Slow Ca2+ Efflux by Ca2+/H+ Exchange in Cardiac Mitochondria Is Modulated by Ca2+ Re-uptake via MCU, Extra-Mitochondrial pH, and H+ Pumping by FOF1-ATPase

Mitochondrial (m) Ca²⁺ influx is largely dependent on membrane potential (ΔΨ_m), whereas mCa²⁺ efflux occurs primarily via Ca²⁺ ion exchangers. We probed the kinetics of Ca²⁺/H⁺ exchange (CHE_m) in guinea pig cardiac muscle mitochondria. We tested if net mCa²⁺ flux is altered during a matrix inward H⁺ leak that is dependent on matrix H⁺ pumping by ATP_m hydrolysis at complex V (F_OF₁-ATPase). We measured [Ca²⁺]_m, extra-mitochondrial (e) [Ca²⁺]_e, ΔΨ_m, pH_m, pH_e, NADH, respiration, ADP/ATP ratios, and total [ATP]_m in the presence or absence of protonophore dinitrophenol (DNP), mitochondrial uniporter (MCU) blocker Ru360, and complex V blocker oligomycin (OMN). We proposed that net slow influx/efflux of Ca²⁺ after adding DNP and CaCl₂ is dependent on whether the ΔpH_m gradient is/is not maintained by reciprocal outward H⁺ pumping by complex V. We found that adding CaCl₂ enhanced DNP-induced increases in respiration and decreases in ΔΨ_m while [ATP]_m decreased, ΔpH_m gradient was maintained, and [Ca²⁺]_m continued to increase slowly, indicating net mCa²⁺ influx via MCU. In contrast, with complex V blocked by OMN, adding DNP and CaCl₂ caused larger declines in ΔΨ_m as well as a slow fall in pH_m to near pH_e while [Ca²⁺]_m continued to decrease slowly, indicating net mCa²⁺ efflux in exchange for H⁺ influx (CHE_m) until the ΔpH_m gradient was abolished. The kinetics of slow mCa²⁺ efflux with slow H⁺ influx via CHE_m was also observed at pH_e 6.9 vs. 7.6 by the slow fall in pH_m until ΔpH_m was abolished; if Ca²⁺ reuptake via the MCU was also blocked, mCa²⁺ efflux via CHE_m became more evident. Of the two components of the proton electrochemical gradient, our results indicate that CHE_m activity is driven largely by the ΔpH_m chemical gradient with H⁺ leak, while mCa²⁺ entry via MCU depends largely on the charge gradient ΔΨ_m. A fall in ΔΨ_m with excess mCa²⁺ loading can occur during cardiac cell stress. Cardiac cell injury due to mCa²⁺ overload may be reduced by temporarily inhibiting F_OF₁-ATPase from pumping H⁺ due to ΔΨ_m depolarization. This action would prevent additional slow mCa²⁺ loading via MCU and permit activation of CHE_m to mediate efflux of mCa²⁺.

HIGHLIGHTS

- We examined how slow mitochondrial (m) Ca²⁺ efflux via Ca²⁺/H⁺ exchange (CHE_m) is triggered by matrix acidity after a rapid increase in [Ca²⁺]_m by adding CaCl₂ in the presence of dinitrophenol (DNP) to permit H⁺ influx, and oligomycin (OMN) to block H⁺ pumping via F_OF₁-ATP synthase/ase (complex V).

- Declines in ΔΨ_m and pH_m after DNP and added CaCl₂ were larger when complex V was blocked.

- [Ca²⁺]_m slowly increased despite a fall in ΔΨ_m but maintained pH_m when H⁺ pumping by complex V was permitted.

- [Ca²⁺]_m slowly decreased and external [Ca²⁺]_e increased with declines in both ΔΨ_m and pH_m when complex V was blocked.

- ATP_m hydrolysis supports a falling pH_m and redox state and promotes a slow increase in [Ca²⁺]_m.

- After rapid Ca²⁺ influx due to a bolus of CaCl₂, slow mCa²⁺ efflux by CHE_m occurs directly if pH_e is low.

Introduction

Mitochondrial (m) Ca²⁺ overload is a damaging consequence of cardiac ischemia-reperfusion (IR) injury and hence is an important subject for potential therapy (Brookes et al., 2004; O’Rourke et al., 2005; Stowe and Camara, 2009; Camara et al., 2010). During IR, mitochondria can consume rather than generate ATP (Chinopoulos and Adam-Vizi, 2010; Chinopoulos et al., 2010), which consequently can augment mCa²⁺ overload (Riess et al., 2002) sufficient to induce cell apoptosis and necrosis (Murphy and Steenbergen, 2008). [Ca²⁺]_m is regulated in part by electrochemical dependent cation flux via Ca²⁺ transporters and by cation exchangers within the inner mitochondrial membrane (IMM) (Gunter and Pfeiffer, 1990; Gunter et al., 1994; Bernardi, 1999; Brookes et al., 2004). The major route for mCa²⁺ uptake is via the ruthenium red (RR) sensitive mitochondrial Ca²⁺ uniporter (MCU), now considered a macromolecular complex composed of two pore components, MCU and MCUb, and MCU regulators MCU1, 2, 3, and EMRE (essential MCU regulator), and other components (De Stefani et al., 2015). Ca²⁺ influx via the MCU is reduced by competition with cytosolic Mg²⁺ (Boelens et al., 2013; Tewari et al., 2014). Additional modes of mCa²⁺ uptake are proposed to occur via a ryanodine type channel (RTC) in the IMM (Ryu et al., 2011; O-Uchi et al., 2013; Tewari et al., 2014) and at the sarcoplasmic reticular-MCU interface where functional Ca²⁺ signaling between the cytoplasmic and mitochondrial compartments is believed to occur (Csordas et al., 2010).

A primary mCa²⁺ efflux pathway is the Na⁺/Ca²⁺ exchanger (NCE_m) (Boyman et al., 2013). In unicellular organisms and in some non-cardiac tissues there is firm evidence (Azzone et al., 1977; Pozzan et al., 1977; Wingrove et al., 1984; Brand, 1985; Rottenberg and Marbach, 1990; Gunter et al., 1991, 1994; Bernardi, 1999; Demaurex et al., 2009; Nishizawa et al., 2013) for slow homeostatic mCa²⁺ efflux through a Na⁺-independent Ca²⁺ exchanger (NICE), i.e., a non-electrogenic Ca²⁺/H⁺ exchanger (CHE) that might be activated when the ΔpH_m gradient across the IMM is altered. The amount of free (ionized) [Ca²⁺]_m available for exchange depends on the extent of dynamic mCa²⁺ buffering (Bazil et al., 2013; Blomeyer et al., 2013; Tewari et al., 2014). mCa²⁺ influx via the MCU and efflux via the NCE_m are largely voltage (ΔΨ_m) dependent, whereas Ca²⁺ transport via the CHE_m, while pH-dependent, may be electrogenic (1 H⁺ for 1 Ca²⁺) or non-electrogenic (2 H⁺ for 1 Ca²⁺). However, CHE_m can be indirectly dependent on the full IMM electrochemical gradient if there is a decrease in the IMM ΔpH_m gradient (Rottenberg and Marbach, 1990; Dash and Beard, 2008; Dash et al., 2009).

There is a well-known direct correlation between ΔΨ_m and mCa²⁺ uptake based on the Nernst equation; a more polarized ΔΨ_m permits greater mCa²⁺ uptake (Wingrove et al., 1984; Gunter et al., 1994). mCa²⁺ uptake via the MCU depends both on the electrical (charge) gradient, ΔΨ_m, and on the concentration gradient for [Ca²⁺] across the IMM. ATP_m hydrolysis with H⁺ pumping can occur at complex V (F_OF₁-ATPsynthase/ase) during cardiac ischemia (Jennings et al., 1991) in an attempt to maintain the ΔpH_m gradient, and along with the ΔΨ_m gradient (Chinopoulos and Adam-Vizi, 2010; Chinopoulos, 2011), equals the proton motive force, pmf. However, it is not known how the magnitude, rate, and route of mCa²⁺ uptake or release in cardiac muscle cell mitochondria is affected by manipulating the IMM Δ[H⁺]_m gradient by allowing mATP hydrolysis, which would result in H⁺ pumping and better maintain the Δ[H⁺] gradient when ΔΨ_m is low, vs. blocking mATP hydrolysis (no H⁺ pumping with collapsing Δ[H⁺]) and lower ΔΨ_m.

Exposure of mitochondria to external (e) CaCl₂ when the IMM is fully charged (high ΔΨ_m), defined here by the presence of substrate in state 2 conditions without an induced inward H⁺ leak, promotes rapid voltage-dependent mCa²⁺ uptake via MCU (Hoppe, 2010). In contrast, decreased net mCa²⁺ uptake might be expected during a protonophore-induced inward H⁺ leak if H⁺ influx leads to Ca²⁺ efflux. However, an inward H⁺ flux that slowly decreases ΔΨ_m can still result in a slow, continued uptake of mCa²⁺ via the MCU if there remains sufficient ΔΨ_m and Ca²⁺ chemical gradient ([Ca²⁺]_e > [Ca²⁺]_m) across the IMM. mCa²⁺ influx via the MCU can partially depolarize ΔΨ_m (Delcamp et al., 1998; Di Lisa and Bernardi, 1998) due to the influx of positive charges without an effect on the Δ[H⁺]_m, and more so with a fall in Δ[H⁺]_m gradient from the added influx of H⁺ in the presence of a protonophore.

