scholarly journals OptoGap is an optogenetics-enabled assay for quantification of cell–cell coupling in multicellular cardiac tissue

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Patrick M. Boyle ◽  
Jinzhu Yu ◽  
Aleksandra Klimas ◽  
John C. Williams ◽  
Natalia A. Trayanova ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Cardiac Fibroblasts ◽  
Direct Method ◽  
Electrical Coupling ◽  
Three Dimensional ◽  
Computational Modelling ◽  
Cell Coupling ◽  
Excitable Cells ◽  
Potential Applicability ◽  
Cell Cell

AbstractIntercellular electrical coupling is an essential means of communication between cells. It is important to obtain quantitative knowledge of such coupling between cardiomyocytes and non-excitable cells when, for example, pathological electrical coupling between myofibroblasts and cardiomyocytes yields increased arrhythmia risk or during the integration of donor (e.g., cardiac progenitor) cells with native cardiomyocytes in cell-therapy approaches. Currently, there is no direct method for assessing heterocellular coupling within multicellular tissue. Here we demonstrate experimentally and computationally a new contactless assay for electrical coupling, OptoGap, based on selective illumination of inexcitable cells that express optogenetic actuators and optical sensing of the response of coupled excitable cells (e.g., cardiomyocytes) that are light-insensitive. Cell–cell coupling is quantified by the energy required to elicit an action potential via junctional current from the light-stimulated cell(s). The proposed technique is experimentally validated against the standard indirect approach, GapFRAP, using light-sensitive cardiac fibroblasts and non-transformed cardiomyocytes in a two-dimensional setting. Its potential applicability to the complex three-dimensional setting of the native heart is corroborated by computational modelling and proper calibration. Lastly, the sensitivity of OptoGap to intrinsic cell-scale excitability is robustly characterized via computational analysis.

scholarly journals OptoGap: an optogenetics-enabled assay for quantification of cell-cell coupling in multicellular cardiac tissue

2017 ◽  
Author(s):  
Jinzhu Yu ◽  
Patrick M. Boyle ◽  
Aleksandra Klimas ◽  
John C. Williams ◽  
Natalia Trayanova ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Quantitative Methods ◽  
Cardiac Tissue ◽  
De Novo ◽  
Cardiac Fibroblasts ◽  
Direct Method ◽  
Electrical Coupling ◽  
Cell Coupling ◽  
Western Blots ◽  
Cell Cell

AbstractIntercellular electrical coupling is an essential means of communication between cells. It is important to obtain quantitative knowledge of such coupling between cardiomyocytes and nonexcitable cells when, for example, pathological electrical coupling between myofibroblasts and cardiomyocytes yields increased arrhythmia risk or during the integration of donor (e.g. cardiac progenitor) cells with native cardiomyocytes in cell-therapy approaches. Currently, there is no direct method for assessing heterocellular coupling within multicellular tissue. Here we demonstrate experimentally and computationally a new contactless assay for electrical coupling, OptoGap, based on selective illumination of inexcitable cells that express optogenetic actuators and optical sensing of the response of coupled excitable cells, e.g. cardiomyocytes, that are light-insensitive. Cell-cell coupling is quantified by the energy required to elicit an action potential via junctional current from the light-stimulated cell(s). The proposed technique is experimentally validated against the standard indirect approach, GapFRAP, using light-sensitive cardiac fibroblasts and non-transformed cardiomyocytes in a two-dimensional setting. It’s potential applicability to the complex three-dimensional setting of the native heart is corroborated by computational modeling and proper calibration.Intercellular coupling is a fundamental form of communication between cells, essential for the synchronization of physiological processes in different organs. Pathologically altered coupling or the emergence of de novo coupling between native and donor cells are problems of interest in many cardiac applications, e.g. during cell delivery and cell integration for cardiac repair therapy1,2. In particular, interactions between cardiomyocytes and fibroblasts are of interest, especially the pro-arrhythmic increase in coupling as the latter transition to myofibroblasts3-6.Electrical coupling in cardiac tissue is mediated primarily by low-resistance paths formed by gap-junctional proteins (connexins), that can link cardiomyocytes (CMs) to each other and to non-cardiomyocytes (nCMs), such as fibroblasts. Qualitative and quantitative methods, e.g. immunofluorescence, messenger RNA and Western blots, are often used to assay connexin expression levels as a surrogate measure of coupling, but they do not provide functional information. A method for direct quantification of cell-cell coupling within the multicellular tissue context is highly desirable.

scholarly journals Mechanisms of endothelial cell coverage by pericytes: computational modelling of cell wrapping and in vitro experiments

