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. (c) 2008 S. Karger AG, Basel.
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. (c) 2008 S. Karger AG, Basel.