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1.
J Mol Cell Cardiol ; 67: 1-11, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24342076

RESUMO

Clinical application of potent anthracycline anticancer drugs, especially doxorubicin (DOX), is limited by a toxic cardiac side effect that is not fully understood and preventive strategies are yet to be established. Studies in genetically modified mice have demonstrated that focal adhesion kinase (FAK) plays a key role in regulating adaptive responses of the adult myocardium to pathological stimuli through activation of intracellular signaling cascades that facilitate cardiomyocyte growth and survival. The objective of this study was to determine if targeted myocardial FAK activation could protect the heart from DOX-induced de-compensation and to characterize the underlying mechanisms. To this end, mice with myocyte-restricted FAK knock-out (MFKO) or myocyte-specific expression of an active FAK variant (termed SuperFAK) were subjected to DOX treatment. FAK depletion enhanced susceptibility to DOX-induced myocyte apoptosis and cardiac dysfunction, while elevated FAK activity provided remarkable cardioprotection. Our mec6hanistic studies reveal a heretofore unappreciated role for the protective cyclin-dependent kinase inhibitor p21 in the repression of the pro-apoptotic BH3-only protein Bim and the maintenance of mitochondrial integrity and myocyte survival. DOX treatment induced proteasomal degradation of p21, which exacerbated mitochondrial dysfunction and cardiomyocyte apoptosis. FAK was both necessary and sufficient for maintaining p21 levels following DOX treatment and depletion of p21 compromised FAK-dependent protection from DOX. These findings identify p21 as a key determinant of DOX resistance downstream of FAK in cardiomyocytes and indicate that cardiac-restricted enhancement of the FAK/p21 signaling axis might be an effective strategy to preserve myocardial function in patients receiving anthracycline chemotherapy.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Doxorrubicina/antagonistas & inibidores , Doxorrubicina/toxicidade , Quinase 1 de Adesão Focal/metabolismo , Miócitos Cardíacos/patologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Miocárdio/enzimologia , Miocárdio/patologia , Miócitos Cardíacos/enzimologia , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos
2.
Arterioscler Thromb Vasc Biol ; 32(4): 924-33, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22383703

RESUMO

OBJECTIVE: We previously reported that cardiac-restricted deletion of focal adhesion kinase (FAK) exacerbated myocyte death following ischemia/reperfusion (I/R). Here, we interrogated whether targeted elevation of myocardial FAK activity could protect the heart from I/R injury. METHODS AND RESULTS: Transgenic mice were generated with myocyte-specific expression of a FAK variant (termed SuperFAK) that conferred elevated allosteric activation. FAK activity in unstressed transgenic hearts was modestly elevated, but this had no discernable effect on anabolic heart growth or cardiac function. Importantly, SuperFAK hearts exhibited a dramatic increase in FAK activity and a reduction in myocyte apoptosis and infarct size 24 to 72 hours following I/R. Moreover, serial echocardiography revealed that the transgenic mice were protected from cardiac decompensation for up to 8 weeks following surgery. Mechanistic studies revealed that elevated FAK activity protected cardiomyocytes from I/R-induced apoptosis by enhancing nuclear factor-κB (NF-κB)-dependent survival signaling during the early period of reperfusion (30 and 60 minutes). Moreover, adenoviral-mediated expression of SuperFAK in cultured cardiomyocytes attenuated H(2)O(2) or hypoxia/reoxygenation-induced apoptosis, whereas blockade of the NF-κB pathway using a pharmacological inhibitor or small interfering RNAs completely abolished the beneficial effect of SuperFAK. CONCLUSIONS: Enhancing cardiac FAK activity attenuates I/R-induced myocyte apoptosis through activation of the prosurvival NF-κB pathway and may represent a novel therapeutic strategy for ischemic heart diseases.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Terapia Genética , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/enzimologia , Animais , Apoptose , Células Cultivadas , Modelos Animais de Doenças , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/genética , Peróxido de Hidrogênio/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Interferência de RNA , Ratos , Fatores de Tempo , Transfecção , Função Ventricular Esquerda , Remodelação Ventricular
3.
Circ Res ; 104(10): 1201-8, 2009 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-19372463

