Calcitriol, the Bioactive Metabolite of Vitamin D, Increases Ventricular K+ Currents in Isolated Mouse Cardiomyocytes

Figures

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  FIGURE 1. Protocol used to isolate the fast transient outward K+ current (Itof), the ultrarapid delayed rectifier K+ current (Ikur) and the non-inactivating steady-state outward current (Iss) in mouse ventricular cardiomyocytes. (A) Superimposed current records of total K+ currents activated by voltage protocol shown on the left. (B) Family of K+ currents obtained from the same cell as in (A) (Cm = 212 pF), but voltage-clamp steps were each preceded by an inactivating pulse at −30 mV, shown in the left panel. Itof was then calculated by substracting the current recording with (A) and without (B) the inactivating prepulse. (C) Superimposed current records of K+ currents obtained after the same cell as those in (B) (Cm = 212 pF) was perfused with 4-AP, which selectively blocks IKur. The current remaining after this protocol is Iss, and Ikur was then obtained by subtracting the current in the absence and in the presence of 4-AP.

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  FIGURE 2. Calcitriol increases total K+ currents in ventricular myocytes. (A) Superimposed current records of total K+ current (Itotal) for 1 myocyte treated with vehicle and 1 myocyte treated with 10 nM calcitriol. (B) Current-voltage curves for Itotal density obtained in myocytes treated with vehicle or calcitriol. (C) Time course of the effect of calcitriol on Itotal by perforated patch-clamp recordings. The black points in the figure are peak outward currents recorded at +50 mV every 10 s. Horizontal line indicates exposure to calcitriol 10 nM and washout. Upper panel shows Itotal traces at +50 mV corresponding to time 0 min (a), 9 min after calcitriol perfusion (b), and ∼10 min after washout (c). Data are expressed as means ± SEM. Cm = membrane capacitance in pF. ∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05.

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  FIGURE 3. The fast transient outward K+ current (Itof) and the ultrarapid delayed rectifier K+ current (Ikur) are significantly increased in calcitriol-treated myocytes. (A) Left panel: representative Itof traces for 1 myocyte treated with vehicle and 1 myocyte treated with 10 nM calcitriol. Right panel: I/V curves for Itof density obtained in myocytes treated with vehicle or with 10 nM calcitriol. (B) Left panel: representative Ikur traces for 1 myocyte treated with vehicle and 1 myocyte treated with calcitriol. Right panel: I/V curves for Ikur density obtained in myocytes treated with vehicle or calcitriol. (C) Left panel: representative Iss traces for 1 myocyte treated with vehicle and 1 myocyte treated with calcitriol. Right panel: IV curves for Issx densities obtained in myocytes treated with vehicle or with calcitri. Data are expressed as means ± SEM. Cm = membrane capacitance in pF. ∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05.

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  FIGURE 4. Effects of calcitriol on Action Potentials Duration (A) Representative traces of action potentials recorded in 1 myocyte treated with vehicle and in 1 myocyte treated with 10 nM calcitriol. (B) Bar graph showing mean action potential duration (APD) values measured at 20, 50, and 90% of repolarization (APD20, APD50, and APD90, respectively). Data are expressed as means ± SEM.

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  FIGURE 5. Effect of calcitriol on Itof and Ikur involves binding to the vitamin D receptor. (A) Left panel: representative traces of Itof obtained in 1 VDR-KO myocyte treated with vehicle and in 1 VDR-KO myocyte treated with 10 nM calcitriol. Right panel: I/V curves for Itof density obtained in VDR-KO myocytes treated with vehicle or calcitriol. (B) Left panel: representative traces of Ikur obtained in 1 VDR-KO myocyte treated with vehicle and in 1 VDR-KO myocyte treated with 10 nM calcitriol. Right panel: I/V curves for Ikur density obtained in VDR-KO myocytes treated with vehicle or calcitriol. (C) Left panel: representative traces of Iss obtained in 1 VDR-KO myocyte treated with vehicle and in 1 VDR-KO myocyte treated with 10 nM calcitriol. Right panel: I/V curves for Iss density obtained in VDR-KO myocytes treated with vehicle or calcitriol. Data are expressed as means ± SEM. Cm = membrane capacitance in pF.

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  FIGURE 6. Calcitriol activates the Akt pathway in ventricular myocytes. Ventricular myocytes were exposed to vehicle or calcitriol 10 nM for 15 min. (A) Representative western blot and (B) average ratio of protein levels expressed as the percentage of phospho (P)-Akt normalized to total Akt. Data are expressed as mean ± SEM. ∗P < 0.05.

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  FIGURE 7. Inhibition of Akt signaling prevents the increase in Itof and Ikur densities induced by calcitriol. (A) Left panel: Itof traces obtained in 1 myocyte treated with 1 μM triciribine and in 1 myocyte treated with 1 μM triciribine plus 10 nM calcitriol. Right panel: I/V curves for Itof density obtained in myocytes treated with 1 μM triciribine or with triciribine plus 10 nM calcitriol. (B) Left panel: Ikur traces obtained in one myocyte treated with 1 μM triciribine and in 1 myocyte treated with 1 μM triciribine plus 10 nM calcitriol. Right panel: I/V curves for Ikur density obtained in myocytes treated with 1 μM triciribine or with triciribine plus 10 nM calcitriol. (C) Left panel: Iss traces obtained in 1 myocyte treated with 1 μM triciribine and in 1 myocyte treated with 1 μM triciribine plus 10 nM calcitriol. Right panel: I/V curves for Iss density obtained in myocytes treated with 1 μM triciribine or with triciribine plus 10 nM calcitriol. Data are expressed as means ± SEM. Cm, membrane capacitance; TCB, triciribine.