Cyclosporin A Increases Mitochondrial Buffering of Calcium: An Additional Mechanism in Delaying Mitochondrial Permeability Transition Pore Opening

Abstract

Regulation of mitochondrial free Ca²⁺ is critically important for cellular homeostasis. An increase in mitochondrial matrix free Ca²⁺ concentration ([Ca²⁺]_m) predisposes mitochondria to opening of the permeability transition pore (mPTP). Opening of the pore can be delayed by cyclosporin A (CsA), possibly by inhibiting cyclophilin D (Cyp D), a key regulator of mPTP. Here, we report on a novel mechanism by which CsA delays mPTP opening by enhanced sequestration of matrix free Ca²⁺. Cardiac-isolated mitochondria were challenged with repetitive CaCl₂ boluses under Na⁺-free buffer conditions with and without CsA. CsA significantly delayed mPTP opening primarily by promoting matrix Ca²⁺ sequestration, leading to sustained basal [Ca²⁺]_m levels for an extended period. The preservation of basal [Ca²⁺]_m during the CaCl₂ pulse challenge was associated with normalized NADH, matrix pH (pH_m), and mitochondrial membrane potential (ΔΨ_m). Notably, we found that in PO₄³⁻ (P_i)-free buffer condition, the CsA-mediated buffering of [Ca²⁺]_m was abrogated, and mitochondrial bioenergetics variables were concurrently compromised. In the presence of CsA, addition of P_i just before pore opening in the P_i-depleted condition reinstated the Ca²⁺ buffering system and rescued mitochondria from mPTP opening. This study shows that CsA promotes P_i-dependent mitochondrial Ca²⁺ sequestration to delay mPTP opening and, concomitantly, maintains mitochondrial function.

1. Introduction

Regulation of intra-mitochondrial free calcium ([Ca²⁺]_m) is critical in cardiac physiology and pathophysiology. Under physiological conditions, a moderate increase in [Ca²⁺]_m is believed to stimulate key enzymes of the Krebs cycle and oxidative phosphorylation and to drive mitochondrial ATP production to match cellular energy demand [1,2]. In contrast, a pathological increase in [Ca²⁺]_m causes opening of the mitochondrial permeability transition pore (mPTP), a key factor in initiation of cell death [3,4]. Pathophysiological dysregulation of [Ca²⁺]_m is a primary mediator in cardiac ischemia and reperfusion (IR) injury, as Ca²⁺ overloading can lead to apoptosis [5,6,7].

[Ca²⁺]_m is regulated by a dynamic balance between mitochondrial Ca²⁺ uptake, intra-mitochondrial Ca²⁺ buffering, and mitochondrial Ca²⁺ release. Mitochondrial Ca²⁺ uptake is mediated primarily through the mitochondrial Ca²⁺ uniporter (MCU) [8,9,10], and is controlled by the large membrane potential (ΔΨ_m: −180 to −200 mV) across the inner mitochondrial membrane (IMM). The ΔΨ_m in turn is generated by the flow of electrons and proton pumping along the respiratory chain complexes [11]. When [Ca²⁺]_m increases, this depolarizes ΔΨ_m, which is compensated by enhanced H⁺ pumping/extrusion to alkalinize the matrix. Therefore, powerful, dynamic buffering of matrix pH (pH_m) and Ca²⁺ are required to enable sufficient recovery of ΔΨ_m and to avoid overloading the matrix with a high [Ca²⁺]. Inorganic phosphate (P_i) has been recognized as a major player in maintaining the trans-matrix pH gradient when accompanied by the effective cotransport of H⁺ [12] and buffering of matrix Ca²⁺ through the formation of amorphous calcium phosphate (Ca–P_i) granules [13,14,15]. The Ca–P_i buffer system sets the free Ca²⁺ at a steady-state level, enabling greater mitochondrial Ca²⁺ loading without impeding the Ca²⁺ uptake and affecting the efflux system [16,17,18]. The efflux systems that regulate [Ca²⁺]_m are the Na⁺/Ca²⁺exchanger (NCLX) [17], and the putative Na⁺-independent Ca²⁺ exchanger/Ca²⁺-hydrogen exchanger (CHE) [19]. Any disruption in the uptake, and or impairment in the buffering or efflux of Ca²⁺ would disrupt the delicate balance of the [Ca²⁺]_m and lead to impaired bioenergetics and to opening of the mPTP [3,4].

The opening of the high conductance mPTP channel is associated with a high degree of mitochondrial swelling, dissipation of ΔΨ_m, uncoupling of oxidative phosphorylation, membrane rupture and release of sequestered Ca²⁺, metabolites, and apoptotic signaling molecules [20,21,22,23]. Although the molecular components of the mPTP and its regulation remain largely unclear, cyclophilin D (Cyp D) is the only unambiguously recognized regulatory component of the mPTP. Cyp D is a mitochondrial matrix peptidyl-prolyl cis-trans isomerase (PPIase) that is translocated to the IMM during high matrix Ca²⁺ conditions; Cyp D is proposed to facilitate conformational changes in the putative mPTP core proteins thereby regulating pore opening [24,25,26].

Adenine nucleotides (AdN: ATP and ADP) have been implicated in the inhibition of Ca²⁺-dependent mPTP opening [27,28]. A previous study from our laboratory suggested that matrix AdN modulate [Ca²⁺]_m, potentially by increased buffering of [Ca²⁺]_m [29]. Oligomycin (OMN), an F₀F₁-ATP synthase inhibitor, influences the AdN (ATP/ADP) pool, and has been shown to modulate mPTP opening [30]. Cyclosporin A (CsA), a potent mPTP inhibitor is also believed to suppress pore opening by inhibiting matrix Cyp D, thereby preventing the Cyp D-induced conformational changes in mPTP core proteins [31,32]. CsA has long been known to desensitize mPTP from early opening during Ca²⁺ challenges by impeding Ca²⁺ interaction with Cyp D; however, the direct effects of CsA on the [Ca²⁺]_m buffering system have not been investigated systematically. It is worth noting that in a previous study from Chalmers and Nicholls [14], it was proposed that CsA enhances the Ca²⁺ loading capacity of mitochondria without changing the relationship between free [Ca²⁺]_m and total [Ca²⁺]_m during continuous Ca²⁺ infusion in isolated rat liver and brain mitochondria. Altschuld et al. [33] proposed that CsA increases mitochondrial Ca²⁺ influx and reduces its efflux. Later, Wei et al. [34] demonstrated that although CsA had no effect on MCU activity, it inhibited NCLX activity at higher concentrations. Altogether, these findings raise important questions about how CsA delays Ca²⁺-induced mPTP opening while increasing net [Ca²⁺]_m accumulation. Our study sought to answer these questions by (i) examining the effect of CsA during repeated CaCl₂ challenges over an extended time-period on mitochondrial Ca²⁺ buffering, and (ii) by examining the underlying changes in bioenergetics during excessive Ca²⁺ overload.

To address our objective, we investigated systematically the effect of CsA on mitochondrial Ca²⁺ buffering and compared its effect with a known matrix buffering component, the AdN pool (OMN+ADP), by monitoring [Ca²⁺]_e, [Ca²⁺]_m, and key mitochondrial bioenergetics variables, ΔΨ_m, pH_m, and NADH (redox state), under conditions of repeated Ca²⁺ loading. Furthermore, we determined the effect of CsA on the rescue of buffering capability and bioenergetics of failing mitochondria just before mPTP opening. We found that CsA enhanced the sequestration of mitochondrial Ca²⁺, maintained [Ca²⁺]_m at a steady-state level, and markedly delayed mPTP opening. In addition, CsA preserved ΔΨ_m, NADH, and pH_m during CaCl₂ bolus challenges. However, in the absence of P_i, this CsA-induced matrix Ca²⁺ sequestration was abrogated, and in turn led to the early mPTP opening. The results described herein reveal a novel way by which CsA modulates matrix Ca²⁺ sequestration to maintain [Ca²⁺]_m, despite increased Ca²⁺ loading. CsA-mediated Ca²⁺ sequestration is likely achieved via a P_i-dependent [Ca²⁺]_m buffering system that delays Ca²⁺-induced mPTP opening.

