br Results br Discussion Adrenergic

Results
Discussion α-Adrenergic receptors play a critical role in the regulation of cellular growth pathways, including hypertrophy and proliferation. G protein-coupled α1ARs are the predominant α-adrenergic subtype in the myocardium of most species (Brückner et al., 1985), and catecholamine receptor agonists strongly induce pathological cardiac hypertrophy both in vivo and in vitro (Zhong and Minneman, 1999; Rokosh et al., 1996). Development of model systems have therefore focused on the response through the α1AR as a means to understand the downstream signaling involved and to develop therapeutic agents against hypertrophic conditions. Expression of ADRA1A subtype mRNAs is tissue and cell specific (Stewart et al., 1994), and control of ADRA1 mRNAs by agonists can be also diverse in different nuclear receptor (Rokosh et al., 1996). It is therefore crucial that the new hESC-CMs and hiPSC-CMs are characterized with respect to their α1AR repertoire. We had previously seen robust and specific effects of the canonical α1AR agonist PE on a wide range of markers of hypertrophy such as cell size, cell volume, ANF expression, sarcomere alignment, and protein/DNA ratio (Földes et al., 2011). While these observations were confirmed here in an hESC-CM from a number of lines, surprisingly we found that hiPSC-CM were in contrast unresponsive to PE. Loss of PE response was independent from the cell line, cell culture conditions, reprogramming, and differentiating protocols we used and was reproduced in different laboratories. Our data confirm that α1AAR (ADRA1A gene) is the dominant receptor subtype of the α1AR expressed in the human adult heart and ventricular myocytes. However, cardiomyocytes differentiated from hESCs or hiPSCs did not measurably express the ADRA1A gene. ADRA1A mRNA was expressed (albeit modestly) in undifferentiated hESCs and hiPSCs but disappeared rapidly during differentiation to either cardiomyocytes or fibroblasts. There was a close correspondence between loss of pluripotency genes and loss of the ADRA1A receptor. Once again, this was independent of line or reprogramming method; in our definitive experiment, we showed this by reprogramming fibroblasts differentiated from an hESC line and comparing the resulting hiPSC-CMs with the hESC-CMs from the same line. Since loss of the α1AAR occurred in both hESC-CMs and hiPSC-CMs, we were now left with the conundrum of why there was an active PE response in hESC-CMs and why the two cell types differed. We further explored alternate αAR subtypes and showed that differentiation activated a unique, nonontogenetic gene program by a marked shift from ADRA1A toward a dominant ADRA1B subtype both in hiPSC-CMs and hESC-CMs. However, expression levels of ADRA1B were considerably increased in both cell types, which again did not explain the difference between hiPSC-CMs and hESC-CMs. Other subtypes (ADRA1D or ADRA2C) are also present both in hiPSC-CMs and hESC-CMs. However, lack of hypertrophic responsiveness to PE suggests that their presence is not sufficient to mediate PE effects in hiPSC-CMs. We then attempted to restore the response to PE by overexpression of ADRA1A, but this too was unsuccessful despite high levels of receptor expression. This strongly suggests that there is either loss of coupling components for the downstream signaling pathway or active repression of hypertrophy by opposing signaling pathways. The next level of regulation for the α1AR is at the G protein-coupling stage, with agonist binding to the receptor allowing activation of the Gαq subunit by dissociation from Gβγ proteins. While levels of Gαq, Gβ, and Gγ were similar between undifferentiated hESCs and hiPSCs (and also similar to adult cardiomyocytes), differences then occurred during differentiation. We found an increase in Gq, Gβ1, and Gγ2 mRNA levels during hESC differentiation, whereas in most differentiating hiPSC lines, the mRNA levels were unchanged or downregulated. This represents a clear difference between the two cell types and would be expected to produce a relative damping of the hypertrophic response in hiPSC-CMs. However, other Gq-coupled hypertrophic agents (ET-1 and angiotensin II) produced only small changes in cell size in either cell type, but robust increases in ANF and B-type natriuretic peptide (BNP) were observed even in hiPSC-CMs. This suggests that G protein coupling is not entirely compromised in these cells. Increases in cell size somewhat larger than this have been reported for iCell hiPSC-CMs with ET-1 (24% versus our 10%) (Carlson et al., 2013), but it is notable that the stronger BNP signal was chosen for the final hypertrophy assay in that study, which would be consistent with our findings.