[Pacemaker current gene expression of rat mesenchymal stem cells and identification of mesenchymal stem cells expressing human pacemaker current gene (hHCN2)].
OBJECTIVE
To study pacemaker current gene expression of mesenchymal stem cells (MSCs) and the electrophysiological property of MSCs expressing human pacemaker current gene.


METHODS
Pacemaker current gene expression of MSCs were studied by real-time quantitative polymerase chain reaction (real-time PCR) and pcDNA3-hHCN2 was transfected with Lipofectin 2000 into MSCs. hHCN2 expression at mRNA and at protein levels in the transfected cells were identified by real-time PCR and Western blot, respectively. The ionic currents of cloned hHCN2 (IhHCN2) were recorded and the current characteristics were studied through the whole-cell patch clamp technique.


RESULTS
mHCN1, mHCN2, mHCN3, mHCN4 represent (0.08+/-0.01)%, (77.16+/-0.03)%, (0.24+/-0.01)%, (22.53+/-0.02)% of total HCN mRNA in MSCs as determined by real-time PCR. Transfected hHCN2 ionic currents were recorded by whole-cell patch clamp and current density-voltage curves were obtained. The threshold for activation of IhHCN2 was approximately -80 mV and this current could be blocked by Cs+ (4 mmol/L). hHCN2 expression in transfected MSCs was detected both at mRNA and protein levels.


CONCLUSIONS
1. mHCN2 and mHCN4 represent the major populations of total HCN mRNA in MSCs. 2. Plasmid pcDNA3-hHCN2 by Lipofectin could be successfully transfected into MSCs with IhHCN2 recorded by whole-cell patch clamp technique, this study provides a basis for future antiarrhythmic gene therapy.
Li
Hong-xia
HX
Department of Cardiology, the First Affiliated Hospital of Soochow University, Suzhou 215006, China.
Yang
Xiang-jun
XJ
Zhao
Xin
X
Jiang
Bin
B
Cheng
Xu-jie
XJ
Chen
Tan
T
Han
Lian-hua
LH
Song
Jian-ping
JP
Liu
Zhi-hua
ZH
Jiang
Wen-ping
WP
D000818:Animals; D054815:Cyclic Nucleotide-Gated Cation Channels; D015870:Gene Expression; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D008564:Membrane Potentials / Q000502:physiology; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
D054815:Cyclic Nucleotide-Gated Cation Channels; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015221:Potassium Channels