Biology subjects

Pardon, G.

Publications and source records attributed to Pardon, G..

5 recordsLinked to original sources

bioRxiv2023

Spatial Patterning of Laminin and N-Cadherin for Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs)

Controlling cellular shape with protein micropatterning can mimic physiological morphologies and has been shown to improve reproducibility, enhancing our ability to collect statistics on single-cell behaviors. It has also advanced efforts in developing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a promising human model for studies of heart structure and function. hiPSC-CMs have key physiological differences from primary human cardiomyocytes (CMs), including lower sarcomere alignment and contractility, smaller area and lower aspect ratio, and lower force production. Protein micropatterning has been demonstrated to make hiPSC-CMs behave more like primary human CMs across these metrics. However, these micropatterned models typically use only extracellular matrix (ECM) proteins and have not investigated whether providing a protein associated with CM-CM interactions, such as N-cadherin, further enhances hiPSC-CM structure and function. Here, we developed a novel dual-protein patterning process to geometrically control single-cell CM placement on deformable hydrogels suitable for traction force microscopy (TFM). The patterns were comprised of rectangular laminin islands for attachment across the majority of the cell area, with N-cadherin "end-caps" imitating cell-cell interactions. We first photopatterned two proteins on a glass coverslip using a two-step process with photomolecular adsorption of proteins. After both photopatterning steps were complete, we transferred the pattern from the coverslip to a physiologically relevant [~]10-kPa polyacrylamide hydrogel. We seeded -actinin-tagged hiPSC-CMs on the dual-protein-patterned hydrogels and verified interaction between the hiPSC-CMs and the N-cadherin end-caps via immunofluorescent staining. We found hiPSC-CMs on dual-protein patterns have a higher cell area and contractility in the direction of sarcomere organization than those on laminin-only patterns, but no difference in sarcomere organization or force production. While N-cadherin modestly improves the single-cell patterned hiPSC-CM model, it is not sufficient to replicate the role of cell-cell contacts in CM development for in vitro hiPSC-CM systems.

bioengineering↗

bioRxiv2021

Insights into single hiPSC-derived cardiomyocyte phenotypes and maturation using ConTraX, an efficient pipeline for tracking contractile dynamics

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in-vitro models to study the mechanisms underlying cardiomyopathies and cardiotoxicity. To understand how cellular mechanisms affect the heart, it is crucial to quantify the contractile function in single hiPSC-CMs over time, however, such measurements remain demanding and low-throughput, and are too seldom considered. We developed an open-access, versatile, streamlined, and highly automated pipeline to address these challenges and enable quantitative tracking of the contractile dynamics of single hiPSC- CMs over time: ConTraX. Three interlocking software modules enable: (i) parameter-based localization and selection of single hiPSC-CMs; (ii) automated video acquisition of >200 cells/hour; and (iii) streamlined measurements of the contractile parameters via traction force microscopy. Using ConTraX, we analyzed >2,753 hiPSC-CMs over time under orthogonal experimental conditions in terms of culture media and substrate stiffnesses. Using undirected high-dimensional clustering, we dissected the complex diversity of contractile phenotypes in hiPSC-CM populations and revealed converging maturation patterns. Our modular ConTraX pipeline empowers biologists with a potent quantitative analytic tool applicable to the development of cardiac therapies.

bioengineering↗

bioRxiv2020

Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super-relaxed state: multiscale measurements and computational modeling

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1000 mutations, many in {beta}-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super-relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned hiPSC-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super-relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease. Significance StatementHeart disease is the leading cause of death worldwide, and hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, affecting over 1 in 200 people. Mutations in myosin, the motor protein responsible for contraction of the heart, are a common cause of HCM but have diverse effects on the biomechanics of the myosin protein. We demonstrate that complex biomechanical effects of mutations associated with heart disease can be effectively studied and understood using a multi-scale experimental and computational modeling approach. This work confirmed an important role for disruption of the super-relaxed state for one particular HCM mutation, and our approach can be extended to aid in the development of new targeted therapies for patients with different mutations.

biophysics↗

bioRxiv2020

A benchmarking model for validation and standardization of traction force microscopy analysis tools

Traction Force Microscopy (TFM) has become a well-established technique to assay the biophysical force produced by cells cultured on soft substrates of controlled stiffness. However, experimental conditions as well as computational implementations can have a large impact on the analysis results accuracy and reproducibility. While this can be alleviated using appropriate controls and a rigorous analytical approach, the comparison of results across studies remains difficult and there is a need for validation and benchmarking tools. To validate the accuracy of and compare various computational TFM analysis algorithms, we developed a virtual in silico model of a cell contracting on a soft substrate of controlled stiffness. The model utilizes user-defined parameters for the cell dimensions as well as for the strength and spatial distribution of a contraction dipole to calculate the deformation that would result on a soft substrate due to the cell contraction. The deformation is computed using the forward analytical stress-strain tensor calculation in the Fourier space. The resulting displacement field is used to apply, using image processing, a deformation on a real or simulated image of fluorescent microspheres embedded into a soft hydrogel, which is normally obtain experimentally by TFM imaging. The deformation field and resulting image then serve as input in the PIV and TFM analysis. The model also enables to create movies of a dynamic cell contraction, such as that of a cardiomyocyte, to validate the time accuracy of the TFM analysis after application of image processing algorithm, such as denoising. Our tool therefore addresses the need for validation and standardization of TFM analytical algorithms and its experimental implementations.

biophysics↗

bioRxiv2019

Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies

Cryogenic electron tomography is the highest resolution tool available for structural analysis of macromolecular organization inside cells. Micropatterning of extracellular matrix (ECM) proteins is an established in vitro cell culture technique used to control cell shape. Recent traction force microscopy studies have shown correlation between cell morphology and the regulation of force transmission. However, it remains unknown how cells sustain increased strain energy states and localized stresses at the supramolecular level. Here, we report a technology to enable direct observation of mesoscale organization in epithelial cells under morphological modulation, using a maskless protein photopatterning method to confine cells to ECM micropatterns on electron microscopy substrates. These micropatterned cell culture substrates can be used in mechanobiology research to correlate changes in nanometer-scale organization at cell-cell and cell-ECM contacts to strain energy states and traction stress distribution in the cell.

bioengineering↗