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1.
Int J Mol Sci ; 20(17)2019 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-31438494

RESUMEN

Human variants in plakophilin-2 (PKP2) associate with most cases of familial arrhythmogenic cardiomyopathy (ACM). Recent studies show that PKP2 not only maintains intercellular coupling, but also regulates transcription of genes involved in Ca2+ cycling and cardiac rhythm. ACM penetrance is low and it remains uncertain, which genetic and environmental modifiers are crucial for developing the cardiomyopathy. In this study, heterozygous PKP2 knock-out mice (PKP2-Hz) were used to investigate the influence of exercise, pressure overload, and inflammation on a PKP2-related disease progression. In PKP2-Hz mice, protein levels of Ca2+-handling proteins were reduced compared to wildtype (WT). PKP2-Hz hearts exposed to voluntary exercise training showed right ventricular lateral connexin43 expression, right ventricular conduction slowing, and a higher susceptibility towards arrhythmias. Pressure overload increased levels of fibrosis in PKP2-Hz hearts, without affecting the susceptibility towards arrhythmias. Experimental autoimmune myocarditis caused more severe subepicardial fibrosis, cell death, and inflammatory infiltrates in PKP2-Hz hearts than in WT. To conclude, PKP2 haploinsufficiency in the murine heart modulates the cardiac response to environmental modifiers via different mechanisms. Exercise upon PKP2 deficiency induces a pro-arrhythmic cardiac remodeling, likely based on impaired Ca2+ cycling and electrical conduction, versus structural remodeling. Pathophysiological stimuli mainly exaggerate the fibrotic and inflammatory response.


Asunto(s)
Calcio/metabolismo , Cardiomiopatías/metabolismo , Haploinsuficiencia/fisiología , Enfermedad Autoinmune Experimental del Sistema Nervioso/etiología , Enfermedad Autoinmune Experimental del Sistema Nervioso/metabolismo , Placofilinas/metabolismo , Animales , Western Blotting , Cardiomiopatías/etiología , Cardiomiopatías/patología , Ecocardiografía , Electrocardiografía , Fibrosis/etiología , Fibrosis/metabolismo , Fibrosis/patología , Haploinsuficiencia/genética , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad Autoinmune Experimental del Sistema Nervioso/patología , Placofilinas/genética , Reacción en Cadena de la Polimerasa
2.
Cell Mol Life Sci ; 72(15): 2899-910, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26100515

RESUMEN

Connexin43 and pannexin1 are found in immune cells. While gap junctional communication has been demonstrated between immune cells, hemichannels have been implicated in many cellular functions. Among the functions involved as being connexin dependent and pannexin dependent are cell migration, phagocytosis, antigen presentation, T-cell reactivity and B-cell responses. Surprisingly, many of these connexin-related and pannexin-related functions are not recapitulated in in vivo models. This is leading to a reevaluation of the role of these proteins in immune function.


Asunto(s)
Linfocitos B/metabolismo , Conexinas/metabolismo , Sistema Inmunológico/metabolismo , Linfocitos T/metabolismo , Animales , Uniones Comunicantes/metabolismo , Humanos
3.
Comp Med ; 63(1): 29-37, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23561935

RESUMEN

BALB/c mice are predisposed to dystrophic cardiac calcinosis-the mineralization of cardiac tissues, especially the right ventricular epicardium. In previous reports, the disease appeared in aged animals and had an unknown etiology. In the current study, we report a substrain of BALB/c mice (BALB/cByJ) that develops disease early and with high frequency. Here we analyzed hearts grossly to identify the presence and measure the severity of disease and to compare BALB/c substrains. Histologic analysis and fluorescent and immunofluorescent microscopy were used to characterize the calcinotic lesions. BALB/cByJ mice exhibited more frequent and severe calcium deposition than did BALB/c mice of other substrains (90% compared with 3% at 5 wk). At this age, lesions covered an average of 30% of the total ventricular surface area in BALB/cByJ mice, compared with less than 1% in other strains. In bone-marrow-chimeric mice, green fluorescent protein was used as a marker to show that the lesions contain an infiltration of cells of bone marrow origin. Lesion histology showed that calcium deposits were surrounded by fibrosis with interspersed immune cells. Lymphocytes, macrophages, and granulocytes were all present. Internalization of the gap-junction protein connexin 43 was observed in myocytes adjacent to lesions. In conclusion, BALB/cByJ mice exhibit more frequent and severe dystrophic cardiac calcinosis than do other BALB/c substrains. Our findings suggest that immune cells are actively recruited to lesions and that myocyte gap junctions are altered near lesions.


Asunto(s)
Calcinosis/veterinaria , Cardiomiopatías/veterinaria , Ratones Endogámicos BALB C , Enfermedades de los Roedores/inmunología , Enfermedades de los Roedores/patología , Animales , Calcinosis/inmunología , Calcinosis/patología , Cardiomiopatías/inmunología , Cardiomiopatías/patología , Conexina 43/metabolismo , Uniones Comunicantes/inmunología , Uniones Comunicantes/patología , Granulocitos/inmunología , Proteínas Fluorescentes Verdes/metabolismo , Técnicas Histológicas/veterinaria , Linfocitos/inmunología , Macrófagos/inmunología , Ratones , Microscopía Fluorescente/veterinaria , Especificidad de la Especie
4.
J Immunol ; 190(9): 4830-5, 2013 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-23554311

RESUMEN

Macrophages that lack connexin43 (Cx43), a gap junction protein, have been reported to exhibit dramatic deficiencies in phagocytosis. In this study, we revisit these findings using well-characterized macrophage populations. Cx43 knockout (Cx43(-/-)) mice die soon after birth, making the harvest of macrophages from adult Cx43(-/-) mice problematic. To overcome this obstacle, we used several strategies: mice heterozygous for the deletion of Cx43 were crossed to produce Cx43(+/+) (wild type [WT]) and Cx43(-/-) fetuses. Cells isolated from 12- to 14-d fetal livers were used to reconstitute irradiated recipient animals. After reconstitution, thioglycollate-elicited macrophages were collected by peritoneal lavage and bone marrow was harvested. Bone marrow cells and, alternatively, fetal liver cells were cultured in media containing M-CSF for 7-10 d, resulting in populations of cells that were >95% macrophages based on flow cytometry. Phagocytic uptake was detected using flow cytometric and microscopic techniques. Quantification of phagocytic uptake of IgG-opsonized sheep erythrocytes, zymosan particles, and Listeria monocytogenes failed to show any significant difference between WT and Cx43(-/-) macrophages. Furthermore, the use of particles labeled with pH-sensitive dyes showed equivalent acidification of phagosomes in both WT and Cx43(-/-) macrophages. Our findings suggest that modulation of Cx43 levels in cultured macrophages does not have a significant impact on phagocytosis.


Asunto(s)
Conexina 43/inmunología , Macrófagos/inmunología , Fagocitosis/inmunología , Animales , Células de la Médula Ósea/metabolismo , Células Cultivadas , Conexina 43/genética , Conexina 43/metabolismo , Eritrocitos/inmunología , Eritrocitos/metabolismo , Femenino , Genes MHC Clase I , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/inmunología , Listeria monocytogenes/metabolismo , Hígado/metabolismo , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Fagocitosis/genética , Fagosomas/genética , Fagosomas/inmunología , Fagosomas/metabolismo , Ovinos , Zimosan/genética , Zimosan/metabolismo
5.
J Am Coll Cardiol ; 60(18): 1799-806, 2012 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-23040566

RESUMEN

OBJECTIVES: This study sought to determine if serum markers for collagen I and III synthesis, the carboxyl terminal peptide from pro-collagen I (PICP) and the amino terminal peptide from pro-collagen III (PIIINP), correlate with left atrial (LA) fibrosis and post-operative atrial fibrillation (AF). BACKGROUND: AF after cardiac surgery is associated with adverse outcomes. We recently demonstrated that LA fibrosis is associated with post-operative AF in patients with no previous history of AF. METHODS: Fifty-four patients having cardiac surgery without a history of AF consented to left and right atrial biopsies and a pre-operative peripheral blood draw. Picrosirius red staining quantified the percentage of fibrosis, and reverse transcriptase polymerase chain reaction assessed atrial tissue messenger ribonucleic acid transcripts involved in the fibrosis pathway. PICP and PIIINP levels were measured using an enzyme immunosorbent assay. RESULTS: Eighteen patients developed AF, whereas 36 remained in normal sinus rhythm. LA fibrosis was higher in patients who developed AF versus normal sinus rhythm (6.13 ± 2.9% vs. 2.03 ± 1.9%, p = 0.03). LA messenger ribonucleic acid transcripts for collagen I, III, transforming growth factor, and angiotensin were 1.5- to 2.0-fold higher in AF patients. Serum PICP and PIIINP levels were highest in AF versus normal sinus rhythm (PICP: 451.7 ± 200 ng/ml vs. 293.3 ± 114 ng/ml, p = 0.006; PIIINP: 379 ± 286 pg/ml vs. 191.6 ± 162 pg/ml, p = 0.01). Furthermore, there was a linear correlation between LA fibrosis and serum PICP levels (R(2) = 0.2; p = 0.01), and of the markers, only PICP was independently associated with AF. CONCLUSIONS: This demonstrates that serum PICP and PIIINP levels correlate with the presence of LA fibrosis and may act as predictors for post-operative AF even in the absence of previous history of AF.


Asunto(s)
Fibrilación Atrial/sangre , Fibrilación Atrial/etiología , Colágeno Tipo III/sangre , Colágeno Tipo I/sangre , Atrios Cardíacos/fisiopatología , Adulto , Anciano , Biopsia/métodos , Electrocardiografía/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/sangre , Complicaciones Posoperatorias , Procolágeno/sangre , Resultado del Tratamiento
6.
Circ Res ; 109(2): 193-201, 2011 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-21617128

RESUMEN

RATIONALE: The early description of the intercalated disc defined 3 structures, all of them involved in cell-cell communication: desmosomes, gap junctions, and adherens junctions. Current evidence demonstrates that molecules not involved in providing a physical continuum between cells also populate the intercalated disc. Key among them is the voltage-gated sodium channel complex. An important component of this complex is the cytoskeletal adaptor protein Ankyrin-G (AnkG). OBJECTIVE: To test the hypothesis that AnkG partners with desmosome and gap junction molecules and exerts a functional effect on intercellular communication in the heart. METHODS AND RESULTS: We used a combination of microscopy, immunochemistry, patch-clamp, and optical mapping to assess the interactions between AnkG, Plakophilin-2, and Connexin43. Coimmunoprecipitation studies from rat heart lysate demonstrated associations between the 3 molecules. With the use of siRNA technology, we demonstrated that loss of AnkG expression caused significant changes in subcellular distribution and/or abundance of PKP2 and Connexin43 as well as a decrease in intercellular adhesion strength and electric coupling. Regulation of AnkG and of Na(v)1.5 by Plakophilin-2 was also demonstrated. Finally, optical mapping experiments in AnkG-silenced cells demonstrated a shift in the minimal frequency at which rate-dependence activation block was observed. CONCLUSIONS: These experiments support the hypothesis that AnkG is a key functional component of the intercalated disc at the intersection of 3 complexes often considered independent: the voltage-gated sodium channel, gap junctions, and the cardiac desmosome. Possible implications to the pathophysiology of inherited arrhythmias (such as arrhythmogenic right ventricular cardiomyopathy) are discussed.


Asunto(s)
Ancirinas/metabolismo , Conexina 43/metabolismo , Corazón/fisiología , Placofilinas/metabolismo , Canales de Sodio/metabolismo , Animales , Comunicación Celular , Desmosomas , Uniones Comunicantes , Activación del Canal Iónico , Unión Proteica/fisiología , Ratas
7.
Heart Rhythm ; 7(10): 1491-8, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20601149

RESUMEN

BACKGROUND: Gap junctions are potential targets for pharmacologic intervention. We previously developed a series of peptide sequences that prevent closure of connexin43 (Cx43) channels, bind to cardiac Cx43, and prevent acidification-induced uncoupling of cardiac gap junctions. OBJECTIVE: The purpose of this study was to identify and validate the minimum core active structure in peptides containing an RR-N/Q-Y motif. Based on that information, we sought to generate a peptidomimetic molecule that acts on the chemical regulation of Cx43 channels. METHODS: Experiments were based on a combination of biochemical, spectroscopic, and electrophysiologic techniques as well as molecular modeling of active pharmacophores with Cx43 activity. RESULTS: Molecular modeling analysis indicated that the functional elements of the side chains in the motif RRXY form a triangular structure. Experimental data revealed that compounds containing such a structure bind to Cx43 and prevent Cx43 chemical gating. These results provided us with the first platform for drug design targeted to the carboxyl terminal of Cx43. Using that platform, we designed and validated a peptidomimetic compound (ZP2519; molecular weight 619 Da) that prevented octanol-induced uncoupling of Cx43 channels and pH gating of cardiac gap junctions. CONCLUSION: Structure-based drug design can be applied to the development of pharmacophores that act directly on Cx43. Small molecules containing these pharmacophores can serve as tools to determine the role of gap junction regulation in the control of cardiac rhythm. Future studies will determine whether these compounds can function as pharmacologic agents for the treatment of a selected subset of cardiac arrhythmias.


Asunto(s)
Conexina 43/metabolismo , Uniones Comunicantes/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Oligopéptidos/farmacología , Peptidomiméticos/farmacología , Animales , Proteínas Portadoras/farmacología , Células Cultivadas , Conexina 43/efectos de los fármacos , Diseño de Fármacos , Uniones Comunicantes/fisiología , Concentración de Iones de Hidrógeno , Ratones , Modelos Moleculares , Miocitos Cardíacos/fisiología , Octanoles/farmacología , Oligopéptidos/síntesis química , Técnicas de Placa-Clamp , Peptidomiméticos/síntesis química , Peptidomiméticos/química , Ratas
8.
J Biol Chem ; 285(36): 28000-9, 2010 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-20530486