RESUMO

Focal adhesion kinase (FAK) is strongly activated by integrins and growth factors and is essential for embryonic development. We previously showed that the C terminus of FAK is expressed as a separate protein termed FAK-related nonkinase (FRNK) in a smooth muscle cell-selective fashion and that FRNK functions to buffer FAK-dependent signals. We now show that FRNK is also transiently expressed in the neonatal myocardium, with peak levels occurring 5 to 7 days postnatal, just before cell cycle withdrawal. Using novel mouse models, we demonstrate that cardiac-selective expression of FRNK (leading to inhibition of FAK) starting at embryonic day 10.5 leads to a severe ventricular noncompaction defect associated with reduced cardiomyocyte proliferation. Remarkably, postnatal expression of nearly identical levels of FRNK is well tolerated and does not affect viability or anabolic cardiac growth. Nonetheless, FRNK expression in the adult heart does attenuate pathological cardiac hypertrophy following aortic banding, confirming and extending our previous data that this compensatory response is blunted in FAK null hearts. Our mechanistic studies in cultured neonatal cardiomyocytes reveal that FRNK expression induces p38/p27(kip)-dependent cell cycle withdrawal and attenuates extracellular signal-regulated kinase-dependent hypertrophic growth. These findings indicate that dynamic expression of FRNK in the neonatal heart may function to promote cardiomyocyte quiescence in an environment that is particularly rich in growth factors and growth promoting extracellular matrices.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Ciclo Celular/fisiologia , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/citologia , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
4.
Circ Res ; 102(12): 1502-11, 2008 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-18497331

RESUMO

Leupaxin is a LIM domain-containing adapter protein belonging to the paxillin family that has been previously reported to be preferentially expressed in hematopoietic cells. Herein, we identified leupaxin in a screen for focal adhesion kinase binding partners in aortic smooth muscle, and we show that leupaxin is enriched in human and mouse vascular smooth muscle and that leupaxin expression is dynamically regulated during development. In addition, our studies reveal that leupaxin can undergo cytoplasmic/nuclear shuttling and functions as an serum response factor cofactor in the nucleus. We found that leupaxin forms a complex with serum response factor and associates with CArG-containing regions of smooth muscle promoters and that ectopic expression of leupaxin induces smooth muscle marker gene expression in both 10T1/2 cells and rat aortic smooth muscle cells. Subsequent studies indicated that enhanced focal adhesion kinase activity (induced by fibronectin or expression of constitutively active focal adhesion kinase) attenuates the nuclear accumulation of leupaxin and limits the ability of leupaxin to enhance serum response factor-dependent gene transcription. Thus, these studies indicate that modulation of the subcellular localization of serum response factor cofactors is 1 mechanism by which extracellular matrix-dependent signals may regulate phenotypic switching of smooth muscle cells.


Assuntos
Moléculas de Adesão Celular/fisiologia , Miócitos de Músculo Liso/metabolismo , Fosfoproteínas/fisiologia , Elemento de Resposta Sérica/fisiologia , Animais , Aorta/citologia , Aorta/embriologia , Aorta/crescimento & desenvolvimento , Transporte Biológico , Moléculas de Adesão Celular/farmacologia , Diferenciação Celular , Células Cultivadas/efeitos dos fármacos , Vasos Coronários/citologia , Feminino , Quinase 1 de Adesão Focal/fisiologia , Adesões Focais/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Miométrio/citologia , Especificidade de Órgãos , Fosfoproteínas/farmacologia , Mapeamento de Interação de Proteínas , Ratos , Proteínas Recombinantes de Fusão/fisiologia , Elemento de Resposta Sérica/efeitos dos fármacos , Fator de Resposta Sérica/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição , Transcrição Gênica
5.
Mol Cell Biol ; 27(15): 5352-64, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17526730

