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Directed fusion of cardiac spheroids into larger heterocellular microtissues enables investigation of cardiac action potential propagation via cardiac fibroblasts.

ABSTRACT: Multicellular spheroids generated through cellular self-assembly provide cytoarchitectural complexities of native tissue including three-dimensionality, extensive cell-cell contacts, and appropriate cell-extracellular matrix interactions. They are increasingly suggested as building blocks for larger engineered tissues to achieve shapes, organization, heterogeneity, and other biomimetic complexities. Application of these tissue culture platforms is of particular importance in cardiac research as the myocardium is comprised of distinct but intermingled cell types. Here, we generated scaffold-free 3D cardiac microtissue spheroids comprised of cardiac myocytes (CMs) and/or cardiac fibroblasts (CFs) and used them as building blocks to form larger microtissues with different spatial distributions of CMs and CFs. Characterization of fusing homotypic and heterotypic spheroid pairs revealed an important influence of CFs on fusion kinetics, but most strikingly showed rapid fusion kinetics between heterotypic pairs consisting of one CF and one CM spheroid, indicating that CMs and CFs self-sort in vitro into the intermixed morphology found in the healthy myocardium. We then examined electrophysiological integration of fused homotypic and heterotypic microtissues by mapping action potential propagation. Heterocellular elongated microtissues which recapitulate the disproportionate CF spatial distribution seen in the infarcted myocardium showed that action potentials propagate through CF volumes albeit with significant delay. Complementary computational modeling revealed an important role of CF sodium currents and the spatial distribution of the CM-CF boundary in action potential conduction through CF volumes. Taken together, this study provides useful insights for the development of complex, heterocellular engineered 3D tissue constructs and their engraftment via tissue fusion and has implications for arrhythmogenesis in cardiac disease and repair.

SUBMITTER: Kim TY

PROVIDER: S-EPMC5929561 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Directed fusion of cardiac spheroids into larger heterocellular microtissues enables investigation of cardiac action potential propagation via cardiac fibroblasts.

Kim Tae Yun TY Kofron Celinda M CM King Michelle E ME Markes Alexander R AR Okundaye Amenawon O AO Qu Zhilin Z Mende Ulrike U Choi Bum-Rak BR

PloS one 20180501 5

Multicellular spheroids generated through cellular self-assembly provide cytoarchitectural complexities of native tissue including three-dimensionality, extensive cell-cell contacts, and appropriate cell-extracellular matrix interactions. They are increasingly suggested as building blocks for larger engineered tissues to achieve shapes, organization, heterogeneity, and other biomimetic complexities. Application of these tissue culture platforms is of particular importance in cardiac research as ...[more]

PMID: 29715271

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Project description:Rationale and objectiveThe use of genetic engineering of unexcitable cells to enable expression of gap junctions and inward rectifier potassium channels has suggested that cell therapies aimed at establishing electrical coupling of unexcitable donor cells to host cardiomyocytes may be arrhythmogenic. Whether similar considerations apply when the donor cells are electrically excitable has not been investigated. Here we tested the hypothesis that adenoviral transfer of genes coding Kir2.1 (I(K1)), Na(V)1.5 (I(Na)) and connexin-43 (Cx43) proteins into neonatal rat ventricular myofibroblasts (NRVF) will convert them into fully excitable cells, rescue rapid conduction velocity (CV) and reduce the incidence of complex reentry arrhythmias in an in vitro model.Methods and resultsWe used adenoviral (Ad-) constructs encoding Kir2.1, Na(V)1.5 and Cx43 in NRVF. In single NRVF, Ad-Kir2.1 or Ad-Na(V)1.5 infection enabled us to regulate the densities of I(K1) and I(Na), respectively. At varying MOI ratios of 10/10, 5/10 and 5/20, NRVF co-infected with Ad-Kir2.1+ Na(V)1.5 were hyperpolarized and generated action potentials (APs) with upstroke velocities >100 V/s. However, when forming monolayers only the addition of Ad-Cx43 made the excitable NRVF capable of conducting electrical impulses (CV?=?20.71±0.79 cm/s). When genetically engineered excitable NRVF overexpressing Kir2.1, Na(V)1.5 and Cx43 were used to replace normal NRVF in heterocellular monolayers that included neonatal rat ventricular myocytes (NRVM), CV was significantly increased (27.59±0.76 cm/s vs. 21.18±0.65 cm/s, p<0.05), reaching values similar to those of pure myocytes monolayers (27.27±0.72 cm/s). Moreover, during reentry, propagation was faster and more organized, with a significantly lower number of wavebreaks in heterocellular monolayers formed by excitable compared with unexcitable NRVF.ConclusionViral transfer of genes coding Kir2.1, Na(V)1.5 and Cx43 to cardiac myofibroblasts endows them with the ability to generate and propagate APs. The results provide proof of concept that cell therapies with excitable donor cells increase safety and reduce arrhythmogenic potential.