2. Materials and Methods

2.1. Materials

All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless stated otherwise. Fluorescent probes Fura-4F, Fura-4F^AM, tetramethylrhodamine methyl ester perchlorate (TMRM) and 2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF^AM) were purchased from Life Technologies (Eugene, OR, USA).

2.2. Animals

Albino Hartley guinea pigs of both sexes weighing between 250 to 350 g were procured from Kuiper Rabbit Farm (Gary, IN, USA). All procedures were carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.

2.3. Mitochondria Isolation

Mitochondria were isolated from guinea pig hearts as described previously [29,35,36]. Briefly, the guinea pig was anesthetized with an intraperitoneal injection of 30 mg ketamine plus 700 units of heparin, for anticoagulation, and the heart was rapidly excised and minced in ice-cold isolation buffer containing 200 mM mannitol, 50 mM sucrose, 5 mM KH₂PO₄, 5 mM MOPS, 1 mM EGTA, and 0.1% bovine serum albumin (BSA) at pH 7.15 (adjusted with KOH). The suspension was homogenized at low speed for 20 s in ice-cold isolation buffer containing 5 U/mL protease (from Bacillus licheniformis) and the homogenate was centrifuged at 8000× g for 10 min. The supernatant was discarded, and the pellet was suspended in 25 mL isolation buffer, and centrifuged at 850× g for 10 min. The supernatant was centrifuged further at 8000× g to yield the final mitochondrial pellet, which was suspended in isolation buffer and kept on ice until experimentation. All isolation procedures were performed at 4 °C and all experiments were conducted at room temperature. Protein concentration was determined by the Bradford method and the final mitochondrial suspension was adjusted to 12.5 mg protein/mL with isolation buffer.

The functional integrity of mitochondria was determined by the respiratory control index (RCI) as described before [29,37]. Mitochondria were energized with pyruvic acid (PA, 0.5 mM; pH 7.15, adjusted with KOH) followed by ADP (250 µM) addition. RCI was defined as the ratio of state 3 (after added ADP) to state 4 respiration (after complete phosphorylation of the added ADP). Only mitochondrial preparations with RCIs ≥ 10 were used to conduct further experiments.

2.4. Experimental Groups and Protocols

Two protocols (Protocol A and Protocol B) were used to assess the effect of CsA and AdN on mitochondrial Ca²⁺ handling and bioenergetics in normal and Ca²⁺-overloaded mitochondria, as shown in Figure 1. Protocol A investigated the ability of CsA and AdN to modulate mitochondrial Ca²⁺ handling and delay mPTP opening. To further substantiate CsA-mediated buffering of matrix Ca²⁺, Protocol B was designed to test the effectiveness of CsA and AdN on rescuing a failing mitochondrial Ca²⁺ buffering system from imminent mPTP opening. There were five experimental groups: vehicle (DMSO), CsA, ADP, OMN, and OMN+ADP. Experiments were also conducted in the presence of deionized H₂O as another vehicle (not shown). Each group was subjected to two different experimental protocols (Protocol A and Protocol B) that differed in the order of treatment and addition of CaCl₂ boluses to the mitochondrial suspension in experimental buffer.

Delayed opening of mPTP (Protocol A): At t = 0 s, the experiment was initiated by suspending 0.5 mg of isolated mitochondria into the experimental buffer containing 130 mM KCl, 5 mM K₂HPO₄, 20 mM MOPS, 1 mM EGTA, 0.1% BSA, and EGTA ~0.036–0.040 µM at pH 7.15 (adjusted with KOH). At t = 30 s, mitochondria were treated with DMSO (1 µM), ADP (250 µM), OMN (10 µM), OMN+ADP or CsA (0.5 µM); at t = 60 s, mitochondria were energized with PA (0.5 mM). At t = 180 s, CaCl₂ bolus (20 μM final concentration) was added and subsequent CaCl₂ boluses added at 5 min intervals until pore opening (Figure 1A). Note that all experiments were conducted under state 2 conditions, except in the ADP-and OMN+ADP-treated groups.

Rescue of mitochondria from mPTP opening (Protocol B): The mitochondrial suspension was exposed to repetitive boluses of CaCl₂ (20 μM) as described in Protocol A; rescue of mitochondria from mPTP opening with the different treatments was carried out at 1 min of the last CaCl₂ bolus in which mitochondria Ca²⁺ uptake was observed before pore opening (Figure 1B). The onset of mPTP opening was predicted based on calcium retention capacity (CRC) of the DMSO (control)-treated group for each day’s experiment. The pulse preceding mPTP opening observed in the control was the pulse chosen for targeted intervention in all subsequent experiments. In all experiments, extrusion of Ca²⁺ via the Na⁺/Ca²⁺ exchanger (NCLX) was prevented by conducting all the experiments in Na⁺-free conditions. That is, the respiration buffer, mitochondrial substrates, and all reagents/drugs were Na⁺-free to prevent activation of the NCLX. Some experiments were conducted in the presence of 10 μM CGP 37157 (Tocris Bioscience), an NCLX inhibitor, which ascertained there was no potential Na⁺ contamination in the respiration buffer from other sources [35,38,39].

2.5. Mitochondrial Function Measurements

Fluorescence spectrophotometry (Qm-8, Photon Technology International, Horiba, Birmingham, NJ, USA) was used to measure mitochondrial function, including mitochondria extra- and intra-matrix free [Ca²⁺] ([Ca²⁺]_e and [Ca²⁺]_m, respectively), ΔΨ_m, redox state (NADH), and pH_m. Fura-4F penta-potassium salt (1 µM, Invitrogen™, Eugene, OR) was used to measure [Ca²⁺]_e. For [Ca²⁺]_m measurements, mitochondria were incubated with Fura-4F AM (5 µM, Invitrogen™, Eugene, OR) for 30 min at room temperature (25 °C) followed by a final spin and resuspension to remove any residual dye. ΔΨ_m was assessed using the cationic lipophilic dye TMRM (1 µM, Invitrogen™, Eugene, OR, USA) in a ratiometric excitation approach [40]. NADH was measured by tissue autofluorescence, and matrix pH (pH_m) was assessed by incubating mitochondria in 5 μM BCECF^AM (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature (25 °C) followed by a final spin and resuspension [29,35,38,39].

2.6. Measurements of Free Ca²⁺

Quantification of [Ca²⁺]_e and [Ca²⁺]_m were made using the fluorescent Ca²⁺ indicator probe Fura-4F with dual-excitation wavelengths (λ_ex) at 340/380 nm and a single emission wavelength (λ_em) at 510 nm. Ca²⁺ fluorescent intensities with Fura-4F are not influenced by background noise (e.g., NADH autofluorescence), so a background subtraction was unnecessary [38]. Fura-4F fluorescence ratios (F₃₄₀/F₃₈₀) were used to calculate [Ca²⁺] using the equation described by Grynkiewicz: [41].

$$[{\mathrm{Ca}}^{2+}]={\mathrm{K}}_{\mathrm{d}}\frac{{\mathrm{S}}_{\mathrm{f}2}}{{\mathrm{S}}_{\mathrm{b}2}}\frac{(\mathrm{R}-{\mathrm{R}}_{\min })}{({\mathrm{R}}_{\max }-\mathrm{R})}.$$

(1)

The K_d value for Fura-4F binding to Ca²⁺ is 890 nM, which was described by us previously [38]. R is the ratio of the fluorescence intensities at λ_ex 340 and 380 nm, S_f2/S_b2 is the ratio of fluorescence intensities measured at λ_ex 380 nm in Ca²⁺-free (f)/Ca²⁺-saturated (Ca²⁺-bound, b) conditions. R_min (Ca²⁺-free) and R_max (Ca²⁺-saturated) are R values for Fura 4F, carried out after mPTP opening, adding 1 mM CaCl₂, followed by 10 mM EGTA, pH 7.1. The free [Ca²⁺] in the buffer was calculated using an online version of MaxChelator program (http://www.stanford.edu/~cpatton/maxc.html) and accordingly, a standard curve was generated for the Fura-4F signal to the free [Ca²⁺] in the experimental solution by fitting to the Grynkiewicz equation, as described above in Equation 1 [41].

2.7. Calculation of Mitochondrial Ca²⁺ Buffering Capacity

The ability of mitochondria to sequester Ca²⁺ is an index of its Ca²⁺ loading capacity, without altering mitochondrial function. Here we calculated mitochondrial Ca²⁺ buffering capacity (mβ_Ca) using the model described by Bazil et al. [42]. Briefly, experimental data for extra-and intra-matrix Ca²⁺ were fit with smooth trend curves satisfying the equation:

$$\mathrm{y}(\mathrm{t})={\mathrm{p}}_1+{\mathrm{p}}_2{\mathrm{e}}^{\raisebox{1ex}{$\left(\mathrm{t}-{\mathrm{p}}_3\right)$}\!\left/ \!\raisebox{-1ex}{${\mathrm{p}}_4$}\right.}+{\mathrm{p}}_5\mathrm{t},$$

(2)

where y(t) was either [Ca²⁺]_e or [Ca²⁺]_m at any given time, t. Global trend-fits were performed in MATLAB (Mathworks, Inc., MA) and parameters p₁ (offset value), p₂ (pre = exponential constant), p₃ (time lag), p₄ (decay time constant), and p₅ (steady-state slope) were estimated and optimized using the lsqnonlin and fmincon functions.

Mitochondrial Ca²⁺ buffering capacity for the second Ca²⁺ pulse (a cumulative of 40 µM added Ca²⁺) was then calculated [42] as:

$${\mathrm{m}\mathsf{\beta }}_{\mathrm{Ca}}=-{\mathsf{\beta }}_{\mathrm{Ca},\mathrm{e}}{\mathrm{V}}_{\mathrm{r}}\frac{\mathrm{d}{[{\mathrm{Ca}}^{2+}]}_{\mathrm{e}}}{\mathrm{dt}}/\frac{\mathrm{d}{[{\mathrm{Ca}}^{2+}]}_{\mathrm{m}}}{\mathrm{dt}},$$

(3)

where, mβ_Ca, is the intra-mitochondrial Ca²⁺ buffering power, β_Ca,e is the extra-mitochondrial Ca²⁺ buffering power determined by:

$${\mathsf{\beta }}_{\mathrm{Ca},\mathrm{e}}=1+\frac{\partial {\left[\mathrm{CaEGTA}\right]}_{\mathrm{e}}}{\partial {[{\mathrm{Ca}}^{2+}]}_{\mathrm{e}}}.$$

(4)

Vr is the volume ratio of the extra-mitochondrial space and matrix space (~2000), d[Ca²⁺]_e/dt and d[Ca²⁺]_m/dt are the rates of change of extra-and intra-mitochondrial free [Ca²⁺], respectively. d[Ca²⁺]_e/dt and d[Ca²⁺]_m/dt were estimated by evaluating the analytical derivative of Equation (2) using parameter estimates obtained from the trend fits [42].

Trend fits for data in Figure S4 were performed in Origin 2017 (OriginLab Corporation, Northampton, MA, USA).

2.8. Measurement of ΔΨ_m, Redox State (NADH) and Matrix pH

Membrane potential was assessed by the dual-excitation ratiometric approach using the fluorescent dye, TMRM, as described by Scaduto and Grotyohann [40] and in our published work [35,38,39]. Fluorescence changes were determined by two excitations, λ_ex 546 and 573 nm, and a single emission λ_em 590 nm. The calculated ratio of λex 573/546 is proportional to ΔΨ_m and has the advantage of a broader dynamic range when compared to a single wavelength technique. Changes in mitochondrial redox state (NADH) were determined by autofluorescence (i.e., by exciting the energized mitochondria at λ_ex 350 nm and collecting data at λ_em 456 nm). An increase in the signal reflects an increase in the redox ratio of NADH to NAD⁺ (i.e., a shift to a more reduced state). Matrix pH was assessed using BCECF^AM (5 μM) at λ_ex 504 nm and λ_em 530 nm. This fluorescent probe emits less fluorescence in an acidic environment, thus a decrease in signal indicates matrix acidification and an increase in signal indicates matrix alkalization [29].

2.9. Depletion of Endogenous Mitochondrial Phosphate

Given the important role of P_i in the mitochondrial Ca²⁺ buffering system [14,29], we tested the effect of P_i in CsA-induced mitochondrial Ca²⁺ buffering. Isolated cardiac mitochondria were depleted of endogenous P_i by pre-incubating mitochondria for 10 min at room temperature with 0.75 units/mL hexokinase, 1 mM glucose, 0.5 mM ADP, 1 mM MgCl₂, and 5 mM PA, as previously described [14,43,44].

2.10. Statistical Analyses

Data were transferred from PTI FelixGX (Version 3) into Microsoft^® Excel^® (2007). An unpaired Student’s t-test was used to evaluate significant differences between means of CsA-treated versus DMSO- and AdN-treated groups on specific variables ([Ca²⁺]_m, [Ca²⁺]_e, ΔΨ_m, NADH, or pH_m) in both Protocols A and B. The final data of a specific variable were expressed as mean ± standard error (SE) over at least 4 replicates of the same variable (n = 4). Comparisons within and between groups were performed by one-way ANOVA (analysis of variance) with Tukey’s post-hoc test to examine differences among individual groups. p < 0.05 (two-tailed) was considered significant. See Figure legends for statistical notations.

3. Results

3.1. Effect of CsA on Extra-Matrix Free [Ca²⁺]

To determine the effect of CsA on matrix Ca²⁺ uptake, we measured [Ca²⁺]_e during repetitive additions of 20 µM CaCl₂ boluses at 5 min (300 s) intervals to allow characterization of the detailed kinetics of steady-state Ca²⁺ dynamics (influx and buffering). Figure 2 shows the dynamics of [Ca²⁺]_e during CaCl₂ pulse challenges, with different treatments. Panels A, B, C and panels D, E, F depict the Ca²⁺ dynamics profile using Protocols A and B, respectively. Each panel consists of five traces representing different treatment groups (DMSO, ADP, OMN, OMN+ADP, and CsA) in the presence of approximately 40 μM EGTA (carried over from the isolation buffer). In response to each CaCl₂ pulse, an increase in Fura-4F fluorescence intensity was observed, which then returned to a baseline; the steady-state (ss) level is marked by the flat response as mitochondria take up and sequester the added Ca²⁺. The opening of mPTP is evident by cessation of mitochondrial Ca²⁺ uptake and a sharp rise in the extra-matrix dye fluorescent intensity. Ca²⁺ concentrations were determined from the fluorescence ratios using Equation (1).