RESUMEN

Synapse-associated protein-97 (SAP97) is a membrane-associated guanylate kinase scaffolding protein expressed in cardiomyocytes. SAP97 has been shown to associate and modulate voltage-gated potassium (Kv) channel function. In contrast to Kv channels, little information is available on interactions involving SAP97 and inward rectifier potassium (Kir2.x) channels that underlie the classical inward rectifier current, I(K1). To investigate the functional effects of silencing SAP97 on I(K1) in adult rat ventricular myocytes, SAP97 was silenced using an adenoviral short hairpin RNA vector. Western blot analysis showed that SAP97 was silenced by approximately 85% on day 3 post-infection. Immunostaining showed that Kir2.1 and Kir2.2 co-localize with SAP97. Co-immunoprecipitation (co-IP) results demonstrated that Kir2.x channels associate with SAP97. Voltage clamp experiments showed that silencing SAP97 reduced I(K1) whole cell density by approximately 55%. I(K1) density at -100 mV was -1.45 +/- 0.15 pA/picofarads (n = 6) in SAP97-silenced cells as compared with -3.03 +/- 0.37 pA/picofarads (n = 5) in control cells. Unitary conductance properties of I(K1) were unaffected by SAP97 silencing. The major mechanism for the reduction of I(K1) density appears to be a decrease in Kir2.x channel abundance. Furthermore, SAP97 silencing impaired I(K1) regulation by beta(1)-adrenergic receptor (beta1-AR) stimulation. In control, isoproterenol reduced I(K1) amplitude by approximately 75%, an effect that was blunted following SAP97 silencing. Our co-IP data show that beta1-AR associates with SAP97 and Kir2.1 and also that Kir2.1 co-IPs with protein kinase A and beta1-AR. SAP97 immunolocalizes with protein kinase A and beta1-AR in the cardiac myocytes. Our results suggest that in cardiac myocytes SAP97 regulates surface expression of channels underlying I(K1), as well as assembles a signaling complex involved in beta1-AR regulation of I(K1).


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Conductividad Eléctrica , Proteínas de la Membrana/metabolismo , Miocardio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Inmunoprecipitación , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Células Musculares/metabolismo , Transporte de Proteínas , Ratas , Receptores Adrenérgicos beta 1/metabolismo
9.
Heart Rhythm ; 6(11): 1632-8, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19879542

RESUMEN

BACKGROUND: Phosphorylation is a key regulatory event in controlling the function of the cardiac gap junction protein connexin43 (Cx43). Three new phosphorylation sites (S296, S297, S306) have been identified on Cx43; two of these sites (S297 and S306) are dephosphorylated during ischemia. The functional significance of these new sites is currently unknown. OBJECTIVE: The purpose of this study was to examine the role of S296, S297, and S306 in the regulation of electrical intercellular communication. METHODS: To mimic constitutive dephosphorylation, serine was mutated to alanine at the three sites and expressed in HeLa cells. Electrical coupling and single channel measurements were performed by double patch clamp. Protein expression levels were assayed by western blotting, localization of Cx43, and phosphorylation of S306 by immunolabeling. Free hemichannels were assessed by biotinylation. RESULTS: Macroscopic conductance in cells expressing S306A was reduced to 57% compared to wild type (WT), whereas coupling was not significantly changed in cells expressing either S296A or S297A. S306A-expressing cells displayed similar protein and free hemichannel abundance compared to WT Cx43, whereas the fractional area of plaques in cell-to-cell interfaces was increased. However, single channel measurements showed a WT Cx43 main state conductance of 119 pS, whereas the main state conductance of S306A channels was reduced to 95 pS. Furthermore, channel gating was affected in S306A channels. CONCLUSION: Lack of phosphorylation at serine 306 results in reduced coupling, which can be explained by reduced single channel conductance. We suggest that dephosphorylation of S306 partly explains the electrical uncoupling seen in myocardial ischemia.


Asunto(s)
Comunicación Celular/fisiología , Conexina 43/metabolismo , Serina/metabolismo , Humanos , Fosforilación
10.
Heart Rhythm ; 6(10): 1415-22, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19656731

RESUMEN

BACKGROUND: The development of atrial fibrillation (AF) after cardiac surgery is associated with adverse outcomes; however, the mechanism(s) that trigger and maintain AF in these patients are unknown. OBJECTIVE: The purpose of this study was to test our hypothesis that postoperative AF is maintained by high-frequency sources in the left atrium (LA) resulting from ion channel and structural features that differ from the right atrium (RA). METHODS: Forty-four patients with no previous history of AF who underwent cardiac surgery consented to LA and RA biopsies. Histologic sections evaluated fatty infiltration, fibrosis, and iron deposition; quantitative reverse transcription-polymerase chain reaction (RT-PCR) assessed ion channel expression. In a subset of 27 patients, LA and RA unipolar recording leads were also placed. In patients who developed AF, the dominant frequency (DF) for each lead was calculated using fast Fourier transform. RESULTS: DFs during AF were LA 6.26 +/- 0.8 Hz, RA 4.56 +/- 0.7 Hz (P <.01). RT-PCR revealed LA-to-RA differences in mRNA abundance for Kir2.3 (1.8:1) and Kir3.4 (2.3:1). While LA fibrosis was greater in patients developing AF compared with those remaining in normal sinus rhythm (10.8% +/- 11% vs. 3.8% +/- 3.5%; P = .03), the amount of LA fibrosis inversely correlated with the LA DF. CONCLUSIONS: This is the first demonstration of LA-to-RA frequency differences during postoperative AF, which are associated with LA-to-RA differences in mRNA levels for potassium channel proteins and LA fibrosis. These results strongly suggest that sources of AF after cardiac surgery are located in the LA and are stabilized by LA fibrosis.


