1Dipartimento di Biologia Cellulare e Molecolare, Università di Perugia, Perugia; and 2Centro Scienze dell'Invecchiamento, Istituto Interuniversitario di Miologia, Dipartimento di Scienze del Farmaco, Laboratorio di Fisiologia Cellulare, Università "G. d'Annunzio" Chieti-Pescara, Chieti, Italy
Submitted 29 July 2004 ; accepted in final form 9 February 2005
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ABSTRACT |
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ATP; cell proliferation
Previous studies have reported three distinct classes of Ca2+-activated K+ channels (3, 32). Large-conductance Ca2+-activated K+ (BKCa) channels are gated by the concerted action of internal Ca2+ and membrane potential and have a unitary conductance from 100 to 220 pS. In contrast, small-conductance Ca2+-activated K+ (SKCa) and IKCa channels, solely gated by internal Ca2+, have a unitary conductance of 220 and 2080 pS, respectively (3, 10, 11, 32). The IKCa channels (KCa3.1; Ref. 7) have a distinct pharmacological profile, being blocked by the scorpion venom toxin charybdotoxin (CTX) and the antifungal imidazole compound clotrimazole (CTL) but resistant to tetraethylammonium (TEA) and D-tubocurarine (D-TC), effective blockers of BKCa channels and of two of the three subtypes (SK2 and SK3) of SKCa channels, respectively (3, 32). These biophysical and pharmacological properties were used to identify the IKCa current in C2C12 myoblasts. The IKCa channel was previously shown to be expressed in the myogenic mesodermal stem cell line C3H10T1/2 expressing the myogenic regulatory factor 4 (MRF4) (C3H10T1/2-MRF4), required for complete myogenic differentiation (24, 25). In this cell line, the upregulation of IKCa channels sustained by basic FGF (bFGF) was shown to prevent myogenic differentiation while maintaining proliferation (2426). The results presented here show that the IKCa channel is abundantly expressed in C2C12 myoblasts and is downregulated during myogenesis. Unlike the C3H10T1/2-MRF4 cell line, the IKCa channel in C2C12 myoblasts was not required for proliferation. The IKCa channel may thus represent a marker for undifferentiated C2C12 myoblasts.
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METHODS |
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Electrophysiology.
Whole cell (perforated), cell-attached, and inside-out patch-clamp configurations were used for electrophysiological recordings on C2C12 myoblasts. Electrode resistance was 35 M for whole cell experiments and 510 M
for cell-attached and inside-out experiments. Single-channel and whole cell currents were amplified with a List EPC-7 amplifier (List Medical, Darmstadt, Germany), and digitized with a 12-bit analog-to-digital converter (TL-1, DMA interface; Axon Instruments, Foster City, CA). The pCLAMP software package (version 7.0; Axon Instruments) was used. For online data collection, macroscopic and single-channel currents were filtered at 5 and 0.5 kHz and sampled at 20 and 200 µs/point, respectively. Membrane capacitance (Cm) measurements were made by using the Membrane Test routine of the pCLAMP software. Whole cell currents were routinely expressed as current densities (pA/pF) calculated with respect to the measured cell Cm. In cell-attached experiments the membrane potential (Vm,patch) sensed by the patch results from the sum of the applied (pipette) potential and the cell membrane potential (Vm,patch = Vpipette + Vm,cell). During the ATP response, Vm,cell was estimated to reach and remain stable for 520 s at about 60 mV (cf. Fig. 4, D and E). The Na+-to-K+ permeability ratio (PNa/PK) for single IKCa channels, bathed in 150 mM internal Na+ and 150 mM external K+ solutions, was assessed with the Goldman-Hodgkin-Huxley (GHK) relationship PNa/PK = exp(RT/ErevF), where Erev is the single-channel current reversal potential and R, T, and F are the gas constant, temperature, and Faraday constant, respectively (8). Experiments were carried out at room temperature (RT; 1822°C), and the resulting data are presented as means ± SE.
