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During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (β2AR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, in vitro experiments with primary cells, and transgenic overexpression models. However, heterogeneous genetic background and non-physiological transgene expression levels confound interpretation, leading to conflicting mechanistic conclusions. To overcome these limitations, we used CRISPR/Cas9 gene editing technology in human pluripotent stem cells (hPSCs) to create a unique suite of four isogenic homozygous variants at amino acid positions 16(G/R) and 27(G/Q), which reside in the N terminus of the β2AR. By producing cardiomyocytes from these hPSC lines, we determined that at a functional level β2AR signaling dominated over β1AR . Examining changes in beat rates and responses to isoprenaline, Gi coupling, cyclic AMP (cAMP) production, downregulation, and desensitization indicated that responses were often heightened for the GE variant, implying differential dominance of both polymorphic location and amino acid substitution. This finding was corroborated, since GE showed hypersensitivity to doxorubicin-induced cardiotoxicity relative to GQ and RQ variants. Thus, understanding the effect of β2AR polymorphisms on cardiac response to anticancer therapy may provide a route for personalized medicine and facilitate immediate clinical impact., Graphical Abstract, During normal- and patho-physiological situations, the behavior of beta2-adrenoreceptor (β2AR) is determined by its polymorphic variants. A human-based isogenic model system represents a promising tool for systematic analysis of mechanisms of β2AR variant-mediated cellular response under normal and stressed conditions. This allows important subtleties of polymorphisms in β2AR to be unraveled.

Summary Cardiomyocytes from human embryonic stem cells (hESC-CMs) and induced pluripotent stem cells (hiPSC-CMs) represent new models for drug discovery. Although hypertrophy is a high-priority target, we found that hiPSC-CMs were systematically unresponsive to hypertrophic signals such as the α-adrenoceptor (αAR) agonist phenylephrine (PE) compared to hESC-CMs. We investigated signaling at multiple levels to understand the underlying mechanism of this differential responsiveness. The expression of the normal α1AR gene, ADRA1A, was reversibly silenced during differentiation, accompanied by ADRA1B upregulation in either cell type. ADRA1B signaling was intact in hESC-CMs, but not in hiPSC-CMs. We observed an increased tonic activity of inhibitory kinase pathways in hiPSC-CMs, and inhibition of antihypertrophic kinases revealed hypertrophic increases. There is tonic suppression of cell growth in hiPSC-CMs, but not hESC-CMs, limiting their use in investigation of hypertrophic signaling. These data raise questions regarding the hiPSC-CM as a valid model for certain aspects of cardiac disease., Highlights • Human iPSC-CMs are unresponsive to α-adrenergic hypertrophic signals • Silenced ADRA1A is accompanied by ADRA1B upregulation during differentiation • ADRA1B signal supports hypertrophy in hESC-CMs but is inhibited in hiPSC-CMs • Similar phenotype of hESC-CMs and hiPSC-CMs may mask differences in signaling, In this article, Földes and colleagues show that hiPSC-derived cardiomyocytes are relatively unresponsive to α-adrenergic hypertrophic signals compared to hESC cardiomyocytes. The main difference in hiPSC-CMs that accounts for the defective response is the suppression of growth by tonic antihypertrophic pathways. Superficial similarities in phenotype between cardiomyocytes derived from hESCs or hiPSCs may mask complex differences in signaling.

Identification of the stem cell niche is crucial for understanding the factors that regulate these cells. Rodent enteric neural crest-derived stem cells have previously been isolated by flow cytometry and culture of cell suspensions from the outer smooth muscle layers or the entire gut wall from postnatal and adult animals. Such cell suspensions contain a mixture of cell types, including smooth muscle, fibroblasts and cells associated with the vasculature and extrinsic innervation. Thus these preparations may be contaminated by stem cells associated with extrinsic sensory and autonomic nerves and by other types of stem cell that reside in the gut. Here we describe a different approach, similar to that recently used for infant human gut, to obtain enteric ganglion-derived cells, with properties of neural progenitor cells, using isolated myenteric ganglia from postnatal rat ileum. Myenteric ganglia were separated from the gut wall, dispersed and resulting cell dissociates were plated in non-adherent culture conditions with EGF and FGF-2. Under these conditions neurosphere-like bodies (NLB) developed. Cells in NLB incorporated BrdU and expressed the stem cell marker nestin but not the pan-neuronal marker PGP 9.5. Upon growth factor withdrawal some BrdU-immunopositive cells assumed the morphology of neurons and expressed PGP 9.5; others were flattened and expressed the glial cell marker GFAP. This work therefore provides evidence that neural crest-derived progenitors in the postnatal rat gut are located in the myenteric plexus, and shows that these cells can be expanded and differentiated in NLB in vitro.

