2853-Pos Board B545 Zebrafish Heart as a Model System to Study Structure-Function Relation- ships Alexey V. Dvornikov 1 , Sukriti Dewan 1 , Olga V. Alekhina 1 , F Bryan Pickett 2 , Pieter P. de Tombe 1 . 1 Loyola University Medical Center, Maywood, IL, USA, 2 Loyola University Chicago, Chicago, IL, USA. The zebrafish (Danio rerio) has been intensively used in cardiovascular biology mainly for study of heart development. This is a promising cost- effective model system to study structure-function due to the ease of genetic manipulations. However, the basic contractile physiology of zebrafish heart is still incompletely understood. Moreover, the applicability of the fish heart as a model system for study mammalian heart contractility needs to be established. The aim of our work was to establish the zebrafish as a model system to approach structure-function cardiac myocyte biology. Accordingly, we per- formed experiments with a focus on the Frank-Starling mechanisms at two levels: cellular (intact cells) and myofilament level (permeabilized cells). In single cell experiments we were able to attach a single enzymatically iso- lated zebrafish ventricular myocytes to myotak coated carbon probes and measure force, intracellular calcium, cell length, and sarcomere length in electrically paced cells over a range (20%) of cell lengths. In skinned cell experiments, we used a single myofibril technique to measure activation/ relaxation kinetics and force-Ca 2þ relations at short and long sarcomere lengths (2.2 and 2.4 um). Zebrafish intact myocytes displayed robust length induced increase in twitch force in the absence of calcium transient alter- ation. In skinned zebrafish muscle we found robust myofilament length dependent activation as well as characteristic activation/relaxation dynamics. We conclude that the zebrafish heart is an appropriate model to study cardiac structure-function relationships. 2854-Pos Board B546 Molecular and Functional Characterization of Uniform-Sized Beating Embryoid Bodies and Cardiomyocytes from Human Embryonic and Induced Pluripotent Stem Cells Martin Pesl 1,2 , Acimovic Ivana 1 , Jan Pribyl 3 , Renata Hezova 3 , Aleksandra Vilotic 1 , Franck Aimond 4 , Jeremy Fauconnier 4 , Jan Vrbsky 1,2 , Peter Kruzliak 2 , Peter Skladal 3,5 , Tomas Kara 2 , Vladimir Rotrekl 1 , Alain Lacampagne 4 , Petr Dvorak 1,2 , Albano C. Meli 4 . 1 Masaryk University - Faculty of Medicine, Brno, Czech Republic, 2 ICRC - St. Anne’s University Hospital, Brno, Czech Republic, 3 CEITEC - Masaryk University, Brno, Czech Republic, 4 Inserm U1046 - University of Montpellier I & II, Montpellier, France, 5 Masaryk University - Faculty of Science, Brno, Czech Republic. In vitro human embryonic stem cells (hESCs) and human induced pluripo- tent stem cells (hiPSCs) are able to differentiate into functional cardiomyo- cytes (CMs). Traditional suspension method for 3D embryoid bodies (EBs) formation results in heterogeneous EB population. Here, we hypothesized that forming homogenous EB in size and shape allows studying the mechano-biological properties of spontaneously beating clusters containing CMs. Using a defined number of single cells in AggreWell plate, we formed ho- mogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors. We checked the expression of cardiac markers by qRT-PCR, immunocytochemistry and western blotting in both beating EBs and enzymatically-dissociated CMs and found increased expression of MYH6, MYH7 and RYR2 genes as well as sarcomeric pattern for cardiac troponin T and alpha-actinin. Using atomic force microscopy- based technique, we measured the beating properties of hESC-EBs and hiPSC-EBs and found a slower beat rate in hESC-CMs when compared to hiPSC-CMs (51 5 5 vs 74 5 7 bpm) and similar contraction force (31 5 7 vs 39 5 9 nN). Both cell types respond upon stimulation or inhi- bition of beta-adrenergic pathway and caffeine. Using Ca 2þ imaging we also demonstrated the functionality of RyR2 to release Ca 2þ from the sarco- plasmic reticulum and evaluated the contribution of IP3R. Thus, upon xes- tospongin C and histamine (IP3R modulators), spontaneous Ca 2þ transients are maintained confirming the role of RyR2. Using the patch-clamp technique, we evaluated the cardiac population constituting the EBs and found that more than 75% of excitable cells exhibit atrial-like action potentials. Our results indicated that the mechano-biological properties of homogenous beating EBs can be investigated. They also indicated that spontaneous beating EBs contain mostly functional and organized atrial CMs. 2855-Pos Board B547 Assessing the Contractility of Human ips Derived Cardiomyocytes with Arrays of Microposts Marita L. Rodriguez 1 , Brandon T. Graham 2 , Lil M. Pabon 3 , Sangyoon J. Han 4 , Charles E. Murry 3 , Nathan J. Sniadecki 1 . 