scholarly journals Human Cardiac Fibroblast Number and Activation State Modulate Electromechanical Function of hiPSC-Cardiomyocytes in Engineered Myocardium

2020 ◽  
Vol 2020 ◽  
pp. 1-16
Author(s):  
Cassady E. Rupert ◽  
Tae Yun Kim ◽  
Bum-Rak Choi ◽  
Kareen L. K. Coulombe
Keyword(s):  
Cardiac Tissue ◽  
Disease Modeling ◽  
Contractile Function ◽  
Cardiac Fibroblasts ◽  
Cell Number ◽  
Cardiac Fibroblast ◽  
Cellular Interactions ◽  
Excitation Rate ◽  
Cardiac Tissues

Cardiac tissue engineering using hiPSC-derived cardiomyocytes is a promising avenue for cardiovascular regeneration, pharmaceutical drug development, cardiotoxicity evaluation, and disease modeling. Limitations to these applications still exist due in part to the need for more robust structural support, organization, and electromechanical function of engineered cardiac tissues. It is well accepted that heterotypic cellular interactions impact the phenotype of cardiomyocytes. The current study evaluates the functional effects of coculturing adult human cardiac fibroblasts (hCFs) in 3D engineered tissues on excitation and contraction with the goal of recapitulating healthy, nonarrhythmogenic myocardium in vitro. A small population (5% of total cell number) of hCFs in tissues improves tissue formation, material properties, and contractile function. However, two perturbations to the hCF population create disease-like phenotypes in engineered cardiac tissues. First, increasing the percentage of hCFs to 15% resulted in tissues with increased ectopic activity and spontaneous excitation rate. Second, hCFs undergo myofibroblast activation in traditional two-dimensional culture, and this altered phenotype ablated the functional benefits of hCFs when incorporated into engineered cardiac tissues. Taken together, the results of this study demonstrate that human cardiac fibroblast number and activation state modulate electromechanical function of hiPSC-cardiomyocytes and that a low percentage of quiescent hCFs are a valuable cell source to advance a healthy electromechanical response of engineered cardiac tissue for regenerative medicine applications.

scholarly journals Myofibrils in Cardiomyocytes Tend to Assemble Along the Maximal Principle Stress Directions

2017 ◽  
Vol 139 (12) ◽  
Author(s):  
Hongyan Yuan ◽  
Bahador Marzban ◽  
Kevin Kit Parker
Keyword(s):  
Rational Design ◽  
Spatial Organization ◽  
Cardiac Tissue ◽  
Contractile Function ◽  
Focal Adhesions ◽  
Cardiac Tissues ◽  
Mechanics Model ◽  
Stress Directions

The mechanisms underlying the spatial organization of self-assembled myofibrils in cardiac tissues remain incompletely understood. By modeling cells as elastic solids under active cytoskeletal contraction, we found a good correlation between the predicted maximal principal stress directions and the in vitro myofibril orientations in individual cardiomyocytes. This implies that actomyosin fibers tend to assemble along the maximal tensile stress (MTS) directions. By considering the dynamics of focal adhesion and myofibril formation in the model, we showed that different patterns of myofibril organizations in mature versus immature cardiomyocytes can be explained as the consequence of the different levels of force-dependent remodeling of focal adhesions. Further, we applied the mechanics model to cell pairs and showed that the myofibril organizations can be regulated by a combination of multiple factors including cell shape, cell–substrate adhesions, and cell–cell adhesions. This mechanics model can guide the rational design in cardiac tissue engineering where recapitulating in vivo myofibril organizations is crucial to the contractile function of the heart.

scholarly journals Loss of Function in Dopamine D3 Receptor Attenuates Left Ventricular Cardiac Fibroblast Migration and Proliferation in vitro

2021 ◽  
Vol 8 ◽  
Author(s):  
Andrew Kisling ◽  
Shannon Byrne ◽  
Rohan U. Parekh ◽  
Deepthy Melit-Thomas ◽  
Lisandra E. de Castro Brás ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Cardiac Fibroblasts ◽  
Fibroblast Cell ◽  
Left Ventricular ◽  
Cardiac Fibroblast ◽  
Loss Of Function ◽  
Fibroblast Migration ◽  
And Migration ◽  
Proliferation And Migration

