Biomedical Microdevices

, Volume 12, Issue 6, pp 1073–1085 | Cite as

Hypertrophy, gene expression, and beating of neonatal cardiac myocytes are affected by microdomain heterogeneity in 3D

  • Matthew W. Curtis
  • Sadhana Sharma
  • Tejal A. Desai
  • Brenda Russell


Cardiac myocytes are known to be influenced by the rigidity and topography of their physical microenvironment. It was hypothesized that 3D heterogeneity introduced by purely physical microdomains regulates cardiac myocyte size and contraction. This was tested in vitro using polymeric microstructures (G′ = 1.66 GPa) suspended with random orientation in 3D by a soft Matrigel matrix (G′ = 22.9 Pa). After 10 days of culture, the presence of 100 μm-long microstructures in 3D gels induced fold increases in neonatal rat ventricular myocyte size (1.61 ± 0.06, p < 0.01) and total protein/cell ratios (1.43 ± 0.08, p < 0.05) that were comparable to those induced chemically by 50 μM phenylephrine treatment. Upon attachment to microstructures, individual myocytes also had larger cross-sectional areas (1.57 ± 0.05, p < 0.01) and higher average rates of spontaneous contraction (2.01 ± 0.08, p < 0.01) than unattached myocytes. Furthermore, the inclusion of microstructures in myocyte-seeded gels caused significant increases in the expression of beta-1 adrenergic receptor (β1-AR, 1.19 ± 0.01), cardiac ankyrin repeat protein (CARP, 1.26 ± 0.02), and sarcoplasmic reticulum calcium-ATPase (SERCA2, 1.59 ± 0.12, p < 0.05), genes implicated in hypertrophy and contractile activity. Together, the results demonstrate that cardiac myocyte behavior can be controlled through local 3D microdomains alone. This approach of defining physical cues as independent features may help to advance the elemental design considerations for scaffolds in cardiac tissue engineering and therapeutic microdevices.


Cardiomyocyte Beat frequency Cell remodeling Focal adhesion Mechanotransduction Microstructure Microenvironment Three dimensions Hypertrophy Spontaneous contraction 