We plotted steady-state [Ca²⁺]_e (ss[Ca²⁺]_e) as a function of cumulative added CaCl₂ at each pulse (Figure 2B). The detailed dynamics of ss[Ca²⁺]_e for the initial four CaCl₂ pulses, in each group, are illustrated in the enlarged scale inset (Figure 2A). The exposure of mitochondria to DMSO and OMN, followed by repeated boluses of CaCl₂, resulted in a gradual increase in ss[Ca²⁺]_e with less mitochondrial Ca²⁺ uptake and rapid Ca²⁺ release by the third or fourth CaCl₂ pulse. The total CRC for the DMSO and OMN were comparable (i.e., 133.3 ± 13.3 nmol Ca²⁺/mg protein and 146.6 ± 13.3 nmol Ca²⁺/mg protein, respectively) (Figure S1). In the presence of ADP, mitochondria took up more Ca²⁺ before pore opening and the CRC was further augmented with OMN+ADP; the CRC value increased from 213.3 ± 13.3 nmol Ca²⁺/mg protein for ADP alone to 373.3 ± 35.3 nmol Ca²⁺/mg protein for OMN+ADP (Figure S1). Mitochondria treated with CsA before the addition of CaCl₂ boluses displayed a more robust Ca²⁺ uptake, with a significantly higher CRC value, 573.3 ± 26.6 nmol Ca²⁺/mg protein, compared with all other groups (Figure 2A and Figure S1). Importantly, in CsA-treated mitochondria, the addition of CaCl₂ pulses (20 µM) did not significantly increase the ss[Ca²⁺]_e until the sixth to seventh pulse (Figure 2B), suggesting enhanced Ca²⁺ uptake. Figure 2C, summarizes the effects of the different treatments on the ss[Ca²⁺]_e for cumulative additions of 80, 140, and 180 µM of Ca²⁺, which corresponds to the fourth, seventh, and ninth CaCl₂ pulses, respectively. The addition of CsA strongly blunted the Ca²⁺-induced increase in ss[Ca²⁺]_e by stimulating faster and more Ca²⁺ uptake. We observed that the ss[Ca²⁺]_e was significantly lower for the CsA-treated mitochondria than for OMN+ADP-treated mitochondria after the cumulative addition of CaCl₂ of 80 µM (0.28 ± 0.0 µM vs. 0.50 ± 0.02 µM), 140 µM (0.57 ± 0.06 µM vs. 0.77 ± 0.06 µM), and 180 µM (0.71 ± 0.02 µM vs. 0.95 ± 0.1 µM) CaCl₂, respectively (Figure 2B,C). The sustained low ss[Ca²⁺]_e for an extended period of CaCl₂ additions in the CsA-treated group indicates a maintained ΔΨ_m for Ca²⁺ uptake, resulting in enhanced Ca²⁺ loading capacity because of improved buffering.

We next examined [Ca²⁺]_e dynamics in the situation in which mitochondrial matrix Ca²⁺ nearly reached threshold, as determined by the predicted opening of mPTP; in this case we added CsA just before the anticipated mPTP opening. Using Protocol B (Figure 1), [Ca²⁺]_e was measured and the kinetics were compared in response to adding either DMSO, ADP, OMN, OMN+ADP, or CsA just before the onset of the mPTP opening (Figure 2D). The dynamic changes in ss[Ca²⁺]_e during the addition of different treatments are illustrated in more detail in the inset of Figure 2D. In the DMSO-treated mitochondria, three to four Ca²⁺ pulses (cumulative addition of 68 ± 4.9 µM CaCl₂) were sufficient to induce the release of matrix Ca²⁺. Addition of ADP or OMN reversed the initial pore opening and delayed matrix Ca²⁺ release by one to two pulses compared to DMSO. Addition of OMN+ADP also showed a significant reversal of Ca²⁺ release with reduction in the ss[Ca²⁺]_e (0.74 ± 0.18 µM). Thus, there was a considerable increase in the CRC by OMN+ADP compared to DMSO (Figure 2D and Figure S1) and a further delay in mPTP opening by one additional CaCl₂ bolus compared to OMN or ADP alone. More impressively, the addition of CsA not only reversed the increasing trend of ss[Ca²⁺]_e to the baseline levels (0.42 ± 0.06 µM) (Figure 2D,E), it further maintained the ss[Ca²⁺]_e at a constant low value for an additional twelve to thirteen Ca²⁺ pulses. This resulted in a four-fold and a two-fold increase in the CRC, compared to DMSO and OMN+ADP, respectively (Figure 2E,F and Figure S1).

Altogether, these results demonstrate that CsA enhances mitochondrial Ca²⁺ uptake, thereby inhibiting a consequent increase in free [Ca²⁺]_e during CaCl₂ pulse challenges, leading to an increase in the CRC of the mitochondria. This sustained low ss[Ca²⁺]_e and concomitant increase in Ca²⁺ uptake are likely explained by enhanced [Ca²⁺]_m buffering to maintain basal [Ca²⁺]_m, which results in a preserved ΔΨ_m for Ca²⁺ uptake and greater CRC. To investigate further the potential for CsA on mediating Ca²⁺ buffering, it was necessary to examine the effects of CsA on matrix [Ca²⁺]_m dynamics in the next set of experiments.

3.2. Effect of CsA on Matrix Free [Ca²⁺] Handling

Matrix Ca²⁺ was assessed with Fura-4 AM as described in Materials and Methods. We explored the effect of CsA on [Ca²⁺]_m, under identical conditions and protocols as shown in Figure 1 (Protocols A,B). Mitochondrial Ca²⁺ buffering was measured as a function of a decrease in Ca²⁺ fluorescence, reaching a steady-state at approximately 270 s after each bolus of CaCl₂ added. The magnitude of mitochondrial Ca²⁺ uptake for the first CaCl₂ pulse (20 µM) was similar in all groups; however, on subsequent additions of CaCl₂, the ADP- and/or OMN-treated groups showed faster declines in [Ca²⁺]_m with lower mitochondrial steady-state [Ca²⁺]_m (ss[Ca²⁺]_m) and delayed mPTP opening compared to DMSO (Figure 3A,B). Interestingly, the CsA-treated group showed a small increase in ss[Ca²⁺]_m with each CaCl₂ pulse, but a gradual decline in ss[Ca²⁺]_m was observed after [Ca²⁺]_m exceeded 3 ± 0.10 µM with the cumulative addition of 100–150 µM CaCl₂ (Figure 3A,B) and a significant increase in CRC up to fifteen to sixteen pulses. This suggested that the buffering effect of CsA on matrix Ca²⁺ is triggered when [Ca²⁺]_m reaches a certain value.

To estimate the mitochondrial Ca²⁺ buffering capacity, the ratio of bound Ca²⁺:free Ca²⁺ was calculated from the change in [Ca²⁺]_m (Figure 3A) to the total amount of Ca²⁺ taken up from the extra-matrix medium (ΣCa²⁺_uptake): (ΣCa²⁺_uptake-[Ca²⁺]_m)/[Ca²⁺]_m, as described previously [45]. Although the extent of bound Ca²⁺:free Ca²⁺ at each Ca²⁺ pulse was comparable in all the treated groups (Figure 3C), the addition of CsA maintained the buffering capacity, with a gradual increase in the capacity to bind Ca²⁺ up to fifteen or sixteen Ca²⁺ pulses (Figure 3C).