Evidence suggests the existence of an intracardiac dopaminergic system that plays a pivotal role in regulating cardiac function and fibrosis through G-protein coupled receptors, particularly mediated by dopamine receptor 3 (D3R). However, the expression of dopamine receptors in cardiac tissue and their role in cardiac fibroblast function is unclear. In this brief report, first we determined expression of D1R and D3R both in left ventricle (LV) tissue and fibroblasts. Then, we explored the role of D3R in the proliferation and migration of fibroblast cell cultures using both genetic and pharmaceutical approaches; specifically, we compared cardiac fibroblasts isolated from LV of wild type (WT) and D3R knockout (D3KO) mice in response to D3R-specific pharmacological agents. Finally, we determined if loss of D3R function could significantly alter LV fibroblast expression of collagen types I (Col1a1) and III (Col3a1). Cardiac fibroblast proliferation was attenuated in D3KO cells, mimicking the behavior of WT cardiac fibroblasts treated with D3R antagonist. In response to scratch injury, WT cardiac fibroblasts treated with the D3R agonist, pramipexole, displayed enhanced migration compared to control WT and D3KO cells. Loss of function in D3R resulted in attenuation of both proliferation and migration in response to scratch injury, and significantly increased the expression of Col3a1 in LV fibroblasts. These findings suggest that D3R may mediate cardiac fibroblast function during the wound healing response. To our knowledge this is the first report of D3R's expression and functional significance directly in mouse cardiac fibroblasts.

scholarly journals 3094 Production of Engineered Cardiac Tissue for Disease Modeling

2019 ◽  
Vol 3 (s1) ◽  
pp. 18-19
Author(s):  
Morgan Ellis ◽  
Elizabeth Lipke
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cardiac Tissue ◽  
Disease Modeling ◽  
Cardiac Regeneration ◽  
Cardiac Markers ◽  
Cardiac Tissues ◽  
Over Time

OBJECTIVES/SPECIFIC AIMS: Cardiovascular diseases (CVD) is the leading cause of death worldwide in both men and women due to lack of cardiac regeneration after disease or damaged is caused. There are many challenges to studying CVD since native cardiomyocytes cannot be cultured in vitro. With the advancements in biomaterial and pluripotent stem cells research, scientists are now able to produce engineered cardiac tissue models in vitro that mimic the native myocardium. This study shows our methods for producing engineered cardiac tissue with potential applications in cardiac regeneration, disease modeling, and scalable production. METHODS/STUDY POPULATION: In this study, human induced pluripotent stem cells (hiPSCs) were combined with two different photocrosslinkable hybrid biomaterials, poly (ethylene)- glycol fibrinogen (PF) and gelatin methacrylate (GelMa), in various tissue geometries to form 3D human engineered cardiac tissues (3D-hECTs). To study tissue growth and contraction, image and video analysis was performed at specific timepoints. To analyze differentiation efficiency and cell population, flow cytometry was performed using cardiac markers. To evaluate gene expression, qPCR was performed using pluripotency and cardiac specific primers. RESULTS/ANTICIPATED RESULTS: Direct cardiac differentiation of encapsulated hiPSCs resulted in synchronously contracting 3D-hECTs in both biomaterials and all tissue geometries. Spontaneous contractions started on Day 7 and increased in velocity, frequency, and synchronicity over time. 3D-hECTs had high cell viability with > 70% of cells positive for cardiac markers. Engineered tissues showed appropriate temporal changes in gene expression over time with pluripotency gene expression decreasing and cardiac gene expression increasing. DISCUSSION/SIGNIFICANCE OF IMPACT: This study shows the potential for direct differentiation of encapsulated hiPSCs to produce physiologically relevant engineered cardiac tissues. Resulting 3D-hECTS showed features of mature myocardium with appropriate cardiomyocyte populations, mechanical motion, and gene expression. Using this platform, we are able to produce engineered cardiac tissue in a variety of biomaterials and tissue geometries to study new therapeutics, mechanism of disease, and scalable tissue culture.

Methomyl induced effect on fortilin and S100A1 in serum and cardiac tissue: Potential biomarkers of toxicity

2018 ◽  
Vol 38 (3) ◽  
pp. 371-377
Author(s):  
SD Nusair ◽  
AN Joukhan ◽  
AH Bani Rashaid ◽  
AM Rababa’h
Keyword(s):  
Toxic Effect ◽  
Cardiac Tissue ◽  
Cardiac Fibroblasts ◽  
Study Group ◽  
Sprague Dawley ◽  
Cardiac Tissues ◽  
Sprague Dawley Rats ◽  
Cervical Dislocation ◽  
Potential Biomarkers

Methomyl toxicity has been reported as a cause of several accidental and suicidal fatalities. The study is evaluating the effect of lethal methomyl toxicity on fortilin and S100A1 in serum and cardiac tissues. Adult 96 female Sprague-Dawley rats were divided equally into a control (euthanized by cervical dislocation) and a study group (overdosed with methomyl). The levels of fortilin and S100A1 in serum were measured antemortem (to establish the basal levels in serum) and postmortem (to evaluate changes after methomyl exposure) using enzyme-linked immunoassay. S100A1 was immunostained in sections from cardiac tissues. Both proteins in the control were not significantly different ( p > 0.05) compared with the antemortem levels. On the contrast, both biomarkers levels in the intoxicated group were remarkably higher ( p < 0.001) than the control and the antemortem levels. Ventricular tissues from the intoxicated rats presented depleted S100A1 immunostain in cardiomyocytes localized mainly in the epicardium with deeply stained adjacent cardiac fibroblasts. The cardiomyocytes were damaged with a prominent loss of striations compared to normal cardiac tissues from the control. The present outcomes explain to a certain degree the potential toxic effect of methomyl poisoning on the cardiac tissue. Both proteins could be added to the currently available battery of markers for assessing methomyl toxicity.

