Calcium Overload Decreases Net Free Radical Emission in Cardiac Mitochondria

Abstract

Elevated calcium and reactive oxygen species (ROS) are responsible for the bulk of cell death occurring in a variety of clinical settings that include acute coronary events, cerebrovascular accidents, and acute kidney injury. It is commonly believed that calcium and ROS participate in a viscous cycle during these events. However, the precise feedback mechanisms responsible are unknown. In this study, we quantitatively demonstrate that, on the contrary, calcium does not stimulate free radical production but suppresses it. Isolated mitochondria from guinea pig hearts were energized with a variety of substrates and exposed to calcium concentrations designed to induce moderate calcium overload conditions associated with ischemia/reperfusion injury but do not elicit the well-known mitochondrial permeability transition phenomenon. Metabolic function and free radical emission were simultaneously quantified using high-resolution respirometry and fluorimetry. In parallel, membrane potential, high amplitude swelling, and calcium dynamics were also quantified. Our results reveal that calcium overload does not lead to excessive ROS emission but does decrease ADP stimulated respiration rates for NADH-dependent pathways. Moreover, we developed an empirical model of mitochondrial free radical homeostasis to identify the processes that are different for each substrate and calcium condition. In summary, we show that in healthy guinea pig mitochondria, calcium uptake and free radical generation do not contribute to a viscous cycle and that the relationship between net free radical production and oxygen concentration is hyperbolic. Altogether, these results lay out an important foundation necessary to quantitatively determine the role of calcium in IR injury and ROS production.

1. Introduction

Ischemia-reperfusion (IR) injuries underlie a variety of pathological settings that are associated with high mortality rates and long-term decreases in organ function. These injuries occur in common clinical settings such as acute coronary artery events, cerebrovascular accidents and acute kidney injury¹. While the pathophysiological events underlying IR injury have been reviewed extensively elsewhere^2,3, they are briefly summarized here. During ischemia, partial or complete cessation of oxygen delivery impedes electron transport system (ETS) activity. As a result, metabolites accumulate, and ETS redox centers become highly reduced. During this ischemic period, the cytoplasm acidifies and is flooded with calcium. Altogether, these factors create an environment conducive to free radical production. Although reperfusion is necessary for tissue salvage, paradoxically, most tissue damage occurs during this period. During reperfusion, exacerbation of calcium overload and a sudden increase in free radical levels trigger a phenomenon known as mitochondrial permeability transition^3–6. After this transition, mitochondria switch from ATP producers to ATP consumers⁷. When the injury is most severe, such as that caused by prolonged or complete ischemia, this transition is irreversible and leads to ATP depletion and cell death. As the main triggers for this phenomenon are attributed to calcium overload and oxidative stress, many studies have suggested a causative relationship between calcium overload and the burst of reactive oxygen species (ROS) occurring in the reperfusion phase. But others have implied a vicious cycle between calcium and ROS production^8–11. Nevertheless, the relationship between calcium and mitochondrial ROS production remain an important topic of investigation.

Oxidative stress during IR is primarily caused by the mitochondrial derived superoxide, a type of ROS. While the formation of superoxide is thermodynamically favorable^12,13, its production is under kinetic control¹³. Kinetic control is set by the mitochondrial membrane potential, the primary regulator of superoxide formation. This bioenergetic variable controls the degree of ETS redox center reduction and thus kinetically limits superoxide production. Under physiological conditions, free radical production is balanced by elimination to maintain free radical homeostasis and prevent oxidative stress. Recent evidence points to complexes I and III as the major ETS sources of ROS^13–15; however, other mitochondrial enzymes also contribute to ROS production^16–19. For example, the dihydrolipoamide dehydrogenase subunit (E3 component) of the matrix enzyme complexes α-ketoglutarate dehydrogenase, pyruvate dehydrogenase and branched-chain alpha-keto acid dehydrogenase has been shown to be a major source of ROS under conditions where NADH/NAD⁺ is maintained at a high level^16,20,21. While many studies have linked calcium overload to excessive ROS formation during reperfusion^8,9,22, the precise mechanism linking these two factors remain unknown.

Metabolic alterations and calcium overload during IR are the major components of IR injury. As such, we tested the metabolite- and calcium-dependence of mitochondrial free radical homeostasis by quantitatively assessing net free radical generation from isolated cardiac mitochondria. The experiments were performed under various calcium challenges designed to reflect mitochondrial calcium loads that occur during IR injury. In addition, the effect of calcium on the metabolism of different substrates that are utilized by different metabolic pathways important for cardiac function were investigated. The respiratory and net free radical production rates were simultaneously measured. In parallel experiments, the membrane potential, high amplitude swelling, and buffer calcium were quantified. We found a modest calcium-dependent stimulation of respiration in the absence of ADP but a profound inhibition of ADP-stimulated respiration with NADH-linked metabolites. Most importantly, we find that there is no explicit calcium-dependent stimulation of net free radical generation. In fact, our data reveal that calcium overload decreases free radical production in mitochondria as opposed to increasing it. These findings have important implications for the role of calcium in IR injury and ROS production.

2. Materials and Methods

2.1. Mitochondria Isolation

Animal care and handling conformed to the National Institutes of Health’s Guild for the Care and Use of Laboratory Animals and was approved by Michigan State University’s Institutional Animal Care and Use Committee. Ventricular mitochondria from Hartley guinea pigs were isolated based on protocol previously described^{23, 24} and will be briefly summarized here. Animals (4-6 weeks, 350-450 grams) were euthanized after being anesthetized with 5% isoflurane and tested to be unresponsive to noxious stimuli. The heart was perfused in chest with ice-cold cardioplegic solution until no blood was observed in the coronary arteries and cardiac veins. The cardioplegic solution (CS) consisted of 25 mM KCl, 100 mM NaCl, 10 mM dextrose, 25 mM MOPS and 1 mM EGTA at pH = 7.15. The heart was then excised and washed with ice-cold isolation buffer (IB). IB consisted of 200 mM mannitol, 50 mM sucrose, 5 mM K₂HPO₄ and 0.1% w/v BSA at pH = 7.15. Connective tissues, thymus and the great vessels were removed. Ventricular tissues were minced in ice-cold IB until small pieces on the order of a mm³ were left. The homogenate was then transferred to a 50 mL canonical tube containing 0.5 U/mL protease (Bacillus licheniformis) in 25 mL IB. Tissue homogenization was done using an Omni handheld homogenizer at 18,000 rpm for 20 sec. Mitochondria were then recovered by gradient centrifugation in IB at 4°C. Mitochondrial protein was quantified using the BCA assay and an Olis DM-245 spectrofluorometer with dual-beam absorbance module. The mitochondrial stock solution was diluted to a working concentration of 40 mg/mL. All reagents were from Sigma unless otherwise noted.

2.2. Experimental Set-up

For all experiments, mitochondria were suspended in a respiratory buffer containing 130 mM KCl, 5 mM K₂HPO₄, 20 mM MOPS, 1 mM MgCl₂ and 0.1% w/v BSA at pH of 7.1 at 37°C. In the absence of EGTA, the free calcium concentration is approximately 4 μM due to chemical impurities²³. Buffer sodium concentration in all experiments at 20 mM was achieved by adding the appropriate amount of NaCl to the standard respiratory buffer. Under physiological conditions, mitochondrial matrix sodium concentration is in the range of 5-10 mM. During ischemia, this value can increase to the 20 mM range. Performing the experiments at this level of sodium yields results that can more accurately delineate the capacity of mitochondria to handle calcium overload and oxidative stress during conditions like ischemia. However, experiments were also performed at lower sodium concentrations to explicitly investigate potential effects of exogenous sodium on mitochondrial bioenergetics. In experiments where a lower oxygen concentration ([O₂]) was needed, N₂ gas was bubbled into respiratory buffer . Oxygen-depleted buffer was added to the respiratory chamber, and the oxygen concentration was allowed to increase to a desired level by diffusion.