Our aim was to mechanistically examine the slow mode kinetics of mCa²⁺ influx/efflux in cardiac cell mitochondria. The conditions under which CHE_m may occur in cardiac mitochondria are unknown. We proposed that an induced, net influx of H⁺ is coupled to net mCa²⁺ efflux by activation of CHE_m in the face of continued mCa²⁺ uptake via the MCU in partially depolarized ΔΨ_m mitochondria. In addition, if the extra-mitochondrial milieu is acidic, pH_m would slowly decrease as mH⁺ entry by mCHE_m is exchanged for mCa²⁺ efflux in Ca²⁺ overloaded mitochondria. We postulated that CHE_m is activated under conditions of slow a H⁺ influx and a high m[Ca²⁺], and especially when H⁺ pumping by complex V, stimulated by the lowered ΔΨ_m, is prevented. To carry out our aim, we examined the time dependent changes in ΔΨ_m, [Ca²⁺]_m and pH_m, and extra-mitochondrial [Ca²⁺]_e and pH_e, after a bolus of CaCl₂ either by inducing an inward H⁺ leak that causes an outward pumping of H⁺ by complex V, or by altering the extra-mitochondrial pH_e.

In one set of experiments, we challenged isolated energized mitochondria with a bolus of CaCl₂ in the absence or presence of increasing concentrations of the protonophore 2,4-dinitrophenol (DNP) in the absence or presence of the complex V inhibitor oligomycin (OMN) to block ATP hydrolysis-induced H⁺ pumping, and or Ru360 to block the reuptake of Ca²⁺ via the MCU. To understand how DNP, OMN, and Ru360 dynamically alter [Ca²⁺]_m or [Ca²⁺]_e after a bolus of CaCl₂, we considered it crucial to also dynamically measure ΔΨ_m, pH_m, and NADH, as well as mitochondrial respiration (extent of uncoupling), total [ATP]_m, and ATP_m/ADP_m ratio. In another set of isolated mitochondrial experiments, we directly induced mCa²⁺ efflux via CHE_m after CaCl₂ loading by altering the Na⁺-free medium from a control pH_e of 7.15 to either pH 7.6 or 6.9. We show that secondary Ca²⁺ influx vs. efflux is Δ[H⁺]_m dependent.

Materials and Methods

Isolated Mitochondrial Experiments

All experiments conformed to the Guide for the Care and Use of Laboratory Animal and were approved by the Medical College of Wisconsin Biomedical Resource Center animal studies committee. Detailed methods for mitochondrial isolation and measurements of ΔΨ_m, [Ca²⁺]_m, NADH redox state, pH_m, [ATP]_m, ADP_m/ATP_m ratio, respiration, and the number of animals per group, are furnished (see section “Supplementary Materials S.1.1–S.1.12”). Briefly, mitochondria were isolated from guinea pig heart ventricles in iced buffer and were suspended in experimental buffer containing in mM: KCl 130, K₂HPO₄ 5, MOPS 20, bovine serum albumin 0.016 and EGTA ∼0.036–0.040 at pH 7.15 (adjusted with KOH) at room temperature (21°C). The experimental buffer had a final protein concentration of 0.5 mg/mL. Specific fluorescent probes and spectrophotometry (Qm-8, Photon Technology International, Birmingham, NJ, United States) were used to measure [Ca²⁺]_m (indo-1AM) and buffer [Ca²⁺]_e (indo-1 or Fura 4 F penta-K⁺ salt), NADH, an indicator of mitochondrial redox state (autofluorescence), pH_m (BCECF-AM), and mitochondrial membrane potential (ΔΨ_m) assessed by rodamine-123 or TMRM (Heinen et al., 2007; Huang et al., 2007; Aldakkak et al., 2010; Haumann et al., 2010) (all fluorescence probes from Invitrogen^TM – Thermo Fisher Scientific). Respiration (Clark electrode) and ATP_m (bioluminescence) and ATP_m/ADP_m ratio (HPLC, luminometry) were also measured. The experimental buffer, mitochondrial substrates, and drugs were Na⁺-free to prevent activation of NCE_m by extra-mitochondrial Na⁺. The inactivity of the NCE was verified by comparing data from these experiments to data from experiments with added CGP-37157, a known mitochondrial NCE_m inhibitor (data not shown).

Experimental Protocols

Medium pH_e-Induced Changes in pH_m

The experimental buffer was identical to that described above except that in addition to the pH 7.15 buffer, buffers at pH 6.9 and 7.6 were prepared by titration with HCl and KOH, respectively. The residual EGTA carried over from the isolation buffer to the experimental buffer resulted in an ionized extra-mitochondrial [Ca²⁺]_e of <200 nM (Figure 1). To measure changes in [Ca²⁺]_e after adding a bolus of 40 μM CaCl₂, each pH buffer contained Fura 4 F penta-K⁺ salt. The K_D’s for Ca²⁺ were calculated and corrected for each buffer pH because pH affects the binding of Ca²⁺ to the fluorescence dye (see section “Supplementary Materials S.1.4, S.1.8”). In other experiments, pH_m and ΔΨ_m were measured using BCECF-AM and TMRM fluorescent dyes, respectively. Experiments were initiated at t = 30 s when mitochondria were added to the buffer; at t = 90 s pyruvic acid (PA, 0.5 mM) was added, followed by a bolus of 40 μM CaCl₂ at t = 210 s to initiate rapid mCa²⁺ uptake via MCU. Note that in guinea pig cardiac mitochondria, the respiratory control index (RCI) is higher in the presence of pyruvate alone (Heinen et al., 2007; Blomeyer et al., 2013; Boelens et al., 2013) than with pyruvate plus malate (Riess et al., 2008). For some experiments, 1 μM Ru360 (or vehicle, 0.1% DMSO) was added at t = 300 s shortly after adding CaCl₂ to block Ca²⁺ reuptake into mitochondria via MCU after the Ca²⁺ was extruded from mitochondria. At the end (1700 s) of each experiment, the potent protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 4 μM) was given to completely abolish the ΔpH gradient and depolarize ΔΨ_m. Data for each pH group were collected in mitochondrial suspensions from the same heart; approximately 8–10 hearts were used for each fluorescent probe. At pH 7.15, adding 40 μM CaCl₂, which increased extra-mitochondrial [Ca²⁺]_e into the 1 μM range and increased the initial [Ca²⁺]_m to approximately 500 nM (Figures 1, 2), is unlikely to induce membrane permeability transition pore (mPTP) opening. However, to test the possibility of mPTP opening, 500 nM cyclosporine A (CsA), a modulator of cyclophilin D required to open mPTP, was given before adding CaCl₂ in several experiments at pH_e 6.9 and 7.15.

FIGURE 1

Figure 1. Changes in buffer [Ca²⁺]_e (A), matrix pH_m (B), and ΔΨ_m (C) over time after adding 40 μM CaCl₂ (210 s) at extra-mitochondrial pH_e 7.6, 7.15, and 6.9 with or without 1 μM Ru360 (300 s) to inhibit additional mCa²⁺ uptake via MCU. Note the rapid fall in [Ca²⁺]_e due to fast mCa²⁺ uptake via the MCU and the following slow rise in [Ca²⁺]_e (Ca²⁺ efflux) (A), slow decline in pH_m (B), and slow depolarization of ΔΨ_m (C) at pH 6.9 (each line = mean of 3–4 replicates from 12 guinea pig hearts for each fluorescence measurement). Note in the pH 6.9 medium the faster rate of mCa²⁺ efflux (A) over time when MCU was blocked, and the faster declines in pH_m (B) and ΔΨ_m (C) over time when MCU was not blocked.

FIGURE 2

Figure 2. Plots of extra-mitochondrial [Ca²⁺]_e as a function of [H⁺]_m from the mean data of Figures 1A,B. Arrows denote the difference in net mCa²⁺ efflux at a given [H⁺]_m when MCU was or was not blocked with Ru360 given 90 s after the CaCl₂ bolus. While mCa²⁺ was extruded by CHE_m, much of it re-entered via the MCU when it was not blocked. At trans-membrane pH equilibrium (pH 6.9) Ca²⁺ uptake via MCU stopped as the ΔΨ_m was nearly depolarized having resulted in a large net extrusion of Ca²⁺.

Protonophore-Induced Changes in pH_m

Experiments were initiated at t = -120 s; at t = -90 s, mitochondria were added to the experimental buffer (time line, Figure 3); external pH_e was 7.15. At t = 0 s, pyruvic acid (PA, 0.5 mM) was added to the mitochondria suspended in the experimental buffer, followed by 0, 10, 20, 30, or 100 μM DNP, a mild protonophore, at t = 90 s, followed by the addition of de-ionized H₂O, 10, or 25 μM CaCl₂ at t = 225 s. The 90 s period allowed for full ΔΨ_m polarization and stabilization of pH_m and NADH. In some experiments (see section “Supplementary Results S.2.4” and Supplementary Figure S.6), 100 nM Ru360 was added at t = 300 s, after the addition of CaCl₂, to block any reuptake of mCa²⁺ by the MCU that was extruded by CHE_m. For the OMN treated groups, 10 μM OMN was added to the experimental buffer at the start of the experimental protocol (Figure 3). At the end of each experiment CCCP was added at t = 760 s to maximally depolarize ΔΨ_m. DNP, Ru360, OMN, and CCCP were each dissolved initially in DMSO and then in buffer to yield a final buffer concentration for DMSO of 0.1 to 0.4% (wt/vol). Each drug or DMSO alone was added to a final volume of 10 μL. To test for mPTP opening, CsA was given before adding 20 or 30 μM DNP and 25 μM CaCl₂ in several experiments conducted at pH_e 7.15.

FIGURE 3

Figure 3. Time line of protonophore-induced experimental protocol: addition of mitochondria, ±oligomycin (OMN), pyruvic acid (PA), dinitrophenol (DNP), CaCl₂, and CCCP to respiratory buffer. Early and late plots refer to time points where means of several variables are plotted to summarize their interrelationships. In Supplementary Experiments, Ru360 (100 nM) was given to block the MCU (see section “Supplementary Results S.2.4” and Supplementary Figures S.6A,B).

Statistical Analyses

Data were summarized at 500, 1000, and 1500 s (for Figures 1, 2) for external buffer-induced changes in pH_m on [Ca²⁺]_e. Data were summarized for protonophore-induced changes in pH_m on [Ca²⁺]_m at 80 s (after adding PA), 215 s (after adding DNP), 275 s (early after adding CaCl₂), and 700 s (late after adding CaCl₂) (e.g., Figure 4). All data points were presented and expressed as average ± SEM. Repeated measure ANOVAs followed by a post hoc analyses using Student-Newman-Keuls’ test was performed to determine statistically significant differences among groups. A P-value < 0.05 (two-tailed) was considered significant. See Figure legends for statistical notations.

FIGURE 4

Figure 4. Change in mitochondrial membrane potential (ΔΨ_m), assessed with rhodamine-123 as % of maximal depolarization, as a function of time after adding dinitrophenol (DNP) and CaCl₂ in the absence (A,B) and presence (C,D) of oligomycin (OMN). Note that adding DNP caused a concentration-dependent fall in ΔΨ_m that was more pronounced in the presence of OMN. Adding CaCl₂ caused a small polarization, while increasing depolarization occurred over time. Adding 25 μM CaCl₂ (B,D) resulted in a more pronounced fall in ΔΨ_m compared to 10 μM CaCl₂ (A,C). Buffer pH = 7.15. Data obtained from 10 hearts with 4–5 replicates per heart. For P < 0.05: ^∗after DNP vs. before DNP; ^#after CaCl₂ vs. before CaCl₂; ^†late (700 s) vs. early (215 s) after CaCl₂.

Results

CHE_m Activation Was Exposed by Efflux of Ca²⁺ With Influx of H⁺ and Was Greater If MCU Was Inhibited

Direct evidence for CHE_m activation was observed by acidifying the extra-mitochondrial buffer (low pH_e), which subsequently decreased the matrix pH_m slowly over time (Figure 1). With NCE_m and Na⁺/H⁺ (NHE_m) inactivated by using Na⁺-free solutions and substrates, fast mCa²⁺ influx via the MCU, induced after adding 40 μM CaCl₂ at pH 6.9, was followed by a slow mCa²⁺ efflux over time ∼(300–1700 s) as shown by the increase in extra-mitochondrial [Ca²⁺]_e from <200 nM to nearly 4500 nM in the absence of Ru360 (Figure 1A). When Ru360 was added 90 s after adding CaCl₂, [Ca²⁺]_e rose even more over the first 1000 s, indicating blockade of Ca²⁺ recycling via the MCU and revealing the total mCa²⁺ effluxed via CHE_m. In the pH 6.9 plus Ru360 group the mean rate (slope) of increase in [Ca²⁺]_e (mCa²⁺ efflux) over time (300–1700 s) was 1.5 ± 0.1 nM/s, ΔpH 0.4 units). This was greater than in the pH 6.9 minus Ru360 group (1.0 ± 0.2 nM/s over 300–1000 s), suggesting that approximately 1/3 of the mCa²⁺ extruded was retaken up across the IMM via the MCU. In contrast, mCa²⁺ efflux was not observed in the pH 7.6 medium without Ru360, and minimally at 1500 s at pH 7.6 with Ru360. There was less mCa²⁺ efflux at pH 7.15 ± Ru360 compared to pH 6.9 ± Ru360. However, even at pH 7.15 ± Ru360, there were similar steady declines in pH_e while net slow Ca²⁺ efflux was noted only in the plus Ru360 groups, indicating Ca²⁺ re-uptake via MCU. Therefore, in the acidic extra-mitochondrial medium, slow decreases in pH_m (H⁺ influx) were accompanied by slow increases in mCa²⁺ efflux, indicating CHE_m activity. Eventually, matrix acidification was more pronounced in the pH 6.9 medium (ΔpH 0.62 units) in the absence of Ru360 than in all other groups so that over time as H⁺ influx was exchanged for Ca²⁺ efflux the IMM ΔpH gradient was eventually obliterated, halting Ca²⁺ efflux (Figure 1B). Eventually, because of mCa²⁺ influx, near complete depolarization of ΔΨ_m occurred in the pH 6.9 medium (Figure 1C), as shown by little change after adding CCCP, and by the complete depolarization of ΔΨ_m when Ca²⁺ recycling via the MCU was permitted (minus Ru360 group). Although adding CaCl₂ at an external pH_e of 6.9 led eventually to near complete dissipation of ΔΨ_m, when CsA was first added to the buffer, CsA prevented the gradual, slow extrusion of mCa²⁺ and declines in pH_m and ΔΨ_m induced by adding CaCl₂ at pH_e 6.9 indicating a complete lack of CHE_m activity (see section “Supplementary Results S.2.1” and Supplementary Figures S.1A–C).

Increasing Matrix Acidification Led to Ca²⁺ Efflux Until Loss of the ΔpH_m Gradient and a Lack of Ca²⁺ Re-uptake via MCU on Full Depolarization of ΔΨ_m

A plot of extra-mitochondrial [Ca²⁺]_e as a function of matrix [H⁺]_m at each extra-mitochondrial pH (Figure 2) indicates maximal mCa²⁺ efflux occurred in the pH_e 6.9 medium (largest IMM (ΔH⁺] gradient), much less so in the pH 7.15 medium, and not at all in the pH 7.6 medium. Ca²⁺ efflux was accentuated in the presence of Ru360 given just after the added CaCl₂ bolus (Figure 2). The difference (arrow) between the absence and presence of Ru360 indicates the rapid reuptake (recycling) of Ca²⁺ via MCU on extrusion via CHE_m. Thus total Ca²⁺ efflux was greater in the pH 6.9 group when MCU was not blocked because [H⁺]_m rose higher than when MCU was blocked. The steep, vertical increase in mCa²⁺ efflux at the highest [H⁺]_m in the pH 6.9 group resulted from cessation of mCa²⁺ reuptake via MCU due to depolarization of ΔΨ_m (Figure 1C). The net amount of H⁺ entering mitochondria per Ca²⁺ exiting mitochondria may be indeterminate because much of the H⁺ entering is pumped out via the respiratory enzyme complexes.

Mitochondrial Membrane Potential (ΔΨ_m) Was Depressed by DNP After Adding CaCl₂

In the protonophore series of experiments (time line, Figure 3), DNP alone decreased ΔΨ_m slightly as assessed by rodamine-123 (R123) (Huang et al., 2007) (Figure 4), in a concentration-dependent manner, except at 100 μM DNP, which alone fully (+OMN) or nearly (-OMN) depolarized ΔΨ_m. ΔΨ_m was estimated as % of maximal depolarization, where the baseline after adding substrate with OMN signifies full polarization (0%) and addition of CCCP denotes complete depolarization (100%). Adding 10 μL of 0.1% DMSO (DNP vehicle) or 10 μM DNP had no significant effect when given before CaCl₂, whereas adding 20, 30, or 100 μM DNP before 10 μM CaCl₂ reduced the R123 ΔΨ_m signals by 12.7, 18.7, and 92.4% vs. DMSO (Figure 4A), respectively. In the presence of OMN (Figure 4C), adding 20, 30, or 100 μM DNP before 10 μM CaCl₂ increased the fluorescence signal intensities (i.e., depolarized ΔΨ_m) by 16.2, 33.0, and 99.0%, respectively, vs. DMSO (0%). Overall, before adding either 10 or 25 μM CaCl₂, 20 and 30 μM DNP moderately decreased ΔΨ_m in the absence of OMN but greatly decreased ΔΨ_m in the presence of OMN, suggesting blocked proton pumping from complex V (Figures 4C,D vs. Figures 4A,B). If no CaCl₂ was given after DNP, the moderate decrease in ΔΨ_m, which was unaffected by CsA, persisted for up to 25 min (see section “Supplementary Results S.2.5” and Supplementary Figure S.7A). After adding 10 and 30 μM DNP, and then CaCl₂, there were large decreases in ΔΨ_m resulting from entry of Ca²⁺. Although ΔΨ_m depolarization by DNP alone was unaffected by CsA, the subsequent slow ΔΨ_m depolarization induced by 25 μM CaCl₂ was delayed by CsA (Supplementary Figure S.7B). Supplementary Results S.2.3 and Supplementary Figures S.3A–D shows statistics on mean ± SEM data for ΔΨ_m replotted from Figure 4 at time points 215, 275, and 700 s.