2020 ◽  
Vol 17 (162) ◽  
pp. 20190739
Author(s):  
Kei Sugihara ◽  
Saori Sasaki ◽  
Akiyoshi Uemura ◽  
Satoru Kidoaki ◽  
Takashi Miura
Keyword(s):  
Cell Adhesion ◽  
Three Dimensional ◽  
Computational Modelling ◽  
Cellular Level ◽  
Cellular Potts Model ◽  
Contributing Factors ◽  
Modelling Framework ◽  
Cell Cell

Pericytes (PCs) wrap around endothelial cells (ECs) and perform diverse functions in physiological and pathological processes. Although molecular interactions between ECs and PCs have been extensively studied, the morphological processes at the cellular level and their underlying mechanisms have remained elusive. In this study, using a simple cellular Potts model, we explored the mechanisms for EC wrapping by PCs. Based on the observed in vitro cell wrapping in three-dimensional PC–EC coculture, the model identified four putative contributing factors: preferential adhesion of PCs to the extracellular matrix (ECM), strong cell–cell adhesion, PC surface softness and larger PC size. While cell–cell adhesion can contribute to the prevention of cell segregation and the degree of cell wrapping, it cannot determine the orientation of cell wrapping alone. While atomic force microscopy revealed that PCs have a larger Young’s modulus than ECs, the experimental analyses supported preferential ECM adhesion and size asymmetry. We also formulated the corresponding energy minimization problem and numerically solved this problem for specific cases. These results give biological insights into the role of PC–ECM adhesion in PC coverage. The modelling framework presented here should also be applicable to other cell wrapping phenomena observed in vivo .

scholarly journals Gq-activated fibroblasts induce cardiomyocyte action potential prolongation and automaticity in a three-dimensional microtissue environment

2017 ◽  
Vol 313 (4) ◽  
pp. H810-H827 ◽  
Author(s):  
C. M. Kofron ◽  
T. Y. Kim ◽  
M. E. King ◽  
A. Xie ◽  
F. Feng ◽  
...  
Keyword(s):  
Action Potential ◽  
Electrical Activity ◽  
Rise Time ◽  
Connexin 43 ◽  
Cardiac Fibroblasts ◽  
Three Dimensional ◽  
Neonatal Rat ◽  
Resting Membrane Potential ◽  
Western Blots ◽  
Cell Cell

Cardiac fibroblasts (CFs) are known to regulate cardiomyocyte (CM) function in vivo and in two-dimensional in vitro cultures. This study examined the effect of CF activation on the regulation of CM electrical activity in a three-dimensional (3-D) microtissue environment. Using a scaffold-free 3-D platform with interspersed neonatal rat ventricular CMs and CFs, Gq-mediated signaling was selectively enhanced in CFs by Gαq adenoviral infection before coseeding with CMs in nonadhesive hydrogels. After 3 days, the microtissues were analyzed by signaling assay, histological staining, quantitative PCR, Western blots, optical mapping with voltage- or Ca2+-sensitive dyes, and microelectrode recordings of CF resting membrane potential (RMPCF). Enhanced Gq signaling in CFs increased microtissue size and profibrotic and prohypertrophic markers. Expression of constitutively active Gαq in CFs prolonged CM action potential duration (by 33%) and rise time (by 31%), prolonged Ca2+ transient duration (by 98%) and rise time (by 65%), and caused abnormal electrical activity based on depolarization-induced automaticity. Constitutive Gq activation in CFs also depolarized RMPCF from –33 to −20 mV and increased connexin 43 and connexin 45 expression. Computational modeling confers that elevated RMPCF and increased cell-cell coupling between CMs and CFs in a 3-D environment could lead to automaticity. In conclusion, our data demonstrate that CF activation alone is capable of altering action potential and Ca2+ transient characteristics of CMs, leading to proarrhythmic electrical activity. Our results also emphasize the importance of a 3-D environment where cell-cell interactions are prevalent, underscoring that CF activation in 3-D tissue plays a significant role in modulating CM electrophysiology and arrhythmias. NEW & NOTEWORTHY In a three-dimensional microtissue model, which lowers baseline activation of cardiac fibroblasts but enables cell-cell, paracrine, and cell-extracellular matrix interactions, we demonstrate that selective cardiac fibroblast activation by enhanced Gq signaling, a pathophysiological trigger in the diseased heart, modulates cardiomyocyte electrical activity, leading to proarrhythmogenic automaticity.

scholarly journals Computational modelling of epidermal stratification highlights the importance of asymmetric cell division for predictable and robust layer formation

2014 ◽  
Vol 11 (99) ◽  
pp. 20140631 ◽  
Author(s):  
Alexander Gord ◽  
William R. Holmes ◽  
Xing Dai ◽  
Qing Nie
Keyword(s):  
Cell Adhesion ◽  
Cell Division ◽  
Asymmetric Cell Division ◽  
Three Dimensional ◽  
Interaction Strength ◽  
Computational Modelling ◽  
Protective Layer ◽  
The Body ◽  
Cell Interactions ◽  
Cell Cell

