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

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…

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…

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…

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.