Genetically Engineered Excitable Cardiac Myofibroblasts Coupled to Cardiomyocytes Rescue Normal Propagation and Reduce Arrhythmia Complexity in Heterocellular Monolayers
Rationale and Objective The use of genetic engineering of unexcitable cells to enable expression of gap junctions and inward rectifier potassium channels has suggested that cell therapies aimed at establishing electrical coupling of unexcitable donor cells to host cardiomyocytes may be arrhythmogenic. Whether similar considerations apply when the donor cells are electrically excitable has not been investigated. Here we tested the hypothesis that adenoviral transfer of genes coding Kir2.1 (IK1), NaV1.5 (INa) and connexin-43 (Cx43) proteins into neonatal rat ventricular myofibroblasts (NRVF) will convert them into fully excitable cells, rescue rapid conduction velocity (CV) and reduce the incidence of complex reentry arrhythmias in an in vitro model. Methods and Results We used adenoviral (Ad-) constructs encoding Kir2.1, NaV1.5 and Cx43 in NRVF. In single NRVF, Ad-Kir2.1 or Ad-NaV1.5 infection enabled us to regulate the densities of IK1 and INa, respectively. At varying MOI ratios of 10/10, 5/10 and 5/20, NRVF co-infected with Ad-Kir2.1+ NaV1.5 were hyperpolarized and generated action potentials (APs) with upstroke velocities >100 V/s. However, when forming monolayers only the addition of Ad-Cx43 made the excitable NRVF capable of conducting electrical impulses (CV = 20.71±0.79 cm/s). When genetically engineered excitable NRVF overexpressing Kir2.1, NaV1.5 and Cx43 were used to replace normal NRVF in heterocellular monolayers that included neonatal rat ventricular myocytes (NRVM), CV was significantly increased (27.59±0.76 cm/s vs. 21.18±0.65 cm/s, p<0.05), reaching values similar to those of pure myocytes monolayers (27.27±0.72 cm/s). Moreover, during reentry, propagation was faster and more organized, with a significantly lower number of wavebreaks in heterocellular monolayers formed by excitable compared with unexcitable NRVF. Conclusion Viral transfer of genes coding Kir2.1, NaV1.5 and Cx43 to cardiac myofibroblasts endows them with the ability to generate and propagate APs. The results provide proof of concept that cell therapies with excitable donor cells increase safety and reduce arrhythmogenic potential.
References
[1]
Choudry FA, Mathur A (2011) Stem cell therapy in cardiology. Regen Med 6: 17–23.
[2]
Cho HC, Kashiwakura Y, Marban E (2007) Creation of a biological pacemaker by cell fusion. Circ Res 100: 1112–1115.
[3]
Plotnikov AN, Shlapakova I, Szabolcs MJ, Danilo P Jr, Lorell BH, et al. (2007) Xenografted adult human mesenchymal stem cells provide a platform for sustained biological pacemaker function in canine heart. Circulation 116: 706–713.
[4]
Feld Y, Melamed-Frank M, Kehat I, Tal D, Marom S, et al. (2002) Electrophysiological modulation of cardiomyocytic tissue by transfected fibroblasts expressing potassium channels: a novel strategy to manipulate excitability. Circulation 105: 522–529.
[5]
Yankelson L, Feld Y, Bressler-Stramer T, Itzhaki I, Huber I, et al. (2008) Cell therapy for modification of the myocardial electrophysiological substrate. Circulation 117: 720–731.
[6]
McSpadden LC, Nguyen H, Bursac N (2012) Size and Ionic Currents of Unexcitable Cells Coupled to Cardiomyocytes Distinctly Modulate Cardiac Action Potential Shape and Pacemaking Activity in Micropatterned Cell Pairs. Circ Arrhythm Electrophysiol.
[7]
Miragoli M, Gaudesius G, Rohr S (2006) Electrotonic modulation of cardiac impulse conduction by myofibroblasts. Circ Res 98: 801–810.
[8]
Zlochiver S, Munoz V, Vikstrom KL, Taffet SM, Berenfeld O, et al. (2008) Electrotonic myofibroblast-to-myocyte coupling increases propensity to reentrant arrhythmias in two-dimensional cardiac monolayers. Biophys J 95: 4469–4480.
[9]
Fozzard HA, Hanck DA (1996) Structure and function of voltage-dependent sodium channels: comparison of brain II and cardiac isoforms. Physiol Rev 76: 887–926.
[10]
Kohl P, Kamkin AG, Kiseleva IS, Noble D (1994) Mechanosensitive fibroblasts in the sino-atrial node region of rat heart: interaction with cardiomyocytes and possible role. Exp Physiol 79: 943–956.
[11]
Milstein ML, Musa H, Balbuena DP, Anumonwo JM, Auerbach DS, et al. (2012) Dynamic reciprocity of sodium and potassium channel expression in a macromolecular complex controls cardiac excitability and arrhythmia. Proc Natl Acad Sci U S A.
