Interaction of background Ca2+ influx, sarcoplasmic reticulum threshold and heart failure in determining propensity for Ca2+ waves in sheep heart

Abstract

Ventricular arrhythmias can cause death in heart failure (HF). A trigger is the occurrence of Ca²⁺ waves which activate a Na⁺ -Ca²⁺ exchange (NCX) current, leading to delayed after-depolarisations and triggered action potentials. Waves arise when sarcoplasmic reticulum (SR) Ca²⁺ content reaches a threshold and are commonly induced experimentally by raising external Ca²⁺ , although the mechanism by which this causes waves is unclear and was the focus of this study. Intracellular Ca²⁺ was measured in voltage-clamped ventricular myocytes from both control sheep and those subjected to rapid pacing to produce HF. Threshold SR Ca²⁺ content was determined by applying caffeine (10 mM) following a wave and integrating wave and caffeine-induced NCX currents. Raising external Ca²⁺ induced waves in a greater proportion of HF cells than control. The associated increase of SR Ca²⁺ content was smaller in HF due to a lower threshold. Raising external Ca²⁺ had no effect on total influx via the L-type Ca²⁺ current, I_Ca-L , and increased efflux on NCX. Analysis of sarcolemmal fluxes revealed substantial background Ca²⁺ entry which sustains Ca²⁺ efflux during waves in the steady state. Wave frequency and background Ca²⁺ entry were decreased by Gd³⁺ or the TRPC6 inhibitor BI 749327. These agents also blocked Mn²⁺ entry. Inhibiting connexin hemi-channels, TRPC1/4/5, L-type channels or NCX had no effect on background entry. In conclusion, raising external Ca²⁺ induces waves via a background Ca²⁺ influx through TRPC6 channels. The greater propensity to waves in HF results from increased background entry and decreased threshold SR content. KEY POINTS: Heart failure is a pro-arrhythmic state and arrhythmias are a major cause of death. At the cellular level, Ca²⁺ waves resulting in delayed after-depolarisations are a key trigger of arrhythmias. Ca²⁺ waves arise when the sarcoplasmic reticulum (SR) becomes overloaded with Ca²⁺ . We investigate the mechanism by which raising external Ca²⁺ causes waves, and how this is modified in heart failure. We demonstrate that a novel sarcolemmal background Ca²⁺ influx via the TRPC6 channel is responsible for SR Ca²⁺ overload and Ca²⁺ waves. The increased propensity for Ca²⁺ waves in heart failure results from an increase of background influx, and a lower threshold SR content. The results of the present study highlight a novel mechanism by which Ca²⁺ waves may arise in heart failure, providing a basis for future work and novel therapeutic targets.

Keywords: Ca2+; heart failure; sarcoplasmic reticulum; threshold; waves.

Figure 1. Effects of increasing external Ca²⁺ concentration in control and heart failure cells

A, specimen records showing effects of elevating Ca²⁺ from 1.8 to 10 mM in a control (a) and heart failure (HF) (b) myocytes. In this and subsequent figures, cells were stimulated with 100 ms duration pulses from a holding potential of −40 to 10 mV, applied at 0.5 Hz. Arrows indicate Ca²⁺ waves. B–D, summary data (normalized to 1.8 mM Ca²⁺) showing the effects of increasing external Ca²⁺ on Ca²⁺ transient amplitude (B), diastolic [Ca²⁺]_i (C) and average [Ca²⁺]_i (D). E, summary data of the proportion of cells showing waves. Mean ± SD shown to the right of data in 10 mM Ca²⁺. For Ca²⁺ transient amplitude; control 11 cells/8 animals one sample t test, HF 19 cells/8 animals Wilcoxon matched pairs signed rank test. For diastolic [Ca²⁺]_i; control 9 cells/6 animals one sample t test, HF 12 cells/7 animals Wilcoxon matched pairs signed rank test. For average Ca²⁺; control 8 cells/5 animals, HF 11 cells/7 animals, one sample t test for both comparisons. For proportion of cells waving; control 1.8 mM Ca²⁺ 117 cells/41 animals, control 10 mM Ca²⁺ 30 cells/16 animals, HF 1.8 mM Ca²⁺ 31 cells/10 animals, HF 10 mM Ca²⁺ 27 cells/10 animals, chi‐squared test for all comparisons. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. Effects of external Ca²⁺concentration on SR Ca²⁺ content and threshold for waves

