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Investigation of cellular localization focal adhesion kinase (FAK) and mutant FAK (FAKY397F) in cardiomiocytes in vitro and in genetically modified mice

Grant number: 10/17086-7
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): March 01, 2011
Effective date (End): December 31, 2012
Field of knowledge:Health Sciences - Medicine - Medical Clinics
Principal researcher:Kleber Gomes Franchini
Grantee:Sílvio Roberto Consonni
Home Institution: Faculdade de Ciências Médicas (FCM). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
Associated research grant:06/54878-3 - Pathogenesis of cardiac hypertrophy and failure: mechanisms activated by mechanical stress, AP.TEM


There is an increasing move towards the development of technologies that allow the localization of proteins in cells by combined electron and light microscopy, with the use of molecular probes such as miniSOG and APEX2. Additionally we seek to understand how proteins are responsible for the cellular and tissue functions. The Focal Adhesion Kinase (FAK) is protein of integrin signaling cascade considered as a potential mediator of mechanical stress in cardiomyocytes. It is known that in cardiomyocytes subjected to hypertrophic stimuli by rapid activation of FAK and its subcellular redistribution, however the mechanisms involved in these processes are poorly understood. Another very important protein in the heart is Calsarcin-1 (CS1), a negative regulator of the Calcineurin pathway which is crucial in the development of cardiac hypertrophy. However the mechanisms involved in the negative regulation as well as the subcellular distribution CS1 are poorly understood. Aiming at the interaction between these areas to study the spatial-temporal distribution of cellular and protein components, and the importance of FAK and CS1 in the sarcomere and its role in hypertrophic signaling under mechanical stimulation, the aim of this study was to explore the ability of FAK and CS1 to incorporate genetically molecular probes that allow monitoring through images the behavior of these key molecules in the Z disc biology in MVRNs subjected to mechanical stretch. To this end, we performed subcellular localization assays using molecular probes applied to the correlative microscopy, biochemical and molecular assays and enzymatic activity. These data confirm the FAK associated with actin and focal adhesions fibers in H9c2 cells and has been shown by light microscopy that FAK wild-type (wt) is partially translocated to the nuclear compartment after stimulation of the agonist phenylephrine, while FAK Y397F (inactive mutant form) did not show the same phenotype. Moreover, despite standardization and expression of FAK and miniSOG or APEX2 in HEK 293T cells and H9c2, it was inconclusive subcellular localization of FAK, through the use of electron microscopy, in MVRN. Probably due to the diffuse distribution of most FAK molecules, it has no conclusive electron-dense region in transmission electron microscopy. Regarding the importance of CS1, it was observed that the cyclic stretch did not induce an increase in protein expression or gene relative CS1 and CnA, as there was no change in the phosphatase activity of CnA. However there was less interaction CS1 and CnA and change in CS1 location in MVRN under mechanical stimulation. CS1 overexpression and silencing corroborate the negative regulation of the CS1 over CnA in MVRN under mechanical stimulation. Based on structural data, it has been speculated that as the NFAT and CS1 binding sites are very close in CnA and at the same time distant from the active site of the phosphatase, it is possible that the role of CS1 in the negative regulation of CnA occur by steric hindrance to the NFAT transcription factor. Therefore, these results may contribute to a possible pharmacological inference, whereas Calcineurin-NFAT pathway is a major mediator of hypertrophy in cardiac myocytes by pathologic stimuli. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
CONSONNI, SILVIO R.; DE CARVALHO, ANNA C. P. V.; MAURO, ARTUR B.; FRANCHINI, KLEBER G.; BAJGELMAN, MARCIO C.. Lentiviral transduction of neonatal rat ventricular myocytes preserves ultrastructural features of genetically modified cells. VIROLOGY, v. 562, p. 190-196, . (17/21720-2, 19/04458-8, 10/17086-7, 12/13132-0)
Academic Publications
(References retrieved automatically from State of São Paulo Research Institutions)
CONSONNI, Sílvio Roberto. Localization and function of focal adhesion kinase and Calsarcin-1 in rat cardiomyocytes: using of molecular probes and lentivirus. 2015. Doctoral Thesis - Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia Campinas, SP.

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