1 Mechanical Engineering, University of Washington, Seattle, WA, USA, 2 Bioengineering, Washington State University, Pullman, WA, USA, 3 Pathology, University of Washington, Seattle, WA, USA, 4 Cell Biology, Harvard University, Cambridge, MA, USA. Stem cell derived cardiomyocytes enable studies on heart repair, disease modeling, drug screening, and questions of fundamental biology. Here, we report on the development of an in vitro platform for assessing the contrac- tile performance of stem cell-derived cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were seeded onto elastomeric micropost arrays coated with laminin, fibronectin, or collagen IV, in order to characterize the contractile force, velocity, and po- wer produced by these cells. We assessed contractile function of the hiPSC- CMs by tracking the deflection of each of the microposts underneath an individual cell, with high-speed optical microscopy (A). Immunofluorescent staining of these cells was used with the microposts to assess their spread area, nucleation, and sarcomeric structure (B). We found that this technique was able to track the twitch movement of individ- ual hiPS-CMs with sufficient temporal resolution to determine maxima in force (C), velocity (D), and power (E); as well as that hiPSC-CMs on laminin-coated posts demonstrated higher attachment (F), spread area (G), and contractile force (H) than those seeded on fibronectin or collagen IV coatings. Therefore, these results suggest that future studies using this platform employ laminin-coated microposts. 2856-Pos Board B548 Mechanical Analysis of Single Myocyte Contraction in a 3D Elastic Matrix John Shaw 1 , Leighton Izu 2 , Ye Chen-Izu 3 . 1 Aerospace Engineering, University of Michigan, Ann Arbor, MI, USA, 2 Pharmacology, University of California, Davis, CA, USA, 3 Biomedical Engineering, Pharmacology, Medicine/Cardiology, University of California, Davis, CA, USA. Objective: Excessive mechanical stress in myocardium, arising from various genetic or acquired pathological conditions, leads to arrhythmias and heart fail- ure, yet little is known about the mechano-chemo-transduction (MCT) mecha- nisms at the single cell level that underlie heart disease development. Recently we developed a novel Cell-in-Gel system by embedding isolated live myocytes in a constraining hydrogel, in order to study MCT mechanisms under well- defined mechanical afterload. Methods: Here we present a mechanical model and analysis of a single con- tracting myocyte embedded in an infinite elastic matrix, which is an exact closed-form 3D elasticity solution based on the classical Eshelby Inclusion problem. Results: The model enables quick parametric studies to gain insight into ex- pected trends. (1) The fractional shortening of a myocyte is dependent on its geometric aspect ratio and the stiffness of the surrounding gel. A typical car- diomyocyte is able to contract only 20% of its load-free fractional shortening in a gel of similar stiffness. (2) The stress state is multi-axial and uniform within the cell. (3) The traction on the cell surface, however, is highly non- uniform with a maximum near the ends, suggesting that mechanosensors on the cell surface will experience various degrees of normal and shear stresses under certain pathological conditions (infarction, fibrosis or asynchronous contraction). Conclusions: The mechanical model provides a baseline prediction of the contractile response of the myocyte during contraction in-gel, assuming the cell is a purely elastic object. Preliminary experimental data show stronger than predicted (baseline) contractility under afterload, and the model allows quantification of this enhancement of contractility (by up- regulation of Ca 2þ transients related to the Anrep effect). Thus, our analyses will quantitatively inform ongoing studies of the MCT mechanisms that link mechanical stress to cardiac function and remodeling in health and disease. Tuesday, February 18, 2014 565a