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10 Pre-clinical studies of sotagliflozin in hypertrophic cardiomyopathy
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- Rebecca Taichman, Benjamin Lee, Kenneth Margulies, Sharlene Day
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- Journal:
- Journal of Clinical and Translational Science / Volume 8 / Issue s1 / April 2024
- Published online by Cambridge University Press:
- 03 April 2024, p. 3
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OBJECTIVES/GOALS: Study the regulatory role of sodium glucose co-transporter 1 (SGLT1) in cardiomyocytes and the therapeutic potential of sotagliflozin in hypertrophic cardiomyopathy (HCM) by (a) quantifying SGLT1 expression in HCM and (b) examining the impact of sotagliflozin on cardiac mechanics. METHODS/STUDY POPULATION: * Use Western Blot in cardiac tissue from HCM and non-HCM patients and pre-existing RNA seq and proteomics datasets to quantify SGLT1 levels in HCM. Hypothesis: SGLT1 is upregulated in HCM * Determine how SGLT1/2 inhibition by sotagliflozin will affect cardiac mechanics using living myocardial slice (LMS) preparations. A vibratome creates 200um-thick slices from (a) failing HCM heart explants, (b) septal myectomy samples from HCM patients, and (c) nonfailing rejected donor hearts. LMS are mounted on a force transducer and work-loops are stimulated under varying pre- and after-loads. Collecting baseline and post-drug work loops allows each slice to function as its own control. Hypothesis: sotagliflozin will improve diastolic mechanics by reducing stiffness in the end-diastolic pressure-volume relationship RESULTS/ANTICIPATED RESULTS: * Preliminary results from RNA seq data indicate that SLC5A1 mRNA (encoding gene for SGLT1) is significantly decreased in HCM. No proteomics study examined thus far has detected SLC5A1, indicating that overall SGLT1 levels in cardiac tissue are quite low. We will examine SGLT1 levels in our own HCM and non-HCM tissue samples with both mass-spectrometry and Western Blot. * We analyze six slices from each heart and expect 15 donor hearts and 15 HCM hearts/myectomy samples. We visualize the work loop by plotting stress/strain. Stress/strain at mitral valve closure represents exponential end diastolic pressure-volume relationship; Stress/strain at aortic valve closure represents linear end systolic pressure volume relationship. A two-sample paired t-test will compare change in stiffness and elastance. DISCUSSION/SIGNIFICANCE: This project contributes to a growing body of research surrounding the currently unknown cardioprotective mechanism of SGLT 1/2 inhibitors, furthers the technique of using living myocardial slices to study cardiac mechanics, and supports a trial examining sotagliflozin in HCM, for which disease modifying therapy remains a prevailing unmet need.
3158 Sunitinib-Induced Cardiotoxicity in an Engineered Cardiac Microtissue Model
- Carissa Livingston, Abhinay Ramachandran, Elise Corbin, Alexia Vite, Alexander Bennett, Kenneth Margulies
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- Journal:
- Journal of Clinical and Translational Science / Volume 3 / Issue s1 / March 2019
- Published online by Cambridge University Press:
- 26 March 2019, pp. 114-115
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OBJECTIVES/SPECIFIC AIMS: The aims of this study are threefold. Firstly, we are examining the effects of increased in vitro afterload (a proxy for hypertension) on human induced pluripotent stem cell cardiomyocyte (hiPSC-CM) response to sunitinib in a durable and dynamic cardiac microtissue culture system. Secondly, we are exploring effects of repeat exposure and recovery of both sunitinib and afterload throughout the lifetime of the hiPSC-CM microtissue. Finally, we are assessing methods to prevent and treat sunitinib induced cardiotoxicity. Primary outcomes for this study are commonly utilized metrics of cardiotoxicity: degree of caspase activation, electrophysiology benchmarks for minimum voltage threshold and maximum capture rate, and microtissue force generation. METHODS/STUDY POPULATION: HiPSC-CMs are cultured and matured as 3D cardiac microtissues (CMTs) on a microtissue array. After maturation, cells are exposed to sunitinib doses of 0µM, 0.5µM, 1µM or 5µM for 12 hours. Concurrently with sunitinib dosing, increases in microtissue array stiffness are created with application of an external magnetic field. Afterload spring constants are fixed at pre-determined physiologic values ranging from 0.5µN/µm, to 5µN/µm. For Aim 1: Half of the CMTs are harvested at 8 hours after sunitinib dosing to conduct the caspase 3/7 assay, and the remainder are examined for 3 days following drug exposure to track temporal changes in electrophysiology and force generation. For Aim 2: After CMT maturation, 12-hour exposures to sunitinib are repeated three times at a fixed dose, with doses separated by one week. Concurrently with sunitinib dosing, increases or decreases in microtissue stiffness are created by changing the strength of an applied external magnetic field to create “ramp up” or “ramp down” stiffness conditions. Caspase assay and contractility metrics are measured at each timepoint. For Aim 3: Experimental conditions are conducted as described in Aim 1. Prior to the introduction of sunitinib, either carvedilol or an AMP-kinase activator is added to the CMT culture media at physiologic concentrations. Primary outcomes are examined as in Aim 1. RESULTS/ANTICIPATED RESULTS: Aim 1: We hypothesize that increases in microtissue afterload, synchronized with sunitinib exposure will augment sunitinib toxicity in cardiomyocytes resulting in elevations of caspase 3/7 activity and minimum voltage capture as well as decreases in maximum capture rate and maximum force generation. Aim 2: We hypothesize that repeat exposures to both sunitinib and to increases in afterload will augment sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. Additionally, we hypothesize that decreases in afterload will decrease effective sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. Aim 3: We hypothesize that exposure to an AMP-kinase activator but not carvedilol will decrease the effects of sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. DISCUSSION/SIGNIFICANCE OF IMPACT: The use of small molecule, targeted chemotherapeutic agents is increasingly common. Many of these agents cause cardiotoxic side effects, the mechanisms of which are incompletely understood. Our lab has developed a novel 3D tissue engineering platform capable of supporting durable in vitro cardiac microtissues that experience dynamic alterations in their biomechanical load. By using this platform to examine the cardiotoxic effects of sunitinib, insight into treatment and prevention of this common problem will be developed.
3299 Dynamic Afterload Cardiac Microtissue Model To Examine Molecular Pathways of Heart Failure
- Abhinay Ramachandran, Carissa Livingston, Elise Corbin, Alexia Vite, Alex Bennett, Kenneth Margulies
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- Journal:
- Journal of Clinical and Translational Science / Volume 3 / Issue s1 / March 2019
- Published online by Cambridge University Press:
- 26 March 2019, p. 9
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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.