Beat-to-beat dynamic regulation of intracellular pH in cardiomyocytes

Abstract

The mammalian heart beats incessantly with rhythmic mechanical activities generating acids that need to be buffered to maintain a stable intracellular pH (pH_i) for normal cardiac function. Even though spatial pH_i non-uniformity in cardiomyocytes has been documented, it remains unknown how pH_i is regulated to match the dynamic cardiac contractions. Here, we demonstrated beat-to-beat intracellular acidification, termed pH_i transients, in synchrony with cardiomyocyte contractions. The pH_i transients are regulated by pacing rate, Cl^-/HCO₃ ^- transporters, pH_i buffering capacity, and β-adrenergic signaling. Mitochondrial electron-transport chain inhibition attenuates the pH_i transients, implicating mitochondrial activity in sculpting the pH_i regulation. The pH_i transients provide dynamic alterations of H⁺ transport required for ATP synthesis, and a decrease in pH_i may serve as a negative feedback to cardiac contractions. Current findings dovetail with the prevailing three known dynamic systems, namely electrical, Ca²⁺, and mechanical systems, and may reveal broader features of pH_i handling in excitable cells.

Keywords: Cardiovascular medicine; Molecular biology; Molecular dynamics.

Graphical abstract

Figure 1

pH_i transients in rabbit and mouse ventricular cardiomyocytes recorded at RT and 36°C (A) Representative traces of pH_i in rabbit (left) and mouse (right) ventricular myocytes in parallel with the sarcomere length measurement at RT. Cardiomyocytes were paced at 0.5 Hz with pHrodo green loading. The averaged traces of pH_i and sarcomere shortening are overlapped and shown in the lower panel. (B) Representative traces of pH_i in rabbit (left) and mouse (right) ventricular myocytes in parallel with the sarcomere length measurement at RT. Cardiomyocytes were paced at 0.5 Hz with SNARF-1 loading. The averaged traces of pH_i and sarcomere shortening are overlapped and shown in the lower panel. (C) Comparisons of the baseline pH_i measured at RT in rabbit (n = 14 and 16 for pHrodo green and SNARF-1, respectively) and mouse cardiomyocytes (n = 13 and 14 for pHrodo green and SNARF-1, respectively). Animal numbers (N) are shown in the bar graphs. (D) Comparisons of the absolute pH_i transient amplitudes (ΔpH_i) in cardiomyocytes using pHrodo green (rabbit: n = 9, mouse: n = 9; ∗p = 0.010), and SNARF-1 (rabbit: n = 12, mouse: n = 10; ∗∗p = 0.012; ∗∗∗p = 0.028, ∗∗∗∗p = 0.021) at RT (one-way ANOVA combined with Tukey's post hoc analyses), where n represents cell numbers. (E) pH_i measurement in rabbit ventricular myocytes using pHrodo green with simultaneous sarcomere length measurement paced at 1 Hz at 36°C. (F and G) Comparisons of baseline pH_i (F, n = 14 and 13 for RT and 36°C, respectively; ∗p = 0.00069) and ΔpH_i (G, n = 9 and 13 for RT and 36°C, respectively; ∗p = 0.0014) in rabbit ventricular myocytes (two-sample t test). Data are represented as mean ± SEM. See also Figures S2 and S3.

Figure 2

Reduction of pH_i transients by the inhibition of cardiomyocyte contractions (A) Inhibition of the contraction by 25 μM BLEB reduced the pH_i transients. (B) Inhibition of the contraction by 10 mM BDM abolished the pH_i transients. The right three panels in A and B showed three fragments of the time course at three conditions as shown on the top. (C) Comparisons of baseline pH_i (∗p = 0.0074, n = 6, paired sample t test) and ΔpH_i (∗∗p = 0.0009; n = 6, paired sample t test) in the absence and presence of 25 μM BLEB. (D) Comparisons of baseline pH_i and ΔpH_i in the absence and presence of 10mM BDM (∗∗p = 0.0055, n = 6; paired sample t test). The open circles represent the individual data points, and the filled circles and the error bars represent Mean ± S.E.M. N and n represent the animal numbers and cell numbers, respectively. See also Figure S4.

Figure 3

Regulation of pH_i transients by alterations of pH_i (A) Effects of cellular alkalization by perfusing solutions containing 10 mM ammonium. (B) Effects of cellular acidification by perfusing solutions containing 20 mM acetate. The right three panels in A and B showed three fragments of the time course at three conditions as shown on the top. (C) Comparisons of baseline pH_i, ΔpH_i and sarcomere fractional shortening (FS) in the absence and presence of 10 mM ammonium (∗p = 0.00072, ∗∗p = 0.00078, ∗∗∗p = 0.0042, n = 7; paired sample t test). (D) Comparisons of pH_i, ΔpH_i and FS in the absence and presence of 20 mM acetate (∗p = 0.036, ∗∗p = 0.00098, ∗∗∗p = 0.000028, n = 11; paired sample t test). N and n represent animal numbers and cell numbers, respectively. Data are represented as mean ± SEM.

