scholarly journals How Localized Z-Disc Damage Affects Force Generation and Gene Expression in Cardiomyocytes

2021 ◽  
Vol 8 (12) ◽  
pp. 213
Author(s):  
Dominik Müller ◽  
Sören Donath ◽  
Emanuel Georg Brückner ◽  
Santoshi Biswanath Devadas ◽  
Fiene Daniel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Troponin I ◽  
Troponin T ◽  
Laser System ◽  
Traction Force ◽  
Force Generation ◽  
Proper Function ◽  
Force Microscopy ◽  
Vitro Model

The proper function of cardiomyocytes (CMs) is highly related to the Z-disc, which has a pivotal role in orchestrating the sarcomeric cytoskeletal function. To better understand Z-disc related cardiomyopathies, novel models of Z-disc damage have to be developed. Human pluripotent stem cell (hPSC)-derived CMs can serve as an in vitro model to better understand the sarcomeric cytoskeleton. A femtosecond laser system can be applied for localized and defined damage application within cells as single Z-discs can be removed. We have investigated the changes in force generation via traction force microscopy, and in gene expression after Z-disc manipulation in hPSC-derived CMs. We observed a significant weakening of force generation after removal of a Z-disc. However, no significant changes of the number of contractions after manipulation were detected. The stress related gene NF-kB was significantly upregulated. Additionally, α-actinin (ACTN2) and filamin-C (FLNc) were upregulated, pointing to remodeling of the Z-disc and the sarcomeric cytoskeleton. Ultimately, cardiac troponin I (TNNI3) and cardiac muscle troponin T (TNNT2) were significantly downregulated. Our results allow a better understanding of transcriptional coupling of Z-disc damage and the relation of damage to force generation and can therefore finally pave the way to novel therapies of sarcomeric disorders.

scholarly journals Advanced in silico validation framework for three-dimensional Traction Force Microscopy and application to an in vitro model of sprouting angiogenesis

2020 ◽  
Author(s):  
J. Barrasa-Fano ◽  
A. Shapeti ◽  
J. de Jong ◽  
A. Ranga ◽  
J.A. Sanz-Herrera ◽  
...  
Keyword(s):  
In Silico ◽  
Inverse Method ◽  
In Vitro Model ◽  
Three Dimensional ◽  
Focal Adhesions ◽  
Traction Force ◽  
Force Microscopy ◽  
Validation Framework ◽  
Vitro Model

AbstractIn the last decade, cellular forces in three-dimensional hydrogels that mimic the extracellular matrix have been calculated by means of Traction Force Microscopy (TFM). However, characterizing the accuracy limits of a traction recovery method is critical to avoid obscuring physiological information due to traction recovery errors. So far, 3D TFM algorithms have only been validated using simplified cell geometries, bypassing image processing steps or arbitrarily simulating focal adhesions. Moreover, it is still uncertain which of the two common traction recovery methods, i.e., forward and inverse, is more robust against the inherent challenges of 3D TFM. In this work, we established an advanced in silico validation framework that is applicable to any 3D TFM experimental setup and that can be used to correctly couple the experimental and computational aspects of 3D TFM. Advancements relate to the simultaneous incorporation of complex cell geometries, simulation of microscopy images of varying bead densities and different focal adhesion sizes and distributions. By measuring the traction recovery error with respect to ground truth solutions, we found that while highest traction recovery errors occur for cases with sparse and small focal adhesions, our implementation of the inverse method improves two-fold the accuracy with respect to the forward method (average error of 23% vs. 50%). This advantage was further supported by recovering cellular tractions around angiogenic sprouts in an in vitro model of angiogenesis. The inverse method recovered more realistic traction patterns than the forward method, showing higher traction peaks and a clearer pulling pattern at the sprout protrusion tips.

scholarly journals In Vitro Feasibility Analysis of a New Sutureless Wound-Closure System Based on a Temperature-Regulated Laser and a Transparent Collagen Membrane for Laser Tissue Soldering (LTS)

2020 ◽  
Vol 21 (19) ◽  
pp. 7104
Author(s):  
Moritz Alexander Birkelbach ◽  
Ralf Smeets ◽  
Imke Fiedler ◽  
Lan Kluwe ◽  
Martin Wehner ◽  
...  
Keyword(s):  
Wound Closure ◽  
Oral Surgery ◽  
Ex Vivo ◽  
Laser System ◽  
Collagen Membrane ◽  
Toxic Substances ◽  
Closure System ◽  
Vitro Model ◽  
Suturing Techniques

