Suppression of ventricular arrhythmias by targeting late L-type Ca2+ current

Figures

• 

  Figure 1.. Proposed antiarrhythmic strategy based on the reduction of the late ICa,L achieved by decreasing the pedestal component of the LTCC steady-state inactivation. (A) ICa,L predictions for a depolarization to 10 mV (red trace) using a modified UCLA AP model (Madhvani et al., 2011; Madhvani et al., 2015). ICa,L activates rapidly, generating a “peak,” and inactivates incompletely, generating a small but sustained current that persists until the end of the depolarization (late ICa,L). Late ICa,L is enhanced under pathological conditions (e.g., under oxidative stress, red trace; Madhvani et al., 2011), promoting EADs. As such, late ICa,L represents an ideal target to suppress EAD-mediated arrhythmias (Madhvani et al., 2015). The antiarrhythmic strategy tested in this study is based on a reduction of the late ICa,L, as shown in A (green trace). (A and B) The two ICa,L current traces (A) have been simulated using different parameter values for the ICa,L steady-state (SS) inactivation (red curve = 10% pedestal; green curve = 4% pedestal) shown in B. In fact, as late ICa,L flows in quasi–steady state, its amplitude is governed by the position and shape of the curves that describe its…

• 

  Figure S1.. Experimental and analytical procedure to isolate nifedipine-sensitive current under AP clamp. (A) AP waveform used as voltage command in AP-clamp recordings shown in B. (B) Three sequential recordings from the same myocyte under ionic conditions suitable for Ca2+ current recording. In gray is the total current measured before the sequential addition of 20 µM roscovitine (red) and 10 µM nifedipine (blue). The selective effect of roscovitine on the late current is already evident in the raw, unsubtracted current density. (C) The difference in current density obtained by subtracting from the total current (B, gray) and from roscovitine (B, red) recordings of the residual nifedipine-insensitive current (B, blue) is shown.

• 

  Figure 2.. Roscovitine selectively reduces the late ICa,L in rabbit ventricular myocytes without affecting ICa,L peak. (A and B) Representative nifedipine-sensitive ICa,L recordings elicited in response to the voltage protocol shown above (HP = −80 mV) in the presence of 600 µM H2O2 (control; A) or in the presence of 20 µM roscovitine + H2O2 (roscovitine; B). (C) Current-voltage relationship for peak and late ICa,L (measured at the end of 200-ms depolarizations) in control (n = 6, N = 6; n/N refers to cells/animals measured in each group) and with 20 µM roscovitine (n = 6, N = 6). (D) ICa,L steady-state (SS) activation and inactivation curves in control (n = 8, N = 6) and in the presence of roscovitine (n = 7, N = 6). Data points are mean ± SEM. Vertical asterisks, P < 0.001; *, P < 0.05.

• 

  Figure 3.. Roscovitine selectively reduces the late ICa,L in rabbit ventricular myocytes under AP clamp. (A) Nifedipine-sensitive current (ICa,L) recorded before (black, control) and after 20 µM roscovitine (green) from the same myocytes under AP clamp. All recordings were performed in the presence of 600 µM H2O2. The vertical dotted lines indicate the times (10 ms and 150 ms) at which the extent of roscovitine reduction of the current was calculated. (B) Plot summarizing the extent of peak and late ICa,L reduction caused by extracellular application of 20 µM roscovitine estimated from experiments as in A. Note that roscovitine selectively reduced the late ICa,L without affecting peak ICa,L. Data points are mean ± SEM. n = 9, N = 7.