Biophysical stimulation for in vitro engineering of functional cardiac tissues

2017 ◽  
Vol 131 (13) ◽  
pp. 1393-1404 ◽  
Author(s):  
Anastasia Korolj ◽  
Erika Yan Wang ◽  
Robert A. Civitarese ◽  
Milica Radisic
Keyword(s):  
Cardiac Tissue ◽  
Culture Media ◽  
Culture Conditions ◽  
Lineage Specification ◽  
Cardiac Tissues ◽  
Cardiac Lineage ◽  
And Function ◽  
Tissue Models

Engineering functional cardiac tissues remains an ongoing significant challenge due to the complexity of the native environment. However, our growing understanding of key parameters of the in vivo cardiac microenvironment and our ability to replicate those parameters in vitro are resulting in the development of increasingly sophisticated models of engineered cardiac tissues (ECT). This review examines some of the most relevant parameters that may be applied in culture leading to higher fidelity cardiac tissue models. These include the biochemical composition of culture media and cardiac lineage specification, co-culture conditions, electrical and mechanical stimulation, and the application of hydrogels, various biomaterials, and scaffolds. The review will also summarize some of the recent functional human tissue models that have been developed for in vivo and in vitro applications. Ultimately, the creation of sophisticated ECT that replicate native structure and function will be instrumental in advancing cell-based therapeutics and in providing advanced models for drug discovery and testing.

Abstract 30: In vitro Fabrication of Human Cardiac Tissues With Porcine Perfusable Blood Vessels

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Akitoshi Inui ◽  
Hidekazu Sekine ◽  
Kazunori Sano ◽  
Izumi Dobashi ◽  
Azumi Yoshida ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Cardiac Tissue ◽  
Severe Heart Failure ◽  
Perfusion Culture ◽  
Cardiac Cell ◽  
Cell Sheets ◽  
Cardiac Tissues ◽  
Bioreactor System ◽  
Human Cardiac Tissue

The definitive treatment of severe heart failure is heart transplantation; however the number of heart transplantation procedures performed in Japan per year ranges from 30-40 due to donor shortage. Therefore, recently other treatments such as ventricular assist device or regenerative therapy by human cardiac tissue engineering have been developed and are considered as appropriate alternatives. We have developed an original technology, which was named cell-sheet based tissue engineering to fabricate functional three-dimensional tissue by layering cell sheets. The utilization of this technique allowed us to successfully engineer thick rat cardiac tissue with perfusable blood vessels in vitro. Here, we demonstrate a technique to engineer human cardiac tissue with perfusable blood vessels using cardiac cell sheets derived from human induced pluripotent stem cells, and porcine small intestine as a vascular bed for perfusion culture. The small intestine was harvested from with a branch of the superior mesenteric artery and vein and underwent mucosal resection after harvested tissue was cut open. To engineer cardiac tissue with perfusable blood vessels, cardiac cell sheets co-cultured with endothelial cells, were triple-layered and then was overlaid on the vascular bed in the bioreactor system. One day after perfusion culture, overlaid cardiac tissues pulsated spontaneously and were synchronized. The cardiac tissue construct was viable tissue without any observable necrosis. Furthermore we examined the possibility of transplantation of the in vitro engineered human cardiac tissue with the connectable host artery and vein. Engineered cardiac tissue was removed from the bioreactor system after 4-day perfusion, and transplanted to another pig heart. The branch of the superior mesenteric artery and vein of the graft were then reconnected to the host internal thoracic artery and vein. When the cardiac tissue reperfused, it began to beat spontaneously after a few minutes. We believe that this method is useful to fabricate functional cardiac tissue and may become an appropriate treatment for severe heart failure.

scholarly journals SPARC regulates collagen interaction with cardiac fibroblast cell surfaces

2011 ◽  
Vol 301 (3) ◽  
pp. H841-H847 ◽  
Author(s):  
Brett S. Harris ◽  
Yuhua Zhang ◽  
Lauren Card ◽  
Lee B. Rivera ◽  
Rolf A. Brekken ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Cardiac Fibroblasts ◽  
Pressure Overload ◽  
Fibroblast Cell ◽  
Immunoblot Analysis ◽  
Collagen I ◽  
Cell Surfaces ◽  
Insoluble Collagen ◽  
Total Collagen

Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [3H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α1(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.

scholarly journals Computational Approaches to Understanding the Role of Fibroblast-Myocyte Interactions in Cardiac Arrhythmogenesis

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Tashalee R. Brown ◽  
Trine Krogh-Madsen ◽  
David J. Christini
Keyword(s):  
Gap Junctions ◽  
Potential Role ◽  
Cardiac Tissue ◽  
Cardiac Fibroblasts ◽  
Cardiac Arrhythmogenesis ◽  
Voltage Dependent ◽  
Underlying Mechanisms ◽  
Slow Propagation

The adult heart is composed of a dense network of cardiomyocytes surrounded by nonmyocytes, the most abundant of which are cardiac fibroblasts. Several cardiac diseases, such as myocardial infarction or dilated cardiomyopathy, are associated with an increased density of fibroblasts, that is, fibrosis. Fibroblasts play a significant role in the development of electrical and mechanical dysfunction of the heart; however the underlying mechanisms are only partially understood. One widely studied mechanism suggests that fibroblasts produce excess extracellular matrix, resulting in collagenous septa. These collagenous septa slow propagation, cause zig-zag conduction paths, and decouple cardiomyocytes resulting in a substrate for arrhythmia. Another emerging mechanism suggests that fibroblasts promote arrhythmogenesis through direct electrical interactions with cardiomyocytes via gap junctions. Due to the challenges of investigating fibroblast-myocyte coupling in native cardiac tissue, computational modeling andin vitroexperiments have facilitated the investigation into the mechanisms underlying fibroblast-mediated changes in cardiomyocyte action potential morphology, conduction velocity, spontaneous excitability, and vulnerability to reentry. In this paper, we summarize the major findings of the existing computational studies investigating the implications of fibroblast-myocyte interactions in the normal and diseased heart. We then present investigations from our group into the potential role of voltage-dependent gap junctions in fibroblast-myocyte interactions.

MiR-150 Attenuates Maladaptive Cardiac Remodeling Mediated by Long Noncoding RNA MIAT and Directly Represses Profibrotic Hoxa4

2022 ◽  
Author(s):  
Tatsuya Aonuma ◽  
Bruno Moukette ◽  
Satoshi Kawaguchi ◽  
Nipuni P. Barupala ◽  
Marisa N. Sepúlveda ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cardiac Remodeling ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Molecular Mechanisms ◽  
Cardiac Fibroblasts ◽  
Cardiac Fibroblast ◽  
Null Mouse ◽  
Increased Risk

Background: MicroRNA-150 (miR-150) plays a protective role in heart failure (HF). Long noncoding RNA, myocardial infarction–associated transcript (MIAT) regulates miR-150 function in vitro by direct interaction. Concurrent with miR-150 downregulation, MIAT is upregulated in failing hearts, and gain-of-function single-nucleotide polymorphisms in MIAT are associated with increased risk of myocardial infarction (MI) in humans. Despite the correlative relationship between MIAT and miR-150 in HF, their in vivo functional relationship has never been established, and molecular mechanisms by which these 2 noncoding RNAs regulate cardiac protection remain elusive. Methods: We use MIAT KO (knockout), Hoxa4 (homeobox a4) KO, MIAT TG (transgenic), and miR-150 TG mice. We also develop DTG (double TG) mice overexpressing MIAT and miR-150. We then use a mouse model of MI followed by cardiac functional, structural, and mechanistic studies by echocardiography, immunohistochemistry, transcriptome profiling, Western blotting, and quantitative real-time reverse transcription-polymerase chain reaction. Moreover, we perform expression analyses in hearts from patients with HF. Lastly, we investigate cardiac fibroblast activation using primary adult human cardiac fibroblasts and in vitro assays to define the conserved MIAT/miR-150/HOXA4 axis. Results: Using novel mouse models, we demonstrate that genetic overexpression of MIAT worsens cardiac remodeling, while genetic deletion of MIAT protects hearts against MI. Importantly, miR-150 overexpression attenuates the detrimental post-MI effects caused by MIAT. Genome-wide transcriptomic analysis of MIAT null mouse hearts identifies Hoxa4 as a novel downstream target of the MIAT/miR-150 axis. Hoxa4 is upregulated in cardiac fibroblasts isolated from ischemic myocardium and subjected to hypoxia/reoxygenation. HOXA4 is also upregulated in patients with HF. Moreover, Hoxa4 deficiency in mice protects the heart from MI. Lastly, protective actions of cardiac fibroblast miR-150 are partially attributed to the direct and functional repression of profibrotic Hoxa4 . Conclusions: Our findings delineate a pivotal functional interaction among MIAT, miR-150, and Hoxa4 as a novel regulatory mechanism pertinent to ischemic HF.

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