To investigate the effects of electron donors from various metabolic pathways on oxygen consumption and hydrogen peroxide emission rates, four combinations of substrates and inhibitors were used: pyruvate/L-malate (P/M), palmitoylcarnitine/L-malate (PC/M), succinate/rotenone (S/R) and succinate (S). Malate is added to pyruvate and palmitoylcarnitine to prevent the depletion of oxaloacetate during the experiments. Pyruvate is primarily an NADH-linked and a complex I substrate. Under the experimental conditions we used in this study, TCA cycle intermediates produced from pyruvate metabolism leak out of mitochondria. Therefore, pyruvate is incompletely oxidized^25,26. Electrons derived from palmitoylcarnitine enter the ETS via QH₂ and NADH. And with succinate, electrons enter the ETS via QH₂ at complex II. Rotenone inhibits complex I and prevents excess free radical production from its quinone-reductase site¹⁵. The appropriate volumes of substrates and/or inhibitor stock solutions were added to each 2 mL respiratory chamber in separate experiments to achieve the desired total concentrations of 5 mM pyruvate/1 mM L-malate, 25 μM palmitoylcarnitine/2 mM L-malate, 10 mM succinate/1 μM rotenone or 10 mM succinate. In the experiments where the total free calcium was set to zero, 1 mM EGTA was added to chelate calcium contamination from reagents and the isolation process.

Mitochondrial respiration, membrane potential and volume dynamics were all quantified using the same experimental protocol timeline. Specifically, substrates were added to the buffer at the beginning of each experiment. Instrumental background signal was allowed to stabilize prior to mitochondria addition. Mitochondria were then added to the final concentration of 0.1 mg/mL. After respiration reached a steady state and achieved leak state (leak), a bolus of CaCl₂ was injected to achieve sodium/calcium (Na⁺/Ca²⁺) cycling state. After respiration stabilized, 1 mM ADP was added to achieve the oxidative phosphorylation respiratory state (oxphos). In classic nomenclature, leak and oxphos states are also known as states 2 and 3, respectively. In some instances, state 4 is used to describe the leak state; however, state 4 is leak state respiration in the presence of high ATP and low ADP concentrations. There is not an equivalence in the classic nomenclature for Na⁺/Ca²⁺ cycling state. Therefore, the descriptive nomenclature will be used in the remaining of this study for consistency and clarity. In high amplitude swelling and membrane potential experiments, 0.5 mM ADP used.

2.3. Oxygen Consumption Rates and Hydrogen Peroxide Emission Rates

Oxygen consumption rates (J_O2, nmol mg⁻¹ min⁻¹) and hydrogen peroxide emission rates (J_H2O2, pmol mg⁻¹ min⁻¹) were simultaneously measured using an Oroboros Oxygraph (O2k) system. J_H2O2was monitored using the Amplex UltraRed assay. Amplex UltraRed (AmpUR) was dissolved to a stock concentration of 10 mM according to the manufacturer’s instructions. Type II horseradish peroxidase (HrP) and superoxide dismutase (SOD) were individually dissolved to the stock concentration of 500 U/mL and stored at the appropriate temperatures. Hydrogen peroxide calibration curves were made using a working solution of 200 μM H₂O₂ prepared fresh on the day of every experiment. An enzyme mixture was added containing HrP/SOD to the final concentrations of 1 U/mL and 0.5 U/mL, respectively, followed by the addition of 10 μM Amplex Ultra Red. After the amperimetric signal reached a steady state, mitochondria, metabolites, and calcium were added as detailed below.

2.4. High Amplitude Swelling Assay

Mitochondrial high amplitude swelling was determined by a decrease in absorbance at 540 nm using the Olis DM-245 spectrofluorometer with dual-beam absorbance module. However, in the presence of sufficient calcium, calcium phosphate granules cause an increase in apparent absorbance caused by light scattering. Mitochondria were challenged with calcium boluses at concentrations designed to elicit moderate calcium overload or at supraphysiological concentrations high enough to trigger permeability transition. The maximum concentrations achieve the upper limits of moderate calcium overload in the J_O2 and J_H2O2 experiments are 20 μM for PC/M; 50 μM for P/M, S/R and S. They are referred to from here on as maximal experimental concentrations. Higher concentrations led to mitochondrial respiratory dysfunction. Permeability transition inducing concentrations are 100 μM for PC/M; 150 μM for P/M; 500 μM for S/R and S. These concentrations were sufficient to cause large amplitude swelling, a classic indicator for mitochondrial permeability transition.

2.5. Membrane Potential Measurement

Membrane potential was monitored by measuring the fluorescence of TMRM using the Olis DM-245 spectrofluorometer. Specifically, TMRM was diluted in 100% EtOH and added to a 2 mL buffer containing cuvette to the final concentration of 0.1 μM. TMRM accumulation into the mitochondrial matrix is a membrane potential driven process. At the concentration used in this study, matrix TMRM accumulation decreases fluorescence due to self-quenching. In these studies, the maximal experimental calcium concentrations were tested, as well as, calcium concentrations that trigger permeability transition. In addition, buffer with 1 mM EGTA was also included as a control. For calibration, nigericin (30 ng/mg mitochondria) was added for maximal membrane potential and alamethicin (50 μg/mg mitochondria) was added to completely depolarize the mitochondria.

2.6. Buffer Calcium Measurement

Buffer calcium was monitored by measuring the fluorescence of CaGreen-5N using the Olis DM-245 Spectrophotometer (excitation wavelength = 506 nm, emission wavelength = 531 nm). The final concentration of CaGreen-5N was 1 μM. After calcium addition, the calcium concentration in the buffer was measured in the steady-state. Then the maximal signal was achieved by adding 300 μM Cal₂. The minimal signal was achieved by adding 1 mM EGTA. The steady-state buffer calcium was calculated using Eq. 1, assuming a K_d value of 30 μM previously determined by our lab²⁴. The matrix calcium was then determined by subtracting the steady-state buffer calcium from the total calcium delivered by the boluses.

$${\left[C{a}^{2+}\right]}_{buffer}=K_d\frac{F-F_{\min }}{F_{\max }-F}$$ Eq. 1

2.7. Data Analysis and Statistical Testing

Data are presented as mean +/− standard deviation with individual data points included. Data were checked and confirmed for normality using the Shapiro-Wilk test. The effects of calcium on J_O2, J_H2O2, membrane potential and volume were tested using the anoval function in Matlab. The effects of calcium boluses and substrates on calcium uptake were tested using the anovan function. A posthoc Tukey’s range test was used to confirm any statistical significance among groups. At least 3 biological replicates were performed for each condition. A post-hoc power analysis was performed on the G*Power software^{27, 28} using the most conservative parameters of sample size, effect size, alpha = 0.05 and power = 0.80. The power analysis confirmed that the study (sample size, alpha value, effect size) is well-designed to detect statistical significance.

The p values associated with statistical significance indicated in the figures are reported either in the figure legend or in separate tables when there are more than a few values to report. However, due to a very large number of possible combinations, a full table of p values are reported separately in the Supporting Material.

3. Results

As shown in Fig. 1, the respiratory dynamic profile for each substrate tested is unique. After the addition of calcium, the respiratory rate generally increased above the leak state rate. This increase in respiration is more pronounced when S/R or S were the substrates. Moreover, when the substrates are P/M or S/R, ADP addition caused the oxygen consumption rate (J_O2) to rapidly rise and reach a plateau regardless of calcium levels. This plateau is not observed when the substrate is PC/M in the presence of calcium or succinate alone with or without calcium. With P/M, the oxphos J_O2 monotonically decreased as a function of calcium bolus; however, calcium had little effect on oxphos J_O2 with S/R. Mitochondria respiring on PC/M displayed the most significant decrease in oxphos J_O2 by calcium. Oxygen consumption rates are essentially zero in experiments where 25 μM or 50 μM CaCl₂ were added to mitochondria respiring on PC/M. Therefore, calcium treatments were lowered to 10 μM and 20 μM in place of 25 μM and 50 μM total CaCl₂ for the PC/M group, respectively. When succinate was the sole substrate, the addition of ADP resulted in a jump in respiration followed by a decrease to a new steady-state respiratory rate. This steady-state respiratory rate is highest when there is no calcium and decreases as a function of calcium.

