Advertisement

Optogenetics pp 303-317 | Cite as

Inscribing Optical Excitability to Non-Excitable Cardiac Cells: Viral Delivery of Optogenetic Tools in Primary Cardiac Fibroblasts

  • Jinzhu Yu
  • Emilia EntchevaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1408)

Abstract

We describe in detail a method to introduce optogenetic actuation tools, a mutant version of channelrhodopsin-2, ChR2(H134R), and archaerhodopsin (ArchT), into primary cardiac fibroblasts (cFB) in vitro by adenoviral infection to yield quick, robust, and consistent expression. Instructions on adjusting infection parameters such as the multiplicity of infection and virus incubation duration are provided to generalize the method for different lab settings or cell types. Specific conditions are discussed to create hybrid co-cultures of the optogenetically modified cFB and non-transformed cardiomyocytes to obtain light-sensitive excitable cardiac syncytium, including stencil-patterned cell growth. We also describe an all-optical framework for the functional testing of responsiveness of these opsins in cFB. The presented methodology provides cell-specific tools for the mechanistic investigation of the functional bioelectric contribution of different non-excitable cells in the heart and their electrical coupling to cardiomyocytes under different conditions.

Key words

Optogenetics Cardiac Non-excitable cells Fibroblasts ChR2 ArchT 

Notes

Acknowledgements

This work was supported by NIH-NHLBI grant R01-HL-111649 and NSF-Biophotonics grant 1511353 (to E.E.), and partially by a NYSTEM grant C026716 to the Stony Brook Stem Cell Center. We thank Christina Ambrosi and Aleks Klimas for helpful discussions.

