Characterization of multiple ion channels in cultured human cardiac fibroblasts - PubMed (original) (raw)

Characterization of multiple ion channels in cultured human cardiac fibroblasts

Gui-Rong Li et al. PLoS One. 2009.

Abstract

Background: Although fibroblast-to-myocyte electrical coupling is experimentally suggested, electrophysiology of cardiac fibroblasts is not as well established as contractile cardiac myocytes. The present study was therefore designed to characterize ion channels in cultured human cardiac fibroblasts.

Methods and findings: A whole-cell patch voltage clamp technique and RT-PCR were employed to determine ion channels expression and their molecular identities. We found that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts. These include a big conductance Ca(2+)-activated K(+) current (BK(Ca)) in most (88%) human cardiac fibroblasts, a delayed rectifier K(+) current (IK(DR)) and a transient outward K(+) current (I(to)) in a small population (15 and 14%, respectively) of cells, an inwardly-rectifying K(+) current (I(Kir)) in 24% of cells, and a chloride current (I(Cl)) in 7% of cells under isotonic conditions. In addition, two types of voltage-gated Na(+) currents (I(Na)) with distinct properties were present in most (61%) human cardiac fibroblasts. One was a slowly inactivated current with a persistent component, sensitive to tetrodotoxin (TTX) inhibition (I(Na.TTX), IC(50) = 7.8 nM), the other was a rapidly inactivated current, relatively resistant to TTX (I(Na.TTXR), IC(50) = 1.8 microM). RT-PCR revealed the molecular identities (mRNAs) of these ion channels in human cardiac fibroblasts, including KCa.1.1 (responsible for BK(Ca)), Kv1.5, Kv1.6 (responsible for IK(DR)), Kv4.2, Kv4.3 (responsible for I(to)), Kir2.1, Kir2.3 (for I(Kir)), Clnc3 (for I(Cl)), Na(V)1.2, Na(V)1.3, Na(V)1.6, Na(V)1.7 (for I(Na.TTX)), and Na(V)1.5 (for I(Na.TTXR)).

Conclusions: These results provide the first information that multiple ion channels are present in cultured human cardiac fibroblasts, and suggest the potential contribution of these ion channels to fibroblast-myocytes electrical coupling.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Families of membrane currents in human cardiac fibroblasts.

A. Noisy current was activated at positive potential. Currents were elicited with the protocol shown in the inset (0.2 Hz). B. A transient outward current was activated in a human cardiac fibroblast by the same protocol as in A. C. A current with inward rectification activated by hyperpolarized potentials (inset) was co-present with the noisy current. D. Voltage-dependent current with outward rectification was recorded with the same protocol as in C. E. An inward current with fast inactivation activated by depolarization voltage steps (inset) was co-present with the noisy current. F. An inward current with slow inactivation (arrow) activated by the same protocol as in E was co-present with the noisy current.

Figure 2

Figure 2. BKCa and IKDR in human cardiac fibroblasts.

A. Voltage-dependent current was reversibly suppressed by the BKCa blocker paxilline (1 µM). Currents were elicited by the voltage protocol as shown in the inset. B. Current-voltage (I-V) relationships of membrane current were recorded by a 2-s ramp protocol (−80 to +80 mV from a holding potential −40 mV) in a representative cell in the absence and presence of 1 µM paxilline. C. Membrane currents recorded in a typical experiment with the same voltage protocol as in A were partially inhibited by 1 µM paxilline. The remaining current was suppressed by co-application of paxilline and 5 mM 4-AP.

Figure 3

Figure 3. Ito in human cardiac fibroblasts.

A. Ito traces recorded in a representative cell with the voltage protocol showed in the inset in the absence and presence of 5 mM 4-AP. B. Normalized mean values of voltage-dependent availability (I/Imax) and activation conductance (g/gmax) of Ito were fitted to the Boltzmann function: y = 1/{1+exp[(Vm−V0.5)/S]}, where Vm is membrane potential, V0.5 is the estimated midpoint, and S is the slope factor. C. Normalized Ito (I2/I1) plotted vs. P1−P2 interval. The recovery curve was fitted to a mono-exponential function. The Ito was measured from the current peak to the ‘quasi’-steady-state level.

Figure 4

Figure 4. Effect of Ba2+ on membrane current in human cardiac fibroblasts.

A. Voltage-dependent currents were reversibly inhibited by 0.5 mM BaCl2 in a representative cell. Currents were recorded with the protocol as shown in the inset (0.2 Hz). B. Voltage-dependent current recorded in another cell with voltage protocol shown in the inset of A was increased by elevating K+ o from 5 to 20 mM. Ba2+ (0.5 mM) remarkably suppressed the current. C. Left panel: I-V relationships of membrane currents recorded in a representative cell with a 2-s ramp protocol (−120 to 0 mV from a holding potential of −40 mV) in 5 mM K+ o, 20 mM K+ o, and after application of 0.5 mM Ba2+. Right panel: Ba2+-sensitive I-V relationships of the membrane current, typical of IKir.

Figure 5

Figure 5. ICl in human cardiac fibroblasts.

A. Voltage-dependent current was inhibited by the Cl− channel blocker DIDS (150 µM). Current was elicited by the voltage steps as shown in the inset (0.2 Hz). B. I-V relation curve of DIDS-sensitive current obtained by subtracting currents before and after DIDS application in A. C. Voltage-dependent current recorded in a representative cells during control, after 20 min 0.7T exposure and application of 100 µM NPPB. D. I-V relationships for control current (1.0T), 0.7T and 0.7T with 100 µM NPPB. The 0.7T-induced current was significantly inhibited by NPPB at all test potentials (n = 5, P<0.01). The arrows in the figure indicate the zero current level.

Figure 6

Figure 6. INa.TTX and INa.TTXR in human cardiac fibroblasts.

A. An inward current with a persistent component (arrow) recorded in a representative cell under K+-free conditions using the voltage steps as shown in the inset. Nifedipine (10 µM) had no effect on the current, while the current disappeared when Na+ o was replaced with equimolar choline, and recovered as restoration of Na+ o. B. Similar inward current with persistent component (arrow) recorded in another cell was highly sensitive to inhibition by low concentrations of TTX. C. An inward current with fast inactivation recorded using the same voltage protocol as shown in the inset of A. The current was not affected by 10 nM TTX, but reversibly inhibited by 10 µM nifedipine. D. Similar current recorded in another cell disappeared with Na+ o removal, and recovered as restoration of Na+ o. The current was suppressed by a high concentration of TTX (10 µM). E. Concentration-dependent response of two types of inward currents to TTX. The data were fitted to the Hill equation: E = Emax/[1+(IC50/C)b], where E is the percentage inhibition of current at concentration C, Emax is the maximum inhibition, IC50 is the concentration for a half inhibitory effect, and b is the Hill coefficient. The IC50 of TTX for inhibiting TTX-sensitive INa was 7.8 nM (n = 5−9 for each concentration), the Hill coefficient was 0.94. The IC50 of TTX for inhibiting TTX-resistant INa was 1.8 µM (n = 6−9 cell for each concentration), the Hill coefficient was 0.58. F. Concentration-dependent relationships of INa.TTX and INa.TTXR to nifedipine. The IC50 of nifedipine for inhibiting INa.TTXR was 56.2 µM (n = 4−7 cells for each concentration) with a Hill coefficient of 0.59.

Figure 7

Figure 7. Kinetics of INa.TTX and INa.TTXR.

A. Mean values of I-V relationships of INa.TTX and INa.TTXR. B. Left panel: inactivation time course of representative INa traces (at 0 mV) was fitted to a monoexponential function with time constant (τ) shown, 4.3 ms for INa.TTX and 1.82 ms for INa.TTXR. Right panel: mean values of voltage dependence of inactivation of INa.TTX (n = 8) and INa.TTXR (n = 10). P<0.05 or P<0.01 at −20 to +60 mV. C. Voltage-dependent availability (I/Imax) of INa was determined with the protocol as shown in the left inset (with 1-s conditioning pulses from voltages between −120 and −10 mV then a 50-ms test pulse to 0 mV). Curves of I/Imax and activation conductance (g/gmax) were fitted to a Boltzmann equation. E. Recovery curves of INa.TTX and INa.TTXR from inactivation were fitted to a monoexponential function.

Figure 8

Figure 8. RT-PCR for detecting ion channels expressed in human cardiac fibroblasts.

A. Images of RT-PCR products corresponding to significant gene expression of KCa1.1 (BKCa), Kv1.5 (IKDR), Kv4.3 (Ito), and Kir2.1 (IKir) and Clcn3 (ICl.vol), and NaV1.2, NaV1.3, NaV1.5, NaV1.6 and NaV1.7 in human cardiac fibroblasts. A weak expression of Kv4.2, Kir2.3, Clcn2 and NaV1.1 was also found in human cardiac fibroblasts. B. No significant bands were observed in the PCR experiment when RT product was replaced by total RNA.

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