Improved Ca2+ release synchrony following selective modification of Itof and phase 1 repolarization in normal and failing ventricular myocytes

Figures

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  Fig. 1. Modifying early AP repolarization with Itof,DyC alters cell contraction in non-failing rabbit ventricular myocytes. A Exemplar control APs recorded from a non-failing rabbit ventricular myocyte before (black line) and after dynamic clamp with a ‘synthetic’ Itof was enabled to enhance (+Itof,DyC, green line) or decrease (-Itof,DyC, magenta line) the phase 1 repolarization notch. The stimulus pulse has been partially removed for clarity. B Exemplar recording of sarcomere shortening during control APs with and without Itof,DyC (no blebbistatin or Fluo-4 in pipette or extracellular solution). C + Itof,DyC increased the magnitude of sarcomere shortening and (D) decreased the time to peak shortening, whereas -Itof,DyC decreased shortening and prolonged the time to peak and E increased the full duration at half maximal shortening (FDHM). F Fluo-4 was included in the pipette in a subset of cells (n = 6) to simultaneously record intracellular Ca2+ during contractions. In non-failing myocytes +Itof,DyC caused a modest increase in Ca2+ transient amplitude, whereas -Itof,DyC decreased Ca2+ transient amplitude more severely, corresponding to an increase and decrease in contraction with…

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  Fig. 2. Effect of subtracting Itof with DyC on AP shape and Ca2+ transients. A Upper panel Opposing native Itof in non-failing rabbit myocytes using DyC abolished the small phase 1 notch and lengthened APD slightly, despite -Itof,DyC fully inactivating within ~50 ms (magenta trace lower panel) during pacing at 1 Hz. Gtof,DyC was 0.1 nS/pF. The stimulus artefact has been suppressed for clarity. B -Itof,DyC decreased the amplitude and increased the duration of the resulting Ca2+ transient. C Confocal Ca2+ line scan recordings reveal -Itof,DyC caused a loss of Ca2+ release uniformity across the cell. Lower panels show processed images [17] revealing LCS activity and an approximate doubling in the number LCS frequency occurred when native Itof was electrically neutralised (white boxes in lower panel). Mean results show -Itof,DyC increased the latency between electrical stimulation and Ca2+ release (D), decreased Ca2+ transient amplitude (E), increased LCS frequency (F) and increased Ca2+ transient duration (G). n/N = 12/4, **P < 0.01, ***P < 0.001 vs Control. D,F,G paired t-test, E Wilcoxon matched-pairs signed rank test. (For interpretation of the references to colour in this figure legend,…

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  Fig. 3. Adding Itof,DyC to guinea pig myocytes synchronises Ca2+ release and shortens APD. A Normal guinea pig ventricular myocytes (black trace) exhibited no phase 1 notch but addition of +Itof,DyC induced a phase 1 notch (green trace) reminiscent of rabbit and human APs as well as a slight shortening of APD90 (stimulation frequency 1 Hz). B The associated Ca2+ transient was larger with a faster rate of rise and decay when Itof,DyC was added. C Confocal Ca2+ line scan recordings show some non-uniformity in Ca2+ release during normal Ca2+ transients (left panels) which was reduced by addition of Itof,DyC (right panels). Lower panels show a reduction in LCS occurred with Itof,DyC addition. In all cells examined (n/N = 14/4), +Itof,DyC decreased the average Ca2+ transient latency (D) and increased the Ca2+ transient amplitude. (E) + Itof,DyC decreased LCS frequency (F). +Itof,DyC did not change Ca2+ transient duration (G; P = 0.32). n/N = 14/4, **P < 0.01, ***P < 0.001 vs control. D,F,G paired t-test, E Wilcoxon matched-pairs signed rank test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 4. Improved Ca2+ release in failing cells with AP phase 1 notch restoration. A Exemplar failing rabbit ventricular myocyte (HF) AP lacking a phase 1 notch before (black) and after introducing +Itof,DyC (green). +Itof,DyC produced a distinct phase 1 notch and slightly shortened APD. The control APD90 in this cell was 366 ms. B The Ca2+ transient amplitude, rate of Ca2+ rise and rate of decay increased when +Itof,DyC was added. C Confocal Ca2+ line scans show +Itof,DyC increased the spatiotemporal synchrony of Ca2+ release throughout the cell (top panels) and decreased the number of LCS (lower panels and D) and reduced the duration of the Ca2+ transient (E). F Fixed patterns of Ca2+ release variation were detected by averaging 5 Ca2+ transient upstrokes before (top panel) and after adding +Itof,DyC (middle panel). Ca2+ release latency across the cell (marked by yellow lines) following electrical stimulation (white lines). Latency (lower panel) decreased in most areas. Vertical scale bar 25 ms. G Despite regional variation, the variability in local latency was reduced by +Itof,DyC. n/N = 19/6, *P < 0.05, ***P < 0.001, D,E,G paired t-test. (For interpretation of the references to colour…

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  Fig. 5. Steady state changes in ICa and Ca2+ release synchrony with Itof,DyC. A The steady state Ca2+ transient was established by pacing cells with a typical control AP or + Itof,DyC AP as the voltage clamp command. The Ca2+ transient responded to a sudden change from an control AP to a + Itof,DyC AP waveform with an immediate increase in amplitude. This was followed by a return to the previous steady state amplitude in 4–5 further beats. When a final control AP was reapplied (indicated by the arrow) there was a 20% reduction in the resulting Ca2+ transient amplitude, indicating a reduction in steady state Ca2+ load with +Itof,DyC. B Improvement in Ca2+ release synchrony with +Itof,DyC AP persisted in steady state, as shown by a reduction in time to 50% peak Ca2+ release (C). D ICa measured as the nifedipine sensitive current was recorded using control and + Itof,DyC AP waveforms. The membrane potential was set to −40 mV just before the AP waveform to prevent voltage escape. The +Itof,DyC AP resulted in a marked increase in ICa amplitude together with a faster inactivation time course (lower panels) so that although peak ICa increased with +Itof,DyC AP (E), the integral of ICa (F) was…

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  Fig. 6. Experiments and computer simulations show a biphasic response of Ca2+ release and APD to increasing Itof,DyC. The links between Ca2+ release and APD90 with increasing Gtof,DyC was investigated experimentally (A-C) and with computer simulations of rabbit cardiac myocyte biophysics (D—H). A Increasing Gtof,DyC shifted the nadir of the AP notch to progressively more negative potentials. APD90 initially shortened (green trace), then lengthened (red trace), before finally collapsing to form a very short AP (blue trace). B Confocal Ca2+ line scans show an increase in Ca2+ release synchrony and (C) amplitude at ~0.2 nS/pF Gtof,DyC (Gtof,DyC values given in figure panels), followed by a decrease in amplitude and synchrony at greater Gtof,DyC. D Simulations also showed a similar biphasic response of phase 1 notch and APD90 to increasing Gtof,DyC (top panel) with ionic currents shown in the lower panels. Moderate Gtof,DyC increased peak ICa, decreased plateau ICa and decreased inward INCX during late repolarization (blue traces). Greater Gtof,DyC caused rapid termination of ICa, followed by reactivation, and delayed IKs and IKr activation (green traces). ICa failed to reactivate when…

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  Fig. 7. Effect of + Itof,DyC on AP morphology, duration and Ca2+ transients in failing cells the presence of β-adrenergic stimulation. A Adding 50 nmol/l isoproterenol (ISO) shortened the AP of failing cells and slightly deepened phase 1 repolarization. Adding +Itof,DyC (0.4 nS/pF) in the presence of ISO caused the phase 1 repolarization notch to deepen further but did not markedly change phase 2/3 or AP duration. B Line scan recordings of the Ca2+ transient upstroke in a failing cell in Tyrode, ISO and ISO + Itof,DyC. ISO improved Ca2+ release synchrony and amplitude which was further improved by adding +Itof,DyC. Compared to ISO alone, +Itof,DyC (0.4 nS/pF) increased Ca2+ transient amplitude (C) and decreased Ca2+ release latency (D) and latency variability (E). Panel F shows AP duration at 90% repolarization (APD90) measured in response to increasing levels of +Itof,DyC with and without ISO. Data are normalized to the control APD90 (Gtof,DyC = 0 nS/pF) in Tyrode's or ISO solutions. APD90 in Tyrode decreased sharply when +Itof,DyC was increased beyond a threshold conductance (~1nS/pF). In ISO this threshold value was increased to >2 nS/pF. n/N = 9/3, *P < 0.05, **P < 0.01 vs ISO, C…

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  Fig S1. Kinetics of the mathematical model used to produce Itof for DyC experiments and simulations. A Itof response to steps to different holding potentials (shown inset). B Current voltage relationship for modelled Itof C Recovery from inactivation. Itof recovery from inactivation consisted of a single fast component and was essentially complete in 100 ms. D Time course of Itof during a simulated AP.

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  Fig S2. Non-failing rabbit myocytes voltage clamped with a failing AP waveform increased LCS rate. A Non-failing rabbit myocytes were voltage clamped with a train of 5 normal APs followed by a failing AP (HF) at 1 Hz to examine the role of loss of the AP notch on Ca2+ release and LCS. B Ca2+ transient amplitude was smaller and decayed more slowly with a failing AP than a normal AP. C Confocal Ca2+ line scans reveal greater heterogeneity in Ca2+ release timing with a failing AP. High-pass image processing shows many more LCS occurred in response to a failing AP (lower panels). D Average Ca2+ transient latency was increased, E Ca2+ transient amplitude decreased, and F LCS frequency and G Ca2+ transient duration increased with a HF AP compared to a normal AP. n/N = 12/4, *P < 0.05, **P < 0.01, ***P < 0.001. D,F paired t-test, E,G Wilcoxon matched-pairs signed rank test.

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  Fig S3. Faster Ca2+ transient decay contributes to action potential duration shortening with enhanced Itof through reduced INCX The role of LCS and INCX in APD90 shortening caused by Itof,DyC (0.2 nS/pF) was investigated by clamping cytosolic Ca2+ ([Ca2+]cyt) during computer simulations. A Traces show the simulated Ca2+ transient in response to the native control AP (black line) and when Itof,DyC was added (green line). After the initial Ca2+ release and when cytosolic Ca2+ levels were approximately equal between simulations, the Itof,DyC cytosolic Ca2+ was clamped to the control Ca2+ transient decay time course (broken red line) B Simulated APs without DyC (black line), with DyC (green line), and with DyC plus cytosolic Ca2+ clamp (green continuing into red dashed line). APD90 increases in the Itof,DyC simulation when Ca2+ is clamped to the normal Ca2+ transient decay. C Inward INCX increased when Ca2+ was clamped to the normal Ca2+ transient decay during the Itof,DyC simulation, likely due to increased cytosolic Ca2+ and slightly more negative plateau potential of the Itof,DyC simulation favouring forward mode Ca2+ removal. D Ca2+ clamp had negligible effect on ICa.