Asunto(s)
Fibrilación Atrial/fisiopatología , Procedimientos Quirúrgicos Cardíacos , Fibrosis/patología , Atrios Cardíacos/patología , Canales de Potasio/análisis , Anciano , Electrocardiografía , Femenino , Análisis de Fourier , Atrios Cardíacos/fisiopatología , Humanos , Proteínas de Interacción con los Canales Kv/análisis , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
11.
Circ Res ; 105(6): 523-6, 2009 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-19661460

RESUMEN

RATIONALE: Plakophilin-2 (PKP2) is an essential component of the cardiac desmosome. Recent data show that it interacts with other molecules of the intercalated disc. Separate studies show preferential localization of the voltage-gated sodium channel (Na(V)1.5) to this region. OBJECTIVE: To establish the association of PKP2 with sodium channels and its role on action potential propagation. METHODS AND RESULTS: Biochemical, patch clamp, and optical mapping experiments demonstrate that PKP2 associates with Na(V)1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes. CONCLUSIONS: These results emphasize the importance of intermolecular interactions between proteins relevant to mechanical junctions, and those involved in electric synchrony. Possible relevance to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy is discussed.


Asunto(s)
Potenciales de Acción , Desmosomas/metabolismo , Miocitos Cardíacos/metabolismo , Placofilinas/metabolismo , Canales de Sodio/metabolismo , Animales , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patología , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Desmosomas/patología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Miocitos Cardíacos/patología , Canal de Sodio Activado por Voltaje NAV1.5 , Ratas , Ratas Sprague-Dawley , Disfunción Ventricular/metabolismo , Disfunción Ventricular/patología
12.
Mol Immunol ; 46(15): 2938-46, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19608277

RESUMEN

Connexin 43 (Cx43) is the predominant gap junction protein expressed in immune cells. Previous manuscripts have stated that gap junctions may play a role in antigen cross-presentation, dendritic cell maturation, T cell development, and regulatory T cell function. Many of these previous studies were performed in vitro. In vivo studies were not directly possible in adult mice because Cx43-/- mice die shortly after birth due to a cardiac malformation. To overcome these drawbacks, we have developed a mouse model that deletes Cx43 in the immune system while maintaining normal cardiac function. In our model, irradiated CD45.1+ wild-type mice were reconstituted with Cx43WT, Cx43+/-, or Cx43-/- hematopoietic fetal liver cells that were derived from CD45.2+ mice. The presence of CD45.2 allowed us to identify and track the donor cells following reconstitution. We determined that Cx43+/- and Cx43-/- hematopoietic cells were able to reconstitute irradiated mice as well as Cx43WT cells. Reconstitution was nearly 100% in the thymus and over 90% in the spleen. There appeared to be no difference in thymocyte development or in the ability of lymphocytes to transmigrate to peripheral lymphoid organs. However in response to inflammation, Cx43+/- radiation chimeras had increased peritoneal infiltration compared to Cx43WT and Cx43-/- groups. IgG responses were normal in all groups but the Cx43-/- reconstituted mice had an elevated IgM response. Our data suggests that Cx43 may not be involved in the normal development of the immune system but may regulate certain effector functions in vivo.


Asunto(s)
Movimiento Celular/inmunología , Conexina 43/metabolismo , Células Madre Hematopoyéticas/inmunología , Quimera por Radiación/inmunología , Timo/inmunología , Animales , Anticuerpos/sangre , Conexina 43/genética , Conexina 43/inmunología , Células Madre Hematopoyéticas/metabolismo , Hemocianinas/inmunología , Sistema Inmunológico , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Quimera por Radiación/metabolismo , Timo/metabolismo , Irradiación Corporal Total
13.
Circ Res ; 105(2): 176-84, 2009 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-19556520

RESUMEN

Gap junction pharmacology is a nascent field. Previous studies have identified molecules that enhance intercellular communication, and may offer potential for innovative antiarrhythmic therapy. However, their specific molecular target(s) and mechanism(s) of action remain unknown. Previously, we identified a 34-aa peptide (RXP-E) that binds the carboxyl terminal domain of Cx43 (Cx43CT) and prevents cardiac gap junction closure and action potential propagation block. These results supported the feasibility of a peptide-based pharmacology to Cx43, but the structure of the core active element in RXP-E, an essential step for pharmacological development, remained undefined. Here, we used a combination of molecular modeling, surface plasmon resonance, nuclear magnetic resonance and patch-clamp strategies to define, for the first time, a unique ensemble of pharmacophores that bind Cx43CT and prevent closure of Cx43 channels. Two particular molecules are best representatives of this family: a cyclized heptapeptide (called CyRP-71) and a linear octapeptide of sequence RRNYRRNY. These 2 small compounds offer the first structural platform for the design of Cx43-interacting gap junction openers. Moreover, the structure of these compounds offers an imprint of a region of Cx43CT that is fundamental to gap junction channel function.


Asunto(s)
Antiarrítmicos/farmacología , Conexina 43/metabolismo , Diseño de Fármacos , Uniones Comunicantes/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Potenciales de Acción , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Antiarrítmicos/química , Antiarrítmicos/metabolismo , Sitios de Unión , Línea Celular , Diseño Asistido por Computadora , Conexina 43/química , Conexina 43/genética , Uniones Comunicantes/metabolismo , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Miocitos Cardíacos/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Técnicas de Placa-Clamp , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Conformación Proteica , Ratas , Ratas Sprague-Dawley , Resonancia por Plasmón de Superficie , Factores de Tiempo , Transfección
14.
Circ Res ; 103(5): 519-26, 2008 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-18669919

RESUMEN

Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called "RXP-E" binds to the carboxyl terminal of connexin43 and prevents chemically induced uncoupling in connexin43-expressing N2a cells. Here, pull-down experiments show RXP-E binding to adult cardiac connexin43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes. Separately, RXP-E was concatenated to a cytoplasmic transduction peptide (CTP) for cytoplasmic translocation (CTP-RXP-E). The effect of RXP-E on action potential propagation was assessed by high-resolution optical mapping in monolayers of neonatal rat ventricular myocytes, containing approximately 20% of randomly distributed myofibroblasts. In contrast to control experiments, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP-RXP-E, action potential propagation was maintained, albeit at a slower velocity. Similarly, intracellular acidification (pH(i) 6.2) caused a loss of action potential propagation in control monolayers; however, propagation was maintained in CTP-RXP-E-treated cells, although at a slower rate. Patch-clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium currents, nor did it alter voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: (1) bind cardiac connexin43; (2) prevent heptanol and acidification-induced uncoupling of cardiac gap junctions; and (3) preserve action potential propagation among cardiac myocytes. RXP-E can be used to characterize the role of gap junctions in the function of multicellular systems, including the heart.


Asunto(s)
Potenciales de Acción/fisiología , Proteínas Portadoras/síntesis química , Proteínas Portadoras/farmacología , Conexina 43/metabolismo , Uniones Comunicantes/fisiología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Ácidos/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Transporte Biológico , Proteínas Portadoras/metabolismo , Células Cultivadas , Diseño de Fármacos , Heptanol/farmacología , Concentración de Iones de Hidrógeno , Miocitos Cardíacos/citología , Canal de Sodio Activado por Voltaje NAV1.5 , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/fisiología , Unión Proteica , Ratas , Canales de Sodio/fisiología
15.
Biophys J ; 95(9): 4469-80, 2008 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-18658226

RESUMEN

In pathological conditions such as ischemic cardiomyopathy and heart failure, differentiation of fibroblasts into myofibroblasts may result in myocyte-fibroblast electrical coupling via gap junctions. We hypothesized that myofibroblast proliferation and increased heterocellular coupling significantly alter two-dimensional cardiac wave propagation and reentry dynamics. Co-cultures of myocytes and myofibroblasts from neonatal rat ventricles were optically mapped using a voltage-sensitive dye during pacing and sustained reentry. The myofibroblast/myocyte ratio was changed systematically, and junctional coupling of the myofibroblasts was reduced or increased using silencing RNAi or adenoviral overexpression of Cx43, respectively. Numerical simulations in two-dimensional models were used to quantify the effects of heterocellular coupling on conduction velocity (CV) and reentry dynamics. In both simulations and experiments, reentry frequency and CV diminished with larger myofibroblast/myocyte area ratios; complexity of propagation increased, resulting in wave fractionation and reentry multiplication. The relationship between CV and coupling was biphasic: an initial decrease in CV was followed by an increase as heterocellular coupling increased. Low heterocellular coupling resulted in fragmented and wavy wavefronts; at high coupling wavefronts became smoother. Heterocellular coupling alters conduction velocity, reentry stability, and complexity of wave propagation. The results provide novel insight into the mechanisms whereby electrical myocyte-myofibroblast interactions modify wave propagation and the propensity to reentrant arrhythmias.


