INTRODUCTION

Section:

ChooseTop of pageABSTRACTINTRODUCTION <<RESULTSDISCUSSIONMETHODSSUPPLEMENTARY MATERIALThe function of the myocardium is highly dependent on its unique structural organization of cells and extracellular matrix (ECM). In the healthy human myocardium, cardiomyocytes (CMs) and quiescent cardiac fibroblasts (cFBs) are arranged as dense, aligned cellular aggregates surrounded by an anisotropic collagen matrix. This spatial arrangement enables electrical and mechanical coupling between CMs and facilitates their synchronous, coordinated contraction.1–31. R. H. Anderson, P. F. Niederer, D. Sanchez‐Quintana, R. S. Stephenson, and P. Agger, J. Anat. 235, 697 (2019). https://doi.org/10.1111/joa.130272. I. J. LeGrice, B. H. Smaill, L. Z. Chai, S. G. Edgar, J. B. Gavin, and P. J. Hunter, Am. J. Physiol.-Heart Circ. Physiol. 269, H571 (1995). https://doi.org/10.1152/ajpheart.1995.269.2.H5713. S. E. Lee, C. Nguyen, J. Yoon, H. J. Chang, S. Kim, C. H. Kim, and D. Li, Sci. Rep. 8, 6640 (2018). https://doi.org/10.1038/s41598-018-24622-6 Following ischemic cardiac injury, such as myocardial infarction (MI), billions of CMs die. The ischemia and subsequent inflammatory response at the site of injury initiate the activation and differentiation of cFBs toward myofibroblasts. These cells not only aid in replacing the damaged tissue with abundant ECM but also mediate adverse remodeling of the anisotropic myocardium into a disorganized fibrotic scar tissue. In addition, the myofibroblasts secrete profibrotic and hypertrophic cytokines, advancing the progression of adverse remodeling in the continuously beating myocardium and ultimately leading to heart failure.4–104. C. Hall, K. Gehmlich, C. Denning, and D. Pavlovic, J. Am. Heart Assoc. 10, e019338 (2021). https://doi.org/10.1161/JAHA.120.0193385. D. Gabriel-Costa, Pathophysiology 25, 277 (2018). https://doi.org/10.1016/j.pathophys.2018.04.0036. N. G. Frangogiannis, Mol. Aspects Med. 65, 70 (2019). https://doi.org/10.1016/j.mam.2018.07.0017. C. Humeres and N. G. Frangogiannis, JACC Basic Transl. Sci. 4, 449 (2019). https://doi.org/10.1016/j.jacbts.2019.02.0068. N. G. Frangogiannis, J. Clin. Invest. 127, 1600 (2017). https://doi.org/10.1172/JCI874919. R. Gaetani, E. A. Zizzi, M. A. Deriu, U. Morbiducci, M. Pesce, and E. Messina, Front. Cell Dev. Biol. 8, 00334 (2020). https://doi.org/10.3389/fcell.2020.0033410. K. M. Herum, J. Choppe, A. Kumar, A. J. Engler, and A. D. McCulloch, Mol. Biol. Cell 28, 1871 (2017). https://doi.org/10.1091/mbc.e17-01-0014The relevance of anisotropic cellular organization for adequate myocardial function has been demonstrated using well-defined in vitro systems. For instance, aligned CMs on two-dimensional (2D) substrates demonstrated improved calcium handling and contractile properties compared to randomly oriented CM monolayers.1111. A. W. Feinberg, P. W. Alford, H. Jin, C. M. Ripplinger, A. A. Werdich, S. P. Sheehy, A. Grosberg, and K. K. Parker, Biomaterials 33, 5732 (2012). https://doi.org/10.1016/j.biomaterials.2012.04.043 In three-dimensional (3D) cardiac microtissues, aligned CMs and ECM were found to generate a homogeneous and coordinated contraction, which was not observed in isotropic tissues.1212. A. C. C. van Spreeuwel, N. A. M. Bax, A. J. Bastiaens, J. Foolen, S. Loerakker, M. Borochin, D. W. J. van der Schaft, C. S. Chen, F. P. T. Baaijens, and C. V. C. Bouten, Integr. Biol. 6, 422 (2014). https://doi.org/10.1039/C3IB40219C Furthermore, alignment of mesenchymal stem cells, induced by gelatin microgrooves, supported differentiation toward the myocardial lineage.1313. A. Tijore, S. A. Irvine, U. Sarig, P. Mhaisalkar, V. Baisane, and S. Venkatraman, Biofabrication 10, 025003 (2018). https://doi.org/10.1088/1758-5090/aaa15d These studies suggest that restoring tissue anisotropy may represent a critical step in the functional regeneration of damaged myocardium. Yet, until today, cardiac regenerative strategies have largely focused on replenishing CMs,14–1614. R. Gong, Z. Jiang, N. Zagidullin, T. Liu, and B. Cai, Signal Transduct. Target. Ther. 6, 31 (2021). https://doi.org/10.1038/s41392-020-00413-215. M. Gyöngyösi, W. Wojakowski, P. Lemarchand, K. Lunde, M. Tendera, J. Bartunek, E. Marban, B. Assmus, T. D. Henry, J. H. Traverse, L. A. Moyé, D. Sürder, R. Corti, H. Huikuri, J. Miettinen, J. Wöhrle, S. Obradovic, J. Roncalli, K. Malliaras, E. Pokushalov, A. Romanov, J. Kastrup, M. W. Bergmann, D. E. Atsma, A. Diederichsen, I. Edes, I. Benedek, T. Benedek, H. Pejkov, N. Nyolczas, N. Pavo, J. Bergler-Klein, I. J. Pavo, C. Sylven, S. Berti, E. P. Navarese, G. Maurer, and ACCRUE Investigators, Circ. Res. 116, 1346 (2015). https://doi.org/10.1161/CIRCRESAHA.116.30434616. B. A. Tompkins, M. Natsumeda, W. Balkan, and J. M. Hare, Circ. Res. 120, 252 (2017). https://doi.org/10.1161/CIRCRESAHA.116.310340 while the restoration of the myocardial structural organization is largely overlooked.In vivo, the mechanically active myocardium presents cyclic deformations—due to cardiac beating—as well as structural cues—imposed by collagen fibers—to the CMs and cFBs. Such microenvironmental biophysical cues are known to affect cell alignment by mediating the anisotropic organization of their internal structures.1717. C. Tamiello, A. B. C. Buskermolen, F. P. T. Baaijens, J. L. V. Broers, and C. V. C. Bouten, Cell. Mol. Bioeng. 9, 12 (2016). https://doi.org/10.1007/s12195-015-0422-7 In vitro, anisotropic structural guidance cues can be mimicked using parallel micro-grooves, adhesive protein patterns, or aligned scaffold fibers, demonstrating that both cFBs18–2018. S. Al-Haque, J. W. Miklas, N. Feric, L. L. Y. Chiu, W. L. K. Chen, C. A. Simmons, and M. Radisic, Macromol. Biosci. 12, 1342 (2012). https://doi.org/10.1002/mabi.20120004219. Y. Liu, T. Xia, J. Wei, Q. Liu, and X. Li, Nanoscale 9, 4950 (2017). https://doi.org/10.1039/C7NR00001D20. H. T. H. Au, I. Cheng, M. F. Chowdhury, and M. Radisic, Biomaterials 28, 4277 (2007). https://doi.org/10.1016/j.biomaterials.2007.06.001 and CMs11,19–2211. A. W. Feinberg, P. W. Alford, H. Jin, C. M. Ripplinger, A. A. Werdich, S. P. Sheehy, A. Grosberg, and K. K. Parker, Biomaterials 33, 5732 (2012). https://doi.org/10.1016/j.biomaterials.2012.04.04319. Y. Liu, T. Xia, J. Wei, Q. Liu, and X. Li, Nanoscale 9, 4950 (2017). https://doi.org/10.1039/C7NR00001D20. H. T. H. Au, I. Cheng, M. F. Chowdhury, and M. Radisic, Biomaterials 28, 4277 (2007). https://doi.org/10.1016/j.biomaterials.2007.06.00121. P. Y. Wang, J. Yu, J. H. Lin, and W. B. Tsai, Acta Biomater. 7, 3285 (2011). https://doi.org/10.1016/j.actbio.2011.05.02122. I. Batalov, Q. Jallerat, S. Kim, J. Bliley, and A. W. Feinberg, Sci. Rep. 11, 11502 (2021). https://doi.org/10.1038/s41598-021-87550-y can sense this 2D micro-environment and align with the anisotropy presented—a phenomenon termed contact guidance. When subjected to uniaxial cyclic strain, adherent cells may (re)orient toward the direction (almost) perpendicular to the strain—a phenomenon called strain avoidance. Dynamic reorganization and remodeling of actin stress fibers in combination with a nucleo-cytoskeletal connection are believed to play an important role in the cell type-dependent response to uniaxial cyclic strain (mechanoresponse).23–2523. C. Tamiello, M. Halder, M. A. F. Kamps, F. P. T. Baaijens, J. L. V. Broers, and C. V. C. Bouten, J. Cell Sci. 130, 779 (2017). https://doi.org/10.1242/jcs.18483824. M. M. Nava, Y. A. Miroshnikova, L. C. Biggs, D. B. Whitefield, F. Metge, J. Boucas, H. Vihinen, E. Jokitalo, X. Li, J. M. García Arcos, B. Hoffmann, R. Merkel, C. M. Niessen, K. N. Dahl, and S. A. Wickström, Cell 181, 800 (2020). https://doi.org/10.1016/j.cell.2020.03.05225. A. Mauretti, N. A. M. Bax, M. H. van Marion, M. J. Goumans, C. Sahlgren, and C. V. C. Bouten, Integr. Biol. 8, 991 (2016). https://doi.org/10.1039/C6IB00117C While cFBs are consistent in their strain avoidance behavior,26,2726. G. S. Ugolini, M. Rasponi, A. Pavesi, R. Santoro, R. Kamm, G. B. Fiore, M. Pesce, and M. Soncini, Biotechnol. Bioeng. 113, 859 (2016). https://doi.org/10.1002/bit.2584727. R. D. H. Tran, T. A. Morris, D. Gonzalez, A. H. S. H. A. Hetta, and A. Grosberg, Cells 10, 3199 (2021). https://doi.org/10.3390/cells10113199 CMs show inconsistent responses,27–3127. R. D. H. Tran, T. A. Morris, D. Gonzalez, A. H. S. H. A. Hetta, and A. Grosberg, Cells 10, 3199 (2021). https://doi.org/10.3390/cells1011319928. A. Salameh, A. Wustmann, S. Karl, K. Blanke, D. Apel, D. Rojas-Gomez, H. Franke, F. W. Mohr, J. Janousek, and S. Dhein, Circ. Res. 106, 1592 (2010). https://doi.org/10.1161/CIRCRESAHA.109.21442929. Y. Qi, Z. Li, C. W. Kong, N. L. Tang, Y. Huang, R. A. Li, and X. Yao, J. Mol. Cell. Cardiol. 87, 65 (2015). https://doi.org/10.1016/j.yjmcc.2015.08.00530. Y. W. Chun, D. E. Voyles, R. Rath, L. H. Hofmeister, T. C. Boire, H. Wilcox, J. H. Lee, L. M. Bellan, C. C. Hong, and H. J. Sung, J. Biomech. 48, 3890 (2015). https://doi.org/10.1016/j.jbiomech.2015.09.02831. J. Kreutzer, M. Viehrig, R.-P. Pölönen, F. Zhao, M. Ojala, K. Aalto-Setälä, and P. Kallio, Biomech. Model. Mechanobiol. 19, 291 (2020). https://doi.org/10.1007/s10237-019-01211-8 probably related to differences in their contractile state and actin organization.32,3332. J. J. Saucerman, P. M. Tan, K. S. Buchholz, A. D. McCulloch, and J. H. Omens, Nat. Rev. Cardiol. 16, 361 (2019). https://doi.org/10.1038/s41569-019-0155-833. M. Ward and T. Iskratsch, Biochim. Biophys. Acta-Mol. Cell Res. 1867, 118436 (2020). https://doi.org/10.1016/j.bbamcr.2019.01.017 To date, CM mechanoresponse remains largely unexplored, contributing to the controversy about how CMs respond to cyclic strain. Moreover, little is known about the collective organization of physiological and pathological combinations of CMs and cFBs, and how this is guided or disrupted by structural and mechanical cues in the myocardium.2222. I. Batalov, Q. Jallerat, S. Kim, J. Bliley, and A. W. Feinberg, Sci. Rep. 11, 11502 (2021). https://doi.org/10.1038/s41598-021-87550-y A better understanding of how biophysical cues in the myocardium can contribute to the singular and collective organization of CMs and cFBs represents a key step for improving in vivo myocardial regeneration, including cell-based therapies.

