Regulation von zyklischem Adenosinmonophosphat durch Phosphodiesterasen in aus humanen induzierten pluripotenten Stammzellen abgeleiteten Kardiomyozyten: Einfluss der Zellkulturbedingungen

Abstract

Although the impact of cardiovascular diseases on human life has been declining yet there are still many open questions. These need to be answered in the field of cardiovascular diseases. These questions focus on the (I).pathophysiology of several lethal cardiovascular diseases (II) the scrutiny of available drug screening models for their safe use in human beings and (III) the potential creation of human cardiovascular tissue for the repair of the heart. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be a bag full of solutions for all hindrances and shortcomings in the research field of cardiovascular diseases. But before they can be utilized, hiPSC-CMs must closely resemble adult cardiomyocytes on a genomic, anatomical, electrophysiological and physiological level. β1-ARs are a key regulator of ECC in the heart. Calcium channels in hiPSC-CMs respond to β1-AR signaling, however the effect size depends upon culture conditions with smaller effects having been observed in conventional 2D than in 3D culture constructs. Since in adult heart β1-AR-mediated effects are spatially and temporally controlled by different PDEs, it was the aim of the presented study to investigate whether smaller effects of NE in ML relate to a different impact of PDEs on β1-AR-dependent cAMP. Suitable generation and propagation of cAMP to its effectors like the L-type calcium channels produces a wide range of physiological and pathophysiological activities in cardiomyocytes. So the study of dynamics of cAMP and its impact on ICa, L can be vital parameters to judge the resemblance between hiPSC-CMs and adult cardiomyocytes. We used hiPSC-CMs produced in our lab and cast them into ML and EHT formats. After 21 days continuous feeding with culture medium we isolated hiPSC-CMs from ML and EHT by using an established protocol (Uzun et al., 2016). These hiPSC-CMs were used to measure ICa, L by patch clamp technique and cAMP by FRET. Effects of inhibition of different PDEs on the basal level of ICa and cAMP as well as on NE-induced ICa, L and cAMP were investigational parameters of this study. HiPSC-CMs show proper sensitivity to β1-adrenergic receptor stimulation with respect to ICa, L and cAMP in Ml construct (Fig 52 A). PDE4 is the dominant regulator of basal ICa, L and cAMP levels in hiPSC-CMs, which is in contrast to adult human atrial cardiomyocytes (Molina et al., 2012; Christ et al., 2006). Furthermore, PDE4 is the major phosphodiesterase which regulates NE-induced ICa, L and cAMP in hiPSC-CMs isolated from ML tissue (Fig 52 A).

NE-induced ICa, L was augmented by the inhibition of PDE4 in isolated hiPSC-CMs from EHTs, while results of NE-induced cAMP in the absence of PDE4 in isolated hiPSC-CMs from EHTs were not conclusive (Fig.52 B). Contribution of PDE3 to define NE-induced ICa, L and cAMP in hiPSC-CMs from EHT (C-25) was divergent (Fig.52 B). In all three experimental designs (ICa, L, global FRET and SL FRET) effects of NE on ICa, L and cAMP were suppressed in the presence of cilostamide. We studied contributions of other PDEs to this discrepancy, until we realized that these results may be a peculiarity of a single cell line, namely C-25, as we do not find this in hiPSC-CMs from other cell lines HiPSC-CMs from both ML and EHT respond to NE with an increase in cAMP, but the maximum effect size is smaller in ML than EHT, whereas the sensitivity is not changed. As in human adult atrial CMs basal cAMP can be increased by inhibition of PDE3 and PDE4. While the impact of PDE3 on cAMP in adult human atrial CMs is clearly larger than with PDE4, the contribution of PDE4 in hiPSC-CMs is at least as large as with PDE3 (ML) or even larger than with PDE3 (EHT). The maximum effect size of NE is not regulated by PDE3 and PDE4 in adult human atrial CMs. In stark contrast, inhibition of either PDE3 or PDE4 can increase the maximum effect size of NE in ML but not EHT. The latter finding could indicate a more mature regulation of β1-AR-mediated cAMP signals in hiPSC-CMs from EHT. Regulation of maximum effects of NE by PDE3 and PDE4 is a strong disadvantage in ML. On the other hand maximum effects of NE not sensitive to PDE4 inhibition in EHT resemble the situation in adult human atrial CMs. Suppression of maximum effects of NE in the presence of PDE3 inhibition as observed in just one cell line serves as a precautionary note to always profile hiPSC-CMs from different origins. Stringent investigational exploration of different cell lines is required, before they are used to make generalized statements about the utilization of hiPSC-CMs as a model for scientific study of cardiovascular diseases and their treatments.