• 

  Figure S2.. Roscovitine reduces late ICa,L (noninactivating component) in human CaV1.2 channels. (A) Superimposed Ba2+ currents from CaV1.2 channels expressed in oocytes (α1C + β2b + α2δ − 1) before (control) and after 100 µM roscovitine. (B) Average steady-state (SS) activation and quasi–steady-state inactivation curves obtained before and after roscovitine extracellular application. Continuous lines are Boltzmann fits to the activation and inactivation data points (fitting parameters are reported in Table S2). Note that roscovitine selectively reduced the late ICa,L, enhancing the extent of the steady-state inactivation (i.e., reduced pedestal of the steady-state inactivation curve, black diamond versus green triangle) without affecting the voltage dependence of activation (black circle versus green square). Data points are mean ± SEM; n = 7; *, P < 0.05.

• 

  Figure 4.. Roscovitine suppresses oxidative stress–induced EADs in ventricular myocytes. (A and B) Time course of APD90 (A) and selected APs (B) from the same myocyte for a representative experiment testing the EAD-suppressing action of roscovitine. A, a and B, a show AP recordings in control condition (Tyrode’s solution). H2O2 perfusion (600 µM) induced a robust EAD regime (B, b) indicated by the sudden and dramatic increases of APD90 (A, b; ∼6 min after H2O2 application). Extracellular addition of roscovitine (20 µM) completely abolished EAD occurrence and restored the normal AP duration despite the continuous presence of H2O2 (A, c and B, c). (C) Plot summarizing the average changes in APD90 and EAD occurrence during experiments performed as in A. Note that roscovitine suppressed EADs and restored a normal APD90, unlike vehicle alone (0.02% ethanol, light blue triangle), which had no significant effect on the EAD regime (roscovitine: n = 7, vehicle: n = 4, N = 9; mean ± SEM).

• 

  Figure 5.. Roscovitine reversibly suppresses hypokalemia and oxidative stress–induced EADs in ventricular myocytes. (A and B) Time course of APD90 (A) and selected APs (B) from the same myocyte for a representative experiment testing the EAD-suppressing action of 20 µM roscovitine. A, a and B, a show AP recordings in control condition (Tyrode’s solution). Hypokalemia (HypoK; 2 mM K+) + 100 µM H2O2 perfusion induced a robust EAD regime (B, b), indicated by the sudden and dramatic increases of APD90 (A, b). Extracellular addition of roscovitine (20 µM) completely abolished EAD occurrence and restored the normal AP duration despite the continuous presence of the stressors (A, c and B, c). The acute effect of roscovitine was reversible: following a washout of the drug from the extracellular solution, the APs were prolonged and EADs reappeared within a few minutes (A, d and B, d). The EAD regime was again abolished by a second application of roscovitine (A, e and B, e). (C) Summary of the average changes in APD90 and EAD occurrence during experiments performed as in A. Note that roscovitine suppressed EADs and restored a normal APD90, despite the continuous presence of stressors (n = 6, N = 5;…

• 

  Figure 6.. EADs are not prevented by intracellular application of roscovitine and are suppressed by extracellularly applied roscovitine. (A and B) Time course of the APD90 (A) and selected APs (B) from a rabbit ventricular myocyte in which 20 µM roscovitine was added to the intracellular (pipette) recording solution. A, a and B, a show AP recordings in control condition (Tyrode’s solution). In spite of the presence of intracellular roscovitine, 600 µM H2O2 was still able to induce a robust EAD regime (A, b and B, b), which was reversibly suppressed by the application of 20 µM extracellular roscovitine (A, c and B, c). The acute effect of roscovitine was reversible: following a washout of the drug from the extracellular solution, the APs were prolonged and EADs reappeared within a minute (A, d and B, d). The EAD regime was again abolished by a second application of roscovitine (A, e and B, e). (C) The plot quantifies the average changes in APD90 and EAD incidence for experiments performed as in A (n = 6, N = 4; mean ± SEM). Note that roscovitine acted reversibly from the extracellular side of the cells to potently suppress EADs.

