Heart failure is one of the leading causes of death worldwide. In part, this is due to the inadequate regenerative capacity of cardiomyocytes post-injury. Modulation of the Hippo signalling pathway in mice has been shown to enhance cardiomyocyte proliferation and improve survival in a myocardial infarction model. While the discovery of the Hippo pathway and its function as a master regulator of cell proliferation has led to greater understanding of its core components such as Yes-associated protein (YAP), the upstream signals that regulate the Hippo pathway have remained elusive. This study was aimed to identify novel upstream regulators of the Hippo pathway in cardiomyocytes that could be targeted pharmacologically to induce regeneration.
We performed a targeted RNAi screen in H9c2 cardiomyoblast cell line using adenovirus-mediated luciferase reporter system to detect the activity of YAP, the major effector of the Hippo pathway. Using this system, 5-hydroxytryptamine receptor 2B (5-HT2B) was identified as a potential regulator of the Hippo pathway. Serotonin-mediated 5-HT2B has previously been shown to play a significant role in cardiac development during embryogenesis; however, a link between 5-HT2B and the Hippo pathway has not yet been documented.
An in vitro model was subsequently established by overexpressing 5-HT2B in primary neonatal rat cardiomyocytes (NRCMs) using an adenoviral system. The activities of different components of the Hippo pathway were investigated with an emphasis on YAP. Immunofluorescence microscopy was utilised to quantify cardiomyocyte proliferation and survival.
Using this system, we found that overexpression of 5-HT2B in cardiomyocytes enhanced YAP activity by 12 folds compared to the control group as indicated by YAP-luciferase assay. In keeping with this, we observed an increase in YAP nuclear translocation following 5-HT2B overexpression, indicating YAP activation. Mechanistically, we found that 5-HT2B expression reduced Large tumour suppressor (LATS) phosphorylation, eventually leading to YAP activation. Since YAP is known to mediate cell proliferation we analysed proliferation rate in cardiomyocytes overexpressing 5-HT2B. We found that cell proliferation was increased by 39.7% compared with control cells as indicated by EdU incorporation assay.
In conclusion, our findings have identified 5-HT2B as a novel upstream regulator of the Hippo pathway in cardiomyocytes. We also observed that in cardiomyocytes, 5-HT2B is a potent stimulator of YAP activity and cell proliferation. Since 5-HT2B is a membrane receptor that can be targeted pharmacologically, this finding may provide new insight for the development of a new approach to induce cardiomyocyte regeneration.
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