Feb 29, 2016 - Theresia Kraft1. 1Molecular- and Cell Physiology, Hannover Medical School, Hannover,. Germany, 2Cardiac, Thoracic, Transplantation and ...
294a
Monday, February 29, 2016
1446-Pos Board B423 Maturation Towards Pure b-Myosin Protein Expression and Corresponding Functional Properties of Individual hESC-Cardiomyocytes Natalie Weber1, Meike Wendland1, Stephan Greten1, Kristin Schwanke2, Bogdan Iorga1, Martin Fischer1, Cornelia Geers-Kno¨rr1, Jan Hegermann3, Christoph Wrede3, Ulrich Martin2, Bernhard Brenner1, Robert Zweigerdt2, Theresia Kraft1. 1 Molecular- and Cell Physiology, Hannover Medical School, Hannover, Germany, 2Cardiac, Thoracic, Transplantation and Vascular Surgery (HTTG), LEBAO, Hannover Medical School, Hannover, Germany, 3Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany. Human embryonic stem cell derived cardiomyocytes (hESC-CMs) represent a powerful tool for analyzing pathogenesis of cardiac diseases such as hypertrophic or dilative cardiomyopathies. Several studies revealed an immature state of CMs after differentiation from pluripotent stem cells. This immature state is indicated, for example, by the high expression level of the fast atrial cardiac myosin heavy chain (a-MyHC). In human ventricular cardiomyocytes, however, the slow b-MyHC predominates. Here we show maturation of hESC-CMs towards exclusive expression of b-MyHC protein in individual cardiomyocytes and their functional characterization. After growing hESC-CMs in cardiac bodies vs. plated on laminin-coated coverslips, myosin heavy chain isoform was determined using a specific antibody against ventricular b-MyHC and a newly generated anti-atrial a-MyHC-specific antibody. A single-cell mapping technique was established to relate functional characteristics of individual cardiomyocytes to their expression of a- vs. b-MyHC-protein isoforms. Cardiomyocytes grown in cardiac bodies for a maximum of 110 days mostly contained a mixture of a- and b-MyHC. Only a minority of about 10% of cardiomyocytes expressed b-MyHC exclusively. However, cardiomyocytes plated on laminin-coated coverslips shifted MyHC-expression towards 66% and 87% of all cardiomyocytes expressing exclusively b-MyHC after 35 and 75 days, respectively. This isoform switch was accompanied by morphological changes towards more elongated cardiomyocytes with highly organized sarcomeres. Surprisingly, twitch kinetics and calcium transients were found unaffected by the MyHCisoform in the sarcomeres while cardiomyocytes grown on laminin-coated coverslips in general displayed faster twitch kinetics and calcium transients. We conclude that cultivating conditions of hESC-CMs during maturation severely affect sarcomeric protein isoform expression in individual cardiomyocytes up to pure b-MyHC expression, thus changing the functional phenotype of the CMs, which is crucial for tissue engineering and cardiac disease models with functional assessment up to single cell level. 1447-Pos Board B424 A Tuned Tension Regulates the Contractility of Cardiomyocytes Differentiated from Induced Pluripotent Stem Cells Alexandre J. Ribeiro1,2, Yen-Sin Ang3,4, Robin E. Wilson1,2, Renee N. Rivas3,4, Deepak Srivastava3,4, Beth L. Pruitt1,2. 1 Mechanical Engineering, Stanford University, Stanford, CA, USA, 2 Stanford Cardiovascular Institute, Stanford, CA, USA, 3Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA, 4University of California San Francisco, San Francisco, CA, USA. The ability to differentiate human cardiomyocytes (heart muscle cells) from induced pluripotent stem cells (iPSCs) presents high potential to model heart contractility. Shortening of sarcomeres in series along intracellular myofibrils enables the beating of cardiomyocytes. We developed a platform with patterned single iPSC-cardiomyocytes in arrays and tested the contractility of these cells as a function of their shape and of substrate stiffness. We fluorescently labelled sarcomeres with LifeAct to analyze their shortening and organization. We measured the mechanical output of contractile cycles with traction force microscopy and adapted cross-correlation algorithms to characterize movement of sarcomeres. We assayed single cells on patterns of 2000 mm2 with aspect ratios (length:width) ranging from 1:1 to 7:1 and on substrates with varied stiffnesses: 6 kPa, 10 kPa and 35 kPa. Preliminary studies suggested that cell morphology and substrate stiffness affect the organization of sarcomeres with a potential impact in cardiomyocyte contractility. We aimed to understand how the contractility of iPSC-cardiomyocytes is affected by the improved organization of sarcomeres induced by these cues. For a substrate stiffness of 10 kPa, we observed that cells with a 7:1 aspect ratio produced higher contractile forces per mm of sarcomere shortening. Improved sarcomere alignment seemed to drive increased contractility of cells with this shape. Substrate stiffness also affected the contractility of 7:1 iPSC-cardiomyocytes. 35 kPa substrates induced sarcomere ruptures and lower contractile forces. Cells on 6 kPa substrates developed sarcomeres that buckled. These results suggested a role of
intracellular tension in contractility. We further tested how tension affects contractility and sarcomere organization in 7:1 cells through cell stretching, culture in high calcium and by inhibiting non-muscle myosin. Results showed evidence that a tuned intracellular tension mechanism drives myofibril alignment and improved contractility in iPSC-cardiomyocytes. 1448-Pos Board B425 Contractile Function of Permeabilized Human Embryonic Stem CellDerived Cardiomyocytes with Defined Myosin Protein Isoform Expression Bogdan Iorga1, Meike Wendland1, Natalie Weber1, Stephan Greten1, Kristin Schwanke2, Ulrich Martin2, Robert Zweigerdt2, Theresia Kraft1, Bernhard Brenner1. 1 Molecular and Cell Physiology, Hannover Medical School, Hannover, Germany, 2Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany. Cardiomyocytes derived from human induced pluripotent or embryonic stem cells (hESC-CMs) are widely used for cellular disease models, tissue engineering and drug testing. However, the phenotype of such cardiomyocytes varies, depending not least on the sarcomeric proteins that are expressed. After differentiation, hESC-CMs typically express high levels of the fast atrial myosin heavy chain (a-MyHC). Yet, in the human ventricle, 95% slow b-MyHC-protein is found. Here we aimed to determine the effect of MyHC-protein isoform on contractile function of individual hESC-CMs with known cardiac myosin isoform composition. hESC-CMs with exclusive b-MyHC-containing sarcomeres were obtained by long-term maturation on laminin-coated glass coverslips. Treatment for one week with T3 (triiodothyronine) yielded pure a-MyHC-positive hESC-CMs. For functional assessment, hESC-CMs were chemically permeabilized and mounted in a nN-sensitive micromechanical setup, allowing precise control of activation and relaxation. hESC-CMs generated maximum isometric force of 42kN/m2, which was independent of MyHC isoform. Yet, the rate constants of force redevelopment (kTR) and of the isometric phase of force relaxation (kLIN) were significantly faster for cardiomyocytes with pure a- vs. pure b-MyHC-protein (kTR: 2.4450.30s-1 and 0.6750.10s-1, kLIN: 1.4750.38s-1 and 0.3050.13s-1 for a-MyHC-CMs and b-MyHC-CMs, respectively). The kinetic values of b-MyHC-CMs were very similar to myofibrils isolated from ventricular cardiomyocytes of human donor hearts (kTR=0.6450.09s-1, kLIN=0.2850.09s-1). The presence of some a-MyHC in the sarcomeres of mainly b-MyHC-positive hESC-CMs accelerated kTR and kLIN significantly. Importantly, similar maximum force but faster kLIN of a-MyHC-CMs yields much higher tension cost of a-MyHC-CMs compared to the energetically more economical b-MyHC-CMs, like in adult myocardium. We conclude that assessment of hESC-CM’s contractile function together with sarcomere protein isoform composition at the single cell level are important for well-defined cardiac disease models and generation of artificial heart tissue. 1449-Pos Board B426 Cross-Bridge Kinetics in Rat Papillary Muscle Fibers that Carry a-MHC and b-MHC by Sinusoidal Analysis Tarek S. Karam1, John J. Michael2, Chandra Murali3, Masataka Kawai1. 1 Anatomy and Cell Biology, University of Iowa, Iowa City, IA, USA, 2 Integrative Physiology and Neuroscience, Washington StateUniversity, Pullman, WA, USA, 3Integrative Physiology and Neuroscience, Washington State University, Pullman, WA, USA. In mammalian heart muscles, two isoforms of MHC were identified. Small animals carry primarily a-MHC (fast), whereas large animals (e.g., bovines and humans) carry mostly b-MHC (slow), resulting in a large difference in the heart rate. Sprague-Dawley rats, which possess 99% a-MHC in their ventricles, were treated with propylthiouracil to result in 100% b-MHC, as demonstrated by SDS-PAGE. Papillary muscles were dissected, skinned, split into small fibers (90-110 mm diameter), and mounted on experimental apparatus. To understand the functional difference between a-MHC and b-MHC, fibers were activated under intracellular ionic conditions of cardiomyocytes: 5mM ATP, 1mM Mg2þ, 8mM phosphate (Pi), 200mM ionic strength, and pH 7.0. These measurements were carried out at 25 C. When steady tension developed, small amplitude sinusoidal length oscillations were applied in the frequency range 0.13-100Hz (corresponding time domain: 1.6-1200 ms), and the effects of Ca2þ, Pi, and ATP were studied. The results show that Ca2þ sensitivity was slightly less (12%) in b-MHC than a-MHC, while cooperativity was not statistically different. Sinusoidal analysis demonstrated that, in b-MHC containing fibers, K1 (ATP association constant) was greater (1.7x), k2 and k-2 (crossbridge detachment and its reversal rate constants) were smaller (0.6x); k4 (rate constant of the force-generation step) and k-4 (its reversal step) were