Greater uptake of Ca²⁺ from the extra-matrix space (indicated by lower ss[Ca²⁺]_e), combined with lower ss[Ca²⁺]_m, indicated a greater Ca²⁺ buffering capacity of mitochondria in the presence of CsA. Consistent with this notion, the calculated matrix Ca²⁺ buffering capacity (mβ_Ca) in CsA-treated mitochondria was about ten-fold higher compared to DMSO and two-fold higher than with OMN+ADP (Figure 4). This CsA-mediated increase in mβ_Ca is possibly due to an effect of CsA in triggering the matrix physiological buffers to enhance sequestration of Ca²⁺.

After observing the high buffering capacity of mitochondria pre-treated with CsA before the CaCl₂ bolus challenges, we next examined the effect of CsA on the rescue of mitochondria from Ca²⁺ release when the matrix Ca²⁺ buffering system (MCBS) becomes overwhelmed by the added boluses of CaCl₂ (Figure 1B). As shown in Figure 3D,E, OMN and ADP, each failed to reverse the mitochondrial Ca²⁺ efflux with added boluses; however, adding CsA or OMN+ADP at similar time points significantly reduced ss[Ca²⁺]_m by reinstating Ca²⁺ sequestration. This reversal was more effective and sustained in the presence of CsA than with OMN+ADP. This observation is consistent with the calculated values of bound Ca²⁺: free Ca²⁺_, which increased two-fold for CsA compared to OMN+ADP (Figure 3F). Taken together, these data demonstrate that CsA increases the mitochondrial Ca²⁺ threshold for mPTP opening by activating [Ca²⁺]_m buffering that results in maintenance of a low ss[Ca²⁺]_m.

3.3. Effect of CsA on Ca²⁺-Mediated Changes in ΔΨ_m, NADH, and Matrix pH

A major driving force for Ca²⁺ uptake, in addition to the chemical gradient, is a high IMM potential gradient (ΔΨ_m); but increased Ca²⁺ uptake without efflux or sequestration can decrease ΔΨ_m by flooding the matrix with positive charges. To strengthen the thesis that CsA increases the capacity of mitochondria to sequester Ca²⁺, we next investigated the effect of CsA on mitochondrial bioenergetics. ΔΨ_m, NADH, and pH_m were assessed using the same protocols as described in Figure 1 for CRC to correlate changes in [Ca²⁺]_m to changes in bioenergetics over time. mPTP opening was marked by a sudden rise in the TMRM signal, indicating maximal depolarization of Ψ_m. Correspondingly, the oxidation of NADH was marked by a decrease in matrix NADH signal intensity when mPTP opens. Figure 5 shows representative traces of ΔΨ_m, NADH, and pH_m for each experimental condition. The rate of ΔΨ_m depolarization and NADH oxidation correlated well with the induction of mPTP, as seen in the CRC data. The loss of CRC coincided with total ΔΨ_m dissipation and NADH oxidation. DMSO-treated mitochondria (control) exhibited rapid Ca²⁺-induced ΔΨ_m depolarization and NADH oxidation (black trace) after only a few CaCl₂ pulses. Addition of OMN+ADP significantly delayed the Ca²⁺ induced ΔΨ_m depolarization and NADH oxidation when compared to DMSO, with 533.3 ± 26.7 nmol Ca²⁺/mg protein vs. 200 ± 23 nmol Ca²⁺/mg protein and 546.7 ± 18.9 nmol Ca²⁺/mg protein vs.173.3 ± 13.3 nmol Ca²⁺/mg protein Ca²⁺ capacity, respectively (Figure 5A,B). Mitochondria treated with CsA maintained ΔΨ_m and NADH for a higher number of CaCl₂ pulses than with OMN+ADP (666.7 ± 13.3 nmol Ca²⁺/mg protein, and 626.6 ± 13.3 nmol Ca²⁺ /mg protein, respectively) (Figure 5A,B). Mitochondrial matrix pH (pH_m) is known to modulate mitochondrial P_i concentration and thus influence the matrix Ca²⁺ buffering [14]. The presence of CsA maintained pH_m at a basal level until mPTP opened (Figure 5C).

In Protocol B, intervention with OMN + ADP or CsA maintained ΔΨ_m, mitochondrial NADH, and pH_m (Figure 5D–F), and contributed to the improved capacity of mitochondria to take up and sequester additional Ca²⁺ after CaCl₂ pulses. However, OMN+ADP was less effective in preserving ΔΨ_m, NADH, and pH_m compared to CsA. This incapacity to sustain the bioenergetic status in the OMN+ADP- vs. CsA-treated mitochondria during CaCl₂ challenges reflects a lower capacity to sequester Ca²⁺ in the matrix for a protracted time.

In summary, maintenance of ΔΨ_m, NADH, and pH_m in the presence of CsA is consistent with changes in [Ca²⁺]_e and [Ca²⁺]_m that reflect greater Ca²⁺ sequestration (Figure S2) and uptake. Collectively, these results indicate that CsA reduced the accumulation of [Ca²⁺]_m, by potentiating matrix Ca²⁺ buffering, which in turn, maintained ΔΨ_m, NADH, and pH_m necessary for normal mitochondrial function. Together, these mitochondrial variables preserve mitochondria and protect against mPTP opening.

3.4. Time Dependent Effect of CsA Addition on Rescue of Mitochondria from Imminent Ca²⁺-Induced mPTP Opening

After demonstrating that CsA can reverse the induction of mPTP opening (Figure 2, Figure 3 and Figure 5), we next investigated the dynamics of [Ca²⁺]_e, [Ca²⁺]_m and ΔΨ_m, by adding CsA at three different time points, before the onset of mPTP opening_. This approach allowed us to determine the threshold at which CsA can effectively restore the mitochondrial sequestration system that will protect mitochondria from Ca²⁺ overload-mediated pore opening. Figure 6, panels A-C, show changes in [Ca²⁺]_e, [Ca²⁺]_m, and ΔΨ_m depolarization_, induced by adding CsA at 1, 2, and 3 min after the last CaCl₂ bolus in which mitochondrial Ca²⁺ uptake was observed before pore opened. Right panels D-F show detailed (close up) comparison of kinetics of [Ca²⁺]_e, [Ca²⁺]_m, and ΔΨ_m after adding CsA at different time points. Adding CsA at all three tested time points, markedly delayed the large increase in [Ca²⁺]_e due to mitochondrial Ca²⁺ release. However, the effect of CsA to prolong Ca²⁺ uptake, which eventually maintains ss[Ca²⁺]_e at baseline, diminished as the interval before CsA addition and [Ca²⁺]_e accumulation was lengthened (Figure 6A). Adding CsA at 1 min caused a decline in [Ca²⁺]_e, with a marked decrease in ss[Ca²⁺]_e (0.39 ± 0.07 µM) of the succeeding Ca²⁺ pulses, compared to adding CsA at 2 min (0.67 ± 0.03 µM) and 3 min (0.84 ± 0.05 µM) (Figure 6D; inset). In addition, we examined for changes in kinetics of [Ca²⁺]_m with CsA added at the same time points (Figure 6B). The rate of maximal Ca²⁺ buffering (i.e., the time to reach steady-state [Ca²⁺]_m) and the Ca²⁺ threshold for pore opening was significantly higher when CsA was added at the early time points (i.e., 1 and 2 min) compared to the late time point of 3 min (Figure 6E, inset).

