , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Síndrome de QT Prolongado/clasificación , Síndrome de QT Prolongado/metabolismo , Síndrome de QT Prolongado/fisiopatología , Masculino , Ratones , Miocardio/metabolismo , Miocardio/patología , Técnicas de Placa-Clamp , Linaje , Fenotipo , Unión Proteica , Receptores Citoplasmáticos y Nucleares/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Although tumor necrosis factor (TNF)-alpha is implicated in numerous cardiac pathologies, the intracellular events leading to its production by heart cells are largely unknown. The goal of the present study was to identify the role of the transcription factor nuclear factor (NF)-kappaB in this process. Among the many inducers of TNF-alpha expression in myeloid cells, only lipopolysaccharide (LPS) led to its induction in cultured neonatal myocytes. LPS also activated the NF-kappaB pathway, as evidenced by the degradation of the inhibitory protein IkappaB and the appearance of NF-kappaB-binding complexes in nuclear extracts. Furthermore, inhibitors of NF-kappaB activation, such as lactacystin, MG132, and pyrrolidine dithiocarbamate, were found to completely block the production of TNF-alpha in response to LPS stimulation, indicating a requirement of NF-kappaB for TNF-alpha expression. However, interleukin-1beta and phorbol 12-myristate 13-acetate also activated NF-kappaB but did not evoke TNF-alpha expression, revealing that this factor is not sufficient for cytokine production. Detailed examination of the NF-kappaB cascade revealed that cardiac cells displayed a unique pattern of IkappaB degradation in response to LPS, with IkappaBbeta but not IkappaBalpha being degraded upon stimulation. Additionally, two specific p65-containing DNA-binding complexes were observed in the nuclear extracts of neonatal cardiomyocytes: an inducible complex that is necessary for TNF-alpha expression and a constitutive species. Taken together, these results reveal that NF-kappaB is not only involved in cytokine production but also may be linked to other pathways that subserve a constitutive, protective mechanism for the heart cell.
Asunto(s)
Acetilcisteína/análogos & derivados , Endotoxinas/toxicidad , Corazón/fisiología , Lipopolisacáridos/toxicidad , FN-kappa B/metabolismo , Pirrolidinas/farmacología , Tiocarbamatos/farmacología , Factor de Necrosis Tumoral alfa/genética , Acetilcisteína/farmacología , Animales , Animales Recién Nacidos , Antioxidantes/farmacología , Western Blotting , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Corazón/efectos de los fármacos , Ratones , Complejos Multienzimáticos/metabolismo , Miocardio/citología , Miocardio/metabolismo , Complejo de la Endopetidasa Proteasomal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés Fisiológico , Transcripción Genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Calyculin A was used to examine the importance of phosphatases in the modulation of cardiac contractile magnitude in the absence of any neural or humoral stimulation. Protein phosphatase (PP)1 and PP2A activity, twitch contractions, intracellular Ca(2+) concentration ([Ca(2+)](i)) transients, action potentials, membrane currents, and myofilament Ca(2+) sensitivity were measured in isolated mouse ventricular myocytes. Calyculin A (125 nM) inhibited PP1 and PP2A by 50% and 85%, respectively, whereas it doubled the twitch magnitude and increased twitch duration by 50% in field-stimulated cells. Calyculin A-evoked increases in L-type Ca(2+) current (70%) and the resulting [Ca(2+)](i) transient (83%) explain the positive inotropic response. However, increases in twitch and action potential durations did not result from increased myofilament Ca(2+) sensitivity or K(+) current inhibition, respectively. Comparison of the effects of calyculin A and isoproterenol on [Ca(2+)](i) transients and twitch contractions revealed that calyculin A had a much smaller lusitropic effect than the beta-agonist, indicating that calyculin A did not significantly increase sarcoplasmic reticulum Ca(2+) reuptake. Thus while cardiac contractile magnitude is controlled by a steady-state kinase/phosphatase balance, this regulation is not equally operative at all of the steps in the excitation-contraction coupling pathway and may in fact be most important to the regulation of the L-type Ca(2+) channel.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Contracción Miocárdica/fisiología , Miocardio/enzimología , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Ventrículos Cardíacos , Técnicas In Vitro , Isoproterenol/farmacología , Cinética , Masculino , Toxinas Marinas , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Contracción Miocárdica/efectos de los fármacos , Miocardio/citología , Fosforilasa a/metabolismo , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiologíaRESUMEN
The delta isoform of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) predominates in the heart. To investigate the role of CaMKII in cardiac function, we made transgenic (TG) mice that express the nuclear delta(B) isoform of CaMKII. The expressed CaMKIIdelta(B) transgene was restricted to the myocardium and highly concentrated in the nucleus. Cardiac hypertrophy was evidenced by an increased left ventricle to body weight ratio and up-regulation of embryonic and contractile protein genes including atrial natriuretic factor, beta-myosin heavy chain, and alpha-skeletal actin. Echocardiography revealed ventricular dilation and decreased cardiac function, which was also observed in hemodynamic measurements from CaMKIIdelta(B) TG mice. Surprisingly, phosphorylation of phospholamban at both Thr(17) and Ser(16) was significantly decreased in the basal state as well as upon adrenergic stimulation. This was associated with diminished sarcoplasmic reticulum Ca(2+) uptake in vitro and altered relaxation properties in vivo. The activity and expression of protein phosphatase 2A were both found to be increased in CaMKII TG mice, and immunoprecipitation studies indicated that protein phosphatase 2A directly associates with CaMKII. Our findings are the first to demonstrate that CaMKII can induce hypertrophy and dilation in vivo and indicate that compensatory increases in phosphatase activity contribute to the resultant phenotype.