RESUMEN
Cardiac fibroblasts play a critical role in extracellular matrix homeostasis, wound healing, and cardiac interstitial fibrosis: the latter being a pathophysiological response to a chronic increase in afterload. Using a standard protocol to isolate cardiac fibroblasts and maintain them in their quiescent phenotype in vitro will enable a better understanding of cardiac fibroblast biology and their role in the response to profibrotic stimuli. Here, we describe an enzymatic method for isolating cardiac fibroblasts. The resulting cells are maintained on either a collagen-coated hydrogel-bound polystyrene (compliant) substrate or standard polystyrene culture dishes (non-compliant) to obtain quiescent fibroblasts and activated fibroblasts (myofibroblasts), respectively. Fibroblasts maintained on a non-compliant substrate developed a myofibroblast phenotype, in which the αSMA immunoreactivity was markedly elevated and incorporated into the stress fibers. In contrast, ventricular and atrial fibroblasts retain their quiescent phenotype for up to 3 passages when maintained on a compliant substrate. Hence, the methodology described herein provides a simple and reproducible way to isolate adult murine atrial and ventricular cardiac fibroblasts from a single animal and, by selecting a substrate with the appropriate compliance, examine the mediators of fibroblast activation or inactivation.
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Miofibroblastos , Poliestirenos , Animales , Diferenciación Celular , Fibroblastos , Corazón , Ventrículos Cardíacos , Ratones , MiocardioRESUMEN
The role of different G protein-coupled receptors (GPCRs) in the cardiovascular system is well understood in cardiomyocytes and vascular smooth muscle cells (VSMCs). In the former, stimulation of Gs-coupled receptors leads to increases in contractility, whereas stimulation of Gq-coupled receptors modulates cellular survival and hypertrophic responses. In VSMCs, stimulation of GPCRs also modulates contractile and cell growth phenotypes. Here, we will focus on the relatively less well-studied effects of GPCRs in cardiac fibroblasts, focusing on key signaling events involved in the activation and differentiation of these cells. We also review the hierarchy of signaling events driving the fibrotic response and the communications between fibroblasts and other cells in the heart. We discuss how such events may be distinct depending on where the GPCRs and their associated signaling machinery are localized in these cells with an emphasis on nuclear membrane-localized receptors. Finally, we explore what such connections between the cell surface and nuclear GPCR signaling might mean for cardiac fibrosis.
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Fibroblastos , Receptores Acoplados a Proteínas G , Núcleo Celular/metabolismo , Fibroblastos/metabolismo , Miocitos Cardíacos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp1, Leu2]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and, to a lesser extent, IP1 production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hormonas Peptídicas/metabolismo , Péptidos/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Regulación Alostérica , Proliferación Celular , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Ligandos , Hormonas Peptídicas/química , Hormonas Peptídicas/genética , Péptidos/química , Conformación Proteica en Hélice alfa , Receptores Acoplados a Proteínas G/genética , Transducción de SeñalRESUMEN
Cardiomyocyte migration represents a requisite event of cardiogenesis and the regenerative response of the injured adult zebrafish and neonatal rodent heart. The present study tested the hypothesis that the appearance of the intermediate filament protein nestin in neonatal rat ventricular cardiomyocytes (NNVMs) was associated in part with the acquisition of a migratory phenotype. The cotreatment of NNVMs with phorbol 12,13-dibutyrate (PDBu) and the p38α/ß mitogen-activated protein kinase inhibitor SB203580 led to the de novo synthesis of nestin. The intermediate filament protein was subsequently reorganized into a filamentous pattern and redistributed to the leading edge of elongated membrane protrusions translating to significant lengthening of NNVMs. PDBu/SB203580 treatment concomitantly promoted the reorganization of nonmuscle myosin IIB (NMIIB) located predominantly at the periphery of the plasma membrane of NNVMs to a filamentous phenotype extending to the leading edge of elongated membrane protrusions. Coimmunoprecipitation assay revealed a physical interaction between NMIIB and nestin after PDBu/SB203580 treatment of NNVMs. In wild-type and heterozygous NMIIB embryonic hearts at E11.5-E14.5 days, nestin immunoreactivity was identified in a subpopulation of cardiomyocytes elongating perpendicular to the compact myocardium, at the leading edge of nascent trabeculae and during thickening of the compact myocardium. In mutant embryonic hearts lacking NMIIB protein expression, trabeculae formation was reduced, the compact myocardium significantly thinner and nestin immunoreactivity undetectable in cardiomyocytes at E14.5 days. These data suggest that NMIIB and nestin may act in a coordinated fashion to facilitate the acquisition of a migratory phenotype in neonatal and embryonic cardiomyocytes.
