Our previous study demonstrated that a large-conductance Ca2+-activated K+ current (BKCa), a voltage-gated TTX-sensitive sodium current (INa.TTX), and an inward rectifier K+ current (IKir) were heterogeneously present in most of human cardiac c-kit+ progenitor cells. The present study was designed to investigate the effects of these ion channels on cell cycling progression and migration of human cardiac c-kit+ progenitor cells with approaches of cell proliferation and mobility assays, siRNA, RT-PCR, Western blots, flow cytometry analysis, etc. It was found that inhibition of BKCa with paxilline, but not INa.TTX with tetrodotoxin, decreased both cell proliferation and migration. Inhibition of IKir with Ba2+ had no effect on cell proliferation, while enhanced cell mobility. Silencing KCa.1.1 reduced cell proliferation by accumulating the cells at G0/G1 phase and decreased cell mobility. Interestingly, silencing Kir2.1 increased the cell migration without affecting cell cycling progression. These results demonstrate the novel information that blockade or silence of BKCa channels, but not INa.TTX channels, decreases cell cycling progression and mobility, whereas inhibition of Kir2.1 channels increases cell mobility without affecting cell cycling progression in human cardiac c-kit+ progenitor cells.
Citation: Zhang Y-Y, Li G, Che H, Sun H-Y, Xiao G-S, Wang Y, et al. (2015) Effects of BKCa and Kir2.1 Channels on Cell Cycling Progression and Migration in Human Cardiac c-kit+ Progenitor Cells. PLoS ONE 10(9): e0138581. https://doi.org/10.1371/journal.pone.0138581
Editor: Shang-Zhong Xu, University of Hull, UNITED KINGDOM
Received: June 6, 2015; Accepted: September 1, 2015; Published: September 21, 2015
Copyright: © 2015 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper.
Funding: This work was supported by a General Research Fund (771712M) from the Research Grant Council of Hong Kong. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
In addition to cardiac myocytes and fibroblasts, cardiac stem cells with high growth potential, clonogenicity and pluripotency have been reported in mammalian hearts. Based on the expression of cell surface markers, cardiac stem cells have been classified into different subgroups, including side population, c-kit+, Sca-1+, Islet 1+, SSEA-1+ [1–5]. Human cardiac c-kit+ progenitor cells are one of the dominant members in human cardiac stem cell family. C-kit, also known as CD117 or stem cell growth factor, is the cell surface marker that has been used for stem cell isolation and enrichment from different sources [3, 6–9]. It has been reported that human cardiac c-kit+ progenitor cells have the capability to differentiate into three cardiac lineages, i.e. cardiomyocytes, smooth muscle and endothelial cells [10–12]. The in situ stimulation of c-kit+ progenitor cell growth or injection of expanded c-kit+ progenitor cells to the infarct area has been reported to improve cardiac repair, heart function and survival after myocardial infarction [13, 14].
It is well recognized that ion channels play a crucial role in controlling electrophysiology and excitation-contraction coupling in cardiomyocytes in the heart. Our recent study has demonstrated that ion channels regulate cell cycling progression in human cardiac fibroblasts . Although we demonstrated that a large conductance Ca2+-activated K+ current (BKCa), an inwardly-rectifying K+ current (IKir), and a voltage-gated tetrodotoxin-sensitive Na+ currents (INa.TTX), were heterogeneously expressed in most (61–86%) of human cardiac c-kit+ progenitor cells , the potential physiological roles of these channels are not understood. The present study was to investigate the roles of these functional ion channels in regulating cell cycling progression and mobility in human cardiac c-kit+ progenitor cells with the approaches including cell proliferation and migration assays, flow cytometry, siRNA, RT-PCR, and Western blot analysis.
Materials and Methods
Human cardiac c-kit+ cells were isolated from atrial specimens obtained from coronary artery bypass surgery with the modified procedure as described previously [3, 11, 16], and the procedure of tissue collection was approved by the Ethics Committee of the University of Hong Kong (UW-10-174, S1 File), with written consent from patients as described previously . In the previous report, we demonstrated that human cardiac c-kit+ cells expressing the stem cell markers CD29 and CD105 were >99%, in which the hematopoietic stem cell markers CD34 and CD45, and adult somatic cell marker CD8A were present in a very limited population (<10%), and hematopoietic stem cell markers CD34 and CD45 were mostly absent , consistent with the previous reports by other research groups [3, 11]. The cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 5 ng/ml human basic fibroblast growth factor, 5 ng/ml human epidermal growth factor .
Chemicals and reagents
Mouse monoclonal anti-KCa1.1 and anti-Kir2.1 antibodies were from UC Davis (www.neuromab.org). Goat anti-mouse IgG horseradish peroxidase (HRP) and mouse monoclonal anti-GAPDH antibodies were from Santa-Cruz Biotechnology Inc. (Santa Cruz, CA http://www.scbt.com). Epithelial growth factor (EGF), basic fibroblast growth factor (bFGF), propidium iodide (PI), lipofectamine 2000, Triton X-100 and Tween 20 were purchased from Invitrogen (Invitrogen, Hong Kong, China). [3H]-thymidine was from GE Healthcare Life Sciences (Hong Kong, China). Other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Whole-cell patch recording
Human cardiac c-kit+ progenitor cells (passages 2–4) were trypsinized when cell grew to 70–80% confluence used for ionic current recordings with a whole-cell patch voltage-clamp technique (at room temperature, 23–25°C) using an EPC-9 amplifier and Pulse software (Heka, Lambrecht, Germany) as described previously .
Cell proliferation assays
Cell proliferation was determined by 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and DNA incorporation with [3H]-thymidine to evaluate the effects of ion channel blockade or ion channel silence on cell proliferation with procedure described previously [17, 18]. For MTT assay, cells were plated into 96-well plates at a density of ~4000 cells per well (2×104 cells/ml) in 200 μl complete culture medium. After 8 h culture, ion channel blockers or vehicle (control) were applied for additional 48 h incubation, and PBS-buffered MTT (5 mg/ml) solution (20 μl) was added for additional 4 h incubation. The mixture of culture medium with MTT was then removed, and DMSO (100 μl) was added to each well to dissolve the formazan crystals formed in cells attached at the bottom. The plates were protected from light with agitation for 30 min at room temperature. The absorbance was read at wavelength 570 nm with a reference filter of 630 nm using a Quant microplate spectrophotometer (Bio-Tek Instruments) for quantitative evaluation. In experiments with siRNA molecules targeting to different ion channels, cells were plated into 96-well plates at a density of ~4000 cells per well. The siRNA molecules were transfected into the cells for 8 h, and the cells were cultured for additional 64 h. MTT solution (20 μl per well) was then added. Results were standardized using control group values.
[3H]-thymidine incorporation assay was performed using 96-well plates with seeding ~4000 cells per well in 200 μl complete culture medium. The cells were cultured for 8 h, and then the culture medium was changed to that containing ion channel blockers, or siRNA molecules. After 48 h incubation with ion channel blockers or 72 h after siRNA transfection, [3H]-thymidine was added into each well at the concentration of 1 μCi (0.037 MBq). [3H]-thymidine (1 μCi) was added into each well with an additional 12 h incubation, and the cells were harvested and transferred to a nitrocellulose-coated 96-well plate via suction. Nitrocellulose membrane was washed with H2O, and the plate was air dried at 50°C overnight. Liquid scintilla (20 μl/well) was then added to each well. Counts per min (CPM) were read by a TopCount microplate scintillation and luminescence counter (PerkinElmer, Waltharn, MA).
Cell mobility determination
Cell migration was determined using a wound healing method and chemotaxis assay with a transwell system to investigate the potential effect of ion channels on cell motivation in human cardiac c-kit+ progenitor cells with procedure described previously [19, 20]. The wound healing assay was conducted when the cells grew to total confluence in 6-well plates. A standard wound was created by scratching the cell monolayer with a sterile 200 μl plastic pipette tip. Line makers were made at the bottom of plates to indicate the wound edges. After removing cell fragments by washing cell monolayer gently with PBS, the cells were incubated at 37°C with the medium containing 1% FBS and ion channel blockers (not for the cells transfected with siRNA molecules) for 8 h. Then the defined areas of the wound gap were photographed under a phase contrast microscope (Olympus, Tokyo, Japan). The migrated cells on the images were counted to assess cell mobility under different conditions of treatments.
Transwell assay with a modified Boyden chamber with 8 μm-pore polycarbonate membranes (Corning Inc., Corning, NY, USA) was made to determine cell migration following the procedure described previously  to exclude the potential contamination of cell migration by proliferated cells. The chambers were pre-coated with 600 μl serum-free medium for at least 1 h. After the pre-coated medium was removed, ~5000 viable human cardiac c-kit+ cells were plated into the upper chamber in 200 μl medium containing 1% FBS with or without ion channel blockers, and the lower chamber was added 600 μl medium with 1% FBS. The plates were incubated at 37°C in 5% CO2 for 8 h. Then the chambers were washed with PBS for three times, fixed with formaldehyde for 15 min at room temperature, and stained with crystal violet for 15 min. After washing with PBS to thoroughly remove the dye, non-migrated cells on the upper surface of the membrane were scraped off by cotton swabs. The migrated cells on the lower surface of the membrane were counted under a microscope.
Cell cycling progression analysis
Flow cytometry (FC500, Beckman Coulter) was used to determine cell cycling progression in human cardiac c-kit+ progenitor cells with procedure as described previously [17, 18]. Briefly, the cells were plated in 100 mm cell culture dishes at a density of 6×103 cells/cm2, cultured for 8 h in complete culture medium, and synchronized to G0/G1 phase with a cultured medium containing 1% FBS for 12 h. The cells were then cultured in normal culture medium with treatment of ion channel blockers for 60 h or siRNA transfection for 72 h. The cells were lifted using 0.125% trypsin, washed with PBS, and fixed with ice-cold ethanol (75%) at −20°C (72 h). Then ethanol was removed by centrifugation, and cell pellets were washed with PBS twice. Cells were incubated in a propidium iodide/PBS staining buffer (20 μg/ml propidium iodide, 100 μg/ml RNase A, and 0.1% Triton X-100) at 37°C for 30 min. Data were acquired using CellQuest software, and the percentages of G0/G1, S, and G2/M phase cells were calculated with MODFIT LT software.
Small interference RNA
Small interference RNA (siRNA) technique was adopted to silence the related ion channels with the procedure described previously [17, 18]. Briefly, siRNA molecules targeting human KCa1.1 (sc-42511) and Kir2.1 (sc-42612) were purchased from Santa Cruz Biotechnology. These siRNA molecules are pools of 3 target-specific 20–25 nucleotides designed to silence corresponding gene expression. Lipofectamine 2000 reagent (Invitrogen) was used for siRNA transfection. Total RNA and protein were extracted and evaluated by RT-PCR and Western-blot respectively after 72 h transfection. Membrane potential and currents were recorded in current clamp mode and voltage-clamp mode, respectively. Proliferation and migration assays, and flow cytometry analysis were conducted after 72 h siRNA transfection.
Reverse transcription and polymerase chain reaction
Total RNA of human cardiac c-kit+ positive progenitor cells was isolated using the TRIzol method (Invitrogen). Reverse transcription (RT) was performed with the RT system (Promega Corp., Madison, WI, USA) protocol in a 20-μ1 reaction mixture with the procedure as described previously [17, 18]. After the RT, the reaction product (cDNA) was used for polymerase chain reaction (PCR). The cDNA was kept at −80°C for long-time storage.
Primers used in the present study were adopted from our previous report . PCR was performed with the Promega PCR Core System I using a DNA thermal cycler (Mycycler; Bio-Rad Laboratories, Hercules, CA) as described previously . The PCR products, amplified cDNA bands, were analyzed by 1.3% agarose gel electrophoresis, and visualized in ethidium bromide-stained gel illuminated with UV light. Quantitative evaluation and imagination was conducted via the Chemi-Genius Bio Imaging System (Syngene, Cambridge, UK).
