Gene transfer of connexin43 mutants attenuates coupling in cardiomyocytes: novel basis for modulation of cardiac conduction by gene therapy.
Modification of electrical conduction would be a useful principle to recruit in preventing or treating certain arrhythmias, notably ventricular tachycardia (VT). Here we pursue a novel gene transfer approach to modulate electrical conduction by reducing gap junctional intercellular communication (GJIC) and hence potentially modify the arrhythmia substrate. The ultimate goal is to develop a nondestructive approach to uncouple zones of slow conduction by focal gene transfer. Lentiviral vectors encoding connexin43 (Cx43) internal loop mutants were produced and studied in vitro. Transduction of neonatal rat ventricular myocytes (NRVMs) revealed the expected subcellular localization of the mutant gene product. Fluorescent dye transfer studies showed a significant reduction of GJIC in NRVMs that had been genetically modified. Additionally, adjacent mutant gene-modified NRVMs displayed delayed calcium transients, indicative of electrical uncoupling. Multi-site optical mapping of action potential (AP) propagation in gene-modified NRVM monolayers revealed a 3-fold slowing of conduction velocity (CV) relative to nontransduced NRVMs. In conclusion, lentiviral vector-mediated gene transfer of Cx43 mutants reduced GJIC in NRVMs. Electrical charge transfer was also reduced as evidenced by delayed calcium transients in adjacent NRVMs and reduced CV in NRVM monolayers. These data validate a molecular tool that opens the prospect for gene transfer targeting gap junctions as an approach to modulate cardiac conduction.
Kizana
Eddy
E
Gene Therapy Research Unit, The Children's Hospital at Westmead and Children's Medical Research Institute, Westmead, Australia.
Chang
Connie Y
CY
Cingolani
Eugenio
E
Ramirez-Correa
Genaro A
GA
Sekar
Rajesh B
RB
Abraham
M Roselle
MR
Ginn
Samantha L
SL
Tung
Leslie
L
Alexander
Ian E
IE
Marbán
Eduardo
E
D000818:Animals; D000831:Animals, Newborn; D020013:Calcium Signaling / Q000235:genetics; D002450:Cell Communication / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D018031:Connexin 43 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D005456:Fluorescent Dyes / Q000378:metabolism / Q000493:pharmacokinetics; D017629:Gap Junctions / Q000235:genetics / Q000502:physiology*; D018014:Gene Transfer Techniques; D005799:Genes, Dominant; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006329:Heart Conduction System / Q000502:physiology*; D006352:Heart Ventricles / Q000166:cytology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D017384:Sequence Deletion
D018031:Connexin 43; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins