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. 2024 May 10;134(10):1292-1305.
doi: 10.1161/CIRCRESAHA.123.323882. Epub 2024 Apr 15.

Inhibition of the mPTP and Lipid Peroxidation Is Additively Protective Against I/R Injury

Arielys Mendoza  1 , Pooja Patel  1 , Dexter Robichaux  1 , Daniel Ramirez  1 , Jason Karch  1   2
Affiliations

Inhibition of the mPTP and Lipid Peroxidation Is Additively Protective Against I/R Injury

Arielys Mendoza et al. Circ Res. .

Abstract

Background: During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca2+ and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability transition pore (mPTP), which causes mitochondrial dysfunction and ultimately necrotic death. However, the mechanisms of how these triggers individually or cooperatively open the pore have yet to be determined.

Methods: Here, we use a combination of isolated mitochondrial assays and in vivo I/R surgery in mice. We challenged isolated liver and heart mitochondria with Ca2+, ROS, and Fe2+ to induce mitochondrial swelling. Using inhibitors of the mPTP (cyclosporine A or ADP) lipid peroxidation (ferrostatin-1, MitoQ), we determined how the triggers elicit mitochondrial damage. Additionally, we used the combination of inhibitors during I/R injury in mice to determine if dual inhibition of these pathways is additivity protective.

Results: In the absence of Ca2+, we determined that ROS fails to trigger mPTP opening. Instead, high levels of ROS induce mitochondrial dysfunction and rupture independently of the mPTP through lipid peroxidation. As expected, Ca2+ in the absence of ROS induces mPTP-dependent mitochondrial swelling. Subtoxic levels of ROS and Ca2+ synergize to induce mPTP opening. Furthermore, this synergistic form of Ca2+- and ROS-induced mPTP opening persists in the absence of CypD (cyclophilin D), suggesting the existence of a CypD-independent mechanism for ROS sensitization of the mPTP. These ex vivo findings suggest that mitochondrial dysfunction may be achieved by multiple means during I/R injury. We determined that dual inhibition of the mPTP and lipid peroxidation is significantly more protective against I/R injury than individually targeting either pathway alone.

Conclusions: In the present study, we have investigated the relationship between Ca2+ and ROS, and how they individually or synergistically induce mitochondrial swelling. Our findings suggest that Ca2+ mediates mitochondrial damage through the opening of the mPTP, although ROS mediates its damaging effects through lipid peroxidation. However, subtoxic levels both Ca2+ and ROS can induce mPTP-mediated mitochondrial damage. Targeting both of these triggers to preserve mitochondria viability unveils a highly effective therapeutic approach for mitigating I/R injury.

Keywords: lipid peroxidation; mitochondrial permeability transition pore; myocardial infarction; necrosis.

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Conflict of interest statement

Disclosures None.