Western blotting analysis
Western blot analysis was performed to determine protein expression of with the procedure as described previously . Briefly, cells lysates were extracted via a modified RIPA buffer, and cell lysates (50 μg) were mixed with sample buffer and denatured by heating to 70°C for 10 min. Samples were resolved via SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in Tris-buffered saline with Tween (TTBS) and then probed with primary antibody (mouse monoclonal anti-KCa1.1, anti-Kir2.1, or anti-GAPDH) at 4°C overnight with agitation. After washing with TTBS, the membranes were incubated with goat anti-mouse IgG-horseradish peroxidase (HRP) at 1:4,000 dilution in TTBS at room temperature for 1 h. Membranes were washed again with TTBS and then processed to develop X-ray film using an enhanced chemiluminescence detection system (GE Healthcare). The expression of GAPDH levels was used as an internal control to standardize the relative levels of target protein. The relative band intensities of Western blot were measured by quantitative scanning densitometer and image analysis software (Bio-1D version 97.04).
Results were expressed as mean ± SEM. Unpaired Student’s t-test was used as appropriate to evaluate the statistical significance of differences between two group means, and analysis of variance was used for multiple groups. A value of P<0.05 was considered statistically significant.
Inhibition of membrane currents by specific blockers in human cardiac c-kit+ progenitor cells
Our previous study demonstrated that BKCa (encoded by KCa1.1), INa.TTX (encoded by Nav1.3 and Nav1.6) and IKir (encoded by Kir2.1) were heterogeneously co-expressed in human cardiac c-kit+ progenitor cells , which are inhibited by corresponding blockers as shown in Fig 1. BKCa and INa.TTX were co-expressed in a human cardiac c-kit+ progenitor cell and inhibited respectively by the BKCa inhibitor paxilline (1 μM) and tetrodotoxin (TTX, 30 nM) (Fig 1A), while IKir and BKCa were co-expressed in another representative cell and suppressed respectively by Ba2+ (500 μM) and paxilline (Fig 1B). Similar results were obtained in other 10 cells for each treatment.
(A): BKCa and INa.TTX were co-expressed in a human cardiac c-kit+ progenitor cell, and inhibited respectively by 1 μM paxilline and 30 nM TTX. (B): IKir and BKCa were co-expressed in a typical human cardiac c-kit+ progenitor cell, and inhibited respectively by 500 μM Ba2+ and 1 μM paxilline.
Effects of ion channel blockers on cell proliferation
To determine whether blockade of ion channels would affect cell proliferation, MTT assay was initially used in human cardiac c-kit+ progenitor cells. The cells were treated with paxilline (0.1–3 M) to block BKCa, TTX (0.1–3 M) to block INa.TTX, and Ba2+ (100–600 M) to block IKir for 48 h. Fig 2A shows the percentage values of cells in the absence or presence of different ion channel blockers. The cell proliferation was inhibited by 1 and 3 M paxilline (n = 8, P<0.05 or P<0.01 vs. control), but not by TTX or Ba2+ (n = 8, P = NS).
(A): cell proliferation was assessed by MTT assay in cells treated with vehicle (V), paxilline, TTX or Ba2+ at concentrations as indicated (n = 8, *P<0.05, **P<0.01 vs. vehicle control). (B): [3H]-thymidine incorporation assay was conducted in cells treated with paxilline, TTX or Ba2+ at different concentrations (n = 6, *P<0.05, **P<0.01 vs. vehicle control).
Similar results were obtained with [3H]-thymidine incorporation assay (Fig 2B). Paxilline (1 and 3 M), but not TTX and Ba2+, significantly reduced DNA synthesis rate in human cardiac c-kit+ progenitor cells (n = 6, P<0.05 or 0.01 vs. control). These results suggest that the inhibition of BKCa decreases the proliferation of human cardiac c-kit+ progenitor cells, while blockade of INa.TTX or IKir had no significant effect on cell proliferation.
Blockade of ion channels on cell migration
To examine whether ion channels would regulate cell migration in human cardiac c-kit+ progenitor cells, wound healing and chemotaxis assays were conducted in cells treated with different ion channel blockers. Fig 3A shows the wound healing images in cells treated with paxilline (1 μM), TTX (1 μM), or Ba2+ (300 μM) for 8 h. Fig 3B illustrates the ratio of migrated cells into the acellular area in different treatments. Blockade of BKCa channels with paxilline, but not INa.TTX with TTX, significantly inhibited cell migration (n = 7, P<0.01 vs. vehicle control). Interestingly, blockade of IKir with Ba2+ increased cell migration (n = 6, P<0.05 vs. control).
(A): Cell images with acellular wound. The broken lines indicate the initial wound produced with a pipette tip in cells treated with vehicle (control), paxilline (1 M), TTX (1 M), or Ba2+ (300 μM). (B): Ratio of migrated cells in human cardiac c-kit+ progenitor cells treated with vehicle, 1 μM paxilline, 1 μM TTX or 300 μM Ba2+ (n = 6; *P<0.05, **P<0.01 vs. vehicle control). (C): Images of migrated human cardiac c-kit+ progenitor cells on lower surface of the transwell membrane in cells treated with vehicle (control), paxilline (1 M), TTX (1 M) or 300 μM Ba2+. (D): Ratio of migrated human cardiac c-kit+ progenitor cells on the lower membrane in cells treated with vehicle (control), paxilline (1 M), TTX (1 M) or 300 μM Ba2+ (n = 5; *P<0.05, **P<0.01 vs. vehicle control).
Fig 3C displays the chemotaxis assay with a HTS Transwell system. The cells migrated to the lower surface of the membrane were reduced with treatment of 1 μM paxilline, whereas increased with treatment of 300 μM Ba2+. TTX treatment did not affect cell mobility. Fig 3D illustrates the ratio of migrated cells on lower surface of the membrane. The ratio of migrated cells was decreased by 1 M paxilline (n = 5, P<0.01 vs. control), while increased by 300 μM Ba2+ (n = 5, P<0.05 vs. control). TTX (1 μM) had no effect on cell migration. These results suggest that blockade of BKCa, but not INa.TTX, decreases cell migration, whereas inhibition of IKir increases cell migration in human cardiac c-kit+ progenitor cells.
