Use of rats mesenchymal stem cells modified with mHCN2 gene to create biologic pacemakers.
The possibility of rats mesenchymal stem cells (MSCs) modified with murine hyperpolarization-activated cyclic nucleotide-gated 2 (mHCN2) gene as biological pacemakers in vitro was studied. The cultured MSCs were transfected with pIRES2-EGFP plasmid carrying enhanced green fluorescent protein (EGFP) gene and mHCN2 gene. The identification using restriction enzyme and sequencing indicated that the mHCN2 gene was inserted to the pIRES2-EGFP. Green fluorescence could be seen in MSCs after transfection for 24-48 h. The expression of mHCN2 mRNA and protein in the transfected cells was detected by RT-PCR and Western blot, and the quantity of mHCN2 mRNA and protein expression in transfected MSCs was 5.31 times and 7.55 times higher than that of the non-transfected MSCs respectively (P<0.05, P<0.05). I(HCN2) was recorded by whole-cell patch clamp method. The effect of Cs+, a specific blocker of pacemaker current, was measured after perfusion by patch clamp. The results of inward current indicated that there was no inward current recording in non-transfected MSCs and a large voltage-dependent inward and Cs+-sensitive current activated on hyperpolarizations presented in the transfected MSCs. I(HCN2) was fully activated around -140 mV with an activation threshold of -60 mV. The midpoint (V50) was -95.1+/-0.9 mV (n=9). The study demonstrates that mHCN2 mRNA and protein can be expressed and the currents of HCN2 channels can be detected in genetically modified MSCs. The gene-modified MSCs present a novel method for pacemaker genes into the heart or other electrical syncytia.
Ma
Jin
J
Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. majin556@126.com
Zhang
Cuntai
C
Huang
Shen
S
Wang
Guoqiang
G
Quan
Xiaoqing
X
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics; D006339:Heart Rate; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000096:biosynthesis / Q000235:genetics*; D008564:Membrane Potentials / Q000502:physiology; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D014162:Transfection
C578712:Hcn2 protein, rat; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D015221:Potassium Channels; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins