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Research Papers

Measuring the Contractile Forces of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Arrays of Microposts

[+] Author and Article Information
Marita L. Rodriguez

Department of Mechanical Engineering,
University of Washington,
Seattle, WA 98195

Brandon T. Graham

Department of Bioengineering,
Washington State University,
Pullman, WA 99164

Lil M. Pabon

Department of Pathology,
Center for Cardiovascular Biology,
Institute for Stem Cell and
Regenerative Medicine,
University of Washington,
Seattle, WA 98109;
Department of Bioengineering,
University of Washington,
Seattle, WA 98195

Sangyoon J. Han

Department of Cell Biology,
Harvard University,
Cambridge, MA 02115

Charles E. Murry

Department of Pathology,
Center for Cardiovascular Biology,
Institute for Stem Cell and
Regenerative Medicine,
University of Washington,
Seattle, WA 98109;
Department of Bioengineering,
University of Washington,
Seattle, WA 98195;
Department of Medicine/Cardiology,
University of Washington,
Seattle, WA 98195

Nathan J. Sniadecki

Department of Mechanical Engineering,
University of Washington,
Seattle, WA 98195;
Department of Bioengineering,
University of Washington,
Seattle, WA 98195
e-mail: nsniadec@uw.edu

1Corresponding author.

Contributed by the Bioengineering Division of ASME for publication in the Journal of Biomechanical Engineering. Manuscript received August 21, 2013; final manuscript received February 21, 2014; accepted manuscript posted March 10, 2014; published online April 10, 2014. Assoc. Editor: Kevin D. Costa.

J Biomech Eng 136(5), 051005 (Apr 10, 2014) (10 pages) Paper No: BIO-13-1381; doi: 10.1115/1.4027145 History: Received August 21, 2013; Revised February 21, 2014; Accepted March 10, 2014

Human stem cell-derived cardiomyocytes hold promise for heart repair, disease modeling, drug screening, and for studies of developmental biology. All of these applications can be improved by assessing the contractility of cardiomyocytes at the single cell level. We have developed an in vitro platform for assessing the contractile performance of stem cell-derived cardiomyocytes that is compatible with other common endpoints such as microscopy and molecular biology. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were seeded onto elastomeric micropost arrays in order to characterize the contractile force, velocity, and power produced by these cells. We assessed contractile function by tracking the deflection of microposts beneath an individual hiPSC-CM with optical microscopy. Immunofluorescent staining of these cells was employed to assess their spread area, nucleation, and sarcomeric structure on the microposts. Following seeding of hiPSC-CMs onto microposts coated with fibronectin, laminin, and collagen IV, we found that hiPSC-CMs on laminin coatings demonstrated higher attachment, spread area, and contractile velocity than those seeded on fibronectin or collagen IV coatings. Under optimized conditions, hiPSC-CMs spread to an area of approximately 420 μm2, generated systolic forces of approximately 15 nN/cell, showed contraction and relaxation rates of 1.74 μm/s and 1.46 μm/s, respectively, and had a peak contraction power of 29 fW. Thus, elastomeric micropost arrays can be used to study the contractile strength and kinetics of hiPSC-CMs. This system should facilitate studies of hiPSC-CM maturation, disease modeling, and drug screens as well as fundamental studies of human cardiac contraction.

