Regional acidosis locally inhibits but remotely stimulates Ca2+ waves in ventricular myocytes

Figure 1. Intracellular acidosis stimulates Ca2+ waves in an NHE1-dependent manner. (Ai) Ca2+ waves (fluo-3) were triggered at 5 mM extracellular [Ca2+]. Images recorded in linescan (xt) mode along the longitudinal axis of the cell at 400 lines per second. F/F0 = fluorescence divided by resting fluorescence (F0 averaged over first 1 s of recording). (Aii) Decreasing pHi (cSNARF-1, mean pHi trace, error bars are SEM, n = 13 cells/2 animals) by 80 mM acetate superfusion initially decreased, then increased wave frequency (n = 18 cells/5 animals). Waves were counted in 10 s bins and normalized to the mean frequency in the first 10 s bin. (Aiii) [Na+]i timecourse, on the same timescale as Aii and taken under comparable experimental conditions, replotted from Figure 2B of reference 18. SBFI ratio acquired every 4 s, n = 6 cells/2 animals, error bars are SEM. (Aiv) Ca2+ wave frequency showed a positive relationship with decreasing pHi. Error bars are SD (pHi), SEM (frequency). Wave frequency and pHi averaged over 1 min, from 30 s after onset of acetate superfusion. For 80 mM acetate n = 18 cells/5 animals (waves), 13 cells/2 animals (pHi); 40 mM acetate n = 5 cells/1 animal (waves), 8 cells/2…

Figure 2. Acidosis-induced stimulation of Ca2+ waves depends on sarcolemmal NCX. (Ai) Ca2+ wave initiation frequency is steeply dependent on [Na+]i. Wave frequency ∝ [Na+]i4. Note the logarithmic axes. (Aii) In the absence of an NHE-driven [Na+]i rise, Ca2+ waves show a linear, inverse relationship with [H+]i. Note the logarithmic axes. Fit using linear regression: 50.5 × [H+]−1. (B) Proposed interactions between intracellular H+, Na+, and Ca2+ modify SR Ca2+ load, and thus Ca2+ wave probability. Pharmacological inhibitors are in red. (C) Wave frequency averaged during 2nd min of acetate superfusion. Inhibiting mitochondrial NCX (‘CGP’: 20 μM CGP-37157) had no effect on acidosis-induced Ca2+ wave stimulation (n = 11 cells/2 animals). Inhibiting both mitochondrial NCX and the mitochondrial Ca2+-uniporter (‘Ru360’: 10 μM ruthenium-360) enhanced wave stimulation (n = 11 cells/5 animals). Inhibiting sarcolemmal NCX (10 μM SN-6) prevented acidosis-induced wave stimulation (n = 4 cells/2 animals). Nested ANOVA followed by pairwise comparison with Holm correction for multiple testing.

Figure 3. Intracellular acidosis alters Ca2+ wave velocity. (A) Intracellular acidosis increases velocity of Ca2+ waves in the presence (n = 18 cells/5 animals) (i) and absence (i.e. with 30 µM DMA; n = 11 cells/2 animals) (ii) of NHE1 activity. (B) Normalized wave velocity measured during 2nd min of 80 mM acetate superfusion. (C) Wave velocity and pHi were averaged over 100 s, starting 20 s after onset of acetate superfusion with 30 μM DMA (80 mM acetate + DMA n = 11 cells/2 animals (waves), 5 cells/1 animals (pHi); 40 mM acetate + DMA n = 4 cells/1 animal (waves), 6 cells/2 animals (pHi); 20 mM acetate + DMA n = 8 cells/2 animals (waves), 8 cells/2 animals (pHi); 20 mM TMA n = 12 cells/2 animals (waves), 8 cells/2 animals (pHi)). Fit: y=-0.5223x + 4.7943 (R2 = 0.996). Paired t-tests.