In this study, we address this knowledge gap by emulating the fibrous ECM organization and beating of the myocardium in a 2D in vitro model of the human myocardial microenvironment. Human fetal epicardial cell derived cFBs and human pluripotent stem cell derived cardiomyocytes (CMs) were cultured on (an)isotropic ECM protein patterns and subsequently subjected to uniaxial cyclic strain. By systematically examining the cellular response of monocultures as well as co-cultures of (varying ratios of) CMs and cFBs to singular and combined environmental cues, this approach provided novel insights into the interplay between the two cell types in regulating organization in physiological and pathological myocardial tissue compositions.

RESULTS

Section:

ChooseTop of pageABSTRACTINTRODUCTIONRESULTS <<DISCUSSIONMETHODSSUPPLEMENTARY MATERIAL

ECM patterning guides CM and cFB orientation

Structurally, the myocardium is a fibrous tissue, implying that cardiac cells receive structural cues from contact with ECM fibers. Similar anisotropic topographical or structural cues from the substrate, such as micropatterns, microfabricated grooves, or collagen fibers, have been shown to guide the orientation of CMs and cFBs—a phenomenon termed “contact-guidance.”11,34,3511. A. W. Feinberg, P. W. Alford, H. Jin, C. M. Ripplinger, A. A. Werdich, S. P. Sheehy, A. Grosberg, and K. K. Parker, Biomaterials 33, 5732 (2012). https://doi.org/10.1016/j.biomaterials.2012.04.04334. E. T. Den Braber, J. E. De Ruijter, L. A. Ginsel, A. F. von Recum, and J. A. Jansen, J. Biomed. Mater. Res. 40, 291 (1998). https://doi.org/10.1002/(SICI)1097-4636(199805)40:2<291::AID-JBM14>3.0.CO;2-P35. G. A. Dunn and J. P. Heath, Exp. Cell Res. 101(1), 1–14 (1976). https://doi.org/10.1016/0014-4827(76)90405-5 Here, to mimic the local anisotropic structure of the healthy myocardium and to allow application of mechanical strain, we created fibronectin (FN) adhesive patterns on deformable polydimethylsiloxane (PDMS) membranes using previously established microcontact printing methods (Fig. 1).3636. M. T. Yang, J. Fu, Y. K. Wang, R. A. Desai, and C. S. Chen, Nat. Protoc. 6, 187 (2011). https://doi.org/10.1038/nprot.2010.189 Two types of patterns were produced: (i) parallel lines with a linewidth (10 μm), identical to the inter-line spacing [Figs. 1(a) and 1(d)], corresponding to the physiological fiber size of perimysial collagen in the myocardium,3737. P. J. Hanley, A. A. Young, I. J. LeGrice, S. G. Edgar, and D. S. Loiselle, J. Physiol. 517, 831 (1999). https://doi.org/10.1111/j.1469-7793.1999.0831s.x and (ii) crosshatch patterns [orthogonal linear features of 5 μm linewidth and 10 μm spacing, Figs. 1(b) and 1(e)] to mimic isotropic ECM. The linewidth for the crosshatch pattern was set to be half that of the parallel line patterns to achieve comparable area density for cell adhesion on the two pattern types. Homogeneously FN-printed PDMS using an unpatterned microstamp served as control substrate [Figs. 1(c) and 1(f)]. All FN patterns remained visually intact and well defined throughout the experimental procedures of 48 h of strained or static cell culture (Fig. S1).Orientation of cells was evaluated using cytoplasmic calcein AM staining, which circumvents drawbacks with cytoskeleton-based staining due to the substantially different cytoskeletal organizations in CMs and cFBs and serves as optimal start for the quantification of cell orientation and shape in the same wells over time. Within 24 h of seeding on the printed ECM patterns, both cFBs [Fig. 1(a)] and CMs [Fig. 1(d)] showed a clear contact guidance behavior. cFBs were aligned in the direction of the parallel lines and consistently displayed an elongated morphology on all FN patterns with an aspect ratio (AR) of 4.5 ± 2.9 (n = 30) on the parallel patterns and 3.5 ± 2.7 (n = 30) on crosshatch patterns. Alignment along the parallel lines was also found for the CMs, although the CMs showed morphological heterogeneity and formed small aggregates on the printed ECM patterns. Such aggregations are