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1.
J Bone Miner Res ; 37(4): 660-674, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-34989027

RESUMEN

This study took advantage of the recent discovery that the EphA4 signaling has anti-catabolic effects on osteoclasts/macrophages/synoviocytes but pro-anabolic effects on articular chondrocytes and sought to develop an EphA4 signaling-based therapeutic strategy for osteoarthritis (OA) using a mouse model of OA/posttraumatic OA (PTOA). The injured joint of C57BL/6J mice received biweekly intraarticular injections of a soluble EphA4-binding ligand (EfnA4-fc) at 1 day after the tibial plateau injury or at 5 weeks post-injury. The animals were euthanized 5 weeks later. The injured right and contralateral uninjured left joints were analyzed for hallmarks of OA by histology. Relative severity was determined by a modified Mankin OA scoring system and serum COMP and CTX-II levels. Tibial plateau injury caused more severe OA in Epha4 null mice than in wild-type (WT) littermates, suggesting a protective role of EphA4 signaling in OA. A prototype strategy of an EphA4 signaling-based strategy involving biweekly injections of EfnA4-fc into injured joints was developed and was shown to be highly effective in preventing OA/PTOA when it was administered at 1 day post-injury and in treating OA/PTOA when it was applied after OA has been established. The efficacy of this prototype was dose- and time-dependent. The effects were not caused by the Fc moiety of EfnA4-fc. Other soluble EfnA ligands of EphA4, ie, EfnA1-fc and EfnA2-fc, were also effective. A prototype of a novel EphA4 signaling-based therapy was developed for OA/PTOA that not only reduces the progressive destruction of articular cartilage but may also promote regeneration of the damaged cartilage. © 2022 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.


Asunto(s)
Cartílago Articular , Osteoartritis , Sinoviocitos , Animales , Cartílago Articular/patología , Condrocitos/patología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Osteoartritis/patología , Sinoviocitos/patología
2.
Calcif Tissue Int ; 107(6): 576-592, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32816052

RESUMEN

The expression and activation of EphA4 in the various cell types in a knee joint was upregulated upon an intraarticular injury. To determine if EphA4 signaling plays a role in osteoarthritis, we determined whether deficient EphA4 expression (in EphA4 knockout mice) or upregulation of the EphA4 signaling (with the EfnA4-fc treatment) would alter cellular functions of synoviocytes and articular chondrocytes. In synoviocytes, deficient EphA4 expression enhanced, whereas activation of the EphA4 signaling reduced, expression and secretion of key inflammatory cytokines and matrix metalloproteases. Conversely, in articular chondrocytes, activation of the EphA4 signaling upregulated, while deficient EphA4 expression reduced, expression levels of chondrogenic genes (e.g., aggrecan, lubricin, type-2 collagen, and Sox9). EfnA4-fc treatment in wildtype, but not EphA4-deficient, articular chondrocytes promoted the formation and activity of acidic proteoglycan-producing colonies. Activation of the EphA4 signaling in articular chondrocytes upregulated Rac1/2 and downregulated RhoA via enhancing Vav1 and reducing Ephexin1 activation, respectively. However, activation of the EphA4 signaling in synoviocytes suppressed the Vav/Rac signaling while upregulated the Ephexin/Rho signaling. In summary, the EphA4 signaling in synoviocytes is largely of anti-catabolic nature through suppression of the expression of inflammatory cytokines and matrix proteases, but in articular chondrocytes the signaling is pro-anabolic in that it promotes the biosynthesis of articular cartilage. The contrasting action of the EphA4 signaling in synoviocytes as opposing to articular chondrocytes may in part be mediated through the opposite differential effects of the EphA4 signaling on the Vav/Rac signaling and Ephexin/Rho signaling in the two skeletal cell types.


