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Study of Extracellular Matrix Remodeling and the Role of Periostin B During Zebrafish Heart Regeneration

出版	Universitat de Barcelona, 2019
URL	http://books.google.com.hk/books?id=LzwkyAEACAAJ&hl=&source=gbs_api

註釋Adult zebrafish (Danio rerio), in contrast to mammals, are able to regenerate their hearts in response to injury or experimental amputation. Our understanding of the cellular and molecular bases that underlie this process, although fragmentary, has increased significantly over the last years. However, the role of the extracellular matrix (ECM) during zebrafish heart regeneration has been comparatively rarely explored. A better understanding of all the mechanisms underlying this complex process would help developing strategies to regenerate the human heart. With this aim, here, we set out to characterize the ECM protein composition in adult zebrafish hearts, whether it changed during the regenerative response, and the role of the matricellular protein periostin b. For this purpose, in the first part of the current thesis, we established a decellularization protocol of adult zebrafish ventricles that significantly enriched the yield of ECM proteins. We then performed proteomic analyses of decellularized control hearts and at different times of regeneration. Our results show a dynamic change in ECM protein composition, most evident at the earliest (7 days post-amputation) time-point analyzed. Regeneration associated with sharp increases in specific ECM proteins, and with an overall decrease in collagens and cytoskeletal proteins. We finally tested by atomic force microscopy that the changes in ECM composition translated to decreased ECM stiffness. Our cumulative results identify changes in the protein composition and mechanical properties of the zebrafish heart ECM during regeneration. In the second part of the current thesis, we have examined the role of periostin b (postnb) during zebrafish heart regeneration. We found that postnb was abundant in the injury of amputated zebrafish ventricles, and we designed two strategies to suppress postnb during regeneration: 1) a shRNA transgenic knock-down, and 2) a CRISPR/Cas9 postnb-/- zebrafish. Knock-down and knock-out fishes did not exhibit any evident developmental defects, but showed a lack complete cardiac regeneration after amputation. The absence of postnb did not have any effect on leukocyte recruitment, matrix metalloproteinase activation or revascularization after amputation. However, postnb-/- zebrafish developed a softer cardiac ECM due to a reduction of collagen cross- linking, and showed an increase of cardiomyocyte proliferation and a decrease on cardiomyocyte cell death after amputation. The stiffness control by postnb seem to be important to correctly modulate stiffness for a complete cardiac regeneration. Stiffness analysis toguether with proliferation analysis suggest that postnb is needed to increase the stiffness in the injury border to instruct cardiomyocytes to stop proliferating and migrate to the injury site.