Matrix Free [Ca²⁺]_m Rose or Fell Slowly Depending on Block of Complex V

Adding 10 μM CaCl₂ without DNP (ΔΨ_m fully polarized) caused [Ca²⁺]_m to increase rapidly from 80 nM (no added CaCl₂) initially to 235 nM at 300 s, whereas after adding 25 μM CaCl₂, [Ca²⁺]_m rose more rapidly to 450 nM (Figures 5A,B); [Ca²⁺]_m remained unchanged over time (300–750 s) after adding 10 μM CaCl₂ but fell slightly and gradually (non-significantly) over time after adding 25 μM CaCl₂ (DMSO group, Figures 5A,B). After adding 10–30 μM DNP, adding 10 μM CaCl₂ promoted a slow, secondary rise in [Ca²⁺]_m (Figure 5A). The secondary, slow increase in [Ca²⁺]_m beginning 300 s after adding 10 μM CaCl₂ plus DNP was accompanied by a slow decrease in extra-mitochondrial [Ca²⁺]_e (see Supplementary Figure S.6A). When ΔΨ_m was nearly or totally depolarized by 100 μM DNP in the absence of OMN, and after adding 10 μM CaCl₂, there was no change in [Ca²⁺]_m over 300–750 s and thus no mCa²⁺ uptake over time (Figure 5A). [Ca²⁺]_m slowly increased over 300–750 s after first adding 10 and 20 μM DNP and then 25 μM CaCl₂ (Figure 5B), which caused the slow declines in ΔΨ_m (Figure 4B). In the 100 μM DNP group [Ca²⁺]_m increased moderately immediately after adding 25 μM CaCl₂, but did not change further over time. Supplementary Results S.2.6 and Supplementary Figures S.4A,B display statistics on mean ± SEM data for [Ca²⁺]_m replotted from Figure 5 (-OMN) at time points 215, 275, and 700 s.

FIGURE 5

Figure 5. Change in [Ca²⁺]_m as a function of time (A,B) after adding DNP and CaCl₂ in the absence of OMN. Adding DNP did not alter [Ca²⁺]_m, per se, but did affect [Ca²⁺]_m depending on [DNP] and the amount of CaCl₂ added in the absence of OMN. Adding 10 μM CaCl₂ (A) caused a smaller increase in [Ca²⁺]_m than 25 μM CaCl₂ (B). In the absence of OMN the effect of DNP on [Ca²⁺]_m was less concentration-dependent and [Ca²⁺]_m continued to increase over time. Initial, rapid rates (averaged over 7 s) of increases in [Ca²⁺]_m (nM/s) as a function of [DNP] (C,D) just after adding CaCl₂ in the absence of OMN. The rate of increase in [Ca²⁺]_m decreased as the degree of ΔΨ_m depolarization increased with increasing [DNP]. Note different Y-axis scales for 10 and 25 μM CaCl₂. See Figure 4 for statistical notation for (A–D) plots. Much slower rates of increase in [Ca²⁺]_m (pM/s) occurred over time (slopes of data between 300 and 750 s) after the initial CaCl₂ bolus (E,F); the additional slow mCa²⁺ uptake was also dependent on ΔΨ_m. Buffer pH = 7.15. Data obtained from seven hearts with 3–4 replicates per heart. For plots (E,F), P < 0.05: ^∗DNP vs. DMSO.

In marked contrast, when complex V was blocked by OMN, adding 10 μM CaCl₂ (Figure 6A) after adding10–30 μM DNP caused a marked decrease in [Ca²⁺]_m over time (300–750 s); after adding 25 μM CaCl₂ in the absence of DNP (Figure 6B), [Ca²⁺]_m rose higher initially, whereas 10–30 μM DNP caused a slow decrease in [Ca²⁺]_m over this period, indicating net mCa²⁺ efflux. Supplementary Results S.2.6 and Supplementary Figures S.4C,D shows statistics on mean ± SEM data for [Ca²⁺]_m replotted from Figure 6 (+OMN) at time points 215, 275, and 700 s. The secondary, slow decrease in [Ca²⁺]_m after adding 20 μM DNP plus 25 μM CaCl₂ was accompanied by an increase in extra-mitochondrial [Ca²⁺]_e (see Supplementary Figure S.6B). Note that additional mCa²⁺ uptake after giving 25 μM CaCl₂ was halted after adding Ru360, 90 s later (at t = 325 s) and converted to mCa²⁺ efflux in the presence of OMN as shown by the increase in [Ca²⁺]_e (see Supplementary Figures S.6B vs. S.6A).

FIGURE 6

Figure 6. Change in [Ca²⁺]_m as a function of time (A,B) after adding DNP and CaCl₂ in the presence of OMN. Adding DNP did not alter [Ca²⁺]_m, per se, but did affect [Ca²⁺]_m depending on [DNP], the amount of CaCl₂ added, and the presence of OMN. Adding 10 μM CaCl₂ (A) caused a smaller increase in [Ca²⁺]_m than 25 μM CaCl₂ (B). In the presence of OMN, DNP caused concentration-dependent decreases in [Ca²⁺]_m over time. Initial, rapid rates (averaged over 7 s) of increase in [Ca²⁺]_m (nM/s) as a function of [DNP] (C,D) just after adding CaCl₂ in the presence of OMN. The rate of increases in [Ca²⁺]_m decreased as the degree of ΔΨ_m depolarization increased with increasing [DNP]. See Figure 4 for statistical notation for (A–D) plots. A slow rate of decrease in [Ca²⁺]_m (pM/s) occurred over time (slopes of data between 300 and 750 s) after the initial CaCl₂ bolus (E,F); the slow mCa²⁺ efflux was dependent on slow mCa²⁺ influx (hidden by OMN treatment) (see Figure 6 vs. Figure 5) and a slow fall in matrix pH and ΔΨ_m. Note different Y-axis scales for 10 and 25 μM CaCl₂. Buffer pH = 7.15. Data obtained from seven hearts with 3–4 replicates per heart. For plots (E,F), P < 0.05: ^∗DNP vs. DMSO. ^¶+OMN vs. —OMN (Figure 5) for same [DNP].

A summary of slope data collected over the first 7 s (1 sample/s) after adding 10 or 25 μM CaCl₂ in the absence (Figures 5C,D) or presence (Figures 6C,D) of OMN shows that the average initial, rapid increase in [Ca²⁺]_m via the MCU was much faster after adding 25 μM CaCl₂ than after 10 μM CaCl₂ in the ± OMN groups; this initial rate of mCa²⁺ uptake decreased as ΔΨ_m fell with added DNP. The initial rate of increase in [Ca²⁺]_m during the first 7 s after adding 10 μM CaCl₂ (Figure 5C) decreased from 8 to 2 nM/s (DNP 0–100 μM). After adding 25 μM CaCl₂ (Figure 5D), the rate decreased from 88 to 20 nM/s. In the presence of OMN (Figures 6C,D), the initial increases in [Ca²⁺]_m in fully coupled mitochondria (no DNP) were larger than those in the absence of OMN (Figures 6C,D vs. Figures 5C,D). With OMN present, the initial increases in [Ca²⁺]_m decreased from 30 to 4 nM/s after adding 10 μM CaCl₂ and from 130 to 13 nM/s after adding 25 μM CaCl₂, Thus the initial rates of increase in [Ca²⁺]_m with 10 μM CaCl₂ were consistently faster in the presence of OMN (Figure 6C vs. Figure 5C), and at 25 μM CaCl₂, with or without 10 μM DNP (Figure 6D vs. Figure 5D).

A summary of slope data collected between 300 and 750 s, i.e., after the initial, rapid increase in [Ca²⁺]_m via the MCU with added 10 μM CaCl₂, demonstrates a much slower and smaller (pM/s) gradual increase in [Ca²⁺]_m over time in the absence of OMN with a threefold greater slope after 30 μM DNP vs. DMSO (Figure 5E). After adding 25 μM CaCl₂, the slow increase in [Ca²⁺]_m was about fourfold higher after 20 μM DNP vs. DMSO (Figure 5F). The secondary slow rise in [Ca²⁺]_m was about 1000 times slower than the initial fast phase and roughly dependent on both the amount of mCa²⁺ that was taken up initially just after adding CaCl₂ and the extent of ΔΨ_m depolarization. In contrast, in the presence of OMN under the same conditions of added CaCl₂ and DNP, the slope data showed slow and small declines (rather than increases) in [Ca²⁺]_m over time (Figures 6E,F). The slow rate of extrusion of mCa²⁺ by CHE_m when complex V was blocked with OMN (Figures 6E,F) became greater when mCa²⁺ entry via the MCU was greater (Figures 6 A,B).

Matrix pH Remained Steady Without OMN but Fell With OMN-Induced Block of Complex V

Baseline matrix pH_m was approximately 7.55 in each group after adding PA and before adding DNP (Figures 7A–D). In the absence of OMN, adding 10–30 μM DNP did not result in a significant net decrease in pH_m; however, 100 μM DNP markedly decreased pH_m (Figures 7A,B). This effect to collapse the ΔpH_m gradient was proportional to the collapse of the ΔΨ_m gradient (Figure 4). In the absence of OMN, adding CaCl₂ had no appreciable effect on pH_m (ΔΨ_m partially depolarized) even in the presence of DNP, except for 100 μM DNP, when pH_m fell markedly (ΔΨ_m fully depolarized) (Figures 7A,B). In the absence of OMN, H⁺ influx was matched by H⁺ pumping as pH_m did not change appreciably. In contrast, in the presence of OMN there was a strong DNP concentration-dependent fall in matrix pH_m (Figures 7C,D) after adding CaCl₂. This fall in pH_m was likely due to blocked H⁺ pumping by complex V in the presence of OMN (see below). Supplementary Figures S.5A–D shows statistics on mean ± SEM data on pH_m replotted from Figure 7 (main text) at time points 215, 275, and 700 s. Supplementary Figure S.8 displays plots of pH_m as a function of [Ca²⁺]_m at 700 s after adding DNP and CaCl₂; these correlations show how [Ca²⁺]_m decreases while pH_m decreases in the presence, but not in the absence of OMN.

