Biomechanics and Modeling in Mechanobiology

, Volume 12, Issue 1, pp 95–109 | Cite as

Microdomain heterogeneity in 3D affects the mechanics of neonatal cardiac myocyte contraction

  • Matthew W. Curtis
  • Elisa Budyn
  • Tejal A. Desai
  • Allen M. Samarel
  • Brenda RussellEmail author
Original Paper


Cardiac muscle cells are known to adapt to their physical surroundings, optimizing intracellular organization and contractile function for a given culture environment. A previously developed in vitro model system has shown that the inclusion of discrete microscale domains (or microrods) in three dimensions (3D) can alter long-term growth responses of neonatal ventricular myocytes. The aim of this work was to understand how cellular contact with such a domain affects various mechanical changes involved in cardiac muscle cell remodeling. Myocytes were maintained in 3D gels over 5 days in the presence or absence of 100−μm-long microrods, and the effect of this local heterogeneity on cell behavior was analyzed via several imaging techniques. Microrod abutment resulted in approximately twofold increases in the maximum displacement of spontaneously beating myocytes, as based on confocal microscopy scans of the gel xy-plane or the myocyte long axis. In addition, microrods caused significant increases in the proportion of aligned myofibrils (≤20° deviation from long axis) in fixed myocytes. Microrod-related differences in axial contraction could be abrogated by long-term interruption of certain signals of the RhoA-/Rho-associated kinase (ROCK) or protein kinase C (PKC) pathway. Furthermore, microrod-induced increases in myocyte size and protein content were prevented by ROCK inhibition. In all, the data suggest that microdomain heterogeneity in 3D appears to promote the development of axially aligned contractile machinery in muscle cells, an observation that may have relevance to a number of cardiac tissue engineering interventions.


Mechanobiology Muscle hypertrophy Digital image correlation Microenvironment Finite element Mechanotransduction 