often found in contracting CMs cultures and involve cell–cell interactions,3838. C. C. Veerman, G. Kosmidis, C. L. Mummery, S. Casini, A. O. Verkerk, and M. Bellin, Stem Cells Dev. 24, 1035 (2015). https://doi.org/10.1089/scd.2014.0533 possibly influencing the amount of CM adhesion to the parallel protein patterns. However, CM density on the substrates did not change during the experiments, suggesting that the vast majority of CMs maintained cell–ECM interactions with the FN patterns. The CMs exhibited an AR of 5.3 ± 1.8 (n = 30) on parallel lines and 2.1 ± 1.0 (n = 30) on crosshatch patterns. The AR found on the crosshatch protein patterns is in line with previous studies using hPSC-CMs3939. H. Y. S. Chan, W. Keung, R. A. Li, A. L. Miller, and S. E. Webb, Stem Cell Int. 2015, 586908. https://doi.org/10.1155/2015/586908 and is lower than that for adult CMs in vivo,3838. C. C. Veerman, G. Kosmidis, C. L. Mummery, S. Casini, A. O. Verkerk, and M. Bellin, Stem Cells Dev. 24, 1035 (2015). https://doi.org/10.1089/scd.2014.0533 which has been attributed to the lower maturity state of hPSC-CMs.Previously, it has been shown that anisotropic ECM induced the alignment of cFBs,18–2018. S. Al-Haque, J. W. Miklas, N. Feric, L. L. Y. Chiu, W. L. K. Chen, C. A. Simmons, and M. Radisic, Macromol. Biosci. 12, 1342 (2012). https://doi.org/10.1002/mabi.20120004219. Y. Liu, T. Xia, J. Wei, Q. Liu, and X. Li, Nanoscale 9, 4950 (2017). https://doi.org/10.1039/C7NR00001D20. H. T. H. Au, I. Cheng, M. F. Chowdhury, and M. Radisic, Biomaterials 28, 4277 (2007). https://doi.org/10.1016/j.biomaterials.2007.06.001 hPSC-CMs,40–4340. J. Han, Q. Wu, Y. Xia, M. B. Wagner, and C. Xu, Stem Cell Res. 16, 740 (2016). https://doi.org/10.1016/j.scr.2016.04.01441. D. Carson, M. Hnilova, X. Yang, C. L. Nemeth, J. H. Tsui, A. S. T. Smith, A. Jiao, M. Regnier, C. E. Murry, C. Tamerler, and D. H. Kim, ACS Appl. Mater. Interfaces 8, 21923 (2016). https://doi.org/10.1021/acsami.5b1167142. J. Macadangdang, H. J. Lee, D. Carson, A. Jiao, J. Fugate, L. Pabon, M. Regnier, C. Murry, and D. H. Kim, J. Vis. Exp. 88, e50039 (2014). https://doi.org/10.3791/500343. M. Khan, Y. Xu, S. Hua, J. Johnson, A. Belevych, P. M. L. Janssen, S. Gyorke, J. Guan, and M. G. Angelos, PLoS One 10, e0126338 (2015). https://doi.org/10.1371/journal.pone.0126338 and neonatal rat CMs.13,42,4413. A. Tijore, S. A. Irvine, U. Sarig, P. Mhaisalkar, V. Baisane, and S. Venkatraman, Biofabrication 10, 025003 (2018). https://doi.org/10.1088/1758-5090/aaa15d42. J. Macadangdang, H. J. Lee, D. Carson, A. Jiao, J. Fugate, L. Pabon, M. Regnier, C. Murry, and D. H. Kim, J. Vis. Exp. 88, e50039 (2014). https://doi.org/10.3791/500344. H. T. Heidi Au, B. Cui, Z. E. Chu, T. Veres, and M. Radisic, Lab Chip 9, 564 (2009). https://doi.org/10.1039/B810034A Consistent with these reports, quantification of cell orientation showed a peak in the direction of the parallel FN lines (0°) and random distribution on crosshatch and homogeneous FN for both cFBs and CMs [Fig. 1(g)–1(i)]. Notably, a direct comparison between the two cell types on the same, well-defined ECM patterns, revealed that CMs exhibited more pronounced anisotropic orientation than the cFBs [Fig. 1(g)]. This is possibly explained by the smaller cell size of the CMs (110 ± 26 μm longitudinal axis) compared to cFBs (242 ± 59 μm longitudinal axis). Indeed, Buskermolen et al. showed that cell size is an important determinant of cell alignment on microscale ECM patterns and found that the alignment of cardiomyocyte progenitor cells (∼100 μm length) occurred at smaller ECM pattern size compared to that of myofibroblasts (∼200 μm length).4545. A. B. C. Buskermolen, T. Ristori, D. Mostert, M. C. van Turnhout, S. S. Shishvan, S. Loerakker, N. A. Kurniawan, V. S. Deshpande, and C. V. C. Bouten, Cell Rep. Phys. Sci. 1, 100055 (2020). https://doi.org/10.1016/j.xcrp.2020.100055 Taken together, these experiments confirmed that ECM patterning is an effective approach to exploit contact guidance to direct the alignment response of both CMs and cFBs.