FIGURE 7

Figure 7. Change in pH_m, measured with BCECF, as a function of time after adding DNP and CaCl₂ in the absence (A,B) or presence (C,D) of OMN. Note that except for 100 μM DNP, neither DNP nor added CaCl₂ altered pH_m in the absence of OMN (A,B). However, in the presence of OMN (C,D) graded decreases of ΔΨ_m by DNP caused a graded increase in matrix acidity that was further increased in the presence of added CaCl₂ (C,D). Buffer pH = 7.15. Data obtained from 10 hearts with 3–4 replicates per heart. For P < 0.05: ^∗after DNP vs. before DNP; ^†late (700 s) vs. early (215 s) after adding CaCl₂.

Mitochondrial Redox State Remained Steady Without OMN but Fell With OMN-Induced Block of Complex V

A reduced redox state is associated with maintenance of pH_m. Adding the substrate PA increased the redox state (more reduced) as determined by high NADH autofluorescence (Figure 8). In the absence of OMN, adding 10 to 30 μM DNP ± 10 or 25 μM CaCl₂ (Figures 8A,B) did not cause a significant change in NADH. NADH was unchanged despite up to 60% decrease in ΔΨ_m fluorescence (Figures 4A,B) after adding DNP and CaCl₂. However, when complex V was blocked by OMN (Figures 8C,D), there was significant oxidation (low NADH) by DNP in a concentration dependent manner. In contrast to the condition without OMN, with OMN present as little as a 20% fall in ΔΨ_m fluorescence (Figures 4C,D) led to a more oxidized NADH state. Moreover, NADH was fully oxidized at 20 μM DNP with OMN present (Figures 8C,D), and the oxidized state was not altered significantly by adding CaCl₂ after DNP. In the absence or presence of CaCl₂, NADH was completely oxidized after adding 100 μM DNP (data not shown).

FIGURE 8

Figure 8. Change in mitochondrial redox state, measured by NADH autofluorescence, as a function of time after adding DNP and CaCl₂ in the absence (A,B) or presence (C,D) of OMN. Note that the redox state was maintained after adding 10–30 μM DNP and CaCl₂ in the absence of OMN (A,B) but that in the presence of OMN (C,D) there was a concentration-dependent decrease in NADH autofluorescence. Adding CaCl₂ did not alter DNP-induced changes in redox state in the presence of OMN. Buffer pH = 7.15. Data obtained from eight hearts with 3–4 replicates per heart. See Figure 4 for statistical notation.

ATP Concentration Fell Without OMN but Remained Steady With OMN-Induced Block of Complex V

Total medium [ATP] was measured and mitochondrial [ATP]_m was estimated (see section “Supplementary Materials S.1.10”). Basal [ATP]_m was measured after adding mitochondria to the experimental buffer in the absence of OMN (Figures 9A,B). There was no change in basal [ATP]_m after adding PA. DNP, at 10 μM, did not significantly change [ATP] before or after adding CaCl₂ (Figures 9A,B). Basal [ATP]_m was unchanged if CaCl₂ was not added (data not displayed). Adding 20 or 30 μM DNP alone had no significant effect on [ATP]_m, but adding CaCl₂ resulted in a decrease in [ATP]_m (Figures 9A,B). In the presence of OMN (Figures 9C,D), adding mitochondria to the buffer did not change [ATP]_m, indicating inhibited complex V activity. [ATP]_m remained at a very low level and was unaffected by DNP or CaCl₂ in the presence of OMN. With OMN present, ATP_m/ADP_m ratios (see section “Supplementary Materials S.1.11, S.1.12 and Supplemental Results S.2.9”) also decreased with added DNP and CaCl₂, along with the progressive declines in ΔΨ_m.

FIGURE 9

Figure 9. [ATP], measured in total solution by the luciferin-luciferase reaction, and expressed as the calculated mitochondrial [ATP], was altered as a function of DNP and added CaCl₂ in the absence (A,B) and presence (C,D) of OMN. Adding mitochondria to the respiration buffer (first arrow) increased [ATP]; adding pyruvic acid (PA) and DNP had no additional effect; but adding CaCl₂ after DNP in the absence of OMN resulted in concentration-dependent decreases in [ATP] as graded depolarization of the IMM occurred (Figure 4). F_OF₁-ATP synthase/ase activity was near zero and remained essentially unchanged after adding PA, DNP, and CaCl₂ in the presence of OMN (C,D). Note that [ATP] was not measured continuously, so the lines between sampling points do not represent averaged data for any given intermediate time period between points. ADP/ATP ratio results are also given (see section “Supplementary Materials S.1.1, S.1.12 and Supplementary Results S.2.9”). Data obtained from 20 hearts. See Figure 4 for statistical notation.

Additional Supplemental Comparisons and Calculations

Supplementary Results S.2.2 and Supplementary Figure S.2 demonstrate the effect of adding DNP and CaCl₂ on respiration. Supplementary Results S.2.7 and Supplementary Figure S.9 furnish values for ΔΨ_m, [Ca²⁺]_m, and pH_m at 700 s, replotted from Figures 4–7, to compare these results in the presence or absence of OMN. The Supplementary Table shows DNP concentrations that produced 50% inhibitions (IC₅₀) of ΔΨ_m, [Ca²⁺]_m, fast (initial) d[Ca²⁺]_m/dt, and pH_m as a linear function of 0–30 μM DNP ± OMN at the 700 s time point. Supplementary Figure S.10 displays calculated mCa²⁺ flux rates (J_CHE) for CHE_m (see section “Supplementary Results S.2.8”) in the absence and presence of OMN.

Discussion

Ca^2+/H⁺ Exchange Activity Is Identified by Manipulating IMM Δ[H⁺] and Δ[Ca²⁺] Gradients

We provide firm support for a role of CHE_m in maintaining homeostasis of Ca²⁺ against H⁺ under certain conditions in cardiac cell mitochondria that may mimic some sequelae of cardiac IR injury. Our results: (1) furnish direct evidence for CHE_m activity by the secondary, slow increases in matrix Ca²⁺ efflux coupled to slow increases in matrix H⁺ influx, when both NCE and NHE activities are blocked, and particularly, when MCU-dependent mCa²⁺ re-uptake is blocked with Ru360; (2) demonstrate that respiration increases while ΔΨ_m decreases mildly, whereas pH_m and redox state are relatively maintained when inducing a matrix inward H⁺ leak with DNP before adding CaCl₂; adding CaCl₂ results in a secondary, slow increase in [Ca²⁺]_m that slowly depolarizes ΔΨ_m; (3) show that with permissive H⁺ influx, but inhibited outward H⁺ pumping at complex V, adding CaCl₂ causes larger decreases in ΔΨ_m, pH_m, and NADH and results in a slow decrease in [Ca²⁺]_m; (4) indicate that blocking complex V with OMN to prevent H⁺ pumping causes ΔΨ_m to further decrease after adding CaCl₂ because the influx of mCa²⁺ via the MCU is not opposed by H⁺ pumping at complex V; (5) suggest that the lack of a slow fall or rise in [Ca²⁺]_m in the presence of 100 μM DNP is due to the loss of ΔΨ_m-dependent mCa²⁺ uptake by MCU; (6) point out that only in partially depolarized mitochondria does added CaCl₂ result in a pH_m-independent gradual increase in [Ca²⁺]_m that is reciprocated by H⁺ pumping to maintain pH_m; preventing matrix acidification is associated with a maintained redox state; and (7) show that the decrease in [ATP] in the absence of OMN supports ATP hydrolysis with H⁺ pumping. These two scenarios, ±OMN, are depicted graphically in Figure 10A vs. Figure 10B.

FIGURE 10

Figure 10. Schema depicting putative role of MCU and CHE_m on slow Ca²⁺ influx and efflux, respectively, during stepwise depolarization with DNP with un-inhibited (i.e., minus OMN) (A) vs. inhibited (B) F_OF₁-ATPsynthase(ase) (i.e., plus OMN) after a bolus addition of CaCl₂ to the mitochondrial medium. (A) (1) DNP permits H⁺ entry that tends to (2) decrease ΔΨ_m, which enhances H⁺ pumping by respiratory complexes, including (3) F_OF₁-ATPase, so that pH_m does not decrease appreciably and ΔΨ_m is partially supported (2). Adding CaCl₂ further depolarizes ΔΨ_m by allowing more cationic (Ca²⁺) charges into the matrix via the MCU (4). Over time, in the range of a 20–60% decline in ΔΨ_m, pH_m remains unchanged as (5) H⁺ is pumped out (3) in exchange for permissive H⁺ entry (1) in triggering additional slow mCa²⁺ uptake by MCU and causing ΔΨ_m to decrease further. CHE_m is inhibited by the lack (pH 7.6) of matrix acidity (5) and NCE_m and NHE_m are inactivated by the lack of substrate and buffer Na⁺ (6). (B) Alternatively, when F_OF₁-ATPase is inhibited (3), matrix acidity gradually increases (pH 6.9), ΔΨ_m is less supported (2) and Ca²⁺ slowly exits (CHE_m) in exchange for slow H⁺ entry due to DNP (5). This sequence triggers a net loss of mCa²⁺ even though uptake of Ca²⁺ via the MCU continues, as shown by a greater efflux of mCa²⁺ by CHE_m when additional mCa²⁺ uptake via MCU is blocked by Ru360 (4). DNP, dinitrophenol; ETC, electron transport chain; IMS, inner membrane space; MCU, mitochondrial Ca²⁺ uniporter; OMN, oligomycin; TCA, tricarboxylic acid.