Asunto(s)
Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patología , Fibroblastos/metabolismo , Células Musculares/metabolismo , Miocardio/metabolismo , Miocardio/patología , Animales , Secuencia de Bases , Diferenciación Celular , Técnicas de Cocultivo , Conexina 43/genética , Conexina 43/metabolismo , Conductividad Eléctrica , Fibroblastos/citología , Expresión Génica , Silenciador del Gen , Células Musculares/citología , Ratas
16.
Heart Rhythm ; 4(9): 1196-205, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17765621

RESUMEN

BACKGROUND: Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by ventricular arrhythmias, sudden death, and fatty or fibrofatty replacement of right ventricular myocytes. Recent studies have noted an association between human ARVD/C and molecular remodeling of intercalated disc structures. However, progress has been constrained by limitations inherent to human studies. OBJECTIVE: We studied the molecular composition of the intercalated disc structure in a naturally occurring animal model of ARVD/C (Boxer dogs). METHODS: We studied hearts from 12 Boxers with confirmed ARVD/C and 2 controls. Ventricular sections from 4 animals were examined by immunofluorescent microscopy. Frozen tissue samples were used for Western blot analysis. Proteins investigated were N-cadherin, plakophilin 2, desmoplakin, plakoglobin, desmin, and connexin 43 (Cx43). RESULTS: In control dogs, all proteins tested by immunofluorescence analysis yielded intense localized signals at sites of end-to-end cell apposition. In contrast, myocardial tissues from ARVD/C-afflicted Boxers showed preservation of N-cadherin staining but loss of detectable signal for Cx43 at the intercalated disc location. Western blots indicated that the Cx43 protein was still present in the samples. Gene sequencing analysis showed no mutations in desmoplakin, plakoglobin, Cx43, or plakophilin 2. CONCLUSION: Mutation(s) responsible for ARVD/C in Boxers lead, directly or indirectly, to severe modifications of mechanical and electrical cell-cell interactions. Furthermore, significant reduction in gap junction formation may promote a substrate for malignant ventricular arrhythmias. This model may help to advance our understanding of the molecular basis, pathophysiology, and potential therapeutic approach to patients with ARVD/C.


Asunto(s)
Displasia Ventricular Derecha Arritmogénica/genética , Cadherinas/genética , Conexina 43/genética , Miocitos Cardíacos/química , Animales , Western Blotting/métodos , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Perros , Femenino , Uniones Comunicantes/química , Uniones Comunicantes/genética , Masculino , Mutación , Miocitos Cardíacos/patología , Miocitos Cardíacos/ultraestructura , Análisis de Secuencia de ADN
17.
Cell Commun Adhes ; 14(2-3): 75-84, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17668351

RESUMEN

Migration of the gap junction protein connexin 43 (Cx43) in SDS-PAGE yields 2 to 4 distinct bands, detectable in the 40-47 kDa range. Here, we show that antibodies against the carboxy-terminal domain of Cx43 recognized an additional 20-kDa product. This protein was detected in some culture cell lysates. The presence of the 20-kDa band was not prevented by the use of protease inhibitors (Complete(R) and phenylmethylsulfonyl fluoride (PMSF), 1-5 mM). The band was absent from cells treated with Cx43-specific RNAi, and from those derived from Cx43-deficient mice, indicating that this Cx43-immunoreactive protein is a product of the Cx43 gene. Treatment of CHO cells with cyclosporin A caused a reduction in the amount of full-length Cx43 and a concomitant increase in the amount of the 20-kDa band. Overall, our data show that a fraction of the Cx43-immunoreactive protein pool within a given cell may correspond to a C-terminal fragment of the protein.


Asunto(s)
Conexina 43/química , Conexina 43/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Animales , Anticuerpos/farmacología , Células CHO , Células Cultivadas , Conexina 43/genética , Conexina 43/inmunología , Cricetinae , Cricetulus , Células HeLa , Humanos , Immunoblotting , Ratones , Ratones Noqueados , Peso Molecular , Octoxinol/farmacología , Inhibidores de Proteasas/farmacología , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Ratas , Solubilidad/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos
18.
Circ Res ; 101(7): 703-11, 2007 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-17673670

RESUMEN

Desmosomes and gap junctions are distinct structural components of the cardiac intercalated disc. Here, we asked whether the presence of plakophilin (PKP)2, a component of the desmosome, is essential for the proper function and distribution of the gap junction protein connexin (Cx)43. We used RNA silencing technology to decrease the expression of PKP2 in cardiac cells (ventricular myocytes, as well as epicardium-derived cells) obtained from neonatal rat hearts. We evaluated the content, distribution, and function of Cx43 gap junctions. Our results show that loss of PKP2 expression led to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intracellular space, and a decrease in dye coupling between cells. Separate experiments showed that Cx43 and PKP2 can coexist in the same macromolecular complex. Our results support the notion of a molecular crosstalk between desmosomal and gap junction proteins. The results are discussed in the context of arrhythmogenic right ventricular cardiomyopathy, an inherited disease involving mutations in desmosomal proteins, including PKP2.