cFB cultures, but not CM cultures, show strain avoidance in response to uniaxial cyclic strain

Mechanically, the myocardium resident cells experience continuous cyclic mechanical strain due to cardiac beating. Thus, we sought to investigate the orientation response of CMs and cFBs to cyclic strain in the presence of guiding ECM structures mimicked by protein patterns. For several cardiovascular cell types, studies have shown a synergistic effect of structural guidance cues and uniaxial cyclic strain when the cyclic strain direction is presented perpendicular to the anisotropic ECM structures.46,4746. C. Tamiello, C. V. C. Bouten, and F. P. T. Baaijens, Sci. Rep. 5, 8752 (2015). https://doi.org/10.1038/srep0875247. K. Balachandran, P. W. Alford, J. Wylie-Sears, J. A. Goss, A. Grosberg, J. Bischoff, E. Aikawa, R. A. Levine, and K. K. Parker, Proc. Natl. Acad. Sci. U. S. A. 108, 19943 (2011). https://doi.org/10.1073/pnas.1106954108 In contrast, we now asked whether ECM alignment and cyclic strain present competing cues when they are applied in the same direction, similar to the mechanical microenvironment in the myocardium. To this end, we seeded CMs and cFBs on printed ECM patterns that were subsequently subjected to uniaxial cyclic strain at a physiological strain magnitude of 10% and a frequency of 0.5 Hz (Fig. 2). In response to such strains, several adherent cell types have been reported to orient perpendicular to the direction of applied cyclic strains—a phenomenon called “strain avoidance.”24,25,48,4924. M. M. Nava, Y. A. Miroshnikova, L. C. Biggs, D. B. Whitefield, F. Metge, J. Boucas, H. Vihinen, E. Jokitalo, X. Li, J. M. García Arcos, B. Hoffmann, R. Merkel, C. M. Niessen, K. N. Dahl, and S. A. Wickström, Cell 181, 800 (2020). https://doi.org/10.1016/j.cell.2020.03.05225. A. Mauretti, N. A. M. Bax, M. H. van Marion, M. J. Goumans, C. Sahlgren, and C. V. C. Bouten, Integr. Biol. 8, 991 (2016). https://doi.org/10.1039/C6IB00117C48. M. Moretti, A. Prina-Mello, A. J. Reid, V. Barron, and P. J. Prendergast, J. Mater. Sci. Mater. Med. 15, 1159 (2004). https://doi.org/10.1023/B:JMSM.0000046400.18607.7249. C. Huang, K. Miyazaki, S. Akaishi, A. Watanabe, H. Hyakusoku, and R. Ogawa, J. Plast. Reconstr. Aesthetic Surg. 66, e351 (2013). https://doi.org/10.1016/j.bjps.2013.08.002 On the anisotropic ECM patterns, however, both the CMs and cFBs showed only a slight disruption of cellular alignment without a complete strain avoidance response within the 48 h of cyclic strain [Figs. 2(a), 2(d), and 2(g)]. Quantification of the cell orientation demonstrated a slight decrease in the fraction of cells oriented at 0° ± 5° (contact-guided response) and a lack of alignment in the direction perpendicular to the strain at 90° ± 5° (strain avoidance response) [Figs. 2(j) and 2(m)], supporting this dominance of cellular contact guidance over strain avoidance. However, when the starting point was a dis- or nonorganized cellular organization (on crosshatch ECM patterns and homogeneous FN), the cFBs displayed a clear strain avoidance response, as most cells oriented toward 90° [Figs. 2(b)–2(c), 2(h)–2(i), blue]. This is consistent with the reported response of cFBs on homogeneous ECM.26,5026. G. S. Ugolini, M. Rasponi, A. Pavesi, R. Santoro, R. Kamm, G. B. Fiore, M. Pesce, and M. Soncini, Biotechnol. Bioeng. 113, 859 (2016). https://doi.org/10.1002/bit.2584750. N. A. M. Bax, S. N. Duim, B. P. T. Kruithof, A. M. Smits, C. V. C. Bouten, and M. J. Goumans, Front. Cardiovasc. Med. 6, 00081 (2019). https://doi.org/10.3389/fcvm.2019.00081 In contrast, uniaxial cyclic strain failed to induce orientation of the CMs away from the direction of the applied dynamic strain, even on dis- or nonorganized ECM [Figs. 2(e) and 2(f), 2(h)–2(i), red]. This dispersity between cell types is further highlighted by the significant increase in the oriented cell fractions at 90° ± 5° for cFBs and the absence of (re)orientation for the CMs [Figs. 2(n) and 2(o)]. These findings suggest that cFBs and CMs have different intrinsic abilities to sense and respond to cyclic strain.

Cardiac co-cultures of varying cell ratios organize in response to uniaxial cyclic strain