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Corazón/crecimiento & desarrollo , Proteína Quinasa 14 Activada por Mitógenos/genética , Nestina/biosíntesis , Miosina Tipo IIB no Muscular/genética , Organogénesis/genética , Animales , Animales Recién Nacidos/genética , Animales Recién Nacidos/crecimiento & desarrollo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/genética , Regulación del Desarrollo de la Expresión Génica/genética , Corazón/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/crecimiento & desarrollo , Humanos , Imidazoles/farmacología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Nestina/genética , Forbol 12,13-Dibutirato/farmacología , Piridinas/farmacología , Ratas , Pez Cebra/genéticaRESUMEN
MK5 is a protein serine/threonine kinase activated by p38 MAPK and the atypical MAPKs ERK3 and ERK4. Although little is known of the physiological role of MK5 in the heart, both hypertrophic growth and the increase in collagen 1-α1 mRNA induced by increased afterload are attenuated in hearts of MK5 haploinsufficient (MK5+/-) mice. MK5 transcripts are detected at high levels in the left ventricular myocardium; however, MK5 immunoreactivity is detected in adult cardiac fibroblasts, but not myocytes. The present study was to determine if MK5 has a potential role in remodeling of the extracellular matrix. Ventricular fibroblasts were isolated from MK5+/+, MK5+/-, or MK5-/- mice and maintained in culture on either compliant (8â¯kPa) or rigid substrates to obtain quiescent fibroblasts or activated myofibroblasts, respectively. In quiescent fibroblasts, reduced MK5 had little effect: BMP7 and TGF-ß1 mRNA was increased in MK5+/- and MK5-/-.cells, respectively. Ang-II altered the abundance of numerous transcripts in an MK5-sensitive manner. Both collagen 1-α1 mRNA and secreted type 1 collagen immunoreactivity were increased by Ang-II in wild type but not MK5-deficient fibroblasts. The effects of deleting MK5 were quite different in myofibroblasts: both the abundance of collagen 1-α1 mRNA and secreted type 1 collagen immunoreactivity elevated in the absence of added Ang-II and addition of Ang-II failed to evoke a further increase in either. In addition, whereas type I collagen immunoreactivity was distributed throughout the cytosol of wild-type myofibroblasts, it was perinuclear in MK5-/- myofibroblasts. Furthermore, in MK5-deficient myofibroblasts the abundance of collagen 3-α2, Timp3, Smad 6, Smad 7, TGF-ß3, and snail homolog 1 transcripts was increased whereas integrin ß3, latent TGF-ß binding protein 1, thrombospondin 1, hepatocyte growth factor, and interleukin 13 were decreased. Finally, fibroblast contraction was decreased upon knocking down MK5. These results indicate that MK5 may be involved in fibroblast-mediated regulation of extracellular matrix homeostasis.
