Roles of Functional Ion Channels In Human Cardiac C-Kit+ Progenitor Cells

Poster PresentationBackground and objectives: Cardiac progenitor cells play an important role in cardiac repair and regeneration; however, cellular biology and electrophysiology are not understood. The present study was to investigate the functional ion channel expression in human cardiac c-kit+ progenitor cells and the 53 potential roles of these ion channels in regulating proliferation and migration. Methods: Multiple experimental approaches were employed in this study, including whole-cell patch voltage-clamp, RT-PCR, Western blots, cell proliferation and migration assays, etc. Results: Several ionic currents were heterogeneously expressed in human cardiac c-kit+ progenitor cells, including a large conductance Ca2+-activated K+ current (BKCa) in most (93%) of cells, an inwardly-rectifying K+ current (IKir) in 87% of cells, a transient outward K+ current (Ito) in 39% of cells, a voltage-gated tetrodotoxin-sensitive Na+ currents (INa,TTX) in 76% of cells. Molecular identities of these ionic currents were determined with RT-PCR and Western blot analysis. KCa.1.1 (for BKCa), Kir2.1 (for IKir), Kv4.2, Kv4.3 (for Ito), NaV1.2, NaV1.3, NaV1.6, NaV1.7 (for INa.TTX) were expressed in human cardiac progenitor cells. Inhibition of BKCa with paxilline, Ito with 4-aminopyridine, but not INa.TTX with TTX and IKir with Ba2+, decreased cell proliferation. Silencing of KCa.1.1, Kv4.2 or Kv4.3, but not Kir2.1, with siRNA targeting corresponding channels reduced proliferation. Inhibition of KCa1.1 or Kv4.2 or Kv4.3 channels accumulated cells at G0/G1 phase. Interestingly, down regulation of KCa1.1, Kv4.2 or Kv4.3 channels decreased, while of Kir2.1 channels increased migration in human c-kit+ progenitor cells. Conclusions: These results demonstrate for the first time that multiple ion channels are expressed in human cardiac c-kit+ cells. KCa1.1, Kv4.2, and Kv4.3 channels, but not Na+ channels and Kir 2.1 channels, participate in regulating proliferation. KCa1.1, Kv4.2 or Kv4.3 channels promote, while Kir2.1 channels reduce cell migration in human cardiac c-kit+ progenitor cells.published_or_final_versio

EGFR Tyrosine kinase regulates small conductance Ca2+-activated K+ (hSKCa1) channels expressed in HEK 293 cells

SKCa (small-conductance Ca(2+)-activated K(+)) channels are widely distributed in different tissues, including the brain, pancreatic islets and myocardium and play an important role in controlling electrical activity and cellular functions. However, intracellular signal modulation of SKCa channels is not fully understood. The present study was designed to investigate the potential regulation of hSKCa1 (human SKCa1) channels by PTKs (protein tyrosine kinases) in HEK (human embryonic kidney)-293 cells expressing the hSKCa1 (KCNN1) gene using approaches of whole-cell patch voltage-clamp, immunoprecipitation, Western blotting and mutagenesis. We found that the hSKCa1 current was inhibited by the broad-spectrum PTK inhibitor genistein, the selective EGFR (epidermal growth factor receptor) kinase inhibitors T25 (tyrphostin 25) and AG556 (tyrphostin AG 556), but not by the Src-family kinases inhibitor PP2. The inhibitory effect of these PTK inhibitors was significantly antagonized by the PTP (protein tyrosine phosphatase) inhibitor orthovanadate. The tyrosine phosphorylation level of hSKCa1 channels was reduced by genistein, T25 or AG556. The reduced tyrosine phosphorylation was countered by orthovanadate. Interestingly, the Y109F mutant hSKCa1 channel lost the inhibitory response to T25 or AG556, and showed a dramatic reduction in tyrosine phosphorylation levels and a reduced current density. These results demonstrate the novel information that hSKCa1 channels are inhibited by genistein, T25 and AG556 via EGFR tyrosine kinase inhibition, which is related to the phosphorylation of Tyr(109) in the N-terminus. This effect may affect electrical activity and cellular functions in brain, pancreatic islets and myocardium.postprin

The Natural Flavone Acacetin Blocks Kv4.3 Current by Interacting With P-Loop Filter Helix of the Channel

Poster presentationBackground and objective: We have recently demonstrated that the natural flavone acacetin is an atrial-selective compound that inhibits ultra-rapid delayed rectifier potassium current (IKur) and transient outward potassium current (Ito) in human atrial myocytes, and also acetylcholine-activated potassium current (IK.ACh). It increased atrial effective refractory period and effectively prevented atrial fibrillation (AF) in anesthetized dogs without prolonging QT interval of ECG. The present study was designed to investigate the potential molecular determinants of hK4.3 channels that encode human cardiac Ito. Methods: Cell culture, mutagenesis and whole-cell patch voltage-clamp techniques were used in the present study. Results: It was found acacetin inhibited hKv4.3 current in HEK 293 cells stably expressing Kv4.3 gene (KCND3) in a concentration-dependent manner. The current inhibition with an increase of time-to-peak and inactivation time constant of the current, suggesting an open channel blockade. However, the stimulation pause during drug administration revealed a strong tonic blocking property. This effect induced a use- or frequency-dependent inhibition at lower concentrations (1 and 3 M), but not at high concentrations. The IC50 of acacetin for inhibiting hKv4.3 was reduced from 6.09 M at 0.2 Hz to 5.80, 4.55, 3.96, and 3.65 M respectively at 1, 2, 3, and 4 Hz. The mutagenesis study showed that the channel blockade by acacetin was dramatically reduced in hKv4.3 mutant T366A and T367A (IC50, 197.8 M for T366A and 166.1 M for T367A) of the P-loop helix, and IC50 was also reduced in V392A, I395A, and V399A (IC50: 25.9 M, 24.1 M, and 9.5 M) of the S6 domain. Conclusion: These results demonstrate the novel information that acacetin is a tonic and open channel blocker of hKv4.3 by binding to T365 and T366 of the P-loop helix, and also interacts with V392, I395, and V399 of the S6 domain of hKv4.3 channels. The use- and rate-dependent blocking property of hKv4.3 by acacetin indicates that this natural compound could exert a strong suppressive effect in the treatment of tachycardiac arrhythmia diseases.published_or_final_versio

Identification of transient receptor potential channels in human atrial myocytes

In Hong Kong Medical Journal, 2011, v. 17, suppl. 1, p. 68, abstract no. 115published_or_final_versionThe 16th Medical Resarch Conference (MRC), The University of Hong Kong, Hong Kong, China, 22 January 2011. In Hong Kong Medical Journal, 2011, v. 17, suppl. 1, p. 68, abstract no. 11

Cyclic ADP ribose is a novel regulator of intracellular Ca 2+ oscillations in human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine. However, the cellular biology of these cells is not fully understood. The present study characterizes the cyclic ADP-ribose (cADPR)-mediated Ca 2+ signals in human MSCs and finds that externally applied cADPR can increase the frequency of spontaneous intracellular Ca 2+ (Ca 2+ i) oscillations. The increase was abrogated by a specific cADPR antagonist or an inositol trisphosphate receptor (IP3R) inhibitor, but not by ryanodine. In addition, the cADPR-induced increase of Ca 2+ i oscillation frequency was prevented by inhibitors of nucleoside transporter or by inhibitors of the transient receptor potential cation melastatin-2 (TRPM2) channel. RT-PCR revealed mRNAs for the nucleoside transporters, concentrative nucleoside transporters 1/2 and equilibrative nucleoside transporters 1/3, IP3R1/2/3 and the TRPM2 channel, but not those for ryanodine receptors and CD38 in human MSCs. Knockdown of the TRPM2 channel by specific short interference RNA abolished the effect of cADPR on the Ca 2+ i oscillation frequency, and prevented the stimulation of proliferation by cADPR. Moreover, cADPR remarkably increased phosphorylated extracellular-signal-regulated kinases 1/2 (ERK1/2), but not Akt or p38 mitogen-activated protein kinase (MAPK). However, cADPR had no effect on adipogenesis or osteogenesis in human MSCs. Our results indicate that cADPR is a novel regulator of Ca 2+ i oscillations in human MSCs. It permeates the cell membrane through the nucleoside transporters and increases Ca 2+ oscillationviaactivation of the TRPM2 channel, resulting in enhanced phosphorylation of ERK1/2 and, thereby, stimulation of human MSC proliferation. This study delineates an alternate signalling pathway of cADPR that is distinct from its well-established role of serving as a Ca 2+ messenger for mobilizing the internal Ca 2+ stores. Whether cADPR can be used clinically for stimulating marrow function in patients with marrow disorders remains to be further studied. © 2011 The Authors © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.postprin

Multiple Ca2+ signaling pathways regulate intracellular Ca 2+ activity in human cardiac fibroblasts