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References

Bonow, R., Mann, D. L., Zipes, D. P., and Libby, P., 2012, Braunwald's Heart Disease: A Textbook of Cardiovascular Medicine, Saunders, Philadelphia, PA.
Karantalis, V., Balkan, W., Schulman, I. H., Hatzistergos, K. E., and Hare, J. M., 2012, “Cell-Based Therapy for Prevention and Reversal of Myocardial Remodeling,” Am. J. Physiol.: Heart Circ. Physiol., 303(3), pp. H256–H270. [CrossRef] [PubMed]
Suncion, V. Y., Schulman, I. H., and Hare, J. M., 2012, “Concise Review: The Role of Clinical Trials in Deciphering Mechanisms of Action of Cardiac Cell-Based Therapy,” Stem Cells Transl. Med., 1(1), pp. 29–35. [CrossRef] [PubMed]
Bellin, M., Marchetto, M. C., Gage, F. H., and Mummery, C. L., 2012, “Induced Pluripotent Stem Cells: The New Patient?,” Nat. Rev. Mol. Cell Biol., 13(11), pp. 713–726. [CrossRef] [PubMed]
Grskovic, M., Javaherian, A., Strulovici, B., and Daley, G. Q., 2011, “Induced Pluripotent Stem Cells-Opportunities for Disease Modelling and Drug Discovery,” Nat. Rev. Drug Discov., 10(12), pp. 915–929. [PubMed]
Dambrot, C., Passier, R., Atsma, D., and Mummery, C. L., 2011, “Cardiomyocyte Differentiation of Pluripotent Stem Cells and Their Use as Cardiac Disease Models,” Biochem. J., 434(1), pp. 25–35. [CrossRef] [PubMed]
Das, A. K., and Pal, R., 2010, “Induced Pluripotent Stem Cells (Ipscs): The Emergence of a New Champion in Stem Cell Technology-Driven Biomedical Applications,” J. Tissue Eng. Regener. Med., 4(6), pp. 413–421.
Freund, C., and Mummery, C. L., 2009, “Prospects for Pluripotent Stem Cell-Derived Cardiomyocytes in Cardiac Cell Therapy and as Disease Models,” J. Cell. Biochem., 107(4), pp. 592–599. [CrossRef] [PubMed]
Mercola, M., Colas, A., and Willems, E., 2013, “Induced Pluripotent Stem Cells in Cardiovascular Drug Discovery,” Circ. Res., 112(3), pp. 534–548. [CrossRef] [PubMed]
Miki, K., Uenaka, H., Saito, A., Miyagawa, S., Sakaguchi, T., Higuchi, T., Shimizu, T., Okano, T., Yamanaka, S., and Sawa, Y., 2012, “Bioengineered Myocardium Derived From Induced Pluripotent Stem Cells Improves Cardiac Function and Attenuates Cardiac Remodeling Following Chronic Myocardial Infarction in Rats,” Stem Cells Transl. Med., 1(5), pp. 430–437. [CrossRef] [PubMed]
Kawamura, M., Miyagawa, S., Miki, K., Saito, A., Fukushima, S., Higuchi, T., Kawamura, T., Kuratani, T., Daimon, T., Shimizu, T., Okano, T., and Sawa, Y., 2012, “Feasibility, Safety, and Therapeutic Efficacy of Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Sheets in a Porcine Ischemic Cardiomyopathy Model,” Circulation, 126(11 Suppl 1), pp. S29–S37. [CrossRef] [PubMed]
Masumoto, H., Matsuo, T., Yamamizu, K., Uosaki, H., Narazaki, G., Katayama, S., Marui, A., Shimizu, T., Ikeda, T., Okano, T., Sakata, R., and Yamashita, J. K., 2012, “Pluripotent Stem Cell-Engineered Cell Sheets Reassembled With Defined Cardiovascular Populations Ameliorate Reduction in Infarct Heart Function Through Cardiomyocyte-Mediated Neovascularization,” Stem Cells, 30(6), pp. 1196–1205. [CrossRef] [PubMed]
Mauritz, C., Martens, A., Rojas, S. V., Schnick, T., Rathert, C., Schecker, N., Menke, S., Glage, S., Zweigerdt, R., Haverich, A., Martin, U., and Kutschka, I., 2011, “Induced Pluripotent Stem Cell (iPSC)-Derived Flk-1 Progenitor Cells Engraft, Differentiate, and Improve Heart Function in a Mouse Model of Acute Myocardial Infarction,” Eur. Heart J., 32(21), pp. 2634–2641. [CrossRef] [PubMed]
Singla, D. K., Long, X., Glass, C., Singla, R. D., and Yan, B., 2011, “Induced Pluripotent Stem (IPS) Cells Repair and Regenerate Infarcted Myocardium,” Mol. Pharm., 8(5), pp. 1573–1581. [CrossRef] [PubMed]
Mosna, F., Annunziato, F., Pizzolo, G., and Krampera, M., 2010, “Cell Therapy for Cardiac Regeneration After Myocardial Infarct: Which Cell Is the Best?,” Cardiovasc. Hematol. Agents Med. Chem., 8(4), pp. 227–243. [CrossRef] [PubMed]
Yin, S., Zhang, X., Zhan, C., Wu, J., Xu, J., and Cheung, J., 2005, “Measuring Single Cardiac Myocyte Contractile Force Via Moving a Magnetic Bead,” Biophys. J., 88(2), pp. 1489–1495. [CrossRef] [PubMed]
Hazeltine, L. B., Simmons, C. S., Salick, M. R., Lian, X., Badur, M. G., Han, W., Delgado, S. M., Wakatsuki, T., Crone, W. C., Pruitt, B. L., and Palecek, S. P., 2012, “Effects of Substrate Mechanics on Contractility of Cardiomyocytes Generated From Human Pluripotent Stem Cells,” Int. J. Cell Biol., 2012, p. 508294. [CrossRef] [PubMed]
Hersch, N., Wolters, B., Dreissen, G., Springer, R., Kirchgebner, N., Merkel, R., and Hoffmann, B., 2013, “The Constant Beat: Cardiomyocytes Adapt Their Forces by Equal Contraction Upon Environmental Stiffening,” Biol. Open, 2, pp. 351–361. [CrossRef] [PubMed]
Jacot, J. G., Mcculloch, A. D., and Omens, J. H., 2008, “Substrate Stiffness Affects the Functional Maturation of Neonatal Rat Ventricular Myocytes,” Biophys. J., 95(7), pp. 3479–3487. [CrossRef] [PubMed]
Jacot, J. G., Martin, J. C., and Hunt, D. L., 2010, “Mechanobiology of Cardiomyocyte Development,” J. Biomech., 43(1), pp. 93–98. [CrossRef] [PubMed]
Iribe, G., Helmes, M., and Kohl, P., 2007, “Force-Length Relations in Isolated Intact Cardiomyocytes Subjected to Dynamic Changes in Mechanical Load,” Am. J. Physiol.: Heart Circ. Physiol., 292(3), pp. H1487–H1497. [CrossRef] [PubMed]
Borbely, A., Van Der Velden, J., Papp, Z., Bronzwaer, J. G., Edes, I., Stienen, G. J., and Paulus, W. J., 2005, “Cardiomyocyte Stiffness in Diastolic Heart Failure,” Circulation, 111(6), pp. 774–781. [CrossRef] [PubMed]
Nishimura, S., Yasuda, S., Katoh, M., Yamada, K. P., Yamashita, H., Saeki, Y., Sunagawa, K., Nagai, R., Hisada, T., and Sugiura, S., 2004, “Single Cell Mechanics of Rat Cardiomyocytes Under Isometric, Unloaded, and Physiologically Loaded Conditions,” Am. J. Physiol.: Heart Circ. Physiol., 287(1), pp. H196–H202. [CrossRef] [PubMed]
Nishimura, S., Nagai, S., Sata, M., Katoh, M., Yamashita, H., Saeki, Y., Nagai, R., and Sugiura, S., 2006, “Expression of Green Fluorescent Protein Impairs the Force-Generating Ability of Isolated Rat Ventricular Cardiomyocytes,” Mol. Cell. Biochem., 286(1–2), pp. 59–65. [CrossRef] [PubMed]
Domke, J., Parak, W. J., George, M., Gaub, H. E., and Radmacher, M., 1999, “Mapping the Mechanical Pulse of Single Cardiomyocytes With the Atomic Force Microscope,” Eur. Biophys. J., 28(3), pp. 179–186. [CrossRef] [PubMed]
Chang, W. T., Yu, D., Lai, Y. C., Lin, K. Y., and Liau, I., 2012, “Characterization of the Mechanodynamic Response of Cardiomyocytes With Atomic Force Microscopy,” Anal. Chem., 85, pp. 1395–1400. [CrossRef]