[12]
Noujaim SF, Pandit SV, Berenfeld O, Vikstrom K, Cerrone M, et al. (2007) Up-regulation of the inward rectifier K+ current (I K1) in the mouse heart accelerates and stabilizes rotors. J Physiol 578: 315–326.
[13]
Munoz V, Grzeda KR, Desplantez T, Pandit SV, Mironov S, et al. (2007) Adenoviral expression of IKs contributes to wavebreak and fibrillatory conduction in neonatal rat ventricular cardiomyocyte monolayers. Circ Res 101: 475–483.
[14]
He Y, Pan Q, Li J, Chen H, Zhou Q, et al. (2008) Kir2.3 knock-down decreases IK1 current in neonatal rat cardiomyocytes. FEBS Lett 582: 2338–2342.
[15]
Auerbach DS, Grzda KR, Furspan PB, Sato PY, Mironov S, et al. (2011) Structural heterogeneity promotes triggered activity, reflection and arrhythmogenesis in cardiomyocyte monolayers. J Physiol 589: 2363–2381.
[16]
Delmar M, Glass L, Michaels DC, Jalife J (1989) Ionic basis and analytical solution of the wenckebach phenomenon in guinea pig ventricular myocytes. Circ Res 65: 775–788.
[17]
Abbaci M, Barberi-Heyob M, Stines JR, Blondel W, Dumas D, et al. (2007) Gap junctional intercellular communication capacity by gap-FRAP technique: a comparative study. Biotechnol J 2: 50–61.
[18]
Walsh KB, Zhang J (2008) Neonatal rat cardiac fibroblasts express three types of voltage-gated K+ channels: regulation of a transient outward current by protein kinase C. Am J Physiol Heart Circ Physiol. 294: H1010–1017.
[19]
Gray RA, Pertsov AM, Jalife J (1998) Spatial and temporal organization during cardiac fibrillation. Nature 392: 75–78.
[20]
Pertsov AM, Davidenko JM, Salomonsz R, Baxter WT, Jalife J (1993) Spiral waves of excitation underlie reentrant activity in isolated cardiac muscle. Circ Res 72: 631–650.
[21]
Cho HC, Marban E (2010) Biological therapies for cardiac arrhythmias: can genes and cells replace drugs and devices? Circ Res 106: 674–685.
[22]
Potapova I, Plotnikov A, Lu Z, Danilo P Jr, Valiunas V, et al. (2004) Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res 94: 952–959.
[23]
Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, et al. (2003) Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 425: 968–973.
[24]
Weimann JM, Johansson CB, Trejo A, Blau HM (2003) Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant. Nat Cell Biol 5: 959–966.
[25]
Xue T, Cho HC, Akar FG, Tsang SY, Jones SP, et al. (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.
[26]
Kirkton RD, Bursac N (2011) Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies. Nat Commun 2: 300.
[27]
Kirkton RD, Bursac N (2012) Genetic engineering of somatic cells to study and improve cardiac function. Europace 14 Suppl 5: v40–v49.
[28]
Kohl P, Camelliti P, Burton FL, Smith GL (2005) Electrical coupling of fibroblasts and myocytes: relevance for cardiac propagation. Journal of electrocardiology 38: 45–50.
[29]
Gaudesius G, Miragoli M, Thomas SP, Rohr S (2003) Coupling of cardiac electrical activity over extended distances by fibroblasts of cardiac origin. Circ Res 93: 421–428.
[30]
Raizman JE, Komljenovic J, Chang R, Deng C, Bedosky KM, et al. (2007) The participation of the Na+-Ca2+ exchanger in primary cardiac myofibroblast migration, contraction, and proliferation. J Cell Physiol 213: 540–551.
[31]
De Maziere AM, van Ginneken AC, Wilders R, Jongsma HJ, Bouman LN (1992) Spatial and functional relationship between myocytes and fibroblasts in the rabbit sinoatrial node. J Mol Cell Cardiol 24: 567–578.
[32]
Camelliti P, Devlin GP, Matthews KG, Kohl P, Green CR (2004) Spatially and temporally distinct expression of fibroblast connexins after sheep ventricular infarction. Cardiovasc Res 62: 415–425.
[33]
Rosker C, Salvarani N, Schmutz S, Grand T, Rohr S (2011) Abolishing myofibroblast arrhythmogeneicity by pharmacological ablation of alpha-smooth muscle actin containing stress fibers. Circ Res 109: 1120–1131.
[34]
Jayawardena TM, Egemnazarov B, Finch EA, Zhang L, Payne JA, et al. (2012) MicroRNA-Mediated In Vitro and In Vivo Direct Reprogramming of Cardiac Fibroblasts to Cardiomyocytes. Circ Res 110: 1465–1473.
[35]
Greener I, Donahue JK (2011) Gene therapy strategies for cardiac electrical dysfunction. J Mol Cell Cardiol 50: 759–765.
