Effects of mitoxantrone on action potential and membrane currents in isolated cardiac myocytes

Abstract

1. The effects of mitoxantrone (MTO), an anticancer drug, on the membrane electrical properties of cardiac myocytes were investigated using the whole-cell clamp technique. 2. In isolated guinea-pig ventricular myocytes, 30 microM MTO induced a time-dependent prolongation of action potential duration (APD) which was occasionally accompanied by early afterdepolarizations. APD prolongation was preserved in the presence of 10 microM tetrodotoxin and showed reverse rate-dependence. 3. Both the inward rectifier K+ current (I(KI)) and the delayed rectifier K+ current (I(K)) of guinea-pig ventricular myocytes were significantly depressed by 30 microM MTO. The rapidly activating component of I(k) (I(Kr)) seemed to be preferentially blocked by MTO. The transient outward current was not affected by MTO in rat ventricular myocytes. 4. Thirty microM MTO had no direct effect on the L-type Ca2+ current (I(Ca(L))), but reversed the inhibitory effect of 1 microM carbamylcholine but not the A1-adenosine receptor agonist (-)-N6-phenylisopropyladenosine (1 microM) on I(Ca(L)) enhanced by 50 nM isoprenaline in guinea-pig ventricular myocytes. In guinea-pig atrial mycotyes, 30 microM MTO inhibited by 93% the muscarinic receptor gated K+ current (I(K,ACh)) evoked by 1 microM carbamylcholine, whereas I(K,ACh) elicited by 100 microM GTPgammaS, a nonhydrolysable GTP analogue, was only decreased by 12%. 5. The specific binding of [3H]QNB, a muscarinic receptor ligand, to human atrial membranes was concentration-dependently displaced by MTO (1-1000 microM). 6. In conclusion, MTO blocks cardiac muscarinic receptors and prolongs APD by inhibition of I(KI) and I(Kr). The occasionally observed early afterdepolarizations may signify a potential cardiac hazard of the drug.

Figure 1

Effect of MTO on the action potential duration (APD) in guinea-pig ventricular myocytes. Action potentials were evoked with current-clamp at a frequency of 0.5 Hz. (a) Recording showing time-dependent prolongation of APD induced by 30 μM MTO. All action potentials were continuously recorded from one cell. (b) Superimposed recordings showing early afterdepolarization caused by superfusion of a myocyte with 30 μM MTO for 1 h. (c) Time-dependent change of APD₉₀ in control cells and in cells superfused with 30 μM MTO. (d) Failure of tetrodotoxin (TTX) to prevent the prolongation of APD₉₀ induced by superfusion of myocytes with 30 μM MTO for 60 min. APD₉₀ was measured in the absence (control) and in the presence of either 30 μM MTO, 10 μM TTX, or 30 μM MTO plus 10 μM TTX. Data from four different cell groups are compared. Numbers of cells in each group are given in parentheses. ^**P<0.01.

Figure 2

Current-voltage relation of the steady-state K⁺ currents of guinea-pig ventricular myocytes in the absence (control) and in the presence of 30 μM MTO. The drug effect was measured 1 h after its addition to the cells. Currents were evoked by applying 1-s depolarizing or hyperpolarizing pulses in 10 mV steps from a holding potential of −40 mV every 10 s. The mean current densities are plotted against the respective test potentials. Data from two different cell groups are compared. ^*P<0.05 and ^**P<0.01 vs control.

Figure 3

Inhibition of I_k by MTO in guinea-pig ventricular myocytes. I_k was elicited every 10 s by voltage protocols indicated in the inset of panel (a) and (b). The drug effect was determined 60 min after its addition to the myocytes. (a) Superimposed traces showing I_k steady-state currents and tail currents of a control cell (C_m=80 pF) and of a cell superfused with 30 μM MTO (C_m=83 pF). (b) I_k tail current-voltage relation in the absence (control) and in the presence of 30 μM MTO. The mean I_k tail current densities from two different cell groups at the respective test potentials are compared.