Liu, J., Sun, N., Bruce, M. A., Wu, J. C., and Butte, M. J., 2012, “Atomic Force Mechanobiology of Pluripotent Stem Cell-Derived Cardiomyocytes,” PLoS One, 7(5), p. e37559. [CrossRef] [PubMed]
Brixius, K., Hoischen, S., Reuter, H., Lasek, K., and Schwinger, R. H., 2001, “Force/Shortening-Frequency Relationship in Multicellular Muscle Strips and Single Cardiomyocytes of Human Failing and Nonfailing Hearts,” J. Card. Failure, 7(4), pp. 335–341. [CrossRef]
Edes, I. F., Czuriga, D., Csanyi, G., Chlopicki, S., Recchia, F. A., Borbely, A., Galajda, Z., Edes, I., Van Der Velden, J., Stienen, G. J. M., and Papp, Z., 2007, “Rate of Tension Redevelopment Is Not Modulated by Sarcomere Length in Permeabilized Human, Murine, and Porcine Cardiomyocytes,” Am. J. Physiol.: Regul., Integr. Comp. Physiol., 293(1), pp. R20–R29. [CrossRef]
Tanaka, Y., Morishima, K., Shimizu, T., Kikuchi, A., Yamato, M., Okano, T., and Kitamori, T., 2006, “Demonstration of a PDMS-Based Bio-Microactuator Using Cultured Cardiomyocytes to Drive Polymer Micropillars,” Lab Chip, 6(2), pp. 230–235. [CrossRef] [PubMed]
Vannier, C., Chevassus, H., and Vassort, G., 1996, “Ca-Dependence of Isometric Force Kinetics in Single Skinned Ventricular Cardiomyocytes From Rats,” Cardiovasc. Res., 32(3), pp. 580–586. [CrossRef] [PubMed]
Borg, T. K., Rubin, K., Lundgren, E., Borg, K., and Obrink, B., 1984, “Recognition of Extracellular Matrix Components by Neonatal and Adult Cardiac Myocytes,” Dev. Biol., 104(1), pp. 86–96. [CrossRef] [PubMed]
Xi, J., Khalil, M., Shishechian, N., Hannes, T., Pfannkuche, K., Liang, H., Fatima, A., Haustein, M., Suhr, F., Bloch, W., Reppel, M., Saric, T., Wernig, M., Janisch, R., Brockmeier, K., Hescheler, J., and Pillekamp, F., 2010, “Comparison of Contractile Behavior of Native Murine Ventricular Tissue and Cardiomyocytes Derived From Embryonic or Induced Pluripotent Stem Cells,” FASEB J., 24(8), pp. 2739–2751. [CrossRef] [PubMed]
Eschenhagen, T., Fink, C., Remmers, U., Scholz, H., Wattchow, J., Weil, J., Zimmermann, W., Dohmen, H. H., Schafer, H., Bishopric, N., Wakatsuki, T., and Elson, E. L., 1997, “Three-Dimensional Reconstitution of Embryonic Cardiomyocytes in a Collagen Matrix: A New Heart Muscle Model System,” FASEB J., 11(8), pp. 683–694. [PubMed]
Pillekamp, F., Reppel, M., Rubenchyk, O., Pfannkuche, K., Matzkies, M., Bloch, W., Sreeram, N., Brockmeier, K., and Hescheler, J., 2007, “Force Measurements of Human Embryonic Stem Cell-Derived Cardiomyocytes in an in Vitro Transplantation Model,” Stem Cells, 25(1), pp. 174–180. [CrossRef] [PubMed]
Kim, J., Park, J., Na, K., Yang, S., Baek, J., Yoon, E., Choi, S., Lee, S., Chun, K., Park, J., and Park, S., 2008, “Quantitative Evaluation of Cardiomyocyte Contractility in a 3D Microenvironment,” J. Biomech., 41(11), pp. 2396–2401. [CrossRef] [PubMed]
Park, J., Ryu, J., Choi, S. K., Seo, E., Cha, J. M., Ryu, S., Kim, J., Kim, B., and Lee, S. H., 2005, “Real-Time Measurement of the Contractile Forces of Self-Organized Cardiomyocytes on Hybrid Biopolymer Microcantilevers,” Anal. Chem., 77(20), pp. 6571–6580. [CrossRef] [PubMed]