Three dimensions


Cytosine β-D-arabino-furanoside


Bovine serum albumin




Dulbecco’s modified Eagle’s medium


Dimethyl sulfoxide


Enhanced chemiluminescence


Extracellular matrix


Glyceraldehyde-3-phosphate dehydrogenase


Horseradish peroxidase


Myosin light chain


Myosin light chain kinase


Myosin light chain phosphatase


Phosphate-buffered saline


Protein kinase C


Polyvinylidene fluoride


Receptor for activated C-kinase-1


Rho-associated kinase


Standard error of measurement


Tris-buffered saline-Tween 20


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Supplementary material

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  1. Ayala P, Lopez JI, Desai TA (2010) Microtopographical Cues in 3D attenuate fibrotic phenotype and extracellular matrix deposition: implications for tissue regeneration. Tissue Eng Part A 16(8): 2519–2527CrossRefGoogle Scholar
  2. Ayala P, Desai TA (2011) Integrin α3 blockade enhances microtopographical down-regulation of α-smooth muscle actin: role of microtopography in ECM regulation. Integr Biol (Camb) 3(7): 733–741CrossRefGoogle Scholar
  3. Bakunts K, Gillum N, Karabekian Z, Sarvazyan N (2008) Formation of cardiac fibers in Matrigel matrix. Biotechniques 44(3): 341–348CrossRefGoogle Scholar
  4. Besson A, Wilson TL, Yong VW (2002) The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility. J Biol Chem 277(24): 22073–22084CrossRefGoogle Scholar
  5. Bian W, Liau B, Badie N, Bursac N (2009) Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues. Nat Protoc 4(10): 1522–1534CrossRefGoogle Scholar
  6. Bischofs IB, Schwarz US (2003) Cell organization in soft media due to active mechanosensing. Proc Natl Acad Sci USA 100(16): 9274–9279CrossRefGoogle Scholar
  7. Bray MA, Sheehy SP, Parker KK (2008) Sarcomere alignment is regulated by myocyte shape. Cell Motil Cytoskeleton 65(8): 641–651CrossRefGoogle Scholar
  8. Budyn E, Hoc T (2010) Analysis of micro fracture in human haversian cortical bone under transverse tension using extended physical imaging. Int J Numer Meth Eng 82(8): 940–965zbMATHCrossRefGoogle Scholar
  9. Chopra A, Tabdanov E, Patel H, Janmey PA, Kresh JY (2011) Cardiac myocyte remodeling mediated by N-cadherin-dependent mechanosensing. Am J Physiol Heart Circ Physiol 300(4): H1252–H1266CrossRefGoogle Scholar
  10. Collins JM, Ayala P, Desai TA, Russell B (2010) Three-dimensional culture with stiff microstructures increases proliferation and slows osteogenic differentiation of human mesenchymal stem cells. Small 6(3): 355–360CrossRefGoogle Scholar
  11. Cukierman E, Pankov R, Stevens DR, Yamada KM (2001) Taking cell-matrix adhesions to the third dimension. Science 294(5547): 1708–1712CrossRefGoogle Scholar
  12. Curtis MW, Sharma S, Desai TA, Russell B (2010) Hypertrophy, gene expression, and beating of neonatal cardiac myocytes are affected by microdomain heterogeneity in 3D. Biomed Microdev 12(6): 1073–1085CrossRefGoogle Scholar
  13. DiMichele LA, Hakim ZS, Sayers RL, Rojas M, Schwartz RJ, Mack CP, Taylor JM (2009) Transient expression of FRNK reveals stage-specific requirement for focal adhesion kinase activity in cardiac growth. Circ Res 104(10): 1201–1208CrossRefGoogle Scholar
  14. Doumalin P, Bornert M, Caldemaison D (1999) Microextensometry by image correlation applied to micromechanical studies using the scanning electron microscopy. In: Proceedings of the international conference on advanced technology in experimental mechanics, Japan Society of Experimental Engineering, pp 81–86Google Scholar
  15. Doumalin P (2000) Microextensométrie locale par corrélation d’images numériques. Dissertation, Ecole PolytechniqueGoogle Scholar
  16. Doumalin P, Bornert M, Crepin J (2003) Characterization of the strain distribution in heterogeneous materials. Mecan Ind 6: 607–617CrossRefGoogle Scholar
  17. Eble DM, Qi M, Waldschmidt S, Lucchesi PA, Byron KL, Samarel AM (1998) Contractile activity is required for sarcomeric assembly in phenylephrine-induced cardiac myocyte hypertrophy. Am J Physiol 274(5 Pt 1): C1226–C1237Google Scholar
  18. 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 heart-like elasticity: scar-like rigidity inhibits beating. J Cell Sci 121(Pt 22): 3794–3802CrossRefGoogle Scholar
  19. Evans HJ, Sweet JK, Price RL, Yost M, Goodwin RL (2003) Novel 3D culture system for study of cardiac myocyte development. Am J Physiol Heart Circ Physiol 285(2): H570–H578Google Scholar
  20. Feng Z, Matsumoto T, Nakamura T (2003) Measurements of the mechanical properties of contracted collagen gels populated with rat fibroblasts or cardiomyocytes. J Artif Organs 6(3): 192–196CrossRefGoogle Scholar
  21. Frey N, Olson EN (2003) Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev Physiol 65(1): 45–79CrossRefGoogle Scholar
  22. Geisse NA, Sheehy SP, Parker KK (2009) Control of myocyte remodeling in vitro with engineered substrates. In Vitro Cell Dev Biol Anim 45(7): 343–350CrossRefGoogle Scholar
  23. Geuzaine C, Remacle JF (2009) Gmsh: a three-dimensional finite element mesh generator with built-in pre- and post-processing facilities. Int J Numer Methods Eng 79(11): 1309–1331MathSciNetzbMATHCrossRefGoogle Scholar
  24. Goldyn AM, Rioja BA, Spatz JP, Ballestrem C, Kemkemer R (2009) Force-induced cell polarisation is linked to RhoA-driven microtubule-independent focal-adhesion sliding. J Cell Sci 122 (Pt 20): 3644–3651CrossRefGoogle Scholar
  25. Gopalan SM, Flaim C, Bhatia SN, Hoshijima M, Knoell R, Chien KR, Omens JH, McCulloch AD (2003) Anisotropic stretch-induced hypertrophy in neonatal ventricular myocytes micropatterned on deformable elastomers. Biotechnol Bioeng 81(5): 578–587CrossRefGoogle Scholar
  26. Grinnell F. (2000) Fibroblast-collagen-matrix contraction: growth-factor signalling and mechanical loading. Trends Cell Biol 10(9): 362–365CrossRefGoogle Scholar
  27. Grosberg A, Kuo PL, Guo CL, Geisse NA, Bray MA, Adams WJ, Sheehy SP, Parker KK (2011) Self-organization of muscle cell structure and function. PLoS Comput Biol 7(2): e1001088CrossRefGoogle Scholar
  28. Hartman TJ, Martin JL, Solaro RJ, Samarel AM, Russell B (2009) CapZ dynamics are altered by endothelin-1 and phenylephrine via PIP2 and PKC-dependent mechanisms. AJP Cell 296(5): C1034–C1039CrossRefGoogle Scholar
  29. Heidkamp MC, Bayer AL, Scully BT, Eble DM, Samarel AM (2003) Activation of focal adhesion kinase by protein kinase C epsilon in neonatal rat ventricular myocytes. Am J Physiol Heart Circ Physiol 285(4): H1684–H1696Google Scholar
  30. Isemura M, Mita T, Satoh K, Narumi K, Motomiya M (1991) Myosin light chain kinase inhibitors ML-7 and ML-9 inhibit mouse lung carcinoma cell attachment to the fibronectin substratum. Cell Biol Int Rep 15(10): 965–972CrossRefGoogle Scholar
  31. Jacot JG, McCulloch AD, Omens JH (2008) Substrate stiffness affects the functional maturation of neonatal rat ventricular myocytes. Biophys J 95(7): 3479–3487CrossRefGoogle Scholar
  32. Kajzar A, Cesa CM, Kirchgessner N, Hoffmann B, Merkel R (2008) Toward physiological conditions for cell analyses: forces of heart muscle cells suspended between elastic micropillars. Biophys J 94(5): 1854–1866CrossRefGoogle Scholar
  33. Kamgoué A, Ohayon J, Usson Y, Riou L, Tracqui P (2009) Quantification of cardiomyocyte contraction based on image correlation analysis. Cytometry A 75(4): 298–308Google Scholar
  34. Karlon WJ, Hsu PP, Li S, Chien S, McCulloch AD, Omens JH (1999) Measurement of orientation and distribution of cellular alignment and cytoskeletal organization. Ann Biomed Eng 27(6): 712–720CrossRefGoogle Scholar