A, original data. Traces show: top, membrane current; bottom, integral of current. Records are taken from representative examples from control (a) and HF (b) myocytes. In both, the left‐hand traces were recorded in 1.8 mM Ca²⁺ and the right‐hand in 10 mM. 10 mM caffeine was applied for the period shown by the horizontal bars. Arrows show inward currents produced by Ca²⁺ waves. B, summary data. In this, and subsequent diagrams, error bars denote ± SD for both control and heart failure, the left‐hand points (open symbols) show SR Ca²⁺ content measured in 1.8 mM Ca²⁺ (in the absence of waves: 21 cells from 15 animals in control and 20 cells from 7 animals in HF). The right‐hand points (+waves) show the SR Ca²⁺ content in those cells which displayed waves in elevated Ca²⁺. This was achieved in 9 control cells (from 7 animals) and 18 HF cells (from 9 animals) by elevating external Ca²⁺ to 10 mM, and in 7 control cells (from 4 animals) by elevating external Ca²⁺ to 15 mM. Cells from control animals which did not display waves in high Ca²⁺ are also shown (grey symbols, marked ‘−waves’, total 6 cells from 5 animals; two of which were in 15 mM Ca²⁺ and 4 in 10 mM Ca²⁺). For control 1.8 Ca vs. control high Ca ‘−waves’, Mann‐Whitney test. For control 1.8 Ca vs. control high Ca ‘+waves’, Mann‐Whitney test. For control high Ca ‘+waves’ vs. control high Ca ‘−waves’, unpaired t test. For control high Ca ‘+waves’ vs. HF high Ca ‘+waves’, mixed effects linear mixed modelling. For HF 1.8 Ca vs. HF high Ca, Mann‐Whitney test. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. The effects of external Ca²⁺ concentration on the L‐type Ca²⁺ current

A, specimen paired records showing the effects of elevating external Ca²⁺ from 1.8 to 10 mM. In all panels 100 ms duration depolarizing pulses were applied at 0.5 Hz to +10 mV from a holding potential of −40 mV. Panels show: a, control; b, heart failure. In both panels the left‐hand trace was obtained in 1.8 and the right‐hand in 10 mM external Ca²⁺ from the same cell. B, integral of the L‐type Ca²⁺ current in 10 mM external Ca²⁺. Black symbols from control cells, red heart failure. In both, data are separated by whether the cells showed waves or not. Control: no waves 20 cells/14 animals, with waves 11 cells/5 animals. Heart failure: no waves 5 cells/3 animals, with waves 19 cells/8 animals. For control no waves vs. with waves, unpaired t test. For HF no waves vs. with waves, Mann‐Whitney test. For control no waves vs. HF no waves, mixed effects linear mixed modelling. For control with waves vs. HF with waves, Mann‐Whitney test. C, time course of mean data (31 control and 24 heart failure cells). Graphs show: a, fraction of cells displaying waves; b, mean peak L‐type Ca²⁺ current; c, mean integral of L‐type current. Black symbols, control; red symbols, heart failure. External Ca²⁺ concentration was increased from 1.8 to 10 mM for the period shown. Shaded areas show 95% confidence limits. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. Measurement of Ca²⁺ efflux in elevated external Ca²⁺