Silence of ion channels with corresponding siRNA molecules
To exclude the potential nonspecific effects of BKCa and IKir blockers on cell proliferation and/or migration, siRNA molecules targeting KCa1.1 gene (for BKCa), and Kir2.1 gene (for IKir) were employed in human cardiac c-kit+ progenitor cells. The experiment of silencing INa.TTX was not performed, because no effect was observed on cell proliferation or migration in cells treated with0.1–3 μM TTX, and the concentration was much higher than that for blocking the current.
Fig 4A and 4C illustrate the RT-PCR images and the relative mRNA levels of KCa1.1 and Kir2.1 in cells transfected with the corresponding siRNA molecules (10 or 40 nM) for 72 h. KCa1.1 siRNA and Kir2.1 siRNA molecules significantly reduced the corresponding mRNA levels. The mRNA was reduced to 7.0 ± 1.8% of control for KCa1.1 and 10.5 ± 5.4% for Kir2.1 in cells transfected with corresponding siRNA molecules (n = 5, P<0.01 vs. control siRNA 40 nM).
(A): PCR images and relative level of KCa1.1 mRNA in cells treated with lipofectamine 2000 (Lipo), control siRNA, or KCa1.1 siRNA. (B): Western blots and relative level of KCa1.1 protein in cell treated with lipofectamine 2000, control siRNA, or KCa1.1 siRNA. (C): PCR images and relative level of Kir2.1 mRNA in cells treated with lipofectamine 2000, control siRNA, or Kir2.1 siRNA. (D): Western blots and relative level of Kir2.1 protein in cell treated with lipofectamine 2000, control siRNA, or Kir2.1 siRNA (n = 6 for each group, **P<0.01 vs. control siRNA).
Western blots of KCa1.1 and Kir2.1 channels were determined in cells transfected with the corresponding siRNA molecules. Significant reduction of protein level was observed in cells transfected with siRNA molecules targeting to KCa1.1 (Fig 4B) or Kir2.1 (Fig 4C). The mean relative protein level was reduced to 17.3 ± 8.2% of control for KCa1.1, and 16.0 ± 2.5% for Kir2.1 in cells transfected with 40 nM corresponding siRNA molecules (n = 6, P<0.01 vs. control siRNA 40 nM).
Membrane potential, BKCa and IKir currents were recorded in human cardiac c-kit+ progenitor cells transfected with siRNA molecules. The membrane potential recorded in current clamp mode. BKCa current recorded with the voltage protocol as shown in Fig 1A, and IKir was recorded with the voltage protocol as shown in Fig 1B. In cells transfected with 40 nM control siRNA, the membrane potential was -51.3 ± 2.5 mV (n = 25, with adjustment of 15 mV liquid junctional potential in pipette solution). BKCa at +60 mV was 4.1 ± 1.2 pA/pF (n = 18), and IKir at -100 mV was -2.8 ± 0.5 pA/pF (n = 17).
In cells transfected with 40 nM KCa1.1 siRNA, BKCa current at +60 mV was reduced to 1.3 ± 0.7 pA/pF (n = 14, P<0.01 vs. control siRNA) and the membrane potential was slightly depolarized (-47.5 ± 3.7 mV, n = 15, P = NS vs. control siRNA). Nonetheless, in cells transfected with 40 nM Kir2.1 siRNA, IKir at -100 mV was decreased (to -0.8 ± 0.7 pA/pF, n = 13, P<0.05 vs. control siRNA) and the membrane potential was significantly depolarized (to -23.9 ± 2.5 mV, n = 16, P<0.01 vs. control siRNA). These results indicate that silencing KCa1.1 channels reduces BKCa current with slight membrane depolarization, whereas silencing Kir2.1 channels decreases IKir with significant membrane depolarization.
Effects of silencing KCa1.1 and Kir2.1 on cell proliferation and cell cycling progression
The effects of silencing KCa1.1 or Kir2.1 channels on cell proliferation were determined with MTT and [3H]-thymidine incorporation assays in human cardiac c-kit+ progenitor cells transfected with the corresponding siRNA molecules. Cell proliferation (Fig 5A) was significantly reduced in cells transfected with KCa1.1 siRNA (n = 6, P<0.05 or P<0.01 vs. control), but not with Kir2.1 siRNA (n = 4, P = NS). Similar results were obtained with [3H]-thymidine incorporation assay (Fig 5B). DNA synthesis rates were significantly reduced by KCa1.1 siRNA, not by Kir2.1 siRNA. These results confirm that BKCa, but not IKir, participates in the regulation of cell proliferation.
(A): Cell proliferation was assessed by MTT assay in cells treated with lipofectamine 2000 (Lipo) or transfected with control siRNA (40 nM), KCa1.1 siRNA or Kir2.1 siRNA (10 nM and 40 nM). (B): [3H]-thymidine incorporation assay was conducted in cells treated with lipofectamine 2000 or transfected with control siRNA, KCa1.1 siRNA, or Kir2.1 siRNA (n = 6 for each group, *P<0.05, **P<0.01 vs. control siRNA). (C). Flow cytometry graphs in cells transfected with control siRNA, KCa1.1 siRNA, or Kir2.1 siRNA. (D): Mean values of different cycle phases in cells treated with lipofectamine, control siRNA, KCa1.1 siRNA, or Kir2.1 siRNA (40 nM each group, n = 6,*P<0.05, **P<0.01 vs. control siRNA).