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Colágeno/metabolismo , Proteínas de la Matriz Extracelular/genética , Fibronectinas/metabolismo , Ventrículos Cardíacos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Miofibroblastos/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Transcriptoma , Animales , Proteínas de la Matriz Extracelular/metabolismo , Ventrículos Cardíacos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miofibroblastos/citologíaRESUMEN
MK5 is a protein serine/threonine kinase activated by p38, ERK3, and ERK4 MAPKs. MK5 mRNA and immunoreactivity are detected in mouse cardiac fibroblasts, and MK5 haplodeficiency attenuates the increase in collagen 1-α1 mRNA evoked by pressure overload. The present study examined the effect of MK5 haplodeficiency on reparative fibrosis following myocardial infarction (MI). Twelve-week-old MK5+/- and wild-type littermate (MK5+/+) mice underwent ligation of the left anterior descending coronary artery (LADL). Surviving mice were euthanized 8 or 21 days post-MI. Survival rates did not differ significantly between MK5+/+ and MK5+/- mice, with rupture of the LV wall being the primary cause of death. Echocardiographic imaging revealed similar increases in LV end-diastolic diameter, myocardial performance index, and wall motion score index in LADL-MK5+/+ and LADL-MK5+/- mice. Area at risk did not differ between LADL-MK5+/+ and LADL-MK5+/- hearts. In contrast, infarct size, scar area, and scar collagen content were reduced in LADL-MK5+/- hearts. Immunohistochemical analysis of mice experiencing heart rupture revealed increased MMP-9 immunoreactivity in the infarct border zone of LADL-MK5+/- hearts compared with LADL-MK5+/+. Although inflammatory cell infiltration was similar in LADL-MK5+/+ and LADL-MK5+/- hearts, angiogenesis was more pronounced in the infarct border zone of LADL-MK5+/- mice. Characterization of ventricular fibroblasts revealed reduced motility and proliferation in fibroblasts isolated from MK5-/- mice compared with those from both wild-type and haplodeficient mice. siRNA-mediated knockdown of MK5 in fibroblasts from wild-type mice also impaired motility. Hence, reduced MK5 expression alters fibroblast function and scar morphology but not mortality post-MI. NEW & NOTEWORTHY MK5/PRAK is a protein serine/threonine kinase activated by p38 MAPK and/or atypical MAPKs ERK3/4. MK5 haplodeficiency reduced infarct size, scar area, and scar collagen content post-myocardial infarction. Motility and proliferation were reduced in cultured MK5-null cardiac myofibroblasts.
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Cicatriz/enzimología , Colágeno/metabolismo , Haploinsuficiencia , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Infarto del Miocardio/enzimología , Miocardio/enzimología , Miofibroblastos/enzimología , Proteínas Serina-Treonina Quinasas/deficiencia , Cicatrización de Heridas , Animales , Movimiento Celular , Proliferación Celular , Células Cultivadas , Cicatriz/patología , Cicatriz/fisiopatología , Modelos Animales de Enfermedad , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/patología , Miofibroblastos/patología , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Función Ventricular Izquierda , Remodelación VentricularRESUMEN
Follicle growth and granulosa cell health are dependent on the secretion of estradiol from granulosa cells. Estradiol is synthesized from androgen precursor by cytochrome P450 aromatase (CYP19A1), and in cattle CYP19A1 messenger RNA has a short half-life but a long (3.5 kb) 3'-untranslated region (3'UTR), suggesting that posttranscriptional regulation may be important for control of enzyme activity. We tested this hypothesis by inserting the CYP19A1 3'UTR and fragments thereof into a reporter vector between the end of the luciferase coding region and the polyadenylation signal. The full-length aromatase 3'UTR suppressed luciferase activity to 10% of control levels, and smaller fragments showed that this inhibitory activity lies between +926 and +1134 of the 3'UTR. Protein-RNA cross-linking experiments revealed that these 3'UTR fragments formed an RNA-protein complex of approximately 70 kDa that was present in granulosa cells but not in corpus luteum, lung, liver, kidney, pancreas, or bladder extracts. The RNA-binding activity was specific to the 3'UTR, as shown by competition experiments with unlabeled RNA, and was present only in 3'UTR constructs that inhibited luciferase activity. These data suggest that posttranscriptional regulation is an important component of the control of CYP19A1 expression and involves protein binding to a specific sequence in the 3'UTR.
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Regiones no Traducidas 3' , Aromatasa/biosíntesis , Células de la Granulosa/metabolismo , Complejos Multiproteicos/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/metabolismo , Animales , Bovinos , Femenino , Células de la Granulosa/citologíaRESUMEN
Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming CaVα1, CaVß, and CaVα2δ1 subunits. The full complement of subunits is required to reconstitute the native-like properties of L-type Ca2+ currents, but the molecular determinants responsible for the formation of the heteromeric complex are still being studied. Enzymatic treatment with phosphatidylinositol-specific phospholipase C, a phospholipase C specific for the cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, disrupted plasma membrane localization of the cardiac CaVα2δ1 prompting us to investigate deletions of its hydrophobic transmembrane domain. Patch-clamp experiments indicated that the C-terminally cleaved CaVα2δ1 proteins up-regulate CaV1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (CaVα2δ1 Δ1059-1063) impaired current up-regulation. CaVα2δ1 mutants G1060I and G1061I nearly eliminated the cell-surface fluorescence of CaVα2δ1, indicated by two-color flow cytometry assays and confocal imaging, and prevented CaVα2δ1-mediated increase in peak current density and modulation of the voltage-dependent gating of CaV1.2. These impacts were specific to substitutions with isoleucine residues because functional modulation was partially preserved in CaVα2δ1 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured immunoblots of CaVα2δ1 G1060I and CaVα2δ1 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, CaVα2δ1 Δ1059-1063, but not CaVα2δ1 G1060A, failed to co-immunoprecipitate with CaV1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the CaVα2δ1 subunit at the predicted membrane interface and further suggest that preventing GPI anchoring of CaVα2δ1 averts its cell-surface expression, its interaction with CaVα1, and modulation of CaV1.2 currents.
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Canales de Calcio Tipo L/metabolismo , Activación del Canal Iónico/fisiología , Miocardio/metabolismo , Sustitución de Aminoácidos , Animales , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/genética , Línea Celular , Humanos , Mutación Missense , Dominios Proteicos , ConejosRESUMEN
There is significant evidence that internal pools of G protein-coupled receptors (GPCRs) exist and may be affected by both endogenous signaling molecules and hydrophobic pharmaceutical ligands, once assumed to only affect cell surface versions of these receptors. Here, we discuss evidence that the biology of nuclear GPCRs in particular is complex, rich, and highly interactive with GPCR signaling from the cell surface. Caging existing GPCR ligands may be an excellent means of further stratifying the phenotypic effects of known pharmacophores such as ß-adrenergic, angiotensin II, and type B endothelin receptor ligands in the cardiovascular system. We describe some synthetic strategies we have used to design ligands to go from in cellulo to in vivo experiments. We also consider how surface and intracellular GPCR signaling might be integrated and ways to dissect this. If they could be selectively targeted, nuclear GPCRs and their associated nucleoligands would represent a completely novel area for exploration by Pharma.
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Fármacos Cardiovasculares/farmacología , Enfermedades Cardiovasculares/tratamiento farmacológico , Sistema Cardiovascular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Diseño de Fármacos , Reposicionamiento de Medicamentos/métodos , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores Acoplados a Proteínas G/efectos de los fármacos , Animales , Fármacos Cardiovasculares/síntesis química , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/fisiopatología , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/fisiopatología , Núcleo Celular/genética , Humanos , Ligandos , Estructura Molecular , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
MAPK-activated protein kinase-5 (MK5) is a protein serine/threonine kinase that is activated by p38 MAPK and the atypical MAPKs ERK3 and ERK4. The physiological function(s) of MK5 remains unknown. Here, we examined the effect of MK5 haplodeficiency on cardiac function and myocardial remodeling. At 12 wk of age, MK5 haplodeficient mice (MK5+/-) were smaller than age-matched wild-type littermates (MK5+/+), with similar diastolic function but reduced systolic function. Transverse aortic constriction (TAC) was used to induce chronic pressure overload in 12-wk-old male MK5+/- and MK5+/+ mice. Two weeks post-TAC, heart weight-to-tibia length ratios were similarly increased in MK5+/- and MK5+/+ hearts, as was the abundance of B-type natriuretic peptide and ß-myosin heavy chain mRNA. Left ventricular ejection fraction was reduced in both MK5+/+ and MK5+/- mice, whereas regional peak systolic tissue velocities were reduced and isovolumetric relaxation time was prolonged in MK5+/+ hearts but not in MK5+/- hearts. The TAC-induced increase in collagen type 1-α1 mRNA observed in MK5+/+ hearts was markedly attenuated in MK5+/- hearts. Eight weeks post-TAC, systolic function was equally impaired in MK5+/+ and MK5+/- mice. In contrast, the increase in E wave deceleration rate and progression of hypertrophy observed in TAC MK5+/+ mice were attenuated in TAC MK5+/- mice. MK5 immunoreactivity was detected in adult fibroblasts but not in myocytes. MK5+/+, MK5+/-, and MK5-/- fibroblasts all expressed α-smooth muscle actin in culture. Hence, reduced MK5 expression in cardiac fibroblasts was associated with the attenuation of both hypertrophy and development of a restrictive filling pattern during myocardial remodeling in response to chronic pressure overload.NEW & NOTEWORTHY MAPK-activated protein kinase-5 (MK5)/p38-regulated/activated protein kinase is a protein serine/threonine kinase activated by p38 MAPK and/or the atypical MAPKs ERK3 and ERK4. MK5 immunoreactivity was detected in adult ventricular fibroblasts but not in myocytes. MK5 haplodeficiency attenuated the progression of hypertrophy, reduced collagen type 1 mRNA, and protected diastolic function in response to chronic pressure overload.
