Glial cells influence cardiac permittivity as evidenced through in vitro and in silico models

Abstract

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

Reconfigurable Microphysiological Systems for Modeling Innervation and Multitissue Interactions

“…Primary Cell Isolation: Primary CMs and aCCs from the ventricles, and primary postganglionic sympathetic neurons from the SCG were isolated from 2 d old (p2) Sprague-Dawley neonatal rats (Taconic Biosciences) using established protocols approved by Northeastern University's Institutional Animal Care and Use Committee (19-0104R). [17,20] In brief, rat pups were euthanized by decapitation and isolated tissue kept on ice in Hibernate-A (BrainBits) while tissue from every pup was collected. Once all tissue was harvested, SCGs were enzymatically dissociated sequentially in collagenase I (305 units mg −1 in Hank's Balanced Salt Solution (HBSS), Gibco) for 60 min and then in 0.5x Trypsin in HBSS for 15 min.…”

“…In our previous work, visible light photocrosslinked GelMA hydrogels were shown to support the 3D culture of primary cardiac cell populations and peripheral neurons. [17,18] Furthermore, to maintain ion channel expression, membrane resting potential, and depolarization characteristics to that of intact tissue in vitro, SNS neurons must be cultured within a 3D microenvironment. [19] Here, we established cardiac microtissues on-chip, using a 7.5% (w/v) GelMA hydrogel.…”

“…A 50/50 mixture of enrichened CMs and adherent cardiac cells (aCCs) from the left and right ventricles was prepared with a 20 m cells mL −1 concentration in a GelMA precursor solution. [17] This solution was carefully loaded into the device so that it filled to the GelPin and gelled in situ with 10 mW cm −2 405 nm light for 60 s. Adjacent to these tissues, primary postganglionic SNS neurons from the superior cervical ganglion (SCG) similarly loaded and gelled in situ at 5 m cells mL −1 . A representative depth-coded, immunofluorescent image of the cardiac SNS MPS demonstrates neurites extend toward the target cardiac cells, both above and below the floating GelPins, crossing the gel interfaces while maintaining physiological compartmentalization of neurons' somas and cardiac cell bodies ( Figure 1F).…”

“…Further, on-chip culture supported similar viability results previously reported using more traditional culture techniques. [17,20] Using a live/dead assay 7 d postencapsulation, cardiac cells and SNS neurons were shown to have 89.5% ± 1.4% and 67.5% ± 6.6% viability, respectively.…”

“…[33] Toward modeling sympathetic innervation in the absence of the parasympathetic activity and neurohormonal regulation, a cardiac SNS MPS were prepared as described above (Figure 1) and beat rate and degree of beating coordination were quantified. [17] Immunofluorescent images of cardiac SNS MPS showed that SNS neurites extend toward CMs, across the GelPin, and exhibit colocalization of neuron and cardiac cell markers with synapsin I, a presynaptic marker (Figure 3A). Cardiac cells in proximity to SNS neurites have a ≈28% increase in beat rate (p < 0.05), with a ≈7% decrease in beating coordination compared to controls (p < 0.05, Figure 3B,C).…”