Skin is a complex organ tasked with, among other functions, protecting the body from the outside world. Its outermost protective layer, the epidermis, is comprised of multiple cell layers that are derived from a single-layered ectoderm during development. Using a new stochastic, multi-scale computational modelling framework, the anisotropic subcellular element method, we investigate the role of cell morphology and biophysical cell–cell interactions in the formation of this layered structure. This three-dimensional framework describes interactions between collections of hundreds to thousands of cells and (i) accounts for intracellular structure and morphology, (ii) easily incorporates complex cell–cell interactions and (iii) can be efficiently implemented on parallel architectures. We use this approach to construct a model of the developing epidermis that accounts for the internal polarity of ectodermal cells and their columnar morphology. Using this model, we show that cell detachment, which has been previously suggested to have a role in this process, leads to unpredictable, randomized stratification and that this cannot be abrogated by adjustment of cell–cell adhesion interaction strength. Polarized distribution of cell adhesion proteins, motivated by epithelial polarization, can however eliminate this detachment, and in conjunction with asymmetric cell division lead to robust and predictable development.

Cell Patterning: Interaction of Cardiac Myocytes and Fibroblasts in Three-Dimensional Culture

2008 ◽  
Vol 14 (2) ◽  
pp. 117-125 ◽  
Author(s):  
Troy A. Baudino ◽  
Alex McFadden ◽  
Charity Fix ◽  
Joshua Hastings ◽  
Robert Price ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Three Dimensional ◽  
Normal Growth ◽  
Cell Types ◽  
Cell Interactions ◽  
Cellular Interactions ◽  
Cell Layers ◽  
Cell Cell

Patterning of cells is critical to the formation and function of the normal organ, and it appears to be dependent upon internal and external signals. Additionally, the formation of most tissues requires the interaction of several cell types. Indeed, both extracellular matrix (ECM) components and cellular components are necessary for three-dimensional (3-D) tissue formationin vitro. Using 3-D cultures we demonstrate that ECM arranged in an aligned fashion is necessary for the rod-shaped phenotype of the myocyte, and once this pattern is established, the myocytes were responsible for the alignment of any subsequent cell layers. This is analogous to thein vivopattern that is observed, where there appears to be minimal ECM signaling, rather formation of multicellular patterns is dependent upon cell–cell interactions. Our 3-D culture of myocytes and fibroblasts is significant in that it modelsin vivoorganization of cardiac tissue and can be used to investigate interactions between fibroblasts and myocytes. Furthermore, we used rotational cultures to examine cellular interactions. Using these systems, we demonstrate that specific connexins and cadherins are critical for cell–cell interactions. The data presented here document the feasibility of using these systems to investigate cellular interactions during normal growth and injury.

scholarly journals Polymeric Biomaterials for the Treatment of Cardiac Post-Infarction Injuries

Pharmaceutics ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1038
Author(s):  
Sonia Trombino ◽  
Federica Curcio ◽  
Roberta Cassano ◽  
Manuela Curcio ◽  
Giuseppe Cirillo ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Cardiac Tissue ◽  
Polymeric Nanoparticles ◽  
Three Dimensional ◽  
Cardiac Regeneration ◽  
Biological Properties ◽  
Positive Influence ◽  
Regenerative Process ◽  
Current Status ◽  
Polymeric Biomaterials

Cardiac regeneration aims to reconstruct the heart contractile mass, preventing the organ from a progressive functional deterioration, by delivering pro-regenerative cells, drugs, or growth factors to the site of injury. In recent years, scientific research focused the attention on tissue engineering for the regeneration of cardiac infarct tissue, and biomaterials able to anatomically and physiologically adapt to the heart muscle have been proposed as valuable tools for this purpose, providing the cells with the stimuli necessary to initiate a complete regenerative process. An ideal biomaterial for cardiac tissue regeneration should have a positive influence on the biomechanical, biochemical, and biological properties of tissues and cells; perfectly reflect the morphology and functionality of the native myocardium; and be mechanically stable, with a suitable thickness. Among others, engineered hydrogels, three-dimensional polymeric systems made from synthetic and natural biomaterials, have attracted much interest for cardiac post-infarction therapy. In addition, biocompatible nanosystems, and polymeric nanoparticles in particular, have been explored in preclinical studies as drug delivery and tissue engineering platforms for the treatment of cardiovascular diseases. This review focused on the most employed natural and synthetic biomaterials in cardiac regeneration, paying particular attention to the contribution of Italian research groups in this field, the fabrication techniques, and the current status of the clinical trials.

