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.