| Junctophilins (JPs) is a novel protein family that contributes to normal formation of the junction membrane complexes (JMCs) between the plasma membrane (PM) and the endoplasmic/sarcoplasmic reticulum (ER/SR) in excitable cells. JP2 is a unique subtype which abundantly expressed in the heart. It has been demonstrated that JP2 is essential for the physiological communication between caveolin channels and ryanodine receptor 2 (RyR2) in cardiac excitation-contraction (EC) coupling, mediated Ca2+ release for cellular Ca2+ homeostasis. Mutant mice lacking JP2 exhibited embryonic lethality, and mutation of JP2 was associated with hypertrophic and dilated cardiomyopathies.Although multiple roles for JP2 have been proposed, little is known of the significance of JP2 in early cardiac development, especially during the differentiation of cardiomyocytes from ES cells. Therefore, we hypothesized that JP2 which plays a critical role in adult cardiomyocytes may also take part in cardiogenesis, and set out to determine the role of JP2 in mouse ES cells undergoing cardiac differentiation with JP2 expression.In the present study, we found that the colocalizations of all the Ca2+ handling proteins and cardiac specilized proteins, a-Actinin and Troponin-T, did appear in the derived cardiomyocytes at the terminal stage. JP2 and RyR2, the definite protein marker for SR/ER, appeared early and expressed gradually during the course of cardiomyocyte differentiation of ES cells. While Caveolin3, the definite protein marker for t-tubules (TTs), did not show expression until terminal stage. Suppression of Jp2 with siRNA against Jp2 to both ES cells and ES cell derived cardiomyocytes (ESC-CMs) at least in part affected the cardiogenesis and the cellular Ca2+ homeostasis evoked by selective RyR agonist caffeine or Ca2+ in cardiomyocyte differentiated. Therefore, we confirmed that the terminal cisternae of cardiomyocytes is assembled from at least two distinct membrane components in the derived cardiomyocytes. JP2 do take a role in the ES cell-derived progeny, and could be an important target in drug discovery or cell-based regenerative strategy for cardiac repair.The significant promoting effects of some nature prenylflavonoids, such as icariin (ICA), icaritin (ICT), and desmethylicaritin, on cardiac differentiation of mouse ES cells in virto had been investigated previously. The three traditional Chinese medicine all belong to flavonoids, with an isopentenyl on the 8-substituent. However, the promoting effect whether or not was affected by the isopentenyl on the 8-substituent isopentenyl, and protein prenylation in cardiac differentiation of ES cells had not been investigated yet. Therefore we decided to design, synthetize and screen a series of flavonoids for drawing structure-activity relationship.Using mouse embryonic carcinoma cell line P19 stably transfected with cardiac-specific atrial natriuretic factor gene promoter-driven luciferase reporter, the compounds from the flavonoids libraries, therefore, were primary screened for their ability to induce cardiac differentiation. As a result, out of thirty flavonoids identified, 1’b-5’b of flavonoids were chosen to evaluate the cardiogenesis promoting effects of prenylflavonoids. Our results showed that the compounds with isopentenyl-compound 3’b displayed more effective facilitating abilities preferentially. We hypothesized that protein prenylation plays an important role in cardiogenesis of ES cells differentiation. We found that inhibition of geranylgeranylation by GGTI-298 inhibited cardiogenesis, while inhibition of farnesylation had no effect on cardiogenesis. The results showed that treatment with ICA, the mRNA expression of Racl, PI3K and were up-regulation, which could be inhibited by PI3K specific inhibitor LY294002, indicating the role of Rac-PI3K signaling cascade in prenylflavonoids induced cardiac differentiation. Inhibitors of isoprenylation by GGTI-298 had the effect on p38 and ERK-MAPK, which had been demonstrated to be a key switch of cardiogenesis. The PI3K-GSK3βalso was affected by GGTI-298 as well as PI3K.Our data indicate that isoprenylation plays a role in cardiogenesis. Addition of prenylflavonoids enhanced the cardiomyocte differentiating ES cells, inhibition of either geranylgeranylation or farnesylation affected cardiomyocyte differentiation and function in a positive manner, respectively. In conclusion, protein isoprenylation is an important component of the cardiomyocyte differentiation process that could constitute a new therapeutic target for cardiogenesis in the future.In the present study, the major findings were that decrease Jp2 expression in embryonic stem (ES) cells are further likely due to an extra non-anchor-dependent pathway:decreasing Jp2 by siRNA in ES cells (ⅰ) reduce gene expression towards mesoderm and cardiogenesis, (ⅱ) abolish sarcomeric structures in ES cell-derived cardiomyocytes (ESC-CMs) (ⅲ) lost JP2 colocalization with sarcomere proteins; while decreasing Jp2 by acute siRNA in ESC-CMs (ⅰ) disrupt the dynamin-related protein mitofusin 2 tethering SR to mitochondria, and (ⅱ) disrupt intact mitochondria and its functional gene expression. |
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