Figure 4

(a) Effect of test pulse duration on the voltage-dependent inhibition of I_k tail currents by 30 μM MTO. Guinea-pig ventricular myocytes were clamped every 10 s from a holding potential of −40 mV to test potentials up to +70 mV in 10 mV increments. Test pulses of 1-s and 250-ms were applied to every cell. Per cent inhibition of the I_k tail current was calculated by dividing the mean current densities of the cells pretreated with 30 μM MTO for 1 h (n=15) by that of control cells (n=31) at the respective test potential and test pulse duration. (b) Inhibition of I_kr by 30 μM MTO. Myocytes were superfused with nominally Ca²⁺-free bath solution. I_kr steady-state current was evoked by a 500-ms depolarization pulse to −10 mV at 0.5 Hz, and I_kr tail current was elicited upon repolarization to a holding potential of −40 mV. The effect of MTO was measured after cells had been treated with the drug for 1 h. The mean current densities of two different cell groups are compared.

Figure 5

Reverse rate-dependence of the prolongation of action potential duration (APD) by MTO in guinea-pig ventricular papillary muscles. The effect of MTO was measured 2 h after its addition to the bath. (a) Superimposed recordings showing the effect of 30 μM MTO on APD in the same muscle at two different stimulation frequencies. (b) Comparison of the reverse rate-dependence of the prolongation of APD₉₀ by 30 μM MTO and 30 μM d-sotalol. The effect of d-sotalol was evaluated 30 min after its addition to the bath. Two different groups are compared.

Figure 6

(a) Failure of 30 μM MTO to influence I_to of rat ventricular myocytes. I_to was elicited by depolarization to various test potentials for 500 ms after a 100 ms prepulse to −60 mV from a holding potential of −80 mV every 10 s. Current-voltage relation of I_to in control cells and in cells pretreated with 30 μM MTO for 1 h are compared. The inset shows representative I_to recordings of a control cell (C_m=135 pF) and of a MTO-treated cell (C_m=129 pF). (b) Superimposed traces showing the time-dependent effect of 30 μM MTO on the action potential of a rat ventricular myocyte, current-clamped at 0.5 Hz.

Figure 7

Reversal by MTO of the inhibitory effect of carbamylcholine on I_Ca(L) activated by isoprenaline in guinea-pig ventricular myocytes. I_Ca(L) was elicited by depolarizing steps from a holding potential of −40 mV to 0 mV for 300 ms at a frequency of 0.2 Hz. MTO: 30 μM mitoxantrone; ISO: 50 nM isoprenaline; CCh: 1 μM carbamylcholine; R-PIA: 1 μM (−)-N⁶-(2-phenylisopropyl)-adenosine. (a) Superimposed current recordings showing the reversing effect of MTO on peak I_Ca(L) inhibited by CCh. The current traces were recorded at the times indicated by the corresponding letters in panel (b). (b) Time course of the change of peak I_Ca in the same cell as in (a). (c) and (d) Summary of the effect of MTO on peak I_Ca(L) inhibitited by CCh and R-PIA, respectively. ^**P<0.01.

Figure 8

Effect of MTO on I_K,ACh of guinea-pig atrial myocytes. The cells were continuously clamped at −50 mV. (a) Typical current recording showing the inhibitory effect of MTO on I_K,ACh induced by carbamylcholine (CCh). (b) Summary of the inhibitory effect of 30 μM MTO on I_K,ACh induced by 1 μM CCh. Data from the same cell group are compared. (c) Typical recording showing the effect of MTO on I_K,ACh activated by loading the cell with GTPγS through a patch pipette.

Figure 9

Displacement of specific [³H]-QNB binding by MTO. MTO was added at concentrations ranging from 0.1–1000 μM in the presence of three different [³H]-QNB concentrations. Fitted curves are representative of two independent experiments carried out in triplicate. Correlation coefficients of the fits are 0.99, 0.96 and 0.97 in the presence of 0.7, 1.48 and 2.91 nM [³H]-QNB, respectively.