Arrhythmogenic late Ca ²⁺ sparks in failing heart cells and their control by action potential configuration

Figures

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  Fig. 1.. Changes in structure, AP time course, t-tubule morphology, and increased LCS in HF. (A) di-8-ANEPPS staining shows reduced tubule regularity and (B) reorientation of t-tubules in HF cells compared to control (CON). n/N = 9/4 CON 12/5 HF. (C) ICa density was unchanged in HF. n/N = 20/4 CON 10/3 HF Representative currents at the indicated test potentials (mV) are shown (Inset). Error bars not shown where error is less than the size of symbols. (Scale bars: 5 pA/pF, 10 ms.) (D) APs in HF myocytes had less AP phase 1 repolarization (arrow). (E) Ca2+ transients in CON myocytes showed synchronous Ca2+ release, while HF showed less uniform Ca2+ release and (F) an increase in latency from electrical stimulation to detectable Ca2+ increase. (G) Image processing reveals increased LCS frequency in HF with some Ca2+ ripples (yellow dashed box). (H) LCS frequency was greater in HF cells than in CON. n/N = 16/6 CON; 14/5 HF. **P < 0.01, ***P < 0.001, unpaired t tests.

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  Fig. 2.. LCS can trigger EADs. (A) EADs evoked by pacing-pause protocol. During repolarization, an initial Vm inflection at ∼ −20 mV generates EADs (Top), and numerous LCS create oscillatory Ca2+ release (Bottom). (B) Calculation of Inet for a typical HF AP. The filled region indicates where the greatest number of LCS occur (A and Fig. 4E) and corresponds to an Inet of ∼0.18 pA/pF. Any balancing current that counteracts Inet (i.e., −Inet, Bottom) will stop repolarization (SI Appendix, Fig. S5). (C) Failing AP with voltage-clamp steps used to probe Ca2+ and currents underlying EADs. (D) During the AP steps, LCS-triggered Ca2+ waves (Top) and inward currents (black trace) appear. Dashed line shows the inward current that balances Inet in B and could therefore stop repolarization. This current occurs when LCS are about to initiate Ca2+ waves. (E) Example of apparently chaotic Ca2+ during EADs in an HF cell (Top). Image processing reveals many LCS and Ca2+ ripples during Vm oscillations (red trace). The filled regions show where the depolarizing current (Inet) is increasing, and this corresponds to increasing Ca2+ (blue) and LCS activity (orange bars).

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  Fig. 3.. Computer simulations of experimental traces using a model that generates LCS (29). Panels are ordered to indicate the flow of causation. (Ai) Increased LCS frequency is in phase with inward INCX and dVm/dt, which precedes Vm and ICa forming an oscillatory feedback pathway as shown by the arrows. (Aii) When Ca2+ load is reduced, Vm oscillates because ICa (and possibly INa) supply the inward current to increase dVm/dt and cause the EAD, as shown. In this case, LCS (and Ca2+) are the consequence of the reactivation of ICa. Note that in this simulation, INCX bears no obvious relation to dVm/dt (unlike the results shown in Ai). Cross-talk between the oscillators shown in Ai and Aii can occur because they contain common mechanisms. (B) Cross-correlograms between Vm and both ICa and INCX from the data shown in part A. The negative peaks in the cross-correlogram (black arrows) reveal both the strength of association (amplitude) and temporal relationship (offset from Vm) between Vm and ICa or INCX (see SI Appendix). (Bi) When Ca2+ load was high, inward INCX preceded Vm oscillations and was more strongly correlated with Vm than ICa. (Bii) Under reduced Ca2+ load, INCX lags behind Vm…

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  Fig. 4.. Applying a normal AP to HF cells improves excitation–contraction coupling and reduces LCS frequency. (A) Ca2+ transient upstroke velocity, amplitude (orange), and Ca2+ release synchrony all increase in an HF cell (Left) when voltage-clamped with a normal AP (Right). (Lower) Improved synchrony in Ca2+ release. (B) Mean latency for Ca2+ release and (C) Ca2+ transient duration were reduced in HF cells clamped with a normal AP. (D) Processed Ca2+ images show fewer LCS in HF after applying normal AP. (E) Number of LCS detected in HF with failing AP waveform (HF AP, 404 LCS) and with a normal AP (+Norm AP, 217 LCS) from 13 HF cells binned by Vm during the AP repolarization. **P < 0.01, paired t test n/N = 13/5.

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  Fig. 5.. Flow diagram for two interacting positive-feedback mechanisms that can drive Vm and Ca2+ oscillations during EADs. The red inner cycle represents the well-established Vm oscillator, wherein an increase in dVm/dt (caused by a relative increase in depolarizing currents compared to repolarizing currents, e.g., text in orange box at Lower Right) leads to voltage-dependent reactivation of ICa, which in turn causes further depolarization. The blue outer cycle represents the stochastic LCS-mediated Ca2+ oscillator mechanism for EAD initiation. Reduced early Ca2+ release and/or increased SR load increases LCS production, and the resulting increase in inward INCX tends to depolarize the membrane which then feeds onto ICa to trigger additional LCS. Green arrows indicate possible factors contributing to EAD initiation. β-AR, beta-adrenoreceptor.