Boudou, T., Legant, W. R., Mu, A., Borochin, M. A., Thavandiran, N., Radisic, M., Zandstra, P. W., Epstein, J. A., Margulies, K. B., and Chen, C. S., 2011, “A Microfabricated Platform to Measure and Manipulate the Mechanics of Engineered Cardiac Microtissues,” Tissue Eng. Part A, 18(9–10), pp. 910–919. [CrossRef]
Legant, W. R., Pathak, A., Yang, M. T., Deshpande, V. S., Mcmeeking, R. M., and Chen, C. S., 2009, “Microfabricated Tissue Gauges to Measure and Manipulate Forces From 3D Microtissues,” Proc. Natl. Acad. Sci. USA, 106(25), pp. 10097–10102. [CrossRef]
Kim, K., Taylor, R., Sim, J. Y., Park, S. J., Norman, J., Fajardo, G., Bernstein, D., and Pruitt, B. L., 2011, “Calibrated Micropost Arrays for Biomechanical Characterisation of Cardiomyocytes,” Micro Nano Lett., 6(5), pp. 317–322. [CrossRef]
Zhao, Y., Lim, C. C., Sawyer, D. B., Liao, R. L., and Zhang, X., 2005, “Cellular Force Measurements Using Single-Spaced Polymeric Microstructures: Isolating Cells From Base Substrate,” J. Micromech. Microeng., 15(9), pp. 1649–1656. [CrossRef]
Zhao, Y., and Zhang, X., 2006, “Cellular Mechanics Study in Cardiac Myocytes Using PDMS Pillars Array,” Sens. Actuators, A, 125(2), pp. 398–404. [CrossRef]
Rodriguez, A. G., Han, S. J., Regnier, M., and Sniadecki, N. J., 2011, “Substrate Stiffness Increases Twitch Power of Neonatal Cardiomyocytes in Correlation With Changes in Myofibril Structure and Intracellular Calcium,” Biophys. J., 101(10), pp. 2455–2464. [CrossRef] [PubMed]
Rodriguez, A. G., Rodriguez, M. L., Han, S. J., Sniadecki, N. J., and Regnier, M., 2013, “Enhanced Contractility With 2 Deoxy-ATP and EMD 57033 Leads to Reduced Myofibril Structure and Twitch Power in Neonatal Cardiomyocytes,” Integr. Biol., 5(11), pp. 1366–1373. [CrossRef]
Taylor, R. E., Kim, K., Sun, N., Park, S. J., Sim, J. Y., Fajardo, G., Bernstein, D., Wu, J. C., and Pruitt, B. L., 2013, “Sacrificial Layer Technique for Axial Force Post Assay of Immature Cardiomyocytes,” Biomed. Microdevices, 15(1), pp. 171–181. [CrossRef] [PubMed]
Terracio, L., Rubin, K., Gullberg, D., Balog, E., Carver, W., Jyring, R., and Borg, T. K., 1991, “Expression of Collagen Binding Integrins During Cardiac Development and Hypertrophy,” Circ. Res., 68(3), pp. 734–744. [CrossRef] [PubMed]
Lundgren, E., Terracio, L., Mardh, S., and Borg, T. K., 1985, “Extracellular Matrix Components Influence the Survival of Adult Cardiac Myocytes in vitro,” Exp. Cell Res., 158(2), pp. 371–381. [CrossRef] [PubMed]
Lundgren, E., Terracio, L., and Borg, T. K., 1985, “Adhesion of Cardiac Myocytes to Extracellular Matrix Components,” Basic Res. Cardiol., 80(Suppl 1), pp. 69–74. [PubMed]
Tan, J. L., Tien, J., Pirone, D. M., Gray, D. S., Bhadriraju, K., and Chen, C. S., 2003, “Cells Lying on a Bed of Microneedles: An Approach to Isolate Mechanical Force,” Proc. Natl. Acad. Sci. USA, 100(4), pp. 1484–1489. [CrossRef]
Sniadecki, N. J., and Chen, C. S., 2007, “Microfabricated Silicone Elastomeric Post Arrays for Measuring Traction Forces of Adherent Cells,” Methods in Cell Biology: Cell Mechanics, Elsevier Inc., San Diego, CA.
Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I., and Thomson, J. A., 2007, “Induced Pluripotent Stem Cell Lines Derived From Human Somatic Cells,” Science, 318(5858), pp. 1917–1920. [CrossRef] [PubMed]
