Wild type (WT) C57BL/6J and Ppif ^−/− 2–3 month old male and female mice were utilized for the ex vivo mitochondrial isolations. Ppif ^−/− mice were previously generated as described (Baines et al., 2005; Nakagawa et al., 2005). All experimental procedures with animals were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine, protocols IACUC AN-7915. All mice were treated humanely as per compliance with the rules and regulations of animal care and euthanasia under this committee. The minimal number of mice were used in this study to attain statistical significance using a two-tailed Student T-test. Both male and female mice were.

WT and Bax/Bak1 double knockout (DKO) SV40 immortalized mouse embryonic fibroblasts (MEFs) were plated on six 245 mm × 245 mm plates (Corning, 431110) and were cultured until reaching 100% confluency. Plates were washed with 10 ml mitochondrial isolation buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, pH 7.4). Cells were then scraped in 7 ml isolation buffer per plate and collected into a 50 ml conical tube. The suspension was pelleted at 800 x g for 5 min at 4°C. The supernatant was discarded and the pellet was suspended in 7 ml of isolation buffer before homogenization by a Teflon/glass tissue grinder (5–10 strokes). The homogenate was brought up to 12 ml isolation buffer before centrifugation at 800 g for 5 min. Then the supernatant was transferred to a fresh tube and centrifuged again at 800 x g for 5 min. Then the supernatant was transferred to another fresh tube and spun at 10,000 x g for 10 min. The supernatant was aspirated, and the pellet was washed with 7 ml of isolation buffer and was then centrifuged at 10,000 x g for 10 min. All centrigfugation was performed at 4°C and samples were kept on ice. The final pellet was suspended in 1 ml of KCl buffer (125 mM KCl, 20 mM HEPES, 2 mM KH₂PO₄, 40 µM EGTA, pH 7.2). Mitochondrial concentration was quantified using a NanoDrop.

Whole livers were isolated from WT and Ppif ^−/− mice and washed in mitochondrial isolation buffer then minced into 1–2 mm pieces in 7 ml of mitochondrial isolation buffer. The tissue was homogenized using a glass and Teflon/glass tissue grinder (8–10 strokes). All steps were performed on ice. The liver homogenates were then centrifuged at 800 x g for 5 min, and the supernatants were then collected and centrifuged at 10,000 x g for 10 min. The supernatant was aspirated, and the pellet was washed with 7 ml of isolation buffer and was then centrifuged at 10,000 x g for 10 min. All centrifugations were performed at 4°C. The pellet from the last spin was suspended in 1 ml KCL buffer and mitochondrial concentration was measured using a NanoDrop.

Two mg of isolated mitochondria were suspended in a total volume of 1 ml consisting KCl buffer, 1 mM Malic acid (Sigma-Aldrich), 7 mM Pyruvate (Sigma-Aldrich), and 50 nM Calcium Green 5N (Invitrogen) in a quartz cuvette, which was placed inside the fluorimeter (PTI QuantaMaster 800, Horiba Scientific). Calcium uptake was measured by fluorescent emission of Calcium Green 5N. Simultaneously, mitochondrial swelling was measured by transmittance light. Each BH3 mimetic was used at concentrations: 0.5, 1, 10, 50, 100 nM, and 1 µM. Final BH3 mimetics concentrations used throughout the study were 200 nM ABT-199 (Selleckchem.com, S8048), 100 nM A-1331852 (abcam, ab218112), 100 nM ABT-737 (EMD Millipore Sigma, 197333), 10 nM S63845 (Selleckchem.com, S8383), 10 nM Obatoclax (Selleckchem, GX15-070). For some experiments ADP (300 uM) (Sigma-Aldrich, A2754) and/or CsA (2 uM) (Sigma-Aldrich, 30024) were used to desensitize the MPTP. CaCl₂ (20 μM, 40 μM, and 80 µM) (Sigma-Adrich, C4901) was added into this system in succession until MPTP opening occurred, indicated by an upturn in Calcium Green 5N fluorescence, or when mitochondria were saturated with Ca²⁺ and were no longer able to take up further additions of CaCl₂, indicated by a stair-stacking in the Ca²⁺ uptake graphs.

WT and Bax/Bak1 DKO MEFs were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) (Cytiva, SH30228.01) supplemented with 10% bovine growth serum (HyClone, SH30541.03), 1 mM penicillin streptomycin (gibco, 15140-122), and 1 mM nonessential amino acids (gibco, 11140-050). For cell death experiments, these cells were plated in 12-well plates and were treated with cell death inducing agents when they were 85–90% confluent. The cells were treated with BH3 mimetics ABT-199 (30 µM), A-1331852 (10 µM), ABT-737 (10 µM), S63845 (10 µM), and Obatoclax (500 nM) with and without ionomycin (5 µM for 20 h) or staurosporine (5 nM for 12 h). Cell death was determined by propidium iodide (PI) positivity (BioVision, Milpitas, CA). When quantifying cell death, the media was collected into a microcentrifuge tube and the cells were trypsinized and placed into their appropriate tube of media. The cells were then centrifuged for 5 min at 5,000 x g and the cell pellet was suspended in Hank’s Balanced Salt solution (HBSS) containing 0.01% bovine serum albumin (BSA) and 0.1% PI and incubated for 10 min. The cells were then quantified for PI positivity at 10,000 counts per sample using a Guava® easyCyte 5HT HPL Benchtop Flow Cytometer (Millipore Sigma).

All Western blots were performed from MEF and liver mitochondrial lysates (isolated as previously described). Following mitochondrial isolations, the mitochondrial pellets were suspended in radioimmunoprecipitation (RIPA) buffer (10 mM Tris-HCl pH7.49, 100 nM NaCl, 1 mM EDTA, 1 nM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate) containing protease inhibitor cocktails (Roche). The samples were then sonicated, and the insoluble fractions were discarded following centrifugation (21,000 x g for 10 min at 4°C). SDS sample buffer (250 mM Tris-HCl pH 7.0, 10% SDS, 5% β-mercaptoethanol, 0.02% bromophenol blue, 30% glycerol) was added to the lysates, and samples were boiled for 5 min at 100°C. The samples were then loaded onto to 12% SDS-PAGE gels and then transferred onto polyvinylidene fluoride transfer membranes (MilliporeSigma). Prior to primary antibody incubation, the membranes were blocked in tris-buffered saline, 0.1% Tween (TBST) containing 4% dry milk. The following primary antibodies were used: Bcl-xL (Cell Signaling Technology; 54H6; 1:2000), Mcl-1 (ROCKLAND, 600-401-394S, 1:500), Bcl-2 (abcam, ab182858,1:500), and Total OXPHOS rodent WB antibody cocktail (that contained antibodies to complexes 1 to 5, ComII, or ComV as labeled in Western blots) (abcam; ab110413; 1:10,000). Following overnight incubation with the primary antibody, the membranes were washed 3 times for 5 min with 1X TBST. These blots were then incubated in their respective secondary antibody, goat-anti-mouse IgG (H + L) Secondary Alkaline phosphatase (NOVUS, NB7536; 1:10,000) or goat anti-rabbit IgG (H + L) Secondary Alkaline phosphatase (NOVUS, NB7157; 1:10,000) for 2 h. Before imaging, the membranes were washed 5 times for 5 min each with 1X TBST. These blots were incubated in ECF substrate for 1 min before imaging using the iBright imaging system (ThermoFisher).

Anti-apoptotic Bcl-2 family (Bcl-2, Bcl-xL, Mcl-1) protein expression was determined in 5 WT liver and 5 MEF mitochondrial crude fractions using quantitative tandem mass tags (TMT10plex #90110, Pierce) (Parks et al., 2019). Protein extracts from each liver or MEF mitochondrial fraction were reduced, alkylated, then digested overnight before being labeled with TMT Reagents before being mixed and fractionated. These labeled samples were analyzed by high resolution Thermo Scientific^TM Orbitrap^TM Mass Spectrometry. Each protein amount was quantified as a percent of total anti-apoptotic Bcl-2 protein amount.

The data is presented as the mean with the error bars representing the standard error of the mean (SEM). When comparing two groups, an unpaired two-tailed Student’s t test was performed and when comparing multiple groups, a one-way ANOVA was performed followed by a Bonferroni Post Hoc analysis for multiple comparisons using GraphPad Prism. All values were considered statistically significant when p < 0.05 or p < 0.005 as labeled in the figure legends. The sample number of biological replicates for each experiment is indicated in the figure legends.