References

  1. 1.
    Lajiness JD, Conway SJ (2013) Origin, development, and differentiation of cardiac fibroblasts. J Mol Cell Cardiol. doi: 10.1016/j.yjmcc.2013.11.003 PubMedPubMedCentralGoogle Scholar
  2. 2.
    Vasquez C, Benamer N, Morley GE (2011) The cardiac fibroblast: functional and electrophysiological considerations in healthy and diseased hearts. J Cardiovasc Pharmacol 57(4):380–388. doi: 10.1097/FJC.0b013e31820cda19 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bursac N (2014) Cardiac fibroblasts in pressure overload hypertrophy: the enemy within? J Clin Invest 124(7):2850–2853. doi: 10.1172/JCI76628 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Doetschman T, Azhar M (2012) Cardiac-specific inducible and conditional gene targeting in mice. Circ Res 110(11):1498–1512. doi: 10.1161/CIRCRESAHA.112.265066 CrossRefPubMedGoogle Scholar
  5. 5.
    Zeisberg EM, Kalluri R (2010) Origins of cardiac fibroblasts. Circ Res 107(11):1304–1312. doi: 10.1161/CIRCRESAHA.110.231910 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Krenning G, Zeisberg EM, Kalluri R (2010) The origin of fibroblasts and mechanism of cardiac fibrosis. J Cell Physiol 225(3):631–637. doi: 10.1002/jcp.22322 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Baudino TA, Carver W, Giles W, Borg TK (2006) Cardiac fibroblasts: friend or foe? Am J Physiol 291(3):H1015–H1026. doi: 10.1152/ajpheart.00023.2006 Google Scholar
  8. 8.
    Nag AC (1980) Study of non-muscle cells of the adult mammalian heart: a fine structural analysis and distribution. Cytobios 28(109):41–61PubMedGoogle Scholar
  9. 9.
    Camelliti P, Borg TK, Kohl P (2005) Structural and functional characterisation of cardiac fibroblasts. Cardiovasc Res 65(1):40–51. doi: 10.1016/j.cardiores.2004.08.020 CrossRefPubMedGoogle Scholar
  10. 10.
    Kohl P, Gourdie RG (2014) Fibroblast-myocyte electrotonic coupling: does it occur in native cardiac tissue? J Mol Cell Cardiol. doi: 10.1016/j.yjmcc.2013.12.024 PubMedPubMedCentralGoogle Scholar
  11. 11.
    Rohr S (2012) Arrhythmogenic implications of fibroblast-myocyte interactions. Circ Arrhythm Electrophysiol 5(2):442–452. doi: 10.1161/circep.110.957647 CrossRefPubMedGoogle Scholar
  12. 12.
    Pedrotty DM, Klinger RY, Kirkton RD, Bursac N (2009) Cardiac fibroblast paracrine factors alter impulse conduction and ion channel expression of neonatal rat cardiomyocytes. Cardiovasc Res 83(4):688–697. doi: 10.1093/cvr/cvp164, cvp164 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Vasquez C, Mohandas P, Louie KL, Benamer N, Bapat AC, Morley GE (2010) Enhanced fibroblast-myocyte interactions in response to cardiac injury. Circ Res 107(8):1011–1020. doi: 10.1161/CIRCRESAHA.110.227421 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Miragoli M, Salvarani N, Rohr S (2007) Myofibroblasts induce ectopic activity in cardiac tissue. Circ Res 101(8):755–758. doi: 10.1161/CIRCRESAHA.107.160549 PubMedGoogle Scholar
  15. 15.
    Miragoli M, Gaudesius G, Rohr S (2006) Electrotonic modulation of cardiac impulse conduction by myofibroblasts. Circ Res 98(6):801–810. doi: 10.1161/01.RES.0000214537.44195.a3 CrossRefPubMedGoogle Scholar
  16. 16.
    Vasquez C, Morley GE (2012) The origin and arrhythmogenic potential of fibroblasts in cardiac disease. J Cardiovasc Transl Res 5(6):760–767. doi: 10.1007/s12265-012-9408-1 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Chen W, Frangogiannis NG (2013) Fibroblasts in post-infarction inflammation and cardiac repair. Biochim Biophys Acta 1833(4):945–953. doi: 10.1016/j.bbamcr.2012.08.023 CrossRefPubMedGoogle Scholar
  18. 18.
    Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, Gottschalk A (2005) Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol 15(24):2279–2284. doi: 10.1016/j.cub.2005.11.032, S0960-9822(05)01407-7 [pii]CrossRefPubMedGoogle Scholar
  19. 19.
    Han X, Chow BY, Zhou H, Klapoetke NC, Chuong A, Rajimehr R, Yang A, Baratta MV, Winkle J, Desimone R, Boyden ES (2011) A high-light sensitivity optical neural silencer: development and application to optogenetic control of non-human primate cortex. Front Syst Neurosci 5:18. doi: 10.3389/fnsys.2011.00018 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Chow BY, Han X, Dobry AS, Qian X, Chuong AS, Li M, Henninger MA, Belfort GM, Lin Y, Monahan PE, Boyden ES (2010) High-performance genetically targetable optical neural silencing by light-driven proton pumps. Nature 463(7277):98–102. doi: 10.1038/nature08652 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Dugue GP, Akemann W, Knopfel T (2012) A comprehensive concept of optogenetics. Prog Brain Res 196:1–28. doi: 10.1016/B978-0-444-59426-6.00001-X CrossRefPubMedGoogle Scholar
  22. 22.
    Entcheva E (2013) Cardiac optogenetics. Am J Physiol Heart Circ Physiol 304(9):H1179–H1191. doi: 10.1152/ajpheart.00432.2012 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Ambrosi CM, Klimas A, Yu J, Entcheva E (2014) Cardiac applications of optogenetics. Prog Biophys Mol Biol 115(2-3):294–304. doi: 10.1016/j.pbiomolbio.2014.07.001 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Yu JZ, Boyle PM, Ambrosi CM, Trayanova NA, Entcheva E (2013) High-throughput contactless optogenetic assay for cellular coupling: illustration by Chr2-light-sensitized cardiac fibroblasts and cardiomyocytes. Circulation 128(22)Google Scholar
  25. 25.
    Pedrotty DM, Klinger RY, Badie N, Hinds S, Kardashian A, Bursac N (2008) Structural coupling of cardiomyocytes and noncardiomyocytes: quantitative comparisons using a novel micropatterned cell pair assay. Am J Physiol Heart Circ Physiol 295(1):H390–H400. doi: 10.1152/ajpheart.91531.2007 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Nguyen H, Badie N, McSpadden L, Pedrotty D, Bursac N (2014) Quantifying electrical interactions between cardiomyocytes and other cells in micropatterned cell pairs. Methods Mol Biol 1181:249–262. doi: 10.1007/978-1-4939-1047-2_21 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Arrenberg AB, Stainier DY, Baier H, Huisken J (2010) Optogenetic control of cardiac function. Science 330(6006):971–974. doi: 10.1126/science.1195929 CrossRefPubMedGoogle Scholar
  28. 28.
    Bruegmann T, Malan D, Hesse M, Beiert T, Fuegemann CJ, Fleischmann BK, Sasse P (2010) Optogenetic control of heart muscle in vitro and in vivo. Nat Methods 7(11):897–900. doi: 10.1038/nmeth.1512 CrossRefPubMedGoogle Scholar
  29. 29.
    Abilez OJ, Wong J, Prakash R, Deisseroth K, Zarins CK, Kuhl E (2011) Multiscale computational models for optogenetic control of cardiac function. Biophys J 101(6):1326–1334. doi: 10.1016/j.bpj.2011.08.004 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Williams JC, Xu J, Lu Z, Klimas A, Chen X, Ambrosi CM, Cohen IS, Entcheva E (2013) Computational optogenetics: empirically-derived voltage- and light-sensitive channelrhodopsin-2 model. PLoS Comput Biol 9(9):e1003220. doi: 10.1371/journal.pcbi.1003220 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Vogt CC, Bruegmann T, Malan D, Ottersbach A, Roell W, Fleischmann BK, Sasse P (2015) Systemic gene transfer enables optogenetic pacing of mouse hearts. Cardiovasc Res. doi: 10.1093/cvr/cvv004 PubMedGoogle Scholar
  32. 32.
    Ambrosi C, Entcheva E (2014) Optogenetic control of cardiomyocytes via viral delivery. In: Radisic M, Black Iii LD (eds) Cardiac tissue engineering, vol 1181, Methods in molecular biology. Springer, New York, pp 215–228. doi: 10.1007/978-1-4939-1047-2_19 Google Scholar
  33. 33.
    Nussinovitch U, Shinnawi R, Gepstein L (2014) Modulation of cardiac tissue electrophysiological properties with light-sensitive proteins. Cardiovasc Res 102(1):176–187. doi: 10.1093/cvr/cvu037 CrossRefPubMedGoogle Scholar
  34. 34.
    Jia Z, Valiunas V, Lu Z, Bien H, Liu H, Wang HZ, Rosati B, Brink PR, Cohen IS, Entcheva E (2011) Stimulating cardiac muscle by light: cardiac optogenetics by cell delivery. Circ Arrhythm Electrophysiol 4(5):753–760. doi: 10.1161/CIRCEP.111.964247 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Entcheva E, Bien H (2009) Mechanical and spatial determinants of cytoskeletal geodesic dome formation in cardiac fibroblasts. Integr Biol 1(2):212–219. doi: 10.1039/B818874b CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringStony Brook UniversityStony BrookUSA
  2. 2.Department of Biomedical Engineering, Institute for Molecular CardiologyStony Brook UniversityStony BrookUSA

Personalised recommendations