LDS BBs were found to respond to

LDS BBs were found to respond to ET-1 stimulation with a decrease in cell shortening. Furthermore, our findings that BQ123, but not BQ3020, blocked the negative shortening response to ET-1 are consistent with those of Izumi and co-workers (Izumi et al., 2000) and indicate an ETA receptor-mediated effect. Curiously, ET-1, in the presence of the ETA receptor antagonist BQ123, produced an increase in cell shortening. Furthermore, ET-1 concentrations above 10nM show a reduced negative response. These findings may indicate that ET-1 acts at both ETA and ETB receptors to elicit negative and positive changes in cell shortening, with the ETA receptor effect predominating at low concentrations. Physiological antagonism between the ETA and the ETB receptors has been observed in several studies (for a review, see Brunner et al. (Brunner et al., 2006). Thus, we conclude that ETA mediates a decrease in cell shortening via activation of Gi/o proteins, but we cannot rule out an ETB-mediated positive effect in these cells. In DMH-1 to previous studies that attributed the ET-1-mediated cell shortening response to PKC activation (Izumi et al., 2000; Namekata et al., 2008), we found that PTX blocked the ET-1-mediated decrease, implicating Gi/o protein coupling, rather than Gq/11. Gi/o coupling of ET receptors is common in other species (Ono et al., 1998; Liu et al., 2003; Kim, 1991). However, even though studies have demonstrated ET receptor coupling to both Gq/11 and Gi/o in adult mouse heart (Jiang et al., 1996; Hilal-Dandan et al., 1992; Hilal-Dandan et al., 2000), it has not been associated with negative cell shortening effects of ET-1 in the mouse. The renin angiotensin system plays a central role in the regulation of cardiac function. Ang II is the system\'s main effector and is known to mediate a wide range of physiological and pathophysiological effects following acute and chronic stimulation (Mehta and Griendling, 2007). In the heart, Ang II mediates both inotropic and chronotropic effects via two G-protein-coupled receptor types, AT1 and AT2. These two receptors have similar affinities for Ang II, and are currently distinguished by affinities for antagonists such as losartan (AT1) and PD123,319 (AT2). The AT1 receptor is thought to be the prime mediator of Ang II effects and has been linked to several signaling cascades, most commonly Gq/11 coupling with subsequent PLC and PKC activation (De Gasparo et al., 2000). The Ang II-mediated effect on the contractility of the mouse heart varies depending on the tissue being examined (Sakurai et al., 2002; Nishimaru et al., 2003). In this study, Ang II induced a slight increase in contraction amplitude, a finding consistent with effects seen in mouse atria (Nishimaru et al., 2003). This effect was blocked by losartan and 2-APB, but not by PTX, PD123,319, or GF109203x, and is consistent with a previous study of Ang II effects in human ESC-CMs acting via Gq/11 proteins with subsequent PLC and IP3 production (Sedan et al., 2008). One of the most interesting aspects of this study is that the PKC-mediated positive chronotropic response to both ET-1 and to Ang II was only observed in a maximum of 50% of BBs. To determine whether this was due to the lack of developed second messenger signaling pathways in the nonresponding population, the effect of the PKC activator PDA was investigated. However, all BBs were found to respond to direct PKC activation, indicating that the second messenger pathway is intact in every culture even though half of the BBs are unresponsive to peptide addition. Previous studies using human ESC-CMs have also reported the presence of nonresponding cultures, which has been attributed to the lack of a functional sarcoplasmic reticulum in cardiomyocytes at varying stages of differentiation(Sedan et al., 2008). However, in our study, this observation appears to be specific to peptide effects since (1) all BBs responded to thapsigargin and ryanodine, indicating the presence of a functional sarcoplasmic reticulum, (2) all BBs responded to isoprenaline and carbachol addition, and (3) beating in all bodies was reduced by the If channel blocker. Furthermore, no correlation was found between a nonresponding BB and other observable properties, such as the initial beating frequency or individual batches of differentiated cells. A recent study in mESC-CMs identified two separate pacemaker populations based on hyperpolarization-activated cyclic nucleotide gated channel expression, activation kinetics, and cAMP sensitivity (Barbuti et al., 2009). We think it possible that an analogous situation exists in our cultures where approximately one-half of our beating body contains pacemakers insensitive to peptide effects. This is consistent with the idea of “functional inhomogeneity” resulting from a significant variation in receptor densities within the sinus node (Opthof, 2007). As investigating the function of individual pacemaker cells within an EB is near impossible, studies at the single cell level are ongoing to determine differences in receptor expression and signaling pathway activation within the pacemaker population.