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1.
Int J Mol Sci ; 22(7)2021 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-33801629

RESUMEN

The Na/K-ATPase is the specific receptor for cardiotonic steroids (CTS) such as ouabain and digoxin. At pharmacological concentrations used in the treatment of cardiac conditions, CTS inhibit the ion-pumping function of Na/K-ATPase. At much lower concentrations, in the range of those reported for endogenous CTS in the blood, they stimulate hypertrophic growth of cultured cardiac myocytes through initiation of a Na/K-ATPase-mediated and reactive oxygen species (ROS)-dependent signaling. To examine a possible effect of endogenous concentrations of CTS on cardiac structure and function in vivo, we compared mice expressing the naturally resistant Na/K-ATPase α1 and age-matched mice genetically engineered to express a mutated Na/K-ATPase α1 with high affinity for CTS. In this model, total cardiac Na/K-ATPase activity, α1, α2, and ß1 protein content remained unchanged, and the cardiac Na/K-ATPase dose-response curve to ouabain shifted to the left as expected. In males aged 3-6 months, increased α1 sensitivity to CTS resulted in a significant increase in cardiac carbonylated protein content, suggesting that ROS production was elevated. A moderate but significant increase of about 15% of the heart-weight-to-tibia-length ratio accompanied by an increase in the myocyte cross-sectional area was detected. Echocardiographic analyses did not reveal any change in cardiac function, and there was no fibrosis or re-expression of the fetal gene program. RNA sequencing analysis indicated that pathways related to energy metabolism were upregulated, while those related to extracellular matrix organization were downregulated. Consistent with a functional role of the latter, an angiotensin-II challenge that triggered fibrosis in the α1r/rα2s/s mouse failed to do so in the α1s/sα2s/s. Taken together, these results are indicative of a link between circulating CTS, Na/K-ATPase α1, ROS, and physiological cardiac hypertrophy in mice under baseline laboratory conditions.


Asunto(s)
Glicósidos Cardíacos/química , Corazón/fisiología , Miocardio/enzimología , ATPasa Intercambiadora de Sodio-Potasio/genética , Angiotensina II/farmacología , Animales , Cardiomegalia/patología , Modelos Animales de Enfermedad , Ecocardiografía , Corazón/efectos de los fármacos , Masculino , Ratones , Mutación , Ouabaína/farmacología , Isoformas de Proteínas , RNA-Seq , Especies Reactivas de Oxígeno , Transducción de Señal/efectos de los fármacos
2.
Immunity ; 34(5): 715-28, 2011 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-21565532

RESUMEN

Precise control of myeloid cell activation is required for optimal host defense. However, this activation process must be under exquisite control to prevent uncontrolled inflammation. Herein, we identify the Kruppel-like transcription factor 2 (KLF2) as a potent regulator of myeloid cell activation in vivo. Exposure of myeloid cells to hypoxia and/or bacterial products reduced KLF2 expression while inducing hypoxia inducible factor-1α (HIF-1α), findings that were recapitulated in human septic patients. Myeloid KLF2 was found to be a potent inhibitor of nuclear factor-kappaB (NF-κB)-dependent HIF-1α transcription and, consequently, a critical determinant of outcome in models of polymicrobial infection and endotoxemia. Collectively, these observations identify KLF2 as a tonic repressor of myeloid cell activation in vivo and an essential regulator of the innate immune system.


Asunto(s)
Infecciones Bacterianas/inmunología , Factores de Transcripción de Tipo Kruppel/inmunología , Choque Séptico/inmunología , Animales , Infecciones Bacterianas/microbiología , Línea Celular , Femenino , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Inmunidad Innata , Factores de Transcripción de Tipo Kruppel/genética , Lipopolisacáridos/inmunología , Masculino , Ratones , Ratones Transgénicos , Células Mieloides/inmunología , FN-kappa B/inmunología
3.
Am J Physiol Endocrinol Metab ; 314(6): E620-E629, 2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-29438630

RESUMEN

The distribution of Na/K-ATPase α-isoforms in skeletal muscle is unique, with α1 as the minor (15%) isoform and α2 comprising the bulk of the Na/K-ATPase pool. The acute and isoform-specific role of α2 in muscle performance and resistance to fatigue is well known, but the isoform-specific role of α1 has not been as thoroughly investigated. In vitro, we reported that α1 has a role in promoting cell growth that is not supported by α2. To assess whether α1 serves this isoform-specific trophic role in the skeletal muscle, we used Na/K-ATPase α1-haploinsufficient (α1+/-) mice. A 30% decrease of Na/K-ATPase α1 protein expression without change in α2 induced a modest yet significant decrease of 10% weight in the oxidative soleus muscle. In contrast, the mixed plantaris and glycolytic extensor digitorum longus weights were not significantly affected, likely because of their very low expression level of α1 compared with the soleus. The soleus mass reduction occurred without change in total Na/K-ATPase activity or glycogen metabolism. Serum analytes including K+, fat tissue mass, and exercise capacity were not altered in α1+/- mice. The impact of α1 content on soleus muscle mass is consistent with a Na/K-ATPase α1-specific role in skeletal muscle growth that cannot be fulfilled by α2. The preserved running capacity in α1+/- is in sharp contrast with previously reported consequences of genetic manipulation of α2. Taken together, these results lend further support to the concept of distinct isoform-specific functions of Na/K-ATPase α1 and α2 in skeletal muscle.