Project description:Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions. Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR). Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture. Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.

Project description:Aim: Composite population of myofibroblasts (MFs) within myocardial tissue is known to alter impulse propagation, leading to arrhythmias. However, it remains unclear whether and how MFs alter their propagation patterns when contacting cardiomyocytes (CMs) without complex structural insertions in the myocardium. We attempted to unveil the effects of the one-sided, heterocellular CM-MF connection on the impulse propagation of CM monolayers without the spatial insertion of MFs as an electrical or mechanical obstacle. Methods and results: We evaluated fluo8-based spatiotemporal patterns in impulse propagation of neonatal rat CM monolayers cultured on the microporous membrane having 8-μm diameter pores with co-culture of MFs or CMs on the reverse membrane side (CM-MF model or CM-CM model, respectively). During consecutive pacing at 1 or 2 Hz, the CM monolayers exhibited forward impulse propagation from the pacing site with a slower conduction velocity (θ) and a larger coefficient of directional θ variation in the CM-MF model than that in the CM-CM model in a frequency-dependent manner (2 Hz >1 Hz). The localized placement of an MF cluster on the reverse side resulted in an abrupt segmental depression of the impulse propagation of the upper CM layer, causing a spatiotemporally non-uniform pattern. Dye transfer of the calcein loaded in the upper CM layer to the lower MF layer was attenuated by the gap-junction inhibitor heptanol. Immunocytochemistry identified definitive connexin 43 (Cx43) between the CMs and MFs in the membrane pores. MF-selective Cx43 knockdown in the MF layer improved both the velocity and uniformity of propagation in the CM monolayer. Conclusion: Heterocellular Cx43 gap junction coupling of CMs with MFs alters the spatiotemporal patterns of myocardial impulse propagation, even in the absence of spatially interjacent and mechanosensitive modulations by MFs. Moreover, MFs can promote pro-arrhythmogenic impulse propagation when in face-to-face contact with the myocardium that arises in the healing infarct border zone.

Project description:The first macrophages that seed the developing heart originate from the yolk sac during fetal life. While murine studies reveal important homeostatic and reparative functions in adults, we know little about their roles in the earliest stages of human heart development due to a lack of accessible tissue. Generation of bioengineered human cardiac microtissues from pluripotent stem cells models these first steps in cardiac tissue development, however macrophages have not been included in these studies. To bridge these gaps, we differentiated human embryonic stem cells (hESCs) into primitive LYVE1+ macrophages (hESC-macrophages; akin to yolk sac macrophages) that stably engrafted within cardiac microtissues composed of hESC-cardiomyocytes and fibroblasts to study reciprocal interactions. Engraftment induced a tissue resident macrophage gene program resembling human fetal cardiac macrophages, enriched in efferocytic pathways. Functionally, hESC-macrophages induced production and maturation of cardiomyocyte sarcomeric proteins, and enhanced contractile force, relaxation kinetics, and electrical properties. Mechanistically, the primary effect of hESC-macrophages was during the stressful events surrounding early microtissue formation, where they engaged in phosphatidylserine dependent ingestion of apoptotic cardiomyocyte cargo, which reinforced core resident macrophage identity, reduced microtissue stress and drove hESC-cardiomyocytes to become more similar to human ventricular cardiomyocytes found in early development, both transcriptionally and metabolically. Inhibiting efferocytosis of hESC-cardiomyocytes by hESC-macrophages led to increased cell stress, impaired sarcomeric protein maturation and reduced cardiac microtissue function (contraction and relaxation). Taken together, macrophage-engineered human cardiac microtissues represent a considerably improved model for human heart development, and reveal a major beneficial, yet previously unappreciated role for human primitive macrophages in enhancing cardiac tissue function.

Dataset Information

Directed fusion of cardiac spheroids into larger heterocellular microtissues enables investigation of cardiac action potential propagation via cardiac fibroblasts.

Publications

Directed fusion of cardiac spheroids into larger heterocellular microtissues enables investigation of cardiac action potential propagation via cardiac fibroblasts.

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