Genetic isolation of stem cell-derived pacemaker-nodal cardiac myocytes

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Dysfunction of the cardiac pacemaker tissues due to genetic defects, acquired diseases, or aging results in arrhythmias. When arrhythmias occur, artificial pacemaker implants are used for treatment. However, the numerous limitations of electronic implants have prompted studies of biological pacemakers that can integrate into the myocardium providing a permanent cure. Embryonic stem (ES) cells cultured as three-dimensional (3D) spheroid aggregates termed embryoid bodies possess the ability to generate all cardiac myocyte subtypes. Here, we report the use of a SHOX2 promoter and a Cx30.2 enhancer to genetically identify and isolate ES cell-derived sinoatrial node (SAN) and atrioventricular node (AVN) cells, respectively. The ES cell-derived Shox2 and Cx30.2 cardiac myocytes exhibit a spider cell morphology and high intracellular calcium loading characteristic of pacemaker-nodal myocytes. These cells express abundant levels of pacemaker genes such as endogenous HCN4, Cx45, Cx30.2, Tbx2, and Tbx3. These cells were passaged, frozen, and thawed multiple times while maintaining their pacemaker-nodal phenotype. When cultured as 3D aggregates in an attempt to create a critical mass that simulates in vivo architecture, these cell lines exhibited an increase in the expression level of key regulators of cardiovascular development, such as GATA4 and GATA6 transcription factors. In addition, the aggregate culture system resulted in an increase in the expression level of several ion channels that play a major role in the spontaneous diastolic depolarization characteristic of pacemaker cells. We have isolated pure populations of SAN and AVN cells that will be useful tools for generating biological pacemakers.

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  1. 1.

    Spooner PM, Albert C, Benjamin EJ, Boineau R, Elston RC, George AL Jr, Jouven X, Kuller LH, MacCluer JW, Marban E, Muller JE, Schwartz PJ, Siscovick DS, Tracy RP, Zareba W, Zipes DP (2001) Sudden cardiac death, genes, and arrhythmogenesis: consideration of new population and mechanistic approaches from a national heart, lung, and blood institute workshop, part I. Circulation 103:2361–2364

  2. 2.

    Rosen MR, Brink PR, Cohen IS, Robinson RB (2004) Genes, stem cells and biological pacemakers. Cardiovasc Res 64:12–23

  3. 3.

    Rajesh G, Francis J (2006) Biological pacemakers. Indian Pacing Electrophysiol J 6:1–5

  4. 4.

    Reinlib L, Field L (2000) Cell transplantation as future therapy for cardiovascular disease?: a workshop of the National Heart, Lung, and Blood Institute. Circulation 101:E182–E187

  5. 5.

    Maltsev VA, Rohwedel J, Hescheler J, Wobus AM (1993) Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types. Mech Dev 44:41–50

  6. 6.

    Kehat I, Khimovich L, Caspi O, Gepstein A, Shofti R, Arbel G, Huber I, Satin J, Itskovitz-Eldor J, Gepstein L (2004) Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol 22:1282–1289

  7. 7.

    Xue T, Cho HC, Akar FG, Tsang SY, Jones SP, Marban E, Tomaselli GF, Li RA (2005) Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes: insights into the development of cell-based pacemakers. Circulation 111:11–20

  8. 8.

    Keith A, Flack M (1907) The form and nature of the muscular connections between the primary divisions of the vertebrate heart. J Anat Physiol 41:172–189

  9. 9.

    Opthof T (1988) The mammalian sinoatrial node. Cardiovasc Drugs Ther 1:573–597

  10. 10.

    Espinoza-Lewis RA, Yu L, He F, Liu H, Tang R, Shi J, Sun X, Martin JF, Wang D, Yang J, Chen Y (2009) Shox2 is essential for the differentiation of cardiac pacemaker cells by repressing Nk2–5. Dev Biol 327:376–385

  11. 11.

    Blaschke RJ, Hahurij ND, Kuijper S, Just S, Wisse LJ, Deissler K, Maxelon T, Anastassiadis K, Spitzer J, Hardt SE, Scholer H, Feitsma H, Rottbauer W, Blum M, Meijlink F, Rappold G, Gittenberger-de Groot AC (2007) Targeted mutation reveals essential functions of the homeodomain transcription factor Shox2 in sinoatrial and pacemaking development. Circulation 115:1830–1838

  12. 12.

    Dobrzynski H, Nikolski VP, Sambelashvili AT, Greener ID, Yamamoto M, Boyett MR, Efimov IR (2003) Site of origin and molecular substrate of atrioventricular junctional rhythm in the rabbit heart. Circ Res 93:1102–1110

  13. 13.

    James TN (2003) Structure and function of the sinus node, AV node and his bundle of the human heart: part II-function. Prog Cardiovasc Dis 45:327–360

  14. 14.

    Munshi NV, McAnally J, Bezprozvannaya S, Berry JM, Richardson JA, Hill JA, Olson EN (2009) Cx30.2 enhancer analysis identifies Gata4 as a novel regulator of atrioventricular delay. Development 136:2665–2674

  15. 15.

    Klug MG, Soonpaa MH, Koh GY, Field LJ (1996) Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest 98:216–224

  16. 16.

    Jackson M, Taylor AH, Jones EA, Forrester LM (2010) The culture of mouse embryonic stem cells and formation of embryoid bodies. Methods Mol Biol 633:1–18

  17. 17.

    White SM, Claycomb WC (2005) Embryonic stem cells form an organized, functional cardiac conduction system in vitro. Am J Physiol Heart Circ Physiol 288:H670–H679

  18. 18.

    Puskaric S, Schmitteckert S, Mori AD, Glaser A, Schneider KU, Bruneau BG, Blaschke RJ, Steinbeisser H, Rappold G (2010) Shox2 mediates Tbx5 activity by regulating Bmp4 in the pacemaker region of the developing heart. Hum Mol Genet 19:4625–4633

  19. 19.

    White SM, Constantin PE, Claycomb WC (2004) Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function. Am J Physiol Heart Circ Physiol 286:H823–H829

  20. 20.

    Lam ML, Hashem SI, Claycomb WC (2011) Embryonic stem cell-derived cardiomyocytes harbor a subpopulation of niche-forming Sca-1+ progenitor cells. Mol Cell Biochem 349:69–76

  21. 21.

    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408

  22. 22.

    DiFrancesco D, Ferroni A, Mazzanti M, Tromba C (1986) Properties of the hyperpolarizing-activated current (if) in cells isolated from the rabbit sino-atrial node. J Physiol 377:61–88

  23. 23.

    Verheijck EE, Wessels A, van Ginneken AC, Bourier J, Markman MW, Vermeulen JL, de Bakker JM, Lamers WH, Opthof T, Bouman LN (1998) Distribution of atrial and nodal cells within the rabbit sinoatrial node: models of sinoatrial transition. Circulation 97:1623–1631

  24. 24.

    Wu J, Schuessler RB, Rodefeld MD, Saffitz JE, Boineau JP (2001) Morphological and membrane characteristics of spider and spindle cells isolated from rabbit sinus node. Am J Physiol Heart Circ Physiol 280:H1232–H1240

  25. 25.

    Mangoni ME, Couette B, Bourinet E, Platzer J, Reimer D, Striessnig J, Nargeot J (2003) Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity. Proc Natl Acad Sci USA 100:5543–5548

  26. 26.

    Mangoni ME, Couette B, Marger L, Bourinet E, Striessnig J, Nargeot J (2006) Voltage-dependent calcium channels and cardiac pacemaker activity: from ionic currents to genes. Prog Biophys Mol Biol 90:38–63

  27. 27.

    Marger L, Mesirca P, Alig J, Torrente A, Dubel S, Engeland B, Kanani S, Fontanaud P, Striessnig J, Shin HS, Isbrandt D, Ehmke H, Nargeot J, Mangoni ME (2011) Functional roles of Ca(v)1.3, Ca(v)3.1 and HCN channels in automaticity of mouse atrioventricular cells: insights into the atrioventricular pacemaker mechanism. Channels (Austin) 5:251–261

  28. 28.

    Halbach M, Egert U, Hescheler J, Banach K (2003) Estimation of action potential changes from field potential recordings in multicellular mouse cardiac myocyte cultures. Cell Physiol Biochem 13:271–284

  29. 29.

    Lakatta EG, Maltsev VA, Bogdanov KY, Stern MD, Vinogradova TM (2003) Cyclic variation of intracellular calcium: a critical factor for cardiac pacemaker cell dominance. Circ Res 92:e45–e50

  30. 30.

    Maltsev VA, Vinogradova TM, Lakatta EG (2006) The emergence of a general theory of the initiation and strength of the heartbeat. J Pharmacol Sci 100:338–369

  31. 31.

    Bogdanov KY, Maltsev VA, Vinogradova TM, Lyashkov AE, Spurgeon HA, Stern MD, Lakatta EG (2006) Membrane potential fluctuations resulting from submembrane Ca2+ releases in rabbit sinoatrial nodal cells impart an exponential phase to the late diastolic depolarization that controls their chronotropic state. Circ Res 99:979–987

  32. 32.

    Vinogradova TM, Maltsev VA, Bogdanov KY, Lyashkov AE, Lakatta EG (2005) Rhythmic Ca2+ oscillations drive sinoatrial nodal cell pacemaker function to make the heart tick. Ann N Y Acad Sci 1047:138–156

  33. 33.

    Vinogradova TM, Lyashkov AE, Zhu W, Ruknudin AM, Sirenko S, Yang D, Deo S, Barlow M, Johnson S, Caffrey JL, Zhou YY, Xiao RP, Cheng H, Stern MD, Maltsev VA, Lakatta EG (2006) High basal protein kinase A-dependent phosphorylation drives rhythmic internal Ca2+ store oscillations and spontaneous beating of cardiac pacemaker cells. Circ Res 98:505–514

  34. 34.

    Maltsev VA, Vinogradova TM, Bogdanov KY, Lakatta EG, Stern MD (2004) Diastolic calcium release controls the beating rate of rabbit sinoatrial node cells: numerical modeling of the coupling process. Biophys J 86:2596–2605

  35. 35.

    Habets PE, Moorman AF, Clout DE, van Roon MA, Lingbeek M, van Lohuizen M, Campione M, Christoffels VM (2002) Cooperative action of Tbx2 and Nkx2.5 inhibits ANF expression in the atrioventricular canal: implications for cardiac chamber formation. Genes Dev 16:1234–1246

  36. 36.

    Garg V, Kathiriya IS, Barnes R, Schluterman MK, King IN, Butler CA, Rothrock CR, Eapen RS, Hirayama-Yamada K, Joo K, Matsuoka R, Cohen JC, Srivastava D (2003) GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature 424:443–447

  37. 37.

    Lee Y, Shioi T, Kasahara H, Jobe SM, Wiese RJ, Markham BE, Izumo S (1998) The cardiac tissue-restricted homeobox protein Csx/Nkx2.5 physically associates with the zinc finger protein GATA4 and cooperatively activates atrial natriuretic factor gene expression. Mol Cell Biol 18:3120–3129

  38. 38.

    Adamo RF, Guay CL, Edwards AV, Wessels A, Burch JB (2004) GATA-6 gene enhancer contains nested regulatory modules for primary myocardium and the embedded nascent atrioventricular conduction system. Anat Rec A Discov Mol Cell Evol Biol 280:1062–1071

  39. 39.

    Boogerd KJ, Wong LY, Christoffels VM, Klarenbeek M, Ruijter JM, Moorman AF, Barnett P (2008) Msx1 and Msx2 are functional interacting partners of T-box factors in the regulation of Connexin43. Cardiovasc Res 78:485–493

  40. 40.

    Choi M, Stottmann RW, Yang YP, Meyers EN, Klingensmith J (2007) The bone morphogenetic protein antagonist noggin regulates mammalian cardiac morphogenesis. Circ Res 100:220–228

  41. 41.

    Christoffels VM, Smits GJ, Kispert A, Moorman AF (2010) Development of the pacemaker tissues of the heart. Circ Res 106:240–254

  42. 42.

    Mangoni ME, Nargeot J (2008) Genesis and regulation of the heart automaticity. Physiol Rev 88:919–982

  43. 43.

    Yunker AM, Sharp AH, Sundarraj S, Ranganathan V, Copeland TD, McEnery MW (2003) Immunological characterization of T-type voltage-dependent calcium channel CaV3.1 (alpha 1G) and CaV3.3 (alpha 1I) isoforms reveal differences in their localization, expression, and neural development. Neuroscience 117:321–335

  44. 44.

    Bogdanov KY, Vinogradova TM, Lakatta EG (2001) Sinoatrial nodal cell ryanodine receptor and Na(+)–Ca(2+) exchanger: molecular partners in pacemaker regulation. Circ Res 88:1254–1258

  45. 45.

    Miake J, Marban E, Nuss HB (2003) Functional role of inward rectifier current in heart probed by Kir2.1 overexpression and dominant-negative suppression. J Clin Invest 111:1529–1536

  46. 46.

    Wolf CM, Berul CI (2006) Inherited conduction system abnormalities: one group of diseases, many genes. J Cardiovasc Electrophysiol 17:446–455

  47. 47.

    Bucchi A, Barbuti A, Difrancesco D, Baruscotti M (2012) Funny current and cardiac rhythm: insights from HCN knockout and transgenic mouse models. Front Physiol 3:240

  48. 48.

    Herrmann S, Hofmann F, Stieber J, Ludwig A (2012) HCN channels in the heart: lessons from mouse mutants. Br J Pharmacol 166:501–509

  49. 49.

    Baruscotti M, Bucchi A, Viscomi C, Mandelli G, Consalez G, Gnecchi-Rusconi T, Montano N, Casali KR, Micheloni S, Barbuti A, DiFrancesco D (2011) Deep bradycardia and heart block caused by inducible cardiac-specific knockout of the pacemaker channel gene Hcn4. Proc Natl Acad Sci USA 108:1705–1710

  50. 50.

    Singh R, Hoogaars WM, Barnett P, Grieskamp T, Rana MS, Buermans H, Farin HF, Petry M, Heallen T, Martin JF, Moorman AF, ‘t Hoen PA, Kisper A, Christoffels VM (2012) Tbx2 and Tbx3 induce atrioventricular myocardial development and endocardial cushion formation. Cell Mol Life Sci 69:1377–1389

  51. 51.

    Aanhaanen WT, Brons JF, Dominguez JN, Rana MS, Norden J, Airik R, Wakker V, de Gier-de Vries C, Brown NA, Kispert A, Moorman AF, Christoffels VM (2009) The Tbx2+ primary myocardium of the atrioventricular canal forms the atrioventricular node and the base of the left ventricle. Circ Res 104:1267–1274

  52. 52.

    Wang Y, Morishima M, Zheng M, Uchino T, Mannen K, Takahashi A, Nakaya Y, Komuro I, Ono K (2007) Transcription factors Csx/Nkx2.5 and GATA4 distinctly regulate expression of Ca2+ channels in neonatal rat heart. J Mol Cell Cardiol 42:1045–1053

  53. 53.

    Cheng G, Hagen TP, Dawson ML, Barnes KV, Menick DR (1999) The role of GATA, CArG, E-box, and a novel element in the regulation of cardiac expression of the Na+–Ca2+ exchanger gene. J Biol Chem 274:12819–12826

  54. 54.

    Yamada M, Revelli JP, Eichele G, Barron M, Schwartz RJ (2000) Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: evidence for BMP2 induction of Tbx2. Dev Biol 228:95–105

  55. 55.

    Harrelson Z, Kelly RG, Goldin SN, Gibson-Brown JJ, Bollag RJ, Silver LM, Papaioannou VE (2004) Tbx2 is essential for patterning the atrioventricular canal and for morphogenesis of the outflow tract during heart development. Development 131:5041–5052

  56. 56.

    Kreuzberg MM, Schrickel JW, Ghanem A, Kim JS, Degen J, Janssen-Bienhold U, Lewalter T, Tiemann K, Willecke K (2006) Connexin30.2 containing gap junction channels decelerate impulse propagation through the atrioventricular node. Proc Natl Acad Sci USA 103:5959–5964

  57. 57.

    Kreuzberg MM, Willecke K, Bukauskas FF (2006) Connexin-mediated cardiac impulse propagation: connexin 30.2 slows atrioventricular conduction in mouse heart. Trends Cardiovasc Med 16:266–272

  58. 58.