RESUMO

To examine a role for focal adhesion kinase (FAK) in cardiac morphogenesis, we generated a line of mice with a conditional deletion of FAK in nkx2-5-expressing cells (herein termed FAKnk mice). FAKnk mice died shortly after birth, likely resulting from a profound subaortic ventricular septal defect and associated malalignment of the outflow tract. Additional less penetrant phenotypes included persistent truncus arteriosus and thickened valve leaflets. Thus, conditional inactivation of FAK in nkx2-5-expressing cells leads to the most common congenital heart defect that is also a subset of abnormalities associated with tetralogy of Fallot and the DiGeorge syndrome. No significant differences in proliferation or apoptosis between control and FAKnk hearts were observed. However, decreased myocardialization was observed for the conal ridges of the proximal outflow tract in FAKnk hearts. Interestingly, chemotaxis was significantly attenuated in isolated FAK-null cardiomyocytes in comparison to genetic controls, and these effects were concomitant with reduced tyrosine phosphorylation of Crk-associated substrate (CAS). Thus, it is possible that ventricular septation and appropriate outflow tract alignment is dependent, at least in part, upon FAK-dependent CAS activation and subsequent induction of polarized myocyte movement into the conal ridges. Future studies will be necessary to determine the precise contributions of the additional nkx2-5-derived lineages to the phenotypes observed.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/deficiência , Deleção de Genes , Cardiopatias Congênitas/enzimologia , Ventrículos do Coração/anormalidades , Ventrículos do Coração/anatomia & histologia , Animais , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Proteína Substrato Associada a Crk/metabolismo , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/enzimologia , Feminino , Ventrículos do Coração/embriologia , Ventrículos do Coração/enzimologia , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Morfogênese , Miócitos Cardíacos/patologia , Miofibrilas/patologia , Fenótipo , Fosforilação , Fatores de Transcrição/metabolismo
6.
J Mol Cell Cardiol ; 46(2): 241-8, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19028502

RESUMO

Myocyte apoptosis is central to myocardial dysfunction following ischemia/reperfusion (I/R) and during the transition from hypertrophy to heart failure. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase regulates adhesion-dependent survival signals and unopposed FAK activation has been linked to tumor development. We previously showed that conditional myocyte-specific deletion of FAK (MFKO) in the adult heart did not affect basal cardiomyocyte survival or cardiac function but led to dilated cardiomyopathy and heart failure following pressure overload. In the present study, we sought to determine if FAK functions to limit stress-induced cardiomyocyte apoptosis. We reasoned that (I/R), which stimulates robust apoptotic cell death, might uncover an important cardioprotective function for FAK. We found that depletion of FAK markedly exacerbates hypoxia/re-oxygenation-induced cardiomyocyte cell death in vitro. Moreover, deletion of FAK in the adult myocardium resulted in significant increases in I/R-induced infarct size and cardiomyocyte apoptosis with a concomitant reduction in left ventricular function. Finally, our results suggest that NF-kappaB signaling may play a key role in modulating FAK-dependent cardioprotection, since FAK inactivation blunted activation of the NF-kappaB survival signaling pathway and reduced levels of the NF-kappaB target genes, Bcl2 and Bcl-xl. Since the toggling between pro-survival and pro-apoptotic signals remains central to preventing irreversible damage to the heart, we conclude that targeted FAK activation may be beneficial for protecting stress-dependent cardiac remodeling.


Assuntos
Quinase 1 de Adesão Focal/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/fisiologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Quinase 1 de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/genética , Camundongos , Camundongos Mutantes , Traumatismo por Reperfusão Miocárdica/genética , NF-kappa B/genética , NF-kappa B/fisiologia , Traumatismo por Reperfusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
7.
Circ Res ; 99(6): 636-45, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16902179

RESUMO

Focal adhesion kinase (FAK) is a ubiquitously expressed cytoplasmic tyrosine kinase strongly activated by integrins and neurohumoral factors. Previous studies have shown that cardiac FAK activity is enhanced by hypertrophic stimuli before the onset of overt hypertrophy. Herein, we report that conditional deletion of FAK from the myocardium of adult mice did not affect basal cardiac performance, myocyte viability, or myofibrillar architecture. However, deletion of FAK abolished the increase in left ventricular posterior wall thickness, myocyte cross-sectional area, and hypertrophy-associated atrial natriuretic factor induction following pressure overload. Myocyte-restricted deletion of FAK attenuated the initial wave of extracellular signal-regulated kinase activation and cFos expression induced by adrenergic agonists and biomechanical stress. In addition, we found that persistent challenge of mice with myocyte-restricted FAK inactivation leads to enhanced cardiac fibrosis and cardiac dysfunction in comparison to challenged genetic controls. These studies show that loss of FAK impairs normal compensatory hypertrophic remodeling without a concomitant increase in apoptosis in response to cardiac pressure overload and highlight the possibility that FAK activation may be a common requirement for the initiation of this compensatory response.


Assuntos
Cardiomegalia/prevenção & controle , Quinase 1 de Adesão Focal/fisiologia , Miócitos Cardíacos/citologia , Animais , Apoptose , Cardiomegalia/etiologia , Fibrose Endomiocárdica/etiologia , Quinase 1 de Adesão Focal/deficiência , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-fos/genética , Estresse Mecânico , Disfunção Ventricular Esquerda/terapia
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