Asunto(s)
Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Regulación Enzimológica de la Expresión Génica , Isoenzimas/genética , Isoenzimas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Contracción Muscular/fisiología , Músculo Esquelético/patología , Tamaño de los Órganos/genética , Condicionamiento Físico Animal , ATPasa Intercambiadora de Sodio-Potasio/genética
4.
Int J Mol Sci ; 19(9)2018 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-30213059

RESUMEN

The potassium affinities of Na,K-ATPase isozymes are important determinants of their physiological roles in skeletal muscle. This study measured the apparent K⁺ and Rb⁺ affinities of the Na,K-ATPase α1 and α2 isozymes in intact, dissociated myofibers obtained from WT and genetically altered mice (α1S/Sα2R/R and skα2-/-). It also validates a new method to quantify cations in intact, dissociated myofibers, using inductively coupled plasma mass spectrometry (ICP-MS). Our findings were that: (1) The extracellular substrate sites of Na,K-ATPase bind Rb⁺ and K⁺ with comparable apparent affinities; however; turnover rate is reduced when Rb⁺ is the transported ion; (2) The rate of Rb⁺ uptake by the Na,K-ATPase is not constant but declines with a half-time of approximately 1.5 min; (3) The apparent K⁺ affinity of the α2 isozymes for K⁺ is significantly lower than α1. When measured in intact fibers of WT and α1S/Sα2R/R mice in the presence of 10 µM ouabain; the K1/2,K of α1 and α2 isozymes are 1.3 and 4 mM, respectively. Collectively, these results validate the single fiber model for studies of Na,K-ATPase transport and kinetic constants, and they imply the existence of mechanisms that dynamically limit pump activity during periods of active transport.


Asunto(s)
Isoenzimas/metabolismo , Potasio/metabolismo , Rubidio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Transporte Biológico , Cinética , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Sodio/metabolismo
5.
J Physiol ; 594(21): 6079-6103, 2016 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-27350568

RESUMEN

Reduced smooth muscle (SM)-specific α2 Na+ pump expression elevates basal blood pressure (BP) and increases BP sensitivity to angiotensin II (Ang II) and dietary NaCl, whilst SM-α2 overexpression lowers basal BP and decreases Ang II/salt sensitivity. Prolonged ouabain infusion induces hypertension in rodents, and ouabain-resistant mutation of the α2 ouabain binding site (α2R/R mice) confers resistance to several forms of hypertension. Pressure overload-induced heart hypertrophy and failure are attenuated in cardio-specific α2 knockout, cardio-specific α2 overexpression and α2R/R mice. We propose a unifying hypothesis that reconciles these apparently disparate findings: brain mechanisms, activated by Ang II and high NaCl, regulate sympathetic drive and a novel neurohumoral pathway mediated by both brain and circulating endogenous ouabain (EO). Circulating EO modulates ouabain-sensitive α2 Na+ pump activity and Ca2+ transporter expression and, via Na+ /Ca2+ exchange, Ca2+ homeostasis. This regulates sensitivity to sympathetic activity, Ca2+ signalling and arterial and cardiac contraction.


Asunto(s)
Sistema Cardiovascular/metabolismo , Hipertensión/metabolismo , Ouabaína/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Angiotensinas/metabolismo , Animales , Sitios de Unión , Cardiotónicos/farmacología , Sistema Cardiovascular/efectos de los fármacos , Humanos , Hipertensión/fisiopatología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/química , Sistema Nervioso Simpático/metabolismo , Sistema Nervioso Simpático/fisiología
6.
Am J Physiol Cell Physiol ; 309(12): C813-22, 2015 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-26468207

RESUMEN

The Na(+)-K(+)-ATPase α2-isoform in skeletal muscle is rapidly stimulated during muscle use and plays a critical role in fatigue resistance. The acute mechanisms that stimulate α2-activity are not completely known. This study examines whether phosphorylation of phospholemman (PLM/FXYD1), a regulatory subunit of Na(+)-K(+)-ATPase, plays a role in the acute stimulation of α2 in working muscles. Mice lacking PLM (PLM KO) have a normal content of the α2-subunit and show normal exercise capacity, in contrast to the greatly reduced exercise capacity of mice that lack α2 in the skeletal muscles. Nerve-evoked contractions in vivo did not induce a change in total PLM or PLM phosphorylated at Ser63 or Ser68, in either WT or PLM KO. Isolated muscles of PLM KO mice maintain contraction and resist fatigue as well as wild type (WT). Rb(+) transport by the α2-Na(+)-K(+)-ATPase is stimulated to the same extent in contracting WT and contracting PLM KO muscles. Phosphorylation of sarcolemmal membranes prepared from WT but not PLM KO skeletal muscles stimulates the activity of both α1 and α2 in a PLM-dependent manner. The stimulation occurs by an increase in Na(+) affinity without significant change in Vmax and is more effective for α1 than α2. These results demonstrate that phosphorylation of PLM is capable of stimulating the activity of both isozymes in skeletal muscle; however, contractile activity alone is not sufficient to induce PLM phosphorylation. Importantly, acute stimulation of α2, sufficient to support exercise and oppose fatigue, does not require PLM or its phosphorylation.


