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Engineering Biosynthetic Excitable Tissues from Unexcitable Cells for Electrophysiological and Cell Therapy Studies

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Engineering Biosynthetic Excitable Tissues from Unexcitable Cells for Electrophysiological and Cell Therapy Studies ARTICLE Received 11 Nov 2010 | Accepted 5 Apr 2011 | Published 10 May 2011 DOI: 10.1038/ncomms1302 Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies Robert D. Kirkton1 & Nenad Bursac1 Patch-clamp recordings in single-cell expression systems have been traditionally used to study the function of ion channels. However, this experimental setting does not enable assessment of tissue-level function such as action potential (AP) conduction. Here we introduce a biosynthetic system that permits studies of both channel activity in single cells and electrical conduction in multicellular networks. We convert unexcitable somatic cells into an autonomous source of electrically excitable and conducting cells by stably expressing only three membrane channels. The specific roles that these expressed channels have on AP shape and conduction are revealed by different pharmacological and pacing protocols. Furthermore, we demonstrate that biosynthetic excitable cells and tissues can repair large conduction defects within primary 2- and 3-dimensional cardiac cell cultures. This approach enables novel studies of ion channel function in a reproducible tissue-level setting and may stimulate the development of new cell-based therapies for excitable tissue repair. 1 Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708, USA. Correspondence and requests for materials should be addressed to N.B. (email: [email protected]). NatURE COMMUNicatiONS | 2:300 | DOI: 10.1038/ncomms1302 | www.nature.com/naturecommunications © 2011 Macmillan Publishers Limited. All rights reserved. ARTICLE NatUre cOMMUNicatiONS | DOI: 10.1038/ncomms1302 ll cells express ion channels in their membranes, but cells a b with a significantly polarized membrane that can undergo e 0 a transient all-or-none membrane depolarization (action A 1 potential, AP) are classified as ‘excitable cells’ . The coordinated –40 function of ion channels in excitable cells governs the generation Membran –80 and propagation of APs, which enable fundamental life processes potential (mV) 0 1.5 3 such as the rapid transfer of information in nerves2 and the synchro- wt HEK-293 Time (s) nized pumping of the heart3. For this reason, genetic or acquired c d alterations in ion channel function or irreversible loss of excitable 0 e + 1 mMBaCl cells through injury or disease (for example, stroke or heart attack) 2 are often life threatening4,5. –40 Numerous ion channels (wild type (wt) or mutated) have been studied in single-cell heterologous expression systems to investigate Membran –80 potential (mV) channel structure–function relationships and link specific channel 0 1.5 3 Time (s) mutations found in patients to associated diseases, such as cardiac Kir2.1 arrhythmias or epilepsy6. Typically, the potential implications of e f +40 these single-cell studies for the observed tissue- or organ-level func- e tion are only speculated, often through the use of tissue-specific 0 7,8 computational models . Similarly, experimental studies of AP con- –40 duction in primary excitable tissues and cell cultures are often lim- Membran ited by low reproducibility, heterogeneous structure and function, potential (mV) –80 0 250 500 a diverse and often unknown complement of endogenous chan- Kir2.1 + Na 1.5 Time (ms) nels, and the non-specific action of applied pharmaceuticals. We v therefore set out to develop and validate a simplified, well-defined g h and reproducible excitable tissue system that would enable direct � quantitative studies of the roles that specific ion channels have in = 23 cm AP conduction. Extensive electrophysiological research over the last century1,9 s –1 has revealed that the initiation, shape and transfer of APs in excit- 0 700 1,400 Kir2.1 + Na 1.5 + Cx43 able cells are regulated by an extraordinarily diverse set of ion chan- v Time (ms) nels, pumps and exchangers. Yet, the classic Hodgkin and Huxley bioelectric model of a giant squid axon10 and other simplified mod- els of biological excitable media11,12 suggest that only a few mem- Figure 1 | Stable coexpression of three genes confers impulse conduction brane channels are sufficient to sustain cellular excitability and AP in unexcitable cells. wt HEK-293 cells (a), like most unexcitable cells, conduction. On the basis of these theoretical concepts, we hypoth- have a relatively depolarized resting potential (b). Scale bar, 10 µm. esized that a small number of targeted genetic manipulations could Stable expression of Kir2.1-IRES-mCherry (c) introduces inward-rectifier transform unexcitable somatic cells into an electrically active tissue potassium current in the cell yielding membrane hyperpolarization (d). Coexpression of Na 1.5–IRES–GFP (e) introduces fast sodium current capable of generating and propagating APs. v In this study, we selected a minimum set of channel genes that, that allows firing of regenerative APs on stimulation (f). The additional upon stable expression in unexcitable cells, would yield significant expression of Cx43–IRES–mOrange (g) enhances cell–cell coupling and hyperpolarization of membrane potential, electrical induction of an enables fast and uniform AP propagation (h) in multicellular tissues. all-or-none AP response and robust intercellular electrical coupling θ, Velocity of AP propagation. to support uniform and fast AP conduction over arbitrarily long distances. We designed experiments to thoroughly characterize the electrophysiological properties of these genetically engineered unique fluorescent reporter18 (mCherry, green fluorescent protein cells including pharmacological manipulations to establish the roles (GFP) or mOrange, respectively) and the puromycin resistance of each of the expressed channels in membrane excitability and (PacR) gene (Supplementary Fig. S1). The human embryonic impulse conduction. Furthermore, we explored whether these cells ­kidney 293 (HEK-293) cell line was utilized as a proof-of-concept could be used to generate biosynthetic excitable tissues with the unexcitable somatic cell source based on its low levels of endo­ ability to restore electrical conduction within large cm-sized gaps in genous membrane currents (for example, outwardly rectifying primary excitable cell cultures. potassium currents19), uniform shape and growth, and extensive use as a heterologous expression system for studies of ion chan- Results nel function20. Like most unexcitable cells, wt HEK-293 cells Experimental approach to engineering excitable cells. We tested (Fig. 1a) exhibited a relatively depolarized resting membrane the hypothesis that human unexcitable somatic cells can be potential (RMP) of − 24.4 ± 0.8 mV ( ± s.e.m.; n = 10 cells; Fig. 1b). genetically engineered to form an autonomous source of electrically In contrast, the RMP of excitable cells is highly negative due to the excitable and conducting cells through the stable expression of three action of constitutively open and/or inwardly rectifying potassium genes encoding: the inward-rectifier potassium channel (Kir2.1 or channels such as Kir2.1 (ref. 21). IRK1, gene KCNJ2)13, the pore-forming α-subunit of the fast voltage- 14 gated cardiac sodium channel (Nav1.5 or hH1, gene SCN5A) and IK1 and INa expression enables AP generation. To induce significant the connexin-43 gap junction (Cx43, gene GJA1)15. These three membrane hyperpolarization in the wt HEK-293 cells, we stably channels have critical roles in the generation and propagation of transfected them with a plasmid encoding Kir2.1–IRES–mCherry electrical activity in the mammalian heart16,17. and derived ‘Kir2.1 HEK-293’ monoclonal lines from cells that To facilitate the visual identification and monoclonal selec- displayed bright mCherry fluorescence Fig.( 1c). These stable cell tion of stable cell lines, we constructed three bicistronic plasmids lines exhibited robust barium-sensitive inward-rectifier potassium designed to express each channel (Kir2.1, Nav1.5 or Cx43) with a ­current, IK1 (Supplementary Fig. S2 and Fig. 2a,b), and hyperpolarized NatUre cOMMUNicatiONS | 2:300 | DOI: 10.1038/ncomms1302 | www.nature.com/naturecommunications © 2011 Macmillan Publishers Limited. All rights reserved. NatURE COMMUNicatiONS | DOI: 10.1038/ncomms1302 ARTICLE a d f i 00 mV + TTX: Im nA I 1 50 µM V m Vmm 4 ms 10 µM 0 nA 5 µM –76-76 mV Kir2.Kir2.11 HEK- HEK-29293 3 1 µM mV mV 20 nA 10 ms 20 nA 0.5 µM 10 ms 4 2 nA 00 mV mV 200 ms 4 4 ms 0 µM 10 ms V e g V mm V (mV) + 1 mM BaCl2 m –70 –30 30 70 –75-75 mV Kir2.1+Na 1.5 HEK-293 Kir2.1 + Navv1.5 HEK-293 –200 ) nA nA 4 2 (pA/pF j 200 ms 10 ms –400 0 mV V –600 V m nA V m V (mV) m b m 4 25 525 mV ms 5 ms 4 ms –800 –130 –30 30 –75-75 mV mV –100 II 1.0 1.0 mm ) h Inactivation 0 nA 0.9 0.9 0 nA –200 Activation 0.8 0.8 (pA/pF 0.7 0.7 Na Na + 1 mM BaCl –300 I + 1mM BaCl 2 G 2 0.6 0.6 0 nA 0.5 0.5 II m HEK-293 cell lines 0.4 0.4 m c nA Normalized 0 –20 mV Normalized wt + + + 0.3 0.3 nA –40 mV 0.5 +40 mV 5 ms –20 Kir2.1 Kir2.1 Kir2.1 ∆ 5 mV 5 0.2 ∆ 5 mV 0.2 0. 5 ms Nav1.5 Nav1.5 –100 mV –40 Cx43 –80 mV 0.1 –130 mV –100 mV 40 ms 0.1 I 500 ms Imm –60 0 0 + +5 1µM µ MTT TTX X RMP (mV) –140 –120 –100 –80 –60 –40 –20 0 20 00 nAnA –80 * * * Test potential (mV) Figure 2 | Stable expression of Kir2.1 and Nav1.5 yields membrane excitability in HEK-293 cells. (a) Kir2.1 + Nav1.5 HEK-293 cells exhibited BaCl2- sensitive IK1. Activation of INa also occurred at the end of several IK1 test pulses (insets). (b) Steady-state IK1–V curves obtained from Kir2.1 + Nav1.5 (black squares; n = 9) and wt (white circles; n = 6) HEK-293 cells.
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