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Cell proliferation assays. C2C12 myoblasts were seeded at 3,000 cells/cm and held for 5 h in a synchronization medium containing low-glucose DMEM supplemented with 2% HS, plus 4 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (DM). As assessed by cytofluorimetric analysis, under this condition the cell cycle distribution was: G0/G1 82 ± 7% and G2/S 15 ± 5% (n = 2). Myoblasts were then stimulated with 200 nM CTX, 20 ng/ml bFGF, or 20 ng/ml bFGF in combination with either 200 nM CTX or the src kinase inhibitor PP-2 at 50 µM. PP-2 was dissolved in DMSO. As control, we verified that DMSO, applied to myoblasts at the same concentration used in our experiments, did not significantly interfere with C2C12 growth. CTX and PP-2 were added 25 min and 15 min, respectively, before bFGF addition. Cell proliferation was assessed by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) incorporation assays. The MTT assay was carried out by incubating the cells for 3 h with 5 mg/ml MTT and solubilizing the tetrazolium salts precipitated on the cells with 200 µl of DMSO for 30 min (23). The absorbance was read on a Titertek Multiscan Microelisa reader (Flow Laboratories). We considered 16 wells for treatment and repeated the experiments twice. BrdU incorporation assay was performed by treating the cells with 50 µM BrdU (Sigma). Coverslips were then washed with PBS, and the cells were fixed with 95% ethanol for 5 min at RT. Cells were then treated with 2 M HCl for 1 min at RT, washed with PBS, and maintained for 1 h in 10% normal goat serum (NGS) plus 0.2% Triton X-100 in PBS. The cells were then incubated with 1:100 monoclonal mouse anti-BrdU (DAKO) in 10% NGS in PBS for 90 min at 37°C. Cells were then subjected to three washes with PBS for 5 min each at RT and incubation with 1:200 anti-mouse secondary antibody (mouse immunoglobulin biotin, DAKO no. E0354) in PBS for 40 min at RT. After three washes with PBS for 5 min each at RT, we amplified the signal by incubating with AB complex for 45 min (StreptABComplex/HRP, no. K0377, DAKO). After 2 washes with PBS, we revealed the staining with diaminobenzidine and H2O2, per kit directions. The pictures of the cells were acquired at x200 (Leica DMIL) and recorded on a host computer. From the pictures (10 random fields on 3 distinct coverslips for each treatment) we calculated the mean and SE of BrdU-immunoreactive (IR) cells. The experiments were done in duplicate. Finally, cell cycle distribution was assessed by washing with PBS and incubating for 30 min at 37°C at a density of 106 cells per milliliter of solution containing 250 mg of Na-citrate, 5 mg/ml RNase, 750 µl of Nonidet P-40, and 16.5 mg of propidium iodide dissolved in 200 ml of deionized H2O. After a wash in PBS, 106 cells resuspended in 1 ml of PBS were scanned with a Beckman Epics-XL Coulter cytofluorimeter interfaced to a PC. The experiments were done in duplicate.
Immunocytochemistry for myosin heavy chain proteins. C2C12 myoblasts were seeded on glass coverslips at 6,000 cells/cm in DM (DMEM with 2% HS, L-glutamine, and antibiotics as described in Cell culture) and allowed to differentiate for 14 days. The cells were washed twice with PBS and fixed with 95% ethanol for 5 min at RT. After two washes with PBS, the cells were treated with 2% fetal calf serum in PBS for 30 min at 37°C. The samples were then incubated with 1:50 monoclonal mouse anti-myosin heavy chain antibody (MF20, Hybridoma Bank) in PBS for 90 min at 37°C, washed three times with PBS, and incubated with 1:200 anti-mouse secondary antibody (mouse immunoglobulin biotin, DAKO no. E0354) in PBS for 40 min at RT. After three further washes with PBS (5 min each at RT) we amplified the signal, and the staining was revealed as described in the StreptABComplex/HRP instructions (DAKO no. K0377; see also Cell proliferation assays). The pictures of the cells were acquired and stored as described above. The percentage (mean ± SE) of MF20-IR cells was calculated from the stored pictures by inspecting 10 random fields on three distinct coverslips. The experiments were done in duplicate. Statistical analysis of the data was carried out with Student's paired t-test derived from Prism 2.0 software (GraphPad Software, San Diego, CA).
