Inhibition of late sodium current suppresses calcium-related ventricular arrhythmias by reducing the phosphorylation of CaMK-II and sodium channel expressions

Figure 1. Prolongation of left ventricular monophasic action potential duration (MAPD90) by Bay K 8644 in the absence and presence of ATX-II. (a) and (b) Representative recordings of MAPs recorded from the epi- (a) and endo- (b) myocardium of the left ventricular wall in serially exposed to no drug (control), ATX-II (3 nM), Bay K 8644 (200 nM), and ATX-II (3 nM) plus Bay K 8644 (200 nM). (c) and (d) Concentration-dependent increases by Bay K 8644 of left ventricular epi- (c) and endo- (d) MAPD90 in absence (n = 11) and presence (n = 7) of ATX-II. * P < 0.05 compared with 0 nM Bay K 8644 either in absence or presence of ATX-II; # P < 0.05, the increase of MAPD90 by Bay K 8644 alone vs ATX-II plus Bay K 8644 at the same concentration. Arrows indicate ventricular tachycardia (VT) occurred at or above the concentration indicated.

Figure 2. Reduction of MAPD90 prolongation by Tetrodotoxin (TTX) and eleclazine (Elec) in hearts treated with Bay K 8644 alone and ATX-II plus Bay K 8644. (a) Representative recordings of MAPs recorded from the epicardium of the left ventricular wall in serially exposed to no drug (control, curve 1), ATX-II (3 nM, curve 2), ATX-II plus Bay K 8644 (200 nM, curve 3), ATX-II plus Bay K 8644 plus TTX (1 µM, curve 4), and ATX-II plus Bay K 8644 plus eleclazine (10 µM, curve 5). (b) Concentration-dependent decreases by TTX of epi-MAPD90 and endo-MAPD90 in the presence of 300 nM Bay K 8644 (n = 7). (c) Effect of TTX (1 µM) and eleclazine (10 µM) on the increase of Δ epi-MAPD90 and Δ endo-epi MAPD90 induced by Bay K 8644 in absence and presence of ATX-II. * P < 0.05 vs. control (ctrl); # P < 0.05 vs. Bay K 8644; † P < 0.05 vs. ATX-II; & P < 0.05 vs. ATX-II plus Bay K 8644.

Figure 3. TTX and eleclazine (Elec) abolished ventricular arrhythmias induced by Bay K 8644 both in absence (a) and presence (b) of ATX-II. Representative recordings of MAPs (upper records in each panel) and ECG (lower records in each panel) were recorded simultaneously in control, Bay K 8644 (200 and 300 nM, a) or ATX-II-treated hearts (b) before and after treatment with TTX. Arrows indicate an episode of VT. The incidence of all ventricular arrhythmias and VT are presented in panel c. * P < 0.05 vs. ctrl; # P < 0.05 vs. Bay K 8644; † P < 0.05 vs. ATX-II; & P < 0.05 vs. ATX-II plus Bay K 8644.

Figure 4. Effects of TTX and eleclazine on the reverse use dependence (RUD) of MAPD90 caused by Bay K 8644. (a) Values of MAPD90 were measured in the absence (Ctrl) and presence of Bay K 8644 (200 nM), Bay K 8644 (200 nM) plus TTX (n = 11). * P < 0.05 vs. cycle length (CL) of 400 ms; # P < 0.05 vs. Ctrl at the same CL; † P < 0.05 vs. Bay K 8644 alone at the same CL. (b) Representative AP traces obtained when hearts were paced at CLs of 400 ms, 1000 ms and 2000 ms in control, Bay K 8644 alone, Bay K 8644 plus TTX, and Bay K 8644 plus eleclazine. (c) and (d) Representative recordings of MAPs (upper records in each panel) and ECG (lower records in each panel) were recorded at pacing CL of 2000 ms in control, Bay K 8644 (200 nM), Bay K 8644 + TTX (1 µM), and Bay K 8644 + eleclazine (10 µM). Arrows indicate an episode of VT.

Figure 5. Effects of TTX (1 µM) on cardiomyocyte contraction function and intracellular calcium transient in the presence of Bay K 8644 (300 nM). (a) and (b) Representative recordings of myocardial sarcomere length (a) and intracellular calcium transients (b) under different conditions. (c) The myocardial contraction amplitude (n = 25), and calcium transient amplitude (n = 16) in control, Bay K 8644, and Bay K 8644 plus TTX. (d) The intracellular calcium concentration ([Ca2+]i) in myocardial cells in control, Bay K 8644, and Bay K 8644 plus TTX, n = 16. * P < 0.05 vs. control; # P < 0.05 vs. Bay K 8644.

Figure 6. Bay K 8644 increased I CaL. TTX and eleclazine reduced both endogenous and enhanced late I Na. (a) and (b) Representative recordings of I CaL in control (Ctrl, a) and in a cell treated with Bay K 8644 (300 nM, b). (c) Summarized data of various concentrations of Bay K 8644 to increase the density of I CaL in rabbit ventricular cells. * P < 0.05 vs. control. (d) and (e) Representative recordings of late I Na in a cell in Ctrl, and after treatment with either TTX (1 µM, d) or eleclazine (Elec, 10 µM, e). (f) and (g) Representative recordings of late I Na in a cell in Ctrl, before (Bay K 8644 300 nM alone) and after treatment with either TTX (f) or Elec (g). (h) and (i) Representative recordings of late I Na in a cell in Ctrl, before (ATX-II 3 nM) and after treatment with either TTX (1 µM, h) or Elec (10 µM, i) in the continued presence of ATX-II. (j) Summarized data of TTX and Elec on the density of late I Na in Ctrl and in cells treated with either Bay K 8644 (300 nM) or ATX-II (3 nM). * P < 0.05 vs. Ctrl; # P < 0.05 vs. Bay K 8644; † P < 0.05 vs. ATX-II.

Figure 7. TTX (at concentration of 1 µM) reduced the phosphorylation of CaMK II-δ and Nav 1.5 in hearts pretreated with Bay K 8644 (200 nM) in both absence and presence of ATX-II (3 nM). (a) Representative protein expression of p-CaMK II-δ, CaMK II-δ, p-Nav 1.5 and Nav 1.5 in rabbit left ventricular myocardium under conditions indicated. (b) and (c) The quantitative analysis of p-CaMK II-δ (b) and p-Nav 1.5 (c) protein expression. The corresponding intensity was normalized to expression in Ctrl. * P < 0.05 vs. Ctrl; # P < 0.05 vs. Bay K 8644; † P < 0.05 vs. ATX-II; & P < 0.05 vs. ATX-II plus Bay K 8644.