Figure 1. Calcium inhibits respiration for P/M, PC/M, and S but not S/R fueled mitochondria.

Mitochondria (0.1mg/mL) respiring on different substrates show distinct respiratory profile dynamics. Following mitochondria addition, J_O2 was allowed to stabilize. Then, a bolus of calcium was added to calcium-challenged groups (P/M, S/R and S: 25 μM and 50 μM CaCl₂; PC/M: 10 μM and 20 μM CaCl₂). In experiments where the total buffer Ca²⁺ was zero, 1 mM EGTA was added to remove trace amount of Ca²⁺ in the buffers. After stabilization, 1 mM ADP was added to stimulate maximal respiration. Leak state respiration is defined as the period between mitochondria addition and calcium bolus. Sodium/Calcium cycling state is between calcium bolus and ADP addition. Oxphos occurs after ADP addition. The sudden drop in J_O2 indicates anoxia. J_O2 of each respiratory state was averaged over 30 seconds, shown as the rectangular boxes on the figure. Boxes are color-matched with the respiratory states in subsequent figures.

Calcium uptake has variable effects on respiration depending on the fuel source and respiratory states (Fig. 2). During leak state (blue bars), the presence of 4 μM CaCl₂ resulted in higher J_O2 averages. Statistical significance was reached with all substrate combinations between 0 and 4 μM CaCl₂ condition in the leak state. This is simply due to the sufficiently high contaminating calcium levels capable of stimulating respiration when this measurement was taken. The addition of calcium (Na⁺/Ca²⁺ cycling, red bars) further increased calcium-stimulated respiration with primarily NADH-linked and Q-linked substrates (P/M, S/R or S). Respiration was either not affected or slightly inhibited by calcium in mitochondria respiring on mixed substrate (PC/M). These effects of calcium on Na⁺/Ca²⁺ cycling respiration were statistically significant when the substrates were P/M, S/R or S and statistically insignificant when PC/M was the substrate. During oxphos, a plateau was observed with the exception of PC/M and S. The maximal oxphos J_O2 in P/M containing buffer is achieved at ~ 450 nmol mg⁻¹ min⁻¹ when the total buffer calcium is approximately 4 μM. When the substrates are S/R, maximal oxphos J_O2 is achieved at ~ 350 nmol mg⁻¹ min⁻¹ at the total buffer calcium of essentially zero or 4 μM. In the absence of rotenone, the maximal succinate-supported J_O2 due to oxphos was ~ 270 nmol mg⁻¹ min⁻¹ at the buffer calcium of 0 μM. When the substrate was PC/M, maximal J_O2 was ~ 290 nmol mg⁻¹ min⁻¹. Therefore, mitochondrial respiratory capacity was maximal with primarily NADH-linked substrates. Oxphos J_O2 was generally inhibitied with increasing CaCl₂. While the inhibitory effect was observed only after calcium exceeded 4 μM in P/M group, no statistical significance exists when calcium concentrations were 0 and 25 μM. The inhibitory effect due to calcium was negligible for Q-linked substrate in the presence of rotenone. Mitochondria respiring on PC/M were exquisitely sensitive to calcium and showed a marked inhibition of oxphos J_O2 as the calcium concentration increased. Even at lower calcium boluses of 10 and 20 μM, the inhibitory effect of calcium on respiration was significant. At lower buffer sodium concentrations, similar effects of calcium on respiration are observed across all respiratory states (Fig. S1). Statistical significance was not achieved between different buffer sodium groups. Selected statistical results are reported in Table 1, and a comprehensive list of statistical results is given in Table S1.

Figure 2. Calcium affects both leak and oxphos state respiration rates.

Effects of calcium on respiration are substrate specific. Leak state (blue) respiration is enhanced in the presence of trace amount of buffer calcium (~ 4 μM) across all substrates. Mitochondria respiring on succinate with or without rotenone (C and D, respectively) had highest leak-state J_O2. Sodium/Calcium cycling state (red) respiration is enhanced in the presence of increasing concentrations of calcium boluses up to 50 μM. The exception is PC/M. Mitochondria respiring on PC/M showed depressed J_O2 as the calcium concentration in the bolus exceeds 10 μM. For oxphos (yellow) respiration rates, buffer calcium above 4 μM depresses J_O2 across all substrates. In the absence of calcium, P/M respiring mitochondria have a lower J_O2 compared to when calcium was ~ 4 μM. However, mitochondria respired on PC/M or S had maximal oxphos when trace amount of calcium was removed. With S/R, respiration was nearly identical between the 0 and 4 μM groups, and the inhibitory effect of calcium on oxphos respiration is negligible. Statistical significance (p<0.05) relative to 0 μM CaCl₂ is indicated by *.

Table 1.

Statistical Significance Between Respiratory Rates

Respiratory States	Leak		Sodium/Calcium Cycling		Oxphos
Substrates	calcium	p values	calcium	p values	calcium	p values
P/M	0 μM	2.00E-04	0 μM	---	0 μM	1.52E-02
	25 μM	---	25 μM	3.39E-02	25 μM	1.26E-03
	50 μM	---	50 μM	2.60E-02	50 μM	3.69E-06
PC/M	0 μM	9.00E-04	0 μM	---	0 μM	---
	10 μM	---	10 μM	---	10 μM	1.61E-07
	20 μM	---	20 μM	---	20 μM	3.78E-09
S/R	0 μM	4.02E-05	0 μM	1.03E-03	0 μM	---
	25 μM	---	25 μM	3.95E-09	25 μM	---
	50 μM	---	50 μM	3.78E-09	50 μM	2.64E-02
Succinate	0 μM	2.04E-05	0 μM	1.31E-03	0 μM	---
	25 μM	---	25 μM	6.84E-08	25 μM	9.99E-03
	50 μM	---	50 μM	4.02E-09	50 μM	9.78E-04

All values are compared to the 4 μM CaCl₂ group within each substrate. The p values not associated with statistical significance are indicated by ---. A complete list can be found in the Supporting Information, Table S1.

To investigate the respiratory dynamics when succinate was the sole substrate, follow up experiments with combinations of succinate, rotenone, and glutamate were performed. In the presence of rotenone, glutamate addition to mitochondria respiring on succinate neither increases nor decreases oxphos J_O2 regardless of calcium concentrations (Fig. 3A, 3C). This is expected since rotenone inhibits complex I, and glutamate can be used to supply complex I with NADH. When rotenone is absent, the addition of glutamate leads to a significant increase in oxphos J_O2 (Fig. 3B, 3D). This glutamate-stimulatory effect is consistent across all calcium groups and statistically significant for all calcium groups when compared to the respiratory rate in the absence of rotenone (Fig. 3 and Table S2). While glutamate leads to an increase in respiration regardless of calcium group, there is a moderate inhibitory effect of calcium.

Figure 3. Effects of glutamate addition on oxphos J_O2 during succinate-fueled respiration.

A-B) Representative respiratory dynamics of mitochondria respiring on succinate/rotenone/glutamate (A) and succinate/glutamate (B). The experiments were done in the same fashion as in Fig. 1. Approximately 1.5 minutes after ADP addition, glutamate was added to the final concentration of 5 mM in the respiratory chamber. C-D) Oxphos J_O2 was quantified before and after glutamate addition. Glutamate addition results in a significant increase in oxphos J_O2 when rotenone is absent. The increase in respiration caused by glutamate is consistent across all calcium concentrations. When rotenone is present, glutamate did not affect respiration as expected. Experiments were performed using at least 3 biological replicates. Asterisks (*) indicate statistical significance (p < 0.01) due to glutamate addition within the same calcium treatment group: 3E-3 for 0 μM CaCl₂, 2E-3 for 4 μM CaCl₂, 7E-3 for 25 μM CaCl₂ and 6E-3 for 50 μM CaCl₂.

