Conventional rigid 2D substrates cause complex contractile signals in monolayers of human induced pluripotent stem cell‐derived cardiomyocytes

Abstract

Human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CM) in monolayers interact mechanically via cell–cell and cell–substrate adhesion. Spatiotemporal features of contraction were analysed in hiPSC‐CM monolayers (1) attached to glass or plastic (Young's modulus (E) >1 GPa), (2) detached (substrate‐free) and (3) attached to a flexible collagen hydrogel (E = 22 kPa). The effects of isoprenaline on contraction were compared between rigid and flexible substrates. To clarify the underlying mechanisms, further gene expression and computational studies were performed. HiPSC‐CM monolayers exhibited multiphasic contractile profiles on rigid surfaces in contrast to hydrogels, substrate‐free cultures or single cells where only simple twitch‐like time‐courses were observed. Isoprenaline did not change the contraction profile on either surface, but its lusitropic and chronotropic effects were greater in hydrogel compared with glass. There was no significant difference between stiff and flexible substrates in regard to expression of the stress‐activated genes NPPA and NPPB. A computational model of cell clusters demonstrated similar complex contractile interactions on stiff substrates as a consequence of cell‐to‐cell functional heterogeneity. Rigid biomaterial surfaces give rise to unphysiological, multiphasic contractions in hiPSC‐CM monolayers. Flexible substrates are necessary for normal twitch‐like contractility kinetics and interpretation of inotropic interventions. Key points Spatiotemporal contractility analysis of human induced pluripotent stem cell‐derived cardiomyocyte (hiPSC‐CM) monolayers seeded on conventional, rigid surfaces (glass or plastic) revealed the presence of multiphasic contraction patterns across the monolayer with a high variability, despite action potentials recorded in the same areas being identical. These multiphasic patterns are not present in single cells, in detached monolayers or in monolayers seeded on soft substrates such as a hydrogel, where only ‘twitch’‐like transients are observed. HiPSC‐CM monolayers that display a high percentage of regions with multiphasic contraction have significantly increased contractile duration and a decreased lusotropic drug response. There is no indication that the multiphasic contraction patterns are associated with significant activation of the stress‐activated NPPA or NPPB signalling pathways. A computational model of cell clusters supports the biological findings that the rigid surface and the differential cell–substrate adhesion underly multiphasic contractile behaviour of hiPSC‐CMs.

Does Enhanced Structural Maturity of hiPSC-Cardiomyocytes Better for the Detection of Drug-Induced Cardiotoxicity?

“…The β-adrenergic agonists dobutamine and isoprenaline caused distinct electrophysiological effects that were paralleled in the two experimental groups. There was an increase in the spontaneous rate (decreased cycle length) in both cell systems, indicating an increased activity of the funny channel ( I f ) by cAMP, as reported in nodal cardiac tissue and previously reported in iPSC-CM systems [ 24 ]. The significant and comparable expression of HCN4 in both FM and MM groups indicates that the degree of electrophysiological differentiation was similar in both groups.…”

“…In both experimental groups, decreased contraction duration suggests that the lustotropic actions of adrenergic stimulation were equally present, but in neither group was there a significant positive inotropic response ( Figure 6 ). This form of adrenergic contractile response has been previously reported in hiPSC-CMs (e.g., [ 24 ]) and is thought to be based on the poorly developed sarcoplasmic reticulum systems within these cells, a feature that is unaltered by culturing on the specialized matrix system (MatrixPlus Cellvo™).…”

“…This was observed in both monolayer models to comparable degrees (and there is full data in Supplementary Tables S3 and S4 ). Isoprenaline is a β1 adrenergic agonist and increases the beat rate of hiPSC-CMs [ 24 ] as well as the intrinsic rate of the sino-atrial and atrio-ventricular nodes. The effect on spontaneous rate was clearly observed in the monolayer models from the voltage signal (see Figure 6 and Table 2 , and full data in Supplementary Tables S3 and S4 ).…”

Acute effects of cardiac contractility modulation stimulation in conventional 2D and 3D human induced pluripotent stem cell-derived cardiomyocyte models

“…A contractility platform and software (CellOPTIQ, Clyde Biosciences), based on video pixel displacement, was used to measure 2D hiPSC-CM contractility ( Sala et al, 2018 ; Saleem et al, 2020b ; Huethorst et al, 2022 ). 2D hiPSC-CMs were imaged directly in plates by an inverted fluorescence microscope (Zeiss) using a ×40 objective.…”

“…HiPSC-CMs are a useful in vitro model to assess the molecular and functional effects of CCM on human cardiac tissue ( Feaster et al, 2021 ). However, contractile studies in hiPSC-CMs have been limited as a result of immature contractile properties when plated as conventional 2D monolayers on stiff substrates (i.e., glass or plastic) ( Feaster et al, 2021 ; Korner et al, 2021 ; Huethorst et al, 2022 ; Narkar et al, 2022 ). We previously demonstrated that 2D monolayer hiPSC-CMs, on flexible substrate, displayed increased contraction and calcium handling properties when acutely stimulated with standard CCM pulse.…”