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

<p><i>A.</i> Images of RT-PCR products corresponding to significant gene expression of KCa1.1 (BK_Ca), Kv1.5 (IK_DR), Kv4.3 (I_to), and Kir2.1 (I_Kir) and Clcn3 (I_Cl.vol), and Na_V1.2, Na_V1.3, Na_V1.5, Na_V1.6 and Na_V1.7 in human cardiac fibroblasts. A weak expression of Kv4.2, Kir2.3, Clcn2 and Na_V1.1 was also found in human cardiac fibroblasts. <i>B.</i> No significant bands were observed in the PCR experiment when RT product was replaced by total RNA.</p

I_to in human cardiac fibroblasts.

<p><i>A.</i> I_to traces recorded in a representative cell with the voltage protocol showed in the <i>inset</i> in the absence and presence of 5 mM 4-AP. <i>B.</i> Normalized mean values of voltage-dependent availability (I/I_max) and activation conductance (g/g_max) of I_to were fitted to the Boltzmann function: y = 1/{1+exp[(V_m−V_0.5)/S]}, where V_m is membrane potential, V_0.5 is the estimated midpoint, and S is the slope factor. <i>C.</i> Normalized I_to (I₂/I₁) plotted vs. P₁−P₂ interval. The recovery curve was fitted to a mono-exponential function. The I_to was measured from the current peak to the ‘quasi’-steady-state level.</p

I_Na.TTX and I_Na.TTXR in human cardiac fibroblasts.

<p><i>A.</i> 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 <i>inset</i>. 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. <i>B.</i> Similar inward current with persistent component (arrow) recorded in another cell was highly sensitive to inhibition by low concentrations of TTX. <i>C.</i> An inward current with fast inactivation recorded using the same voltage protocol as shown in the <i>inset</i> of A. The current was not affected by 10 nM TTX, but reversibly inhibited by 10 µM nifedipine. <i>D.</i> 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). <i>E.</i> Concentration-dependent response of two types of inward currents to TTX. The data were fitted to the Hill equation: E = E_max/[1+(IC₅₀/C)^b], where E is the percentage inhibition of current at concentration C, E_max 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 I_Na was 7.8 nM (n = 5−9 for each concentration), the Hill coefficient was 0.94. The IC₅₀ of TTX for inhibiting TTX-resistant I_Na was 1.8 µM (n = 6−9 cell for each concentration), the Hill coefficient was 0.58. <i>F.</i> Concentration-dependent relationships of I_Na.TTX and I_Na.TTXR to nifedipine. The IC₅₀ of nifedipine for inhibiting I_Na.TTXR was 56.2 µM (n = 4−7 cells for each concentration) with a Hill coefficient of 0.59.</p

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

<p><i>A.</i> 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 <i>inset</i> (0.2 Hz). <i>B.</i> Voltage-dependent current recorded in another cell with voltage protocol shown in the <i>inset</i> of A was increased by elevating K⁺_o from 5 to 20 mM. Ba²⁺ (0.5 mM) remarkably suppressed the current. <i>C.</i> Left panel: <i>I-V</i> 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>I-V</i> relationships of the membrane current, typical of I_Kir.</p

I_Cl in human cardiac fibroblasts.

<p>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 <i>inset</i> (0.2 Hz). <i>B. </i><i> I-V</i> relation curve of DIDS-sensitive current obtained by subtracting currents before and after DIDS application in A. <i>C.</i> Voltage-dependent current recorded in a representative cells during control, after 20 min 0.7T exposure and application of 100 µM NPPB. <i>D.</i><i> I-V</i> 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<i> = </i>5, P<0.01). The arrows in the figure indicate the zero current level.</p

BK_Ca and IK_DR in human cardiac fibroblasts.

<p><i>A.</i> Voltage-dependent current was reversibly suppressed by the BK_Ca blocker paxilline (1 µM). Currents were elicited by the voltage protocol as shown in the <i>inset</i>. <i>B.</i> Current-voltage (<i>I-V</i>) 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. <i>C.</i> 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.</p

Families of membrane currents in human cardiac fibroblasts.

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

Kinetics of I_Na.TTX and I_Na.TTXR.

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