The effect of calcium on the ETS downstream of Complex I was tested by measuring FCCP-stimulated respiration in the presence of increased calcium concentrations (Fig 4). Mitochondria respiring on P/M reached maximal J_O2 of ~ 785 ± 100 nmol mg⁻¹ min⁻¹ in the absence of calcium. Even at the lower calcium bolus of 25 μM, FCCP addition caused as significant drop P/M-supported respiration. This is due to mitochondrial permeability transition as shown below in Fig. 5A and also previously determined by Petronilli et al.²⁹. While the maximal succinate-supported J_O2 is also reached in the absence of calcium (~ 755 ± 21 nmol mg⁻¹ min⁻¹), calcium addition at both 25 and 50 μM minimally inhibited respiration. Results are reported with p values in Table S3. There are no significant differences between the low and high calcium treatment groups within the P/M or S/R group, respectively.

Figure 4. Calcium does not significantly affect the electron transport system downstream of Complex I.

Oxygen consumption rates were measured in a similar experimental course as described in Fig. 1 except that ADP was replaced with FCCP (1 μM). A-B) Representative oxygen consumption dynamics of P/M-supported respiration (A) and S/R-supported respiration (B). C) Quantified oxygen consumption rates. In the presence of 25 and 50 μM CaCl₂, P/M-supported respiration was abolished upon FCCP addition. However, S/R-supported respiration showed no such decrease in respiration after FCCP addition. Experiments were performed using at least 3 biological replicates. Error bars are standard deviations. Asterisks (*) indicate statistical significance (p < 0.01) between calcium treatments compared to 0 μM CaCl₂. P/M 8.16E-6 for 25μM CaCl₂ and 7.96E-6 for 50 μM CaCl₂. S/R: 1.66E-2 for 25 μM CaCl₂ and 3E-3 for 50 μM CaCl₂.

Figure 5. Moderate calcium overload does not trigger mitochondrial permeability transition.

Representative absorbance dynamics at 540 nm was normalized to the signal following alamethicin addition (A-D). After an initial signal stabilization, mitochondria were added, and the signal was allowed to stabilize for 5 minutes (leak state absorbance). At this point, calcium was added at the maximal experimental bolus concentrations or at the concentrations that would induce high amplitude swelling. Absorbance was monitored for 5 minutes, followed by FCCP addition. Finally, alamethicin was added to completely permeabilize mitochondria (minimal absorbance). The maximal experimental boluses contain 20 μM Ca²⁺ for PC/M and 50 μM for P/M, S/R and S. The calcium bolus containing 10 μM Ca²⁺ was included for PC/M. The amounts of calcium necessary to induce high amplitude swelling depend on the substrates: 100 μM Ca²⁺ for PC/M, 150 μM for P/M and 50 μM for S/R and S.

High amplitude swelling results in a rapid decrease in absorbance and is used as a gold-standard to monitor mitochondrial permeability transition. Representative absorbance dynamics at 540 nm show that absorbance either increased or did not change upon adding the highest calcium concentrations tested (20 μM CaCl₂ for PC/M group; 50 μM CaCl₂ for P/M, S/R and succinate groups) (Fig. 5A–D). Therefore, high amplitude swelling due to mitochondrial permeability transition does not occur under our highest experimental calcium concentrations. At significantly higher calcium concentrations, high amplitude swelling results in a rapid decrease in absorbance to the level that is unchanged upon FCCP addition (Fig. 4A–D). Interestingly, different calcium concentrations are necessary to induce high amplitude swelling in a substrate-dependent manner. The addition of 150 μM and 100 μM CaCl₂ induced high amplitude swelling in P/M and PC/M groups, respectively. A significantly higher calcium bolus of 500 μM was necessary to induce high amplitude swelling in S/R and S groups. The precise threshold for each substrate required to elicit mitochondrial permeability transition was not further investigated in this study. The relative absorbance (%) confirms that high amplitude swelling did not occur upon adding the maximal calcium boluses in our experiments (Fig. 6). A complete table of averaged values, standard deviations and p values are reported in the Supporting Material (Table S4).

Figure 6. Relative absorbance (%) following calcium addition.

The experimental course is as described in Fig. 4. The absorbance at 540 nm show that high amplitude swelling did not occur at the maximal experimental boluses. Statistical significance (p<0.01) relative to maximal experimental calcium concentrations is indicated by *. The p values are 1.22E-7 for P/M, 1.14E-8 for PC/M, 4.19E-6 for S/R and 3.58E-8 for S.

The substrate-specific trends in free radical emission (J_H2O2) are consistent with respiratory profiles (Fig. 7). The greatest spread among data points is associated with succinate. This is attributed to the variable nature of reverse electron transport. Hydrogen peroxide emission rates are highest during leak (blue) and lowest during oxphos (yellow). The Na⁺/Ca²⁺ cycling hydrogen peroxide emission rates (red) are in the middle range. Leak state J_H2O2 was highest in the S group, followed by the S/R group, and the leak state J_H2O2 was comparable among P/M and PC/M groups. While the presence of ~ 4 μM free calcium resulted in a slight decrease in J_H2O2 in these two groups, the differences were not statistically significant. The Na⁺/Ca²⁺ cycling J_H2O2 decreased as calcium concentrations were increased regardless of the substrates. During Na⁺/Ca²⁺ cycling state, the order of decreasing J_H2O2 is S > S/R > P/M « PC/M. Note that the comparison between P/M and PC/M groups is based only on the order of magnitude in J_H2O2 since calcium treatments were lowered in PC/M group. Although the differences in J_H2O2 between S and other substrate groups were significant during leak and Na⁺/Ca²⁺ cycling, oxphos J_H2O2 values were on the same order of magnitude across substrate groups. Overall, oxphos resulted in further decreases in J_H2O2 with overall no appreciable differences due to calcium across substrates except for S/R group. A complete list of averaged values, standard deviations and p values are reported in Table S5.

Figure 7. Net hydrogen peroxide emission rates are inhibited by calcium.

The reported J_H2O2 measurements were obtained simultaneously as J_O2 reported in Figs. 1 and 2. Overall, the S group is associated with the greatest variation among data points. Leak state (blue) J_H2O2 was highest across respiratory states, regardless of the substrates. Of these rates, group S had the highest J_H2O2. The presence of rotenone decreased J_H2O2 by an order of magnitude. P/M and PC/M respiring mitochondria had similar and the lowest leak state J_H2O2 levels. The presence of free calcium at ~ 4 μM did not affect J_H2O2 to a statistically significant level. Sodium/Calcium ycling state (red) J_H2O2 is depressed in the presence of increasing calcium concentrations although the amount of calcium to achieve statistical significance depends on the substrates. Oxphos (yellow) J_H2O2 was lowest across respiratory states, regardless of the substrates. Calcium does not alter oxphos J_H2O2 with the exception of PC/M where 20 μM calcium bolus resulted in a J_H2O2 that is highest in this group. Statistical significance (p<0.05) relative to 4 μM CaCl₂ is indicated by *. Leak state: 2.00E-4 (S). Na⁺/Ca²⁺ cycling state: 3.44E-6 (PM, 0 μM CaCl₂), 4.74E-3 (P/M, 25 μM CaCl₂), 5.35E-5 (P/M, 50 μM CaCl₂), 5.63E-4 (PC/M, 20 μM CaCl₂), 7.29E-4 (S/R, 25 μM CaCl₂), 2.40E-6 (50 μM CaCl₂). Oxphos state. 3.8E-2 (S/R, 0 μM CaCl₂), 6.34E-3 (S/R, 25 μM CaCl₂), 1.34E-5 (S/R, 50 μM CaCl₂).

