3299 Dynamic Afterload Cardiac Microtissue Model To Examine Molecular Pathways of Heart Failure

Abstract

OBJECTIVES/SPECIFIC AIMS: This project aims to determine the key molecular pathways that link increased myocardial wall stress to cardiomyocyte hypertrophy and subsequent heart failure. We will use a cardiac microtissue (CMT) model with dynamically tunable cantilever stiffness to examine changes in CMT hypertrophy and electro-mechanical properties in response to increased afterload (cantilever stiffness). Subsequently, we will determine if inhibition of pro-hypertrophic or anti-hypertrophic pathways alter the hypertrophic response to increased afterload. Primary outcomes for this study are static/dynamic force, minimum electric field strength (VT), maximum capture rate (MCR), average cell area, and tissue cross-sectional thickness, and secondary outcomes are degree of myoblast activation and apoptosis. METHODS/STUDY POPULATION: CMT platforms will be fabricated using iron-doped polydimethylsiloxane (PDMS) to create magnetically tunable cantilevers. Cantilever stiffness will be increased with the application of an external magnetic field. Cantilever stiffness will be measured using a capacitance probe, where the force required to deflect both the cantilever and calibration probe is in accordance with Hooke’s Law. Human induced pluripotent stem cell cardiomyocyte (hiPSC-CMs) will be cultured and matured as 3D CMTs. In-vitro static/dynamic force generation will also be calculated by measuring the deflection of the cantilevers and applying Hooke’s law. CMTs will be paced using carbon electrodes to obtain VT and MCR. Structural data will be obtained using immunostaining and confocal microscopy. Finally, we will use pharmacologic inhibitors to inhibit molecular pathways that we identified in prior genetic screens such as ABCC8 (anti-hypertrophic mediator) and C1QTNF9 (pro-hypertrophic mediator). We will examine each of these pathways in low- and high-stiffness conditions. RESULTS/ANTICIPATED RESULTS: We believe increased afterload will cause significant hypertrophy, measured by increases in CMT cross-sectional thickness, cardiac myocyte area, myofibroblast activation, and myocyte apoptosis. In addition, we expect to see increases in static/dynamic force, increased voltage threshold, and decreased maximum capture rate. Preliminary results show a 64.3% increase in force generation when stiffness is increased by approximately 30%, and a 44.4% decrease in force generation when stiffness is decreased by approximately 30%. Finally, we expect that inhibiting a pro- or anti-hypertrophic molecular pathway will weaken or strengthen the hypertrophic response to increased afterload, respectively. DISCUSSION/SIGNIFICANCE OF IMPACT: To our knowledge, our lab is the first to create a dynamically tunable afterload system in the cantilever CMT model. This advance provides us with a robust platform to determine the molecular pathways that cause increased myocardial wall stress to result in cardiomyocyte hypertrophy and heart failure, which remain a critical knowledge gap in our understanding of cardiovascular disease. With more precise understanding of these pathways, we will equip ourselves with the knowledge to develop novel therapeutic agents to prevent the development or progression of heart failure.