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RESULTS |
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The effect of ionomycin-induced [Ca2+]i increase was also investigated at the single-channel level, in the cell-attached configuration, with 150 mM K+ solution in the pipette. No single-channel activity could be detected in the absence of ionomycin (control conditions) at the resting membrane potential (0 mV applied potential; Fig. 1C). Ionomycin (0.5 µM) activated a single-channel activity in four of the five patches examined, the mean unitary current being 2.24 ± 0.14 pA (at 0 mV applied potential; n = 4). With a mean zero-current potential of 63 mV, as obtained in the presence of ionomycin (see above), a single-channel conductance of 35 pS results. In accordance with the macroscopic current, the ionomycin-activated unitary current response was transient, with single-channel activity ceasing after 50250 s, suggesting that the macroscopic current is underpinned by this unitary activity (Fig. 1C). The results from Fig. 1, namely Ca2+ dependence, K+ selectivity, and small unitary current, provide compelling evidence to identify the ionomycin-activated current as sustained by either the SKCa or the IKCa channel.
Coapplication of the SKCa/IKCa channel activator DCEBIO (30) together with ionomycin activated a stable K+ current that displayed pharmacological properties congruent with the IKCa current (Fig. 2). A typical experiment illustrating the effects of coapplication of DCEBIO (100 µM) and ionomycin (0.5 µM) to a C2C12 myoblast in the perforated-patch configuration is presented in Fig. 2A. Individual data points in Fig. 2A represent the current assessed at 0 mV with the ramp protocol stimulation under control conditions and after application of DCEBIO + ionomycin and CTL (2 µM; in the continuous presence of DCEBIO + ionomycin; see Fig. 2B, inset). The average current obtained from 11 such experiments was 28.4 ± 8.2 pA/pF at 0 mV (n = 11). Figure 2B illustrates the results of the pharmacological test, showing that the DCEBIO + ionomycin-activated K+ current was insensitive to either TEA (1 mM) or D-TC (300 µM), indicating that neither SK2/SK3 nor BKCa channels underlie this current. In contrast, the DCEBIO + ionomycin-activated K+ current was markedly inhibited by CTX (200 nM), CTL (2 µM), and the newly identified IKCa channel antagonist NPPB (300 µM; see Ref. 4), suggesting that it was sustained by IKCa channels. Figure 2C shows that the ionomycin-activated current block by both CTL and CTX was dose dependent. Fit of the data with the Hill relationship gave an IC50 of 23.7 nM for CTX and 631 nM for CTL (Fig. 2C). The ionomycin-activated K+ current displayed the same pharmacological properties as the DCEBIO + ionomycin-activated K+ current (data not shown), indicating that combined DCEBIO + ionomycin activates the same current activated by ionomycin alone. No significant change of the holding current (measured at 0 mV) was recorded after 10-min CTX incubation [holding current density was 2.5 ± 0.4 pA/pF in control myoblasts (n = 10) and 3.3 ± 0.8 pA/pF in the presence of 200 nM CTX (n = 5)]. These results suggest that the IKCa channels in C2C12 myoblasts have a low basal activity under our recording conditions.
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The number of functional IKCa channels on C2C12 myoblasts was estimated by applying noise analysis to the inactivation phase of the ionomycin-induced macroscopic currents similar to that shown in Fig. 1A. Variance (2) and mean current (Im) of the ionomycin-induced macroscopic current were assessed for each adjacent 100-ms interval (Fig. 3E). The resulting data were then fitted with the parabolic relationship
2 = Im·i Im2/N, where i is single-channel current, which allowed an estimate of the total number of functional IKCa channels (N) of the cell (Fig. 3F). This analysis was performed on four cells, giving a mean number of functional IKCa channels per cell of 8,722 ± 1,132. Using the Cm assessed for these cells (22.5 ± 3.9 pF) and assuming a specific Cm of 1 µF/cm2 (8), we calculated a mean IKCa channel density of 3.9 ± 0.7 channels/µm2.
IKCa channel in C2C12 myoblasts responds to ATP.
