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1.
JCI Insight ; 1(20): e90220, 2016 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-27942593

RESUMO

Noonan syndrome (NS) is a common autosomal dominant disorder that presents with short stature, craniofacial dysmorphism, and cardiac abnormalities. Activating mutations in the PTPN11 gene encoding for the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) causes approximately 50% of NS cases. In contrast, NS with multiple lentigines (NSML) is caused by mutations that inactivate SHP2, but it exhibits some overlapping abnormalities with NS. Protein zero-related (PZR) is a SHP2-binding protein that is hyper-tyrosyl phosphorylated in the hearts of mice from NS and NSML, suggesting that PZR and the tyrosine kinase that catalyzes its phosphorylation represent common targets for these diseases. We show that the tyrosine kinase inhibitor, dasatinib, at doses orders of magnitude lower than that used for its anticancer activities inhibited PZR tyrosyl phosphorylation in the hearts of NS mice. Low-dose dasatinib treatment of NS mice markedly improved cardiomyocyte contractility and functionality. Remarkably, a low dose of dasatinib reversed the expression levels of molecular markers of cardiomyopathy and reduced cardiac fibrosis in NS and NSML mice. These results suggest that PZR/SHP2 signaling is a common target of both NS and NSML and that low-dose dasatinib may represent a unifying therapy for the treatment of PTPN11-related cardiomyopathies.


Assuntos
Dasatinibe/administração & dosagem , Miócitos Cardíacos/efeitos dos fármacos , Síndrome de Noonan/tratamento farmacológico , Animais , Dasatinibe/farmacologia , Fibrose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Miocárdio/patologia , Fosfoproteínas/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transdução de Sinais
2.
Sci Rep ; 6: 32068, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27572147

RESUMO

We have developed an engineered heart tissue (EHT) system that uses laser-cut sheets of decellularized myocardium as scaffolds. This material enables formation of thin muscle strips whose biomechanical characteristics are easily measured and manipulated. To create EHTs, sections of porcine myocardium were laser-cut into ribbon-like shapes, decellularized, and mounted in specialized clips for seeding and culture. Scaffolds were first tested by seeding with neonatal rat ventricular myocytes. EHTs beat synchronously by day five and exhibited robust length-dependent activation by day 21. Fiber orientation within the scaffold affected peak twitch stress, demonstrating its ability to guide cells toward physiologic contractile anisotropy. Scaffold anisotropy also made it possible to probe cellular responses to stretch as a function of fiber angle. Stretch that was aligned with the fiber direction increased expression of brain natriuretic peptide, but off-axis stretches (causing fiber shear) did not. The method also produced robust EHTs from cardiomyocytes derived from human embryonic stem cells and induced pluripotent stem cells (hiPSC). hiPSC-EHTs achieved maximum peak stress of 6.5 mN/mm(2) and twitch kinetics approaching reported values from adult human trabeculae. We conclude that laser-cut EHTs are a viable platform for novel mechanotransduction experiments and characterizing the biomechanical function of patient-derived cardiomyoctyes.


Assuntos
Miocárdio , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Anisotropia , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Lasers de Gás , Mecanotransdução Celular , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Politetrafluoretileno , Ratos , Suínos , Tomografia de Coerência Óptica , Tri-Iodotironina/farmacologia
3.
Proc Natl Acad Sci U S A ; 111(46): 16604-9, 2014 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-25368166

RESUMO

Cardiac disorders are the main cause of mortality in autosomal-dominant polycystic kidney disease (ADPKD). However, how mutated polycystins predispose patients with ADPKD to cardiac pathologies before development of renal dysfunction is unknown. We investigate the effect of decreased levels of polycystin 2 (PC2), a calcium channel that interacts with the ryanodine receptor, on myocardial function. We hypothesize that heterozygous PC2 mice (Pkd2(+/-)) undergo cardiac remodeling as a result of changes in calcium handling, separate from renal complications. We found that Pkd2(+/-) cardiomyocytes have altered calcium handling, independent of desensitized calcium-contraction coupling. Paradoxically, in Pkd2(+/-) mice, protein kinase A (PKA) phosphorylation of phospholamban (PLB) was decreased, whereas PKA phosphorylation of troponin I was increased, explaining the decoupling between calcium signaling and contractility. In silico modeling supported this relationship. Echocardiography measurements showed that Pkd2(+/-) mice have increased left ventricular ejection fraction after stimulation with isoproterenol (ISO), a ß-adrenergic receptor (ßAR) agonist. Blockers of ßAR-1 and ßAR-2 inhibited the ISO response in Pkd2(+/-) mice, suggesting that the dephosphorylated state of PLB is primarily by ßAR-2 signaling. Importantly, the Pkd2(+/-) mice were normotensive and had no evidence of renal cysts. Our results showed that decreased PC2 levels shifted the ßAR pathway balance and changed expression of calcium handling proteins, which resulted in altered cardiac contractility. We propose that PC2 levels in the heart may directly contribute to cardiac remodeling in patients with ADPKD in the absence of renal dysfunction.


