Electric pulse current stimulation increases electrophysiological properties of I current reconstructed in mHCN4-transfected canine mesenchymal stem cells.

The 'funny' current, also known as the I current, play a crucial role in the spontaneous diastolic depolarization of sinoatrial node cells. The I current is primarily induced by the protein encoded by the hyperpolarization-activated cyclic nucleotide-gated channel 4 () gene. The functional I channel can be reconstructed in canine mesenchymal stem cells (cMSCs) transfected with mouse (m). 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 I current reconstructed in m-transfected cMSCs remains unclear. The present study aimed to investigate the effects of EPCS on the I current reconstructed in m-transfected cMSCs. The cMSCs were transfected with the lentiviral vector pLentis-m-GFP. Following transfection, these cells were divided into two groups: m-transfected cMSCs (group A), and m-transfected cMSCs induced by EPCS (group B). Using a whole cell patch-clamp technique, the I current 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 I current 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 of , 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 I channels may have a positive regulatory role in EPCS induction. The cMSCs transfected with m induced by EPCS may be a source of effective biological pacemaker cells.