Asunto(s)
Cartílago Articular , Condrocitos/metabolismo , Receptor EphA4/metabolismo , Sinoviocitos/metabolismo , Animales , Células Cultivadas , Colágeno Tipo II , Ratones , Ratones Noqueados , Transducción de Señal
3.
Calcif Tissue Int ; 106(2): 158-171, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31559470

RESUMEN

This study sought to develop a noninvasive, reliable, clinically relevant, and easy-to-implement mouse model that can be used for investigation of the pathophysiology of PTOA and for preclinical testing of new therapies of PTOA. Accordingly, we have established a closed intraarticular tibial plateau compression loading-induced injury model of PTOA in C57BL/6J mice. In this model, a single application of a defined loading force was applied with an indenter to the tibial plateau of the right knee to create injuries to the synovium, menisci, ligaments, and articular cartilage. The limiting loading force was set at 55 N with the loading speed of 60 N/s. This loading regimen limits the distance that the indenter would travel into the joint, but still yields substantial compression loading energy to cause significant injuries to the synovium, meniscus, and articular cartilage. The joint injury induced by this loading protocol consistently yielded evidence for key histological hallmarks of PTOA at 5-11 weeks post-injury, including loss of articular cartilage, disorganization of chondrocytes, meniscal hyperplasia and mineralization, osteophyte formation, and degenerative remodeling of subchondral bone. These arthritic changes were highly reproducible and of a progressive nature. Because 50% of patients with meniscal and/or ligament injuries without intraarticular fractures developed PTOA over time, this intraarticular tibial plateau compression loading-induced injury model is clinically relevant. In summary, we have developed a noninvasive intraarticular tibial plateau compression loading-induced injury model in the mouse that can be used to investigate the pathophysiology of PTOA and for preclinical testing for new therapies.


Asunto(s)
Osteoartritis/patología , Estrés Mecánico , Tibia , Fracturas de la Tibia/patología , Animales , Cartílago Articular/patología , Cartílago Articular/fisiología , Fuerza Compresiva/fisiología , Modelos Animales de Enfermedad , Femenino , Traumatismos de la Rodilla/complicaciones , Traumatismos de la Rodilla/patología , Articulación de la Rodilla/patología , Articulación de la Rodilla/fisiología , Traumatismos de la Pierna/complicaciones , Traumatismos de la Pierna/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoartritis/etiología , Tibia/patología , Tibia/fisiología , Fracturas de la Tibia/complicaciones , Soporte de Peso/fisiología
4.
JBMR Plus ; 1(2): 73-85, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-29082358

RESUMEN

This study sought to understand the regulation of an osteoclastic protein-tyrosine phosphatase (PTP-oc), a positive regulator of osteoclast activaty. Our past studies suggested that PTP-oc is regulated post-transcriptionally. The 3'-UTR of PTP-oc mRNA contains a target site for miR17. During osteoclastic differentiation, there was an inverse relationship between the cellular levels of miR17 (expressed as one of the six cluster genes of miR17~92) and PTP-oc mRNA. Overexpression of pre-miR17~92 in mouse osteoclast precursors reduced PTP-oc mRNA level and the size of the derived osteoclasts; whereas deletion of miR17~92 or inhibition of miR17 resulted in the formation of larger osteoclasts containing more nuclei that expressed higher PTP-oc mRNA levels and created larger resorption pits. Thus, PTP-oc-mediated osteoclast activation is modulated in part by miR17~92, particularly miR17. The miR17~92 osteoclast conditional knockout (cKO) mutants, generated by breeding miR17~92loxp/loxp mice with Ctsk-Cre mice, had lower Tb.BV/TV, Tb.BMD, Tb.Conn-Dens, Tb.N, and Tb.Th, but larger Tb.Sp, and greater bone resorption without a change in bone formation compared to littermate controls. The cKO marrow-derived osteoclasts were twice as large, contained twice as many nuclei, and produced twice as large resorption pits as osteoclasts of littermate controls. The expression of genes associated with osteoclast activation was increased in cKO osteoclasts, suggesting that deletion of miR17~92 in osteoclasts promotes osteoclast activation. The cKO osteoblasts did not show differences in cellular miR17 level, alkaline phosphatase activity, and bone nodule formation ability. In conclusion, miR17-92 negatively regulates the osteoclast activity, in part via the miR17-mediated suppression of PTP-oc in osteoclasts.

5.
J Cell Biochem ; 116(8): 1785-96, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25676701

RESUMEN

We have previously shown that PTP-oc is an enhancer of the functional activity of osteoclasts and that EphA4 is a suppressor. Here, we provide evidence that PTP-oc enhances osteoclast activity in part through inactivation of EphA4 by dephosphorylating key phosphotyrosine (pY) residues of EphA4. We show that EphA4 was pulled down by the PTP-oc trapping mutant but not by the wild-type (WT) PTP-oc and that transgenic overexpression of PTP-oc in osteoclasts drastically decreased pY602 and pY779 residues of EphA4. Consistent with the previous findings that EphA4 deficiency increased pY173-Vav3 level (Rac-GTP exchange factor [GEF]) and enhanced bone resorption activity of osteoclasts, reintroduction of WT-Epha4 in Epha4 null osteoclasts led to ∼50% reduction in the pY173-Vav3 level and ∼2-fold increase in bone resorption activity. Overexpression of Y779F-Epha4 mutant in WT osteoclasts markedly increased in pY173-Vav3 and reduced bone resorption activity, but overexpression of Y602F-Epha4 mutant had no effect, suggesting that pY779 residue plays an important role in the EphA4-mediated suppression of osteoclast activity. Deficient EphA4 in osteoclasts has been shown to up-regulate Rac-GTPase and down-regulate Rho-GTPase. PTP-oc overexpression in osteoclasts also increased the GTP-Rac level to 300% of controls, but decreased the GTP-Rho level to ∼50% of controls. PTP-oc overexpression or deficient Epha4 each also reduced pY87-Ephexin level, which is a Rho GEF. Thus, PTP-oc may differentially regulate Rac signaling versus Rho signaling through dephosphorylation of EphA4, which has shown to have opposing effects on Rac-GTPase versus Rho-GTPase through differential regulation of Vav3 versus Ephexin.


Asunto(s)
Resorción Ósea/metabolismo , Osteoclastos/fisiología , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Receptor EphA4/metabolismo , Tirosina/metabolismo , Animales , Resorción Ósea/genética , Línea Celular , Masculino , Ratones , Ratones Transgénicos , Mutación , Fosforilación , Proteínas Tirosina Fosfatasas no Receptoras/química , Receptor EphA4/genética , Transducción de Señal
6.
PLoS One ; 9(5): e98004, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24848992

RESUMEN

This study sought to determine if direct application of the lentiviral (LV)-cyclooxygenase 2 (COX2) vector to the tendon-bone interface would promote osteointegration of the tendon graft in a rat model of biceps tenodesis. The LV-COX2 gene transfer strategy was chosen for investigation because a similar COX2 gene transfer strategy promoted bony bridging of the fracture gap during bone repair, which involves similar histologic transitions that occur in osteointegration. Briefly, a 1.14-mm diameter tunnel was drilled in the mid-groove of the humerus of adult Fischer 344 rats. The LV-COX2 or ßgal control vector was applied directly into the bone tunnel and onto the end of the tendon graft, which was then pulled into the bone tunnel. A poly-L-lactide pin was press-fitted into the tunnel as interference fixation. Animals were sacrificed at 3, 5, or 8 weeks for histology analysis of osteointegration. The LV-COX2 gene transfer strategy enhanced neo-chondrogenesis at the tendon-bone interface but with only marginal effect on de novo bone formation. The tendon-bone interface of the LV-COX2-treated tenodesis showed the well-defined tendon-to-fibrocartilage-to-bone histologic transitions that are indicative of osteointegration of the tendon graft. The LV-COX2 in vivo gene transfer strategy also significantly enhanced angiogenesis at the tendon-bone interface. To determine if the increased osteointegration was translated into an improved pull-out mechanical strength property, the pull-out tensile strength of the LV-COX2-treated tendon grafts was determined with a pull-out mechanical testing assay. The LV-COX2 strategy yielded a significant improvement in the return of the pull-out strength of the tendon graft after 8 weeks. In conclusion, the COX2-based in vivo gene transfer strategy enhanced angiogenesis, osteointegration and improved return of the pull-out strength of the tendon graft. Thus, this strategy has great potential to be developed into an effective therapy to promote tendon-to-bone healing after tenodesis or related surgeries.


Asunto(s)
Huesos/fisiología , Ciclooxigenasa 2/genética , Lentivirus/genética , Tendones/trasplante , Tenodesis/métodos , Resistencia a la Tracción , Extremidad Superior/cirugía , Animales , Huesos/metabolismo , Huesos/cirugía , Condrogénesis , Técnicas de Transferencia de Gen , Oseointegración , Osteogénesis , Ratas , Tendones/cirugía , Cicatrización de Heridas
7.
J Bone Miner Res ; 29(4): 804-19, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23983218

RESUMEN

Of the ephrin (Eph) receptors, mature osteoclasts express predominantly EphA4. This study sought to determine if EphA4 has a regulatory role in osteoclasts. Treatment of RAW/C4 cells with Epha4 small interfering RNAs (siRNAs) increased average size, Ctsk mRNA expression level, and bone resorption activity of the derived osteoclast-like cells. Activation of the EphA4 signaling in osteoclast precursors with EfnA4-fc chimeric protein reduced cell size and resorption activity of the derived osteoclasts. Homozygous Epha4 null mice had substantially less trabecular bone in femur and vertebra compared to wild-type controls. The bone loss was due to a decrease in trabecular number and an increase in trabecular spacing, but not to an increase in osteoclast-lined bone surface or an increase in the number of osteoclasts on bone surface. Dynamic histomorphometry and serum biomarker analyses indicate that bone formation in Epha4 null mice was reduced slightly but not significantly. Osteoclasts of Epha4 null mice were also larger, expressed higher levels of Mmp3 and Mmp9 mRNAs, and exhibited greater bone resorption activity than wild-type osteoclasts in vitro. Deficient Epha4 expression had no effects on the total number of osteoclast formed in response to receptor activator of NF-κB ligand nor on apoptosis of osteoclasts in vitro. It also did not affect the protein-tyrosine phosphorylation status of its ligands, EfnB2, EfnA2, and EfnA4, in osteoclasts. Deficient Epha4 expression in Epha4 null osteoclasts activated the ß3 -integrin signaling through reduced phosphorylation of the tyr-747 residue, which led to increased binding of the stimulatory talin and reduced binding of the inhibitory Dok1 to ß3 -integrin. This in turn activated Vav3 and the bone resorption activity of osteoclasts. In conclusion, we demonstrate for the first time that EphA4 is a potent negative regulator of osteoclastic activity, mediated in part through increased Dok1 binding to ß3 -integrin via an increase in EphA4-dependent tyr-747 phosphorylation.


Asunto(s)
Osteoclastos/citología , Receptor EphA4/fisiología , Animales , Remodelación Ósea , Línea Celular , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Receptor EphA4/metabolismo , Transducción de Señal , Microtomografía por Rayos X
8.
Am J Physiol Cell Physiol ; 302(11): C1676-86, 2012 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-22460711