A, original records: top, [Ca²⁺]_i, bottom, membrane current. a, control; b, heart failure; c, expanded, averaged (five sweeps) membrane current records. All data obtained in 10 mM external Ca²⁺. Arrows denote a Ca²⁺ wave and accompanying inward current. B, average Ca²⁺ efflux on NCX during the Ca²⁺ transient (tail). Data shown from both 1.8 and 10 mM external Ca²⁺ in control and HF. Control: 31 cells/18 animals, HF 24 cells/9 animals. For comparisons between 1.8 and 10 mM Ca²⁺ (in both HF and control), paired t tests. For comparison between HF and control, mixed effects linear mixed modelling. C, average Ca²⁺ efflux on NCX per cycle during waves. Control: 31 cells/18 animals, HF 24 cells/9 animals, Mann‐Whitney test. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5. Estimation of background flux

A, time course of mean data showing the effects of elevating external Ca²⁺ from 1.8 to 10 mM: a, efflux during Ca²⁺ transient (tail); b, efflux on waves; c, total efflux (filled symbols) compared with Ca²⁺ influx on L‐type current (open symbols). Shaded areas show 95% confidence limits. B, background influx in the steady state in 10 mM external Ca²⁺ in control (left) and heart failure (right). Control 31 cells/18 animals, HF 24 cells/9 animals, mixed effects linear mixed modelling. C, background influx as a function of both whether waves are present and cell type. Control: no waves 20 cells/14 animals, with waves 11 cells/5 animals. HF: no waves 5 cells/4 animals, with waves 19 cells/9 animals. For control, no waves vs. with waves, Mann‐Whitney test. For HF, no waves vs. with waves, Mann‐Whitney test. For comparisons between HF and control, mixed effects linear mixed modelling. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6. Effect of NCX block on Ca²⁺waves and background Ca²⁺ entry

Aa, representative recordings from a single cell under voltage clamp. Panels show (from left to right): Ni²⁺ (10 mM) and 1.8 mM Ca²⁺; Ni²⁺ and 15 mM Ca²⁺; Ni²⁺ washout in 15 mM Ca²⁺. Ab, summary data for effects of Ni²⁺ (10 mM) on waves in unpatched cells in 10 mM Ca²⁺. Unpaired data. B, representative increases in [Ca²⁺]_i when cells were exposed to 15 mM external Ca²⁺. The cells had been in Ca²⁺‐free solution for at least 2 min before raising Ca²⁺, and caffeine (10 mM) was present throughout to prevent Ca²⁺ uptake into the SR. Records show: a, control; b, Na‐free (a different cell). c, summary data for the maximum rate of rise. For Ab, n = 13 cells/4 animals Ctrl vs. n = 18 cells/3 animals Ni²⁺, unpaired t test. For Bc, n = 11 cells/3 animals in 140 mM Na⁺, n = 17 cells/3 animals in 0 mM Na⁺, Mann‐Whitney test.

Figure 7. Effect of inhibitors on spontaneous Ca²⁺ waves

Waves were induced in control cells by raising external Ca²⁺ to 15 mM. A, representative Ca²⁺ recordings in waving cells exposed to Gd³⁺ and its washout (a) and BI 749327 (b). B, mean effects of inhibitors on wave frequency: a, mean effect of Gd, paired data from n = 18 cells/3 animals. Wilcoxon matched‐pairs signed rank test; b, mean effect of BI 749327, Wilcoxon matched‐pairs signed rank test on paired data from n = 20 cells/4 animals; c, mean effect of nicardipine, paired t test from n = 11 cells/3 animals; d, mean effect of Pico145 on wave frequency, paired t test from n = 13 cells/3 animals; e, mean effect of pre‐incubation with β‐glycrrhetinic acid on wave frequency, unpaired t test from n = 13 cells/4 animals (control) and 11 cells/3 animals (β‐GA).

Figure 8. Assessment of background influx with Mn²⁺ quench

A, representative recordings of Fura signal quench (F ₃₆₅) in single cells exposed to Mn²⁺ (1 mM). The effects of Mn²⁺ were tested in control cells (left), and when exposed to gadolinium, BI 749327, Pico 145 and β‐glycrrhetinic acid. B, summary mean data. For analysis, cells were randomly paired with control cells from the same animal and the rate of quench normalized to the control value. For each inhibitor Ba–d, n = 6 cells/3 animals, Wilcoxon ranked pairs signed rank test.