For the post-surgical treatment of oral wounds and mucosal defects beyond a certain size, the gold standard is still an autologous skin or mucosal graft in combination with complex suturing techniques. A variety of techniques and biomaterials has been developed for sutureless wound closure including different tissue glues or collagen patches. However, no wound covering that enables for sutureless fixation has yet been introduced. Thus, a new system was developed that allows for sutureless wound covering including a transparent collagen membrane, which can be attached to the mucosa using a specially modified 2λ laser beam with integrated temperature sensors and serum albumin as bio-adhesive. The sutureless wound closure system was tested for its applicability and its cytocompatibility by an established in vitro model in the present study. The feasibility of the laser system was tested ex vivo on a porcine palate. The in vitro cytocompatibility tests excluded the potential release of toxic substances from the laser-irradiated collagen membrane and the bio-adhesive. The results of the ex vivo feasibility study using a porcine palate revealed satisfactory mean tensile strength of 1.2–1.5 N for the bonding of the membrane to the tissue fixed with laser of 980 nm. The results suggest that our newly developed laser-assisted wound closure system is a feasible approach and could be a first step on the way towards a laser based sutureless clinical application in tissue repair and oral surgery.

Iron-related transcriptomic variations in CaCo-2 cells, an in vitro model of intestinal absorptive cells

2006 ◽  
Vol 26 (1) ◽  
pp. 55-67 ◽  
Author(s):  
Céline Chicault ◽  
Bertrand Toutain ◽  
Annabelle Monnier ◽  
Marc Aubry ◽  
Patricia Fergelot ◽  
...  
Keyword(s):  
Gene Expression ◽  
Functional Annotation ◽  
In Vitro Model ◽  
Iron Absorption ◽  
Intracellular Iron ◽  
Absorptive Cells ◽  
Number Of Genes ◽  
Vitro Model ◽  
First Time

Regulation of iron absorption by duodenal enterocytes is essential for the maintenance of homeostasis by preventing iron deficiency or overload. Despite the identification of a number of genes implicated in iron absorption and its regulation, it is likely that further factors remain to be identified. For that purpose, we used a global transcriptomic approach, using the CaCo-2 cell line as an in vitro model of intestinal absorptive cells. Pangenomic screening for variations in gene expression correlating with intracellular iron content allowed us to identify 171 genes. One hundred nine of these genes are clustered into five types of expression profile. This is the first time that most of these genes have been associated with iron metabolism. Functional annotation of these five clusters indicates potential links between the immune response, proteolysis processes, and iron depletion. In contrast, iron overload is associated with cellular metabolism, especially that of lipids and glutathione involving redox function and electron transfer.

scholarly journals Different Effects of Cisplatin and Transplatin on the Higher-Order Structure of DNA and Gene Expression

2019 ◽  
Vol 21 (1) ◽  
pp. 34 ◽  
Author(s):  
Toshifumi Kishimoto ◽  
Yuko Yoshikawa ◽  
Kenichi Yoshikawa ◽  
Seiji Komeda
Keyword(s):  
Gene Expression ◽  
Nuclear Dna ◽  
Biological Effects ◽  
Inhibitory Effect ◽  
Higher Order ◽  
Luciferase Assay ◽  
Order Structure ◽  
Force Microscopy ◽  
Order Of Magnitude

Despite the effectiveness of cisplatin as an anticancer agent, its trans-isomer, transplatin, is clinically ineffective. Although both isomers target nuclear DNA, there is a large difference in the magnitude of their biological effects. Here, we compared their effects on gene expression in an in vitro luciferase assay and quantified their effects on the higher-order structure of DNA using fluorescence microscopy (FM) and atomic force microscopy (AFM). The inhibitory effect of cisplatin on gene expression was about 7 times that of transplatin. Analysis of the fluctuation autocorrelation function of the intrachain Brownian motion of individual DNA molecules showed that cisplatin increases the spring and damping constants of DNA by one order of magnitude and these visco-elastic characteristics tend to increase gradually over several hours. Transplatin had a weaker effect, which tended to decrease with time. These results agree with a stronger inhibitory effect of cisplatin on gene expression. We discussed the characteristic effects of the two compounds on the higher-order DNA structure and gene expression in terms of the differences in their binding to DNA.

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