Abstract 020: Cell-sheet-based Bioengineered Human Cardiac Tissue Using Pluripotent Stem Cells For Heart Repair And Disease Models

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Katsuhisa Matsuura ◽  
Tatsuya Shimizu ◽  
Nobuhisa Hagiwara ◽  
Teruo Okano
Keyword(s):  
Cardiac Tissue ◽  
Subcutaneous Tissue ◽  
Cell Sheet ◽  
Three Dimensional ◽  
Embryoid Bodies ◽  
Cardiac Differentiation ◽  
Cardiac Cell ◽  
Cell Sheets ◽  
High Efficient ◽  
Human Cardiac Tissue

We have developed an original scaffold-free tissue engineering approach, “cell sheet engineering”, and this technology has been already applied to regenerative medicine of various organs including heart. As the bioengineered three-dimensional cardiac tissue is expected to not only function for repairing the broad injured heart but also to be the practicable heart tissue models, we have developed the cell sheet-based perfusable bioengineered three-dimensional cardiac tissue. Recently we have also developed the unique suspension cultivation system for the high-efficient cardiac differentiation of human iPS cells. Fourteen-day culture with the serial treatments of suitable growth factors and a small compound in this stirring system with the suitable dissolved oxygen concentration produced robust embryoid bodies that showed the spontaneous beating and were mainly composed of cardiomyocytes (~80%). When these differentiated cells were cultured on temperature-responsive culture dishes after the enzymatic dissociation, the spontaneous and synchronous beating was observed accompanied with the intracellular calcium influx all over the area even after cell were detached from culture dishes as cell sheets by lowering the culture temperature. The cardiac cell sheets were mainly composed of cardiomyocytes (~80%) and partially mural cells (~20%). Furthermore, extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid, and this propagation was inhibited by the treatment with some anti-arrhythmic drugs. When the triple layered cardiac tissue was transplanted onto the subcutaneous tissue of nude rats, the spontaneous pulsation was observed over 2 months and engrafted cardiomyocytes were vascularized with the host tissue-derived endothelial cells. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue. Now we are developing the vascularized thickened human cardiac tissue by the repeated layering of cardiac cell sheets on the artificial vascular bed in vitro.

Abstract 336: A 14-3-3 Mode-1 Binding Motif Promotes Gap Junction Internalization During Acute Cardiac Ischemia

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
James W Smyth ◽  
Jose M Sanchez ◽  
Samy Lamouille ◽  
Ting-Ting Hong ◽  
Jacob M Vogan ◽  
...  
Keyword(s):  
Gap Junction ◽  
Protein Trafficking ◽  
Connexin 43 ◽  
Electrical Coupling ◽  
Acute Ischemia ◽  
Binding Motif ◽  
Wild Type ◽  
Gap Junction Coupling ◽  
Junction Coupling ◽  
Cell Cell

During each heartbeat, robust cell-cell electrical coupling via connexin 43 (Cx43) gap junctions allows billions of individual cardiomyocytes to contract in synchrony. Cx43 turns over rapidly, and altered Cx43 trafficking during disease contributes to the arrhythmias of sudden cardiac death. The overall phosphorylation status of the Cx43 protein is known to regulate gap junction coupling, but the role of many residue specific phosphorylation events remains unknown. One such residue, Ser373, forms a mode-1 14-3-3 binding motif upon phosphorylation. Given that 14-3-3 proteins are known to regulate protein trafficking, we hypothesized a role for Cx43 Ser373 phosphorylation in regulation of Cx43 gap junction coupling. Using Langendorff-perfused mouse hearts we find robust phosphorylation of Cx43 at Ser373 and Ser368 after 30 min of no-flow ischemia. In human cell lines, a S373A mutation ablated Cx43/14-3-3 complexing and 35 S pulse-chase revealed Cx43 S373A also experiences a longer half-life than wild-type Cx43. Previous reports have implicated phosphorylation of Cx43 Ser368 in PKC mediated Cx43 internalization. We find that upon activation of PKC, the Cx43 S373A mutant undergoes lower and more transient levels of phosphorylation at Ser368 than wild-type Cx43. Consistent with these data, siRNA-mediated ablation of 14-3-3 expression results in enlargement of gap junction plaque formation at cell-cell borders. In conclusion, we propose that phosphorylation of Cx43 Ser373 results in 14-3-3 binding which promotes and maintains phosphorylation of Cx43 Ser368 and the subsequent internalization of gap junction channels. These results identify for the first time a specific role for 14-3-3 proteins in regulation of Cx43 internalization during acute ischemia and contribute to the development of therapies aimed at preserving or enhancing gap junction coupling in the heart.

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