Asunto(s)
Conexina 43/biosíntesis , Regulación de la Expresión Génica/fisiología , Miocitos Cardíacos/metabolismo , Placofilinas/antagonistas & inhibidores , Placofilinas/biosíntesis , Animales , Animales Recién Nacidos , Células Cultivadas , Conexina 43/análisis , Conexina 43/genética , Miocitos Cardíacos/citología , Placofilinas/análisis , Ratas
19.
Circ Res ; 101(5): 475-83, 2007 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-17626898

RESUMEN

Previous studies have shown that the gating kinetics of the slow component of the delayed rectifier K(+) current (I(Ks)) contribute to postrepolarization refractoriness in isolated cardiomyocytes. However, the impact of such kinetics on arrhythmogenesis remains unknown. We surmised that expression of I(Ks) in rat cardiomyocyte monolayers contributes to wavebreak formation and facilitates fibrillatory conduction by promoting postrepolarization refractoriness. Optical mapping was performed in 44 rat ventricular myocyte monolayers infected with an adenovirus carrying the genomic sequences of KvLQT1 and minK (molecular correlates of I(Ks)) and 41 littermate controls infected with a GFP adenovirus. Repetitive bipolar stimulation was applied at increasing frequencies, starting at 1 Hz until loss of 1:1 capture or initiation of reentry. Action potential duration (APD) was significantly shorter in I(Ks)-infected monolayers than in controls at 1 to 3 Hz (P<0.05), whereas differences at higher pacing frequencies did not reach statistical significance. Stable rotors occurred in both groups, with significantly higher rotation frequencies, lower conduction velocities, and shorter action potentials in the I(Ks) group. Wavelengths in the latter were significantly shorter than in controls at all rotation frequencies. Wavebreaks leading to fibrillatory conduction occurred in 45% of the I(Ks) reentry episodes but in none of the controls. Moreover, the density of wavebreaks increased with time as long as a stable source sustained the fibrillatory activity. These results provide the first demonstration that I(Ks)-mediated postrepolarization refractoriness can promote wavebreak formation and fibrillatory conduction during pacing and sustained reentry and may have important implications in tachyarrhythmias.


Asunto(s)
Sistema de Conducción Cardíaco/fisiología , Canal de Potasio KCNQ1/metabolismo , Miocitos Cardíacos/fisiología , Canales de Potasio con Entrada de Voltaje/metabolismo , Función Ventricular , Potenciales de Acción/fisiología , Adenoviridae/genética , Animales , Animales Recién Nacidos , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatología , Células Cultivadas , ADN Complementario/genética , Electrofisiología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/virología , Canal de Potasio KCNQ1/genética , Miocitos Cardíacos/citología , Miocitos Cardíacos/virología , Canales de Potasio con Entrada de Voltaje/genética , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta/fisiología
20.
Heart Rhythm ; 4(4): 487-96, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17399639

RESUMEN

BACKGROUND: Data on pH regulation of the cardiac potassium current I(K1) suggest species-dependent differences in the molecular composition of the underlying Kir2 channel proteins. OBJECTIVE: The purpose of this study was to test the hypothesis that the presence of the Kir2.3 isoform in heterotetrameric channels modifies channel sensitivity to pH. METHODS: Voltage clamp was performed on HEK293 cells stably expressing guinea pig Kir2.1 and/or Kir2.3 isoforms and on sheep cardiac ventricular myocytes at varying extracellular pH (pH(o)) and in the presence of CO(2) to determine the sensitivity of macroscopic currents to pH. Single-channel activity was recorded from the HEK293 stables to determine the mechanisms of the changes in whole cell current. RESULTS: Biophysical characteristics of whole-cell and single-channel currents in Kir2.1/Kir2.3 double stables displayed properties attributable to isoform heteromerization. Whole-cell Kir2.1/Kir2.3 currents rectified in a manner reminiscent of Kir2.1 but were significantly inhibited by extracellular acidification in the physiologic range (pK(a) approximately 7.4). Whole-cell currents were more sensitive to a combined extracellular and intracellular acidification produced by CO(2). At pH(o) = 6.0, unitary conductances of heteromeric channels were reduced. Ovine cardiac ventricular cell I(K1) was pH(o) and CO(2) sensitive, consistent with the expression of Kir2.1 and Kir2.3 in this species. CONCLUSION: Kir2.1 and Kir2.3 isoforms form heteromeric channels in HEK293. The presence of Kir2.3 subunit(s) in heteromeric channels confers pH sensitivity to the channels. The single and double stable cells presented in this study are useful models for studying physiologic regulation of heteromeric Kir2 channels in mammalian cells.


Asunto(s)
Miocitos Cardíacos/fisiología , Canales de Potasio de Rectificación Interna/fisiología , Análisis de Varianza , Animales , Western Blotting , Línea Celular , Electrofisiología , Cobayas , Ventrículos Cardíacos/citología , Concentración de Iones de Hidrógeno , Modelos Animales , Oocitos/fisiología , Técnicas de Placa-Clamp , Isoformas de Proteínas , Proyectos de Investigación , Ovinos , Xenopus
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