Given the stark contrast between the collective cellular responses of CMs and cFBs to environmental cues, we next asked how their difference in mechanoresponsiveness would dictate the organization of the intracellular F-actin and sarcomeres (α-actinin) structures in myocardial co-cultures. To assess how co-culture composition influences the overall mechanoresponse, we created co-cultures of CMs and cFBs with either 70:30 (CM-rich) or 30:70 (cFB-rich) cell seeding ratio. In vivo, adult CMs account for ∼70% of myocardial volume, although they comprise ∼30% of myocardial cell number.51,5251. P. Zhou and W. T. Pu, Circ. Res. 118, 368 (2016). https://doi.org/10.1161/CIRCRESAHA.116.30813952. A. R. Pinto, A. Ilinykh, M. J. Ivey, J. T. Kuwabara, M. L. D'antoni, R. Debuque, A. Chandran, L. Wang, K. Arora, N. A. Rosenthal, and M. D. Tallquist, Circ. Res. 118, 400 (2016). https://doi.org/10.1161/CIRCRESAHA.115.307778 However, the cellular volume of adult CMs largely differs from the CM volume used in this study, suggesting that co-cultures consisting of ≥30% cFBs are of relevance to mimic the cellular distribution of the myocardium. We note that impurities in the CM culture and an increase in number of viable cFBs led to an increased number of non-myocyte cells in the co-culture during the experimental procedure, resulting in co-cultures with 55.3% ± 16.5% α-actinin positive cells (indicative for CMs) for the CM-rich co-cultures and 18.7% ± 9.4% for the cFB-rich co-cultures after 48 h [Fig. S2(a)]. In other words, although the cell composition changed throughout the experimental procedure, the number of CMs was always significantly higher in the CM-rich co-culture. Cell viability remained constant between experimental conditions and time points [Figs. S2(b)–S2(e)].Under static culture conditions, both co-cultures demonstrated cellular alignment on the linear ECM patterns [Figs. 3(a) and 3(e)] and a random cellular orientation on the crosshatch ECM patterns [Figs. 3(c) and 3(g)], similar to the response of monocultures of cFBs and CMs. This is further demonstrated by the frequency distribution histograms for F-actin and sarcomere orientation, both showing a peak ∼0° for the parallel patterns and no preferred orientation for the crosshatch patterns [Figs. 3(i)–3(p)].To address how the mechanical environment in the myocardial niche, where ECM guidance and cyclic strain together influence the organization of cardiac cells, affects the organization of cardiac co-cultures, we studied the combinatory effect of uniaxial cyclic strain and (an)isotropic ECM on cFB-rich and CM-rich co-cultures. When uniaxial cyclic strain was applied in the same direction as the anisotropic ECM, both co-cultures lacked a distinct strain avoidance behavior [Figs. 3(b), 3(f), 3(i), 3(k), 3(m), and 3(o)], although a slight disruption in preferred orientation was found for the CM-rich co-culture in both F-actin and sarcomere organization [Figs. 3(k) and 3(o)]. These results show that contact-guided cellular organization of cardiac co-cultures cannot be easily disrupted by cyclic strain induced strain avoidance, at least with the pattern dimensions and strain protocols used in this study.Next, we analyzed whether cyclic strain could overpower the disorder that results from disorganized ECM. To do so, the cardiac co-cultures were strained after culture on crosshatch patterns. Both the CM-rich and cFB-rich co-cultures showed anisotropic organization of the cellular monolayers after 48 h of dynamic culture [Figs. 3(d) and 3(h)], together with realignment of both their F-actin and sarcomere structures [Figs. 3(j), 3(l), 3(n), and 3(p)]. This was unexpected, given that CM monocultures did not show such strain avoidance response. Together, these findings suggest that cFBs guide the orientation of CMs when they align in response to uniaxial cyclic strain, either by direct cell–cell contact or via paracrine signaling. Moreover, these results suggest that uniaxial cyclic strain can be used to induce linear organization of cardiac cells on crosshatch ECM patterns.To assess the specific contribution of the CMs to the collective reorientation response of the co-cultures, we used a double fluorescent hPSC reporter of mRubyIIACTN2 and GFP-NKX2.5 (DRRAGN).5353. M. C. Ribeiro, R. H. Slaats, V. Schwach, J. M. Rivera-Arbelaez, L. G. J. Tertoolen, B. J. van Meer, R. Molenaar, C. L. Mummery, M. M. A. E. Claessens, and R. Passier, J. Mol. Cell. Cardiol. 