Figures

Figure 1.
Figure 1.
Heart mitochondria are more resistant to tert-butyl hydroperoxide (tBHP) but not Ca2+- or Fe2+-induced mitochondrial swelling compared with liver mitochondria. A and B, Representative mitochondrial swelling assays (n=3) using 2 mg of mitochondria isolated from WT heart (A) and liver (B) treated with a range of total Ca2+ concentrations as labeled. Each arrow indicates a bolus of one-third of the total Ca2+. C and D, Representative mitochondrial swelling assays (n=3) using 2 mg of mitochondria isolated from WT heart (C) and liver (D) treated with a range of tBHP concentrations as labeled. E and F, Representative mitochondrial swelling assays (n=3) using 2 mg of mitochondria isolated from WT heart (E) and liver (F) treated with a range of total Fe2+ concentrations as labeled. Each arrow indicates a bolus of one-fourth of the total Fe2+.
Figure 2.
Figure 2.
Ca2+-mediated mitochondrial dysfunction is distinct from reactive oxygen species–mediated dysfunction. A, Representative images of transmission electron microscopy images of isolated WT (wild-type) heart mitochondria treated with 120 µmol/L Ca2+ incubated for 10 minutes, 160 µmol/L Fe2+ incubated for 50 minutes, 200-mmol/L tert-butyl hydroperoxide (tBHP) incubated for 50 minutes, or untreated (control). Images were selected based on most consistent display of mitochondrial morphology B, Representative mitochondrial swelling assays (n=3) using heart mitochondria treated with 120 μM Ca2+, 160 μM Fe2+, or 200-mmol/L tBHP. C, Quantification of change in absorbance from B. D, Representative mitochondrial swelling assays (n=3) using liver mitochondria treated with 120 μM Ca2+, 160 μM Fe2+, or 60 mmol/L tBHP. E, Quantification of change in absorbance from D. F, Representative mitochondrial swelling assays (n=3) using WT liver mitochondria treated with or without EDTA (5 mmol/L) before (pretreatment) and after (posttreatment) the treatment of Ca2+ (120 µmol/L). G, Representative mitochondrial swelling assays (n=3) using WT liver mitochondria treated with or without N-acetyl-l-cysteine (NAC; 5 mmol/L) before (pretreatment) and after (posttreatment) the treatment of tBHP (60 mmol/L). H, Quantification of mitochondrial shrinkage were calculated by subtracting the absorbance value at the end of the assay from the absorbance value at the time of the posttreatment of EDTA or NAC from F and G.
Figure 3.
Figure 3.
Tert-butyl hydroperoxide (tBHP)-induced mitochondrial swelling is not prevented by mitochondrial permeability transition pore (mPTP) inhibition. A, Mitochondrial swelling assays of 120 μM total Ca2+-treated WT (wild-type) heart mitochondria with or without mPTP desensitizers 2 μM cyclosporine A (CsA) or 300-μM ADP. B, Quantification of maximum swelling (delta absorbance) from A. C, Mitochondrial swelling assays of 200-mmol/L tBHP-treated WT heart mitochondria with or without mPTP desensitizers 2-μM CsA or 300-μM ADP. D, Quantification of maximum swelling from C. E, Mitochondrial swelling assays of 120-μM total Ca2+-treated WT liver mitochondria with or without mPTP desensitizers 2-μM CsA or 300-μM ADP. F, Quantification of maximum swelling from E. G, Mitochondrial swelling assays of 60 mmol/L tBHP-treated WT liver mitochondria with or without mPTP desensitizers 2-μM CsA or 300-μM ADP. H, Quantification of maximum swelling from G. mPTP indicates mitochondrial permeability transition pore.
Figure 4.
Figure 4.
Lipid peroxidation (LIPOX) inhibitors are effective against reactive oxygen species (ROS) but not Ca2+-induced mitochondrial swelling. A, Representative trace of BODIPY-C11 excitation assays (n=3) of 200-mmol/L tert-butyl hydroperoxide (tBHP)-treated WT (wild-type) heart mitochondria with or without mPTP desensitizers or LIPOX inhibitors (2-μM cyclosporine A [CsA] and 300-μM ADP, or 10-μM Fer-1 [ferrostatin-1], and 400-μM MitoQ). B, Representative trace of BODIPY-C11 excitation assays (n=3) of 160 μM total Fe2+ or 120 μM total Ca2+ treated WT heart mitochondria, with or without mPTP desensitizers or LIPOX inhibitors (2-μM CsA and 300-μM ADP, or 10-μM Fer-1 and 10-μM MitoQ). C, Representative mitochondrial swelling assays (n=3) of 120-μM total Ca2+ treated WT heart mitochondria with or without LIPOX inhibitors (10-μM Fer-1 and 10-μM MitoQ). D, Quantification of maximum swelling (delta absorbance) of C. E, Representative mitochondrial swelling assays (n=3) of 200-mmol/L tBHP-treated WT heart mitochondria with or without LIPOX inhibitors 10-μM Fer-1 and 400-μM MitoQ. F, Quantification of maximum swelling of E. G, Representative mitochondrial swelling assays (n=3) of 160-μM total Fe2+ treated WT heart mitochondria with or without LIPOX inhibitors 10-μM Fer-1 and 10-μM MitoQ. H, Quantification of maximum swelling of G.
Figure 5.
Figure 5.
Subtoxic levels of reactive oxygen species (ROS) and Ca2+ synergize to induce mitochondrial swelling. A, Representative mitochondrial swelling assays (n=3) of WT (wild-type) liver mitochondria treated with 10-mmol/L tert-butyl hydroperoxide (tBHP) and 45-μM Ca2+. B, Quantification of maximum swelling of A. C, Dosage response of mitochondrial swelling assays using varying concentrations of tBHP (ranging from 250 μM to 20 mmol/L) in combination with a constant level of 45-μM Ca2+ on WT liver mitochondria. D, Representative dosage response (n=3) of mitochondrial swelling assays using varying concentrations of Ca2+ (ranging from 10 to 80 μM) in combination with a constant level of 10-mmol/L tBHP on WT liver mitochondria. E, Representative dosage response (n=3) of varying concentrations of tBHP (ranging from 10 to 200 mmol/L) in combination with a constant concentration of 45-μM Ca2+ on WT heart mitochondria. F, Representative dosage response (n=3) of varying concentrations of Ca2+ (ranging from 20 to 80 μM) in combination with a constant concentration of 50-mmol/L tBHP on WT heart mitochondria.
Figure 6.
Figure 6.
Reactive oxygen species (ROS) and Ca2+ synergistic swelling is dependent upon mitochondrial permeability transition pore (mPTP) opening. A, Representative mitochondria swelling assays (n=3) of WT (wild-type) liver mitochondria treated with subtoxic levels of ROS and Ca2+ (10-mmol/L tert-butyl hydroperoxide [tBHP] and 45-μM Ca2+), with or without mPTP desensitizers (2-μM cyclosporine A [CsA] or 300-μM ADP), or lipid peroxidation (LIPOX) inhibitors (10-μM Fer-1 [ferrostatin-1] or 200-μM MitoQ). B, Quantification of maximal swelling from A. C, Representative mitochondria swelling assays (n=3) of WT heart mitochondria treated with subtoxic levels of ROS and Ca2+ (50-mmol/L tBHP and 60-μM Ca2+), with or without mPTP desensitizer (2-μM CsA), or LIPOX inhibitors (200 μM MitoQ). D, Quantification of maximal swelling from C. E, Representative mitochondria swelling assays (n=3) using CypD (cyclophilin D) null liver mitochondria treated with subtoxic levels ROS and Ca2+ (500-μM tBHP and 240-μM Ca2+), with or without 300-μM ADP, 10-μM Fer-1, or 200-μM MitoQ. F, Quantification of maximal swelling from E.
Figure 7.
Figure 7.
Dual inhibition of mitochondrial permeability transition pore (mPTP)-dependent necrosis and lipid peroxidation (LIPOX) additively protects against cardiac ischemia-reperfusion (I/R) injury. A, Schematic of I/R injury and processing with pretreatment intraperitoneal (IP) injection timing of Veh, 10 mg/kg cyclosporine A (CsA), 5 mg/kg MitoQ, or in combination. B, Quantitation of the area at risk (AAR). C, Representative cross sectional images of TCC and Evan’s blue dye stained hearts from each labeled cohort. Infarcts are circled by white dotted lines. Images are representative of the mean infarct sizes. D, Quantification of infarct region (IR) over AAR (n=12 per cohort). MitoQ indicates mitoquinone.
Figure 8.
Figure 8.
The roles of Ca2+ and reactive oxygen species (ROS) in the promotion of mitochondrial dysfunction during ischemia/reperfusion (I/R) injury. Schematic representation of our hypothesis on the concentration-dependent contribution of Ca2+ and ROS in mitochondrial permeability transition pore (mPTP)- or lipid peroxidation (LIPOX)-dependent mitochondrial dysfunction. High levels of Ca2+ in the absence of ROS induces mPTP opening regulated by both CypD (cyclophilin D) and ANTs (adenine nucleotide translocators). High ROS levels in the absence of Ca2+ induces LIPOX- dependent mitochondrial rupture. Low levels of ROS can sensitize the mPTP to Ca2+-dependent mPTP opening. These diverse forms of mitochondrial stress each contribute to cardiomyocyte death during I/R injury, and dual inhibition of these pathways is additively protective.
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