We then tested whether the IKCa channel in this cell line could be activated by a physiological stimulus such as ATP, given that previous studies demonstrated that this agonist is capable of releasing Ca2+ from intracellular stores in C2C12 myoblasts (5, 17). A brief Picospritzer-driven application of ATP (3 s, 100 µM) on perforated myoblasts evoked, after a latency of several seconds (515 s), an outward current in 28 of the 55 cells tested. The current response took the form of either a single transient peak current (10 of 28; Fig. 4A, left) or current oscillations (15 of 28; Fig. 4A, right). The remaining three current responses could not be classified. With regard to the single transient peak current responses (which we deal with in this study), these could be evoked by successive applications of the agonist and displayed amplitude and time course similar to those of the first application (cf. Fig. 4B). To determine whether the ATP-induced transient outward current was a IKCa current, the agonist was applied while the myoblast was being superfused with the IKCa-specific inhibitor CTL (2 µM). Under these conditions, no response to ATP was observed (cf. Fig. 4B, third ATP application; n = 4). In two cells tested we found that pretreatment with CTX (200 nM) similarly prevented the ATP-induced response (data not shown). The ATP response was also absent after myoblast preincubation with the PLC inhibitor U-73122 (10 µM, 10 min; data not shown), indicating the involvement of inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores in the ATP-activated metabotropic cascade (13). Analysis of the ATP response at the single-channel level (cell attached) confirmed that the IKCa channel underlies this response. Cell-attached patches (with 150 mM K+ in the pipette, PSS in the bath, and no applied voltage) were subjected to brief Picospritzer-driven pulses of ATP (3 s, 100 µM). This protocol evoked transient, unitary inward currents after a delay of several seconds, which had a time course similar to that of the ATP-induced macroscopic currents shown in Fig. 4, A and B (cf. Fig. 4C). Reapplication of the agonist normally evoked a second transient unitary current episode similar to the first episode. In contrast, the response was absent when ATP was applied in the presence of the IKCa channel inhibitor CTL (2 µM; data not shown). The ATP-induced unitary current had an average amplitude (estimated from amplitude histograms) of 3.5 pA, as expected for IKCa channels at a Vm,patch of approximately 110 mV (i.e., Vm,patch = Vpipette + Vm,cell; see METHODS for details).
We then evaluated the effect of ATP-induced IKCa channel activation on Vm. Brief applications of ATP (3 s, 100 µM) to perforated myoblasts under current-clamp recording conditions induced, after a short delay, a large, transient hyperpolarization (cf. Fig. 4D) that had a mean value of 35 mV (n = 8; Fig. 4E). The ATP-evoked hyperpolarizations closely matched the evoked transient currents we observed under voltage-clamp conditions, with regard to both the time course and latency of the response. The correspondence of the membrane voltage and current responses extended to the repeatability of the ATP-induced hyperpolarizations with successive applications (data not shown).
IKCa channel is downregulated during myogenesis.
In the engineered myogenic-like model C3H10T1/2-MRF4, it has been demonstrated that IKCa channels are downregulated during myogenesis and downregulation was prevented by the myogenesis-suppressing agents bFGF and transforming growth factor (24, 25). We have tested whether these findings apply to our myogenic model, i.e., whether myogenic progression in C2C12 myoblasts would likewise be paralleled by downregulation of the IKCa channel, and whether preventing myogenesis would prevent the IKCa channel downregulation. Differentiation of C2C12 myoblasts was induced by plating the cells in DM for 14 days. The induction of the myogenic program was confirmed by a marked increase in the percentage of IR myoblasts for the myosin heavy chain (Fig. 5C). In line with previous data, we found that IKCa current amplitude decreased during myogenesis induced by serum withdrawal (Fig. 5, A and B). Figure 5A illustrates representative DCEBIO + ionomycin-activated IKCa currents from C2C12 myoblasts kept in DM for either 1 or 4 days. As shown in Fig. 5B, the decrease in IKCa current usually started 2448 h after myoblasts had been placed in DM, and after 4 days the IKCa current had decreased on average by 90%, compared with control myoblasts kept in GM. When bFGF was added to the DM to prevent myogenesis, downregulation was likewise removed, with the IKCa current remaining stable in amplitude at the levels found in myoblasts kept in GM (Fig. 5B). Previous reports showed that myoblasts placed in differentiation medium start expressing the DRK current, which is absent in myoblasts kept in growth medium (14). We monitored the DRK current in C2C12 myoblasts (kept in DM) over the time range in which the IKCa current was downregulated. We found that the DRK current, absent at day 1 in DM, normally appeared after 4872 h, and at day 4 it reached a mean amplitude of 22.5 ± 8.8 pA/pF (Fig. 5C). Figure 5D illustrates representative DRK current families activated by depolarizing pulses, in DM at day 1 and day 4. Notably, no DRK current was ever observed in GM, regardless of the time for which myoblasts were kept in culture. The upregulation of the DRK current thus seems to correlate inversely with the downregulation of the IKCa current (cf. Fig. 5C, taken from the DM data of Fig. 5B).
