Poster P6 - 2D Finite Element Modeling of hiPSC-Derived Cardiomyocytes to Study Myofibril Remodeling and its Implications on Hypertrophic Cardiomyopathy

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Primary Investigator(s)

AV

Alison Vander Roest (she/her/hers)

Assistant Professor
Department of Biomedical Engineering, University of Michigan, Ann Arbor, USA, Michigan, United States

Presenting Author(s)

NF

Nathan French (he/him/his)

Master's Student
Department of Biomedical Engineering, University of Michigan, Ann Arbor, USA
Ann Arbor, Michigan, United States

Introduction: Hypertrophic Cardiomyopathy (HCM) is the most common form of genetic heart disease affecting nearly 1 in 500 people worldwide. Patients with HCM are afflicted by poor cardiovascular function which limits their physical capabilities and negatively impacts their quality of life. On the cellular and tissue level, HCM is often characterized by cardiomyocyte hypertrophy and disarray, along with myocardial interstitial fibrosis. It has been noted in previous studies that phenotypic-HCM cardiomyocytes exhibit myofibril disarray which alters contractile dynamics. Understanding the relationship between myofibril organization and remodeling with corresponding force transmission is an area of current investigation for understanding the cellular mechanics underlying HCM. In understanding the implications of altered cellular mechanics on HCM disease progression, cardiac fibroblasts are a noteworthy subject of study given the activation of quiescent cardiac fibroblasts towards a myofibroblast state is a principal driver of interstitial fibrosis development. Fibrosis increases stiffness thereby decreasing tissue compliance which can initiate a positive feedback mechanism leading to increased fibroblast to myofibroblast transition. Different pharmaceutical treatment strategies have emerged targeting particular molecular structures such the cardiac-myosin inhibitor mavacamten. Here the effects of mavacamten and para-nitro blebbistatin are studied to understand modulation of the actomyosin complex on cellular contractility.

Materials and

Methods: Human induced pluripotent stem cells (hiPSC) with an endogenous green fluorescent protein (GFP) reporter for α-actinin were differentiated into cardiomyocytes. On day 30 of the differentiation, cells were plated onto micropatterned 10 kPa polyacrylamide (PA) gels doped with red fluorescent microspheres to allow traction force microscopy measurements of cell force. Beating cardiomyocytes on the polyacrylamide gels were imaged in brightfield and simultaneous dual-channel GFP/RFP imaging at 30 frames per second using the Hamamatsu W-View Gemini. Cardiomyocytes were treated using two different drugs (mavacamten, para-nitro blebbistatin) that have an intended effect on actin-myosin interactions; DMSO was used as the loading control for the study. Post-experiment, the samples were stained for β1-integrin which will allow for visualization of integrin clustering for understanding the cellular and extracellular matrix adhesion. The data collected during the experiment was used to validate a custom MATLAB 2D finite element model, where the myofibrils are segmented using the GFP-α-actinin videos to provide internal cytoskeletal structures of different stiffness and compliance to enforce the anisotropic nature of the cardiomyocyte. hiPSCs were differentiated into cardiac fibroblasts to study myofibroblast activation and mechanics. Cardiac fibroblasts activation was studied at physiological stiffness (10kPa) on deformable PA gels and on stiff glass surfaces which were fixed after 5 days and immunostained for α-Smooth Muscle Actin (αSMA) and other cytoskeletal and matrix adhesion proteins. Preliminary 3D work has been performed by culturing cardiomyocytes and cardiac fibroblasts in a fibrin-based scaffolding in a deformable PDMS mold to recapitulate more of the native cellular environment.

Results, Conclusions, and Discussions: Cardiomyocytes treated with actomyosin modulators blebbistatin and mavacamten exhibited significant changes in contraction rate and intensity as seen in the kymographs along cardiomyocyte sarcomeres in Figure 1. Cardiomyocytes treated with mavacamten at a 100 nM concentration exhibited myofibril remodeling within a 26 hr time course indicating the potent effects of pharmaceutical therapies in altering cellular contractility and intracellular mechanics. Cardiomyocytes were then fixed after imaging to stain for Integrin-β1 to understand extracellular adhesion and mechanical transduction from focal adhesions towards the intracellular myofibrils as seen in Figure 2. Immunostaining of Integrin-β1 did not demonstrate the expected punctate structures as seen in previous published literature indicating the potential immunostaining permeabilization method causes excessive membrane disruption leading to diffuse antibody staining. However, integrin clustering can be predicted as an output of finite element modeling that segments myofibrils contraction videos to understand forces mechanical properties of internal cytoskeletal proteins. Figure 3 demonstrates the various graphical predictions of stress, strain, and integrin clustering of the custom MATLAB finite element code where punctate integrin clustering is seen representing the focal axial focal adhesions securing the cardiomyocyte to the substrate. An interesting note in Figure 4 can be found where there is a slight time-lag between the sarcomere shortening and corresponding bead displacement within the PA hydrogel indicating the compliance of both the cell and the hydrogel construct which can be used to further refine the finite element model’s compliance parameter. To understand cardiac fibroblast activation levels, cardiac fibroblasts were differentiated and plated onto glass coverslips and cultured with B27 media to minimize modulators inhibiting myofibroblast transition. Figure 5 demonstrates myofibroblast transition by the presence of α-Smooth Muscle Actin. Cardiac fibroblasts were cultured in various medias with proprietary fibroblasts growth media demonstrating the least amount of myofibroblast transition presumably due to the higher levels of fibroblast growth factor present in the media compared to B27. Cells were successfully cultured in a 3D fibrin construct seen in Figure 6 that will help elucidate new understandings on myofibril remodeling and fibroblast activation in a 3D environment in the future.

Acknowledgements (Optional):