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Hipertrofia Ventricular Izquierda/fisiopatología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Disfunción Ventricular Izquierda/fisiopatología , Remodelación Ventricular/fisiología , Animales , Haplotipos/genética , Hipertrofia Ventricular Izquierda/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Miocárdica , Volumen Sistólico , Disfunción Ventricular Izquierda/complicacionesRESUMEN
MAP kinase-activated protein kinases (MKs), protein serine/threonine kinases downstream of the MAPKs, regulate a number of biological functions. MK5 was initially identified as a substrate for p38 MAPK but subsequent studies revealed that MK5 activity is regulated by atypical MAPKs ERK3 and ERK4. However, the roles of these MAPKs in activating MK5 remain controversial. The interactome and physiological function of MK5 are just beginning to be understood. Here, we provide an overview of the structure-function of MK5 including recent progress in determining its role in cardiac structure and function. The cardiac phenotype of MK5 haplodeficient mice, and the effect of reduced MK5 expression on cardiac remodeling, is also discussed. © 2017 IUBMB Life, 69(10):785-794, 2017.
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Fibroblastos/enzimología , Ventrículos Cardíacos/enzimología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína Quinasa 6 Activada por Mitógenos/genética , Miocardio/enzimología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Animales , Fibroblastos/citología , Regulación de la Expresión Génica , Ventrículos Cardíacos/citología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Proteína Quinasa 6 Activada por Mitógenos/metabolismo , Miocardio/citología , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/metabolismo , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Remodelación Ventricular/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
In addition to cell surface membranes, numerous G protein-coupled receptors (GPCRs) are located on intracellular membranes including the nuclear envelope. Although the role of numerous GPCRs at the cell surface has been well characterized, the physiological function of these same receptors located on intracellular membranes remains to be determined. Here, we employ a novel caged Ang-II analog, cAng-II, to compare the effects of the activation of cell surface versus intracellular angiotensin receptors in intact cardiomyocytes. When added extracellularly to HEK 293 cells, Ang-II and photolysed cAng-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R). In contrast unphotolysed cAng-II did not. Cellular uptake of cAng-II was 6-fold greater than that of Ang-II and comparable to the HIV TAT(48-60) peptide. Intracellular photolysis of cAng-II induced an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n) that was greater than that induced by extracellular application of Ang-II. We conclude that cell-permeable ligands that can access intracellular GPCRs may evoke responses distinct from those with access restricted to the same receptor located on the cell surface.
Asunto(s)
Membranas Intracelulares/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Perros , Células HEK293 , Humanos , Membranas Intracelulares/efectos de los fármacos , Ligandos , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
First characterized in neuronal tissues, the multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) is a key signaling component in several mammalian biological systems. Its unique capacity to integrate various Ca(2+) signals into different specific outcomes is a precious asset to excitable and nonexcitable cells. Numerous studies have reported roles and mechanisms involving CaMKII in brain and heart tissues. However, corresponding functions in vascular cell types (endothelium and vascular smooth muscle cells) remained largely unexplored until recently. Investigation of the intracellular Ca(2+) dynamics, their impact on vascular cell function, the regulatory processes involved and more recently the spatially restricted oscillatory Ca(2+) signals and microdomains triggered significant interest towards proteins like CaMKII. Heteromultimerization of CaMKII isoforms (four isoforms and several splice variants) expands this kinase's peculiar capacity to decipher Ca(2+) signals and initiate specific signaling processes, and thus controlling cellular functions. The physiological functions that rely on CaMKII are unsurprisingly diverse, ranging from regulating contractile state and cellular proliferation to Ca(2+) homeostasis and cellular permeability. This review will focus on emerging evidence of CaMKII as an essential component of the vascular system, with a focus on the kinase isoform/splice variants and cellular system studied.