Assuntos
Sinalização do Cálcio/fisiologia , Acoplamento Excitação-Contração/fisiologia , Miócitos Cardíacos/fisiologia , Receptores Adrenérgicos beta 1/fisiologia , Receptores Adrenérgicos beta 2/fisiologia , Canais de Cátion TRPP/deficiência , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Pressão Sanguínea/genética , Cafeína/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Estimulação Cardíaca Artificial , Acoplamento Excitação-Contração/efeitos dos fármacos , Acoplamento Excitação-Contração/genética , Heterozigoto , Isoproterenol/farmacologia , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/biossíntese , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais/fisiologia , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/fisiologia , Troponina I/metabolismo , Remodelação Ventricular/efeitos dos fármacos , Remodelação Ventricular/genética , Remodelação Ventricular/fisiologia
4.
Calcif Tissue Int ; 95(5): 446-56, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25190190

RESUMO

The influence of the macroscale material properties of bone on its mechanical competence has been extensively investigated, but less is known about possible contributions from bone's nanoscale material properties. These nanoscale properties, particularly the collagen network and the size and shape of hydroxyapatite mineral crystals, may be affected by aging, mechanical loading, and diseases including osteoporosis. Here, changes to the collagen and mineral properties of cortical bone induced by osteoporosis and subsequent pharmaceutical treatments were investigated by simultaneous small- and wide-angle X-ray scattering (SAXS/WAXS) microbeam mapping. Adult rats (6 months old) were ovariectomized and treated with alendronate, parathyroid hormone, or sodium fluoride, and compared to untreated ovariectomized and age-matched controls. Scattering data from tibial cortical bone showed that osteoporosis increased collagen alignment in existing intracortical bone, while this effect was mitigated in the alendronate and sodium fluoride groups though by different mechanisms. Further, mineral crystal lengths in newly formed cortical bone were smaller in animals with osteoporosis, but existing cortical bone was not altered. Subsequent treatment with alendronate mitigated changes in crystal lengths. Together, these results suggest that osteoporosis may alter the collagen alignment and mineral geometry in bone formed before and after the onset of this disease, and that osteoporosis treatments may differentially rescue these changes.


Assuntos
Conservadores da Densidade Óssea/farmacologia , Osso e Ossos/ultraestrutura , Colágeno/ultraestrutura , Minerais , Osteoporose Pós-Menopausa/patologia , Alendronato/farmacologia , Animais , Osso e Ossos/efeitos dos fármacos , Colágeno/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Minerais/química , Ovariectomia , Ratos , Ratos Sprague-Dawley , Espalhamento a Baixo Ângulo , Fluoreto de Sódio/farmacologia , Difração de Raios X
5.
Bone ; 66: 296-305, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24929241

RESUMO

Spaceflight provides a unique environment for skeletal tissue causing decrements in structural and densitometric properties of bone. Previously, we used the adult hindlimb unloaded (HU) rat model to show that previous exposure to HU had minimal effects on bone structure after a second HU exposure followed by recovery. Furthermore, we found that the decrements during second HU exposure were milder than the initial HU cycle. In this study, we used a moderate intensity resistance exercise protocol as an anabolic stimulus during recovery to test the hypothesis that resistance exercise following an exposure to HU will significantly enhance recovery of densitometric, structural, and, more importantly, mechanical properties of trabecular and cortical bone. We also hypothesized that resistance exercise during recovery, and prior to the second unloading period, will mitigate the losses during the second exposure. The hypothesis that exercise during recovery following hindlimb unloading will improve bone quality was supported by our data, as total BMC, total vBMD, and cancellous bone formation at the proximal tibia metaphysis increased significantly during exercise period, and total BMC/vBMD exceeded age-matched control and non-exercised values significantly by the end of recovery. However, our results did not support the hypothesis that resistance exercise prior to a subsequent unloading period will mitigate the detrimental effects of the second exposure, as the losses during the second exposure in total BMC, total vBMD, and cortical area at the proximal tibia metaphysis for the exercised animals were similar to those of the non-exercised group. Therefore, exercise did not mitigate effects of the second HU exposure in terms of pre-to-post HU changes in these variables, but it did produce beneficial effects in a broader sense.


Assuntos
Transtornos Musculares Atróficos/fisiopatologia , Condicionamento Físico Animal , Tíbia/fisiopatologia , Animais , Biomarcadores/metabolismo , Fenômenos Biomecânicos , Peso Corporal , Reabsorção Óssea/sangue , Reabsorção Óssea/diagnóstico por imagem , Reabsorção Óssea/patologia , Reabsorção Óssea/fisiopatologia , Densitometria , Masculino , Transtornos Musculares Atróficos/sangue , Transtornos Musculares Atróficos/diagnóstico por imagem , Transtornos Musculares Atróficos/patologia , Ratos Sprague-Dawley , Tíbia/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Microtomografia por Raio-X
6.
Bone ; 56(2): 461-73, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23871849

RESUMO

Extended periods of inactivity cause severe bone loss and concomitant deterioration of the musculoskeletal system. Considerable research has been aimed at better understanding the mechanisms and consequences of bone loss due to unloading and the associated effects on strength and fracture risk. One factor that has not been studied extensively but is of great interest, particularly for human spaceflight, is how multiple or repeated exposures to unloading and reloading affect the skeleton. Space agencies worldwide anticipate increased usage of repeat-flier crewmembers, and major thrust of research has focused on better understanding of microgravity effects on loss of bone density at weightbearing skeletal sites; however there is limited data available on repeat microgravity exposure. The adult hindlimb unloaded (HU) rat model was used to determine how an initial unloading cycle will affect a subsequent exposure to disuse and recovery thereafter. Animals underwent 28 days of HU starting at 6 months of age followed by 56 days of recovery, and then another 28 days of HU with 56 days of recovery. In vivo longitudinal pQCT was used to quantify bone morphological changes, and ex vivo µCT was used to quantify trabecular microarchitecture and cortical shell geometry at the proximal tibia metaphysis (PTM). The mechanical properties of trabecular bone were examined by the reduced platen compression mechanical test. The hypothesis that the initial HU exposure will mitigate decrements in bone mass and density for the second HU exposure was supported as pre- to post-HU declines in total BMC, total vBMD, and cortical area by in vivo pQCT at the proximal tibia metaphysis were milder for the second HU (and not significant) compared to an age-matched single HU (3% vs. 6%, 2% vs. 6%, and 2% vs. 6%, respectively). In contrast, the hypothesis was not supported at the microarchitectural level as losses in BV/TV and Tb.Th. were similar during 2nd HU exposure and age-matched single HU. Recovery with respect to post-HU values and compared to aging controls for total BMC, vBMD and cortical area were slower in older animals exposed to single or double HU cycles compared to recovery of younger animals exposed to a single HU bout. Despite milder recovery at the older age, there was no difference between unloaded animals and controls at the end of second recovery period. Therefore, the data did not support the hypothesis that two cycles of HU exposure with recovery would have a net negative effect. Mechanical properties of trabecular bone were affected more severely than densitometric measures (35% loss in trabecular bone ultimate stress vs. 9% loss in trabecular vBMD), which can be attributed most prominently to reductions in trabecular bone density and tissue mineral density. Together, our data demonstrate that initial exposure to mechanical unloading does not exacerbate bone loss during a subsequent unloading period and two cycles of unloading followed by recovery do not have a cumulative net negative effect on total bone mineral content and density as measured by pQCT at the proximal tibia metaphysis.


Assuntos
Elevação dos Membros Posteriores/fisiologia , Tíbia/fisiologia , Animais , Densidade Óssea/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Ratos , Simulação de Ausência de Peso
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