Human pluripotent stem cell-derived cardiomyocytes align under cyclic strain when guided by cardiac fibroblasts

The human myocardium is a mechanically active tissue typified by anisotropic organization of the resident cells (cardiomyocytes (CMs) and cardiac fibroblasts (cFBs)) and the extracellular matrix (ECM). Upon ischemic injury, the anisotropic tissue is replaced by disorganized scar tissue, eventually resulting in loss of coordinated contraction. Efforts to re-establish tissue anisotropy in the injured myocardium are hampered by a lack of understanding on how CM and/or cFB structural organization is affected by the two major physical cues inherent in the myocardium: ECM organization and cyclic mechanical strain. Herein, we investigate the singular and combined effect of ECM (dis)organization and cyclic strain in a 2D human in vitro co-culture model of the myocardial microenvironment. We show that (an)isotropic ECM protein patterning can guide the orientation of CMs and cFBs, both in mono- and co-culture. Subsequent application of uniaxial cyclic strain – mimicking the local anisotropic deformation of beating myocardium – causes no effect when applied parallel to the anisotropic ECM. However, when cultured on isotropic substrates, cFBs, but not CMs, orient away from the direction of cyclic uniaxial strain (strain avoidance). In contrast, CMs show strain avoidance via active remodeling of their sarcomeres only when co-cultured with at least 30% cFBs. Paracrine signaling or N-cadherin-mediated communication between CMs and cFBs were no contributing factors, but our findings suggest that the mechanoresponsive cFBs provide structural guidance for CM orientation and elongation. Our study therefore highlights a synergistic mechanobiological interplay between CMs and cFBs in shaping tissue organization, which is of relevance for regenerating functionally organized myocardium.

consistently displayed an elongated morphology on all FN patterns with an aspect ratio (AR) 4.5 ± 2.9 127 on the parallel patterns and 3.5 ± 2.7 on crosshatch patterns. Alignment along the parallel lines was also 128 found for the CMs, although the CMs showed morphological heterogeneity and formed small aggregates 129 on the printed ECM patterns. Such aggregations are often found in contracting CMs cultures and involve 130 cell-cell interactions [22], possibly influencing the amount of CM adhesion to the parallel protein patterns.

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However, CM density on the substrates did not change during the experiments, suggesting that the vast 132 majority of CMs maintained cell-ECM interactions with the FN patterns. The CMs exhibited an AR of 5.3 133 ± 1.8 on parallel lines and 2.1 ± 1.0 on crosshatch patterns. The low AR found on the crosshatch protein 134 patterns suggests a relatively immature state of the CMs, derived from stem cells, as opposed to adult 135 CMs in vivo [22], which is in line with previous studies using hPSC-CMs [23,24]. 136 Previously, it has been shown that anisotropic ECM induced the alignment of cFBs [10, 23], hPSC-CMs 137 [25 -29], and neonatal rat CMs [8,30]. Consistent with these reports, quantification of cell orientation 138 showed a peak in the direction of the parallel FN lines (0º) and random distribution on crosshatch and 139 homogeneous FN for both cFBs and CMs ( Figure 1G-I). Notably, a direct comparison between the two 140 cell types on the same, well-defined ECM patterns, revealed that CMs exhibited more pronounced 141 anisotropic orientation than the cFBs ( Figure 1G). This is possibly explained by the smaller cell size of 5  showing FN patterns (TRITC-fibronectin, gray) and cFBs (A-C) and CMs (D-F) (alcein AM, green). G -I) Frequency distribution graphs display the cell orientation with respect to the horizontal direction (0º) for cFBs (blue) and CMs (red). Scale bar indicates 100 μm. Results are expressed as mean ± SEM (n = 9 from 3 independent experiments).
Mechanically, the myocardium and resident cells within experience continuous cyclic mechanical strain 155 due to cardiac beating. Thus, we sought to investigate the orientation response of CMs and cFBs to 156 cyclic strain in the presence of guiding ECM structures mimicked by protein patterns. For several 157 cardiovascular cell types, studies have shown a synergistic effect of structural guidance cues and 158 uniaxial cyclic strain when the cyclic strain direction is presented perpendicular to the anisotropic ECM 159 structures [32,33]. In contrast, we now asked whether ECM alignment and cyclic strain present 160 competing cues when they are applied in the same direction, similar to the mechanical 161 microenvironment in the myocardium. To this end, we seeded CMs and cFBs on printed ECM patterns 162 that were subsequently subjected to uniaxial cyclic strain at a physiological strain magnitude of 10% and 163 a frequency of 0.5 Hz (Figure 2). In response to such strains, several adherent cell types have been 164 reported to orient perpendicular to the direction of applied cyclic strainsa phenomenon called "strain  and CMs (red) display a clear peak at 90º for cFBs on crosshatch and homogeneous ECM while this is not observed for CMs. J -O) Oriented cell fractions at 0º ± 5º and 90º ± 5º, showing significant reorganization of the cFBs (blue) upon administration of uniaxial cyclic strain whereas this is not observed for CMs (red). ns = non-strained, s=strained. Scale bar indicates 100 μm. Results are expressed as mean ± SEM (n = 9 from 3 independent experiments); * = P < 0.05; ** = P < 0.01.   [45,46]. To this end, we used a 2D in vitro approach of micropatterned ECM protein patterns on 275 deformable substrates, which allowed controlled presentation of structural cues and mechanical strains 276 independently and simultaneously to monocultures and co-cultures of cFBs and CMs. We show that 277 CMs in co-culture with ≥ 30% cFBs exhibit a collective strain avoidance response concurrent with sarcomere alignment in the same direction, which was not found for the CM monocultures. Moreover, 279 we observed that uniaxial cyclic strain could not induce alignment of CMs via solely paracrine signaling 280 of the cFBs or when the cFBs lacked a strain avoidance response. Given this evidence, we hypothesize 281 that uniaxial cyclic strain promotes strain avoidance in cFBs, which in turn act as guidance cues for

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Our findings indicate that CMs and cFBs, the main cellular components of the myocardium, differentially 323 respond to structural and mechanical cues that typify the myocardial tissue. Importantly, an interplay 324 between the two cell types is necessary for the overall strain-induced response that leads to anisotropic 325 cell organization as is expected in healthy, contractile myocardium. Therefore, our results propose an  Top row: co-cultures of various seeding densities show random organization under static culture conditions. Bottom row: Upon cyclic strain application, strain-avoidance behavior is found for cFBs, which in turn serve as guidance cues for hPSC-CM orientation. We propose the formation of anisotropic guidance cues, resulting from the strain-avoidance response of cFBs, which steer hPSC-CM alignment and sarcomere organization in the direction almost perpendicular to the applied cyclic strain.

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Cardiac fibroblast conditioned medium: cFB conditioned medium was obtained from cFBs after 48 418 hours of mechanical uniaxial cyclic strain to allow secreting of paracrine factors. 50.000 cFBs were 419 plated on homogenous substrates 24 hours before mechanical stimulation. 3mL cFB culture medium 420 (as described in 2.3) was added to every well and the cFBs were strained (as described in 2.6) for 48 421 hours. After mechanical stimulation the conditioned medium was retrieved from the cFBs and filtered 422 using a 0,2 μm syringe filter to remove cells and other large particles. The medium was frozen immediately and thawed just before it was mixed 1:1 with fresh TDI medium and added to monocultures 424 of 100.000 hPSC-CMs, which were mechanically loaded or statically cultured as control.

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Quantification of cell and sarcomere orientation: The orientation response of the cardiac cells was 426 determined from triplicates of three independent experiments (n = 9). Cells were incubated with 1 μg/mL 427 calcein AM (Sigma-Aldrich) for 20 min and the orientation of their long axis was analyzed after medium 428 renewal with an inverted microscope (Zeiss Axiovert 200m equipped with an AxioCam HR camera; 429 Zeiss, Sliedrecht, the Netherlands). Only the central parts of the wells, where the strain field was 430 homogeneous as determined using our in-house calibrations, were considered (1 cm 2 , 50,000 cells).

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Cell orientation was quantified with ImageJ, using the Fiji plug-in "directionality", based on Fourier 432 spectrum analysis ( Figure S8).      Oriented cell fractions at 0º ± 5º and 90º ± 5º show significant reorganization of the cFB-rich (blue) and CM-rich (red) co-cultures upon administration of uniaxial cyclic strain on the crosshatch patterns. Ns = non-strained, s = strained. Results are expressed as mean ± SEM (n = 9 from 3 independent experiments); * = P < 0.05.   Figure S8: Representative fluorescent images and directionality histograms of linear and randomly oriented cFB to validate directionality quantification method. A -B) Orientation maps of cFBs cultured on either parallel or crosshatch protein patterns are colored according to the local directionality, or location orientation. C -D) Directionality histograms demonstrating either a distinct peak or approaching a flat line for cells cultured on parallel and crosshatch protein patterns, respectively.