The effects of silencing KCa1.1 and Kir2.1 channels on cell cycling progression were determined with flow cytometry analysis (Fig 5C) in human cardiac c-kit+ progenitor cells transfected with corresponding siRNA molecules. Fig 5D illustrates the mean percentage values of cycling phases in cells transfected with 40 nM siRNA molecules targeting to KCa1.1 or Kir2.1. Portion of G0/G1 population was increased from 59.7 ± 4.0% of cells transfected with control siRNA to 69.5 ± 3.9% with KCa1.1 siRNA (n = 8, P<0.01 vs. control siRNA), while Kir2.1 siRNA molecules had no effect on cell cycling progression. These results indicate that BKCa, but not IKir, regulates cell cycling progression by accumulating cells at G0/G1 phase in human cardiac c-kit+ progenitor cells.
Effects of silencing KCa1.1 and Kir2.1 channels on cell migration
The effects of BKCa and IKir on cell migration were confirmed with wound healing assay Fig 6A) and transwell assay (Fig 6B) in cells transfected with 40 nM siRNA molecules targeting to KCa1.1 or Kir2.1. The mean values of the ratio of cells migrated to the acellular area or the lower membrane surface of transwell were reduced in human cardiac c-kit+ progenitor cells transfected with KCa1.1 siRNA (n = 5, P<0.01 vs. control siRNA), while increased in cells transfected with Kir2.1 siRNA molecules (n = 5, P<0.05 vs. control siRNA). These results indicate that silencing BKCa (KCa1.1) inhibits cell mobility, while silencing IKir (Kir2.1) increases cell mobility in human cardiac c-kit+ progenitor cells.
(A): Images of human cardiac c-kit+ progenitor cells with wound-healing migration assay in confluent cells transfected with control siRNA, KCa1.1 siRNA or Kir2.1 siRNA (40 nM for each group). (B): Images of migrated human cardiac c-kit+ progenitor cells to the lower membrane in cells transfected with control siRNA, KCa1.1 siRNA or Kir2.1 siRNA (40 nM for each group). (C): Mean values of ratio of migrated cells in cells transfected with control siRNA, KCa1.1 siRNA or Kir2.1 siRNA (40 nM for each group, n = 5 for each group, *P<0.05, **P<0.01 vs. control siRNA).
It is generally recognized that ion channels play important roles in maintaining physiological homeostasis. In excitable cells, ion channels initiate action potentials and conduct the excitation impulse in excitable cells (e.g. neuronal cells, muscle cells, etc.) to generate the excitation-contraction coupling in muscle cells, and the excitation-secretion coupling in gland cells. However, in proliferative cells, ion channels are considered to participate in regulating cell proliferation and mobility in different types of cells [17, 21].
Early in 1984, DeCoursey and colleagues first reported the regulation of cell growth by ion channels in human T lymphocytes . Afterwards, the roles of specific ion channels in modulating cell proliferation are gradually established. Blockade of Kv and/or KCa channels is demonstrated to inhibit proliferation in glial cells, lymphocytes, endothelium, breast and prostate cancer cells , and in bone marrow-derived MSCs from mouse , rat  and human , mouse cardiac c-kit+ progenitor cells , rat vascular smooth muscle cells [20, 26], and also in rat and human cardiac fibroblasts [15, 27]. Moreover, ion channels are found to regulate cell motility .
Our previous study reported that BKCa, IKir, and INa.TTX, are heterogeneously expressed in most (61–86%) of human cardiac c-kit+ cells . In the present study, we demonstrated the new information that blocking or silencing BKCa channels inhibited both cell proliferation and migration, while inhibiting or silencing Kir2.1 channels increased cell migration without affecting cell proliferation. However, blockade of INa.TTX had no effect on either cell proliferation or migration in human cardiac c-kit+ progenitor cells.
Although BKCa channels have been demonstrated to participate in the regulation of cell proliferation in several types of cells, including human cardiac fibroblast , human preadipocytes , endothelial cells , and human cardiac c-kit+ progenitor cells observed in the present study, inhibition of BKCa is found to have little effect on cell proliferation in human bronchial smooth muscle cells , or MCF-7 cells , indicating that regulation of cell proliferation by BKCa channels is cell-type dependent.
The Ca2+-activated K+ (KCa) channels, including BKCa (KCa1.1), SKCa (e.g. KCa2.3), and IKCa (KCa3.1) channels are reported to regulate cell mobility. Inhibition of KCa channels usually reduces cell migration; however, the epithelial restitution is accelerated when KCa3.1 channel is inhibited in intestinal epithelial cells . KCa1.1 channels are only required for migration in gloma cells, but not in microglia cells [34, 35]. In the present study, we demonstrated that blockade of BKCa with paxilline or silencing BKCa with specific siRNA molecules inhibited cell migration in human cardiac c-kit+ progenitor cells. The reports from ours and others suggest that the contribution of different KCa channels to cell migration is also cell-type specific. The modulation of cell migration by ion channels is believed to be related to the regulation of cell membrane potential and cell volume and/or the nonconductive properties of ion channels.
It is well recognized that the Kir2 inward-rectifier K+ channel family including Kir2.1 is expressed in both excitable and non-excitable cells and the primary function of Kir2 maintains a hyperpolarized membrane potential. Cardiac IK1 (mainly encoded by Kir2.1) has been well studied in human cardiac myocytes [36–39]. Dysfunction of IK1/Kir2.1 channels depolarized the resting membrane potential, caused a delayed repolarization of action potential, thus induced serious cardiac arrhythmia [40, 41]. Patients with Andersen Syndrome are characterized with Kir2.1 mutation . The effects of Kir2.1 on cellular functions in non-excitable cells are somewhat controversial. Kir2.1 was reported to be necessary for differentiation of myoblasts  and play a role in the fusion of mono-nucleated myoblasts to form a multinucleated skeletal muscle fiber . In human endothelial progenitor cells, inhibition of Kir2.1 was found to enhance cell proliferation . A recent report demonstrated that blocking Kir2.1 increased proliferation, and decreased the migration induced by IL-4, IL-10 or ATP in cultured rat microglial cells . Interestingly, we demonstrated the blockade of IKir with Ba2+ or silencing Kir2.1 channels with siRNA depolarized the membrane potential, and stimulated cell migration without affecting proliferation in human cardiac c-kit+ progenitor cells. The effect is similar to the inhibition of KCa3.1 channels in intestinal epithelial cells . The results from ours and others support the notion that the Kir2.1 regulation of non-excitable cell functions depends on cell types.