RESUMEN

This study utilized the glutathione transferase (GST) pull-down assay to identify novel substrates of an osteoclastic protein-tyrosine phosphatase, PTP-oc. Consistent with the previous findings that the phosphorylated tyr-527 (pY527) of Src is a substrate of PTP-oc, the major protein pulled down with the phosphatase-deficient (PD)-PTP-oc-GST trapping mutant in RAW264.7 cells was Src. The GST-PD-PTP-oc also pulled down pY-Syk and pY-ß(3)-integrin, but not after PP2 pretreatment. However, PTP-oc transgenic osteoclasts or PTP-oc-overexpressing RAW264.7 cells had elevated, and not reduced, levels of pY525/526-Syk and pY759-ß(3) integrin, and the PTP-oc siRNA treatment drastically reduced levels of pY525/526 Syk and pY759-ß(3)-integrin in RAW264.7 cells. These findings are incompatible with the premise that they are substrates of PTP-oc. The PTP-oc-dependent increases in pY525/526-Syk and pY759-ß(3)-integrin levels were completely blocked by PP2, indicating that these effects are secondary to PTP-oc-mediated activation of the Src protein-tyrosine kinase (PTK). Overexpression of PTP-oc increased, and siRNA-mediated suppression of PTP-oc reduced, pY160-Vav1, pY173-Vav3, and pY783-PLCγ levels, and Rac1 activation, which are downstream mediators of the ITAM/Syk signaling. Overexpression of PTP-oc also increased, and PTP-oc siRNA treatment decreased, the pY-Shp1 levels, which were blocked by PP2. Since Shp1 is a negative regulator of osteoclast activity and is a key mediator of the ITIM signaling, these findings suggest that PTP-oc is an upstream suppressor of the ITIM/Shp1 signaling through PTP-oc-induced Src-dependent Shp1 phosphorylation. In summary, PTP-oc plays a central regulatory role in the concerted regulation of the ß(3)-integrin, the ITAM/Syk, and the ITIM/Shp1 signaling indirectly through activation of Src PTK.


Asunto(s)
Integrina beta3/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Osteoclastos/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Familia-src Quinasas/metabolismo , Animales , Resorción Ósea , Línea Celular , Integrina beta3/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Neuropéptidos/biosíntesis , Neuropéptidos/metabolismo , Osteoclastos/fisiología , Fosfolipasa C gamma/biosíntesis , Fosforilación , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-vav/biosíntesis , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Quinasa Syk , Proteínas de Unión al GTP rac/biosíntesis , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1
9.
Reprod Sci ; 16(4): 364-72, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19336695

RESUMEN

OBJECTIVE: The purpose of this study was to investigate the mechanisms of tension and intracellular calcium regulation following stimulation with the thromboxane A(2) receptor agonist U46619 in the left anterior descending coronary artery of fetal sheep exposed to long-term hypoxia. We hypothesized that there would be a reduction in intracellular calcium responses in long-term hypoxic left anterior descending coronary artery accompanied by an increase in calcium sensitivity of the contractile mechanism. METHODS: Pregnant sheep were kept at altitude (3820 m) from day 30 of gestation until day 140. Fetal hearts from long-term hypoxic and from a control, normoxic group were obtained and the left anterior descending coronary artery of the fetus was dissected, cleaned, and mounted in a bath (Jasco) in which tension and intracellular calcium [Ca(2+)](i), using Fura-2, could be measured simultaneously following stimulation of the thromboxane A(2) receptor with U46619. The role of intracellular calcium and the Rho kinase and protein kinase C pathways in the tension responses were investigated by maintaining intracellular calcium constant or by using the Rho kinase blocker, Y27632, or the protein kinase C blocker, GF109203-X. RESULTS: There was no difference in the tension dose-response to U46619 between the normoxic fetal and hypoxic fetal left anterior descending, although [Ca(2+)](i) was lower in the hypoxic fetal than normoxic fetal at the highest doses. When [Ca(2+)]( i) was maintained constant at baseline levels, U46619 produced the same tension dose-response in both normoxic fetal and hypoxic fetal left anterior descending as when [Ca(2+)](i) was allowed to rise. The tension response was abolished in both groups when the Rho kinase inhibitor, Y27632, was given either during or before stimulation with U46619. The protein kinase C blocker, GF109203-X, had no effect on the tension response in either group. CONCLUSIONS: Long-term hypoxia did not alter the tension response to thromboxane A(2) receptor stimulation in fetal left anterior descending. The contractions in response to U46619 were produced apparently completely by changes in calcium sensitivity through the Rho kinase pathway.


