Enhanced resistance to Ca²⁺-induced mitochondrial permeability transition in the long-lived red-footed tortoise Chelonoidis carbonaria

Intracellular Ca²⁺ modulates many physiological processes through transient increases in its free concentration across various cellular compartments (Berridge et al., 2003; Bagur and Hajnóczky, 2017). Mitochondria from vertebrates can take up cytosolic Ca²⁺ across its transmembrane proton electrochemical gradient (Deluca and Engstrom, 1961; Vasington and Murphy, 1962; Mammucari et al., 2018). Mitochondrial Ca²⁺ influx occurs via a calcium uniporter (MCU), present in the inner membrane, which is highly conserved among eukaryotes (Baughman et al., 2011; De Stefani et al., 2011; Docampo et al., 2014). Mitochondrial Ca²⁺ efflux occurs via two independent pathways: a Ca²⁺/H⁺ antiporter and a Na⁺/Ca²⁺ exchanger (Chweih et al., 2015; De Stefani et al., 2016; Nicholls, 1978; Nicholls and Crompton, 1980). Mitochondrial Ca²⁺ uptake was demonstrated in situ at physiological conditions after stimulation of Ca²⁺ release from the endoplasmic reticulum, generating a transient increase in Ca²⁺ concentration (∼10–20 μmol l⁻¹) at points of close contact between the endoplasmic reticulum and mitochondria, defined as microdomains, kinetically allowing mitochondrial Ca²⁺ influx (Rizzuto and Pozzan, 2006; Rizzuto et al., 1993).

Intramitochondrial Ca²⁺ is essential for controlling oxidative phosphorylation by activating mitochondrial Ca²⁺-sensitive enzymes: pyruvate dehydrogenase phosphatase, and α-ketoglutarate and NAD⁺-dependent isocitrate dehydrogenases (McCormack and Denton, 1979; Denton et al., 1980; Hansford and Castro, 1985; Denton, 2009). Thus, changes in cytosolic Ca²⁺ concentrations are associated with the regulation of Krebs cycle activity, providing reducing equivalents to the electron transport chain under increased cell ATP demand (Glancy and Balaban, 2012; Heineman and Balaban, 1990). Intramitochondrial Ca²⁺ also signals reactive oxygen generation (Kowaltowski et al., 2001; Feissner et al., 2009; Figueira et al., 2013), and there is extensive evidence of a synergistic association between mitochondrial Ca²⁺ overload and oxidative imbalance in the promotion of the phenomenon of the mitochondrial permeability transition (MPT) (Crompton, 1999; Kowaltowski et al., 2001; Vercesi et al., 2018).

The MPT is characterized by the permeabilization of the mitochondrial inner membrane through the opening of a non-specific pore, causing the loss of ions and proteins less than 1.5 kDa and the influx of water as a result of trapped large proteins in the mitochondrial matrix, leading to the swelling of mitochondria, loss of membrane potential and energy-linked functions (Gunter and Pfeiffer, 1990; Haworth and Hunter, 2000; Vercesi et al., 2018; Zoratti and Szabò, 1995). Mitochondria are more susceptible to MPT when the antioxidant systems are overwhelmed, especially when NADPH is exhausted (Kowaltowski et al., 2009; Lehninger et al., 1978; Ronchi et al., 2013; Vercesi, 1987). Studies showed that prooxidants can induce MPT while antioxidants can prevent or even reverse MPT (Castilho et al., 1995; Kowaltowski et al., 2001; Valle et al., 1993), suggesting that the phenomenon is redox sensitive. MPT is related to several pathophysiological states, including anoxia–reperfusion injury, neurodegenerative diseases and aging (Figueira et al., 2013; Rottenberg and Hoek, 2017, 2021); MPT was shown to be favored in aged animals and humans (Crompton, 2004; Di Lisa and Bernardi, 2005; Goodell and Cortopassi, 1998; Mather and Rottenberg, 2000; Paradies et al., 2013; Rottenberg and Wu, 1997; Toman and Fiskum, 2011). MPT is believed to accelerate aging processes because it increases the release of reactive oxygen species (ROS), which in excess damage proteins, lipids and DNA, and deplete cellular NAD (Rottenberg and Hoek, 2017, 2021), leading to global mitochondrial dysfunction, one hallmark of aging (López-Otín et al., 2013).

Despite enormous efforts to discover the structural constituents of the MPT pore, there remains no consensus regarding its molecular identity. Several models have been proposed, suggesting many matrix, inner membrane and outer membrane proteins as the structural components, including the adenine nucleotide transporter (ANT), ATP synthase, cyclophilin D (CyPD), hexokinase complex, the phosphate carrier (PiC), the spastic paraplegia 7 protein, and the voltage-dependent anion channel (VDAC) (Crompton et al., 1998; Giorgio et al., 2010, 2013; Halestrap et al., 1997a; Leung et al., 2008; Shanmughapriya et al., 2015). However, there remains no unequivocal evidence for considering these proteins essential for MPT pore formation (Baines et al., 2005; Basso et al., 2005; Gutiérrez-Aguilar et al., 2014; He et al., 2017; Kokoszka et al., 2004). The growing body of literature regarding the redox sensitivity of the MPT led to the proposal of a model in which a non-selective pore would be formed by oxidation of membrane protein thiols, resulting in disulfide bonds and protein aggregation (Fagian et al., 1990; He and Lemasters, 2002; Kowaltowski et al., 2001). Although the proteins that oxidize and assemble to form the MPT pore have not yet been determined conclusively, several pieces of evidence suggest that either ANT (Carrer et al., 2021a; Halestrap and Davidson, 1990; Woodfield et al., 1998) or ATP synthase (Burstein et al., 2018; Gauba et al., 2019; Giorgio et al., 2009, 2013) interact with CyPD and participate in the process of MPT pore formation. Supporting studies revealed the critical role of redox-sensitive cysteines present in ANT that are subjected to thiol cross-linking, favoring CyPD binding and facilitating MPT pore opening (Halestrap and Brenner, 2005; McStay et al., 2002).

Turtles and tortoises that comprise the Testudines group exhibit negligible senescence; with age, they exhibit decreased rates of mortality and increased rates of fecundity (Cayuela et al., 2019; Congdon et al., 2003; Gibbons, 1987; Jones et al., 2014), suggesting that these animals would be attractive models for the study of senescence (Hoekstra et al., 2020; Krivoruchko and Storey, 2010; Quesada et al., 2019). Chelonoidis carbonaria (Spix 1824), popularly known as the red-footed tortoise, is widely distributed throughout South America and possesses a maximum lifespan of 49 years (AnAge database; De Magalhães and Costa, 2009). We hypothesized that properties of mitochondrial function could partially explain the longevity of tortoises; among those, a lower susceptibility to MPT in comparison to well-known models of short-lived species, such as rats. Despite interesting literature available for turtle mitochondria, especially anoxic-tolerant species (Bundgaard et al., 2018, 2020; Galli and Richards, 2014; Galli et al., 2013; Pamenter et al., 2016), we still have a poor knowledge on mitochondrial function in this group. Therefore, we evaluated C. carbonaria liver mitochondrial oxygen consumption and susceptibility to Ca²⁺-induced MPT.

Juvenile C. carbonaria tortoises (pre-reproductive maturity; 3–4 years old; mean body mass 0.50 kg, range 0.35–0.73 kg, N=7) were obtained from a scientific breeding facility (IBAMA 673766) at the University of São Paulo State (Rio Claro, SP, Brazil). Adult tortoises (males, undetermined age, mean body mass 5.2 kg, range 4.1–6.7 kg, N=4) were used only for one of the assays, because of a shortage of juvenile individuals. Adult tortoises were kept in outdoor enclosures, and juvenile tortoises were maintained in containers (1.60 m long×0.94 m wide) with light:dark cycles of 12 h:12 h provided by incandescent light bulbs. Animals were fed daily ad libitum with fresh vegetables and fruits and eventually supplemented with ground meat and dog food pellets. Freshwater was provided daily. For experiments, animals were transported to the Laboratory of Bioenergetics at UNICAMP, Campinas, SP, Brazil. Tortoises were sedated with midazolam (2 mg kg⁻¹ i.m.), anesthetized with ketamine (40 mg kg⁻¹ i.m.) and propofol (15 mg kg⁻¹ i.v.), and killed by exsanguination. Male Wistar rats (HanUnib:WH; 4–6 months old; mean body mass 0.58 kg, range 0.50–0.65 kg, N=11) were obtained from CEMIB UNICAMP (Campinas, SP, Brazil) and used as a reference standard. Rats were maintained at the animal facility of our department, housed at 22±2°C on a 12 h:12 h light:dark cycle with free access to a standard laboratory rodent chow (Nuvital CR1; Nuvital, Curitiba, PR, Brazil) and tap water. Rats were anesthetized with vaporized isoflurane and immediately exsanguinated. A local committee approved all experimental procedures according to Ethics in Animal Experimentation (CEUA/UNICAMP: 4745-1/2017 for tortoises and 4691-1/2017 for rats). The Brazilian Institute for Environment authorized the use of C. carbonaria tortoises (SISBIO; number 58792-1).

Liver mitochondria were isolated by conventional differential centrifugation as described previously (Ronchi et al., 2013). Briefly, fractions of tortoise and rat liver (approximately 10 g) were rapidly removed, finely minced, and homogenized in an ice-cold isolation medium containing 250 mmol l⁻¹ sucrose, 1 mmol l⁻¹ EGTA and 10 mmol l⁻¹ Hepes buffer (pH 7.2). The homogenates were centrifuged for 10 min at 800 g and the supernatants collected. To remove residual fat from the supernatants, we used a nylon mesh filter for tortoises, and careful aspiration with a fine glass pipette for rats, before a second centrifugation at 7750 g for 10 min. The resulting pellet was resuspended in buffer containing 250 mmol l⁻¹ sucrose, 0.3 mmol l⁻¹ EGTA and 10 mmol l⁻¹ Hepes buffer (pH 7.2) and centrifuged again at 7750 g for 10 min. The final pellets containing liver mitochondria were resuspended in an EGTA-free buffer at approximately 60 mg ml⁻¹ protein concentration. Mitochondrial protein content was determined by the Bradford method with bovine serum albumin as standard, and used to normalize data in mg protein ml⁻¹. Mitochondrial suspensions were maintained in ice baths and used within 4 h. Liver mitochondria from juvenile tortoises were used for most of the experiments, except for the titration of inhibitors of oxidative phosphorylation components, in which mitochondria from adults were used.

Measurements of mitochondrial Ca²⁺ retention capacity in suspensions of intact mitochondria (0.5 mg protein ml⁻¹ in standard reaction medium) were performed at 28°C with continuous magnetic stirring. A standard reaction medium (125 mmol l⁻¹ sucrose, 65 mmol l⁻¹ KCl, 2 mmol l⁻¹ KH₂PO₄, 1 mmol l⁻¹ MgCl₂, 10 mmol l⁻¹ Hepes buffer with pH adjusted to 7.2 with KOH) was supplemented with 10 μmol l⁻¹ EGTA, 0.2 μmol l⁻¹ of a calcium indicator (Calcium Green™-5N), and NADH-linked respiratory substrates (1 mmol l⁻¹ malate, 2.5 mmol l⁻¹ pyruvate and 2.5 mmol l⁻¹ glutamate). The reaction medium contained approximately 28 μmol l⁻¹ contaminant Ca²⁺, provenient from its components, as KCl. Also, the ultrapure water used in our laboratory contains 12–15 μmol l⁻¹ contaminant Ca²⁺. Fluorescence was continuously monitored in a fluorescence spectrophotometer (Hitachi F-4500, Tokyo, Japan) using excitation and emission wavelengths of 506 and 532 nm, respectively, and slit widths of 5 nm.

Multiple pulses of CaCl₂ (30 μmol l⁻¹ for tortoises and 2.5 μmol l⁻¹ for rats) were added after inclusion of mitochondria in the system. The sum of free Ca²⁺ initially present in the reaction medium plus the amount of CaCl₂ added before the start of mitochondrial Ca²⁺ release into the medium (i.e. MPT pore opening) was considered the Ca²⁺ retention capacity. The influence of cyclosporine A (CsA) and ADP as inhibitors of MPT was also tested by evaluating the Ca²⁺ retention capacity under the following conditions: 1 μmol l⁻¹ CsA, 300 μmol l⁻¹ ADP, and 1 μmol l⁻¹ CsA plus 300 μmol l⁻¹ ADP. When ADP was present, the medium was also supplemented with 1 μg ml⁻¹ oligomycin to inhibit ATP synthase. Because there was an increase in mitochondrial Ca²⁺ accumulation in the presence of CsA or ADP, pulses with higher Ca²⁺ concentrations were added at 60 μmol l⁻¹ for tortoises and 20 μmol l⁻¹ for rats. The dissociation constant (K_d) of 26.8 μmol l⁻¹ for the probe Calcium Green™-5N was experimentally determined in the incubation condition, as previously described (Sartori et al., 2021). Raw fluorescence readings were converted into Ca²⁺ concentration levels (expressed as micromolar) according to a hyperbolic equation: [Ca²⁺]=26.8×[(F−F_min)/(F_max−F)], where F is any given fluorescence, F_min is the lowest fluorescence reading after addition of 0.5 mmol l⁻¹ EGTA, and F_max is the maximal fluorescence obtained after two sequential additions of 1 mmol l⁻¹ CaCl₂. F_min and F_max were determined at the end of each experiment.

Respiration measurements in suspensions of intact liver mitochondria were performed using a high-resolution oxygraph (OROBOROS, Innsbruck, Austria) at 28°C with continuous magnetic stirring. Mitochondria (0.5 mg protein ml⁻¹) were suspended in 2 ml of standard reaction medium supplemented with 200 μmol l⁻¹ EGTA. We used the respiratory substrates 1 mmol l⁻¹ malate, 2.5 mmol l⁻¹ glutamate and 2.5 mmol l⁻¹ pyruvate, which generate NADH, promoting respiration through respiratory complex I. After measuring the basal O₂ consumption, 300 μmol l⁻¹ of ADP was added to elicit oxidative phosphorylation (OXPHOS). Then, 1 μg ml⁻¹ of oligomycin was added to cease the phosphorylation by ATP synthase (respiratory state 4_o). Finally, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was titrated until reaching maximal oxygen consumption to estimate electron transport system (ETS) capacity. The coupling of mitochondria was calculated by the ratio of OXPHOS/state 4_o, known as the respiratory control ratio, and the limitation of the phosphorylation system by the ratio of OXPHOS/ETS.

Flux control coefficient (FCC) and estimated maximal inhibitory concentration for ANT, PiC and ATP synthase were determined by the titration of each respective inhibitor (carboxyatractyloside, mersalyl, or oligomycin) on ADP-stimulated respiration (OXPHOS) (Fig. S1A,C). The percentage of ADP-stimulated oxygen consumption was plotted against increasing concentrations of the inhibitor, and a graphical method was applied for FCC estimation (Fig. S1B,D), considering the linear regression of the initial data points and the maximal inhibition of ADP respiration, as previously described (Fell, 1992; Gellerich et al., 1990; Small, 1993). A FCC value close to one indicates that this component of the phosphorylation system exerts robust control over oxidative phosphorylation. Using the same plots, we estimated the maximal inhibitory concentration for each inhibitor. For this, the intersection of the minimal respiration rate with the maximal inhibition rate was used to estimate the amount of inhibitor that elicits the maximal inhibition of the respiratory rate (Fig. S1). Because of difficulties in obtaining juvenile tortoise specimens, this set of data was obtained from adult tortoises, which exhibited similar OXPHOS/ETS ratios (M.R.S., R.F.C. and A.E.V., unpublished data).

Current models for MPT suggests that opening of the pore requires the cyclophilin peptidyl-prolyl cis–trans isomerase (PPIase) activity of CyPD (Baines et al., 2005; Gutiérrez-Aguilar and Baines, 2015; Halestrap and Davidson, 1990). Catalytic PPIase activity was monitored as the cis-to-trans isomerization of prolyl peptide bonds using a PPIase fluorogenic substrate (Suc-Ala-Leu-Pro-Phe-AMC, Sigma #SCP0217) by measurement of AMC fluorescence. The assay was based on Fischer et al. (1989) with modifications for use of a fluorescent product (García-Echeverría et al., 1992; Uchida et al., 2003). Because the peptide bond can only be cleaved by chymotrypsin when in the trans conformation, the rate of cleavage of the cis bond depends upon the rate of cis–trans isomerization. The PPIase substrate was dissolved in trifluoroethanol containing 480 mmol l⁻¹ LiCl to increase the proportion of cis bonds. The fluorescence was continuously monitored in a fluorescence spectrophotometer (Hitachi F-7100, Tokyo, Japan) using excitation and emission wavelengths of 365 and 460 nm, respectively, and slit widths of 2.5 nm at a controlled temperature maintained by a water bath at 12°C. Mitochondria (5 µg protein ml⁻¹) were suspended in a medium containing 40 mmol l⁻¹ Hepes and 0.05% Triton X-100 pH 7.8 adjusted with KOH in a final volume of 2 ml. After 100 s, 10 µmol l⁻¹ α-chymotrypsin (Sigma #C4129) was added, followed by 10 µmol l⁻¹ of the PPIase substrate Suc-Ala-Leu-Pro-Phe-AMC at 130 s. Fluorescence changes were measured every 0.1 s, and each sample was measured in the presence and absence of 1 µmol l⁻¹ CsA. CsA binds to CyPD, inhibiting its catalytic effect, leaving only the uncatalyzed isomerization activity. Activity was determined as the slope of the curves using a 100 data-point interval (a linear range) 10 s after substrate addition, which caused an initial steep increase in fluorescence as a result of the rapid hydrolysis of the trans peptide. CyPD activity was reported as the difference of the uncatalyzed activity (in the presence of CsA) from the catalyzed activity (absence of CsA) of each sample.

H₂O₂ release by isolated liver mitochondria was measured by converting Amplex™ UltraRed (Thermo Fisher Scientific, Eugene, OR, USA) to fluorescent resorufin in the presence of horseradish peroxidase (HRP). Phenylmethyl sulfonyl fluoride (PMSF) was present in the assays to inhibit the conversion of the Amplex™ UltraRed to resorufin by carboxylesterases independently of H₂O₂ (Miwa et al., 2016). Suspensions of mitochondria (0.5 mg protein ml⁻¹) were incubated in standard reaction medium containing 1 mmol l⁻¹ malate, 2.5 mmol l⁻¹ glutamate and 2.5 mmol l⁻¹ pyruvate as respiratory substrates, 10 μmol l⁻¹ Amplex™ UltraRed, 1 U ml⁻¹ HRP, 75 U ml⁻¹ superoxide dismutase and 100 μmol l⁻¹ PMSF. Fluorescence was monitored over time with a temperature-controlled spectrofluorometer (Hitachi F-4500, Tokyo, Japan) using excitation and emission wavelengths of 563 and 586 nm, respectively, and slit widths of 5 nm. For calibration, known amounts of H₂O₂ were added to the mitochondrial reaction medium without samples.

GSH and GSSG were determined in isolated mitochondria using an enzymatic recycling method (Rahman et al., 2007; Teare et al., 1993). Briefly, mitochondrial proteins were suspended to a final volume of 25 μl in water (1:5 ratio), and then a solution (1:1 ratio) of 11% sulfosalicylic acid and 0.1% Triton X-100 was added to these samples. After 5 min incubation at 4°C under intermittent vortexing, the samples were centrifuged at 10,000 g for 10 min (4°C), and the supernatants were saved for subsequent analysis of total glutathione levels. To measure only GSSG, 10 μl of this supernatant was added to 110 μl of a GSH masking buffer containing 100 mmol l⁻¹ phosphate buffer, 1 mmol l⁻¹ EDTA, 1.1% 2-vinylpyridine, pH 7.4, and incubated for 1 h at room temperature. The samples prepared for total glutathione and GSSG were then subjected to enzymatic recycling analysis in a recycling buffer system containing 300 μmol l⁻¹ NADPH, 225 μmol l⁻¹ DTNB, 1.6 U ml⁻¹ glutathione reductase and 1 mmol l⁻¹ EDTA in 100 mmol l⁻¹ phosphate buffer (pH 7.4). The linear increase in absorbance at 412 nm over time was monitored using a microplate reader (PowerWave XS 2, BioTek Instruments, Winooski, VT, USA). A standard curve was generated using known amounts of GSH and GSSG.

Two-tailed unpaired Student's t-tests were used to analyze differences between tortoise and rat data. Data are means±s.d.; sample sizes are indicated in figure legends. Tests were considered significant at the 95% level of confidence (P<0.05). GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis and plotting graphs.

Ca²⁺ retention capacity in tortoise and rat liver mitochondria was used to assess their susceptibility to MPT. Tortoise liver mitochondria exhibited a significantly higher Ca²⁺ retention capacity than rat mitochondria, as depicted in Fig. 1A,B, which shows representative experiments of mitochondria oxidizing NADH-linked substrates subjected to successive Ca²⁺ pulses until MPT pore opening-mediated Ca²⁺ release. Because of the increased capacity of Ca²⁺ uptake in tortoises, the concentration of added Ca²⁺ in each pulse was higher (30 μmol l⁻¹) than in rats (2.5 μmol l⁻¹). In control conditions, tortoise mitochondria retained 273±99 nmol Ca²⁺ mg⁻¹ protein versus 65±7 nmol Ca²⁺ mg⁻¹ protein in rats (Fig. 1C). The inhibitory effects of CsA and ADP against MPT pore opening, well documented in mammalian literature (Halestrap et al., 1997b; Saito and Castilho, 2010; Woodfield et al., 1998; Zoratti and Szabò, 1995), were also observed in tortoise mitochondria. The presence of ADP or CsA increased the Ca²⁺ retention capacity in tortoise and rat mitochondria while maintaining a significantly higher Ca²⁺ retention capacity in tortoises (Fig. 1C). In the presence of CsA plus ADP, both mitochondria exhibited the highest Ca²⁺ retention capacity; however, the difference between tortoises and rats was no longer observed (1005±215 nmol Ca²⁺ mg⁻¹ protein in tortoises and 1232±294 nmol Ca²⁺ mg⁻¹ protein in rats) (Fig. 1C).

