Drosophila melanogaster possess a simple linear heart tube and constitute an excellent genetic model system with which to investigate the effects of cardi- omyopathic mutation. The Mhc5 myosin heavy chain mutation is located in the ‘transducer’domain and elicits hypercontractile function at the molecular level characterized by high ATPase activity and enhanced in vitro motility proper- ties.Additionally, its expression impairs diastolic relaxation of the cardiac tube reminiscent of restrictive cardiomyopathy in humans. We have investi- gated the effect of the Mhc5 mutation on cardiac structure/function by quanti- tative proteomics using isobaric tags for relative quantification (iTRAQ). Ex- cised fly hearts from yw (control)and Mhc5 strainswere digested with trypsin,reduced, alkylated and labeled with ITRAQ reagent. Peptides from each pool were mixed together prior to fractionation by strong cation exchange chromatography and subsequent reversed-phase HPLC coupled to tandem mass spectrometry. This approach identified approximately 600 proteins, of which 94 were upregulated and 86 were downregulated in Mhc5 hearts relative to yw hearts (p<0.05). Ontological cluster analysis of the genes encoding the reg- ulated proteins revealed that myofibrillar disarray in Mhc5 hearts likely stems from overexpression of actin with concomitant reduction of myofibrillar assem- bly proteins such as spectrin, and other actin-binding proteins. Structural re- modeling was also characterized by increased expression of extracellular ma- trix proteins. Upregulation of proteins involved in mitochondrial oxidative phosphorylation and fatty acid catabolism suggests further bioenergetic remod- eling.The proteomic, structural and ultrastructural data are consistent with a model whereby the elevated ATPase activity caused by Mhc5 mutation in- creases energetic demand, thereby stimulating a concerted compensatory met- abolic response to maintain energetic homeostasis. Ongoing protein-network/ interactome analysis will help to further refine the model. 3733-Pos Mesenchymal Stem Cells Protect Cardiomyocytes Dushon DeVere Riley 1,2 , Terry B. Rogers 1 , Aarif Y. Khakoo 3 , Cecilia A. Frederick 2 , Shibani Pati 3 , Shirley A. Gaa 1 , W. Jonathan Lederer 1,2 . 1 University of Maryland School of Medicine, University of Maryland, Baltimore, Baltimore, MD, USA, 2 University of Maryland Biotechnology Institute, Baltimore, MD, USA, 3 University of Texas MD Anderson Cancer Center, Houston, TX, USA. Possible therapeutic benefits of stem cell treatments have been widely investi- gated recently. We have presented initial reports that co-culturing mesenchy- mal stem cells (MSC, Lonza) with rat heart cells in primary culture can prevent the consequences of the treatment with a inflammatory bacterial endotoxin (LPS, Lipopolysaccharide-A). We now investigate how the MSC produce their beneficial actions. Using sparse primary cultures of neonatal rat ventricular or adult rat ventricular myocytes with either MSC or control cells (fibroblasts), we examine cardiac Ca 2þ signaling. LPS causes Ca 2þ signaling anomalies which include delayed afterdepolarizations (DADs) and Ca 2þ -enhanced early afterde- polarizations (EADs). We find that co-cultures with cells co-mingled can pre- vent the untowards actions of LPS on the cardiac myocytes. The negative con- sequences of LPS are alterations in the normal[Ca 2þ ] i transienthatis stimulated by field shocks as described above. Since the benefit of MSC co-cul- ture are found even when a solute per- meable / cell impermeant membrane separates the MSC from the LPS treated cardiac myocytes, we con- clude that a paracrine action of the MSC can account for the treatment at- tributed to the MSCs. We continue to investigate possible beneficial signal- ing pathways thatmay explain the paracrine effect of MSCs. 3734-Pos Mechanical and Biochemical Characteristics of Human Stem Cell-Derived Cardiomyocytes Scott D. Lundy 1 , Marieke J. Bloemink 2 , Samantha C. Lynn 2 , Michael A. Geeves 2 , Michael A. Laflamme 1 , Michael Regnier 1 . 1 University of Washington, Seattle, WA, USA, 2 University of Kent, Canterbury, United Kingdom. Cell-based cardiac repair following myocardial infarction has gained consider- able interest recently, and the human pluripotent stem cell is an attractive cell source due its efficient differentiation into immature but functional cardiomyo- cytes. We examined the biophysical characteristics of cardiomyocytes gener- ated from human embryonic stem cells (hESC-CMs) by measuring calcium transients, single cell contractions, and actomyosin interactions via flash pho- tolysis.Furthermore, we compared these characteristics with those obtained from a second promising but still poorly characterized cell type, the human in- duced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM). We