Next, in a parallel study, we monitored the corresponding changes in ΔΨ_m profile at the same rescue time points (1, 2, or 3 min). Adding CsA reversed the Ca²⁺-induced ΔΨ_m depolarization even after a large depolarization (i.e., at 3 min; Figure 6C,F). Similar to its effect on [Ca²⁺]_e and [Ca²⁺]_m, CsA restored and maintained ΔΨ_m for a longer period at rescue points of 1 min vs. 2 and 3 min. Thus, at these points of intervention, CsA suppressed mPTP opening by increasing matrix Ca²⁺ buffering capacity, which maintained ΔΨ_m and the driving force for further Ca²⁺ uptake (Figure 6C). Together, these results demonstrate that the magnitude of CsA-mediated increase in Ca²⁺ threshold for mPTP opening and maintenance of mitochondrial integrity is dependent on the [Ca²⁺]_m level before CsA intervention.

3.5. Role of Inorganic Phosphate in CsA-Induced [Ca²⁺]_m Regulation.

Inorganic phosphate (P_i) is a required component for mitochondrial matrix Ca²⁺ buffering [14,29]. To gain insight into the mechanism that underlies CsA-mediated activation of the MCBS, we monitored mitochondrial Ca²⁺ handling and ΔΨ_m during repeated boluses of 20 µM CaCl₂ every 5 min, as described in Materials and Methods, but now in the absence of P_i. With mitochondria depleted of P_i, and in P_i-free media, the CRC of mitochondria treated with CsA before the CaCl₂ pulses was not different from DMSO (control). In addition, these mitochondria showed a gradual increase in ss[Ca²⁺]_e and interestingly, after cumulative additions of CaCl₂ to 80 ± 15 µM, there was a significant decrease in mitochondrial Ca²⁺ uptake during additional CaCl₂ pulses (Figure 7A). These results implicated a P_i-dependent mechanism in the CsA-mediated delay in mPTP opening. In contrast, ADP and OMN+ADP, but not OMN alone, caused a significant delay in mPTP opening (Figure 7A) in the absence of P_i.

Along with observing the P_i-mediated effect of CsA on [Ca²⁺]_e dynamics, we also measured [Ca²⁺]_m under identical conditions. In the absence of P_i, mitochondria showed a gradual increase in ss[Ca²⁺]_m; matrix Ca²⁺ sequestration was strongly blunted in both DMSO-and CsA-treated groups. This reflected diminished buffering capacity with the increase in [Ca²⁺]_m (Figure 7B)_. However, in the presence of ADP and OMN+ADP in the P_i-depleted condition, mitochondria displayed robust CRC and enhanced Ca²⁺ buffering and thus decreased [Ca²⁺]_m (Figure 7B). Intriguingly, this effect was stronger than in the P_i replete condition (Figure 3). Mitochondria also showed an increased ratio of bound Ca²⁺:free Ca²⁺ in the OMN+ADP-treated group, but not in the DMSO and CsA groups (Figure 7D). These data further support the premise that P_i is crucial in CsA-induced matrix Ca²⁺ buffering and P_i is a requisite component of matrix calcium sequestration.

Since we observed significant attenuation of Ca²⁺ uptake and buffering by CsA in the absence of P_i, we addressed how the altered mitochondrial Ca²⁺ dynamics impacted ΔΨ_m. Analysis of ΔΨ_m in mitochondria depleted of P_i during CaCl₂ bolus challenges revealed a gradual depolarization with each Ca²⁺ pulse over time in the DMSO-, OMN-, and CsA-treated groups (Figure 7C); this was consistent with the low CRC in these three groups due to the poor buffering after additional CaCl₂ pulses. In contrast, mitochondria exposed to ADP or OMN+ADP in the P_i-depleted state exhibited restored and sustained ΔΨ_m, which supported a robust CRC (Figure 7C).

To further confirm the requisite role of P_i in mediating CsA-induced activation of the MCBS, a rescue experiment with 5 mM P_i was performed with DMSO-and CsA-treated groups in P_i-depleted condition. With addition of deionized H₂O (vehicle), pore opening was not prevented in either group (data not shown). The addition of exogenous P_i to the buffer triggered a rapid reversal of Ca²⁺ release (decrease in ([Ca²⁺]_e) in parallel with complete restoration of ΔΨ_m (Figure 8). In contrast, additional Ca²⁺ pulses in the P_i free DMSO-treated group failed to maintain ss[Ca²⁺]_e and basal Ψ_m, and induced rapid Ca²⁺ efflux (Figure 8). However, the CsA-treated mitochondria showed a robust uptake of [Ca²⁺]_e with low ss[Ca²⁺]_e and sustained ΔΨ_m maintenance with additional CaCl₂ boluses (Figure 8). Taken together, these results establish that P_i is required for CsA-mediated mitochondrial Ca²⁺ buffering that maintains low [Ca²⁺]_m and preserves ΔΨ_m; this in turn contributes to the capacity for more Ca²⁺ uptake and thus increases the Ca²⁺ threshold for mPTP opening.

4. Discussion

Matrix free [Ca²⁺] ([Ca²⁺]_m) plays two important roles: (i) Activation of Ca²⁺-dependent dehydrogenases for oxidative phosphorylation at low concentrations [46]; and (ii) regulation of cytosolic Ca²⁺ by sequestration of excess Ca²⁺ at high concentrations [47]. Excessive accumulation of free [Ca²⁺]_m is a leading factor in inducing mPTP opening. It is well established that repetitive mitochondrial Ca²⁺ loading triggers a gradual increase in [Ca²⁺]_m, leading to a loss of IMM integrity that results in dissipation of ΔΨ_m and release of Ca²⁺. CsA is known to delay pore opening, in part, by inhibiting the PPIase activity of Cyp-D [31]. Whether CsA-mediated delay in mPTP opening involves regulation of [Ca²⁺]_m by P_i-induced matrix Ca²⁺ buffering has not been addressed before. In this study, we investigated the effects of CsA on [Ca²⁺]_m regulation during repeated Ca²⁺ loading and its functional significance in mPTP opening. Additionally, we determined if changes in [Ca²⁺]_m induced by CsA correlated with changes in mitochondrial bioenergetics under identical experimental conditions and if matrix P_i was required for the observed CsA effects.

Since the key postulate was that CsA contributes to mitochondrial Ca²⁺ buffering, all experiments were performed in Na⁺-free condition to completely block NCLX as a route for efflux of excess matrix Ca²⁺. This allowed us to directly assess mitochondrial Ca²⁺ buffering capacity under different treatments. Our major findings during repetitive CaCl₂ bolus challenges are: (i) CsA maintained basal ss[Ca²⁺]_m owing to increased mitochondrial Ca²⁺ buffering capacity; (ii) the effectiveness of CsA to maintain basal ss[Ca²⁺]_m correlates well with preserved mitochondrial bioenergetics; (iii) the buffering effect of CsA in a P_i-replete buffer was more pronounced than the known buffering effect of OMN+ADP; (iv) CsA-induced buffering was abolished in P_i-depleted mitochondria and P_i-free experimental medium. We conclude that the CsA-mediated delay in mPTP opening could, in large part, be attributed to CsA-induced activation of a P_i-dependent mitochondrial Ca²⁺ buffering system (MCBS), which maintains a low free [Ca²⁺]_m and preserves mitochondrial bioenergetics.