  35. Kaunas R, Nguyen P, Usami S, Chien S (2005) Cooperative effects of Rho and mechanical stretch on stress fiber organization. Proc Natl Acad Sci USA 102(44): 15895–15900CrossRefGoogle Scholar
  36. Liu WF, Chen CS (2007) Cellular and multicellular form and function. Adv Drug Deliv Rev 59(13): 1319–1328CrossRefGoogle Scholar
  37. Miyamoto S, Del Re DP, Xiang SY, Zhao X, Florholmen G, Brown JH (2010) Revisited and revised: is RhoA always a villain in cardiac pathophysiology?. J Cardiovasc Transl Res 3(4): 330–343CrossRefGoogle Scholar
  38. Mochly-Rosen D, Khaner H, Lopez J (1991) Identification of intracellular receptor proteins for activated protein kinase C. Proc Natl Acad Sci USA 88(9): 3997–4000CrossRefGoogle Scholar
  39. Moorman AF, Vermeulen JL, Koban MU, Schwartz K, Lamers WH, Boheler KR (1995) Patterns of expression of sarcoplasmic reticulum Ca(2+)-ATPase and phospholamban MRNAs during rat heart development. Circ Res 76(1): 616–625CrossRefGoogle Scholar
  40. Motlagh D, Hartman TJ, Desai TA, Russell B (2003) Microfabricated grooves recapitulate neonatal myocyte connexin43 and N-cadherin expression and localization. J Biomed Mater Res A 67(1): 148–157CrossRefGoogle Scholar
  41. 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): 2463–2476CrossRefGoogle Scholar
  42. Norman JJ, Collins JM, Sharma S, Russell B, Desai TA (2008) Microrods in 3D biological gels affect cell proliferation. Tissue Eng Part A 14(3): 379–390CrossRefGoogle Scholar
  43. Okada J, Sugiura S, Nishimura S, Hisada T (2005) Three-dimensional simulation of calcium waves and contraction in cardiomyocytes using the finite element method. Am J Physiol Cell Physiol 288(3): C510–C522CrossRefGoogle Scholar
  44. Otani H, Yoshioka K, Nishikawa H, Inagaki C, Nakamura T (2011) Involvement of protein kinase C and RhoA in protease-activated receptor 1-mediated F-actin reorganization and cell growth in rat cardiomyocytes. J Pharmacol Sci 115(2): 135–143CrossRefGoogle Scholar
  45. Pan J, Singh US, Takahashi T, Oka Y, Palm-Leis A, Herbelin BS, Baker KM (2005) PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes. J Cell Physiol 202(2): 536–553CrossRefGoogle Scholar
  46. Parker KK, Tan J, Chen CS, Tung L (2008) Myofibrillar architecture in engineered cardiac myocytes. Circ Res 103(4): 340–342CrossRefGoogle Scholar
  47. Parrag IC, Zandstra PW, Woodhouse KA (2011) Fiber alignment and coculture with fibroblasts improves the differentiated phenotype of murine embryonic stem cell-derived cardiomyocytes for cardiac tissue engineering. Biotechnol Bioeng, doi: 10.1002/bit.23353
  48. Patel AA, Thakar RG, Chown M, Ayala P, Desai TA, Kumar S (2010) Biophysical mechanisms of single-cell interactions with microtopographical cues. Biomed Microdev 12(2): 287–296CrossRefGoogle Scholar
  49. Pedersen JA, Swartz MA (2005) Mechanobiology in the third dimension. Ann Biomed Eng 33(11): 1469–1490CrossRefGoogle Scholar
  50. Qin L, Huang J, Xiong C, Zhang Y, Fang J (2007) Dynamical stress characterization and energy evaluation of single cardiac myocyte actuating on flexible substrate. Biochem Biophys Res Commun 360(2): 352–356CrossRefGoogle Scholar
  51. Ren J, Fang CX (2005) Small guanine nucleotide-binding protein Rho and myocardial function. Acta Pharmacol Sin 26(3): 279–285MathSciNetCrossRefGoogle Scholar
  52. Ross RS, Borg TK (2001) Integrins and the myocardium. Circ Res 88(11): 1112–1119CrossRefGoogle Scholar
  53. Ruwhof C, van Wamel JT, Noordzij LA, Aydin S, Harper JC, van der Laarse A (2001) Mechanical stress stimulates phospholipase C activity and intracellular calcium ion levels in neonatal rat cardiomyocytes. Cell Calcium 29(2): 73–83CrossRefGoogle Scholar
  54. Samarel AM (2005) Costameres, focal adhesions, and cardiomyocyte mechanotransduction. Am J Physiol Heart Circ Physiol 289(6): H2291–H2301CrossRefGoogle Scholar
