The development of L-type Ca2+ current mediated alternans does not depend on the restitution slope in canine ventricular myocardium

Figures

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  Figure 1. Relationship between action potential alternans and S1S2 restitution. (a) Illustrates the applied protocol for S1S2 restitution. The basic cycle length for restitution was 1000 and 500 ms. The diastolic intervals increased from − 50 to 1000 ms (relative to APD90) and the slope of APD90 restitution was calculated at both 500 and 1000 ms BCLs. After the restitution protocols, alternans were measured from a cycle length of 250 ms to 160 ms. The magnitude of alternans was measured at APD90, APD50, and APD25. (b) Demonstrates correlation tests between S1S2 restitution slope and alternans. The left 6 panels show S1S2 restitutions at BCL of 500 ms, in the right 6 panels the BCL was 1000 ms. The first row demonstrates alternans at APD90 level, middle row at APD50, bottom row at APD25. Our results indicate that alternans developed even if the restitution slope was smaller than 1, furthermore, data show mainly weak and in some cases moderate correlations between variables.

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  Figure 2. Relationship between action potential alternans and dynamic restitution. (a) Illustrates the applied protocol for dynamic restitution. The basic cycle length for restitution was progressively decreased from 1000 to 160 ms. When BCL was 250 ms or shorter the action potential started to alternate. APD90 was plotted against the preceding diastolic interval in both long and short AP during alternans. (b) The slope of APD90 restitution was calculated and was plotted against the magnitude of alternans. The first row demonstrates alternans at APD90 level, middle row at APD50, bottom row at APD25. Similarly to the S1S2 restitution, we found that alternans developed even if the slope was smaller than 1, and data exerted weak or no correlations between restitution slope and alternans.

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  Figure 3. Relationship between APD and CaT amplitude alternans. (a) Illustrates simultaneous measurements of AP and CaT recorded at 250, 230 and 210 ms of BCL. At the left side a moderate AP alternans (black) coupled with marginal alternans of the CaT (blue curve), measured at 250 ms. (a) Right side illustrates marked AP alternans (black) associated with pronounced CaT alternans (blue) monitored at 210 ms. (b) Illustrates the APD90 alternans amplitude plotted against the extent of CaT alternans defined as CaT peak alternans ratio, at various BCLs. The grey line represents the linear regression of the points. (c) Depicts rapid pacing by non-alternating membrane potential, (upper curve) failed to induce CaT alternans under perforated patch-clamp condition. Parallel measured membrane current (middle curve) and CaT (lower curve) traces at a basic cycle length of 210 ms are shown. The evoked membrane currents were identical, and the evoked CaTs exerted negligible alternans.

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  Figure 4. The role of ICaL alternans in ignition of CaT alternans. ICaL were evoked by 3 subsequently applied alternating voltage clamp pulses, having a basic cycle length of 220 ms in all cases (a). (b) Illustrates a membrane current and CaT measurement evoked by a 140/80 ms voltage protocol. The ICaL peaks as well as the corresponding CaT did not show notable alternans. (c) Represents alternating ICaL and CaT during application of 180/40 ms protocol. (d) Illustrates a close relationship when the peak ICaL alternans were plotted against the corresponding CaT amplitude alternans. The black circles represent experiments evoked by 140/180 ms protocol, the blue circles demonstrate measurements under 180/40 ms protocol, while red circles indicates recordings during 200/20 protocol.

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  Figure 5. Analysis of the ICaL recovery in the presence of unbuffered Ca2+ handling. (a) (upper traces) demonstrates original representative current traces of recovery kinetics of the ICaL, Inset shows the applied voltage protocol. The lower panel demonstrates the parallel Ca2+ releases. The (b) depicts the extent of ICaL recovery plotted against the respective diastolic intervals, while (c) highlights the first 160 ms of the ICaL recovery.

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  Figure 6. Effect of 1 µM nisoldipine on APD alternans and restitution curve measured on multicellular tissue. (a) Illustrates representative original traces in control (red curve) and in the presence of 1 µM nisoldipine (green curve). Nisoldipine reduced the steady-state APD90 (bar graphs) measured at 500 ms BCL. (b) Represents a comparison between nisoldipine effect on restitution steepness (left y-axis) and on APD25 alternans (right y-axis) at a BCL of 500 ms. The nisoldipine reduced the slope of the restitution (green versus red curves) only in a slow phase, at DIs larger than 70 ms (significance marked with *). The APD25 alternans were significantly reduced at 250, 230, and 210 ms (green versus red symbols, significance marked with #). (c) Depicts APD90 restitution under control (red curve) and in the presence of 1 µM nisoldipine (green curve) at a BCL of 500 ms. However, the APD90 shortening after nisoldipine administration is clear, we can observe that the steepness of the curve primarily reduced at the slow phase.