Net Mitochondrial Ca²⁺ Influx Occurs via MCU and Net Ca²⁺ Efflux Can Occur via Ca²⁺/H⁺ Exchange

The dependence of rapid MCU-mediated mCa²⁺ uptake on ΔΨ_m has been examined extensively (Gunter and Pfeiffer, 1990; Gunter et al., 1994; Dash et al., 2009; Haumann et al., 2010). But our study demonstrates that net m[Ca²⁺] can additionally increase slowly via the MCU, and that this happens when pH_m is relatively maintained despite a decline in ΔΨ_m resulting from the DNP-mediated inward H⁺ flux and after the initial rapid Ca²⁺ influx via MCU. A gradual increase in [Ca²⁺]_m at the expense of maintaining the ΔpH_m may be deleterious to mitochondrial function. We propose that this secondary rise in net [Ca²⁺]_m results from an adequate ΔΨ_m with Ru360-dependent slow mCa²⁺ influx, which eventually leads to a slow, continued fall in ΔΨ_m. Because H⁺ pumping at complex V maintains the Δ[H⁺]_m gradient, mCa²⁺ efflux via CHE_m in exchange for mH⁺ influx due to the H⁺ leak is likely masked by mCa²⁺ re-uptake. Thus, the DNP-induced H⁺ leak and the concomitant dissipation of the IMM Δ[H⁺] gradient, when countered by H⁺ pumping at complex V (in addition to other complexes), can maintain the ΔpH_m and support the pmf (ΔΨ_m + RT/FΔpH_m) (Dzbek and Korzeniewski, 2008). This view is especially supported by the smaller decline in extra-mitochondrial [Ca²⁺]_e in the presence of 20 μM DNP, 25 μM CaCl₂, and OMN, as well as in the presence of Ru360, by the gradual increase in [Ca²⁺]_e due to CHE_m mediated Ca²⁺ efflux. These results are reinforced by the exaggerated effect of added CaCl₂ to enhance the decline in ΔΨ_m over time and by the slow decreases in [Ca²⁺]_m linked to slow decreases in pH_m. Blocking outward H⁺ pumping by complex V prevented compensation for DNP-mediated H⁺ influx. Consistent with our observations, it was reported that matrix acidification may reduce Ca²⁺ uptake in cardiac mitochondria by its effect on decreasing ΔΨ_m (Gursahani and Schaefer, 2004). In contrast, when ATP_m hydrolysis is prevented, pH_m slowly decreases toward pH_e with a greater fall in ΔΨ_m; the slow H⁺ influx is accompanied by a slow net fall in [Ca²⁺]_m mediated by CHE_m even though the extruded Ca²⁺ is recycled via the MCU. Since H⁺ influx (DNP-induced leak) is not countered by reciprocal H⁺ pumping to restore ΔpH_m, the slow influx of H⁺ is exchanged for slow Ca²⁺ efflux via CHE_m until the ΔpH gradient is dissipated.

Ca²⁺ and H⁺ gradients across the IMM are largely dependent on ΔΨ_m and ΔpH gradients resulting from H⁺ pumping by respiratory complexes. Ionic homeostasis requires one cation efflux pathway to oppose another cation influx pathway and vice versa. Cation exchangers fulfill this need. Unlike mCa²⁺ uptake via MCU, which is dependent on ΔΨ_m and on the chemical gradient, exchange of Ca²⁺ and H⁺ via CHE_m may or may not be dependent on ΔΨ_m (Rottenberg and Marbach, 1990; Gunter et al., 1991). But the direction of Ca²⁺ and H⁺ flux mediated solely by CHE_m is dependent on a large IMM [H⁺] or [Ca²⁺] gradient to shuttle Ca²⁺ or H⁺ across the IMM. This can be expressed by an electroneutral J_CHE flux equation (Tewari et al., 2014), calculated here in the presence and absence of OMN (see section “Supplementary Results S.2.8” and Supplementary Figure S.10). J_CHE flux analysis of our data suggests that slow mCa²⁺ influx could have occurred via CHE_m in the absence of OMN, whereas mCa²⁺ efflux could have occurred in the presence of OMN. Indeed, we have provided strong support for slow net mCa²⁺ efflux mediated by CHE_m (despite slow mCa²⁺ uptake by MCU) when complex V cannot pump H⁺ in the presence of OMN.

Although CHE_m likely occurs both in the absence or presence of OMN, our results suggest that the observed secondary, slow influx of mCa²⁺ influx (minus OMN) is due primarily to re-uptake by a Ru360 sensitive mechanism, presumably MCU, that may overwhelm any CHE_m activity. This is because Ru360 blocked the slow rise in [Ca²⁺]_m and the slow fall in [Ca²⁺]_e, thus supporting MCU as the mediator of the slow mCa²⁺ influx. The J_CHE flux equation only monitors differences in [H⁺] and [Ca²⁺] on either side of the IMM and does not rely on effects of the ΔpH_m gradient on H⁺ pumping or the ΔΨ_m gradient on mCa²⁺ uptake via MCU. Thus the secondary, slow mCa²⁺ uptake after the initial CaCl₂ bolus (Figures 5A,B,E,F) appears to be a direct effect of H⁺ pumping by complex V (minus OMN) to maintain the ΔpH_m charge gradient and support the pmf although ΔΨ_m continues to fall due to the continued mCa²⁺ influx. On the other hand, inhibiting ATP_m hydrolysis (Figures 9C,D) to prevent H⁺ pumping not only enhances the fall in ΔΨ_m (Figures 4C,D) to retard further mCa²⁺ loading by the MCU, but also permits slow CHE_m-mediated mCa²⁺ efflux (Figures 6A,B,E,F) in exchange for mH⁺ influx until the diminishing ΔpH_m gradient is abolished (Figures 7C,D).

Alternatively, we demonstrated CHE_m activity by acidifying the external medium before adding CaCl₂, while blocking NCE_m and NHE_m activities by using Na⁺ free buffer and substrates. We observed a slowly increasing [Ca²⁺]_e coupled to a slowly increasing [H⁺]_m. We used Ru360 to expose the net amount of mCa²⁺ efflux via CHE_m by blocking the effluxed Ca²⁺ from re-entering via MCU (Figures 1, 2). It is unlikely that 0.1–1 μM Ru360 inhibits CHE_m because Ru360 did not block mCa²⁺ efflux (Figures 1, 2), only mCa²⁺ influx. Of course, Ru360 might block another mode of non-MCU Ca²⁺ uptake. Our proposed mechanism is described schematically in Figures 10A,B. We postulate that CHE_m activity is completely inhibited if the matrix remains alkaline (large ΔpH_m gradient), thus exposing net Ca²⁺ uptake via MCU. The slow increases in [Ca²⁺]_m that we observed previously (Haumann et al., 2010) likely represent net slow mCa²⁺ via MCU (reference Figure 5).

A leucine zipper EF hand-containing trans-membrane protein (LETM1) found in non-mammalian cells is thought to be a molecular component of CHE_m (Jiang et al., 2009; Shao et al., 2016). Knockdown and expression of LETM1 in a number of cell lines support its role in Ca²⁺/H⁺ exchange, particularly in mitochondria (Jiang et al., 2013; Doonan et al., 2014). Alternatively, other studies (Nowikovsky et al., 2004, 2012; Froschauer et al., 2005; Malli and Graier, 2010; Austin et al., 2017) support that LETM1 either does not mediate Ca²⁺ efflux (De Marchi et al., 2014) or that it mediates K⁺/H⁺ and/or Na⁺/H⁺ exchange, so conclusive genetic evidence for CHE requires more study. It is important to note that the elusive CHE protein appears to be insensitive to MCU inhibitors, i.e., ruthenium red (RR) compounds (Bernardi et al., 1984), and to CGP-37157, the NCE inhibitor (Tsai et al., 2014). The present study explores for the first time the kinetics of CHE_m activity in relation to MCU activity in cardiac cell mitochondria.

ΔΨ_m < E_REV-ATPase Promotes ATP Hydrolysis

F_OF₁-ATPsynthase/ase directionality is governed by ΔΨ_m and its “reversal potential” E_{REV -ATPase}, which in turn is dependent on the concentration of the reactants ATP/ADP, and H⁺ (Metelkin et al., 2009; Chinopoulos and Adam-Vizi, 2010; Chinopoulos et al., 2010). Additional factors of E_REV that affect the direction and rate of ATP synthesis/hydrolysis are the free [P_i] and the H⁺_m/ATP_m coupling ratio, n (Cross and Muller, 2004). When ΔΨ_m becomes less negative than E_REV, which depends on a high [ATP]_m and ΔpH_m, but a low [ADP]_m, H⁺ ejection by complex V becomes thermodynamically favorable (Metelkin et al., 2009; Chinopoulos and Adam-Vizi, 2010; Chinopoulos et al., 2010; Chinopoulos, 2011). E_REV-ATPase can occur when ΔΨ_m falls between -130 and -100 mV, depending on matrix [ATP]_m/[ADP]_m, [P_i]_m, ΔpH_m, and the coupling ratio (Chinopoulos et al., 2010; Chinopoulos, 2011). Others (Leyssens et al., 1996; Bains et al., 2006; Chinopoulos and Adam-Vizi, 2010) have observed that a fall in ΔΨ_m caused by a protonophore, such as DNP or CCCP, can induce ATP hydrolysis through reversal of F_OF₁-ATPsynthase. The consequent H⁺ pumping by complex V would tend to partially restore ΔΨ_m to offset the protonophore-induced decreases in pH_m and ΔΨ_m as discussed above. The electrical gradient ΔΨ_m and the H⁺ chemical gradient Δ[H⁺]_m together contribute to the total pmf that powers the synthesis of ATP; when pmf is not maintained, hydrolysis of matrix ATP occurs. Previous studies have also furnished indirect evidence for reversal of F_OF₁-ATPsynthase under conditions of reduced mCa²⁺ uptake and a fully depolarized ΔΨ_m with CCCP (Leyssens et al., 1996; Bains et al., 2006). ATP_m hydrolysis has been reported to occur in vivo during cardiac ischemia (Grover et al., 2004), but the in vivo ΔΨ_m at which this occurs is not known. Here we show how a DNP-induced fall in ΔΨ_m induces ATP hydrolysis.