In addition to the swelling data (Figs. 5 and 6), representative membrane potential dynamics (Fig. 8) and relative membrane potential (Fig. 9) exclude the hypothesis that irreversible mitochondrial permeability transition causes calcium-induced mitochondrial dysfunction in this study. Compared to leak state membrane potential, mitochondria were able to maintain a membrane potential after calcium had been added at the maximal calcium concentrations tested. These concentrations are 20 μM for PC/M and 50 μM for P/M, S/R and S. In contrast, the calcium concentrations that resulted in high amplitude swelling (Figs. 5 and 6) caused a complete loss of membrane potential as shown in Fig. 8. At the highest experimental calcium concentrations (20 μM for PC/M and 50 μM for P/M, S/R and S), ADP addition caused a further decrease in membrane potential. These results are further evidence that mitochondria loaded with these levels of calcium remained well-coupled and were able to utilize the proton motive force to synthesize ATP. When ADP was added after excessive calcium boluses (150 μM for P/M, 100 μM for PC/M, and 500 μM for S/R and S), no further drop in membrane potential was observed. This shows that these calcium concentrations caused complete membrane depolarization. The relative membrane potential values due to respiratory state and calcium are summarized in Fig. 9. Complete list of averages and p values are reported in Table S6. Together, results from the high-amplitude swelling and membrane potential assays exclude that irreversible mitochondrial permeability transition occurred at the concentrations of calcium used to test the effect of calcium on respiration and hydrogen peroxide emission rates.

Figure 8. Mitochondria remain energized during moderate calcium overload.

Representative membrane potential dynamics monitored using TMRM. The presence of trace amount of calcium caused a slight depression in leak state membrane potential when P/M or PC/M were the substrates (A and B, respectively). The slight depression due to trace amount of calcium was not observed when the substrate was succinate with or without rotenone (C and D, respectively). In all cases, the maximal calcium boluses used to test the effect of calcium on respiration and hydrogen peroxide emission rates did not result in complete depolarization. Specifically, mitochondria were able to maintain a membrane potential following 50 μM CaCl₂ bolus when they respired on P/M (A). In the presence of succinate with or without rotenone, the membrane potential was much higher compared to P/M and PC/M (C and D, respectively). Mitochondria respiring on PC/M appeared to be able to maintain a membrane potential following a 20 μM CaCl₂ bolus although the decrease in membrane potential did not reach a steady state (B). Depolarizing calcium concentrations are significantly higher than the maximal experimental calcium concentrations and are substrate specific: P/M + 150 μM CaCl₂, PC/M + 100 μM CaCl₂, S/R + 500 μM CaCl₂, Succinate + 500 μM CaCl₂. The maximal experimental calcium concentrations for J_O2 and J_H2O2 measurements do not inhibit oxphos as membrane potential is further decreased upon ADP addition. However, the extent of decrease depends on the substrates. No further decrease in membrane potential upon ADP addition was observed at depolarizing calcium concentrations, confirming that mitochondria have been depolarized prior to ADP addition.

Figure 9. Mitochondrial membrane potential is affected by calcium loading.

Relative membrane potential (%) obtained by dividing the averaged fluorescence over the fluorescence range. Mitochondria respired on succinate in the presence and absence of rotenone (G and H, respectively) were able to maintain a higher partial membrane potential compared to those respire on pyruvate/L-malate (E) after a 50 μM CaCl₂ bolus had been added. The ability of mitochondria respiring on palmitoylcarnitine/L-malate to maintain a membrane potential was compromised by calcium in a concentration-dependent manner (B). In all cases, the maximal experimental calcium boluses did not cause complete loss of membrane potential. The depolarizing calcium concentrations were significantly higher and resulted in a membrane potential that could not be further decreased by ADP. * indicates statistical significance between relative membrane potential due to maximal experimental calcium and depolarizing calcium concentrations. Sodium/Calcium cycling: 1.45E-7 for P/M, 5.94E-4 for PC/M, 1.61E-8 for S/R, and 7.26E-9 for S. Oxphos: 5.82E-5for P/M, 6.16E-3 for PC/M, 5.37E-8 for S/R, and 1.37E-6 for S.

Buffer calcium dynamics was monitored using CaGreen-5N. The representative calcium dynamics resemble the representative oxygen consumption dynamics (Fig. 10A–D). Steady-state buffer calcium levels after calcium injection for both low and high calcium treated groups are the same as when no additional calcium was added (low calcium: 10 μM for PC/M, 25 μM for P/M, S and S/R; high calcium: 20 μM for PC/M, 50 μM for P/M, S and S/R). Regardless of substrate or calcium bolus addition, mitochondria set the buffer calcium concentration to approximately 3-4 μM. This set point is a direct result of the calcium-dependence on the rate of calcium uptake by the calcium uniporter and the relative constant calcium efflux rate as described in detail by Chalmers and Nicholls³⁰ and further quantitatively characterized by Bazil et al.^{24, 31}. These data confirm that isolated mitochondria can sequester large amounts of exogenous calcium without triggering mitochondrial permeability transition. The steady-state buffer calcium concentrations are quantified using the relationship between buffer calcium and fluorescence intensity, as given in Eq. 1 (Fig. 10E). Matrix calcium content was then obtained by subtracting the steady-state buffer calcium from the total calcium taken up by mitochondria and quantified with respect to the mitochondrial concentration in the assay conditions (Fig. 10F).

Figure 10. Buffer and steady-state matrix calcium.

A-D) Representative fluorescence dynamics using CaGreen-5N. A) Pyruvate/L-malate (P/M). B) Palmitoylcarnitine/L-malate (PC/M). C) Succinate/Rotenone (S/R). D) Succinate (S). The steady-state buffer calcium levels in the low calcium bolus are similar to those delivered by the maximal calcium bolus. Mitochondria take up calcium from the maximal bolus without being permeabilized. The steady-state concentration of buffer calcium was quantified from the fluorescence signal averaged over shaded region where indicated. E) Steady-state buffer calcium 5 minutes (2.5 minutes for S and S/R) following calcium bolus injection was determined using Eq. 1. Regardless of the calcium bolus, the averaged steady-state buffer calcium concentration was approximately the same within a substrate and across substrate groups. F) Mitochondrial calcium content at the corresponding 5-minute mark (2.5 minutes for S and S/R) was determined by subtracting the remaining steady-state calcium concentration in the buffer from the initial calcium concentration plus the bolus addition. The total mitochondrial calcium content was calculated based on a mitochondrial concentration of 0.1 mg/ml. No statistical difference exists between P/M, S/R and S group. Comparisons cannot be made with the PC/M group since the calcium boluses deliver much lower calcium at 10 and 20 μM CaCl₂.

The effects of calcium on J_H2O2 over a range of [O₂] were monitored at 5 minutes and 10 minutes (Fig. 11). There is a monotonic relationship between J_H2O2 and [O₂], and calcium decreased the J_H2O2 at all [O₂]. At 5 minutes, no differences in J_H2O2 were observed whether the initial buffer calcium was 0 or 4 μM regardless of the substrates (Fig. 11A–C). At 10 minutes, the presence of as much as 4 μM Ca²⁺ resulted in a downward shift of the curves when P/M and PC/M were the substrates (Fig. 11D, E). The relationship between J_H2O2 and [O₂] was preserved at 10 minutes when the substrates were S/R (Fig. 11F).

Figure 11. Hydrogen peroxide emission rates are dependent on oxygen concentration.

A-C) Measurements taken at 5 minutes showed no differences in J_H2O2 between 0 and 4 μM CaCl₂. The best-fit lines were produced using nonlinear coefficients assuming the empirical model of steady-state J_H2O2 where J_H2O2 = V_max [O₂]/(K_m + [O₂]). D-F) Measurements taken at 10 minutes showed substrate-dependent effects of calcium on J_H2O2 over a range of [O₂]. The presence of CaCl₂ ≥ 4 μM (for P/M) or CaCl₂ ≥ 10 μM (PC/M) depresses J_H2O2; however, calcium does not affect J_H2O2 for S/R. The same values of V_max and K_m from the fitting procedure at 5 minutes were used to construct the best-fit line at 10 minutes when calcium does not affect the curves. The V_max values are 175.7 (P/M), 174.1 (PC/M) and 353.1 (S/R). The K_m values are 120.7 (P/M), 102.8 (PC/M) and 92.8 (S/R). When present, the effect of calcium on the nonlinearity relationship was accounted for by redetermining V_max and K_m. The Vmax values are 107.1 (P/M), 137 (PC/M) and 353.1 (S/R). Km values are 106 (P/M), 102.5 (PC/M) and 92.8 (S/R).