INa plays an important role in determining rapid upstroke of cardiac action potential. TTX-insensitive Nav1.5 channels are predominantly present in the heart and code for INa in cardiomyocytes, while TTX-sensitive NaV1.2, NaV1.3, NaV1.6, or NaV1.7 channels are mainly reported in neuronal cells and code for INa in brain. We found that INa.TTX (encoded by Nav1.3 and Nav1.6) was also present in human cardiac c-kit+ progenitor cells . Although the previous studies reported that blockade of TTX-insensitive voltage-gated sodium channels (INa, encoded by Nav1.5) was found to reduce proliferation or migration in gastrointestinal epithelial cells [47, 48], we did not find any effect of inhibiting INa.TTX (with concentrations much higher than that for inhibiting the current) on cell proliferation or migration in human cardiac c-kit+ progenitor cells in the present study. These results suggest that TTX-sensitive INa (e.g. NaV1.2, NaV1.3, NaV1.6, or NaV1.7 channels), unlike the TTX-insensitive INa (e.g. Nav1.5), may not have effect on cell cycling progression and/or mobility.
It is believed that cell proliferation and mobility are strictly regulated by multiple mechanisms. Thus, ion channel-mediated regulation of cell cycling progression may not be the sole determinant. During cell proliferation, an increase of cell volume is required, which needs the active participation of ion transport through appropriate ion channels across the cell membrane [21, 49]. Though the detailed mechanisms underlying cell growth regulation by ion channels remain to be further studied, ion channels are generally believed to modulate cell proliferation by regulating cell volume, membrane potential and/or driving force for Ca2+, and also protein-protein interaction . A number of studies demonstrate that membrane potential changes during cell cycling progression [17, 46, 51, 52]; however, in the present study, proliferation was not affected in cells with depolarized membrane potential by silencing Kir2.1 channels, in which, however, cell mobility was increased.
The ability of homing to areas of acute or chronic myocardial injury is very important for human cardiac c-kit+ progenitor cells in the treatment of injury therapy. Recent studies have reported that ion channels are closely involved in the regulation of cell migration in many types of cells, including human mesenchymal stem cells , monocytes , colon cancer cells , pancreatic cancer cells , glioma cells [57, 58]. In the present study, we demonstrated that inhibition or silence of BKCa decreased, while inhibition of IKir enhanced migrating ability of human cardiac c-kit+ progenitor cells, indicating that BKCa promotes, while IKir inhibits, the cell migration in human cardiac c-kit+ progenitor cells under physiological conditions. It should be noted that the observed effects of the BKCa channel or Kir2.1 channel blocker tested here may be related to the experimental condition used in this particular set of experiments (for instance the incubation time), however, the results from experiments with specific siRNA molecules indicate the more specific effects in this specific cell type.
Collectively, the present study provided the novel information that under physiological conditions BKCa, but not IKir, may promote cell proliferation and cell mobility, while IKir could inhibit cell migration without affecting proliferation. INa.TTX has no effect on cell proliferation or migration. The information provides a base for the further understanding of cellular physiology and biology in human cardiac c-kit+ progenitor cells.
Conceived and designed the experiments: YYZ YW GRL. Performed the experiments: YYZ GL HC HYS GSX. Analyzed the data: YYZ HYS. Contributed reagents/materials/analysis tools: YW GSX. Wrote the paper: YYZ GRL.
- 1. Bollini S, Smart N, Riley PR. Resident cardiac progenitor cells: At the heart of regeneration. J Mol Cell Cardiol. 2011;50:296–303. pmid:20643135
- 2. Barile L, Chimenti I, Gaetani R, Forte E, Miraldi F, Frati G, et al. Cardiac stem cells: isolation, expansion and experimental use for myocardial regeneration. Nat Clin Pract Cardiovasc Med. 2007;4 Suppl 1:S9–S14. ncpcardio0738 pmid:17230222
- 3. Bearzi C, Rota M, Hosoda T, Tillmanns J, Nascirnbene A, De Angelis A, et al. Human cardiac stem cells. P Natl Acad Sci USA. 2007;104:14068–73.
