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Electric Pulse Current Stimulation Increases Electrophysiological EXPERIMENTAL AND THERAPEUTIC MEDICINE 11: 1323-1329, 2016 Electric pulse current stimulation increases electrophysiological properties of If current reconstructed in mHCN4-transfected canine mesenchymal stem cells YUANYUAN FENG1,2, SHOUMING LUO1, PAN YANG1 and ZHIYUAN SONG1 1Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing 400038; 2Department of Cardiology, The 401 Hospital of PLA, Qingdao, Shandong 266071, P.R. China Received December 7, 2014; Accepted January 28, 2016 DOI: 10.3892/etm.2016.3072 Abstract. The 'funny' current, also known as the If current, transcription-quantitative polymerase chain reaction and play a crucial role in the spontaneous diastolic depolarization western blot analyses. Transmission electron micrographs also of sinoatrial node cells. The If current is primarily induced by confirmed the increased gap junctions in group B. Collectively, the protein encoded by the hyperpolarization-activated cyclic these results indicated that reconstructed If channels may have nucleotide-gated channel 4 (HCN4) gene. The functional If a positive regulatory role in EPCS induction. The cMSCs channel can be reconstructed in canine mesenchymal stem cells transfected with mHCN4 induced by EPCS may be a source (cMSCs) transfected with mouse HCN4 (mHCN4). Biomimetic of effective biological pacemaker cells. studies have shown that electric pulse current stimulation (EPCS) can promote cardiogenesis in cMSCs. However, Introduction whether EPCS is able to influence the properties of the If current reconstructed in mHCN4-transfected cMSCs remains The sinoatrial node (SAN) is located in the right dorsal wall unclear. The present study aimed to investigate the effects of of the right atrium, and serves as the primary pacemaker for EPCS on the If current reconstructed in mHCN4-transfected initiating the heartbeat and controlling the rate and rhythm of cMSCs. The cMSCs were transfected with the lentiviral vector contraction (1). Deficiencies in such function due to congenital pLentis-mHCN4-GFP. Following transfection, these cells defects, acquired diseases, gene mutation and aging may lead were divided into two groups: mHCN4-transfected cMSCs to sinus node dysfunction (sick sinus syndrome), necessitating (group A), and mHCN4-transfected cMSCs induced by pacemaking therapy (2). The present primary therapy is elec- EPCS (group B). Using a whole cell patch-clamp technique, tronic pacemaker implantation. However, such devices are not the If current was recorded, and group A cMSCs showed optimal because of the limited battery life, biological respon- significant time and voltage dependencies and sensitivity to siveness deficiency and other shortages (2). With respect to + extracellular Cs . The half-maximal activation (V1/2) value these concerns, on-going research is focused on developing a was -101.2±4.6 mV and the time constant of activation was biological pacemaker that is able to integrate into the myocar- 324±41 msec under -160 mV. In the group B cells the If current dium and provide a permanent cure (2-5). increased obviously and activation curve moved to right. The The 'funny' current, also called the If current, is absolute value of V1/2 increased significantly to -92.4±4.8 mV crucially involved in the spontaneous diastolic depolar- (P<0.05), and the time constant of activation diminished under ization of SAN cells. The If current is generated by the the same command voltage (251±44 vs. 324±41, P<0.05). hyperpolarization-activated cyclic nucleotide-gated channel In addition, the mRNA and protein expression levels of (HCN) family. Among the four known HCN isoforms, HCN4, connexin 43 (Cx43) and Cx45 were upregulated in isoform 4 (encoded by HCN4) strongly contributes to pace- group B compared with group A, as determined by reverse maker function (3). In previous studies, the present authors showed that mouse HCN4 (mHCN4)-transfected mesen- chymal stem cells (MSCs) are able to generate a functional If current (3-5). The heart is the largest source of bioelectricity in the human Correspondence to: Professor Zhiyuan Song, Department of body (6,7). Electrical signals are correlated with cardiac depo- Cardiology, Southwest Hospital, The Third Military Medical University, 30 Gaotanyan Road, Chongqing 400038, P.R. China larization and contraction, and electrical currents have been E-mail: [email protected] administered to cells in order to mimic native heart conditions in a number of prior biomimetic studies (7-9). Furthermore, Key words: HCN4, mesenchymal stem cells, electric pulse current an electrical stimulation system has been designed to deliver stimulation, If current, gap junction electrical signals mimicking those in the native heart by the present authors (10). This electric pulse current stimulation (EPCS) had been shown to be safe in canine MSCs (cMSCs), 1324 FENG et al: EPCS INCREASES ELECTROPHYSIOLOGICAL PROPERTIES OF If CURRENT IN mHCN4-cMSCs and may be able to promote cardiogenesis in cMSCs in our tions (rectangular, 2 msec; current intensity, 40 µA; current previous study (10). However, it is not clear whether EPCS is frequency, 2 Hz) (10). For EPCS, the transfected GFP-positive able to influence the properties of If current reconstructed in cells in group B were reseeded in α-MEM at a density of HCN4-transfected cMSCs. 2x105 cells into 60-mm culture dishes (430166; Corning In the present study, cMSCs with high-level expression of Incorporated, Corning, NY, USA) with platinum electrode mHCN4 were produced using exogenous transfection. The (Chow Sang Sang Jewellery Corporation, Hong Kong, China). kinetic characteristics of the resulting If current were analyzed. The EPCS treatment was initiated on the next day after seeding, In addition, the mRNA and protein expression levels of HCN4, lasting 3-6 h per day for 5 days until the cells reached ~95% connexin 43 (Cx43) and Cx45 were evaluated using reverse tran- confluence. α-MEM was changed once during this treatment scription-quantitative polymerase chain reaction (RT-qPCR) period. Each experiment was repeated three times. and western blot assays, and the ultrastructures of the cardiac connective proteins were characterized using transmission elec- Patch clamp studies for If current detection in transfected tron microscopy (TEM). cMSCs. Following EPCS induction for 5 days, the cMSCs in group B and parallel cMSCs in group A were subjected to Materials and methods whole-cell patch clamp detection. The If current was recorded using a voltage clamp (Axon Instruments. Foster City, CA, cMSC culture. Under 30 mg/kg sodium pentobarbital (i.v.) USA) with an Axopatch 200B amplifier (Molecular Devices, and 1‑2% isoflurane anesthesia (Abbott Laboratories Co., Ltd., LLC, Sunnyvale, CA, USA) (3-5). The pipette solution Shanghai, China), cMSCs were harvested from bone marrow of contained the following (in mmol/l): 5.0 Na2-phosphocreatine, three adult Chinese rural canines (two males and one female; 5.0 Mg2-adenosine triphosphate, 110 K aspartate, 0.1 GTP Third Military Medical University (Chongqing, Chian), weighing (Sigma-Aldrich), 1.0 MgCl2, 20 KCl, 0.05 EGTA and 10 HEPES 10-14 kg, as previously described (5) . cMDCs were cultured in (Sangon Biotech Co., Ltd., Shanghai, China); pH was adjusted to α-minimum essential medium (α-MEM) with L-glutamine and 7.2 with KOH (Zhengzhongyi Biotech Co., Ltd., Beijing, China). ribo- and deoxyribonucleosides (SH30265.01B; Hyclone; GE The extracellular solution contained the following (in mmol/l): Healthcare Life Sciences, Logan, UT, USA). cMSC purity was 0.33 NaH2PO4, 1.0 MgCl2, 1.8 CaCl2, 5.4 KCl, 136 NaCl, evaluated using flow cytometry, as previously described (5), in 10 glucose and 10 HEPES; pH was adjusted to 7.4 with NaOH order to detect the markers CD29+, CD44+, CD34- and CD45-, (Sangon Biotech Co., Ltd.). When required, 4 mmol/l CsCl was and by the ability to differentiate into adipogenic, chondrogenic added into the bath solution. Cs+ is a specific pacemaker current and osteogenic lineages, using Oil Red O, Alcian Blue and blocker to block If current and has been used to confirm the fI Toluidine Blue, and Alizarin Red staining, respectively (5). Third current. The sealing resistance was >4 mΩ and the temperature generation cMSCs were transfected with pLentis-mHCN4-GFP was maintained at 25±1˚C. in the presence of 2 µg/ml polybrene (H9268; Sigma-Aldrich, In preparation for patch clamping, cells were trypsinized St. Louis, MO, USA) at a multiplicity of infection (MOI) of 20 (SH30042.01; Hyclone; GE Healthcare Life Sciences) for for 24 h. Following 48 h of transfection, 90% of the cMSCs cells 10-20 sec, while remaining attached to the coverslips. The exhibited green fluorescent protein (GFP) expression, which membrane capacity was measured by the application of a indicated successful transfection. These cMSCs were expanded voltage clamp step, and current density was shown as the value using standard techniques (3-5,10). In order to produce consis- of the peak current per capacity. If current was detected using tent comparisons between mHCN4-transfected cMSCs with or custom voltage clamp methods, as previously described (3-5). without EPCS induction, the cells were divided into two groups: Normalized tail currents were recorded to construct the fully mHCN4-transfected cMSCs (group A), and mHCN4-transfected activated current-voltage relation. The slope factor (k) and the cMSCs induced with EPCS (group B). Ethical approval for the half-maximal activation voltage (V1/2) were obtained by fitting present
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