• 

  Figure 7.. Roscovitine at EAD-suppressing concentration abolishes oxidative stress–induced EATs without affecting the normal Ca2+ transient duration and cell shortening of ventricular myocytes. (A) Representative Ca2+ transients from field-stimulated ventricular myocytes loaded with the Ca2+ indicator Fluo-4 AM in control (Tyrode's solution), after the addition of 200 µM H2O2 and in the presence of roscovitine (20 µM). Note that roscovitine suppressed EATs. (B) Box plot summarizing the occurrence of EATs in control, with H2O2, or the combination of roscovitine with H2O2 (n = 27, N = 8; grey triangle indicates experiments with 40 µM roscovitine; vertical asterisks, P < 0.001). (C) Box plot showing that cell shortening was not impaired by roscovitine (vehicle: n = 21, N = 4; roscovitine: n = 24, N = 4). (D) Representative Ca2+ transients from ventricular myocytes exposed to vehicle (0.02% DMSO) or roscovitine (20 µM). (E) Duration of the Ca2+ transient at half-maximal amplitude for vehicle (n = 16, N = 7) or roscovitine (n = 18, N = 9). (F) Average change in Ca2+ transient amplitude for vehicle (n = 16, N = 7) or roscovitine (n = 18, N = 9; *, P < 0.05). a.u, arbitrary unit. Box plots…

• 

  Figure 8.. Roscovitine terminates VT/VF in aged rat hearts. (A) Experimental protocol used to test the ability of roscovitine to suppress VT/VF in ex vivo perfused aged rat hearts. (B and C) Bipolar electrograms and microelectrode recordings from the indicated chambers of the heart. p-ECGs were obtained from right atrial–left ventricular leads. Single-cell APs were measured by a glass microelectrode inserted in the left ventricle epicardium. (B) The recordings show initiation of VF, which occurred 38 min after exposure to 100 µM H2O2. Note the initiation of VF by cellular EAD-mediated triggered activity, which arose suddenly during sinus rhythm (microelectrode, *). (C) Recordings from the same heart as in B, showing suppression of VF 16 min after the addition of roscovitine (20 µM) to the perfusate, in the continuous presence of H2O2.

• 

  Figure S3.. Roscovitine suppresses hypokalemia-induced VT/VF in young rat hearts. (A) Experimental protocol used to test the ability of roscovitine to suppress VT/VF in ex vivo perfused young rat hearts. (B and C) Bipolar electrograms and microelectrode recordings from the indicated chambers of the heart. p-ECGs were obtained from right atrial–left ventricular leads. Single-cell APs were measured by a glass microelectrode inserted in the left ventricle epicardium. (B) The recordings show the initiation of VF after 12 min of exposure to hypokalemia (HypoK; Tyrode’s with 2 mM K+). Note the initiation of VF by cellular EAD-mediated triggered activity, which arose suddenly during sinus rhythm (microelectrode, *). (C) Recordings from the same heart as in B, showing suppression of VF 8 min after the addition of roscovitine (20 µM) to the perfusate in the continuous presence of hypokalemia.

• 

  Figure 9.. Roscovitine prevents the induction of VT/VF by hypokalemia and oxidative stress in ex vivo rabbit hearts. (A) Experimental protocol used to test the ability of roscovitine to prevent VT/VF in ex vivo perfused aged rabbit hearts. (B) Bipolar electrograms from the indicated chambers of the heart. p-ECGs were obtained from right atrial–left ventricular leads. Left panel shows a representative experiment in control condition (Tyrode’s solution). Right panel shows the initiation of VT/VF in the same heart 14 min after perfusion of a hypokalemic (HypoK; 1 mM K+) Tyrode’s containing 100 µM H2O2. (C) Representative bipolar electrograms in control condition (left), after 15-min pretreatment with 50 µM roscovitine (middle), and 60 min after the perfusion of hypokalemia + 100 µM H2O2 in the presence of roscovitine (right). Note that roscovitine prevented the initiation of VT/VF. (D) Kaplan–Meier plot comparing time to onset of VT/VF for hearts exposed to hypokalemia and H2O2 in control (black) or in the presence of 20 or 50 µM roscovitine (n = 5 hearts per condition; *, P < 0.05).