| Summary: | 碩士 === 國立臺灣大學 === 藥理學研究所 === 101 === Introduction: Cardiac stem/progenitor cells were found resident in the heart and possess the potential for the self-repair. Wnt signaling plays a pivotal role in embryonic cardiogenesis and was found to be activated under diseases, including myocardial infarction, heart failure, and cardiac hypertrophy. It is still unclear in the different functioning of Wnts in cardiac cells. Aim: The present study aimed to clarify the different functional role of Wnt3a in regulating the self-renewal of Nkx2.5+ cardiomyoblasts, the growth and the transdifferentiation of cardiac fibroblast, and the survival of cardiomyocytes.
Material and Method: Nkx2.5-GFP+ cardiomyoblasts were isolated by enzymatic method from the ventricle of a reporter mice expressing GFP driven by an Nkx2.5-enhancer, and were sorted by flow cytometry. Enzyme digestion was also used to isolate fibroblasts and adult cardiomyocytes. CyQuant asssay or BrdU-pulse-labeling assay was performed to examine the effect of Wnts on cell proliferation. The differentiation pattern was evaluated by immunostaining and transcriptional profile by qPCR.
Results: Wnt3a, but not Wnt4, could dose-dependently inhibit the self-renewal of Nkx2.5+ cardiomyoblast, and the effect could be attenuated in the presence of Wnt inhibitors, including KY02111, DKK1, and a ROCK inhibitor (Y27632). In cardiac fibroblasts, Wnt3a could inhibit fibroblast entering cell cycle revealed by BrdU-pulse-labeling assay, which could not be blocked by KY02111. Furthermore, Wnt3a could decrease the cells expressing α-smooth muscle actin (α-SMA) and the effect could be attenuated by KY02111. CHIR (a GSK3β inhibitor) could also reduce α-SMA+ cells of cultured fibroblast. In cultured adult cardiomyocytes, Wnt3a did not alter myocyte survival in vitro. Co-culture of myocytes with Nkx2.5+ cardiomyoblast could enhance myocyte survival after culture for 5days, which could be attenuated by Wnt3a. The transcriptional profile of Wnt-receptor subtypes (Fzds) was analyzed by qPCR. High transcriptional levels of Fzd2 and Fzd1, the binding receptors of Wnt3a, were found in both Nkx2.5+ cardiomyoblast and cardiac fibroblast, and Nkx2.5+ cardiomyoblast expressed higher Fzd2 than fibroblast.
Conclusion: Wnt3a could inhibit the proliferation of Nkx2.5+ cardiomyoblast via both canonical Wnt-signaling (blocked by KY02111) and non-canonical pathway (blocked by Y27632). It was different from the inhibitory effect of Wnt3a in cardiac fibroblast proliferation that was insensitive to KY02111. KY02111 could reverse Wnt3a-induced reduction of myofibroblast transdifferentiation. Wnt3a did not alter adult cardiomyocyte survival or induce cell hypertrophy after in-vitro 3-day-culture, but attenuated the paracrine benefit of Nkx2.5+ cardiomyoblast to enhance myocyte survival after 5-day-culture under co-culture condition. It needs further study to clarify the detailed Wnt mechanisms corresponding to the regulation in cell proliferation, differentiation and the synthesis of paracrine factors in Nkx2.5+ cardiomyoblast.
Key words：cardiac progenitor cell, Nkx2.5, Wnt3a, Wnt signaling pathway, cardiac fibroblasts, cardiomyocytes
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