We next assessed the effects of electron turn-over on free radical homeostasis with P/M, PC/M and S/R. It has been argued that increased electron turn-over can increase the rate of superoxide formation. If true, there should be a non-zero correlation between J_H2O2 and J_O2. However, we found that J_H2O2 and J_O2 are essentially independent as shown on Fig. 12. Data from Fig. 2 and 7 for the 0 and 4 μM calcium conditions were combined and plotted together as shown.

Figure 12.

Hydrogen peroxide emission rate is independent of oxygen consumption rate. The slopes of J_H2O2 versus J_O2 are −2.8227 (−4.715, −0.9299) for P/M, 0.3428 (−1.276, 1.962) for PC/M and 0.0736 (−0.5598, 0.707) for S/R. The y-intercepts are 204.1453 (139.7, 268.6) for P/M, 92.0105 (37.45, 146.6) for PC/M and 69.6132 (151.4, 298.9) for S/R. Values in parenthesis represent 95 % confidence intervals. These slopes indicate negligible dependency, if any, between J_H2O2 and J_O2. Filled circles represent individual data points. Colors are for different substrates: blue for P/M, red for PC/M and yellow for S/R.

4. Discussion

Mitochondrial respiration

4.1. Mitochondrial respiration depends on the fuel source.

Overall, these data confirm that the absolute J_O2 rates are dependent on the fuel sources. Succinate in the presence or absence of rotenone results in the greatest J_O2 for leak and Na⁺/Ca²⁺ cycling states. As a Q-linked substrate, succinate donates electrons to the ETS at complex II and bypass complex I. Electrons from pyruvate enter the ETS at complex I via NADH. Under our experimental conditions, pyruvate is not fully metabolized, so the number of electrons entering complex II is low^25,26. Those from palmitoylcarnitine enter the ETS at both complex I and II. Since complex I is an energy-conserving site by the virtue of its proton pumping activity, a loss of protons per pair of electrons is inevitable when electrons enter the ETS downstream of complex I. Therefore, the maintenance of the membrane potential when mitochondria respiring on Q-linked substrates (succinate ± rotenone in this study) results in an increased electron turnover (reflected as JO₂) to maintain the membrane potential. Moreover, since complex II does not pump protons and complexes III and IV are capable of supporting a higher membrane potential than complex I, higher membrane potentials and thus leak rates are maintained with substrates that bypass complex I.

While the primary Q-linked substrates yield the highest leak- and Na⁺/Ca²⁺ cycling J_O2, they had the lowest oxphos J_O2 rates compared to the primary NADH-linked substrate P/M. This is most likely due to the accumulation of inhibitory levels of oxaloacetate (OAA) and succinate transport limitations. OAA is a potent inhibitor of complex II³². It has a high binding affinity for complex II (K_d ~ 10⁻⁸ M), and the OAA-CII complex has a slow rate of dissociation (k_off ~ 10⁻² min⁻¹)³³. Under physiological TCA cycle turnover conditions, OAA is converted to either citrate or aspartate. The former reaction requires acetyl-coA from pyruvate or fatty acids, and the latter requires glutamate. Because neither acetyl-coA nor glutamate is added in the succinate-supported groups, inhibitory levels of OAA accumulate in the matrix. The relatively low activity of the dicarboxylate carrier has also been demonstrated as another limiting factor on the maximum respiratory capacity of succinate fueled mitochondria¹. Therefore, succinate cannot support high oxphos-state respiration rates compared to P/M.

Leak-state J_O2 is essentially identical whether the substrate is PC/M or P/M. During Na⁺/Ca²⁺ cycling state, J_O2 is smaller when PC/M is the substrate. Respiration was essentially not affected by calcium up to 10 μM CaCl₂ bolus. Beyond this level respiration was compromised (Figs. 1B and 2B). From a bioenergetic perspective, palmitoylcarnitine yields significantly more reducing equivalents per molecule compared to pyruvate. Each pyruvate completely going through the TCA cycle as acetyl-coA yields 3 NADH and 1 QH₂. A palmitoylcarnitine molecule is completely oxidized after 8 cycles of β-oxidation, each of which yields 4 NADH and 2 QH₂ per acetyl-CoA. Therefore, the complete turn-over of a palmitoylcarnitine yields a total of 32 NADH and 16 QH₂ molecules. Three consequences result from this higher yield of reducing equivalents by PC. First, a slower oxidation rate of PC is sufficient to maintain the membrane potential. Second, excess reducing equivalents not utilized by the ETS can inhibit β-oxidation enzymes³⁴. Third, substrate competition among β-oxidation enzymes leads to accumulation of acyl-CoA intermediates³⁵. These culminate in a plausible mechanism explaining the data shown in Figs. 1 and 2 that calcium above 4 μM inhibits complex I-supported respiration, leading to higher levels of NADH and lower PC/M-dependent respiration. It is also possible that uncoupling by fatty acid contributes to the lower PC/M dependent respiration. As uncoupling enables more fatty acids to enter the matrix, it leads to greater amounts of acyl-CoA intermediates that impedes β-oxidation enzymes. The uncoupling nature of fatty acids is distinct from uncoupling mediated by the ATP/ADP nucleotide translocase and has been extensively studied^36–44 . On the other hand, whether calcium directly regulates β-oxidation enzymes remains unknown. Thus, it is a potential mechanism that can also explain our data.

While calcium addition dissipates membrane potential and results in enhanced respiration, increasing calcium concentrations inhibits respiration during oxphos (Fig. 2). The inhibition by calcium is also substrate-dependent. When the substrate was P/M, respiratory depression was observed at calcium levels above 4 μM. With PC/M and S, respiration during oxphos was depressed in the presence of as much as 4 μM CaCl₂. Interesting, when respiration was supported by S/R, the depressive effect of calcium on respiration was not seen. To validate the potential inhibitory effect of calcium on the ETS downstream of complex I, we measured J_O2 in the presence of 1 μM FCCP. We found that FCCP slightly depressed the maximum succinate-supported J_O2 in the presence of 25 and 50 μM CaCl₂ compared to control (0 μM CaCl₂). However, when mitochondria respired on P/M, adding FCCP to the calcium-loaded mitochondria completely abolished oxygen consumption rates. These results support a prior study which found that high calcium primarily inhibits complex I-dependent respiration and minimally affects downstream ETS components²⁴.

4.2. Oxaloacetate accumulation depresses maximal succinate-supported respiration.

Current evidence suggests that OAA accumulation is responsible for the discrepancies in oxphos J_O2 between S and S/R groups after ADP addition. In the absence of rotenone, a higher level of OAA accumulates and inhibits complex II³². This causes a decrease in respiration as shown in Figs. 1D and 2D. In this condition, complex I maintains a low NADH/NAD⁺ ratio and facilitates even more OAA accumulation with the assumption that malate dehydrogenase is near equilibrium. Lowering OAA levels, therefore, by adding acetyl-CoA or glutamate will relieve this inhibitory effect of OAA on oxphos J_O2 of the S group. In a similar fashion, with rotenone present, complex I is inhibited and higher NADH levels necessarily decrease OAA levels via malate dehydrogenase reaction near equilibrium. The key reactions leading to these results are given in a pathway diagram in the Supporting Material (Fig. S8). This effect of rotenone on NADH levels in mitochondria respiring on succinate has been previously shown by Aldakkak et al.⁴⁵ Our data show that glutamate addition led to a significant respiratory increase at each calcium concentration (Fig. 3). A similar result has also been recently reported by Fink et al.⁴⁶ For the lower calcium boluses, the increase in respiration can be explained by a combination of NADH production by glutamate metabolism in addition to a decrease in the OAA concentration. However, at the highest calcium bolus, the contribution to respiration by glutamate metabolism is essential zero as shown in Fig. S7. Specifically, there is an approximately 96% drop in respiration on glutamate between the 0 and 50 μM calcium bolus. In absolute terms, this is a change from ~ 362 to ~ 15 nmol mg⁻¹ min⁻¹. However, there is only a 11 % drop in respiration in the S/G group between the 0 and 50 μM calcium bolus, or in absolute terms, a change from ~ 578 to ~ 470 nmol/mg/min. Therefore, glutamate does not contribute to complex I activity in this calcium condition. This leaves only the decrease OAA concentration as the viable mechanism that can explain the significant increase respiration rates after glutamate addition.

