Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling.
BACKGROUND
Cardiac conduction occurs in an electrical syncytium of excitable cells connected by gap junctions. Disruption of these electrophysiological properties causes conduction slowing or block. Depending on the location of affected cells within the heart, this has the potential to result in clinical syndromes such as atrioventricular block. With a view to developing gene therapy strategies for repairing cardiac conduction defects, we sought to establish whether the phenotype of fibroblasts can be modified by gene transfer to produce cells capable of electrical excitation and coupling.


RESULTS
High-titer lentiviral vectors encoding MyoD, a myogenic transcription factor, and connexin43, a gap junction protein, were produced by established methods. Human dermal fibroblasts (HDFs) were efficiently (>80%) transduced at a multiplicity of infection of 50. HDFs transduced with the MyoD-encoding vector underwent myogenic conversion, as evidenced by myotube formation and detection of muscle-specific proteins. Importantly, calcium transients indicative of membrane excitability were observed in MyoD-induced myotubes after loading with a calcium-sensitive dye and electrical stimulation. Transients from adjacent myotubes displayed different excitation thresholds, indicating an absence of coupling between cells, consistent with skeletal muscle biology. In contrast, simultaneous transduction of HDFs with MyoD and connexin43-encoding vectors resulted in the appearance of transients in adjacent myotubes with identical thresholds, indicative of electrical coupling. Notably, dye transfer studies confirmed gap junctional intercellular communication.


CONCLUSIONS
Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling. These observations strengthen the prospect of developing gene-based strategies for repairing cardiac conduction defects.
Kizana
Eddy
E
Department of Cardiology, Westmead Hospital, Westmead, Australia.
Ginn
Samantha L
SL
Allen
David G
DG
Ross
David L
DL
Alexander
Ian E
IE
D000818:Animals; D020013:Calcium Signaling; D002450:Cell Communication; D002454:Cell Differentiation; D002478:Cells, Cultured / Q000166:cytology / Q000502:physiology; D004396:Coloring Agents / Q000032:analysis; D018031:Connexin 43 / Q000235:genetics / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D017629:Gap Junctions / Q000502:physiology / Q000648:ultrastructure; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D015726:Giant Cells / Q000502:physiology; D006367:HeLa Cells / Q000166:cytology; D006329:Heart Conduction System / Q000166:cytology* / Q000502:physiology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D017570:MyoD Protein / Q000235:genetics / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000502:physiology; D012867:Skin / Q000166:cytology; D014161:Transduction, Genetic
D004396:Coloring Agents; D018031:Connexin 43; D018076:DNA, Complementary; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D011993:Recombinant Fusion Proteins