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  • Of the population that does respond to the peptides

    2018-10-31

    Of the population that does respond to the peptides with changes in chronotropy ET-1, acting via ETA receptors, elicited a PKC-dependent, pertussis toxin- and 2-APB-insensitive positive chronotropic response. This finding is broadly consistent with a previous study in mESC-CMs (Kapur and Banach, 2007). However, in contrast to our findings, Kapur and Banach (Kapur and Banach, 2007) showed that the positive chronotropic effects were mediated by phospholipase C (PLC) with subsequent IP3 receptor activation. In this study the IP3 receptor antagonist 2-APB histone deacetylase inhibitors was without effect, indicating either that we have not used enough 2-APB or that the majority of ETA signaling in these histone deacetylase inhibitors is mediated via PKC effects. The former hypothesis is unlikely since the same concentration of 2-APB reduced the effects of Ang II, leaving us to conclude that, in these cardiomyocytes, the ET-1 effect is largely attributable to the action of PKC. In this study the AT2 receptor antagonist PD312,319 blocked the GF109203x-, 2-APB-sensitive Ang II-mediated increase in contraction frequency. Previous studies have demonstrated an AT2 receptor (PKC-dependent)-mediated effect on contractility (Allen et al., 1988; Kohout and Rogers, 1995); however, the AT2 receptor is normally highly expressed in the fetus and dramatically reduced in the adult; where it is predominantly located on the vascular endothelium (Grady et al., 1991; Wang et al., 1998). In these ESC-CMs, onset of AT2 receptor expression does not occur until at least Day 8 postdifferentiation and is definitively present in LDS CMs. Together with its demonstrated effect on contractile activity, this could imply a less developed Ang II system compared to ET-1, an observation consistent with what is known about Ang II signaling in rodents (Kang et al., 2007). Current studies are investigating the developmental progression of Ang II signaling in these ESC-CMs in more detail.
    Conclusion Use of ESC-CMs for cell-based therapy and in vitro applications during drug discovery necessitates comprehensive characterization of their functional activity. In this study, we have used video microscopy to (i) compare the modulation of cardiac contractile activity by various agonists vital for the physiological function of cardiomyocyte in vivo, (ii) investigate which second messenger systems the peptide agonists activate to produce their responses, and (iii) used these data to establish just how like normal adult heart tissue these ESC-CMs are. We have demonstrated that these mESC-CM cultures respond to stimulation through functional adrenoceptor, muscarinic, Ang II, and ET-1 receptors, and possess intact Gq/ll and Gi/o signaling cascades in a manner comparable to previous studies in the native mouse myocardium. However, even though we demonstrate the progressive maturation of these CMs by the reversal of their cell shortening response to ET-1 between EDS and LDS BBs, and that all BBs have functional sarcoplasmic reticulum, the pacemaker population within the BBs can be divided on the incidence of their response to Ang II and ET-1. Further investigations are required to better characterize the pacemaker populations in these differentiated cultures.
    Materials and methods
    Acknowledgments
    Introduction Articular cartilage of the vertebrate skeleton consists of chondrocytes suspended in rigid extracellular matrix (ECM) and provides a resilient barrier between bones while facilitating load-bearing and joint articulations. Articular cartilage damages are triggered by pathological degradation from enzymes and inflammatory cues in osteoarthritis and rheumatoid arthritis, or they can be caused by physical trauma like intraarticular fractures and ligament injuries (Beris et al., 2005). The avascularity and low metabolic rate of articular cartilage limit the repair capacity of partial-thickness defects due to the inability of progenitor cells to travel through the ECM to the injury site (Vinatier et al., 2009; van Osch et al., 2009). The search for new therapeutic approaches has led to the use of ESCs as a potential renewable chondrogenic cell source in standardized platforms for novel drug screens or cellular therapy for articular cartilage repair. Therefore, it is imperative to fully understand the molecular mechanisms governing germ layer induction and tissue patterning to efficiently generate ESC-derived chondrocytes.