4.3. Respiratory changes are driven by membrane potential.

While the calcium concentrations in this study are in the ischemic range, they are above the range that stimulates maximal metabolism and below those triggering mitochondrial permeability transition (Figs. 1, 2, 5 and 6). At low mitochondrial micromolar concentrations, calcium is a known activator of FAD-glycerolphosphate dehydrogenase, pyruvate dehydrogenase phosphatase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase⁴⁷. Given the mitochondrial concentration of 0.1 mg/mL in the respiratory chambers, the calcium per mitochondria in this study was well above the saturating levels⁴⁸. In addition, after bulk calcium uptake by the mitochondria, the steady-state buffer free calcium is in the 3-4 μM range when the calcium added is less than or equal to 50 μM (Fig. 10). Therefore, the respiratory increase in the presence of calcium above 4 μM is not due to the stimulatory effect of calcium on matrix dehydrogenases. The increase in respiration induced by calcium addition is rather a membrane potential driven process. As calcium entering and leaving the matrix in a steady-state fashion dissipates membrane potential, mitochondria turn over more oxygen to re-establish the membrane potential. This results in an increase of the respiratory rates following calcium addition. The addition of ADP further drops the membrane potential and stimulates respiration. These results are consistent with classic bioenergetic principles.

Hydrogen Peroxide Emission Rates

4.4. Increased calcium concentrations do not increase ROS emission rates.

Our results using isolated cardiac mitochondria show that increasing calcium concentrations either does not affect or has an inhibitory effect on J_H2O2. Our observation is consistent with those reported by several other groups^{49, 50}. However, there are studies suggesting that calcium enhances ROS generation^9,22,51. Therefore, it is important to be aware that the effects of calcium on ROS homeostasis are still highly debatable as many lines of opposing evidence exist. Discrepancies are likely to arise from differences in experimental designs including tissue choice, developmental stage, treatment and detection system. For example, the calcium concentrations employed in our study are in a similar range to those in Starkov et al. and Komary et al.^49,50. Our results are consistent with these studies. However, in one study where calcium is suggested to enhanced ROS, the calcium concentrations used are much higher ranging between 300 and 500 μM CaCl₂⁵¹. As our study demonstrate that these calcium levels permeabilize mitochondria, the net ROS increase in these studies is likely due to the scavenging system being compromised and/or the release of non-neutralized matrix ROS. Another difference is that mitochondria in the calcium-enhanced ROS experiment are de-energized while ours, as well as those in Starkov et al. and Komary et al. are energized with different substrates^{49, 50}. That experimental design can influence results; therefore, interpretations must be made within the context of the experiment. It is also necessary to be mindful of what can and cannot be generalized from the results. Our study does not perturb the mitochondrial metabolic network with inhibitors or supraphysiological concentrations of calcium. Thus, it serves a solid foundation from which to build from regarding the complex relationship between calcium and mitochondrial ROS homeostasis.

4.5. Net hydrogen peroxide emission rates are driven by membrane potential.

During leak state, membrane potentials and net hydrogen peroxide emission rates are maximal across substrates (Figs. 7–9). Calcium addition partially depolarizes membrane potential and slightly decreases J_H2O2. During oxidative phosphorylation, the membrane potential was further decreased, followed by a corresponding decrease in J_H2O2. Data from the S and S/R groups clearly demonstrate the causal effect of membrane potential on J_H2O2. When succinate was the substrate, calcium overload dissipates membrane potential by a smaller degree compared to P/M and PC/M groups. Therefore, the J_H2O2 levels are higher and consistent with this observation. During oxidative phosphorylation, all substrate groups have similar membrane potential. As a result, the J_H2O2 are similar.

Although a definitive conclusion cannot be reached that calcium decreases ROS production since the AmpUR assay detects the net ROS emission. However, it is very likely that calcium lowers ROS production by decreasing membrane potential. The membrane potential dependence of net ROS production was first appreciated by Skulachev³² and subsequently demonstrated by independent works by Liu⁵³ and Korshunov et al.⁵⁴ Liu showed this relationship in state 4 respiration by titrating malonate (0-5 mM) to succinate-respiring mitochondria from rat heart. In addition to malonate titration, Korshunov and colleagues manipulated membrane potential by adding ADP + P_i or SF6847 to rat heart mitochondria pre-treated with H₂O₂ to deplete antioxidants. Several independent studies performed in later years also yield results that support this concept^55–58. Our group has reached the same conclusion by titrating different calcium boluses and triggering oxidative phosphorylation to alter membrane potential. We found that the membrane potential dependence of ROS homeostasis is also true in mitochondria not depleted of antioxidants and irrespective of the substrate sources. Therefore, our study serves as another independent line of evidence that supports the monotonic relationship between net ROS homeostasis and the membrane potential component of the protonmotive force.

4.6. Electron flux does not appreciably influence hydrogen peroxide emission rates.

As electrons derived from succinate bypass complex I, increased electron turn-over downstream of complex I is necessary to maintain the membrane potential. Because H₂O₂ is formed by a redox reaction between O₂ and a redox center, increased electron turnover has been linked to proportional increase in J_H2O2. However, when J_H2O2 is plotted against J_O2, the slopes are essentially zero (Fig. 12). Therefore, our results suggest that [O₂] is a more influential determinant in the net hydrogen peroxide formation in healthy mitochondria.

4.7. Hydrogen peroxide emission rates are non-linearly dependent on oxygen concentration.

The hydrogen peroxide emission rate is a non-linear function of [O₂] regardless of the substrates (Fig. 10). The nonlinear relationship between J_H2O2 and [O₂] has been previously reported^59,60. However, J_H2O2 reaches the maximum values at higher O₂ concentrations in our study. The contribution of our study is that we also determined how calcium affects this relationship. We further proposed an empirical model to relate the effects of oxygen, substrate and calcium on free radical homeostasis. Specifically, our empirical model relates the fluorescence of resorufin to the steady-state matrix hydrogen peroxide (Fig. 13). Mathematical derivation of steady-state matrix hydrogen peroxide further relates the free radical production, elimination and transportation processes together. This derivation is described below.

Figure 13. Schematic of resorufin formation.

The schematic relates the resorufin formation to hydrogen peroxide production, elimination and transportation. The term E_R represents mitochondrial sites available for oxygen reduction. The terms k_p, k_e, and k_t are described in the text.

Because AmpUR and HrP/SOD are used in excess amounts, hydrogen peroxide emitted by mitochondria is immediately converted to resorufin. Therefore, the change in resorufin concentration detected fluorometrically corresponds directly to rate of hydrogen peroxide emission (Eq 2).

$$\frac{\text{d}\left[\mathrm{resorufin}\right]}{\mathrm{dt}}=-\left(\frac{\text{d}\left[{\text{H}}_2{\text{O}}_2\right]}{\mathrm{dt}}\right)\mathrm{buffer}$$ Eq. 2

Buffer hydrogen peroxide is proportional to the level of matrix hydrogen peroxide ([H₂O₂]_m) and the rate of hydrogen peroxide transport (k_t) across the mitochondrial membranes (Eq 3).

$$\left(\frac{\text{d}\left[{\text{H}}_2{\text{O}}_2\right]}{\mathrm{dt}}\right)\mathrm{buffer}={\text{k}}_{\text{t}}{\left[{\text{H}}_2{\text{O}}_2\right]}_{\mathrm{matrix}}$$ Eq. 3

In turn, the steady-state matrix hydrogen peroxide concentration is dependent on the total capacity of electron donors (E_R) and three primary processes including hydrogen peroxide production, elimination and transport across the mitochondrial membranes (Eq 4). The rates of these individual factors are denoted k_p, k_e and k_t, respectively.

$${\left[{\text{H}}_2{\text{O}}_2\right]}_{\mathrm{matrix}}=\frac{{\text{E}}_{\text{R}}\left[{\text{O}}_2\right]}{\frac{{\text{k}}_{\text{e}}+{\text{k}}_{\text{t}}}{{\text{k}}_{\text{p}}}+\left[{\text{o}}_2\right]}$$ Eq. 4

The scavenging system comprises of the Mn-SOD, peroxiredoxin (Prx) and glutathione peroxidase (GPX). However, because hydrogen peroxide is the ROS species detected using the HrP assay (as opposed to superoxide), the Mn-SOD reaction becomes part of the production rather than elimination processes. The rate of hydrogen peroxide production (k_p) is dependent on the superoxide concentration (substrate), which in turn is dependent on the [O₂] and the redox state of an electron donor. Although not explicitly described by Eq. 4, the redox state of an electron donor is dependent on respiratory states and therefore the membrane potential. For the sake of clarity, the reduction potential of the electron donor refers to its tendency to be reduced. Given this definition, a donor’s reduction potential is most positive during oxphos, most negative during leak state and somewhere in between during Na⁺/Ca²⁺ cycling state. Therefore, the hydrogen peroxide production rates are expected to be lowest during oxphos, highest during leak state and somewhere in between during Na⁺/Ca²⁺ cycling state. And this is exactly what our data show.