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Arterias/metabolismo , Encéfalo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Corazón/fisiología , Animales , Calcio/metabolismo , Proliferación Celular/fisiología , Homeostasis/fisiología , Humanos , Isoformas de Proteínas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
KEY POINTS: The renin-angiotensin system plays a key role in cardiovascular physiology and its overactivation has been implicated in the pathogenesis of several major cardiovascular diseases. There is growing evidence that angiotensin II (Ang-II) may function as an intracellular peptide to activate intracellular/nuclear receptors and their downstream signalling effectors independently of cell surface receptors. Current methods used to study intracrine Ang-II signalling are limited to indirect approaches because of a lack of selective intracellularly-acting probes. Here, we present novel photoreleasable Ang-II analogues used to probe intracellular actions with spatial and temporal precision. The photorelease of intracellular Ang-II causes nuclear and cytosolic calcium mobilization and initiates the de novo synthesis of RNA in cardiac cells, demonstrating the application of the method. ABSTRACT: Several lines of evidence suggest that intracellular angiotensin II (Ang-II) contributes to the regulation of cardiac contractility, renal salt reabsorption, vascular tone and metabolism; however, work on intracrine Ang-II signalling has been limited to indirect approaches because of a lack of selective intracellularly-acting probes. Here, we aimed to synthesize and characterize cell-permeant Ang-II analogues that are inactive without uncaging, but release active Ang-II upon exposure to a flash of UV-light, and act as novel tools for use in the study of intracrine Ang-II physiology. We prepared three novel caged Ang-II analogues, [Tyr(DMNB)(4)]Ang-II, Ang-II-ODMNB and [Tyr(DMNB)(4)]Ang-II-ODMNB, based upon the incorporation of the photolabile moiety 4,5-dimethoxy-2-nitrobenzyl (DMNB). Compared to Ang-II, the caged Ang-II analogues showed 2-3 orders of magnitude reduced affinity toward both angiotensin type-1 (AT1R) and type-2 (AT2R) receptors in competition binding assays, and greatly-reduced potency in contraction assays of rat thoracic aorta. After receiving UV-irradiation, all three caged Ang-II analogues released Ang-II and potently induced the contraction of rat thoracic aorta. [Tyr(DMNB)(4)]Ang-II showed the most rapid photolysis upon UV-irradiation and was the focus of subsequent characterization. Whereas Ang-II and photolysed [Tyr(DMNB)(4)]Ang-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R), caged [Tyr(DMNB)(4)]Ang-II did not. Cellular uptake of [Tyr(DMNB)(4)]Ang-II was 4-fold greater than that of Ang-II and significantly greater than uptake driven by the positive-control HIV TAT(48-60) peptide. Intracellular photolysis of [Tyr(DMNB)(4)]Ang-II induced an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n), and initiated 18S rRNA and nuclear factor kappa B mRNA synthesis in adult cardiac cells. We conclude that caged Ang-II analogues represent powerful new tools for use in the selective study of intracrine signalling via Ang-II.
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Angiotensina II/análogos & derivados , Señalización del Calcio , Receptores de Angiotensina/metabolismo , Rayos Ultravioleta , Antagonistas de Receptores de Angiotensina/farmacología , Animales , Fluoresceínas/efectos de la radiación , Colorantes Fluorescentes/efectos de la radiación , Células HEK293 , Humanos , Masculino , Microscopía Fluorescente/métodos , Ratas , Ratas Sprague-Dawley , Receptores de Angiotensina/agonistasRESUMEN
G protein-coupled receptors (GPCRs) play key physiological roles and represent a significant target for drug development. However, historically, drugs were developed with the understanding that GPCRs as a therapeutic target exist solely on cell surface membranes. More recently, GPCRs have been detected on intracellular membranes, including the nuclear membrane, and the concept that intracellular GPCRs are functional is become more widely accepted. Nuclear GPCRs couple to effectors and regulate signaling pathways, analogous to their counterparts at the cell surface, but may serve distinct biological roles. Hence, the physiological responses mediated by GPCR ligands, or pharmacological agents, result from the integration of their actions at extracellular and intracellular receptors. The net effect depends on the ability of a given ligand or drug to access intracellular receptors, as dictated by its structure, lipophilic properties, and affinity for nuclear receptors. This review will discuss angiotensin II, endothelin, and ß-adrenergic receptors located on the nuclear envelope in cardiac cells in terms of their origin, activation, and role in cardiovascular function and pathology.
