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.

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Comments to the Author: Both reviewers thought this an interesting and useful study but had some reservations. Please address the comments of reviewer 1.

Senior Editor:
Comments for Authors to ensure the paper complies with the Statistics Policy: As per reviewing editors comments Comments to the Author: Careful study on a topic of current interest in the field. Please address the concerns raised by reviewer one. In particular, the claim that substrate stiffness does not play a role in stress pathway activation needs to be toned down. This was not the goal of the current study, and has been examined by others in much more detail. Given the tenting that occurred in the 2D monolayer on the "stiff" substrate, the substrate stiffness is not defined.
Huethorst and colleagues report differential mechanical properties of iPSC-derived cardiomyocyte monolayers. If cultured at high cell density on rigid substrates CM monolayers display multiphasic motion. Which is absent in single cells and in high density monolayers on soft substrates. The study comprises experimental results obtained from two commercial iPSC-CM sources and describes a model for linear arrays of cardiomyocytes on stiff and soft substrates. Finally, the authors conclude that basic research and cardiotoxicity studies would benefit from iPSC-CM cultures on appropriate, i.e., flexible, substrates.

Comments:
Motion in monolayers depends on pacemaker activity and spread of excitation. Dense cardiomyocyte monolayers tend to detach from stiff substrates, whereas adhesion on soft substrates tends to be more uniform. Accordingly, synchronicity in monolayers on soft substrates is higher. Please, discuss potential differences in cardiomyocyte adhesion and in particular the contribution to monolayer detachment to the multiphasic motion. Alternatively, provide data in support of similar adhesion on soft and stiff substrates.  Fig 1E this is 20-50% of the seeded cells. Please specify whether the company-provided cell number is total, viable or platable cells. The finding of multiphasic contractions on stiff substrates is clearly dependent on cell density and culture conditions. Please also add information on cell purity in the methods section.
The claim that substrate stiffness does not play a role in stress pathway activation requires a re-consideration. It is quite likely that stress pathway activation will differ depending on cell density.

Minor points:
Page 7: "However, these studies were mostly done with single cells, and overlooked the additional role of cell-cell coupling and intercellular force transmission (Yonemura et al., 2010;Monemian Esfahani et al., 2019)." -I would not state that this was "overlooked". Single cell studies are done to avoid the ensemble phenomena described in the dense monolayers on stiff substrates by Huethorst et al.; in addition, hypertrophy/stress pathway induction studies are typically performed in responsive low density cultures Page 8, line 10: "... or hydrogel substrates (10 μg/ml, bovine, Gibco) in ..." -please specify that you were using bovine collagen Referee #2: Since there is interest in the effects of drugs on stem cell derived cardiomycyte contractility, it is important to know what conditions will give the best contractile behavior of the cardiomyocytes. In these studies, they show that the desired twitchlike transients are present in monolayers on soft substrates. The imaging patterns of the undesirable multiphasic contractions are quite distinct from those of the twitch-like behaviors and the documentation of the differences on soft vs. rigid substrates is quite clear. Thus, this is a well-documented paper that identifies which substrates can be used for drug studies of stem-cell derived cardiomyocytes. If this is an important finding for the field, I can recommend publication.

Response to reviewers:
We would like to thank all reviewers for their time and their comments in order to improve the manuscript. We have amended the manuscript accordingly and all responses are written below in red underneath the corresponding comment.
In addition to this, we have amended the figures to show all raw data points along with the means and SDs to comply with the Statistics Policy of the Journal of Physiology. Furthermore, we have reanalysed the data using a nested method where applicable. This is all written down in the Statistical Summary Table that is included with the submission.

Motion in monolayers depends on pacemaker activity and spread of excitation. Dense
cardiomyocyte monolayers tend to detach from stiff substrates, whereas adhesion on soft substrates tends to be more uniform. Accordingly, synchronicity in monolayers on soft substrates is higher. Please, discuss potential differences in cardiomyocyte adhesion and in particular the contribution to monolayer detachment to the multiphasic motion. Alternatively, provide data in support of similar adhesion on soft and stiff substrates.
The reviewer makes a good point and one we did consider. The measurements would need to assay the density of Cell Adhesion Complexes, and also assess the extent to which CACs were attached to the substrate. This is currently beyond our technology, but we have complied with the reviewer's request and added discussion on the role of cell adhesion and its contribution to multiphasic motion as described. (page 27, lines 14-15 and lines 24-29) 2. Table 1: please add the quantities of truly retained cells after plating; according to Suppl. Fig 1E  this is 20-50% of the seeded cells. Please specify whether the company-provided cell number is total, viable or platable cells. The finding of multiphasic contractions on stiff substrates is clearly dependent on cell density and culture conditions. Please also add information on cell purity in the methods section.
We have complied with the reviewer's request and supplied the quantities of retained cells after plating in the modified version of Table1 (page 38). Furthermore, we clarified that we are stating the plated cells and the viable cells, based on the percentages of Supplementary Figure 1. We also amended the legend accordingly. Then, we stated the purity of the two cell lines on page 5, lines 23-25.
3. The claim that substrate stiffness does not play a role in stress pathway activation requires a reconsideration. It is quite likely that stress pathway activation will differ depending on cell density.
We agree with the reviewer and have edited the Results section and Discussion to reflect this point. Thank you for your report. We are pleased you find the study clear and of importance.

12-Oct-2021 1st Revision -Editorial Decision
Dear Dr Smith, Re: JP-RP-2021-282228R1 "Conventional rigid 2D substrates cause multiphasic contractile signals in monolayers of human induced pluripotent stem cell derived cardiomyocytes" by Eline Huethorst, Peter Mortensen, Radostin D Simitev, Hao Gao, Lotta Pohjolainen, Virpi Talman, Heikki Ruskoaho, Francis L Burton, Nikolaj Gadegaard, and Godfrey L Smith Thank you for submitting your manuscript to The Journal of Physiology. It has been assessed by a Reviewing Editor and by 2 expert referees and I am pleased to tell you that it is considered to be acceptable for publication following satisfactory revision.
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21-Sep-2021
Both reviewers and the reviewing editor were satisfied with the revision. I concur. However, supplemental figures and tables are not allowed per journal policy. Please revise the MS to incorporate all supplemental material (except videos) into the main MS. I would encourage the authors to try to consolidate some of the supplemental figures, or combine some of them with the main MS figures. I realize that this may not be possible for every figure.

Response to referees
We would like to thank the Senior Editor, and all referees for their time to consider this manuscript for publication. We have made all changes as proposed by the Senior Editor and we hope that you find these changes satisfactory for our manuscript to be published. Our replies are written in red below each recommendation.

REQUIRED ITEMS:
Please could authors add... Both reviewers and the reviewing editor were satisfied with the revision. I concur. However, supplemental figures and tables are not allowed per journal policy. Please revise the MS to incorporate all supplemental material (except videos) into the main MS. I would encourage the authors to try to consolidate some of the supplemental figures, or combine some of them with the main MS figures. I realize that this may not be possible for every figure.
We have included all supplementary figures and the table into the main body of the manuscript in a logical order. To minimize the number of figures, some panels have been combined with other figures and some data has been excluded. All figure numbers have been changed in the text accordingly. Changes are summarized in the following list.