The empirical model resembles the Michaelis-Menten relationship. Here, the apparent V_max is the product of total enzyme reducing capacity (E_R) and the rate of H₂O₂ transportation across the mitochondrial membranes (k_t). The term K_m is the ratio of the sum of hydrogen peroxide elimination and transport over the production rates (k_e, k_t and k_p, respectively). The rate of hydrogen peroxide transport across the mitochondrial membranes has been determined to approximate that of water^61,62, so it is likely that different V_max values are determined by E_R. As different substrates produce different number of reducing equivalents that donate electrons to the ETS at various points, the total capacity reducing sites is substrate dependent. Our experimental data suggest that E_R is highest with Q-linked substrate (succinate ± rotenone) and comparable between NADH-linked and mixed substrates. Assuming that k_t is independent of substrates and calcium, K_m relates k_p to k_e terms. Our data suggest that free radical elimination (k_e) is unable to balance free radical production (k_p) when the substrate is Q-linked, which results in a smaller K_m for this substrate. However, an additional equation is necessary to quantitatively determined k_p and k_e. It is known that ROS production and elimination are both modulated by the mitochondrial matrix NADH/NAD⁺ poise although they appear as opposing factors in determining net ROS^63,64. This concept is recapitulated as Redox Optimized ROS Balance wherein mitochondria ROS emission is minimized when the organelles operate somewhere between the extremities of complete oxidation and reduction⁶³. The unique aspect of the measurements shown in Fig. 11 is that they lead to an empirical model enabling insight into processes upstream of the final fluorescence measurements using the AmpUR assay. To our best knowledge, we are also the first to comprehensively investigate the effect of calcium on this Michaelis-Menten like relationship between J_H2O2 and [O₂].

4.8. Strengths and Limitations of Study

Our primary goal of this study was to comprehensively determine how substrates and calcium affect mitochondrial respiration and net ROS production rates in healthy mitochondria. As such, we performed experiments using mitochondria isolated from the ventricular cardiomyocytes of guinea pigs in the absence of injury or disease. Among rodents, guinea pigs are the animals to use in studies related to human cardiovascular diseases. The cardiomyocyte of guinea pigs shares many similarities with that of humans compared to rats and mice. Some of the most important features are the plateau phase of action potential⁶⁵, calcium handling^66–70, purine nucleotide metabolism⁷¹, complement IF1^72,73 and expression of the β-myosin heavy chain isoform^69,74. Therefore, our results will be particularly useful as a reference for future studies of cardiovascular diseases in human that use guinea pigs as the animal model.

To delineate the capacity of mitochondrial calcium handling, we utilized calcium boluses in the ischemic range but do not trigger irreversible mitochondrial permeability transition. Using a healthy model, various substrates and sub-permeability transition calcium levels enables us to define the full capacity of mitochondria in neutralizing ROS. We demonstrate that mitochondria not only are capable of taking up a large amount of calcium but do so in a substrate-dependent manner. Specifically, mitochondria respiring on PC/M have a significantly lower ability to take up calcium. We also provide evidence to support that high calcium inhibits the ETS primarily at complex I. In addition to characterizing how ROS homeostasis and respiratory dynamics response to calcium, our study serves as an additional, independent line of evidence that net ROS production depends on membrane potential. In this study, membrane potential is altered as calcium enters the mitochondrial matrix and ADP stimulates oxidative phosphorylation. In the other studies, membrane potential was manipulated by adding different amounts of inhibitors or malonate. That we used a different system and approach to alter the membrane potential yet arrive at the same conclusion irrefutably supports this relationship

Finally, we contribute an empirical model that relates oxygen, substrates and calcium to the fluorescence measurement. To our best knowledge, previous studies attempting to link upstream processes to the net hydrogen peroxide measurement did not cover the range of calcium. However, in developing the empirical model, we realized that a more detailed, sophisticated model is necessary to further determine not only the relative contribution of individual ROS producers and scavengers but also how they operate under these conditions. In fact, the precise mechanism responsible for mitochondrial free radical homeostasis in the healthy state remains an important standing scientific question. In studies that utilize the AmpUR assay such as ours, H₂O₂ is used as a surrogate for O₂^·−. While the longer half-life of H₂O₂ improves reproducibility for quantitative measurements, it is important to note that the fluorescence obtained is a net measurement. Thus, the measurement itself is not informative of H₂O₂ formation or H₂O₂ elimination, only the net production of H₂O₂. As such, the contributions of individual mitochondrial enzyme that produce or consume ROS cannot be determined using this approach. Traditionally, site-specific inhibitors are used to quantify site-specific ROS production with the underlying assumption that the use of inhibitors does not alter the redox state of mitochondria (at least not to an appreciable degree). However, it is well-known that inhibiting electron flow causes the upstream sites to be more reduced and the downstream to be more oxidized. Therefore, when the mitochondrial redox state is perturbed, interpretation of data calls for certain degree of precaution. And while it is tempting to speculate on the origins of free radicals from these data, a computational model that is comprehensive and biophysically detailed is needed to pinpoint the specific sites and their relative contribution to total ROS emission rates.

5. Summary

The data show that mitochondrial respiration and hydrogen peroxide emission rates are dependent on substrates and calcium treatments which can be readily understood from the bioenergetic perspective. Seemingly contradictory data such as the maximal J_O2 of mitochondria respiring on S/R and S being lower than that of P/M are consistent with OAA inhibition and substrate transport limitations. We also demonstrated that calcium does not enhance net ROS emission in healthy cardiac mitochondria. We further showed that net ROS emission is strongly dependent on membrane potential, which is consistent with results from several previously reported studies. In addition, our data on the calcium sensitivity of PC/M supported metabolism helps explain the heart is more susceptible to IR injury when fatty acids are the sole substrate⁷⁵.

One theme we consistently encountered during our analysis is how to study individual variables that affect mitochondrial respiration and ROS emission without disturbing the mitochondrial redox landscape. In studying free radical production, using inhibitors to block certain sites of the ETS is a common approach aimed to dissect site-specific contributions to the net pool of free radical produced. While a wealth of invaluable information has been accumulated using this approach, using inhibitors inevitably induces systemic alterations to the mitochondrial redox landscape. The extent of deviation cannot be quantified since the native state is unknown. Moreover, data interpretation from many site-specific inhibitor experiments assumes a linear relationship among variables, but this relationship cannot be verified. To our knowledge, site-specific contributions to free radical production and elimination in the absence of inhibitors remain elusive. Thus, the quest for the native redox state of a system cannot be experimentally accomplished with available technologies. With the results of this study, our goal is to use the insights of the empirical model to develop a more detailed computational model that can explain data from this study and other studies.

Highlights.

• In the absence of mitochondrial permeability transition, calcium overload decreases net reactive oxygen species (ROS) emission from healthy cardiac mitochondria.

• Calcium overload inhibits NADH-dependent pathways.

• ROS emission is primarily controlled by membrane potential.

• The relationship between net ROS emission and oxygen concentration is hyperbolic.