three dimensions


cytosine β-D-arabino-furanoside


beta-1 adrenergic receptor


beta-2 microglobulin


2,3-butanedione monoxime


beats per minute


bovine serum albumin


cardiac ankyrin repeat protein


cytochrome c oxidase subunit VIII heart/muscle




differential interference contrast


Dulbecco’s modified Eagle’s medium




extracellular matrix


phosphate buffered saline




poly(ethylene glycol) dimethacrylate


sodium dodecyl sulfate


standard error of measurement


sarcoplasmic reticulum calcium-ATPase


  1. Y. Aihara, M. Kurabayashi, Y. Saito, Y. Ohyama, T. Tanaka, S. Takeda, K. Tomaru, K. Sekiguchi, M. Arai, T. Nakamura, R. Nagai, Cardiac ankyrin repeat protein is a novel marker of cardiac hypertrophy: role of M-CAT element within the promoter. Hypertension 36, 48–53 (2000)Google Scholar
  2. P. Ayala, J.I. Lopez, T.A. Desai, Microtopographical cues in 3D attenuate fibrotic phenotype and extracellular matrix deposition: implications for tissue regeneration. Tissue Eng. Part A. (2010) [Epub ahead of print]Google Scholar
  3. P. Bagnato, V. Barone, E. Giacomello, D. Rossi, V. Sorrentino, Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles. J. Cell Biol. 160, 245–253 (2003)CrossRefGoogle Scholar
  4. A.F. Brown, Neutrophil granulocytes: adhesion and locomotion on collagen substrata and in collagen matrices. J. Cell Sci. 58, 455–467 (1982)Google Scholar
  5. B.M. Cadre, M. Qi, D.M. Eble, T.R. Shannon, D.M. Bers, A.M. Samarel, Cyclic stretch down-regulates calcium transporter gene expression in neonatal rat ventricular myocytes. J. Mol. Cell. Cardiol. 30, 2247–2259 (1998)CrossRefGoogle Scholar
  6. C.S. Chen, M. Mrksich, S. Huang, G.M. Whitesides, D.E. Ingber, Geometric control of cell life and death. Science 276, 1425–1428 (1997)CrossRefGoogle Scholar
  7. J.M. Collins, P. Ayala, T.A. Desai, B. Russell, Three-dimensional culture with stiff microstructures increases proliferation and slows osteogenic differentiation of human mesenchymal stem cells. Small 6, 355–360 (2010)CrossRefGoogle Scholar
  8. G. Cooper 4th, W.E. Mercer, J.K. Hoober, P.R. Gordon, R.L. Kent, I.K. Lauva, T.A. Marino, Load regulation of the properties of adult feline cardiocytes. The role of substrate adhesion. Circ. Res. 58, 692–705 (1986)Google Scholar
  9. J. Deutsch, D. Motlagh, B. Russell, T.A. Desai, Fabrication of microtextured membranes for cardiac myocyte attachment and orientation. J. Biomed. Mater. Res. 53, 267–275 (2000)CrossRefGoogle Scholar
  10. A.C. Durieux, D. Desplanches, D. Freyssenet, M. Flück, Mechanotransduction in striated muscle via focal adhesion kinase. Biochem. Soc. Trans. 35, 1312–1313 (2007)CrossRefGoogle Scholar
  11. D.M. Eble, M. Qi, S. Waldschmidt, P.A. Lucchesi, K.L. Byron, A.M. Samarel, Contractile activity is required for sarcomeric assembly in phenylephrine-induced cardiac myocyte hypertrophy. Am. J. Physiol. 274, C1226–C1237 (1998)Google Scholar
  12. A.J. Engler, S. Sen, H.L. Sweeney, D.E. Discher, Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006)CrossRefGoogle Scholar
  13. A.J. Engler, C. Carag-Krieger, C.P. Johnson, M. Raab, H.Y. Tang, D.W. Speicher, J.W. Sanger, J.M. Sanger, D.E. Discher, Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating. J. Cell Sci. 121, 3794–3802 (2008)CrossRefGoogle Scholar
  14. A. Fraticelli, R. Josephson, R. Danziger, E. Lakatta, H. Spurgeon, Morphological and contractile characteristics of rat cardiac myocytes from maturation to senescence. Am. J. Physiol. 257, H259–H265 (1989)Google Scholar
  15. N.A. Geisse, S.P. Sheehy, K.K. Parker, Control of myocyte remodeling in vitro with engineered substrates. In Vitro Cell. Dev. Biol. Anim. 45, 343–350 (2009)CrossRefGoogle Scholar
  16. S.M. Gopalan, C. Flaim, S.N. Bhatia, M. Hoshijima, R. Knoell, K.R. Chien, J.H. Omens, A.D. McCulloch, Anisotropic stretch-induced hypertrophy in neonatal ventricular myocytes micropatterned on deformable elastomers. Biotechnol. Bioeng. 81, 578–587 (2003)CrossRefGoogle Scholar
  17. M. Hoshijima, Mechanical stress-strain sensors embedded in cardiac cytoskeleton: Z disk, titin, and associated structures. Am. J. Physiol. Heart Circ. Physiol. 290, H1313–H1325 (2006)CrossRefGoogle Scholar
  18. H. Huang, R.D. Kamm, R.T. Lee, Cell mechanics and mechanotransduction: pathways, probes, and physiology. Am. J. Physiol. Cell Physiol. 287, C1–C11 (2004)CrossRefGoogle Scholar
  19. M. Iemitsu, T. Miyauchi, S. Maeda, S. Sakai, T. Kobayashi, N. Fujii, H. Miyazaki, M. Matsuda, I. Yamaguchi, Physiological and pathological cardiac hypertrophy induce different molecular phenotypes in the rat. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R2029–R2036 (2001)Google Scholar
  20. D.E. Ingber, Tensegrity-based mechanosensing from macro to micro. Prog. Biophys. Mol. Biol. 97, 163–179 (2008)CrossRefGoogle Scholar
  21. J.G. Jacot, A.D. McCulloch, J.H. Omens, Substrate stiffness affects the functional maturation of neonatal rat ventricular myocytes. Biophys. J. 95, 3479–3487 (2008)CrossRefGoogle Scholar
  22. A.M. Katz, Maladaptive growth in the failing heart: the cardiomyopathy of overload. Cardiovasc. Drugs Ther. 16, 245–249 (2002)CrossRefGoogle Scholar
  23. P. Kim, D.H. Kim, B. Kim, S.K. Choi, S.H. Lee, A. Khademhosseini, R. Langer, K.Y. Suh, Fabrication of nanostructures of polyethylene glycol for applications to protein adsorption and cell adhesion. Nanotechnology 16, 1–7 (2005)CrossRefGoogle Scholar
  24. H. Kögler, P. Schott, K. Toischer, H. Milting, P.N. Van, M. Kohlhaas, C. Grebe, A. Kassner, E. Domeier, N. Teucher, T. Seidler, R. Knöll, L.S. Maier, A. El-Banayosy, R. Körfer, G. Hasenfuss, Relevance of brain natriuretic peptide in preload-dependent regulation of cardiac sarcoplasmic reticulum Ca2+ ATPase expression. Circulation 113, 2724–2732 (2006)CrossRefGoogle Scholar
  25. C.J. Lee, M.S. Blumenkranz, H.A. Fishman, S.F. Bent, Controlling cell adhesion on human tissue by soft lithography. Langmuir 20, 4155–4161 (2004)CrossRefGoogle Scholar
  26. G.Y. Lee, P.A. Kenny, E.H. Lee, M.J. Bissell, Three-dimensional culture models of normal and malignant breast epithelial cells. Nat. Methods 4, 359–365 (2007)CrossRefGoogle Scholar
  27. E.J. Lee, E. Kim do, E.U. Azeloglu, K.D. Costa, Engineered cardiac organoid chambers: toward a functional biological model ventricle. Tissue Eng. Part A. 14, 215–225 (2008)CrossRefGoogle Scholar
  28. C. Li, W. Hung Wong, Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application. Genome Biol. 2, 1–11 (2001)Google Scholar
  29. D. Motlagh, T.J. Hartman, T.A. Desai, B. Russell, Microfabricated grooves recapitulate neonatal myocyte connexin43 and N-cadherin expression and localization. J. Biomed. Mater. Res. A 67, 148–157 (2003a)CrossRefGoogle Scholar
  30. D. Motlagh, S.E. Senyo, T.A. Desai, B. Russell, Microtextured substrata alter gene expression, protein localization and the shape of cardiac myocytes. Biomaterials 24, 2463–2476 (2003b)CrossRefGoogle Scholar
  31. S.F. Nagueh, G. Shah, Y. Wu, G. Torre-Amione, N.M. King, S. Lahmers, C.C. Witt, K. Becker, S. Labeit, H.L. Granzier, Altered titin expression, myocardial stiffness, and left ventricular function in patients with dilated cardiomyopathy. Circulation 110, 155–162 (2004)CrossRefGoogle Scholar
  32. S. Nishimura, S. Yasuda, M. Katoh, K.P. Yamada, H. Yamashita, Y. Saeki, K. Sunagawa, R. Nagai, T. Hisada, S. Sugiura, Single cell mechanics of rat cardiomyocytes under isometric, unloaded, and physiologically loaded conditions. Am. J. Physiol. Heart Circ. Physiol. 287, H196–H202 (2004)CrossRefGoogle Scholar
  33. J.J. Norman, J.M. Collins, S. Sharma, B. Russell, T.A. Desai, Microstructures in 3D biological gels affect cell proliferation. Tissue Eng. A 14, 379–390 (2008)CrossRefGoogle Scholar
  34. E. Ogawa, Y. Saito, M. Harada, S. Kamitani, K. Kuwahara, Y. Miyamoto, M. Ishikawa, I. Hamanaka, N. Kajiyama, N. Takahashi, O. Nakagawa, I. Masuda, I. Kishimoto, K. Nakao, Outside-in signalling of fibronectin stimulates cardiomyocyte hypertrophy in cultured neonatal rat ventricular myocytes. J. Mol. Cell. Cardiol. 32, 765–776 (2000)CrossRefGoogle Scholar
  35. J.A. Pedersen, M.A. Swartz, Mechanobiology in the third dimension. Ann. Biomed. Eng. 33, 1469–1490 (2005)CrossRefGoogle Scholar
  36. R.J. Pelham Jr., Y. Wang, Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. U. S. A. 94, 13661–13665 (1997)CrossRefGoogle Scholar
  37. S. Pérez, L.J. Royo, A. Astudillo, D. Escudero, F. Alvarez, A. Rodríguez, E. Gómez, J. Otero, Identifying the most suitable endogenous control for determining gene expression in hearts from organ donors. BMC Mol. Biol. 8, 114 (2007)CrossRefGoogle Scholar
  38. S.R. Peyton, A.J. Putnam, Extracellular matrix rigidity governs smooth muscle cell motility in a biphasic fashion. J. Cell. Physiol. 204, 198–209 (2005)CrossRefGoogle Scholar
  39. A.M. Raskin, M. Hoshijima, E. Swanson, A.D. McCulloch, J.H. Omens, Hypertrophic gene expression induced by chronic stretch of excised mouse heart muscle. Mol. Cell. Biomech. 6, 145–159 (2009)Google Scholar
  40. S.A. Ruiz, C.S. Chen, Emergence of patterned stem cell differentiation within multicellular structures. Stem Cells 26, 2921–2927 (2008)CrossRefGoogle Scholar
  41. B. Russell, D. Motlagh, W.W. Ashley, Form follows function: how muscle shape is regulated by work. J. Appl. Physiol. 88, 1127–1132 (2000)Google Scholar
  42. B. Russell, M.W. Curtis, Y.E. Koshman, A.M. Samarel, Mechanical stress-induced sarcomere assembly for cardiac muscle growth in length and width. J. Mol. Cell. Cardiol. 48, 817–823 (2010)CrossRefGoogle Scholar
  43. A. Saez, M. Ghibaudo, A. Buguin, P. Silberzan, B. Ladoux, Rigidity-driven growth and migration of epithelial cells on microstructured anisotropic substrates. Proc. Natl. Acad. Sci. U. S. A. 104, 8281–8286 (2007)CrossRefGoogle Scholar
  44. A.M. Samarel, Costameres, focal adhesions, and cardiomyocyte mechanotransduction. Am. J. Physiol. Heart Circ. Physiol. 289, H2291–H2301 (2005)CrossRefGoogle Scholar
  45. S.E. Senyo, Y.E. Koshman, B. Russell, Stimulus interval, rate and direction differentially regulate phosphorylation for mechanotransduction in neonatal cardiac myocytes. FEBS Lett. 581, 4241–4247 (2007)CrossRefGoogle Scholar
  46. K. Shapira-Schweitzer, D. Seliktar, Matrix stiffness affects spontaneous contraction of cardiomyocytes cultured within a PEGylated fibrinogen biomaterial. Acta Biomater. 3, 33–41 (2007)CrossRefGoogle Scholar
  47. C.C. Strøm, M. Kruhøffer, S. Knudsen, F. Stensgaard-Hansen, T.E. Jonassen, T.F. Orntoft, S. Haunsø, S.P. Sheikh, Identification of a core set of genes that signifies pathways underlying cardiac hypertrophy. Comp. Funct. Genomics 5, 459–470 (2004)CrossRefGoogle Scholar
  48. S.L. Tao, K.C. Popat, J.J. Norman, T.A. Desai, Surface modification of SU-8 for enhanced biofunctionality and nonfouling properties. Langmuir 24, 2631–2636 (2008)CrossRefGoogle Scholar
  49. R.G. Thakar, M.G. Chown, A. Patel, L. Peng, S. Kumar, T.A. Desai, Contractility-dependent modulation of cell proliferation and adhesion by microscale topographical cues. Small 4, 1416–1424 (2008)CrossRefGoogle Scholar
  50. E.W. Thompson, T.A. Marino, C.E. Uboh, R.L. Kent, G. Cooper 4th, Atrophy reversal and cardiocyte redifferentiation in reloaded cat myocardium. Circ. Res. 54, 367–377 (1984)Google Scholar
  51. N. Wang, J.P. Butler, D.E. Ingber, Mechanotransduction across the cell surface and through the cytoskeleton. Science 260, 1124–1127 (1993)CrossRefGoogle Scholar
  52. Y.C. Wang, C.C. Ho, Micropatterning of proteins and mammalian cells on biomaterials. FASEB J. 18, 525–527 (2004)Google Scholar
  53. J. Weisser-Thomas, H. Kubo, C.A. Hefner, J.P. Gaughan, B.S. McGowan, R. Ross, M. Meyer, W. Dillmann, S.R. Houser, The Na+/Ca2+ exchanger/SR Ca2+ ATPase transport capacity regulates the contractility of normal and hypertrophied feline ventricular myocytes. J. Card. Fail. 11, 380–387 (2005)CrossRefGoogle Scholar
  54. S.H. Witt, D. Labeit, H. Granzier, S. Labeit, C.C. Witt, Dimerization of the cardiac ankyrin protein CARP: implications for MARP titin-based signaling. J. Muscle Res. Cell Motil. 26, 401–408 (2005)CrossRefGoogle Scholar
  55. W.H. Zimmermann, K. Schneiderbanger, P. Schubert, M. Didié, F. Münzel, J.F. Heubach, S. Kostin, W.L. Neuhuber, T. Eschenhagen, Tissue engineering of a differentiated cardiac muscle construct. Circ. Res. 90, 223–230 (2002)CrossRefGoogle Scholar
  56. O. Zolk, M. Frohme, A. Maurer, F.W. Kluxen, B. Hentsch, D. Zubakov, J.D. Hoheisel, I.H. Zucker, S. Pepe, T. Eschenhagen, Cardiac ankyrin repeat protein, a negative regulator of cardiac gene expression, is augmented in human heart failure. Biochem. Biophys. Res. Commun. 293, 1377–1382 (2002)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Matthew W. Curtis
    • 1
  • Sadhana Sharma
    • 2
  • Tejal A. Desai
    • 3
  • Brenda Russell
    • 2
  1. 1.Department of BioengineeringUniversity of Illinois at ChicagoChicagoUSA
  2. 2.Department of Physiology and Biophysics (MC 901)University of Illinois at ChicagoChicagoUSA
  3. 3.Department of Physiology and Division of BioengineeringUniversity of California at San FranciscoSan FranciscoUSA

Personalised recommendations