title: Electric pulse current stimulation increases electrophysiological properties of Ifcurrent reconstructed in mHCN4-transfected canine mesenchymal stem cells.
abstract: The 'funny' current, also known as the Ifcurrent, play a crucial role in the spontaneous diastolic depolarization of sinoatrial node cells. The Ifcurrent is primarily induced by the protein encoded by the hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) gene. The functional Ifchannel can be reconstructed in canine mesenchymal stem cells (cMSCs) transfected with mouseHCN4(mHCN4). Biomimetic studies have shown that electric pulse current stimulation (EPCS) can promote cardiogenesis in cMSCs. However, whether EPCS is able to influence the properties of the Ifcurrent reconstructed in mHCN4-transfected cMSCs remains unclear. The present study aimed to investigate the effects of EPCS on the Ifcurrent reconstructed in mHCN4-transfected cMSCs. The cMSCs were transfected with the lentiviral vector pLentis-mHCN4-GFP. Following transfection, these cells were divided into two groups: mHCN4-transfected cMSCs (group A), and mHCN4-transfected cMSCs induced by EPCS (group B). Using a whole cell patch-clamp technique, the Ifcurrent was recorded, and group A cMSCs showed significant time and voltage dependencies and sensitivity to extracellular Cs+. The half-maximal activation (V1/2) value was -101.2±4.6 mV and the time constant of activation was 324±41 msec under -160 mV. In the group B cells the Ifcurrent increased obviously and activation curve moved to right. The absolute value of V1/2 increased significantly to -92.4±4.8 mV (P<0.05), and the time constant of activation diminished under the same command voltage (251±44 vs. 324±41, P<0.05). In addition, the mRNA and protein expression levels ofHCN4, connexin 43 (Cx43) and Cx45 were upregulated in group B compared with group A, as determined by reverse transcription-quantitative polymerase chain reaction and western blot analyses. Transmission electron micrographs also confirmed the increased gap junctions in group B. Collectively, these results indicated that reconstructed Ifchannels may have a positive regulatory role in EPCS induction. The cMSCs transfected with mHCN4induced by EPCS may be a source of effective biological pacemaker cells.
authors.affiliation: Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, P.R. China; Department of Cardiology, The 401 Hospital of PLA, Qingdao, Shandong 266071, P.R. China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, P.R. China.
authors.forename: Shouming
authors.initials: S
authors.lastname: Luo
authors.affiliation: Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, P.R. China.
authors.forename: Pan
authors.initials: P
authors.lastname: Yang
authors.affiliation: Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, P.R. China.
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Song
keywords: HCN4; If current; electric pulse current stimulation; gap junction; mesenchymal stem cells