To determine if the inhibition of various anti-apoptotic Bcl-2 family members through the treatment of BH3 mimetics can exacerbate necrotic cell death, we first determined the amount of each anti-apoptotic Bcl-2 family member expressed at baseline within the mitochondria in WT MEFs by using tandem mass tag-mass spectrometry (TMT-MS) (Figure 1A). The expression of each anti-apoptotic Bcl-2 family member detected by TMT-MS was confirmed by western blot analysis (Figure 1B). Bcl-xL and Mcl-1 were found to be the most abundant anti-apoptotic Bcl-2 family members followed by Bcl-2 in WT MEF mitochondria. WT MEFs were treated with low concentrations of BH3 mimetics (mimetics ABT-199 (Bcl-2 inhibitor), A-1331852 (Bcl-xL inhibitor), S63845 (Mcl-1 inhibitor), ABT-737 (Bcl-2 and Bcl-xL inhibitor), or Obatoclax (Bcl-2, Bcl-xL, and Mcl-1 inhibitor) in the presence or absence of low dose of ionomycin (iono) (5 µM for 20 h) or staurosporine (St) (5 nM for 12 h). Alone, each treatment led to a small increase in cell death, however, the BH3 mimetics exacerbated both apoptosis induced by staurosporine and necrosis induced by ionomycin in WT MEFs (Figure 1C). These data suggest that the anti-apoptotic Bcl-2 family members play a protective role against Ca²⁺ overload-induced cell death, which is indicative of these members playing a protective role against MPTP-dependent necrosis.

To investigate the effects of BH3 mimetics on the MPTP, we utilized ex vivo liver mitochondrial isolations due to their accessibility and abundance. Prior to examining the effect of the mimetics on MPTP kinetics, we performed TMT-MS on isolated liver mitochondrial fractions to elucidate the abundance of the anti-apoptotic Bcl-2 family members in liver mitochondria at baseline (Figure 2A). Bcl-xL is the most abundantly expressed anti-apoptotic Bcl-2 protein, followed by Mcl-1 and then Bcl-2. The expression of each anti-apoptotic member detected by TMT-MS was confirmed by western blot analysis (Figure 2B). To determine whether BH3 mimetics affect mitochondrial Ca²⁺ uptake and/or retention and MPTP sensitivity, WT liver mitochondria were incubated with increasing concentrations of each BH3 mimetic (0.5, 1, 10, 50, 100 nM, 1 µM) prior to being challenged with Ca²⁺ boluses. Following incubation, mitochondrial Ca²⁺ retention capacity (CRC) and swelling were analysed (Supplementary Figures S1A–K). All BH3 mimetics tested were able to sensitize MPTP opening in a dose-dependent manner. However, the multi-targeted BH3 mimetics sensitize the MPTP greater than the single targeted mimetics (Supplementary Figure S1K). Ultimately, we decided to utilize the lowest possible dose of each mimetic that was able to decrease CRC and sensitize MPTP opening to ensure target specificity. These chosen concentrations of each BH3 mimetic are represented in Figures 2C–E.

CypD and the ANT family are two major regulators of the MPTP, whereas CypD triggers pore opening from the matrix of mitochondria via its isomerase activity, while the ANT family can function as a pore-forming component of the MPTP itself (Brustovetsky and Klingenberg, 1996; Rück et al., 1998; Brustovetsky et al., 2002; Karch and Molkentin, 2014). When either regulator is deleted or inhibited, the MPTP becomes desensitized to Ca²⁺-induced opening. However, when both regulators are inhibited against their MPTP functions, Ca²⁺-induced MPTP opening is completely inhibited (Karch et al., 2019). Here, we wanted to probe how the inhibition of the anti-apoptotic Bcl-2 family members may lead to MPTP sensitization by determining how they affect the MPTP when the critical regulators are deleted or inhibited. CRC/swelling assays were performed on WT liver mitochondria pre-treated with with cyclosporine A (CsA), an inhibitor of CypD, and BH3 mimetics to determine whether anti-apoptotic Bcl-2 proteins sensitize the MPTP independently of CypD. Mitochondrial CRC was significantly decreased and MPTP opening occurred with less Ca²⁺ when the BH3 mimetics were present when CypD was inhibited by CsA (Figures 3A–C). We genetically confirmed these results, using liver mitochondria isolated from Ppif ^−/− mice (Ppif being the gene that encodes CypD) and treating these mitochondria with the various BH3 mimetics. Similar to pharmacological inhibition, the CypD null mitochondria had reduced CRC due to sensitization of the MPTP in the presence of all BH3 mimetics (Figures 3D–F). Notably, there was a correlative trend for the multi-targeted BH3 mimetics (ABT-737 and Obatoclax) to have a greater effect than the single targeted mimetics.

In order to determine whether BH3 mimetics sensitizes MPTP opening through the ANT family, WT liver mitochondria were isolated and pre-treated with ADP followed by BH3 mimetics treatment before performing CRC analysis. ADP is a substrate of the ANT family that has an inhibitory effect on the ability of the ANT family to contribute to MPTP opening by stabilizing the location of their nucleotide binding in the matrix (“m”-state) (Buchanan et al., 1976; Haworth and Hunter, 2000). The amount of Ca²⁺ required for MPTP opening decreased significantly in WT liver mitochondria pre-treated with ADP in the presence of each BH3 mimetics (Figures 4A–C). Again, BH3 mimetics targeting multiple anti-apoptotic Bcl-2 family members induced mitochondrial swelling prior to single targeting BH3 mimetics. To determine if the mimetics were able to sensitize the MPTP when both the ANTs and CypD were antagonized, we treated WT liver mitochondria with a combination of CsA and ADP, followed by BH3 mimetics before challenging them with Ca²⁺ boluses and measuring mitochondrial CRC/swelling. As we have previously reported, this combination treatment impressively increases CRC to the point of Ca²⁺ saturation with the absence of a large magnitude-swelling event (Figures 5A,C). Under this MPTP-inhibited state, treatment with all the various BH3 mimetics was unable to reduce CRC and restore mitochondrial swelling (Figures 5A–C). To confirm these results genetically, we isolated mitochondria from Ppif null mice and treated them with ADP to inhibit the MPTP. Similarly, to the combined treatment of CsA and ADP, ADP treated CypD null mitochondria do not engage MPTP opening and MPTP activity is not sensitized or restored in the presence of any of the BH3 mimetics (Figures 5D–F). Thus, BH3 mimetic-dependent sensitization of the MPTP cannot supersede inhibition of the MPTP by dual treatment of ADP and CsA or CypD deletion with ADP.

The inhibition of anti-apoptotic Bcl-2 family members by BH3 mimetics pushes the Bcl-2 family towards the activation and oligomerization of Bax and Bak to sensitize cells to apoptotic cell death (Villalobos-Ortiz et al., 2020). To determine if the exacerbation of necrotic cell death by BH3 mimetic treatment in WT MEFs is dependent on the expression of Bax and Bak, we treated Bax/Bak1 DKO MEFs with BH3 mimetics in the presence of staurosporine (5 nM) or ionomycin (5 µM) before measuring cell death using PI staining and flow cytometry. Bax/Bak1 DKO MEFs were resistant to BH3 mimetic exacerbation of both apoptotic and necrotic cell death compared to WT MEFs, except for Obatoclax, which induced a low level of toxicity when combined with ionomycin (Figures 1A, 6A). Mitochondria isolated from WT and Bax/Bak1 DKO MEFs treated with BH3 mimetics prior to Ca²⁺ boluses demonstrated that BH3 mimetics sensitize MPTP opening in WT mitochondria, while Bax/Bak null mitochondria were refractory to the treatment of mimetics and Ca²⁺ (Figures 6B–G). Thus, the inhibition of the anti-apoptotic Bcl-2 family members function through Bax and Bak to sensitize mitochondria to Ca²⁺-dependent MPTP opening and necrotic cell death. Surprisingly, the treatment of ADP and/or CsA on Bax/Bak null mitochondria did not enhance mitochondrial CRC or alter the kinetics of mitochondrial swelling compare to WT control mitochondria (Supplementary Figures 2A–F). These data confirm that Bax and Bak are critical outer membrane mitochondrial regulators of the MPTP, as previously suggested (Karch et al., 2013).