Assessment of liver mitochondrial oxygen consumption revealed a remarkably reduced OXPHOS capacity of tortoise mitochondria. As shown in the representative traces of Fig. 2A, ADP elicited only a modest increase in the respiration rate of tortoises compared with rats. For tortoise mitochondria, a second addition of 0.3 mmol l⁻¹ ADP was performed to check whether the ADP concentration was limiting OXPHOS, but respiration did not increase further (data not shown). ADP-stimulated respiration in tortoise mitochondria was also much lower than the maximal respiration rates (i.e. ETS capacity) observed in both tortoise and rat after the addition of FCCP. As a result, the OXPHOS capacity of tortoises was 75% lower than that of rats (Fig. 2B). Basal and state 4_o respiratory states in tortoises were 56% and 37% lower than in rats, respectively, while ETS capacity was similar between the species (Fig. 2B). Because of a low OXPHOS capacity, tortoises also exhibited a lower respiratory control ratio (4.8±0.9 in tortoises and 12.0±1.4 in rats; Fig. 2C) and a lower OXPHOS/ETS ratio (0.32±0.04 in tortoises and 1.13±0.21 in rats; Fig. 2D) than rats.

Based on the limitation observed in tortoise OXPHOS capacity, which led to a low OXPHOS/ETS ratio, we then determined the flux control coefficient (FCC) of the putative limiting enzymatic steps of the phosphorylation system, i.e. ANT, PiC and ATP synthase. FCC is one of the fundamental concepts of metabolic control analysis (Small, 1993), which establishes that metabolic control is shared by enzymes rather than one limiting step in a pathway. FCC can be determined experimentally and applied to understand how an enzyme exerts low or high control over a metabolic flux, and in the case of mitochondria, over respiration and oxidative phosphorylation (Moreno-Sánchez and Torres-Márquez, 1991; Rossignol et al., 2000).

Fig. 3A–C depicts representative titration curves of mitochondrial ADP-stimulated respiration with the respective inhibitors of the phosphorylation system. FCC values close to 1 indicate a rate-limiting enzyme. Rats and tortoises exhibited similar FCC values for ATP synthase and PiC (Fig. 3D). For ANT, the shape of the carboxyatractyloside titration curve of tortoise mitochondria showed a sharp fold at a low concentration of the inhibitor (Fig. 3A), which is consistent with a high FCC value (∼1), demonstrated by the model of Gellerich et al. (1990). The steepness of the titration curves in the two species was similar, suggesting no change in dissociation constants of the carboxyatractyloside inhibition between the species. For ANT, the FCC calculated for tortoises was 0.97±0.05 in contrast to rats which was 0.42±0.14 (Fig. 3D), revealing that ANT seems to be the main limiting factor of OXPHOS in tortoise liver mitochondria. The estimated concentrations of each inhibitor that elicited the maximal inhibition of OXPHOS are described in Table 1. Carboxyatractyloside and mersalyl concentrations were respectively 38% and 23% lower in tortoises than in rats.

Next, the PPIase activity of CyPD was measured. Representative traces of the increase in fluorescence due to cleavage of the PPIase peptide substrate in the presence and absence of CsA in tortoise and rat liver mitochondria are shown in Fig. 4A. In the presence of CsA, CyPD is inhibited, and the increase of fluorescence corresponds to uncatalyzed activity, resulting from the slow-rate spontaneous isomerization of the substrate peptide in the cis form. In the absence of CsA, CyPD catalyzes the cis–trans isomerization. CyPD activity was determined by subtracting the respective slopes of fluorescence increase over time from the catalyzed and uncatalyzed isomerase activities. The results showed that tortoise CyPD activity was 68% lower than that of rats (Fig. 4B).

The influence of redox control on MPT was examined in tortoises and rats by measuring mitochondrial H₂O₂ release and glutathione redox status (GSH/GSSG). H₂O₂ release refers to the balance between the amount of H₂O₂ generated and the amount that is scavenged by the antioxidant system. The glutathione system, composed of GSH/GSSG, is a significant mitochondrial antioxidant defense mechanism. GSH is a tripeptide containing cysteine residues that directly scavenge ROS or act as a cofactor for glutathione peroxidase, which oxidizes glutathione to reduce H₂O₂. GSH is subsequently reduced by glutathione reductase, which uses NADPH as a substrate. The results showed that tortoise mitochondrial H₂O₂ release during basal conditions (12.3±2.3 pmol mg⁻¹ protein min⁻¹) was similar to that for rats (12.6±1.1 pmol mg⁻¹ protein min⁻¹) (Fig. 5A). A similar GSH/GSSG ratio was also observed, with 6.1±1.0 for tortoises and 6.0±1.3 for rats (Fig. 5B).

MPT is highly conserved across taxa (Azzolin et al., 2010a; Vianello et al., 2012), occurring with similar properties to those of mammals in a range of vertebrates from fishes (Azzolin et al., 2010b; Belyaeva et al., 2014; Toninello et al., 2000) to amphibians (Hanada et al., 2003), reptiles (da Mota Araujo et al., 2021; Dubinin et al., 2019; Hawrysh and Buck, 2013; Pamenter et al., 2016) and birds (Dubinin et al., 2016; Su et al., 2011; Vedernikov et al., 2015). The present study found that tortoise liver mitochondria exhibited similar features to mammalian MPT, responding to inhibitors such as CsA and ADP similarly to rat liver mitochondria. CsA binds to mitochondrial CyPD, strongly inhibiting MPT pore opening (Broekemeier et al., 1989; Javadov et al., 2017), while ADP decreases the sensitivity to Ca²⁺-induced pore opening after binding to ANT (Halestrap et al., 1997a). Tortoise mitochondria exhibited a remarkably higher Ca²⁺ retention capacity than rats, implying a lower susceptibility to MPT when challenged with Ca²⁺ overload. Surprisingly, the significant difference in Ca²⁺ retention capacity between tortoises and rats found under control conditions or in the presence of either ADP or CsA was no longer observed after MPT inhibition with ADP plus CsA (Fig. 1C). These findings suggest that the mechanism for the lower susceptibility to MPT of tortoise mitochondria involves ANT and CyPD, and a possible interaction between these two proteins, as discussed below.

Remarkable differences were also observed in respiration rates between tortoise and rat mitochondria. The very low OXPHOS/ETS ratio of tortoise liver mitochondria (Fig. 2D) indicated that only approximately 30% of the maximal respiration capacity was used for ADP oxidative phosphorylation. This is a very low level compared with the OXPHOS/ETS ratios reported in the literature (Lemieux and Warren, 2012). The observed OXPHOS limitation raises the question of whether one or more of the phosphorylation system components (i.e. ANT, PiC and ATP synthase) were restricted in number or activity level. Taken together, the similar Ca²⁺ uptake capacities in the presence of CsA plus ADP between tortoises and rats and the OXPHOS limitation presented by tortoises suggest functional differences in proteins considered putative components of the MPT pore. In fact, ATP synthase, ANT, PiC and CyPD have been identified as components or regulators of the MPT pore (Elrod and Molkentin, 2013; Vercesi et al., 2018).

FCC analysis indicated that ANT was a more crucial limiting component of OXPHOS in tortoise (Fig. 3) than in rat mitochondria, while there were no significant differences in FCC for PiC and ATP synthase between the species. Indeed, the maximal inhibitory concentration of carboxyatractyloside (ANT inhibitor) was also significantly lower for tortoises than for rats, corroborating the evidence that ANT is present in a lower amount in tortoise liver mitochondria. The maximal inhibitory concentration of mersalyl (PiC inhibitor) also differed between tortoises and rats, but to a smaller magnitude. However, mersalyl may react with thiol groups from other proteins present in mitochondria (Fonyo and Vignais, 1980; Kowaltowski et al., 1997), which could modify its concentration for maximal OXPHOS inhibition. Therefore, our results strongly suggest that ANT is one important constraint for the oxidative phosphorylation of liver tortoise mitochondria.

ANT, a transmembrane protein that mediates ADP/ATP exchange between the cytosol and matrix, has long been suggested to be a structural component of the MPT pore (Haworth and Hunter, 2000; LêQuôc and LêQuôc, 1988). However, mitochondria from mice knockouts of isoforms ANT1 and ANT2 still triggered MPT pore opening, although requiring higher Ca²⁺ concentrations (Kokoszka et al., 2004). A recent study suggested that this phenomenon may represent the compensatory effects of an increase of ANT4, which was shown to occur in Ant1/Ant2-knockouts (Karch et al., 2019). Nevertheless, another pathway for MPT pore formation involving ATP synthase cannot be discarded (Carrer et al., 2021a,b). Considering the redox state, ANT is subject to oxidation of critical cysteine residues (McStay et al., 2002; Woodfield et al., 1998), and there is evidence of ANT thiol oxidation via the mechanism of NADP⁺-stimulated Ca²⁺ release from mitochondria (Vercesi, 1984). Regardless of the debate, ANT is still considered a major component of the MPT pore, and our findings suggest that a constraint in ANT, through either lower activity or lower content, is an important mechanism involved in the enhanced resistance to Ca²⁺-induced MPT in tortoise mitochondria.

MPT pore opening is inhibited by CsA, which targets cyclophilins, a class of proteins possessing peptidyl-prolyl cis–trans isomerase activity (Broekemeier et al., 1989; Halestrap and Davidson, 1990; Takahashi et al., 1989). The isomerase domain of CyPD was shown to be essential for modulation of the MPT pore (Baines et al., 2005), demonstrating that CyPD probably regulates the MPT pore while not directly involved in the molecular pore structure (De Marchi et al., 2006). Notably, CyPD is highly conserved in eukaryotes. The ppif gene (encoding CyPD) from zebrafish possesses 78% homology with human ppif, while it is also associated with the MPT pore (Azzolin et al., 2010b; Elrod and Molkentin, 2013). Mitochondria from mice lacking the ppif gene required twice the amount of Ca²⁺ for MPT pore opening than wild-type mitochondria (Baines et al., 2005; Basso et al., 2005; Nakagawa et al., 2005; Schinzel et al., 2005). Thus, the lower CyPD activity found in tortoises strongly suggests that a constraint on CyPD might mediate MPT resistance in this species.

Interestingly, CyPD modulation is also implicated in aging processes. In the filamentous fungus Podospora anserina, a model organism for the study of aging, overexpression of PaCyPD increased mitochondrial dysfunction, accelerated the process of aging, and induced cell death in comparison to wild-type organisms (Brust et al., 2010). In addition, inhibition of PaCyPD by treatment with CsA promoted increased lifespan of P. anserina (Brust et al., 2010). Studies in mice showed that partial (heterozygous) deletion of CyPD resulted in a longer lifespan than wild-type or mice with a complete deletion of CyPD (Vereczki et al., 2017). The results of the present study, combined with these other findings, suggest the potential beneficial effects of low CyPD activity on the susceptibility to MPT pore opening and lifespan modulation.

A CyPD–ANT interaction was recently demonstrated in a study showing the lack of Ca²⁺-induced MPT in liver mitochondria from mice with deletion of the three isoforms of ANT (Ant1, Ant2, Ant4) and treated with CsA or with deletion of the ppif gene (Karch et al., 2019). Therefore, despite the mechanistic function of ANT and CyPD in MPT pore formation not being completely understood, and the fact that they might not be the sole entities comprising the pore, various proposed models of MPT pore opening consider essential roles for these proteins, particularly those induced by mitochondrial redox imbalance (Algieri et al., 2021; Carrer et al., 2021a,b; Karch et al., 2019; Kowaltowski et al., 2001). Oxidative stress can trigger conformational changes in the putative MPT pore proteins, facilitating CyPD binding and triggering pore opening (Halestrap and Brenner, 2005; Vercesi et al., 2018; Carraro et al., 2020).

Finally, we assessed the redox state of mitochondria. Tortoise mitochondria displayed similar levels of H₂O₂ release and glutathione redox state to rats. The combination of these results suggests that the mitochondrial redox status of tortoises and rats is similar and therefore is not directly involved in the observed differences of MPT pore opening between them. Our findings suggest that constraints on ANT, an essential target of oxidative modifications, and CyPD, which binds to ANT after oxidation, are critical factors underlying the differences in MPT susceptibility demonstrated between these species. A previous study from our group revealed that liver mitochondria from the short-lived marsupial Gracilianus microtarsus, which demonstrated a higher susceptibility to MPT, possessed similar coupling and OXPHOS respiration rates but a lower content and state of reduction of NADP than mice (Ronchi et al., 2015). Despite no investigations of the putative MPT pore proteins in that study, those results illustrate important species-specific differences in MPT sensitivity in the context of lifespan, which could be further investigated.

Overall, our data suggest that ANT constraint and the lower activity of CyPD in tortoise mitochondria may lead to fewer proteins that can assemble to form the pore structure after oxidative modifications. Furthermore, the enhanced resistance to MPT found in red-footed tortoises could be associated with the slower senescence rates and increased lifespan of the species. Nevertheless, more comparative studies investigating mitochondrial function and the susceptibility to MPT in several species of contrasting lifespans are necessary to provide more insights into the role of mitochondria in the field of comparative aging biology.

We are thankful to Prof. Augusto Shinya Abe for providing animals and to Dr Rafael Campos for assistance with animal handling.