Excitation-contraction (EC) coupling in the heart has, until recently, been solely accredited to 9 cardiomyocytes. The inherent complexities of the heart make it difficult to examine non-muscle 10 contributions to contraction in vivo, and conventional in vitro models fail to capture multiple 11 features and cellular heterogeneity of the myocardium. Here, we report on the development of a 12 3D cardiac µTissue to investigate changes in the cellular composition of native myocardium in 13 vitro. Cells are encapsulated within micropatterned gelatin-based hydrogels formed via visible 14 light photocrosslinking. This system enables spatial control of the microarchitecture, perturbation 15 of the cellular composition, and functional measures of EC coupling via video microscopy and a 16 custom algorithm to quantify beat frequency and degree of coordination. To demonstrate the 17 robustness of these tools and evaluate the impact of altered cell population densities on cardiac 18 µTissues, contractility and cell morphology were assessed with the inclusion of exogenous non-19 myelinating Schwann cells (SCs). Results demonstrate that the addition of exogenous SCs alter 20 cardiomyocyte EC, profoundly inhibiting the response to electrical pacing. Computational 21 modeling of connexin-mediated coupling suggests that SCs impact cardiomyocyte resting 22 Ischemic heart disease remains a leading cause of death worldwide. While pharmacological 1 interventions have improved life expectancy by mitigating key risk factors, therapeutic strategies 2 for repairing the damaged myocardium have yet to become the clinical standard (Björnson et al., 3 2016; Hashimoto et al., 2018). Following infarct, delivery of autologous cells, including 4 mesenchymal stem cells, cardiac stem cells, and endothelial progenitor cells, have yielded 5 promising results at the benchtop, but inconsistent benefits in clinical trials (Hatzistergos and 6

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Excitation-contraction (EC) coupling in the heart has, until recently, been solely accredited to cardiomyocytes. The inherent complexities of the heart make it difficult to examine nonmuscle contributions to contraction in vivo, and conventional in vitro models fail to capture multiple features and cellular heterogeneity of the myocardium. Here, we report on the development of a 3D cardiac µTissue towards recapitulating the architecture and composition of native myocardium in vitro. Cells are encapsulated within micropatterned gelatin-based hydrogels formed via visible light photocrosslinking. This system enables spatial control of cardiac microarchitecture, perturbation of the cellular composition, and functional measures of EC coupling via video microscopy and a custom algorithm to quantify beat frequency and degree of coordination. To demonstrate the robustness of these tools and evaluate the impact of altered cell population densities on cardiac µTissues, contractility and cell morphology were assessed with the inclusion of exogenous non-myelinating Schwann cells (SCs). Results demonstrate that the addition of exogenous SCs alter cardiomyocyte EC, profoundly inhibiting the response to electrical pacing. Computational modeling of connexin-mediated coupling suggests that SCs impact cardiomyocyte resting potential and rectification following depolarization. Cardiac µTissues hold potential for examining the role of cellular heterogeneity in heart health, pathologies, and cellular therapies. 2 GLIAL CELLS IN THE HEART ARTICLE I Figure 1 | Cardiac μTissue development. (A) Schematic representation of the in vitro co-culture system to investigate the heterogeneity of the myocardium. (B) Schematic of photopatterning hydrogels using visible light. (C) Quantification of live/dead images show >90% cardiac cell viability in GelMA crosslinked using LAP with visible light (** p < 0.05). (D) Representative images demonstrating an ability to photopattern a range of geometries (scale = 1000 μm).