hypoth sized that understanding fundamental biochemical and mechanical charac tics of these cells would provide insight into potential strategies to induce further cell maturation in vitro. Our results suggest that hESC-CMs and hiPSC-CMs exhibit spontaneous con tractions and calcium transients with similar kinetics, including time to peak [Ca 2þ ] i (116534msvs. 155540ms)and time to 50% [Ca 2þ ] i decay (352587ms vs. 296549ms). Furthermore, quantitative videomicroscopy of resulting single cell contractions suggests that cardiomyocytes from both s ces demonstrate similar resting cell size (17.151.4um vs. 16.554.6um), con traction amplitude (4.251.6% vs. 4.452.1%),time to peak contraction (0.34650.135secvs.0.33950.214sec), maximum contraction velocity (6.3453.50um/sec vs. 7.4654.81um/sec), and maximum relaxation velocity (3.2152.49um/sec vs. 3.4052.49um/sec). We have also successfully isolated and purified 20 ug of myosin per million hESC-CMs. Using flash photolysis to liberate ATP in a solution of actomyosin we have shown that the myosin binds actin and is dissociated from the com by ATP with the expected 2 nd order rate constant (~1 uM 1 sec 1 ). In summary, the contractile properties of hESC-CMs and hiPSC-CMs are sim ilar to each other but differ from values published for adult human cardiom cytes,suggesting that they are functionally immature and may benefit from in vitro maturation efforts. Support appreciated from NIH HL064387/HL080431, NHLBI T32GMO7266- 35S1, and the Wellcome Trust. 3735-Pos Cell-Seeded Fibrin Scaffolds for Cardiac Tissue Engineering Kassandra S. Thomson, Gabrielle Robinson, F. Steven Korte, Cecilia Giachelli, Buddy D. Ratner, Marta Scatena, Michael Regnier. University of Washington, Seattle, WA, USA. Cellular cardiomyoplasty to replace non-functional tissue following cardiac farction appears clinically viable. Current strategies utilizing direct injection cell suspensions are limited by low cell retention, poor cell localization, and high celldeath.Synthetic biomaterials developed to enhance cell delivery can lead to problems with immune rejection, degradation, and mechanical match, preventing functional integration of constructs with host myocardiu The goalof this project is to develop a functional cardiac tissue construct with enhanced host integration capabilities as a novel strategy to replace d aged myocardium. We have developed a novel templated fibrin scaffold se with cells to promote functional integration. Fibrin is an ideal scaffold mate because it can be autologous, improves cell attachment and growth, and de- grades into natural byproducts that can induce angiogenesis. The novel sc architecture includes 1) microchannels spanning the length of the scaffold, lowing alignment and organization of cells to mimic native cardiac tissue st ture, and 2) micropores to enhance construct survival by improving nutrien livery and waste removal. The dense fibrin scaffolds (stiffness ¼ 16.0 5 3.0 kPa) had mechanical properties closer to native myocardium than fibrin ge (0.5 to < 7 kPa). Centrifuge seeding with a tri-cell mixture of cardiomyocyt endothelial cells, and fibroblasts increased scaffold stiffness (38.3 5 8.9 kPa to values near neonatal myocardial tissue (~40 kPa). Stiffness decreased o time in culture (25.2 5 3.1 kPa, Day 6), which may indicate ECM formation and scaffold degradation. Patches of beating cells were observed inside ch nels within two days in culture. After three days in culture, histology showe cardiomyocyte and fibroblast alignment and immature lumen formation. Th results indicate micro-templated fibrin scaffolds are a unique and viable pla form for cardiac tissue engineering. This work supported by NIH HL064387 (MR, MS, BR) and NSF GRFP (KT). 3736-Pos Cardiac Specific Overexpression of N-RAP in Transgenic Mice Garland Crawford, Shajia Lu, Justin Dore, Robert Horowits. National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA. The muscle specific protein NRAP plays a role in myofibril assembly and is regulated in mouse models of dilated cardiomyopathy. We sought to deter if increased N-RAP expression would directly lead to a cardiomyopathy phe type. Novel transgenic lines were developed using the tet-off system with t genic N-RAP expression requiring the tetracycline transactivator (tTA). tTA was introduced by mating the N-RAP transgenic animals with well-characte ized animals carrying the tTA transgene controlled by the cardiac specific a pha-myosin heavy chain promoter. Multiple founder lineswere examined and lines showing the most significant increase in NRAP expression were used for further investigation. N-RAP expression in theses animals was up t 2.5 times greater than control littermates as determined by western blot a sis. Histological examination of hearts from ~12 week old transgenic mice 718a Wednesday, February 24, 2010