Figure 4

Effects of sarcolemma H⁺-equivalent transporter activities on baseline pH_i and pH_i transients (A) The time course of pH_i measurement when the cell was perfused with Tyrode's solutions either using HCO₃^- or HEPES as pH buffer (left). (B) The time course of pH_i measurement when the cell was perfused with Tyrode's solutions containing higher (130 mM) and lower (10 mM) Cl⁻ (left). (C) The time course of pH_i measurement when the cell was perfused by Tyrode's containing 10 μM EIPA (left). The right three panels in A, B, and C showed three fragments of the time course at three conditions as shown on the top of the panel. (D) Comparisons of baseline pH_i and ΔpH_i of A (∗p = 0.0039, n = 8; ∗∗p = 0.0039, n = 9; paired Wilcoxon signed-rank test). (E) Comparisons of baseline pH_i and ΔpH_i of B (∗p = 0.022, n = 6; ∗∗p = 0.0046, n = 9; paired sample t test). (F) Comparisons of baseline pH_i and ΔpH_i of C (n = 5). N and n represent animal numbers and cell numbers, respectively. Data are represented as mean ± SEM.

Figure 5

Increasing the pacing rate reduced the pH_i transients (A) The time course of pH_i measurement was shown on the left, and the right three panels showed three fragments of the time course at three pacing rates. (B) Comparisons of ΔpH_i at different pacing rates (The p values for a: 0.018; b: 0.0087; c: 0.01. n = 6, one-way repeated measures ANOVA combined with Tukey's post hoc analyses). Animal numbers (N) are shown in the bar graphs. (C) The relationship between the time-averaged sarcomere length and the time-averaged pH_i during pacing at different pacing rates (n = 6). (D) The relationship between ΔpH_i and ΔSarcomere length (including the residual diastolic shortening) at different pacing rates (n = 6). N and n represent animal numbers and cell numbers, respectively. Data are represented as mean ± SEM. See also Figures S5 and S6.

Figure 6

Activation of β-adrenergic signaling by isoproterenol increased the pH_i transients (A) The time course of pH_i measurement with the perfusion of Tyrode's solutions containing 0.05 μM isoproterenol (Iso) was shown on the left, and the right three panels showed three fragments of the time course at three conditions. (B) The time course of pH_i measurement with the perfusion of Tyrode's solutions containing 2.5 μM verapamil was shown on the left, and the right three panels showed three fragments of the time course at three conditions. (C) Comparisons of baseline pH_i (∗p = 0.0024, n = 18), ΔpH_i and sarcomere fractional shortening (FS) (∗∗p = 0.00084, n = 18; ∗∗∗p = 0.0000076, n = 18) in the absence and presence of 0.05 μM of isoproterenol (paired sample t test). (D) Comparisons of baseline pH_i (∗p = 0.035, n = 8), ΔpH_i and FS (∗∗p = 0.00010, n = 8; ∗∗∗p = 0.00000017, n = 8) in the absence and presence of 2.5 μM of verapamil (paired sample t test). N and n represent animal numbers and cell numbers, respectively. Data are represented as mean ± SEM.

Figure 7

Inhibition of mitochondrial ETC reduced the pH_i transients (A–C) Time courses of pH_i measurement when the cell was perfused by Tyrode's containing 2 μM rotenone (A, left), or containing 4 μM antimycin A (B, left), or containing 4 μM FCCP (C, left). The right three panels showed three fragments of the time course at three conditions as shown on the top. (D) Comparisons of baseline pH_i (∗∗p = 0.000083, n = 11, paired sample t test) and ΔpH_i of A (∗p = 0.0078, n = 9; ΔpH_i: 0.046 ± 0.008 in control vs 0.038 ± 0.006 with rotenone, paired Wilcoxon signed-rank test). After washout, ΔpH_i = 0.045 ± 0.007, and is not statistically different compared to control (p = 0.86). (E) Comparisons of baseline pH_i (∗∗p = 0.00056, n = 17, paired sample t test) and ΔpH_i of B (∗p = 0.00031, n = 15. ΔpH_i: 0.046 ± 0.007 in control vs 0.037 ± 0.006 with antimycin A, paired Wilcoxon signed-rank test). After washout, ΔpH_i = 0.045 ± 0.005, and is not statistically different compared to control (p = 0.67). (F) Comparisons of baseline pH_i (∗∗p = 0.0002, n = 7, paired sample t test) and ΔpH_i of C (∗p = 0.016, n = 7, paired Wilcoxon signed-rank test. ΔpH_i: 0.057 ± 0.006 in control vs 0.003 ± 0.002 with FCCP). After washout, ΔpH_i = 0.055 ± 0.005, and is not statistically different compared to control (p = 0.63). N and n represent animal numbers and cell numbers, respectively. Data are represented as mean ± SEM. (G) A summary diagram (created using BioRender.com) showing the beat-to-beat pH_i regulatory system that dovetails with the prevailing dynamic electrical, Ca²⁺, and mechanical regulatory systems. SR: sarcoplasmic reticulum; RYR: ryanodine receptor; SERCA: sarcoplasmic reticulum Ca²⁺ ATPase; PMCA: plasma membrane Ca²⁺-ATPase; NCX: Na⁺/Ca²⁺ exchanger; AP: action potential; CBE: Cl⁻/HCO₃^- exchanger; E_m: membrane potential; ETC: electron transport chain.