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Cardiomyocyte subdomain contractility arising from microenvironmental stiffness and topography

Abstract

Cellular structure and function are interdependent. To understand this relationship in beating heart cells, individual neonatal rat ventricular myocytes (NRVMs) were analyzed one and 3 days after plating when cultured on different stiffness (100, 400 kPa) and surface structures (flat or \(15\,\upmu \hbox {m}\) high, \(15\,\upmu \hbox {m}\) diameter, microposts spaced \(75 \,\upmu \hbox {m}\) apart) manufactured from polydimethylsiloxane. Myofibril structure seen by immunohistochemistry was organized in three dimensions when NRVMs were attached to microposts. On day three, paxillin distribution near the post serving as cellular anchorage was quantified on both soft posts (12.04 % of total voxel count) and stiff posts (8.16 %). Living NRVMs were analyzed using line scans for sarcomeric shortening and shortening velocity, and traction force microscopy for surface stress and surface tension. One day after plating, NRVMs shortened more on soft posts (\(1.08\,\upmu \hbox {m}\) at \(4.75 \,\upmu \hbox {m}/\hbox {s}\)) compared to either soft flat (\(0.84 \,\upmu \hbox {m}\) at \(3.41 \,\upmu \hbox {m}/\hbox {s}\)), stiff posts (\(0.66 \,\upmu \hbox {m}\) at \(2.88 \,\upmu \hbox {m}/\hbox {s}\)) or stiff flat (\(0.48 \,\upmu \hbox {m}\) at \(1.44 \,\upmu \hbox {m}/\hbox {s}\)). NRVMs have decreased shortening and shortening velocity on soft posts (\(1.04 \,\upmu \hbox {m}\) at \(3.85 \,\upmu \hbox {m}/\hbox {s}\)) compared to soft flat (\(0.72 \,\upmu \hbox {m}\) at \(2.36 \,\upmu \hbox {m}/\hbox {s}\)) substrates. The surface stress and surface tension increased over time for both soft post (\(29.12\,\hbox {kN}/\hbox {m}^{2}\) and \(30.10\,\upmu \hbox {N}/\hbox {mm}\) to \(42.87\,\hbox {kN}/\hbox {m}^{2}\) and \(51.68 \,\upmu \hbox {N}/\hbox {mm}\)) and flat (\(15.36\,\hbox {kN}/\hbox {m}^{2}\) and \(19.00\,\upmu \hbox {N}/\hbox {mm}\) to \(32.87\,\hbox {kN}/\hbox {m}^{2}\) and \(37.38\,\upmu \hbox {N}/\hbox {mm}\)) substrates. Paxillin displacement during contraction on day three was significantly greater in NRVMs attached to soft posts \((1.39\,\upmu \hbox {m})\) compared to flat \((1.16\,\upmu \hbox {m})\) substrates. The volume and time creating four-dimensional data, interpreted by structural engineering theory, demonstrate subdomain structure is maintained by the counterbalance between the external load acting upon and the internal forces generated by the cardiomyocyte. These findings provide further insight into localized regulation of cellular mechanical function.

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References

  1. Aratyn-Schaus Y, Oakes PW, Stricker J, Winter SP, Gardel ML (2010) Preparation of complaint matrices for quantifying cellular contraction. J Vis Exp 46(Pii):2173

  2. Berk BC, Fujiwara K, Lehoux S (2007) ECM remodeling in hypertensive heart disease. J Clin Invest 117(3):568–575

  3. Berry MF, Engler AJ, Woo YJ, Pirolli TJ, Bish LT, Jayasankar V, Morine KJ, Gardner TJ, Discher DE, Sweeney HL (2006) Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance. Am J Physiol Heart Circ Physiol 290(6):H2196–H2203

  4. Bhana B, Iyer RK, Chen WL, Zhao R, Sider KL, Likhitpanichkul M, Simmons CA, Radisic M (2010) Influence of substrate stiffness on the phenotype of heart cells. Biotechnol Bioeng 105(6):1148–1160

  5. Boateng SY, Hartman TJ, Ahluwalia N, Vidula H, Desai TA, Russell B (2003) Inhibition of fibroblast proliferation in cardiac myocyte cultures by surface microtopography. Am J Physiol Cell Physiol 285(1):C171–C182

  6. Borbely A, van der Velden J, Papp Z, Bronzwaer JGF, Edes I, Stienen GJM, Paulus WJ (2005) Cardiomyocyte stiffness in diastolic heart failure. Circulation 111:774–781

  7. Buck D, Smith JE III, Chung CS, Ono Y, Sorimachi H, Labeit S, Granzier HL (2014) Removal of immunoglobulin-like domains from titin’s spring segment alters titin splicing in mouse skeletal muscle and causes myopathy. J Gen Physiol 143(2):215–230

  8. Chapin LM, Edgar LT, Blankman E, Beckerle MC, Shiu YT (2014) Mathematical modeling of the dynamic mechanical behavior of neighboring sarcomeres in actin stress fibers. Cell Mol Bioeng 7(1):73–85

  9. Cheng H, Lederer WJ (2008) Calcium sparks. Physiol Rev 88(4):1491–1545

  10. Chu M, Iyengar R, Koshman YE, Kim T, Russell B, Martin JL, Heroux AL, Robia SL, Samarel AM (2011) Serine-910 phosphorylation of focal adhesion kinase is critical for sarcomere reorganization in cardiomyocyte hypertrophy. Cardiovasc Res 92:409–419

  11. Curtis MW, Budyn E, Desai TA, Samarel AM, Russell B (2013) Microdomain heterogeneity in 3D affects the mechanics of neonatal cardiac myocyte contraction. Biomech Model Mechanobiol 12(1):95–109

  12. Discher DE, Janmey P, Wang YL (2005) Tissue cells feel and respond to the stiffness of their substrate. Science 310:1139–1143

  13. Engler AJ, Carag-Krieger C, Johnson CP, Raab M, Tang HY, Speicher DW, Sanger JW, Sanger JM, Discher DE (2008) Embryonic cardiomyocytes beat best on a matrix with heartlike elasticity: scar-like rigidity inhibits beating. J Cell Sci 121(Pt 22):3794–3802

  14. Fomovsky GM, Holmes JW (2010) Evolution of scar structure, mechanics, and ventricular function after myocardial infarction in the rat. Am J Physiol Heart Circ Physiol 298(1):H221–H228

  15. Fomovsky GM, Thomopoulos S, Holmes JW (2010) Contribution of extracellular matrix to the mechanical properties of the heart. J Mol Cell Cardiol 48(3):490–496

  16. Go AS, Mozaffarina D, Roger VL, Benjamin EJ, Berry JD, Blaha MJ, Stroke SS (2014) Heart disease and stroke statistics—2014 update: a report from the American Heart Association. Circulation 129(3):e28

  17. Hanft LM, Korte FS, McDonald KS (2008) Cardiac function and modulation of sarcomeric function by length. Cardiovasc Res 77(4):627–636

  18. Hazeltine LB, Simmons CS, Salick MR, Lian X, Badur MG, Han W, Delgado SM, Wakatsuki T, Crone WC, Pruitt BL, Palecek SP (2012) Effects of substrate mechanics on contractility of cardiomyocytes generated from human pluripotent stem cells. Int J Cell Biol 2012:508294

  19. Hersh N, Wolters B, Dreissen G, Springer R, Kirchgebner N, Merkel R, Hoffman B (2013) The constant beat: cardiomyocytes adapt their forces by equal contraction upon environmental stiffening. Biol Open 2(3):351–361

  20. Heyman J (2008) Basic structural theory. Cambridge University Press, Cambridge

  21. Holmes JW, Borg TK, Covell JW (2005) Structure and mechanics of healing myocardial infarcts. Annu Rev Biomed Eng 7:223–253

  22. Jacot JG, McCulloch AD, Omens JH (2008) Substrate stiffness affects the functional maturation of neonatal rat ventricular myocytes. Biophys J 95(7):3479–3487

  23. Lateef SS, Boateng S, Ahluwalia N, Hartman TJ, Russell B, Hanley L (2005) Three-dimensional chemical structures by protein functionalized micron-sized beads bound to polylysine-coated silicone surfaces. J Biomed Mater Res A 72(4):373–380

  24. Lewinter MM, Granzier HL (2014) Cardiac titin and heart disease. J Cardiovasc Pharmacol 63(3):207–212

  25. Melendez J, Turner C, Avraham H, Steinberg SF, Schaefer E, Sussman MA (2004) Cardiomyocyte apoptosis triggered by RAFTK/pyk2 via Src kinase is antagonized by paxillin. J Biol Chem 279(51):53516–53523

  26. Motlagh D, Senyo SE, Desai TA, Russell B (2003) Microtextured substrata alter gene expression, protein localization and the shape of cardiac myocytes. Biomaterials 24(14):2463–2476

  27. Oakes PW, Beckham Y, Stricker J, Gardel ML (2012) Tension is required but not sufficient for focal adhesion maturation without a stress fiber template. J Cell Biol 196(3):363–374

  28. Omens JH (1998) Stress and strain as regulators of myocardial growth. Prog Biophys Mol Biol 69(2–3):559–572

  29. Pelham RJ, Wang YL (1997) Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci 94:13661–13665

  30. Rodriquez AG, Han SJ, Regnier M, Sniadecki NJ (2011) Substrate stiffness increases twitch power of neonatal cardiomyocytes in correlation with changes in myofibril structure and intracellular calcium. Biophys J 101(10):2455–2464

  31. Russell B, Curtis MW, Koshman YE, Samarel AM (2010) Mechanical stress-induced sarcomere assembly for cardiac muscle growth in length and width. J Mol Cell Cardiol 48(5):817–823

  32. Samarel AM (2005) Costameres, focal adhesions, and cardiomyocyte mechanotransduction. Am J Physiol Heart Circ Physiol 289(6):H2291–H2301

  33. Solaro RJ, deTombe P (2008) Review focus series: sarcomeric proteins as key elements in integrated control of cardiac function. Cardiovasc Res 77(4):616–618

  34. Wang PY, Yu J, Lin JH, Tsai WB (2011) Modulation of alignment, elongation and contraction of cardiomyocyte through a combination of nanotopography and rigidity of substrates. Acta Biomater 7(9):3285–3293

  35. Williams CD, Salcedo MK, Irving TC, Regnier M, Daniel TL (2013) The length-tension curve in muscle depends on lattice spacing. Proc Biol Sci 280(1776):20130697

  36. Yu J, Russell B (2005) Cardiomyocyte remodeling and sarcomere addition after uniaxial static strain in vitro. J Histochem Cytochem 53(7):839–844

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Acknowledgments

We gratefully acknowledge Drs Shen Sun and Michael Cho for providing assistance with AFM experiments. Gratitude is also extended to Dr. Mark Sussman for providing paxillin-GFP used in these experiments. We also thank Dr. Matthew W. Curtis for his help to customize the method of adhering beads to the substratum surface for the traction force microscopy experiments. Support to conduct this research was provided by NIH NHLBI T32/HL07692, PO/HL62426.

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Correspondence to Brenda Russell.

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Broughton, K.M., Russell, B. Cardiomyocyte subdomain contractility arising from microenvironmental stiffness and topography. Biomech Model Mechanobiol 14, 589–602 (2015) doi:10.1007/s10237-014-0624-2

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Keywords

  • 4D Imaging
  • Anisotropic elastic deformation
  • Kymograph
  • Shortening
  • Shortening velocity
  • Traction force
  • Surface stress
  • Surface tension