Enhanced inhibition of L-type calcium currents by troglitazone in streptozotocin-induced diabetic rat cardiac ventricular myocytes

Abstract

1. Troglitazone, an insulin-sensitizing agent shown to improve cardiac function in both experimental animals and patients with diabetes, inhibits voltage-dependent L-type Ca(2+) currents (I(Ca,L)) in cardiac myocytes, which may underlie its cardioprotective effects. However, inhibition by troglitazone of I(Ca,L) in diabetic cardiac myocytes has not been characterized. 2. Using whole-cell voltage-clamp techniques, I(Ca,L) was measured in ventricular myocytes isolated from 4-6 weeks streptozotocin (STZ)-induced diabetic rats and age-matched control rats. 3. Under control conditions with CsCl internal solution, diabetic myocytes did not differ from control myocytes in membrane capacitance, current density or voltage-dependent properties of I(Ca,L). 4. Troglitazone decreased amplitude of I(Ca,L) in both control and diabetic myocytes in a concentration-dependent manner. This inhibition was more potent in diabetic than in control myocytes; half-maximum inhibitory concentrations of troglitazone measured at a holding potential of -50 mV were 4.3 and 9.5 micromol l(-1), respectively. 5. Troglitazone at 5 micromol l(-1) did not significantly influence the voltage dependency of steady-state inactivation or the inactivation time course of I(Ca,L) in either control or diabetic myocytes. 6. Since troglitazone inhibits I(Ca,L) more effectively in STZ-induced diabetic ventricular myocytes, this agent may prevent cardiac dysfunction in diabetes.

Figure 1

L-type Ca²⁺ currents (I_Ca,L) in control (CNT) and diabetic (DM) myocytes. (A), representative tracings of I_Ca,L. I_Ca,L were elicited in 300 ms steps from a holding potential of −50 mV to a range between −60 and +60 mV in increments of 10 mV applied at a frequency of 0.2 Hz. (B), voltage-dependency of I_Ca,L. (C), steady-state inactivation of I_Ca,L as demonstrated with a double-pulse voltage-clamp protocol. After applying conditioning holding potentials at different voltage levels between −60 mV and +60 mV for 1 s, a test pulse to +0 mV with a duration of 300 ms was used to elicit I_Ca,L. Normalized current amplitudes (I/I_max) were plotted against conditioning holding potentials. The steady-state inactivation curve was drawn by fitting data to the Boltzmann distribution. (D), steady-state activation of I_Ca,L determined as ratio of conductances (G) to maximum conductance (G_max). The derived activation curve was fitted to data with a Boltzmann equation. Data are expressed as the mean±s.e.m.

Figure 2

Effects of troglitazone (TRO) on L-type Ca²⁺ currents (I_Ca,L) in control (CNT) and diabetic (DM) myocytes. Cells were held at −50 mV, and command pulses 300 ms in duration causing voltage elevation to +0 mV were applied at 0.2 Hz. A and B, representative tracings of I_Ca,L obtained at times indicated by a through c in C and D. C and D, time courses of alterations of I_Ca,L amplitude. Drug administration sequences also are shown.

Figure 3

Concentration-dependent inhibition of L-type Ca²⁺ currents (I_Ca,L) by troglitazone in control (CNT) and diabetic (DM) myocytes. Amplitude of I_Ca,L after application of troglitazone was compared with the control value. Percentage inhibition of troglitazone on I_Ca,L is plotted against concentration of troglitazone. Data are expressed as the mean±s.e.m. and fit to a Hill equation.

Figure 4

Effects of troglitazone (TRO) on the current-voltage relationships of L-type Ca²⁺ currents (I_Ca,L) in control (CNT) and diabetic (DM) myocytes. (A), current-voltage relationships of I_Ca,L in the absence and presence of troglitazone 5 μmol l⁻¹ in control myocytes. (B), current-voltage relationships of I_Ca,L in the absence and presence of troglitazone 5 μmol l⁻¹ in diabetic myocytes. Data are the mean±s.e.m. NT, normal Tyrode's solution.

Figure 5

Effects of troglitazone (TRO) on steady-state inactivation of L-type Ca²⁺ currents (I_Ca,L) in control (CNT) and diabetic (DM) myocytes. (A), steady-state inactivation of I_Ca,L in the absence and presence of troglitazone 5 μmol l⁻¹ in control myocytes. (B), steady-state inactivation of I_Ca,L in the absence and presence of troglitazone 5 μmol l⁻¹ in diabetic myocytes. Data are the mean±s.e.m. NT, normal Tyrode's solution.