Neurturin (NTN) is a member of the glial cell line-derived (GDNF) family of neurotrophic factors, which act via a receptor complex composed of a signal transducing receptor, c-Ret and a glycosylphosphatidylinositol (GPI)-linked ligand binding receptor, GFRalpha. Different members of the GDNF family bind preferentially to one of four different GFRalpha receptors; NTN binds preferentially to the GFRalpha-2 receptor. Recent evidence has shown that three alternatively spliced isoforms of GFRalpha-2 occur in rodent tissues, including the rat brain, myenteric plexus and kidney, and several mouse tissues. Here we have examined the occurrence of GFRalpha-2 isoforms in the adult male rat urinary bladder by RT-PCR, in parallel with samples from the muscularis externa of the rat ileum. In contrast to the ileum, only a single GFRalpha-2 isoform, the smallest isoform, known as GFRalpha-2c, was detected in the rat urinary bladder. This differential expression of GFRalpha-2 transcripts in bladder and intestine may be related to differences in the roles of NTN in the two tissues and its actions on the neurons that innervate them.

Glial cell line-derived neurotrophic factor family receptor alpha-2 (GFR alpha-2) is a GPI-linked receptor that preferentially binds neurturin (NTN), a member of the glial cell line-derived neurotrophic factor (GDNF) family. Three splice isoforms of GFR alpha-2 have been identified previously in mouse tissues, but the occurrence of splice isoforms in rats has not been described. The aim of this study was therefore to identify GFR alpha-2 splice isoforms in rat tissues using reverse transcription-polymerase chain reaction (RT-PCR) and gene cloning. Three isoforms were identified and sequenced, and named GFR alpha-2(a), (b) and (c), according to the nomenclature used for the previously identified mouse isoforms. The GFR alpha-2(a) and (b) isoforms were identical to those previously described in mice. The GFR alpha-2(c) isoform was novel. Sequences for GFR alpha-2(b) and (c) were deposited in the GenBank database (accession numbers GI: 16797788 and 16797786, respectively). All three isoforms were expressed in the brain, kidney, and intestine of both postnatal and adult rats.

Background Ageing is associated with gastrointestinal dysfunction, which can have a major impact on quality of life of the elderly. A number of changes in the innervation of the gut during ageing have been reported, including neuronal loss and degenerative changes. Evidence indicates that reactive oxygen species (ROS) are elevated in ageing enteric neurons, but that neurotrophic factors may reduce generation of neuronal ROS. Two such factors, glial cell line derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) have also been found to protect enteric neurons against oxidative stress induced cell death of enteric ganglion cells in vitro. We have investigated the possible roles of neurotrophic factors further, by examining their expression in the gut during ageing, and by analysing their effects on antioxidant enzyme production in cultures of enteric ganglion cells. Results Analysis of the expression of GDNF and its receptors c-Ret and GFR α − 1 in rat gut by RT-PCR showed that expression continues throughout life and into ageing, in both ad libitum(AL) and calorically-restricted (CR) animals. Levels of expression of GDNF and GFR α − 1 were elevated in 24 month AL animals compared to 24 month CR animals, and to 24 CR and 6 month control animals respectively. The related factor Neurturin and its receptor GFR α − 2 were also expressed throughout life, the levels of the GFR – α-2(b) isoform were reduced in 24 m AL animals. Immunolabelling showed that c-Ret and GFR α − 1 proteins were expressed by myenteric neurons in ageing animals. GDNF, but not NT-3, was found to increase expression of Cu/Zn superoxide dismutase and catalase by cultured enteric ganglion cells. Conclusions The neurotrophic factors GDNF and neurturin and their receptors continue to be expressed in the ageing gut. Changes in the levels of expression of GDNF , GFR α-1 and GFR α-2(b) isoform occurred in 24 m AL animals. GDNF, but not NT-3, increased the levels of antioxidant enzymes in cultured enteric ganglion cells, indicating a possible mechanism for the reported protective effect of GDNF against menadione-induced neuronal apoptosis in the ageing gut. Together these data suggest that GDNF family members may play a protective role in the gut throughout life, and support the suggestion that dysregulation of neurotrophic factor support could contribute to neuronal ageing in the gut.