Figure 4. An acidic microdomain affects Ca2+ wave initiation both locally and remotely. (A) A longitudinal pHi gradient was generated by regional superfusion of the myocyte with 80 mM acetate. Top: schematic of dual microperfusion apparatus; middle; representative pHi gradient along a myocyte, xy image with SNARF-1; bottom; representative Ca2+ wave recorded in linescan mode during an imposed pHi gradient. (B) Magnitude of pHi gradient (n = 12 cells/4 animals) was unaffected by inhibiting NHE1 in the acidic microdomain (n = 11 cells/2 animals). (Ci) Exemplar linescans recorded from 2 rat ventricular myocytes, under control conditions (left panel of each cell), and during imposition of a longitudinal pHi gradient by regional superfusion of the myocyte with 80 mM acetate (right panel of each cell). In order to compress the results, the timecourse for the line-scans has been broken into three parallel columns. Note that, during control conditions, Ca2+ waves initiate randomly along the length of the myocyte, whereas, during imposition of the pHi gradient, waves initiate almost exclusively in the non-acidic microdomain (represented by the blue bar above each column of line-scans), and their…

Figure 5. Inhibition of RyR channels by H+ ions can account for regional Ca2+ wave inhibition during pHi heterogeneity. (Ai) 3D surface plots of Ca2+ sparks (recorded in linescan mode) illustrate a uniform distribution under resting conditions. Imposed pHi gradient inhibits spark events in acidic (acetate, A) microdomain. (Aii) Sparks were inhibited in the acidic microdomain in the presence (n = 9 cells/2 animals) and absence (n = 5 cells/1 animal) of NHE1 activity. (Bi) Top panel represents dual microperfusion apparatus. Linescan of a Ca2+ wave during regional superfusion with tetracaine. (Bii) Wave initiation and propagation were suppressed in the tetracaine-exposed microdomain (remote effect is not significant; n = 12 cells/3 animals, P = 0.1175). Paired t-tests.

Figure 6. Ca2+ wave velocity and propagation map on to local pHi non-uniformities. (Ai) During a stable longitudinal pHi gradient, Ca2+ wave is faster in the acidic (acetate) microdomain. (Aii) Wave propagation velocity in the acidic (acetate, A) and non-acidic (control, C) microdomains in the presence (n = 8 cells/4 animals) or absence (n = 5 cells/3 animals) of NHE1 activity. (B) Wave propagation was suppressed in the acidic microdomain, while remaining unaffected in the non-acidic microdomain (n = 8 cells/4 animals). (C) NHE1 inhibition had no effect on the success of wave propagation in the acidic microdomain (n = 9 cells/2 animals). Dashed lines represent control. Paired t-tests.

Figure 7. pHi gradients are sustained in multicellular syncytial networks, and generate Nai+ gradients. (A) Exemplar cSNARF-1 fluorescence ratio map, calibrated in units of pH, of a confluent monolayer of neonatal rat ventricular myocytes under (i) control conditions and (ii) during regional superfusion with 40 mM lactate. (B) Exemplar fluorescence map showing [Na+]i and SBFI ratio during regional superfusion with 40 mM lactate, normalized to starting levels. (C) SBFI ratio normalised to control conditions, absolute [Na+]i (uniform exposure to lactate-free solution), and the change in pHi (calculated from cSNARF-1 ratio) plotted across an axis perpendicular to the boundary between microstreams recorded at three different time-points during dual microperfusion. Measurements obtained from four monolayers. The rise in [Na+]i in cells under the lactate-containing microstream is due to Na+ entry via NHE1, stimulated by the ensuing intracellular acidosis. The pHi gradient across the inter-stream boundary is sharp due to slow H+ ion diffusion and rapid transmembrane H+-equivalent fluxes (lactate-H+ transport by MCT). The [Na+]i gradient, in contrast, is shallower due to fast Na+ permeation…

Figure 8. Local and remote pH-Ca2+ interactions. (A) Comparing spatial Ca2+ wave initiation data demonstrates that local inhibition of waves is NHE1-independent, and can be attributed to RyR inhibition, while remote stimulation only occurs in the presence of locally activated NHE1. For clarity, data from Figures 4 and 5 replotted here to permit comparison. (B) The frequency of wave initiation depends on the balance between inhibition by H+ and stimulation by Na+. Top: the experimentally measured pHi gradient illustrated here (cf. Figure 4B) was used in the model. Bottom: Results of a simulation based on the numerical relationships described in Supplementary material online, Figures 1 and 2. The model was used to predict wave initiation frequency (color-coded: red = wave inhibition, white = no change, blue = wave stimulation) during a pHi gradient, for a range of [Na+]i (y-axis, with representative values indicated). The simulation predicts (i) for resting [Na+]i (8 mM, bottom white dashed line, i.e. when NHE is inhibited with DMA), waves are inhibited in the acidic region and remain at control frequency in the non-acidic region; (ii) for a rise in [Na+]i comparable to the load during…