    Herrmann S, Layh B, Ludwig A (2011) Novel insights into the distribution of cardiac HCN channels: an expression study in the mouse heart. J Mol Cell Cardiol 51:997–1006

  59. 59.

    Vicente-Steijn R, Passier R, Wisse LJ, Schalij MJ, Poelmann RE, Gittenberger-de Groot AC, Jongbloed MR (2011) Funny current channel HCN4 delineates the developing cardiac conduction system in chicken heart. Heart Rhythm 8:1254–1263

  60. 60.

    Liu J, Dobrzynski H, Yanni J, Boyett MR, Lei M (2007) Organisation of the mouse sinoatrial node: structure and expression of HCN channels. Cardiovasc Res 73:729–738

  61. 61.

    Yamamoto M, Dobrzynski H, Tellez J, Niwa R, Billeter R, Honjo H, Kodama I, Boyett MR (2006) Extended atrial conduction system characterised by the expression of the HCN4 channel and connexin45. Cardiovasc Res 72:271–281

  62. 62.

    Boyett MR, Inada S, Yoo S, Li J, Liu J, Tellez J, Greener ID, Honjo H, Billeter R, Lei M, Zhang H, Efimov IR, Dobrzynski H (2006) Connexins in the sinoatrial and atrioventricular nodes. Adv Cardiol 42:175–197

  63. 63.

    Moorman AF, Christoffels VM (2003) Cardiac chamber formation: development, genes, and evolution. Physiol Rev 83:1223–1267

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We thank Dr. Gudrun Rappold, University of Heidelberg, Germany for the SHOX2 promoter; Dr. Eric Olson, UT Southwestern for the Cx30.2 enhancer, and Dr. YiPing Chen, Tulane University for CJ-7 (wild type) and Shox2lacZ/+ ES cells.

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Correspondence to William C. Claycomb.

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Supplementary material 3 (AVI 13080 kb). Supplemental online video 1 A representative contracting Shox2lacZ/+ EB at day 14 of differentiation. Fluorescent image of this representative EB is shown in Fig. 1A delineating the area of Shox2 expression (green). The movie was acquired following live fluorescein digalactoside staining. The contracting region is directly adjacent to the Shox2 cell cluster located at the bottom left of this video

Supplementary material 4 (AVI 13080 kb). Supplemental online video 2 A representative contracting Cx30.2-RFP EB at day 14 of differentiation. Fluorescent image of this representative EB is shown in Fig. 1B delineating the area of Cx30.2 expression (red). The contracting region is located at the center of this video

Supplementary material 1 (TIFF 2102 kb). Supplemental Fig. 1 Fold change in gene expression in 3D aggregates versus 2D monolayer cultures. (A) A histogram plot of the fold change in gene expression in Shox2 cells passage 5 cultured as 3D aggregates compared to Shox2 cells passage 5 cultured as 2D monolayers. (B) A histogram plot of the fold change in gene expression in Cx30.2 cells passage 7 cultured as 3D aggregates compared to Cx30.2 cells passage 7 cultured as 2D monolayers. All cultures were allowed to grow for 10 days before RNA was collected. Please note the difference in the Y-axis in (A) and (B)

Supplementary material 2 (DOC 47 kb)

Supplementary material 3 (AVI 13080 kb). Supplemental online video 1 A representative contracting Shox2lacZ/+ EB at day 14 of differentiation. Fluorescent image of this representative EB is shown in Fig. 1A delineating the area of Shox2 expression (green). The movie was acquired following live fluorescein digalactoside staining. The contracting region is directly adjacent to the Shox2 cell cluster located at the bottom left of this video

Supplementary material 4 (AVI 13080 kb). Supplemental online video 2 A representative contracting Cx30.2-RFP EB at day 14 of differentiation. Fluorescent image of this representative EB is shown in Fig. 1B delineating the area of Cx30.2 expression (red). The contracting region is located at the center of this video

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Hashem, S.I., Claycomb, W.C. Genetic isolation of stem cell-derived pacemaker-nodal cardiac myocytes. Mol Cell Biochem 383, 161–171 (2013) doi:10.1007/s11010-013-1764-x

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  • Atrioventricular
  • Embryoid body
  • Embryonic stem cell
  • Sinoatrial
  • Pacemaker