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Membrana Nuclear , Receptores Adrenérgicos/metabolismo , Receptores de Endotelina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Fármacos Cardiovasculares/metabolismo , Fármacos Cardiovasculares/farmacología , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patología , Sistema Cardiovascular/fisiopatología , Descubrimiento de Drogas/métodos , Humanos , Ligandos , Miocitos Cardíacos/fisiología , Membrana Nuclear/efectos de los fármacos , Membrana Nuclear/fisiología , Receptores de Angiotensina/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
G protein-coupled receptors (GPCRs) have historically been associated with signalling events driven from the plasma membrane. More recently, signalling from endosomes has been recognized as a feature of internalizing receptors. However, there was little consideration given to the notion that GPCRs can be targeted to distinct subcellular locations that did not involve an initial trafficking to the cell surface. Here, we focus on the evidence for and the potential impact of GPCR signalling specifically initiated from the nuclear membrane. We also discuss the possibilities for selectively targeting this and other internal pools of receptors as novel venues for drug discovery.
RESUMEN
ERK3/MAPK6 activates MAP kinase-activated protein kinase (MK)-5 in selected cell types. Male MK5 haplodeficient mice show reduced hypertrophy and attenuated increase in Col1a1 mRNA in response to increased cardiac afterload. In addition, MK5 deficiency impairs cardiac fibroblast function. This study determined the effect of reduced ERK3 on cardiac hypertrophy following transverse aortic constriction (TAC) and fibroblast biology in male mice. Three weeks post-surgery, ERK3, but not ERK4 or p38α, co-immunoprecipitated with MK5 from both sham and TAC heart lysates. The increase in left ventricular mass and myocyte diameter was lower in TAC-ERK3+/- than TAC-ERK3+/+ hearts, whereas ERK3 haploinsufficiency did not alter systolic or diastolic function. Furthermore, the TAC-induced increase in Col1a1 mRNA abundance was diminished in ERK3+/- hearts. ERK3 immunoreactivity was detected in atrial and ventricular fibroblasts but not myocytes. In both quiescent fibroblasts and "activated" myofibroblasts isolated from adult mouse heart, siRNA-mediated knockdown of ERK3 reduced the TGF-ß-induced increase in Col1a1 mRNA. In addition, intracellular type 1 collagen immunoreactivity was reduced following ERK3 depletion in quiescent fibroblasts but not myofibroblasts. Finally, knocking down ERK3 impaired motility in both atrial and ventricular myofibroblasts. These results suggest that ERK3 plays an important role in multiple aspects of cardiac fibroblast biology.
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Fibroblastos , Animales , Masculino , Ratones , Fibroblastos/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Miocardio/metabolismo , Miocardio/citología , Proteína Quinasa 6 Activada por Mitógenos/metabolismo , Proteína Quinasa 6 Activada por Mitógenos/genética , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Células Cultivadas , Cardiomegalia/metabolismo , Cardiomegalia/patología , Cardiomegalia/genética , Miocitos Cardíacos/metabolismoRESUMEN
Glutamine, the most abundant amino acid in plasma, has attracted considerable interest for its cardioprotective properties. The primary effect of glutamine in the heart is commonly believed to be mediated via its anaplerotic metabolism to citric acid cycle (CAC) intermediates; however, there is little direct evidence to support this concept. Another potential candidate is the hexosamine biosynthetic pathway (HBP), which has recently been shown to modulate cardiomyocyte function and metabolism. Therefore, the goal of this study was to evaluate the contribution of anaplerosis and the HBP to the acute metabolic effects of glutamine in the heart. Normoxic ex vivo working rat hearts were perfused with (13)C-labeled substrates to assess relevant metabolic fluxes either with a physiological mixture of carbohydrates and a fatty acid (control) or under conditions of restricted pyruvate anaplerosis. Addition of a physiological concentration of glutamine (0.5mM) had no effect on contractile function of hearts perfused under the control condition, but improved that of hearts perfused under restricted pyruvate anaplerosis. Changes in CAC intermediate concentrations as well as (13)C-enrichment from [U-(13)C]glutamine did not support a major role of glutamine anaplerosis under any conditions. Under the control condition, however, glutamine significantly increased the contribution of exogenous oleate to ß-oxidation, 1.6-fold, and triglyceride formation, 2.8-fold. Glutamine had no effect on malonyl-CoA or AMP kinase activity levels; however, it resulted in a higher plasma membrane level of the fatty acid transporter CD36. These metabolic effects of glutamine were reversed by azaserine, which inhibits glucose entry into the HPB. Our results reveal a metabolic role of physiological concentration of glutamine in the healthy working heart beyond anaplerosis. This role appears to involve the HBP and regulation of fatty acid entry and metabolism via CD36. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".
