Submitted on April 20, 2007
Accepted on July 31, 2007
Lentiviral Vector Mediated Expression of GFP or Kir2.1 Alters the Electrophysiology of Neonatal Rat Ventricular Myocytes without Inducing Cytotoxicity
Rajesh Babu Sekar1, Eddy Kizana2, Rachel Ruckdeschel Smith3, Andreas S Barth4, Yibing Zhang5, Eduardo Marban4, and Leslie Tung5*
1 Department of Biomedical Engineering, The Johns Hopkins University - School of Medicine, Baltimore, Maryland, United States
2 Division of Cardiology, The Johns Hopkins University - School of Medicine, Baltimore, Maryland, United States
3 Baltimore, Maryland, United States; Department of Biomedical Engineering, The Johns Hopkins University - School of Medicine, Baltimore, Maryland, United States; Division of Cardiology, The Johns Hopkins University - School of Medicine, Baltimore, Maryland, United States
4 Baltimore, Maryland, United States; Division of Cardiology, The Johns Hopkins University - School of Medicine, Baltimore, Maryland, United States
5 Baltimore, Maryland, United States; Department of Biomedical Engineering, The Johns Hopkins University - School of Medicine, Baltimore, Maryland, United States
* To whom correspondence should be addressed. E-mail: ltung{at}jhu.edu.
Recombinant lentiviral vectors (LVs) are capable of transducing neonatal rat ventricular myocytes (NRVMs) and providing stable, long-term transgene expression. The goal of the present study was to comprehensively test whether transduction of NRVMs by LVs results in cytotoxicity and to examine the electrophysiological consequences of gene-modification of NRVM monolayers by two vectors, one encoding a putatively inert enhanced green fluorescent protein (eGFP) and the other a major ion channel protein, Kir2.1. Freshly isolated NRVMs were transduced and cultured in monolayers. Immunohistochemistry, Trypan Blue exclusion, Annexin V binding followed by flow cytometry, and Terminal transferase dUTP Nick End Labeling (TUNEL) assays were performed to assess for cytotoxicity. Optical mapping studies of action potential propagation in NRVM monolayers were performed to characterize the electrophysiological alterations following transduction. The cytotoxicity assays revealed that transduction had no adverse effects on NRVM cultures. However, eGFP-transduced monolayers exhibited a decrease in conduction velocity (CV) and action potential duration (APD) when compared with monolayers transduced with LVs encoding LacZ or devoid of a transgene. In addition, siRNA-mediated knockdown of eGFP expression corrected this phenotype. In contrast, Kir2.1 gene-modified monolayers showed an increase in CV and a predictable decrease in APD. This study demonstrates that LVs transduce NRVMs without cytotoxic effects. However, eGFP has a significant effect on APD and CV in this experimental system, and calls into question the widely held belief that GFP is physiologically inert. In addition, LV-mediated overexpression of Kir2.1 opens up the prospect of studying the functional role of IK1 current in cardiac arrhythmias.