In the absence of OMN, the lack of a fall in ATP levels after adding 10 μM DNP indicated that ATP_m hydrolysis (Figure 9) did not occur because ΔΨ_m remained relatively stable before adding CaCl₂. However, adding CaCl₂ resulted in a gradual, but large, fall in ΔΨ_m over time. In the presence of 20 μM DNP and 25 μM CaCl₂, ATP hydrolysis occurred (20–25% of maximum) with a decrease in ΔΨ_m at an IMM gradient of approximately 0.35 ΔpH_m units (Figures 7A,B). A faster rate of ATP hydrolysis was indicated by the additional fall in [ATP]_m over time after adding 30 μM DNP and CaCl₂. The DNP-induced falls in ΔΨ_m were accompanied by reduced ATP_m/ADP_m ratios (see section “Supplementary Materials S.1.11, S1.12 and Supplementary Results S.2.9”) indicating consumption of ATP, as also shown by the lower [ATP]_m (Figures 9A,B). A calculation of available matrix ATP is given (see section “Supplementary Results S.2.10”). In the presence of 100 μM DNP and added CaCl₂, ΔΨ_m was maximally depolarized (Figures 4A,B), the ΔpH_m gradient was abolished (Figures 7A,B), and NADH was oxidized (Figures 8A,B), indicating that ATP_m hydrolysis was insufficient to maintain the pmf. This contrasts to the situation with 10–30 μM DNP where pmf was supported largely by the ΔpH_m gradient, as also reflected by the maintained NADH redox state.

ΔΨ_m is normally fully polarized when complex V is blocked by OMN (Valdez et al., 2006; Brand and Nicholls, 2011); however, the effect of DNP to slightly decrease ΔΨ_m was intensified when OMN was present, particularly after adding 25 μM CaCl₂ that intensifies the depolarization of ΔΨ_m in the presence of DNP. This effect of DNP in the absence of OMN indicates that ATP hydrolysis indeed supported the ΔpH_m via H⁺ pumping even at a relatively small decline in ΔΨ_m with DNP. With OMN present, ATP hydrolysis cannot occur (Figures 9C,D) and so complex V cannot contribute to maintaining pH_m; therefore, the low pH_m accompanied by a high [Ca²⁺]_m must have activated CHE_m.

Changes in pH_m, [Ca²⁺]_m, and NADH Are Larger With OMN Than Without OMN

An interesting observation of our study is the contribution of complex V to maintain the ΔpH_m gradient (and thus supporting the pmf) whereby the H⁺ leak is compensated by augmented H⁺ pumping by complex V; this resulted in slow mCa²⁺ influx (“Ca²⁺ leak”) that could be blocked by Ru360, which indicates the influx likely occurred via MCU. But if compensatory H⁺ pumping is blocked by OMN, the matrix becomes acidic, the ΔpH_m gradient falls lower, and slow mCa²⁺ efflux occurs via CHE_m thus masking the slow mCa²⁺ influx (Figure 10B). Evidence for H⁺ pumping during ATP hydrolysis during DNP-mediated H⁺ influx was provided by the maintenance of an alkaline pH_m; moreover, pH_m indeed fell when H⁺ pumping was blocked by OMN. Similarly, if mitochondria reside in an acidic environment (Figures 1, 2), [H⁺]_m falls as [Ca²⁺]_e rises, indicating CHE_m. Indeed, in a previous study it was reported that adding lactic acid to a Na⁺ free mitochondrial suspension increased buffer Ca²⁺ by 43% (Gambassi et al., 1993); it was suggested that Ca²⁺ was extruded as H⁺ influx caused H⁺ ions to compete with Ca²⁺ ions for mitochondrial binding sites (Gambassi et al., 1993). We furnish direct evidence for a link between Ca²⁺ efflux with H⁺ influx in mammalian cardiac muscle mitochondria, when Na⁺ is absent and the MCU is blocked after adding CaCl₂.

NADH levels remained unchanged after adding DNP and CaCl₂ (Figures 8A,B); this likely reflects the faster state 2 respiration (Supplementary Figure S2) since the inward H⁺ leak by DNP was balanced by H⁺ pumping from complex V as well as from complexes I, III, and IV. Only at 100 μM DNP with CaCl₂, which fully depolarized ΔΨ_m (Figures 4A,B), did DNP result in a lower pH_m (Figures 7A,B) and a more oxidized redox state, i.e., a decrease in NADH (Figures 8A,B). It is likely that an increase in F_OF₁-ATPase activity plus a faster TCA cycle turnover (increased NADH/NAD⁺ ratio) can result in maintained NADH levels despite the DNP-induced H⁺ leak. In the presence of OMN, however, NADH was gradually oxidized (Figures 8C,D) along with the fall in pH_m (Figures 7C,D); this scenario likely occurred because the additional H⁺ pumping by complex V to support ΔΨ_m was blocked. We observed that adding CaCl₂ alone did not significantly change NADH levels in this model, which is consistent with our earlier study (Haumann et al., 2010). Although an increase in [Ca²⁺]_m can stimulate NADH producing dehydrogenases (Denton et al., 1980; McCormack and Denton, 1980; Wan et al., 1989; Brandes and Bers, 1997), our experiments were conducted at maximal [Ca²⁺]_m values below the K_0.5 of 1 μM Ca²⁺ at which these dehydrogenases are reported to be activated (Denton et al., 1980; McCormack and Denton, 1980).

What Is the Functional Role of CHE_m: How Is Net mCa²⁺ Efflux Modified by mCa²⁺ Influx via MCU?

The net Ca²⁺ driving force for ions across the IMM can be estimated by Nernst equilibrium potentials for given estimates of ΔΨ_m. Under conditions of 20 μM DNP, 25 μM CaCl₂, and in the absence of OMN, when [Ca²⁺]_m slowly increased, we calculated Nernst equilibrium potentials of approximately -8 and +18 mV, respectively, for [Ca²⁺] and [H⁺] at 700 s. We estimated ΔΨ_m as -110 to -120 mV at 700 s (based on our values for % of minimal and maximal depolarization (R-123 fluorescence) and curve fitting for approximating conversion to ΔΨ_m (Huang et al., 2007)). This indicated that the driving force for both Ca²⁺ and H⁺ would remain inward despite H⁺ pumping at complex V to attempt to re-establish the ΔpH_m gradient by compensating for the DNP-mediated H⁺ influx. Based on our estimated ΔΨ_m and the calculated Ca²⁺ and H⁺ equilibrium potentials driving both Ca²⁺ and H⁺ inward, we conclude that the outward H⁺ pumping by complex V (in addition to complexes I, III, IV) was sufficient to compensate for the continued inward influx of H⁺ mediated by DNP thus restoring the ΔpH_m gradient, but not the pmf, and thus preventing activation of CHE_m. Ru360 blocked this additional uptake of mCa²⁺ by the MCU so that [Ca²⁺]_e did not continue to fall.

We predict that the major conduit for both fast and slow mCa²⁺ influx under our experimental conditions occurs primarily via the MCU. The efflux of Ca²⁺ via the CHE_m is slow so we expect the re-uptake of Ca²⁺ via the MCU also would be slow. Although the J_CHE flux equation alone predicted that slow mCa²⁺ influx could have occurred via CHE_m this is unsustainable if [H⁺]_m < [H⁺]_e. It is likely that voltage-dependent transport of net Ca²⁺ inward is mostly responsible if there is at least a partially maintained ΔΨ_m (Nernst potentials) despite mCa²⁺ extrusion via CHE_m. Interestingly, under the condition of a fully polarized ΔΨ_m (no DNP and no OMN) (Figures 4A–D), [Ca²⁺]_m did not rise as it did in the presence of DNP (Figures 5A,B) when pH_m was maintained (Figures 7A,B). This suggests that the secondary, slow uptake of mCa²⁺ is indirectly related to H⁺ pumping due to the decline in [H⁺]_m to support the pmf; the additional, slow mCa²⁺ uptake by the MCU occurs because of the remaining charge gradient (ΔΨ_m) and Ca²⁺ chemical gradient.

In contrast, in the presence of OMN the kinetics of the delayed, slow mCa²⁺ efflux via CHE_m under conditions of reduced ΔΨ_m and low pH_m are different. Our estimates of ΔΨ (Huang et al., 2007) of -60 to -70 mV at 700 s with OMN present are much lower than without OMN; this is likely due to dissipation of both ΔpH_m and ΔΨ_m gradients because H⁺ pumping by complex V to support ΔpH_m (and ΔΨ_m) was blocked. With OMN present, we estimated Nernst potentials of +13 and +6 mV, respectively, for Ca²⁺ and H⁺ (calculated at 700 s). Based on these Nernst potentials the driving forces for both Ca²⁺ and H⁺ would remain inward with OMN present, although their Nernst potentials are reversed compared to those in the absence of OMN. With the slow inward driving force for H⁺, unmatched by H⁺ pumping at complex V, pH_m approached pH_e and net [Ca²⁺]_m became lowered due to CHE_m. Because inhibiting the MCU with Ru360 caused a robust increase in [Ca²⁺]_e, this indicated the Ca²⁺ effluxed via CHE_m re-enters via the MCU unless this pathway is blocked. Under the unique condition of collapsed ΔΨ_m (100 μM DNP) and ΔpH_m gradients, the secondary, slow uptake of mCa²⁺ is absent (Figures 5A,B, black lines) so that the decline in [Ca²⁺]_m via CHE_m is fully observed (Figures 6A,B). Thus, a fall in pH_e strongly supports net mCa²⁺ efflux via CHE_m even though the Nernst potentials indicate continued slow mCa²⁺ influx (via MCU), which indeed occurs if there is remaining ΔΨ_m. This means that net Ca²⁺ efflux due to CHE_m (Figures 1, 2 and Supplementary Figure S.6) can be exposed by blocking the MCU after the initial bolus of CaCl₂ to prevent further mCa²⁺ uptake. CHE_m is predicted by the J_CHE equation to favor mCa²⁺ efflux in exchange for mH⁺ influx based on matrix and buffer ion concentrations obtained with OMN present (Supplementary Figure S.10). Our prediction assumes that Ca²⁺ is exchanged for 2H⁺ with equal affinities for both cations, or a higher affinity for H⁺.

Does Transient, Low Conductance mPTP Also Shuttle Ca²⁺ Across the IMM in These Experiments?

Inducing a partial ΔΨ_m depolarization was reported to cause a slow influx of mCa²⁺ through low conductance mPTP opening (Saotome et al., 2005). CsA prevented both an increase in mCa²⁺ and the release of the small molecule calcein during simulated ischemia in cardiomyocytes suggesting that transient mPTP opening during ischemia allowed mCa²⁺ influx (Seidlmayer et al., 2015). In the present study adding CaCl₂ in the presence of DNP or an acidic buffer caused falls in ΔΨ_m, so could low conductance mPTP opening have contributed to the secondary, slow increase or decrease in _m[Ca²⁺] we observed in the absence or presence of OMN? We doubt this for the following reasons: (1) ROS, adenine nucleotide levels, and other factors are believed to contribute to mPTP formation during IR injury. But in our study we did not utilize IR to induce increases in Ca²⁺ and ROS or decreases in pH_m or ΔΨ_m; (2) Altering just the driving force for protons across the IMM using DNP or external pH to exchange Ca²⁺ ion for H⁺ ions is not compatible for a mechanism to cause or prevent formation of mPTP but it is for inducing mCHE activity; (3) Transient mPTP formation is controversial and based largely on the utility of calcein or other small particles to mark mitochondrial release of small molecules with free flowing ions such as Ca²⁺ (Petronilli et al., 1999); (4) CsA-sensitive transient mPTP opening in individual mitochondria of cardiac myocytes is quite rare even with elevated m[Ca²⁺] or exposure to H₂O₂ (Lu et al., 2016); (4) CsA, or its inhibition of the peptidyl prolyl cis–trans isomerase activity of cyclophilin D, has known and unknown effects on mitochondrial function that may be unrelated to mPTP formation (Giorgio et al., 2010). Some interpretations on effects of cyclophilin D, via CsA, may pertain to changes in Ca²⁺ flux due to mCHE rather than transitional mPTP opening.

CSA Ceases Activation of CHE_m

CsA unexpectedly stopped the secondary CaCl₂-induced effects attributed to CHE_m. CsA ceased all apparent CHE_m activity after adding CaCl₂ when pH_e was 6.9 or 7.15, as assessed by measurements of extra-matrix [Ca²⁺]_e, pH_m, and ΔΨ_m (Supplementary Figures S.1A–C). CsA did not blunt the partial ΔΨ_m depolarization induced by DNP alone at pH_e 7.15, but did delay full ΔΨ_m depolarization induced by adding CaCl₂ after DNP (Supplementary Figures S.7A,B). We do not believe the slow, attenuated decreases in extrusion of Ca²⁺ or slow fall in matrix pH observed in the presence of CsA are directly related to inhibition of permanent or transient mPTP opening. CsA did not directly prevent the ΔΨ_m depolarization that occurs during CHE_m or with addition of DNP alone. In the absence of CsA (Figures 1A–C), the observed changes in pH_m, external [Ca²⁺]_e, and ΔΨ_m, induced by adding CaCl₂ at extra-matrix pH 6.9, occurred very slowly over 25–30 min; this is indicative of slow cation exchange activity, not mPTP. Moreover, full ΔΨ_m depolarization was incomplete. CsA or its inhibition of cyclophilin D may obviate the conditions for matrix H⁺ influx or mCa²⁺ efflux as well as Ca²⁺ recycling via the MCU. CsA may prevent dissipation of the ΔpH gradient when the external pH is low. Since the results obtained in the presence of CsA are not compatible with preventing or delaying mPTP opening, the effects of CsA in this setting are unclear. Additional experiments will be needed to delineate the mechanism of CsA on preventing CHE_m.

Other Potential Limitations of the Study

One important limitation of our study is the lack of a selective inhibitor of CHE_m to aid in defining a more precise mechanism of action. Since the gene code for LETM1 and its protein sequence are known, point mutations (Tsai et al., 2014) and knockdowns (Jiang et al., 2013; Doonan et al., 2014) in mammalian models will be helpful to assess mechanisms and kinetics of this cation antiporter; but it remains unclear if LETM1 mediates CHE_m exclusively, or at all. Another limitation is that mitochondria were examined outside their normal milieu so that the contributions of ATP synthesis by glycolysis and ATP hydrolysis for cellular metabolic support could not be assessed. Experiments were conducted at room temperature at which metabolism would be lower and buffering capacity different than at 37°C. The activity of CHE_m during cardiac IR is unknown and mCa²⁺ efflux in cardiac mitochondria may occur primarily via the NCE_m and not CHE_m. Nevertheless, induction of CHE_m could occur in vivo during IR injury under very specific circumstances of trans-IMM cationic imbalance. Evaluation of CHE_m activity in cardiac myocytes after IR injury should be helpful to design protective strategies using this mechanism.

Conclusion

This study furnishes new insights into the bioenergetic and dynamic mechanisms in cardiac cell mitochondria of delayed, slow mCa²⁺ influx via the MCU, and mCa²⁺ efflux via the pH_m-dependent CHE_m. We demonstrate the kinetics of slow changes in mCa²⁺ loading/unloading that are linked to unblocked vs. blocked ATP_m hydrolysis to decrease vs. increase pH_m, respectively, after partial depolarization by DNP. We found that after an initial CaCl₂ bolus there is slow mCa²⁺ influx (Ca²⁺ leak) through a Ru360-sensitive pathway if H⁺ pumping counteracts a H⁺ leak; however, there is net slow mCa²⁺ efflux that overrides ΔΨ_m-mediated Ca²⁺ influx that is activated via CHE_m if there is a high ΔpH_m gradient. In cardiac mitochondria, the rapid and slow mode of uptake of mCa²⁺ appears to be dependent primarily on the trans-membrane [Ca²⁺] and ΔΨ_m gradients if outward H⁺ pumping counteracts inward H⁺ entry. In contrast, slow extrusion of mCa²⁺ by CHE_m appears to be dependent primarily on the [ΔH⁺]_m gradient induced by H⁺ influx/leak by DNP or by an acidic pH_e. Importantly, if NCE_m and NHE_m are inactivated, blocking complex V might prevent delayed Ca²⁺ overload and instead stimulate Ca²⁺ extrusion via CHE_m if there is an inward H⁺ leak. In intact cells, this can also serve to preserve TCA cycle-generated ATP, i.e., substrate level phosphorylation. Such passive homeostatic balance of Δ[Ca²⁺]_m may occur during cardiac injury when there is mCa²⁺ loading accompanied by declines in NADH redox state, pH_m and Ψ_m. We conclude that the differences in the rate and magnitude of mCa²⁺ influx/efflux in partially depolarized mitochondria, in the presence or absence of F_OF_1-ATPase activity, can be ascribed to the underlying changes in pmf components, ΔpH_m and ΔΨ_m, after rapid mCa²⁺ loading.

Author Contributions

DS proposed the study and its initial design. JH conducted most experiments, carried out initial statistical analysis, constructed initial figures, and participated in design, interpretation and writing. AG, AB, CB, CN, and MB conducted supporting experiments. AC, W-MK, and RD participated in theoretical interpretation of the results and text editing. DS and AC supervised the team in subsequent experimental designs, interpretation of results, and manuscript construction and writing.

Funding

This project was supported by grants from the National Institutes of Health (R01HL089514, R01HL095122 and 5T35HL072483) and the Veterans Administration (Merit Review BX820405P and BX002539).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors wish to thank Drs. Mohammed Aldakkak, Jason Bazil, Shivendra G. Tewari, Venkat Pannala, Kalyan C. Vinnakota, and Gayathri Natarajan for their help and advice, laboratory manager James S. Heisner for technical assistance and supporting experiments, and medical student David S. Lambert for discussions on follow up studies.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys.2018.01914/full#supplementary-material

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