Laflamme, M. A., Chen, K. Y., Naumova, A. V., Muskheli, V., Fugate, J. A., Dupras, S. K., Reinecke, H., Xu, C., Hassanipour, M., Police, S., O'Sullivan, C., Collins, L., Chen, Y., Minami, E., Gill, E. A., Ueno, S., Yuan, C., Gold, J., and Murry, C. E., 2007, “Cardiomyocytes Derived From Human Embryonic Stem Cells in Pro-Survival Factors Enhance Function of Infarcted Rat Hearts,” Nat. Biotechnol., 25(9), pp. 1015–1024. [CrossRef] [PubMed]
Han, S. J., Bielawski, K. S., Ting, L. H., Rodriguez, M. L., and Sniadecki, N. J., 2012, “Decoupling Substrate Stiffness, Spread Area, and Micropost Density: A Close Spatial Relationship Between Traction Forces and Focal Adhesions,” Biophys. J., 103(4), pp. 640–648. [CrossRef] [PubMed]
Kresh, J. Y., and Chopra, A., 2011, “Intercellular and Extracellular Mechanotransduction in Cardiac Myocytes,” Pflugers Arch., 462(1), pp. 75–87. [CrossRef] [PubMed]
Parker, K. K., and Ingber, D. E., 2007, “Extracellular Matrix, Mechanotransduction and Structural Hierarchies in Heart Tissue Engineering,” Philos. Trans. R. Soc., B, 362(1484), pp. 1267–1279. [CrossRef]
Wu, X., Sun, Z., Foskett, A., Trzeciakowski, J. P., Meininger, G. A., and Muthuchamy, M., 2010, “Cardiomyocyte Contractile Status Is Associated With Differences in Fibronectin and Integrin Interactions,” Am. J. Physiol.: Heart Circ. Physiol., 298(6), pp. H2071–H2081. [CrossRef] [PubMed]
Prowse, A. B., Chong, F., Gray, P. P., and Munro, T. P., 2011, “Stem Cell Integrins: Implications for ex-Vivo Culture and Cellular Therapies,” Stem Cell Res., 6(1), pp. 1–12. [CrossRef] [PubMed]
Ross, R. S., and Borg, T. K., 2001, “Integrins and the Myocardium,” Circ. Res., 88(11), pp. 1112–1119. [CrossRef] [PubMed]
Maitra, N., Flink, I. L., Bahl, J. J., and Morkin, E., 2000, “Expression of Alpha and Beta Integrins During Terminal Differentiation of Cardiomyocytes,” Cardiovasc. Res., 47(4), pp. 715–725. [CrossRef] [PubMed]
Van Laake, L. W., Van Donselaar, E. G., Monshouwer-Kloots, J., Schreurs, C., Passier, R., Humbel, B. M., Doevendans, P. A., Sonnenberg, A., Verkleij, A. J., and Mummery, C. L., 2010, “Extracellular Matrix Formation After Transplantation of Human Embryonic Stem Cell-Derived Cardiomyocytes,” Cell. Mol. Life Sci., 67(2), pp. 277–290. [CrossRef] [PubMed]
Wang, I. N., Wang, X., Ge, X., Anderson, J., Ho, M., Ashley, E., Liu, J., Butte, M. J., Yazawa, M., Dolmetsch, R. E., Quertermous, T., and Yang, P. C., 2012, “Apelin Enhances Directed Cardiac Differentiation of Mouse and Human Embryonic Stem Cells,” PLoS One, 7(6), e38328. [CrossRef] [PubMed]
Kita-Matsuo, H., Barcova, M., Prigozhina, N., Salomonis, N., Wei, K., Jacot, J. G., Nelson, B., Spiering, S., Haverslag, R., Kim, C., Talantova, M., Bajpai, R., Calzolari, D., Terskikh, A., Mcculloch, A. D., Price, J. H., Conklin, B. R., Chen, H. S., and Mercola, M., 2009, “Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes,” PLoS One, 4(4), e5046. [CrossRef] [PubMed]
Sun, N., Yazawa, M., Liu, J., Han, L., Sanchez-Freire, V., Abilez, O. J., Navarrete, E. G., Hu, S., Wang, L., Lee, A., Pavlovic, A., Lin, S., Chen, R., Hajjar, R. J., Snyder, M. P., Dolmetsch, R. E., Butte, M. J., Ashley, E. A., Longaker, M. T., Robbins, R. C., and Wu, J. C., 2012, “Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Dilated Cardiomyopathy,” Sci. Transl. Med., 4(130), 130ra47. [CrossRef] [PubMed]
