Effect of intracellular pH on spontaneous Ca2+ sparks in rat ventricular myocytes

Abstract

1. A fall of intracellular pH (pHi) typically depresses cardiac contractility. Among the many mechanisms underlying this depression, an inhibitory effect of acidosis upon the sarcoplasmic reticulum (SR) Ca2+ release channel has been predicted, but not so far demonstrated in the intact cardiac myocyte. In the present work, pHi was manipulated experimentally while confocal imaging was used to record spontaneous 'Ca2+ sparks' (local SR Ca2+ release events) in rat isolated myocytes loaded with the fluorescent Ca2+ indicator fluo-3. In other experiments, whole cell (global) pHi or [Ca2+]i was measured by microfluorimetry (using, respectively, intracellular carboxy SNARF-1 and indo-1). 2. Reducing pHi (i) increased whole cell intracellular [Ca2+] transients induced either electrically or by addition of caffeine, whereas (ii) it decreased spontaneous Ca2+ spark frequency. Conversely, raising pHi increased spontaneous Ca2+ spark frequency. 3. Blocking sarcolemmal Ca2+ influx with 10 mM Ni2+, or reducing external pH by 1.0 unit, had no effect on the pHi-dependent changes in spontaneous Ca2+ spark frequency. 4. Decreasing pHi over the range 7.78-7.20, decreased Ca2+ spark frequency exponentially as a function of pHi, with frequency declining by approximately 33 % for a 0.2 unit fall in pHi. In contrast, over the same pHi range, Ca2+ spark amplitude was unaffected. Intracellular acidosis produced a slight slowing of Ca2+ spark relaxation. 5. The results indicate that, in the intact myocyte, a reduced pHi decreases the probability of opening of the SR Ca2+ release channel. This phenomenon may contribute to the negative inotropic effects of acidosis.

Figure 1

Effect of pH_i on intracellular pH and Ca²⁺ Traces of pH_i, indo-1 fluorescence ratio and images of Ca²⁺ sparks. A, representative recording of whole cell pH_i, showing the introduction of an intracellular acid load by withdrawal of 10 mm NH₄Cl. B, recording of intracellular indo-1 fluorescence ratio indicating changes in global cytoplasmic [Ca²⁺] at control pH_i (left trace) and during an acidosis (right trace). The left-hand and right-hand recordings were made at the times indicated by a and b in A. The [Ca²⁺]_i transients shown in B were induced either by field stimulation (0.33 Hz), or by application of 10 mm caffeine. After caffeine application, the cell was allowed to recover for 40 s before stimulation was continued (indicated by the break in the indo-1 recording). C, examples of confocal line-scan images of Ca²⁺ sparks measured at control pH_i (left image) and during an acidosis (right trace); the images were taken at times corresponding to a and b in A. Images have been false coloured by computer with blue representing low fluorescence intensity (i.e. low [Ca²⁺]_i) and green, yellow and red representing respectively higher fluorescence intensities. Recordings in A, B and C were made in different cells.

Figure 2

Effect of pH_i on spontaneous Ca²⁺ sparks Confocal line-scan images of Ca²⁺ sparks at control pH_i and during acidosis or alkalosis. Parts A and B, illustrated on the left-hand and right-hand pages, respectively, were taken from different experiments. In both A and B, the top panels show 20 consecutive images taken at the control pH_i of 7.46 (Ai and Bi). The lower panels show the effect of reducing pH_i (left; Aii) and raising pH_i (right; Bii) by the amounts indicated. Data collection in the two lower panels commenced 1 min after removal of either 10 mm NH₄Cl (Aii) or 80 mm acetate (Bii) from the superfusate. False colouring by computer indicates low to high fluorescence intensity spectrally as blue to red. Scale bar in B also applies to A.

Figure 3

Effect of an intracellular acidosis on Ca²⁺ spark frequency Ca²⁺ spark frequency was calculated from a gallery of 20 images taken at the beginning of the experiment (basal frequency) and then again (A) after a control period of 10 min (or 1 min when superfusate pH was 6.4), and (B) 1 min after the introduction of an intracellular acid load (5 min, 10 mm NH₄Cl prepulse). Ca²⁺ spark frequency is expressed as a percentage of the basal frequency. The experiment was performed in NT solution (□), in NT solution containing 10 mm NiCl₂ (), and in NT solution of pH 6.4 (). Decrease in pH_i (±s.e.m.) relative to basal pH_i is shown beneath columns on x-axis. ^*Significant fall of spark frequency (P < 0.01).

Figure 4

Relationship between pH_i and Ca²⁺ spark frequency Change in Ca²⁺ spark frequency is expressed as a fraction of the frequency at control pH_i (n/n₀) and plotted on a log scale versus pH_i (n = 11 at pH_i 7.61; n = 12 at all other points). A linear regression line of best fit has been drawn through the data points. Inset shows the exponential shape of the relationship, revealed when n/n₀ is plotted on a linear axis.

Figure 5

Effect of pH_i on Ca²⁺ spark duration and amplitude A, image of a Ca²⁺ spark, rotated so that time is displayed on the horizontal axis and fluo-3 fluorescence intensity with distance along the cell is displayed on the vertical axis. False colouring by computer indicates fluo-3 fluorescence intensity spectrally from blue (low) to red (high). The pixel values along the dotted line have been plotted below the image and show the fluorescence intensity profile of the Ca²⁺ spark. Bi, mean of 11 Ca²⁺ spark fluorescence intensity profiles for Ca²⁺ sparks (normalised to the same peak intensity) at both control (black trace) and acidotic pH_i (red trace, ΔpH_i = −0.19). Bii, normalised histograms of Ca²⁺ spark amplitudes, measured as Ca²⁺ spark fluorescence intensity divided by background fluorescence intensity (F/F₀) and binned at intervals of 0.1 F/F₀ units, at control pH_i (grey shaded bars) and during an intracellular acidosis of 0.19 pH units (red open bars). Ci, mean of nine Ca²⁺ spark fluorescence intensity profiles for Ca²⁺ sparks (normalised to the same peak intensity) at both control (black trace) and alkaline pH_i (blue trace; ΔpH_i = 0.32). Cii, normalised histograms of Ca²⁺ spark amplitudes at control pH_i (grey shaded bars) and during an intracellular alkalosis of 0.32 pH units (blue open bars).