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DISCUSSION |
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IKCa channel is downregulated during myogenesis. Ion channels are centrally involved in myogenesis, with several types being either upregulated or downregulated during differentiation. A major finding of this study is that the induction of differentiation by serum withdrawal results in downregulation of IKCa channel expression. In myoblasts placed in DM, the IKCa current decrease could be observed after 2448 h, and by day 4 IKCa current had dropped below 10% of its value in control myoblasts (i.e., myoblasts kept in GM). When bFGF, a growth factor shown to prevent myogenesis, was added to the DM, the downregulation of the IKCa current was likewise prevented (Fig. 5B). These results, consistent with previous reports for the C3H10T1/2-MRF4 myogenic cell line (24), indicate that IKCa channel expression is modulated by a regulatory signaling pathway that also controls cell differentiation. This could be the bFGF receptor tyrosine kinase superfamily and its downstream signaling cascade, primarily involving the activation of the Ras/Raf/MEK/ERK pathway, as reported in another myogenic model (24).
During human myoblast differentiation a specific sequential expression of K+ currents [i.e., the ether-à-go-go (eag) and Kir currents] has been reported to be critical for myogenic progression and acquisition of cell excitability (1). Other K+ currents, including the DRK current, were shown to appear 24 h after C2C12 myoblasts had been placed in differentiation medium (14). We found that the DRK current, absent in myoblasts kept in GM, begins to appear after serum withdrawal, at about the same time the IKCa current starts to decrease. These results may indicate that the expressions of these two currents are sequentially ordered during myogenesis of the C2C12 cell line, although a causal link between these two currents has not been established. With regard to IKCa channel downregulation during myoblast differentiation, it is worthy of note that another current, ICl,sw, has been reported to disappear with a similar time course (33). The functional meaning, if any, of this simultaneous downregulation is likewise unknown. However, because these two currents (IKCa current and ICl,sw) are coexpressed in many cell models and coactivated in several important cellular processes including volume regulation (where their combined action mediates KCl efflux), their parallel downregulation may lead to inhibition of those processes that depend on cell volume changes, such as cell motility (21, 29, 36).
Functional role of IKCa channel in C2C12 myoblasts. It has been shown that IKCa channel activity is essential for bFGF-sustained myoblast proliferation in the C3H10T1/2-MFR4 cell line (25). We therefore tested whether the IKCa current is also instrumental for sustaining cell proliferation in C2C12 myoblasts (6, 22, 25, 31). To this end, we used the IKCa channel inhibitor CTX (200 nM), which did not show any effect on the growth rate of C2C12 myoblasts (Fig. 6), indicating that the IKCa channel is not involved in this process. These observations allow us to conclude that the previously described modulatory role of IKCa channels on C3H10T1/2-MFR4 cell proliferation (25) cannot be generalized to all myoblastic cell lines.
The lack of a CTX-sensitive holding current under our resting conditions indicates that the IKCa channel does not contribute to the resting Vm. This conclusion is consistent with the relatively low Vm of this cell line (20 mV, cf. Fig. 4E; also see Refs. 14, 33). We further showed that the IKCa current can be activated by a physiological agent such as ATP that leads to an increase in intracellular Ca2+ (5, 17). Our results indicate that the ATP response occurs via a PLC-mediated Ca2+ release from internal stores. The IKCa current activation induced by ATP occurred in intact myoblasts (perforated and cell-attached configurations) and resulted in a membrane hyperpolarization of
35 mV. As Vm plays an important modulatory role in a number of cellular responses, by linking changes in intracellular Ca2+ to Vm the IKCa channels may serve critical functions in several cellular processes of C2C12 myoblasts, including myogenesis (12, 16, 20).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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