Development of functional I f channels in mMSCs after transfection with mHCN4: effects on cell morphology and mechanical activity in vitro.
OBJECTIVE
To study the functional properties of I(f) channels and the changes in mechanical activity of mouse mesenchymal stem cells (mMSCs) transfected with mHCN4.


METHODS
mMSCs were purified by using CD11b-immunomagnetic microbeads and transfected with pMSCV-mHCN4-EGFP or pMSCV-EGFP. We examined the kinetic characteristics of the mHCN4 channel. The morphological changes of positively transfected mMSCs were investigated at the same time.


RESULTS
The I(f) current recorded from the experimental group was sensitive to extracellular Cs(+) (-44.5 +/- 4.2 vs. -5.5 +/- 1.0 pA/pF, p < 0.001). The half-maximal activation was -99.0 +/- 5.8 mV. The time constant of activation was 451 +/- 61 ms under -140 mV. The control cells did not show the current under the same conditions. The absolute values of half-maximal activation decreased in the presence of cAMP or cGMP in the experimental group (-78.6 +/- 10.4 and -85.7 +/- 8.6 vs. -99.0 +/- 5.8 mV, respectively, p < 0.05). mMSCs transfected with pMSCV-mHCN4-EGFP could form spontaneous beating cells. Extracellular Cs(+) decreased the beating rate significantly (196 +/- 50 vs. 66 +/- 23 bmp, p < 0.01).


CONCLUSIONS
Functional I(f) channels can be reconstructed in mMSCs infected with mHCN4. mMSCs modified by successful transfection with mHCN4 can differentiate so as to develop spontaneous mechanical activity.
Tong
Shifei
S
Department of Cardiology, Southwest Hospital, Third Military Medical Academy, Chongqing, China.
Yao
Qing
Q
Wan
Ying
Y
Zhou
Jianzhi
J
Shu
Maoqin
M
Zhong
Li
L
Li
Yonghua
Y
Zhang
Qian
Q
Yindai
Jiajia
J
Song
Zhiyuan
Z
D000818:Animals; D001683:Biological Clocks / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D002586:Cesium / Q000493:pharmacokinetics; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000502:physiology*; D051379:Mice; D008815:Mice, Inbred Strains; D018408:Patch-Clamp Techniques; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
D054815:Cyclic Nucleotide-Gated Cation Channels; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D002586:Cesium; D000242:Cyclic AMP; D006152:Cyclic GMP