Asunto(s)
Calcio/análisis , Vasos Coronarios/embriología , Hipoxia Fetal/fisiopatología , Contracción Muscular/fisiología , Músculo Liso Vascular/fisiopatología , Receptores de Tromboxano A2 y Prostaglandina H2/fisiología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Altitud , Animales , Calcio/fisiología , Vasos Coronarios/química , Vasos Coronarios/fisiopatología , Femenino , Hipoxia Fetal/etiología , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Embarazo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Receptores de Tromboxano A2 y Prostaglandina H2/agonistas , Receptores de Tromboxano A2 y Prostaglandina H2/efectos de los fármacos , Ovinos/embriología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/fisiología
10.
J Biol Chem ; 284(17): 11531-45, 2009 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-19244239

RESUMEN

This study evaluated whether transgenic expression of PTP-oc (osteoclastic transmembrane protein-tyrosine phosphatase) in cells of the osteoclast lineage would affect bone resorption and bone density in young adult mice. Transgenic mice were generated with a transgenic construct using a tartrate-resistant acid phosphatase exon 1C promoter to drive expression of rabbit PTP-oc in osteoclastic cells. pQCT evaluation of femurs of young adult male progeny of three lines showed that transgenic mice had reduced bone volume and area, cortical and trabecular bone mineral content, and density. Histomorphometric analyses at secondary spongiosa of the femur and at metaphysis of the L4 vertebra confirmed that male transgenic mice had decreased trabecular surface, reduced percentage of trabecular area, decreased trabecular number, increased trabecular separation, and increased osteoclast number per bone surface length. Consistent with an increase in bone resorption, the serum C-telopeptide level was 25% higher in transgenic mice than in wild-type littermates. However, the bone phenotype was not readily observed in female young adult transgenic mice. This could in part be due to potential interactions between estrogen and PTP-oc signaling, since the bone loss phenotype was seen in young adult ovariectomized transgenic mice by microcomputed tomography analysis. In vitro, the average pit area per resorption pit created by marrow-derived transgenic osteoclasts was approximately 50% greater than that created by wild-type osteoclasts. Transgenic osteoclasts showed a lower c-Src phosphotyrosine 527 level, greater c-Src kinase activity, and increased tyrosine phosphorylation of paxillin. In summary, this study provides compelling in vivo evidence that PTP-oc is a positive regulator of osteoclasts.


Asunto(s)
Resorción Ósea , Osteoclastos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/fisiología , Animales , Membrana Celular/metabolismo , Colágeno Tipo I/metabolismo , Fémur/metabolismo , Masculino , Ratones , Ratones Transgénicos , Péptidos/metabolismo , Regiones Promotoras Genéticas , Conejos , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Transducción de Señal , Familia-src Quinasas/metabolismo
11.
J Soc Gynecol Investig ; 13(1): 11-8, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16303320

RESUMEN

OBJECTIVES: We have previously shown that after exposure to long-term hypoxia, fetal coronary flow is maintained at control levels despite a 25% reduction in cardiac output. We also demonstrated that coronary vascular rings isolated from the long-term hypoxic fetuses and studied in well-oxygenated bath system displayed significantly reduced depolarization-induced contraction strength in response to KCl. To study the mechanism of reduced fetal coronary vascular responses to KCl-induced contractions following exposure to long-term hypoxia, we measured tension and intracellular calcium simultaneously, as well as L-type Ca2+ channel density and sensitivity. METHODS: Pregnant ewes were housed at altitude (3820 m) for approximately 110 days. At 138 to 141 days of gestation, long-term hypoxic and control animals were killed and fetal and adult left anterior descending coronary artery (LAD) was isolated and studied in a well-oxygenated bath system. Tension and intracellular calcium ([Ca2+]i) were measured simultaneously in response to increasing concentrations of KCl and, in addition, the sensitivity to the calcium channel blocker nifedipine was measured at a half maximal concentration of KCl. We also measured L-type Ca2+ channel density with (+)-[3H]PN200-110. RESULTS: L-type Ca2+ channel density was decreased by approximately 31% in the long-term hypoxic fetal, but not adult, LAD. Tension in the long-term hypoxic fetal and adult LAD was significantly lower at all concentrations of KCl. [Ca2+]i was lower at rest in both fetal and adult LAD from long-term hypoxic animals and increased to lower levels at all concentrations of KCl. The ratio of tension to [Ca2+]i was also lower at all concentrations of KCl. Sensitivity to nifedipine was unchanged. CONCLUSIONS: The reduced L-type Ca2+ channel density and the reduced [Ca2+]i response to KCl, as well as the reduced tension response to [Ca2+]i, could potentially be involved in the reduction in depolarization-induced contractions in LAD from long-term hypoxic fetuses. In hypoxic adults, reduced [Ca2+]i and reduced tension response to [Ca2+]i may be involved in the lower tension response to KCl-induced contractions.


Asunto(s)
Calcio/metabolismo , Vasos Coronarios/fisiología , Hipoxia Fetal/fisiopatología , Adaptación Fisiológica , Altitud , Animales , Calcio/análisis , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L , Vasos Coronarios/química , Feto/fisiología , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Nifedipino/farmacología , Cloruro de Potasio/farmacología , Flujo Sanguíneo Regional , Ovinos , Vasodilatación/fisiología
12.
J Soc Gynecol Investig ; 11(1): 1-8, 2004 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-14706676

RESUMEN

OBJECTIVE: We studied the effects of long-term high-altitude hypoxia and protein kinase A (PKA) phosphorylation on calcium (Ca2+) responses of skinned cardiac papillary muscles from fetal and adult sheep. METHODS: Fetal and nonpregnant adult sheep were exposed to high-altitude (3820 m), long-term (approximately 110 days) hypoxia. Papillary muscles were isolated and mounted in well-oxygenated, temperature-controlled baths. After the papillary muscles were stimulated electrically to establish the diastolic tension that produced the maximum active contraction, the electrical stimulation was stopped, and the muscles were skinned with 1% vol/vol Triton-X-100. In protocol 1, the skinned muscles were exposed to activating solutions containing different calcium concentrations (pCa; from pCa 8.0 to pCa 4.0), which were prepared by varying the Ca-EGTA/EGTA ratio, and the steady-state tension was measured at each pCa. In protocol 2, the skinned muscles were contracted with activating solution containing a pCa of 5.0. After equilibration, the solution in some baths was changed to activating solution at the same pCa of 5.0 but also containing the catalytic subunit of PKA. The other baths were exchanged with activating solution at a pCa of 5.0 containing no PKA. We then measured the degree of tension reduction caused by PKA until tension reached a new steady state. RESULTS: In the long-term hypoxic fetal heart, the maximum tension response of right, but not left, ventricular skinned papillary muscle to Ca2+ was significantly less than that in control muscles. In the long-term hypoxic adult heart, the left ventricle, but not the right ventricle, displayed an increased maximum tension response to Ca2+ compared with control. Phosphorylation of troponin I (TnI) with PKA reduced active tension in both fetal ventricles of the long-term hypoxic group more than in hearts from control fetuses. In the adult, phosphorylation with PKA resulted in a larger decrease in tension in the left ventricle and a smaller decrease in tension in the right ventricle in the long-term hypoxic group, although the differences were small. CONCLUSION: In the long-term hypoxic fetal right ventricle, the decreased maximum tension response to Ca2+ is consistent with the decrease in myofibrillar magnesium-activated adenosine triphosphatase activity observed previously. The larger decrease in tension after PKA phosphorylation of TnI in the long-term hypoxic fetal left ventricle indicates a larger reduction in Ca2+ binding to troponin C.


Asunto(s)
Altitud , Calcio/farmacología , Corazón Fetal/efectos de los fármacos , Hipoxia/fisiopatología , Músculos Papilares/efectos de los fármacos , Troponina I/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/fisiología , Animales , Calcio/administración & dosificación , Gasto Cardíaco , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ácido Egtácico/administración & dosificación , Estimulación Eléctrica , Femenino , Corazón Fetal/fisiopatología , Hipoxia Fetal/fisiopatología , Edad Gestacional , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/fisiopatología , Músculos Papilares/embriología , Músculos Papilares/fisiopatología , Fosforilación , Embarazo , Ovinos/embriología
13.
J Soc Gynecol Investig ; 10(4): 189-93, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12759146

RESUMEN

OBJECTIVE: In fetal sheep, we found that the augmentation of cardiac contractility by beta-adrenergic receptor (beta-AR) stimulation was reduced after exposure to long-term hypoxia. However, cyclic adenosine monophosphate (cAMP) production after beta-AR stimulation was higher in long-term hypoxic fetal sheep than in normoxic ones. Therefore, we studied the potential role of changes in myocardial protein kinase A (PKA) activity and troponin I (TnI) isoforms in fetal and nonpregnant sheep exposed to approximately 112 days of hypoxia at high altitude (3820 m). METHODS: Resting and maximally stimulated (by cAMP) PKA activity was measured by phosphorylation of the artificial peptide, Kemptide. Specificity was confirmed by inhibition with PKI, a specific PKA inhibitor. For TnI isoforms, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to resolve the proteins. We used monoclonal anti-cardiac TnI antibody (clone C5), which also cross-reacted with slow skeletal muscle TnI, to identify TnI isoforms. RESULTS: For the fetal hearts, resting PKA activity was significantly higher in the high-altitude group than the control group, but total PKA activity was not different between the normoxic and hypoxic groups. In the adult hearts, no significant difference was observed in either resting or total PKA activity between normoxic and hypoxic groups. For both the fetal and adult sheep, the predominant TnI was the cardiac isoform, and hypoxic exposure produced no change in the TnI isoform composition. CONCLUSIONS: Neither a reduction in PKA activity nor a change in TnI isoforms could explain the reduction in beta-receptor augmentation of cardiac contractility in fetal sheep exposed to long-term hypoxia.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Corazón Fetal/fisiopatología , Feto/fisiopatología , Hipoxia/enzimología , Miocardio/enzimología , Ovinos/fisiología , Troponina I/metabolismo , Altitud , Animales , Femenino , Embarazo , Isoformas de Proteínas , Distribución Aleatoria , Ovinos/embriología
14.
J Soc Gynecol Investig ; 9(6): 335-41, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-12445597

RESUMEN

OBJECTIVE: We studied the effect of long-term, high-altitude hypoxia on cardiac myosin, actin, and troponin T (TnT) isoforms and Ca(2+)- and Mg(2+)-activated myofibrillar adenosine triphosphatase (ATPase) activities in fetal and adult sheep. METHODS: We exposed pregnant (beginning at day 30 of gestation) and nonpregnant sheep to high altitude (3820 m) for 110 days. Myosin, actin, and TnT isoforms were analyzed by Western analysis. In purified myofibrillar preparations, Ca(2+)(-) and Mg(2+)-ATPase activities were measured by the appearance of inorganic phosphate after the addition of NaATP and various concentrations of either calcium or magnesium to the reaction mixture. RESULTS: We found no change in myosin, actin, or TnT isoform composition after exposure to long-term hypoxia in either fetal or adult sheep. However, Mg(2+)-activated myofibrillar ATPase activity decreased significantly in the right ventricle of both fetus and adult after hypoxic exposure. There was also a significant maturational increase in both Ca(2+)- and Mg(2+)-ATPase activity in control animals. CONCLUSION: The decrease in Mg(2+)-activated myofibrillar ATPase activity might affect the decrease in cardiac contractility previously noted in the right ventricle of fetal sheep after exposure to long-term hypoxia. Likewise, the increase in Ca(2+)- and Mg(2+)-activated ATPase activities from the fetus to adult could partially explain the previously found maturational increase in cardiac contractility.


Asunto(s)
Proteínas Contráctiles/análisis , Feto/metabolismo , Hipoxia/metabolismo , Miocardio/química , Actinas/análisis , Altitud , Animales , Western Blotting , ATPasa de Ca(2+) y Mg(2+)/análisis , Calcio/administración & dosificación , ATPasas Transportadoras de Calcio/análisis , Electroforesis en Gel de Poliacrilamida , Femenino , Edad Gestacional , Magnesio/administración & dosificación , Miofibrillas/enzimología , Miosinas/análisis , Embarazo , Ovinos , Troponina T/análisis
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