141, 54 (2020). https://doi.org/10.1016/j.yjmcc.2020.03.008 This cell line demonstrates identical behavior as our earlier findings above, both as monoculture and in co-culture with cFBs (Fig. S3). Specifically, quantification of stress fiber (cFBs) and sarcomere (CM) orientation demonstrated the lack of a strain avoidance response in CMs, which is typical for the cFBs [Figs. S3(d) and S3(i)]. Assessment of nuclear orientation and aspect ratio showed a significant increase in nuclear aspect ratio for cFBs when strain was applied as opposed to unstrained cFBs, which was not the case for CMs. Moreover, clear nuclear alignment was found in the direction perpendicular to the strain for cFBs, which was not observed for CMs when uniaxial cyclic strain was applied [Figs. S3(e) and S3(j)].To assess the amount of cFBs necessary to induce the strain-induced alignment of CMs, we used this reporter cell line to systematically create co-cultures with CM-cFB ratios of 20:80, 50:50, 80:20 and subject them to uniaxial cyclic strain for 48 h on homogenous FN. Qualitative assessment of α-actinin expression by immunofluorescent staining demonstrated aligned sarcomere structures in the direction almost perpendicular to the strain in the 20:80 and 50:50 co-cultures [Figs. 4(n) and 4(r)], similar to what was observed in the 70:30 and 30:70 co-cultures. Surprisingly, both CM and cFBs did not reorient in the 80:20 co-culture after strain application [Figs. 4(u)–4(x)]. Quantification of F-actin and sarcomere orientation, which can be linked to the direction of CM contraction, indeed, showed a distinct preferred sarcomere orientation in the 90° direction in contrast to unstrained controls [Figs. 5(a), 5(b), 5(d), and 5(e)]. In addition, as opposed to the more rounded morphologies in CM monocultures and the 80:20 co-culture [Figs. 4(i)–4(l) and 4(u)–4(x)], CMs demonstrated elongated morphologies and sarcomere alignment both under dynamic and static conditions when enough cFBs were present. Notably, a lack of strain-induced alignment was found for CMs in the 80:20 culture [Figs. 5(c) and 5(e)], suggesting a threshold of cFBs, that is, needed in the co-culture to induce strain-induced alignment of CMs.Since sarcomere length is greater with increased CM contractility54–5654. A. G. Rodriguez, S. J. Han, M. Regnier, and N. J. Sniadecki, Biophys. J. 101, 2455 (2011). https://doi.org/10.1016/j.bpj.2011.09.05755. D. E. Rassier, Am. J. Physiol.-Cell Physiol. 313, C134 (2017). https://doi.org/10.1152/ajpcell.00050.201756. F. de Souza Leite and D. E. Rassier, Biophys. J. 119, 2372 (2020). https://doi.org/10.1016/j.bpj.2020.11.005 and can, therefore, serve as a preliminary assessment of CM function, we quantified sarcomere length in the co-cultures of various ratios under static and strained conditions [Fig. 5(g)]. Although the exact values of sarcomere length obtained using this approach might not be as accurate as quantified with other techniques,57,5857. S. M. O'Connor, E. J. Cheng, K. W. Young, S. R. Ward, and R. L. Lieber, J. Exp. Biol. 219, 1432 (2016). https://doi.org/10.1242/jeb.13208458. J. M. Stein, U. Arslan, M. Franken, J. C. de Greef, S. E. Harding, N. Mohammadi, V. V. Orlova, M. Bellin, C. L. Mummery, and B. J. van Meer, Curr. Protoc. 2, e462 (2022). https://doi.org/10.1002/cpz1.462 our results clearly indicate that cardiomyocytes in the 20:80 and 50:50 co-cultures demonstrated sarcomeres of greater length when cyclic strain was applied as opposed to unstrained conditions, indicative of an increased contraction force. Increased sarcomere length with cyclic strain is in line with Zhang et al. who reported lengthening of sarcomeres when engineered heart tissues were loaded cyclically.5959. W. Zhang, C. W. Kong, M. H. Tong, W. H. Chooi, N. Huang, R. A. Li, and B. P. Chan, Acta Biomater. 49, 204 (2017). https://doi.org/10.1016/j.actbio.2016.11.058 Interestingly, sarcomere length was found highest in the 50:50 co-culture, both in the strained and unstrained condition, suggesting that a 1:1 ratio is more favorable for the CM contractile properties as opposed to 1:5 and 5:1. Future studies should be done to probe this further at the cardiac functional level.Together, these results suggest a threshold of the cFB number (>30%) required for inducing strain avoidance in CMs as well as aligned sarcomere structures with greater sarcomere length, indicative of CM maturity.60,6160. Y. Guo and W. T. Pu, Circ. Res. 126, 1086 (2020). https://doi.org/10.1161/CIRCRESAHA.119.31586261. E. Karbassi, A. Fenix, S. Marchiano, N. Muraoka, K. Nakamura, X. Yang, and C. E. Murry, Nat. Rev. Cardiol. 17, 341 (2020). https://doi.org/10.1038/s41569-019-0331-x