Asunto(s)
Proteínas de la Membrana/metabolismo , Fatiga Muscular/fisiología , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Western Blotting , Estimulación Eléctrica , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Muscular/fisiología , Fosforilación , Condicionamiento Físico Animal/fisiología , Espectrofotometría Atómica
7.
J Physiol ; 593(6): 1361-82, 2015 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-25772291

RESUMEN

This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na(+)/Ca(2+) exchange (NCX) and Na(+)/K(+)-ATPase (NKA). While the relevance of Ca(2+) homeostasis in cardiac function has been extensively investigated, the role of Na(+) regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na(+) content have multiple effects on the heart by influencing intracellular Ca(2+) and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na(+) homeostasis. Among the proteins that accomplish this task are the Na(+)/Ca(2+) exchanger (NCX) and the Na(+)/K(+) pump (NKA). By transporting three Na(+) ions into the cytoplasm in exchange for one Ca(2+) moved out, NCX is one of the main Na(+) influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na(+) ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na(+) and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na(+) homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)/K(+) pump and the controversies that still persist in the field.


Asunto(s)
Potenciales de Acción , Arritmias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Congresos como Asunto , Humanos , Miocitos Cardíacos/fisiología
8.
J Biol Chem ; 289(45): 31638-46, 2014 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-25248747

RESUMEN

Previous studies have shown that the myeloid-specific deficiency of the transcription factor Krüppel-like factor 2 (KLF2) accelerates atherosclerosis in hypercholesterolemic Ldlr(-/-) mice due to the enhanced adhesion of myeloid cells to activated endothelial cells in the vessel wall. This study revealed elevated basal inflammation with elevated plasma levels of Ccl2, Ccl4, Ccl5, and Ccl11 in the myeloid-specific KLF2 knock-out (myeKlf2(-/-)) mice. Peritoneal macrophages isolated from myeKlf2(-/-) mice showed increased mRNA levels of several inflammatory mediators, including Ccl2, Ccl5, Ccl7, Cox-2, Cxcl1, and IL-6. In contrast, the levels of two microRNAs, miR-124a and miR-150, were lower in Klf2(-/-) macrophages compared with Klf2(+/+) macrophages. Additional studies showed a direct inverse relationship between miR-124a levels with Ccl2 expression, with anti-miR-124a increasing Ccl2 mRNA levels in Klf2(+/+) macrophages, whereas the restoration of miR-124a levels in Klf2(-/-) macrophages significantly reduced Ccl2 mRNA expression. Likewise, the inverse relationship was observed between miR-150 levels and Cxcl1 expression in Klf2(+/+) and Klf2(-/-) mice. Moreover, miR150 likely regulates the miR124a expression and thus augments expression of inflammatory mediators in myeKlf2(-/-) macrophages. This study documented that the transcription factor KLF2 modulates inflammatory chemokine production via regulation of microRNA expression levels in immune cells.


Asunto(s)
Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , Macrófagos Peritoneales/metabolismo , MicroARNs/sangre , Animales , Aterosclerosis/genética , Secuencia de Bases , Sitios de Unión , Quimiocinas/metabolismo , Femenino , Inflamación , Factores de Transcripción de Tipo Kruppel/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Células Mieloides/citología , Células Mieloides/metabolismo
9.
J Biol Chem ; 288(2): 1226-37, 2013 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-23192345

RESUMEN

The Na,K-ATPase α2 isozyme is the major Na,K-ATPase of mammalian skeletal muscle. This distribution is unique compared with most other cells, which express mainly the Na,K-ATPase α1 isoform, but its functional significance is not known. We developed a gene-targeted mouse (skα2(-/-)) in which the α2 gene (Atp1a2) is knocked out in the skeletal muscles, and examined the consequences for exercise performance, membrane potentials, contractility, and muscle fatigue. Targeted knockout was confirmed by genotyping, Western blot, and immunohistochemistry. Skeletal muscle cells of skα2(-/-) mice completely lack α2 protein and have no α2 in the transverse tubules, where its expression is normally enhanced. The α1 isoform, which is normally enhanced on the outer sarcolemma, is up-regulated 2.5-fold without change in subcellular targeting. skα2(-/-) mice are apparently normal under basal conditions but show significantly reduced exercise capacity when challenged to run. Their skeletal muscles produce less force, are unable to increase force to match demand, and show significantly increased susceptibility to fatigue. The impairments affect both fast and slow muscle types. The subcellular targeting of α2 to the transverse tubules is important for this role. Increasing Na,K-ATPase α1 content cannot fully compensate for the loss of α2. The increased fatigability of skα2(-/-) muscles is reproduced in control extensor digitorum longus muscles by selectively inhibiting α2 enzyme activity with ouabain. These results demonstrate that the Na,K-ATPase α2 isoform performs an acute, isoform-specific role in skeletal muscle. Its activity is regulated by muscle use and enables working muscles to maintain contraction and resist fatigue.