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Glutamina/metabolismo , Corazón/fisiología , Miocardio/metabolismo , Animales , Vías Biosintéticas , Metabolismo Energético , Ácidos Grasos/metabolismo , Glutamina/farmacología , Corazón/efectos de los fármacos , Hexosaminas/biosíntesis , Técnicas In Vitro , Masculino , Oxidación-Reducción , Ácido Pirúvico/metabolismo , RatasRESUMEN
Endothelin receptors are present on the nuclear membranes in adult cardiac ventricular myocytes. The objectives of the present study were to determine 1) which endothelin receptor subtype is in cardiac nuclear membranes, 2) if the receptor and ligand traffic from the cell surface to the nucleus, and 3) the effect of increased intracellular ET-1 on nuclear Ca(2+) signaling. Confocal microscopy using fluorescently-labeled endothelin analogs confirmed the presence of ETB at the nuclear membrane of rat cardiomyocytes in skinned-cells and isolated nuclei. Furthermore, in both cardiac myocytes and aortic endothelial cells, endocytosed ET:ETB complexes translocated to lysosomes and not the nuclear envelope. Although ETA and ETB can form heterodimers, the presence or absence of ETA did not alter ETB trafficking. Treatment of isolated nuclei with peptide: N-glycosidase F did not alter the electrophoretic mobility of ETB. The absence of N-glycosylation further indicates that these receptors did not originate at the cell surface. Intracellular photolysis of a caged ET-1 analog ([Trp-ODMNB(21)]ET-1) evoked an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n) that was attenuated by inositol 1,4,5-trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and prevented by pre-treatment with ryanodine. A caged cell-permeable analog of the ETB-selective antagonist IRL-2500 blocked the ability of intracellular cET-1 to increase [Ca(2+)]n whereas extracellular application of ETA and ETB receptor antagonists did not. These data suggest that 1) the endothelin receptor in the cardiac nuclear membranes is ETB, 2) ETB traffics directly to the nuclear membrane after biosynthesis, 3) exogenous endothelins are not ligands for ETB on nuclear membranes, and 4) ETB associated with the nuclear membranes regulates nuclear Ca(2+) signaling.
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Calcio/metabolismo , Endotelinas/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Aorta/citología , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunoprecipitación , Microscopía Confocal , Miocitos Cardíacos/efectos de los fármacos , Membrana Nuclear/metabolismo , Ratas , Receptores de Endotelina/metabolismo , Rianodina/farmacologíaRESUMEN
At the cell surface, ßARs and endothelin receptors can regulate nitric oxide (NO) production. ß-adrenergic receptors (ßARs) and type B endothelin receptors (ETB) are present in cardiac nuclear membranes and regulate transcription. The present study investigated the role of the NO pathway in the regulation of gene transcription by these nuclear G protein-coupled receptors. Nitric oxide production and transcription initiation were measured in nuclei isolated from the adult rat heart. The cell-permeable fluorescent dye 4,5-diaminofluorescein diacetate (DAF2 DA) was used to provide a direct assessment of nitric oxide release. Both isoproterenol and endothelin increased NO production in isolated nuclei. Furthermore, a ß3AR-selective agonist, BRL 37344, increased NO synthesis whereas the ß1AR-selective agonist xamoterol did not. Isoproterenol increased, whereas ET-1 reduced, de novo transcription. The NO synthase inhibitor l-NAME prevented isoproterenol from increasing either NO production or de novo transcription. l-NAME also blocked ET-1-induced NO-production but did not alter the suppression of transcription initiation by ET-1. Inhibition of the cGMP-dependent protein kinase (PKG) using KT5823 also blocked the ability of isoproterenol to increase transcription initiation. Furthermore, immunoblotting revealed eNOS, but not nNOS, in isolated nuclei. Finally, caged, cell-permeable isoproterenol and endothelin-1 analogs were used to selectively activate intracellular ß-adrenergic and endothelin receptors in intact adult cardiomyocytes. Intracellular release of caged ET-1 or isoproterenol analogs increased NO production in intact adult cardiomyocytes. Hence, activation of the NO synthase/guanylyl cyclase/PKG pathway is necessary for nuclear ß3ARs to increase de novo transcription. Furthermore, we have demonstrated the potential utility of caged receptor ligands in selectively modulating signaling via endogenous intracellular G protein-coupled receptors.