Investigation of βAR-dependent cAMP signalling in induced pluripotent stem cell-derived cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great potential for drug testing, cell therapy, and as models of genetic aberrations such as mutations, polymorphisms etc. However, current differentiation protocols do not produce cells which are as mature, both structurally and functionally, as adult cardiomyocytes. In mature cells, β-adrenergic (βAR) pathway regulates the rate and strength of myocyte contraction. This function is strictly regulated by locating βAR in microdomains such as caveolae, and by regulating pools of second messenger cAMP (particularly, via degradation by phosphodiesterases). Also, βAR activity depends on genetic variations within the population. In our study, we assessed βAR signalling in iPSC-CMs, depending on their genetic background and maturation state. Changes in βAR-dependent cAMP production were assessed in cells with different polymorphic alleles of βAR and at different states of maturation by FRET microscopy and myocyte contractility was measured by the IonOptix technique. We found that single nucleotide polymorphisms within the β2ARs influence receptor sensitivity and desensitisation upon stimulation with isoprenaline, leading to differing myocyte beating rate and cAMP responses. Three methods of increasing iPSC-CM maturation were explored: peptidomimetic technology, microcontact printing for making cell shape more adult-like and prolonged cell culture. Treatment with a mimetic peptide of Cavβ2 (a subunit of L-type calcium channel) showed enhanced development of T-tubules, though further investigation remains to be conducted. Microcontact printing positively influenced hiPSC-CMs shape and structural protein alignment, although little change in functional activity was noted. Prolonging time in culture led to an increase in the number of membrane caveolae and increased β2AR compartmentation in these microdomains, restricting cytoplasmic β2AR-dependent cAMP levels through localised phosphodiesterase activity. Interestingly, limited change of β1AR-dependent cAMP activity was observed. We conclude that β2AR signalling pathway compartmentation approaches the adult phenotype in iPSC-CMs with increased time in culture.Open Acces

CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of β2AR in isogenic hPSC-derived cardiomyocytes

© 2020 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. 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