Lin, G., Pister, K. S. J., and Roos, K. P., 2000, “Surface Micromachined Polysilicon Heart Cell Force Transducer,” J. Microelectromech. Syst., 9(1), pp. 9–17. [CrossRef]
Yasuda, S. I., Sugiura, S., Kobayakawa, N., Fujita, H., Yamashita, H., Katoh, K., Saeki, Y., Kaneko, H., Suda, Y., Nagai, R., and Sugi, H., 2001, “A Novel Method to Study Contraction Characteristics of a Single Cardiac Myocyte Using Carbon Fibers,” Am. J. Physiol.: Heart Circ. Physiol., 281(3), pp. H1442–H1446. [PubMed]
Granzier, H. L., and Irving, T. C., 1995, “Passive Tension in Cardiac Muscle: Contribution of Collagen, Titin, Microtubules, and Intermediate Filaments,” Biophys. J., 68(3), pp. 1027–1044. [CrossRef] [PubMed]
Kass, D. A., Bronzwaer, J. G. F., and Paulus, W. J., 2004, “What Mechanisms Underlie Diastolic Dysfunction in Heart Failure?,” Circ. Res., 94(12), pp. 1533–1542. [CrossRef] [PubMed]
Hamdani, N., Kooij, V., Van Dijk, S., Merkus, D., Paulus, W. J., Dos Remedios, C., Duncker, D. J., Stienen, G. J. M., and Van Der Velden, J., 2008, “Sarcomeric Dysfunction in Heart Failure,” Cardiovasc. Res., 77(4), pp. 649–658. [CrossRef] [PubMed]
Shinozawa, T., Imahashi, K., Sawada, H., Furukawa, H., and Takami, K., 2012, “Determination of Appropriate Stage of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Screening and Pharmacological Evaluation in Vitro,” J. Biomol. Screen, 17(9), pp. 1192–1203. [CrossRef] [PubMed]
Korte, F. S., and McDonald, K. S., 2007, “Sarcomere Length Dependence of Rat Skinned Cardiac Myocyte Mechanical Properties: Dependence on Myosin Heavy Chain,” J. Physiol., 581(Pt 2), pp. 725–739. [CrossRef] [PubMed]
Spach, M. S., Heidlage, J. F., Barr, R. C., and Dolber, P. C., 2004, “Cell Size and Communication: Role in Structural and Electrical Development and Remodeling of the Heart,” Heart Rhythm, 1(4), pp. 500–515. [CrossRef] [PubMed]
Feinberg, A. W., Alford, P. W., Jin, H., Ripplinger, C. M., Werdich, A. A., Sheehy, S. P., Grosberg, A., and Parker, K. K., 2012, “Controlling the Contractile Strength of Engineered Cardiac Muscle by Hierarchal Tissue Architecture,” Biomaterials, 33(23), pp. 5732–5741. [CrossRef] [PubMed]
Lundy, S. D., Zhu, W. Z., Regnier, M., and Laflamme, M. A., 2013, “Structural and Functional Maturation of Cardiomyocytes Derived From Human Pluripotent Stem Cells,” Stem Cells Dev., 22(14), pp. 1991–2002. [CrossRef] [PubMed]
Gordon, A. M., Homsher, E., and Regnier, M., 2000, “Regulation of Contraction in Striated Muscle,” Physiol. Rev., 80(2), pp. 853–924. [PubMed]
Campbell, K. S., 2009, “Interactions Between Connected Half-Sarcomeres Produce Emergent Mechanical Behavior in a Mathematical Model of Muscle,” PLoS Comput. Biol., 5(11), e1000560. [CrossRef] [PubMed]
Walker, C. A., and Spinale, F. G., 1999, “The Structure and Function of the Cardiac Myocyte: A Review of Fundamental Concepts,” J. Thorac. Cardiovasc. Surg., 118(2), pp. 375–382. [CrossRef] [PubMed]
Olivetti, G., Cigola, E., Maestri, R., Corradi, D., Lagrasta, C., Gambert, S. R., and Anversa, P., 1996, “Aging, Cardiac Hypertrophy and Ischemic Cardiomyopathy Do Not Affect the Proportion of Mononucleated and Multinucleated Myocytes in the Human Heart,” J. Mol. Cell. Cardiol., 28(7), pp. 1463–1477. [CrossRef] [PubMed]