- 4. Bolli P, Chaudhry HW. Molecular physiology of cardiac regeneration. Ann N Y Acad Sci. 2010;1211:113–26. pmid:21062300
- 5. Kuhn EN, Wu SM. Origin of cardiac progenitor cells in the developing and postnatal heart. J Cell Physiol. 2010;225:321–5. pmid:20568226
- 6. Messina E, De Angelis L, Frati G, Morrone S, Chimenti S, Fiordaliso F, et al. Isolation and expansion of adult cardiac stem cells from human and murine heart. Circ Res. 2004;95:911–21. pmid:15472116
- 7. Itzhaki-Alfia A, Leor J, Raanani E, Sternik L, Spiegelstein D, Netser S, et al. Patient characteristics and cell source determine the number of isolated human cardiac progenitor cells. Circulation. 2009;120:2559–66. pmid:19996019
- 8. Mishra R, Vijayan K, Colletti EJ, Harrington DA, Matthiesen TS, Simpson D, et al. Characterization and functionality of cardiac progenitor cells in congenital heart patients. Circulation. 2011;123:364–73. CIRCULATIONAHA.110.971622 pmid:21242485
- 9. Davis DR, Kizana E, Terrovitis J, Barth AS, Zhang YQ, Smith RR, et al. Isolation and expansion of functionally-competent cardiac progenitor cells directly from heart biopsies. J Mol Cell Cardiol. 2010;49:312–21. pmid:20211627
- 10. D'Amario D, Fiorini C, Campbell PM, Goichberg P, Sanada F, Zheng HQ, et al. Functionally competent cardiac stem cells can be isolated from endomyocardial biopsies of patients with advanced cardiomyopathies. Circ Res. 2011;108:857–61. pmid:21330601
- 11. He JQ, Vu DM, Hunt G, Chugh A, Bhatnagar A, Bolli R. Human cardiac stem cells isolated from atrial appendages stably express c-kit. Plos One. 2011;6:e27719. pmid:22140461
- 12. Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003;114:763–76. pmid:14505575
- 13. Williams AR, Hatzistergos KE, Addicott B, McCall F, Carvalho D, Suncion V, et al. Enhanced effect of combining human cardiac stem cells and bone marrow mesenchymal stem cells to reduce infarct size and to restore cardiac function after myocardial infarction. Circulation. 2013;127:213–23. pmid:23224061
- 14. Yaniz-Galende E, Chen J, Chemaly E, Liang L, Hulot JS, McCollum L, et al. Stem cell factor gene transfer promotes cardiac repair after myocardial infarction via in situ recruitment and expansion of c-kit+ cells. Circ Res. 2012;111:1434–45. pmid:22931954
- 15. He ML, Liu WJ, Sun HY, Wu W, Liu J, Tse HF, et al. Effects of ion channels on proliferation in cultured human cardiac fibroblasts. J Mol Cell Cardiol. 2011;51:198–206. pmid:21620856
- 16. Zhang YY, Li G, Che H, Sun HY, Li X, Au WK, et al. Characterization of functional ion channels in human cardiac c-kit+ progenitor cells. Basic Res Cardiol. 2014;109:407. pmid:24691761
- 17. Deng XL, Lau CP, Lai K, Cheung KF, Lau GK, Li GR. Cell cycle-dependent expression of potassium channels and cell proliferation in rat mesenchymal stem cells from bone marrow. Cell Prolif. 2007;40:656–70. pmid:17877608
- 18. Tao R, Lau CP, Tse HF, Li GR. Regulation of cell proliferation by intermediate-conductance Ca2+-activated potassium and volume-sensitive chloride channels in mouse mesenchymal stem cells. Am J Physiol Cell Physiol. 2008;295:C1409–16. pmid:18815226
- 19. Chen JB, Liu WJ, Che H, Liu J, Sun HY, Li GR. Adenosine-5'-triphosphate up-regulates proliferation of human cardiac fibroblasts. Br J Pharmacol. 2012;166:1140–50. pmid:22224416
- 20. Su XL, Wang Y, Zhang W, Zhao LM, Li GR, Deng XL. Insulin-mediated upregulation of K(Ca)3.1 channels promotes cell migration and proliferation in rat vascular smooth muscle. J Mol Cell Cardiol. 2011;51:51–7. pmid:21463632
- 21. Pardo LA. Voltage-gated potassium channels in cell proliferation. Physiology (Bethesda). 2004;19:285–92.
- 22. DeCoursey TE, Chandy KG, Gupta S, Cahalan MD. Voltage-gated K+ channels in human T lymphocytes: a role in mitogenesis? Nature. 1984;307:465–8. pmid:6320007.
- 23. Lang F, Shumilina E, Ritter M, Gulbins E, Vereninov A, Huber SM. Ion channels and cell volume in regulation of cell proliferation and apoptotic cell death. Contrib Nephrol. 2006;152:142–60. 96321 [pii] pmid:17065810.
- 24. Zhang YY, Yue J, Che H, Sun HY, Tse HF, Li GR. BKCa and hEag1 channels regulate cell proliferation and differentiation in human bone marrow-derived mesenchymal stem cells. J Cell Physiol. 2014;229:202–12. pmid:23881642.
- 25. Han Y, Chen JD, Liu ZM, Zhou Y, Xia JH, Du XL, et al. Functional ion channels in mouse cardiac c-kit(+) cells. Am J Physiol Cell Physiol. 2010;298:C1109–17. pmid:20130208
- 26. Zhao LM, Su XL, Wang Y, Li GR, Deng XL. KCa3.1 channels mediate the increase of cell migration and proliferation by advanced glycation endproducts in cultured rat vascular smooth muscle cells. Lab Invest. 2013;93:159–67. pmid:23212096
- 27. Wang LP, Wang Y, Zhao LM, Li GR, Deng XL. Angiotensin II upregulates K(Ca)3.1 channels and stimulates cell proliferation in rat cardiac fibroblasts. Biochem Pharmacol. 2013;85:1486–94. pmid:23500546
- 28. Schwab A, Hanley P, Fabian A, Stock C. Potassium channels keep mobile cells on the go. Physiology (Bethesda). 2008;23:212–20. pmid:18697995.
- 29. Hu H, He ML, Tao R, Sun HY, Hu R, Zang WJ, et al. Characterization of ion channels in human preadipocytes. J Cell Physiol. 2009;218:427–35. pmid:18942098
- 30. Kuhlmann CRW, Most AK, Li F, Munz BM, Schaefer CA, Walther S, et al. Endothelin-1-induced proliferation of human endothelial cells depends on activation of K+ channels and Ca2+ influx. Acta Physiol Scand. 2005;183:161–9. pmid:15676057
- 31. Wang K, Xue T, Tsang SY, Van Huizen R, Wong CW, Lai KW, et al. Electrophysiological properties of pluripotent human and mouse embryonic stem cells. Stem Cells. 2005;23:1526–34. pmid:16091557.
- 32. Ouadid-Ahidouch H, Roudbaraki M, Delcourt P, Ahidouch A, Joury N, Prevarskaya N. Functional and molecular identification of intermediate-conductance Ca2+-activated K+ channels in breast cancer cells: association with cell cycle progression. Am J Physiol Cell Physiol. 2004;287:C125–34. pmid:14985237
- 33. Lotz MM, Wang H, Song JC, Pories SE, Matthews JB. K+ channel inhibition accelerates intestinal epithelial cell wound healing. Wound Repair Regen. 2004;12:565–74. pmid:15453839.
- 34. Schilling T, Stock C, Schwab A, Eder C. Functional importance of Ca2+-activated K+ channels for lysophosphatidic acid-induced microglial migration. Eur J Neurosci. 2004;19:1469–74. pmid:15066143.