Strain avoidance of CMs in the presence of cFBs is not mediated by paracrine signaling or N-cadherin

We next asked what mechanisms are underlying the strain avoidance of CMs when in co-culture with a threshold value of cFBs. In particular, we wondered if the observed effect could be explained by paracrine signaling of the cFBs or by direct cell–cell communication via adherens junctional protein N-cadherin. To assess whether the impact of cFBs on CMs alignment could be attributed to paracrine factors, conditioned medium was collected from cFB monocultures after 48 h of uniaxial cyclic strain and added to the CMs before strain application. After 48 h cyclic strain of CMs in the conditioned medium, no significant differences in cell orientation were found (p = 0.28) in comparison to untreated monocultures of CMs [Figs. S4(a), S4(b), S4(e), and S4(f)]. Moreover, limited sarcomere formation was observed in the CMs in the conditioned medium, similar to the CM monocultures, suggesting that the improved maturation state of CMs in co-culture with cFBs is not attributable to paracrine signaling [Figs. S4(c) and S4(d)].

To investigate whether adherens-junction-mediated cell–cell communications play a role in the strain-induced alignment of CMs in cardiac co-cultures, we analyzed the expression and localization of N-cadherin (Fig. S5). While N-cadherin expression was clearly observed at the cell membrane between CMs, N-cadherin was absent between cFBs, and merely cytoplasmic N-cadherin was observed in CMs when in direct contact with cFBs, lacking the clear membrane bound N-cadherin expression found between CMs [Figs. S5(g), S5(j), and S5(m)]. Moreover, no preferential organization of N-cadherin mediated cell–cell interactions was found when CMs are organized via cyclic strain [Figs. S5(n) and S5(m)]. These results suggest that the strain-induced alignment of CMs in co-culture with cFBs is caused neither by paracrine signaling nor by N-cadherin mediated cell–cell communication between the CMs and cFBs. This is in line with a recent report showing a lack of Cx43 and N-cadherin-mediated cell–cell communication in the co-culture organization of neonatal rat CMs and cFBs.2727. R. D. H. Tran, T. A. Morris, D. Gonzalez, A. H. S. H. A. Hetta, and A. Grosberg, Cells 10, 3199 (2021). https://doi.org/10.3390/cells10113199 Future experiments using targeted inhibition of Cx43 and N-cadherin in co-cultures under cyclic strain together with transcriptional analysis should be performed to validate these findings.

DISCUSSION

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