4.1. CsA-Mediated Inhibition of mPTP Opening Relates to the ss[Ca²⁺]_m

Using the two protocols (Figure 1A,B), we examined the changes in [Ca²⁺]_e and [Ca²⁺]_m in response to boluses of CaCl₂ in the presence of vehicle (DMSO), CsA, ADP, OMN, or OMN+ADP over time. Our experimental approaches allowed us to define the contribution of CsA in the regulation of [Ca²⁺]_m when CsA was given before the CaCl₂ boluses (Protocol A) and at the threshold for pore opening under condition of increased free [Ca²⁺]_m accumulation (Protocol B). Our results clearly indicate that the effect of CsA on delaying mPTP opening is due largely to its efficacy in maintaining free ss[Ca²⁺]_m by activating the MCBS in a P_i-dependent manner, and thereby preclude early mitochondrial Ca²⁺ overload and delay induction of mPTP opening. Sustained low ss[Ca²⁺]_e in the CsA-treated group indicated increasing mitochondrial Ca²⁺ uptake driven by the enhanced sequestration of free [Ca²⁺]_m to maintain a transmembrane Ca²⁺ gradient and a charged ΔΨ_m that facilitated additional Ca²⁺ uptake (Figure 2). Unlike previous studies [14,33,34], NCLX was blocked under our experimental conditions, to prevent Ca²⁺ efflux during the repetitive CaCl₂ additions; therefore, the net free ss[Ca²⁺]_m in our study was determined by the balance between Ca²⁺ uptake and Ca²⁺ sequestration.

Notably, the CsA-induced buffering of mitochondrial Ca²⁺ resulted in greater Ca²⁺ uptake to attain a steady-state, as shown by the gradual decrease in ss[Ca²⁺]_m with each added CaCl₂ pulse (Figure 3). Insofar as Ca²⁺–P_i precipitation is a major mechanism for mitochondrial Ca²⁺ buffering, the sustained ss[Ca²⁺]_m after each CaCl₂ bolus indicated matrix Ca²⁺ storage, likely in the form of various inorganic Ca–P_i complexes [14]. The low and maintained ss[Ca²⁺]_m during continuous matrix Ca²⁺ uptake is consistent with formation of these complexes. Although our study did not provide direct experimental evidence for CsA-induced matrix Ca–P_i complex formation, the continuous rise in estimated bound Ca²⁺:free Ca²⁺ ratio with each CaCl₂ bolus as well as the ten-fold increase in mβ_Ca clearly reflects a CsA effect on [Ca²⁺]_m buffering capacity (Figure 3).

The protective effect of CsA in delaying mPTP opening has long been reported [28,31,32]. Our findings; however, provide the first direct evidence for a novel effect of CsA to enhance the capacity of mitochondria to sequester Ca²⁺ by which it obviates Ca²⁺-induced mPTP formation. Moreover, the effect of CsA in mediating greater matrix Ca²⁺ buffering explains the sustained free [Ca²⁺]_m reported by Chalmers and Nicholls [14] and the CsA-induced inhibition of mitochondrial Ca²⁺ efflux observed in other prior studies [33,34].

4.2. Underlying Mechanism of the CsA-Mediated [Ca²⁺]_m Regulation

It is well established that mitochondria are able to sequester large amounts of Ca²⁺, while maintaining free [Ca²⁺]_m over a range of 0.1 and 10 μM depending on the Ca²⁺ load [14]; however, the mechanism and kinetics for this are unclear. Matrix Ca²⁺ buffering capacity is determined by: i) The quantity of Ca²⁺ that can be retained, and ii) the Ca²⁺ threshold level for release when Ca²⁺ exchangers are blocked or maximally operated [48]. The role of P_i as a physiological buffer in regulation of [Ca²⁺]_m has been extensively studied [14,44,45,49]. The major mechanism of P_i-mediated Ca²⁺ sequestration in mitochondria is believed to be achieved by formation of amorphous Ca²⁺–P_i complexes in the matrix [48,50,51], which in turn maintain the free [Ca²⁺]_m at a low level. Hence, sustained [Ca²⁺]_m cyclically promotes more Ca²⁺ uptake via the MCU due to better preservation of both the Ca²⁺ gradient and ΔΨ_m.

Though P_i plays an essential role in matrix Ca²⁺ buffering, P_i has also been suggested to induce mPTP opening [52]. A recent study associated Ca²⁺–P_i precipitation with complex I inhibition and reduced ATP synthase rate during Ca²⁺ overload [53]. Another report demonstrated that increasing [P_i] decreased the mitochondrial Ca²⁺ loading capacity [14]. It was suggested that the mPTP-sensitizing effects of P_i was likely due to its effect in decreasing matrix-free Mg²⁺, an mPTP inhibitor [20]. In addition, formation of polyphosphate, a known inducer of mPTP, could be a factor in regulating the Ca²⁺ threshold for mPTP activation [54,55]. Interestingly, two prior studies [56,57] indicated that P_i is necessary for the inhibitory effect of CsA on mPTP opening. However, two other studies reported that CsA inhibits mPTP opening even in the absence of P_i [58,59]. Conversely, in our study, the CsA-induced enhancement of matrix Ca²⁺ buffering was completely annulled when both mitochondria and the experimental medium were depleted of P_i (Figure 7). This loss of Ca²⁺ sequestration by CsA was reinstated when exogenous P_i was added just before activation of the mPTP (Figure 8). These observations provide the essential explanation for the requirement of P_i in the CsA-mediated MCBS and delay in mPTP opening.

The importance of mitochondrial matrix Ca²⁺ buffering via P_i is underscored by the studies of Wei et al. [44,45]. They reported that P_i modulates the total amount of Ca²⁺ uptake with smaller CaCl₂ boluses, whereas P_i modulates Ca²⁺ buffering capacity with larger CaCl₂ boluses. Since we had P_i in our experimental medium and the mitochondria were replete with exogenous P_i, the observation that CsA induced low ss[Ca²⁺]_e and ss[Ca²⁺]_m could be explained by the following: (i) CsA activates P_i-dependent matrix Ca²⁺ buffering potentially by maintaining the rate of Ca²⁺–P_i complex formation; and (ii) CsA may activate P_i transport processes (via H⁺/P_i transporter and/or phosphate carrier) that help to maintain both the IMM pH_m and ΔΨ_m gradients. These processes would limit the increase in free [Ca²⁺]_m, which in turn would contribute to more Ca²⁺ uptake and retention by increasing the electrochemical driving force for Ca²⁺ influx.

4.3. CsA vs. ADP; As a Regulator of [Ca²⁺]_m

AdN are implicated as one of the multiple matrix factors responsible for sequestering Ca²⁺ by mitochondria [29,60,61,62]. AdN can potentiate mitochondrial Ca²⁺ buffering by maintaining high matrix P_i concentrations that can facilitate precipitation of AdN-Ca–P_i complexes, including, ATP-Mg²⁻/P_i²⁻ and HADP²⁻/P_i²⁻, and thereby increase the Ca²⁺ threshold for mPTP opening [60,63]. In a study by Carafoli et al. [60], it was reported that mitochondrial Ca²⁺-buffering is proportional to mitochondrial ADP uptake. In our P_i-replete study, OMN+ADP had a relatively small effect on Ca²⁺ buffering compared to CsA, but it had a significantly larger effect than ADP or OMN alone (Figure 2, Figure 3 and Figure 5). A reasonable explanation could be that OMN, an ATP synthase (Complex V) inhibitor [64], could contribute towards augmenting the AdN pool and thus enhance matrix Ca²⁺ buffering. Consistent with our findings, a previous study also showed a greater CRC with a low-concentration of ADP with OMN compared to 10-fold larger concentration of ADP alone [30]. Thus, in agreement with Sokolova et al. [30], the observed high buffering capacity and expanded CRC with OMN+ADP is largely attributed to the ADP component of the matrix AdN pool. However, a previous study [62] reported that AdN also prevent mitochondrial Ca²⁺ influx by directly chelating Ca²⁺ by a Ca–ATP complexation [61]. Contrary to this observation, in our study, the direct effect of ADP on binding free Ca²⁺ was negligible, as assessed by adding ADP and CaCl₂ together in mitochondria-free experimental buffer (Figure S4). Additionally, carboxyatractyloside-mediated inhibition of ADP uptake via adenine nucleotide translocase precluded matrix Ca²⁺ buffering and blunted the CRC by OMN+ADP or ADP alone (Figure S5). In this case, the extra-matrix ADP that accumulated did not chelate the Ca²⁺ added to the buffer. Altogether, these observations indicate that a direct sequestration of Ca²⁺ outside the mitochondria does not explain the effect of ADP alone or OMN+ADP on the enhanced CRC in our study.

A previous study [42] from our group proposed that the MCBS relies on at least two classes of Ca²⁺ buffers. The first class could represent classical Ca²⁺ buffers, including mostly metabolites (ATP, ADP, and P_i) and mobile proteins that bind a single Ca²⁺ ion at a single binding site. A second class of buffers could be associated with the formation of amorphous Ca²⁺ phosphates, which may be capable of binding multiple Ca²⁺ ions at a single site in a cooperative fashion [35,38,39,42]. Genge et al. [65] showed, in an in vitro study, that annexins, a diverse class of proteins, are required for Ca²⁺-phosphate nucleation. Additionally, many studies have suggested an AdN-dependent Ca²⁺-binding property of annexins [66]. Interestingly, mitochondria exposed to ADP alone or OMN+ADP retained their ability to maintain low ss[Ca²⁺]_e and ss[Ca²⁺]_m for an extended period of cumulative CaCl₂ additions, and showed a higher Ca²⁺ threshold for mPTP opening without P_i compared to with P_i (Figure 7). This extended delay in mPTP opening in the P_i-depleted state compared to the P_i-replete state reflects the ability of P_i to induce early mPTP opening under certain conditions [52]. In this case, the presence of P_i appears to counteract the ADP delay effect and induce a much earlier pore opening compared to the P_i-depleted state. The mechanisms for this AdN-mediated massive matrix Ca²⁺ loading capacity in the absence of exogenous P_i is unclear and needs to be further investigated. A plausible hypothesis could be that, in the absence of P_i, a significant Ca²⁺ loading capacity of AdN might be mediated via direct interaction with annexins. CsA, on the other hand, might function as a mediator that activates a P_i-dependent Ca²⁺ buffering system. Another possibility is that Cyp D, as a PPIase, reduces free phosphate levels in the matrix or blocks the Ca²⁺ binding property of annexins; this then would be relieved by CsA’s effect to block Cyp D.

4.4. Implication of CsA-Mediated Ca²⁺ Buffering on Mitochondrial Bioenergetics

Elevated [Ca²⁺]_m over the nanomolar range is reported to increase NADH generation in part by stimulating Ca²⁺-sensitive dehydrogenases of the TCA cycle [67,68] and activating the F₀F₁-ATP synthase [69], thereby accelerating oxidative phosphorylation (OXPHOS). However, excess mitochondrial free Ca²⁺ can dissipate ΔΨ_m and impede OXPHOS. The IMM ΔΨ_m is the key factor in generating the proton motive force across the IMM; it is also one of the primary driving forces for Ca²⁺ uptake via the MCU [70] and triggers Ca²⁺ efflux via the NCLX [71,72]. Therefore, if mitochondria continue to take up Ca²⁺ under increased extra-matrix Ca²⁺ exposure, the Ca²⁺ would have to be buffered or ejected to prevent excess free [Ca²⁺]_m accumulation that could dissipate ΔΨ_m and increase oxidation of NADH.

The stability of Ca²⁺–P_i precipitates inside the mitochondrial matrix largely depends on pH_m [50]. It is also proposed that the matrix [P_i] depends on the pH gradient (e.g., a change in pH from 7 to 8 has been estimated to increase [P_i] by a factor of 1000 [14,50]). Thus, matrix alkaline conditions could facilitate Ca²⁺–P_i precipitation, whereas matrix acidification could lead to a destabilization of the Ca²⁺–P_i precipitate and so enhance matrix free Ca²⁺ levels [14]. Consequently, we correlated the changes in [Ca²⁺]_m with indices of mitochondrial bioenergetics (ΔΨ_m, NADH, and pH) (Figure 5) to have a better understanding of the CsA-mediated MCBS. Mitochondria exposed to CsA before the repetitive CaCl₂ boluses, exhibited robust mitochondrial Ca²⁺ uptake and rapid [Ca²⁺]_m buffering while maintaining basal ΔΨ_m, NADH, and an alkalinized pH_m until mPTP opened (Figure 5). Maintaining ΔΨ_m during excess Ca²⁺ uptake in the absence of functioning NCLX suggests a strong matrix buffering effect that is induced by CsA.

In Protocol B, when CsA was added just before the onset of pore opening, NADH and ΔΨ_m levels transiently increased but immediately returned to baseline with each added CaCl₂ bolus. This transient depolarization and NADH oxidation with each addition of CaCl₂ was not observed in Protocol A. The reason for this is unclear. Nonetheless, the observed transient oxidation of NADH helped to restore ΔΨ_m after Ca²⁺ induced transient depolarization before the next CaCl₂ bolus (Figure 5D,E). The transient redox oxidation and ΔΨ_m depolarization suggest that the CsA added at the point just before mPTP opening activated MCBS more slowly compared to Protocol A. In addition, CsA maintained the pH_m gradient during prolonged Ca²⁺ pulse challenges (Figure 5C). This finding also likely excludes a contribution of the mitochondrial calcium–hydrogen exchange (mCHE) to the Ca²⁺ extrusion in the absence of NaCl. We have recently reported that CsA obviates mCHE activity at low extra-matrix pH [19]. However, based on our current results, it is likely that CsA triggered an enhancement of mitochondrial Ca²⁺ buffering so that the resulting low [Ca²⁺]_m and maintained ΔΨ_m and pH_m accounted for the inactivity of mCHE.

5. Conclusions

The salient observation of this study is that CsA mitigated mPTP opening by promoting the maintenance of a low [Ca²⁺]_m, by stimulating and/or potentiating MCBS. Specifically, we showed that the presence of CsA, (i) significantly delayed the mPTP opening when compared to ADP or OMN+ADP (Protocol A); (ii) overturned the high amplitude increase in [Ca²⁺]_m (Protocol B); (iii) maintained pH_m, redox state (NADH) and basal ΔΨ_m, which maintains the driving force for more Ca²⁺ uptake and sequestration; and (iv) activates P_i-dependent mitochondrial Ca²⁺ sequestration to delay mPTP opening.

Our study provides a novel insight into how CsA-mediates a delay in mPTP opening by activating the MCBS, which lowers ss[Ca²⁺]_m below the threshold for mPTP activation. This concept is shown in the scheme presented in Figure 9. Our finding supports the notion that CsA facilitates P_i-dependent matrix Ca²⁺ buffering, which maintains matrix free Ca²⁺ and enables massive Ca²⁺ loading capacity, without diminishing the driving force for Ca²⁺ influx by maintaining ΔΨ_m. CsA may delay mPTP opening by enhancing P_i-dependent matrix Ca²⁺ buffering and by inhibiting Cyp D [31,32]. The culmination of these two mechanisms, and possibly others not yet identified, might be responsible for CsA protection against mitochondrial Ca²⁺ overload. Together, these findings add to our understanding of the mechanism of CsA-mediated modulation of mPTP. Importantly, we believe that therapeutic approaches targeted at regulating [Ca²⁺]_m homeostasis represent a promising strategy to reduce cardiac injury due to Ca²⁺ overload by delaying mPTP opening and preventing induction of apoptosis.