  55. Shapira-Schweitzer K, Seliktar D (2007) Matrix stiffness affects spontaneous contraction of cardiomyocytes cultured within a PEGylated fibrinogen biomaterial. Acta Biomater 3(1): 33–41CrossRefGoogle Scholar
  56. Sharp WW, Simpson DG, Borg TK, Samarel AM, Terracio L (1997) Mechanical forces regulate focal adhesion and costamere assembly in cardiac myocytes. J Physiol 273(2 Pt 2): H546–H556Google Scholar
  57. Simpson DG, Majeski M, Borg TK, Terracio L (1999) Regulation of cardiac myocyte protein turnover and myofibrillar structure in vitro by specific directions of stretch. Circ Res 85(10): e59–e69CrossRefGoogle Scholar
  58. Slack-Davis JK, Martin KH, Tilghman RW, Iwanicki M, Ung EJ, Autry C, Luzzio MJ, Cooper B, Kath JC, Roberts WG, Parsons JT (2007) Cellular characterization of a novel focal adhesion kinase inhibitor. J Biol Chem 282(20): 14845–14852CrossRefGoogle Scholar
  59. Smith R (2007) Plot digitizer user’s manual (version 2.4.1). Accessed 7 June 2010
  60. Soonpaa MH, Kim KK, Pajak L, Franklin M, Field LJ (1996) Cardiomyocyte DNA synthesis and binucleation during murine development. Am J Physiol 271(5 Pt 2): H2183–H2189Google Scholar
  61. Sutton MA, Cheng MQ, Peters WH, Chao YJ, McNeill SR (1986) Application of an optimized digital correlation method to planar deformation analysis. Image Vis Comput 4(1): 143–150CrossRefGoogle Scholar
  62. Tiburcy M, Didié M, Boy O, Christalla P, Döker S, Naito H, Karikkineth BC, El-Armouche A, Grimm M, Nose M, Eschenhagen T, Zieseniss A, Katschinksi DM, Hamdani N, Linke WA, Yin X, Mayr M, Zimmermann WH (2011) Terminal differentiation, advanced organotypic maturation, and modeling of hypertrophic growth in engineered heart tissue. Circ Res 109(10): 1105–1114CrossRefGoogle Scholar
  63. Torsoni AS, Marin TM, Velloso LA, Franchini KG (2005) RhoA/ROCK signaling is critical to FAK activation by cyclic stretch in cardiac myocytes. Am J Physiol Heart Circ Physiol 289(4): H1488–H1496CrossRefGoogle Scholar
  64. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F (1991) The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 266(24): 15771–15781Google Scholar
  65. Tracqui P, Ohayon J, Boudou T (2008) Theoretical analysis of the adaptive contractile behaviour of a single cardiomyocyte cultured on elastic substrates with varying stiffness. J Theor Biol 255(1): 92–105CrossRefGoogle Scholar
  66. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S (1997) Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389(6654): 990–994CrossRefGoogle Scholar
  67. Zhang J, Jin G, Ma S, Meng L (2003) Application of an improved subpixel registration algorithm on digital speckle correlation measurement. Opt Laser Technol 35: 533–542CrossRefGoogle Scholar
  68. Zhao Y, Lim CC, Sawyer DB, Liao R, Zhang X (2007) Simultaneous orientation and cellular force measurements in adult cardiac myocytes using three-dimensional polymeric microrods. Cell Motil Cytoskeleton 64(9): 718–725CrossRefGoogle Scholar
  69. Zhou JJ, Bian JS, Pei JM, Wu S, Li HY, Wong TM (2002) Role of protein kinase C-epsilon in the development of kappa-opioid receptor tolerance to U50,488H in rat ventricular myocytes. Br J Pharmacol 135(7): 1675–1684CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Matthew W. Curtis
    • 1
  • Elisa Budyn
    • 2
  • Tejal A. Desai
    • 3
  • Allen M. Samarel
    • 4
  • Brenda Russell
    • 1
    Email author
  1. 1.Department of Physiology and BiophysicsUniversity of Illinois at ChicagoChicagoUSA
  2. 2.Department of Mechanical and Industrial EngineeringUniversity of Illinois at ChicagoChicagoUSA
  3. 3.Department of Physiology and Division of BioengineeringUniversity of California at San FranciscoSan FranciscoUSA
  4. 4.The Cardiovascular InstituteLoyola University Medical CenterMaywoodUSA

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