- 35. Weaver AK, Bomben VC, Sontheimer H. Expression and function of calcium-activated potassium channels in human glioma cells. Glia. 2006;54:223–33. pmid:16817201
- 36. Li GR, Lau CP, Leung TK, Nattel S. Ionic current abnormalities associated with prolonged action potentials in cardiomyocytes from diseased human right ventricles. Heart Rhythm. 2004;1:460–8. pmid:15851200
- 37. Gaborit N, Le Bouter S, Szuts V, Varro A, Escande D, Nattel S, et al. Regional and tissue specific transcript signatures of ion channel genes in the non-diseased human heart. J Physiol. 2007;582:675–93. pmid:17478540
- 38. Beuckelmann DJ, Nabauer M, Erdmann E. Alterations of K+ currents in isolated human ventricular myocytes from patients with terminal heart-failure. Circ Res. 1993;73:379–85. pmid:8330380
- 39. Li GR, Sun HY, Chen JB, Zhou Y, Tse HF, Lau CP. Characterization of multiple ion channels in cultured human cardiac fibroblasts. Plos One. 2009;4.
- 40. Li J, McLerie M, Lopatin AN. Transgenic upregulation of IK1 in the mouse heart leads to multiple abnormalities of cardiac excitability. Am J Physiol Heart Circ Physiol. 2004;287:H2790–802. Epub 2004/07/24. pmid:15271672.
- 41. Priori SG, Pandit SV, Rivolta I, Berenfeld O, Ronchetti E, Dhamoon A, et al. A novel form of short QT syndrome (SQT3) is caused by a mutation in the KCNJ2 gene. Circ Res. 2005;96:800–7. pmid:15761194
- 42. Donaldson MR, Yoon G, Fu YH, Ptacek LJ. Andersen-Tawil syndrome: a model of clinical variability, pleiotropy, and genetic heterogeneity. Ann Med. 2004;36 Suppl 1:92–7. pmid:15176430.
- 43. Konig S, Hinard V, Arnaudeau S, Holzer N, Potter G, Bader CR, et al. Membrane hyperpolarization triggers myogenin and myocyte enhancer factor-2 expression during human myoblast differentiation. J Biol Chem. 2004;279:28187–96. pmid:15084602
- 44. Fischer-Lougheed J, Liu JH, Espinos E, Mordasini D, Bader CR, Belin D, et al. Human myoblast fusion requires expression of functional inward rectifier Kir2.1 channels. J Cell Biol. 2001;153:677–86. pmid:11352930.
- 45. Jang SS, Park J, Hur SW, Hong YH, Hur J, Chae JH, et al. Endothelial progenitor cells functionally express inward rectifier potassium channels. Am J Physiol Cell Physiol. 2011;301:C150–61. pmid:21411724.
- 46. Lam D, Schlichter LC. Expression and contributions of the Kir2.1 inward-rectifier K(+) channel to proliferation, migration and chemotaxis of microglia in unstimulated and anti-inflammatory states. Frontiers in cellular neuroscience. 2015;9:185. pmid:26029054.
- 47. Wu WK, Li GR, Wong HP, Hui MK, Tai EK, Lam EK, et al. Involvement of Kv1.1 and Nav1.5 in proliferation of gastric epithelial cells. J Cell Physiol. 2006;207:437–44. pmid:16331678.
- 48. Wu WK, Li GR, Wong TM, Wang JY, Yu L, Cho CH. Involvement of voltage-gated K+ and Na+ channels in gastric epithelial cell migration. Mol Cell Biochem. 2008;308:219–26. pmid:17978865.
- 49. Lang F, Foller M, Lang K, Lang P, Ritter M, Vereninov A, et al. Cell volume regulatory ion channels in cell proliferation and cell death. Methods Enzymol. 2007;428:209–25. pmid:17875419.
- 50. Urrego D, Tomczak AP, Zahed F, Stuhmer W, Pardo LA. Potassium channels in cell cycle and cell proliferation. Philos Trans R Soc Lond B Biol Sci. 2014;369:20130094. pmid:24493742.
- 51. Barghouth PG, Thiruvalluvan M, Oviedo NJ. Bioelectrical regulation of cell cycle and the planarian model system. Biochim Biophys Acta. 2015. pmid:25749155.
- 52. Yang M, Brackenbury WJ. Membrane potential and cancer progression. Front Physiol. 2013;4:185. pmid:23882223.
- 53. Ding F, Zhang G, Liu L, Jiang L, Wang R, Zheng Y, et al. Involvement of cationic channels in proliferation and migration of human mesenchymal stem cells. Tissue Cell. 2012;44:358–64. pmid:22771012.
- 54. Zhao ZG, Ni YX, Chen J, Zhong J, Yu H, Xu XS, et al. Increased migration of monocytes in essential hypertension is associated with increased transient receptor potential channel canonical type 3 channels. Plos One. 2012;7: e32628. pmid:22438881
- 55. Wang P, Zhang C, Yu P, Tang B, Liu T, Cui H, et al. Regulation of colon cancer cell migration and invasion by CLIC1-mediated RVD. Mol Cell Biochem. 2012;365:313–21. pmid:22426742.
- 56. Rybarczyk P, Gautier M, Hague F, Dhennin-Duthille I, Chatelain D, Kerr-Conte J, et al. Transient receptor potential melastatin-related 7 channel is overexpressed in human pancreatic ductal adenocarcinomas and regulates human pancreatic cancer cell migration. Int J Cancer. 2012;131:E851–E61. pmid:22323115
- 57. Zhang Y, Zhang J, Jiang D, Zhang D, Qian Z, Liu C, et al. Inhibition of T-type Ca(2+) channels by endostatin attenuates human glioblastoma cell proliferation and migration. Br J Pharmacol. 2012;166:1247–60. pmid:22233416
- 58. Rooj AK, McNicholas CM, Bartoszewski R, Bebok Z, Benos DJ, Fuller CM. Glioma-specific cation conductance regulates migration and cell cycle progression. J Biol Chem. 2012;287:4053–65. pmid:22130665.