Asunto(s)
Isoenzimas/metabolismo , Músculo Esquelético/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN , Inmunohistoquímica , Ratones , Ratones Noqueados , Contracción Muscular , Músculo Esquelético/fisiología , Reacción en Cadena de la Polimerasa
10.
Circ Res ; 110(10): 1294-302, 2012 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-22474254

RESUMEN

RATIONALE: Hemizygous deficiency of the transcription factor Krüppel-like factor 2 (KLF2) has been shown previously to augment atherosclerosis in hypercholesterolemic mice. However, the cell type responsible for the increased atherosclerosis due to KLF2 deficiency has not been identified. This study examined the consequence of myeloid cell-specific KLF2 inactivation in atherosclerosis. METHODS AND RESULTS: Cell-specific knockout mice were generated by Cre/loxP recombination. Macrophages isolated from myeloid-specific Klf2 knockout (myeKlf2(-/-)) mice were similar to myeKlf2(+/+) macrophages in response to activation, polarization, and lipid accumulation. However, in comparison to myeKlf2(+/+) macrophages, myeKlf2(-/-) macrophages adhered more robustly to endothelial cells. Neutrophils from myeKlf2(-/-) mice also adhered more robustly to endothelial cells, and fewer myeKlf2(-/-) neutrophils survived in culture over a 24-hour period in comparison with myeKlf2(+/+) neutrophils. When myeKlf2(-/-) mice were mated to Ldlr(-/-) mice and then fed a high fat and high cholesterol diet, significant increase in atherosclerosis was observed in the myeKlf2(-/-)Ldlr(-/-) mice compared with myeKlf2(+/+)Ldlr(-/-) littermates. The increased atherosclerosis in myeKlf2(-/-)Ldlr(-/-) mice was associated with elevated presence of neutrophils and macrophages, with corresponding increase of myeloperoxidase as well as chlorinated and nitrosylated tyrosine epitopes in their lesion areas compared with myeKlf2(+/+)Ldlr(-/-) mice. CONCLUSIONS: This study documents a role for myeloid KLF2 expression in modulating atherosclerosis. The increased neutrophil accumulation and atherosclerosis progression with myeloid-specific KLF2 deficiency also underscores the importance of neutrophils in promoting vascular oxidative stress and atherosclerosis. Collectively, these results suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of atherosclerosis.


Asunto(s)
Aterosclerosis/inmunología , Adhesión Celular/inmunología , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Macrófagos/inmunología , Neutrófilos/inmunología , Animales , Aterosclerosis/patología , Muerte Celular/inmunología , Células Endoteliales/citología , Células Endoteliales/inmunología , Femenino , Hipercolesterolemia/inmunología , Hipercolesterolemia/patología , Recuento de Linfocitos , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/citología , Vasculitis/inmunología , Vasculitis/patología
11.
Annu Rev Physiol ; 72: 395-412, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20148682

RESUMEN

The Na,K-ATPase is the membrane "pump" that generates the Na(+) and K(+) gradients across the plasma membrane that drives many physiological processes. This enzyme is highly sensitive to inhibition by cardiotonic steroids, most notably the digitalis/ouabain class of compounds, which have been used for centuries to treat congestive heart failure and arrhythmias. The amino acids that constitute the ouabain-binding site are highly conserved across the evolutionary spectrum. This could be fortuitous or could result from this site being conserved because it has an important biological function. New physiological approaches using genetically engineered mice are being used to define the biological significance of the "receptor function" of the Na,K-ATPase and its regulation by potential endogenous cardiotonic steroid-like compounds. These studies extend the reach of earlier studies involving the biochemical purification of endogenous regulatory ligands.


Asunto(s)
Glicósidos Cardíacos/metabolismo , Ouabaína/metabolismo , Receptores de Esteroides/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Sitios de Unión , Evolución Biológica , Humanos , Isoenzimas/metabolismo , Ligandos , Receptores de Esteroides/efectos de los fármacos
12.
J Biol Chem ; 287(2): 1448-57, 2012 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-22110137

RESUMEN

Although gram-positive infections account for the majority of cases of sepsis, the molecular mechanisms underlying their effects remains poorly understood. We investigated how cell wall components of gram-positive bacteria contribute to the development of sepsis. Experimental observations derived from cultured primary macrophages and the cell line indicate that gram-positive bacterial endotoxins induce hypoxia-inducible factor 1α (HIF-1α) mRNA and protein expression. Inoculation of live or heat-inactivated gram-positive bacteria with macrophages induced HIF-1 transcriptional activity in macrophages. Concordant with these results, myeloid deficiency of HIF-1α attenuated gram-positive bacterial endotoxin-induced cellular motility and proinflammatory gene expression in macrophages. Conversely, gram-positive bacteria and their endotoxins reduced expression of the myeloid anti-inflammatory transcription factor Krüppel-like transcription factor 2 (KLF2). Sustained expression of KLF2 reduced and deficiency of KLF2 enhanced gram-positive endotoxins induced HIF-1α mRNA and protein expression in macrophages. More importantly, KLF2 attenuated gram-positive endotoxins induced cellular motility and proinflammatory gene expression in myeloid cells. Consistent with these results, mice deficient in myeloid HIF-1α were protected from gram-positive endotoxin-induced sepsis mortality and clinical symptomatology. By contrast, myeloid KLF2-deficient mice were susceptible to gram-positive sepsis induced mortality and clinical symptoms. Collectively, these observations identify HIF-1α and KLF2 as critical regulators of gram-positive endotoxin-mediated sepsis.


Asunto(s)
Endotoxinas/toxicidad , Bacterias Grampositivas , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Macrófagos/metabolismo , Choque Séptico/metabolismo , Animales , Línea Celular , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Factores de Transcripción de Tipo Kruppel/genética , Macrófagos/patología , Ratones , Ratones Transgénicos , Choque Séptico/inducido químicamente , Choque Séptico/genética , Choque Séptico/patología
13.
J Biol Chem ; 287(20): 16390-8, 2012 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-22451662

RESUMEN

Decreases in cardiac Na/K-ATPase have been documented in patients with heart failure. Reduction of Na/K-ATPase α1 also contributes to the deficiency in cardiac contractility in animal models. Our previous studies demonstrate that reduction of cellular Na/K-ATPase causes cell growth inhibition and cell death in renal proximal tubule cells. To test whether reduction of Na/K-ATPase in combination with increased cardiotonic steroids causes cardiac myocyte death and cardiac dysfunction, we examined heart function in Na/K-ATPase α1 heterozygote knock-out mice (α1(+/-)) in comparison to wild type (WT) littermates after infusion of marinobufagenin (MBG). Adult cardiac myocytes were also isolated from both WT and α1(+/-) mice for in vitro experiments. The results demonstrated that MBG infusion increased myocyte apoptosis and induced significant left ventricle dilation in α1(+/-) mice but not in their WT littermates. Mechanistically, it was found that in WT myocytes MBG activated the Src/Akt/mTOR signaling pathway, which further increased phosphorylation of ribosome S6 kinase (S6K) and BAD (Bcl-2-associated death promoter) and protected cells from apoptosis. In α1(+/-) myocytes, the basal level of phospho-BAD is higher compared with WT myocytes, but MBG failed to induce further activation of the mTOR pathway. Reduction of Na/K-ATPase also caused the activation of caspase 9 but not caspase 8 in these cells. Using cultures of neonatal cardiac myocytes, we demonstrated that inhibition of the mTOR pathway by rapamycin also enabled MBG to activate caspase 9 and induce myocyte apoptosis.


Asunto(s)
Apoptosis/efectos de los fármacos , Bufanólidos/efectos adversos , Inhibidores Enzimáticos/efectos adversos , Cardiopatías/enzimología , Proteínas Musculares/metabolismo , Miocitos Cardíacos/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Antibacterianos/farmacología , Apoptosis/genética , Bufanólidos/farmacología , Caspasa 8/genética , Caspasa 8/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Inhibidores Enzimáticos/farmacología , Cardiopatías/inducido químicamente , Cardiopatías/genética , Ratones , Ratones Mutantes , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Miocitos Cardíacos/patología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sirolimus/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo
14.
Am J Physiol Heart Circ Physiol ; 304(8): H1147-58, 2013 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-23436327

RESUMEN

The α2-isoform of the Na,K-ATPase (α2) is the minor isoform of the Na,K-ATPase expressed in the cardiovascular system and is thought to play a critical role in the regulation of cardiovascular hemodynamics. However, the organ system/cell type expressing α2 that is required for this regulation has not been fully defined. The present study uses a heart-specific knockout of α2 to further define the tissue-specific role of α2 in the regulation of cardiovascular hemodynamics. To accomplish this, we developed a mouse model using the Cre/loxP system to generate a tissue-specific knockout of α2 in the heart using ß-myosin heavy chain Cre. We have achieved a 90% knockout of α2 expression in the heart of the knockout mice. Interestingly, the heart-specific knockout mice exhibit normal basal cardiac function and systolic blood pressure, and in addition, these mice develop ACTH-induced hypertension in response to ACTH treatment similar to control mice. Surprisingly, the heart-specific knockout mice display delayed onset of cardiac dysfunction compared with control mice in response to pressure overload induced by transverse aortic constriction; however, the heart-specific knockout mice deteriorated to control levels by 9 wk post-transverse aortic constriction. These results suggest that heart expression of α2 does not play a role in the regulation of basal cardiovascular function or blood pressure; however, heart expression of α2 plays a role in the hypertrophic response to pressure overload. This study further emphasizes that the tissue localization of α2 determines its unique roles in the regulation of cardiovascular function.


Asunto(s)
Hormona Adrenocorticotrópica/efectos adversos , Hipertensión/metabolismo , Hipertrofia Ventricular Izquierda/metabolismo , Miocitos Cardíacos/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Disfunción Ventricular Izquierda/metabolismo , Animales , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Presión Sanguínea/genética , Presión Sanguínea/fisiología , Técnicas de Inactivación de Genes/métodos , Hipertensión/inducido químicamente , Hipertensión/genética , Hipertrofia Ventricular Izquierda/diagnóstico por imagen , Hipertrofia Ventricular Izquierda/genética , Integrasas , Ratones , Ratones Noqueados , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Péptido Natriurético Encefálico/genética , Péptido Natriurético Encefálico/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/análisis , ATPasa Intercambiadora de Sodio-Potasio/genética , Ultrasonografía , Vasoconstricción , Disfunción Ventricular Izquierda/diagnóstico por imagen , Disfunción Ventricular Izquierda/genética
15.
J Biol Chem ; 286(31): 27836-47, 2011 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-21680747

RESUMEN

Differentiation of preadipocytes into mature adipocytes capable of efficiently storing lipids is an important regulatory mechanism in obesity. Here, we examined the involvement of histone deacetylases (HDACs) and histone acetyltransferases (HATs) in the regulation of adipogenesis. We find that among the various members of the HDAC and HAT families, only HDAC9 exhibited dramatic down-regulation preceding adipogenic differentiation. Preadipocytes from HDAC9 gene knock-out mice exhibited accelerated adipogenic differentiation, whereas HDAC9 overexpression in 3T3-L1 preadipocytes suppressed adipogenic differentiation, demonstrating its direct role as a negative regulator of adipogenesis. HDAC9 expression was higher in visceral as compared with subcutaneous preadipocytes, negatively correlating with their potential to undergo adipogenic differentiation in vitro. HDAC9 localized in the nucleus, and its negative regulation of adipogenesis segregates with the N-terminal nuclear targeting domain, whereas the C-terminal deacetylase domain is dispensable for this function. HDAC9 co-precipitates with USF1 and is recruited with USF1 at the E-box region of the C/EBPα gene promoter in preadipocytes. Upon induction of adipogenic differentiation, HDAC9 is down-regulated, leading to its dissociation from the USF1 complex, whereas p300 HAT is up-regulated to allow its association with USF1 and accumulation at the E-box site of the C/EBPα promoter in differentiated adipocytes. This reciprocal regulation of HDAC9 and p300 HAT in the USF1 complex is associated with increased C/EBPα expression, a master regulator of adipogenic differentiation. These findings provide new insights into mechanisms of adipogenic differentiation and document a critical regulatory role for HDAC9 in adipogenic differentiation through a deacetylase-independent mechanism.


Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/fisiología , Histona Desacetilasas/fisiología , Proteínas Represoras/fisiología , Células 3T3-L1 , Animales , Regulación hacia Abajo , Histona Desacetilasas/genética , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Proteínas Represoras/genética
16.
Nature ; 442(7100): 299-302, 2006 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-16855590

RESUMEN

Mammalian Kruppel-like transcription factors are implicated in regulating terminal differentiation of several tissue types. Deficiency in Kruppel-like factor (KLF) 2 (also known as LKLF) leads to a massive loss of the peripheral T-cell pool, suggesting KLF2 regulates T-cell quiescence and survival. Here we show, however, that KLF2 is essential for T-cell trafficking. KLF2-deficient (Klf2-/-) thymocytes show impaired expression of several receptors required for thymocyte emigration and peripheral trafficking, including the sphingosine-1-phosphate (S1P) receptor S1P1, CD62L and beta7 integrin. Furthermore, KLF2 both binds and transactivates the promoter for S1P1--a receptor that is critical for thymocyte egress and recirculation through peripheral lymphoid organs. Our findings suggest that KLF2 serves to license mature T cells for trafficking from the thymus and recirculation through secondary lymphoid tissues.


Asunto(s)
Movimiento Celular , Factores de Transcripción de Tipo Kruppel/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Timo/citología , Traslado Adoptivo , Animales , Línea Celular Tumoral , Quimera/metabolismo , Feto , Humanos , Células Jurkat , Factores de Transcripción de Tipo Kruppel/deficiencia , Factores de Transcripción de Tipo Kruppel/genética , Hígado/embriología , Ratones , Regiones Promotoras Genéticas/genética , Receptores de Lisoesfingolípidos/genética , Linfocitos T/trasplante , Activación Transcripcional
17.
Am J Physiol Renal Physiol ; 301(3): F615-21, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21632957

RESUMEN

Endogenous cardiotonic steroids, through their interaction with the ouabain-binding site of the Na-K-ATPase α-subunit, have been implicated in a variety of cardiovascular disease states including hypertension. We have previously shown that ACTH-induced hypertension is abolished in mutant mice expressing ouabain-resistant α1- and α2-subunits. To further evaluate hypertension resistance in these mutant mice, we examined blood pressure changes in a modified model of 2-kidney, 1-clip (2K1C) renovascular hypertension. To reliably generate 2K1C hypertension, we used polyvinyl tubing (inner diameter: ∼0.27 mm) to accurately gauge the degree of renal artery stenosis. Using this method, virtually all of the clipped mice became hypertensive and there was no incidence of apparent renal ischemia. By telemetry, in response to renal artery clipping, blood pressure in wild-type mice (α1 ouabain-resistant, α2 ouabain-sensitive) increased from 97 ± 3 to 136 ± 7 mmHg. In α1-resistant, α2-resistant mice, pressure increased from 93 ± 2 to 123 ± 4 mmHg, and in α1-sensitive, α2-resistant mice, blood pressure increased from 95 ± 2 to 139 ± 5 mmHg. Blood pressure changes were equivalent in all three groups. In sham mice, blood pressure did not change (96 ± 1 to 95 ± 2 mmHg). Renin mRNA expression was dramatically elevated in the left vs. the right kidney, and plasma renin concentration was elevated similarly in all genotypes. These data indicate that sensitivity of the α1- or α2-Na-K-ATPase binding site to cardiotonic steroids is not a prerequisite for the development of 2K1C renovascular hypertension. In addition, use of a polyurethane cuff to constrict the renal artery provides a reliable method for producing 2K1C hypertension in mice.


Asunto(s)
Hipertensión Renovascular/etiología , Hipertensión Renovascular/fisiopatología , Riñón/fisiopatología , Ouabaína/metabolismo , Subunidades de Proteína/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Instrumentos Quirúrgicos/efectos adversos , Animales , Sitios de Unión/fisiología , Presión Sanguínea/fisiología , Modelos Animales de Enfermedad , Femenino , Riñón/irrigación sanguínea , Riñón/cirugía , Masculino , Ratones , Ratones Endogámicos , Ratones Mutantes , Poliuretanos , Unión Proteica/fisiología , Arteria Renal/fisiopatología
18.
Am J Physiol Heart Circ Physiol ; 300(1): H347-55, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20952666

RESUMEN

The Na,K-ATPase is a ubiquitous transmembrane pump and a specific receptor for cardiac glycosides such as ouabain and digoxin, which are used in the management of congestive heart failure (CHF). A potential role for these so-called endogenous cardiotonic steroids (CS) has been explored, and it has become apparent that such compounds are elevated and may play an important role in a variety of physiological and pathophysiological conditions such as hypertension and CHF. Recent evidence suggests that the Na,K-ATPase may act as a signal transducer upon CS binding and induce nonproliferative cardiac growth, implicating a role for endogenous CS in the development of cardiac hypertrophy and progressive failure of the heart. In the present study, we tested whether hypertrophic responses to pressure overload would be altered in mutant mice that specifically express ouabain-sensitive or ouabain-resistant α1- and α2-Na,K-ATPase subunits, as follows: α1-resistant, α2-resistant (α1(R/R)α2(R/R)); α1-sensitive, α2-resistant (α1(S/S)α2(R/R)); and α1-resistant, α2-sensitive (α1(R/R)α2(S/S), wild-type). In α1(S/S)α2(R/R) mice, pressure overload by transverse aortic coarctation induced severe left ventricular (LV) hypertrophy with extensive perivascular and replacement fibrosis at only 4 wk. Responses in α1(R/R)α2(S/S) and α1(R/R)α2(R/R) mice were comparatively mild. Mutant α1(S/S)α2(R/R) mice also had LV dilatation and depressed LV systolic contractile function by 4 wk of pressure overload. In separate experiments, chronic Digibind treatment prevented the rapid progression of cardiac hypertrophy and fibrosis in α1(S/S)α2(R/R) mice. These data demonstrate that mice with a ouabain-sensitive α1-Na,K-ATPase subunit have a dramatic susceptibility to the development of cardiac hypertrophy, and failure from LV pressure overload and provide evidence for the involvement of endogenous CS in this process.


Asunto(s)
Cardiomegalia/metabolismo , Ouabaína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Análisis de Varianza , Animales , Western Blotting , Cardiomegalia/genética , Ecocardiografía , Genotipo , Hemodinámica , Ratones , Ratones Transgénicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Función Ventricular Izquierda/genética
19.
Am J Physiol Heart Circ Physiol ; 301(5): H2147-53, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21856907

RESUMEN

A chronic increase in the concentration of sodium chloride in the cerebrospinal fluid (CSF) (↑CSF [NaCl]) appears to be critically important for the development of salt-dependent hypertension. In agreement with this concept, increasing CSF [NaCl] chronically by intracerebroventricular (icv) infusion of NaCl-rich artificial CSF (aCSF-HiNaCl) in rats produces hypertension by the same mechanisms (i.e., aldosterone-ouabain pathway in the brain) as that produced by dietary sodium in salt-sensitive strains. We first demonstrate here that icv aCSF-HiNaCl for 10 days also causes hypertension in wild-type (WT) mice. We then used both WT and gene-targeted mice to explore the mechanisms. In WT mice with a ouabain-sensitive Na,K-ATPase α(2)-isoform (α2(S/S)), mean arterial pressure rose by ~25 mmHg within 2 days of starting aCSF-HiNaCl (0.6 nmol Na/min) and remained elevated throughout the study. Ouabain (171 pmol/day icv) increased blood pressure to a similar extent. aCSF-HiNaCl or ouabain given at the same rates subcutaneously instead of intracerebroventricularly had no effect on blood pressure. The pressor response to icv aCSF-HiNaCl was abolished by an anti-ouabain antibody given intracerebroventricularly but not subcutaneously, indicating that it is mediated by an endogenous ouabain-like substance in the brain. We compared the effects of icv aCSF-HiNaCl or icv ouabain on blood pressure in α2(S/S) versus knockout/knockin mice with a ouabain-resistant endogenous α(2)-subunit (α2(R/R)). In α2(R/R), there was no pressor response to icv aCSF-HiNaCl in contrast to WT mice. The α2(R/R) genotype also lacked a pressor response to icv ouabain. These data demonstrate that chronic ↑CSF [NaCl] causes hypertension in mice and that the blood pressure response is mediated by the ouabain-like substance in the brain, specifically by its binding to the α(2)-isoform of the Na,K-ATPase.


Asunto(s)
Presión Sanguínea , Encéfalo/enzimología , Cardenólidos/metabolismo , Hipertensión/enzimología , Saponinas/metabolismo , Cloruro de Sodio/líquido cefalorraquídeo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Sitios de Unión , Monitoreo Ambulatorio de la Presión Arterial , Modelos Animales de Enfermedad , Frecuencia Cardíaca , Hipertensión/líquido cefalorraquídeo , Hipertensión/inducido químicamente , Hipertensión/fisiopatología , Infusiones Intraventriculares , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación , Ouabaína/administración & dosificación , Cloruro de Sodio/administración & dosificación , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/deficiencia , ATPasa Intercambiadora de Sodio-Potasio/genética , Telemetría , Factores de Tiempo
20.
Am J Physiol Heart Circ Physiol ; 301(4): H1396-404, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21856916

RESUMEN

The α(2)-isoform of Na,K-ATPase (α(2)) is thought to play a role in blood pressure regulation, but the specific cell type(s) involved have not been identified. Therefore, it is important to study the role of the α(2) in individual cell types in the cardiovascular system. The present study demonstrates the role of vascular smooth muscle α(2) in the regulation of cardiovascular hemodynamics. To accomplish this, we developed a mouse model utilizing the Cre/LoxP system to generate a cell type-specific knockout of the α(2) in vascular smooth muscle cells using the SM22α Cre. We achieved a 90% reduction in the α(2)-expression in heart and vascular smooth muscle in the knockout mice. Interestingly, tail-cuff blood pressure analysis reveals that basal systolic blood pressure is unaffected by the knockout of α(2) in the knockout mice. However, knockout mice do fail to develop ACTH-induced hypertension, as seen in wild-type mice, following 5 days of treatment with ACTH (Cortrosyn; wild type = 119.0 ± 6.8 mmHg; knockout = 103.0 ± 2.0 mmHg). These results demonstrate that α(2)-expression in heart and vascular smooth muscle is not essential for regulation of basal systolic blood pressure, but α(2) is critical for blood pressure regulation under chronic stress such as ACTH-induced hypertension.


Asunto(s)
Hormona Adrenocorticotrópica , Presión Sanguínea/genética , Presión Sanguínea/fisiología , Sistema Cardiovascular/enzimología , Hipertensión/genética , Hipertensión/prevención & control , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Western Blotting , Cardiomegalia/metabolismo , Fenómenos Fisiológicos Cardiovasculares/genética , Separación Celular , Hipertensión/inducido químicamente , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Microsomas/metabolismo , Proteínas Musculares/metabolismo , Mutagénesis Insercional , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/fisiología , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/fisiología , Recombinación Genética , Flujo Sanguíneo Regional/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasa Intercambiadora de Sodio-Potasio/genética , Resistencia Vascular/fisiología
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