Ca2+ signaling pathways are well studied in cardiac myocytes, but not in cardiac fibroblasts. The aim of the present study is to characterize Ca2+ signaling pathways in cultured human cardiac fibroblasts using confocal scanning microscope and RT-PCR techniques. It was found that spontaneous intracellular Ca2+ (Cai 2+) oscillations were present in about 29% of human cardiac fibroblasts, and the number of cells with Cai 2+ oscillations was increased to 57.3% by application of 3% fetal bovine serum. Cai 2+ oscillations were dependent on Ca2+ entry. Cai2+ oscillations were abolished by the store-operated Ca2+ (SOC) entry channel blocker La3+, the phospholipase C inhibitor U-73122, and the inositol trisphosphate receptors (IP3Rs) inhibitor 2-aminoethoxydiphenyl borate, but not by ryanodine. The IP3R agonist thimerosal enhanced Ca2+ i oscillations. Inhibition of plasma membrane Ca2+ pump (PMCA) and Na+-Ca2+ exchanger (NCX) also suppressed Ca i 2+ oscillations. In addition, the frequency of Ca i 2+ oscillations was reduced by nifedipine, and increased by Bay K8644 in cells with spontaneous Cai 2+ oscillations. RT-PCR revealed that mRNAs for IP3R1-3, SERCA1-3, CaV1.2, NCX3, PMCA1,3,4, TRPC1,3,4,6, STIM1, and Orai1-3, were readily detectable, but not RyRs. Our results demonstrate for the first time that spontaneous Cai 2+ oscillations are present in cultured human cardiac fibroblasts and are regulated by multiple Ca2+ pathways, which are not identical to those of the well-studied contractile cardiomyocytes. This study provides a base for future investigations into how Ca2+ signals regulate biological activity in human cardiac fibroblasts and cardiac remodeling under pathological conditions. © 2009 Wiley-Liss, Inc.postprin

Regulation of human cardiac KCNQ1/KCNE1 channel by epidermal growth factor receptor kinase

The aim of the present study was to investigate whether/how the recombinant human cardiac I Ks could be regulated by epidermal growth factor receptor kinase in HEK 293 cells stably expressing hKCNQ1/hKCNE1 genes using the approaches of perforated patch clamp technique, immunoprecipitation and Western blot analysis. It was found that the broad spectrum isoflavone tyrosine kinase inhibitor genistein and the selective epidermal growth factor receptor kinase inhibitor tyrphostin AG556 suppressed the recombinant I Ks, and their inhibition was countered by the protein tyrosine phosphatase inhibitor orthovanadate. The Src-family kinase inhibitor PP2 reduced the current, but the effect was not antagonized by orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hKCNQ1 protein was decreased by genistein or AG556, but not by PP2. These results provide the novel information that epidermal growth factor receptor kinase, but not Src-family kinases, regulates the recombinant cardiac I Ks stably expressed in HEK 293 cells via phosphorylating KCNQ1 protein of the channel. © 2009 Elsevier B.V. All rights reserved.postprin

Electrospun gelatin nanofibers loaded with vitamins A and E as antibacterial wound dressing materials

Vitamin A palmitate and vitamin E TPGS, common derivatives of the unstable vitamins A and E, were successfully incorporated into biodegradable gelatin nanofibers via electrospinning. Electron microscopy showed that smooth cylindrical fibers were produced, albeit with a small amount of beading visible for the vitamin-loaded systems. The diameters of the fibers decrease with the addition of vitamins. The presence of the vitamins in the fibers was confirmed by IR spectroscopy, and X-ray diffraction showed them to exist in the amorphous physical form post-electrospinning. The addition of vitamins did not affect the hydrophilic properties of the gelatin nanofibers. Fibers containing vitamin A or E alone showed a sustained release profile over more than 60 hours, and those incorporating both vitamins showed similar release characteristics, except that the extent of release for vitamin A was increased. Antibacterial tests demonstrated that materials loaded with vitamin E were effective in inhibiting the growth of E. coli and S. aureus. The fibers could promote the proliferation of fibroblasts during the early stages of culture, and enhance the expression of collagen-specific genes. In vivo tests determined that the fibers loaded with vitamins have better wound healing performance than a commercially used antiseptic gauze and casting films

Human ether-à-go-go gene potassium channels are regulated by EGFR tyrosine kinase

Human ether á-go-go gene potassium channels (hEAG1 or Kv10.1) are expressed in brain and various human cancers and play a role in neuronal excitement and tumor progression. However, the functional regulation of hEAG channels by signal transduction is not fully understood. The present study was therefore designed to investigate whether hEAG1 channels are regulated by protein tyrosine kinases (PTKs) in HEK 293 cells stably expressing hEAG1 gene using whole-cell patch voltage-clamp, immunoprecipitation, Western blot, and mutagenesis approaches. We found that the selective epidermal growth factor receptor (EGFR) kinase inhibitor AG556 (10μM), but not the platelet growth factor receptor (PDGFR) kinase inhibitor AG1295 (10μM) or the Src-family inhibitor PP2 (10μM), can inhibit hEAG1 current, and the inhibitory effect can be reversed by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hEAG1 channels was reduced by AG556, and the reduction was significantly countered by orthovanadate. The hEAG1 mutants Y90A, Y344A and Y485A, but not Y376A and Y479A, exhibited reduced response to AG556. Interestingly, the inhibition effect of AG556 was lost in triple mutant hEAG1 channels at Y90, Y344, and Y485 with alanine. These results demonstrate for the first time that hEAG1 channel activity is regulated by EGFR kinase at the tyrosine residues Tyr 90, Try 344, and Try 485. This effect is likely involved in regulating neuronal activity and/or tumor growth. © 2011 Elsevier B.V.postprin