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Grahic Jump Location
Fig. 1

Technique the twitch force, velocity, and power of a single hiPSC-CM is determined by seeding cells onto a micropost array, selecting a beating cell of interest, and then taking a high-speed video of the post tips, as well as a reference image of the post bases (a). This video is taken in the phase contrast setting (b), while the reference image is taken in the red fluorescent channel (c). A custom matlab code is then used to determine the location of each post’s centroid in the reference plane (plus sign), as well as it is used to track the location of the post’s centroid in the video plane (d). The difference in location between these two centroids over time gives the deflection of the post over multiple twitch events, which can be multiplied by the post stiffness to yield the twitch force (e). Here, the dashed line represents the passive tension measured for this post. This deflection data can then be used to determine the twitch velocity (f), and power (g) of that post. Scale bars represent 6 μm.

Grahic Jump Location
Fig. 2

Twitch force representative images of hiPSC-CMs on fibronectin (a), laminin (b), and collagen IV (c) at peak twitch force indicate that the highest forces are present along the edges of the cell, and that the majority of the cell’s force is directed towards the cell center. Here, the arrows indicate the direction and magnitude of the force at each post. When the magnitude of these individual force vectors are summed, the total force produced by the cell can be plotted over multiple twitch cycles. Representative force traces for hiPSC-CMs on fibronectin (d), laminin (e), and collagen IV (f) demonstrate that the micropost array platform is capable of capturing these twitch cycles with high temporal resolution. Here, the text on the left of the graph indicates the twitch force, while that on the right indicates total force, and the dashed line indicates the passive force produced by the cell. Quantification of the passive force (g), maximum twitch force (h), and force per area (i), for hiPSC-CMs on different ECM proteins revealed that the quantities are statistically similar across all three conditions. Additionally, there was no significant difference in the spontaneous beating rate of the cells on the three different ECM proteins (j). Scale bar represents 6 μm and scale arrow indicates 6 nN.

Grahic Jump Location
Fig. 3

Twitch velocity representative maximum velocity traces for hiPSC-CMs on fibronectin (a), laminin (b), and collagen IV (c) indicate that the micropost platforms is capable of capturing both the contraction and relaxation velocity produced by spontaneously beating cells. Quantification of these traces revealed higher overall contraction and relaxation velocities for cells on collagen IV, but no significant difference between any of the treatments (d) and (e).

Grahic Jump Location
Fig. 4

Twitch power representative power traces for cells seeded on fibronectin (a), laminin (b), and collagen IV (c) demonstrate the ability of this technique to effectively resolve the contraction and relaxation power produced by hiPSC-CMs. Quantification of these power traces revealed higher overall contraction and relaxation velocities for cells on collagen IV, but no significant difference between any of the treatments (d) and (e).

Grahic Jump Location
Fig. 5

Maturation Immunofluorescent imaging of hiPSC-CMs seeded onto fibronectin (a), laminin (b), and collagen IV (c) coated microposts revealed punctate, unorganized sarcomeres within cells on fibronectin and collagen IV post, and more highly organized sarcomere structure within cells on laminin posts. Here, α-actinin is indicated by green, the cell nuclei is blue, and the microposts are red. The figure inset demonstrates how measurements of sarcomere length (dark dashed line) and Z-band width (light dashed line) were performed, Cell attachment (d) and spread area (e) are significantly higher on laminin posts than fibronectin posts, as well as attachment is significantly higher on laminin when compared to collagen IV. The circularity (f), Z-band widths (g), sarcomere lengths (h), and percentage of multinucleated cells (i) of the cells was not significantly different based on treatment. Color figures are available in the online version of this publication. Scale bar indicates 6 μm.

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