ROS-mediated PARP activity undermines mitochondrial function after permeability transition pore opening during myocardial ischemia-reperfusion

Abstract

Background: Ischemia-reperfusion (I/R) studies have implicated oxidant stress, the mitochondrial permeability transition pore (mPTP), and poly(ADP-ribose) polymerase (PARP) as contributing factors in myocardial cell death. However, the interdependence of these factors in the intact, blood-perfused heart is not known. We therefore wanted to determine whether oxidant stress, mPTP opening, and PARP activity contribute to the same death pathway after myocardial I/R.

Methods and results: A murine left anterior descending coronary artery (LAD) occlusion (30 minutes) and release (1 to 4 hours) model was employed. Experimental groups included controls and antioxidant-treated, mPTP-inhibited, or PARP-inhibited hearts. Antioxidant treatment prevented oxidative damage, mPTP opening, ATP depletion, and PARP activity, placing oxidant stress as the proximal death trigger. Genetic deletion of cyclophilin D (CypD(-/-)) prevented loss of total NAD(+) and PARP activity, and mPTP-mediated loss of mitochondrial function. Control hearts showed progressive mitochondrial depolarization and loss of ATP from 1.5 to 4 hours of reperfusion, but not outer mitochondrial membrane rupture. Neither genetic deletion of PARP-1 nor its pharmacological inhibition prevented the initial mPTP-mediated depolarization or loss of ATP, but PARP ablation did allow mitochondrial recovery by 4 hours of reperfusion.

Conclusions: These results indicate that oxidant stress, the mPTP, and PARP activity contribute to a single death pathway after I/R in the heart. PARP activation undermines cell survival by preventing mitochondrial recovery after mPTP opening early in reperfusion. This suggests that PARP-mediated prolongation of mitochondrial depolarization contributes significantly to cell death via an energetic crisis rather than by mitochondrial outer membrane rupture.

Figure 1.

Identification of the area at risk (AAR) and the area of necrosis after I/R. A, AAR in wild‐type (WT) and experimental groups subjected to ischemia (30 minutes) followed by 4 hours of reperfusion: WT, n=8; EUK, n=6; CypD, n=6; 3AB, n=6; WT/3AB/EUK, n=6; CypD/3AB, n=7; CypD/EUK, n=8; PARP WT, n=6; PARP KO, n=6. No significant differences were detected. B, Area of necrosis in WT and experimental groups after ischemia (30 minutes) followed by 4 hours reperfusion. Representative slices from each group showing the LV not at risk (purple), the AAR (red+white), and the area of necrosis (white). *P<0.05 compared with WT. Values are means±SEMs. I/R indicates ischemia–reperfusion; EUK, EUK134, SODII, and catalase mimetic; CypD, cyclophilin D; 3AB, 3‐aminobenzamide; PARP, poly(ADP‐ribose) polymerase; KO, knockout; LV, left ventricle.

Figure 2.

Protein oxidation and DNA damage after I/R. A, Analysis of dot blots of total cell protein from the AAR after derivatization of protein carbonyls. Representative dots from a panel blot are shown: sham, n=9; WT, n=8; EUK, n=7; CypD, n=9; 3AB, n=7; PARP WT, n=7; PARP KO, n=5. B, Nuclear and mitochondrial DNA amplification in sham, WT, and experimental groups after ischemia (30 minutes) followed by reperfusion for 90 minutes: sham, n=8; WT, n=6; EUK, n=5; CypD, n=5; 3AB, n=5; PARP WT, n=7; PARP KO, n=7. Values expressed relative to sham. Representative ethidium bromide–stained gels of the long and short mitochondrial products are shown. *P<0.05 compared with sham, # different compared with WT. Values are means±SEMs. I/R indicates ischemia–reperfusion; AAR, area at risk; WT, wild type; EUK, EUK134, SODII, and catalase mimetic; CypD, cyclophilin D; 3AB, 3‐aminobenzamide; PARP, poly(ADP‐ribose) polymerase; KO, knockout.

Figure 3.

Poly(ADP‐ribosylation) (PAR) after I/R. Immunoblotting of total cell protein from AAR of hearts. A, Wild‐type (WT) hearts subjected to sham occlusion, ischemia (30 minutes) without reperfusion, or ischemia (30 minutes) followed by 15, 60, or 90 minutes of reperfusion, n=5 all groups. B, WT, EUK, CypD‐KO, 3AB, and PARP‐KO hearts after ischemia (30 minutes) and 90 minutes of reperfusion: sham, n=12; WT, n=11; EUK, n=9; CypD, n=10; 3AB, n=10; PARP WT, n=6; PARP KO, n=6. Gel bands were analyzed by densitometry and normalized to tubulin as a loading control. Total PAR protein levels are expressed relative to sham levels. *P<0.05 compared with sham and # compared with WT controls. Values are means±SEMs. I/R indicates ischemia–reperfusion; AAR, area at risk; EUK, EUK134, SODII, and catalase mimetic; CypD, cyclophilin D; 3AB, 3‐aminobenzamide; PARP, poly(ADP‐ribose) polymerase; KO, knockout.

Figure 4.

NAD(H) depletion after I/R. NAD(H) levels in the AAR. A, Wild‐type (WT) hearts subjected to sham occlusion, ischemia (30 minutes) without reperfusion, or ischemia (30 minutes) followed by 15, 60, or 90 minutes of reperfusion, n=5 all groups. B, WT, EUK, CypD‐KO, 3AB, and PARP‐KO hearts after 30 minutes of ischemia and 90 minutes of reperfusion: sham, n=6; WT, n=9; EUK, n=7; CypD, n=7; 3AB, n=8; PARP WT, n=6; PARP KO, n=6. NADH levels were normalized to total protein concentration and then normalized to sham levels. *P<0.05 compared with sham and # compared with WT controls. Values are means±SEMs. I/R indicates ischemia–reperfusion; AAR, area at risk; EUK, EUK134, SODII, and catalase mimetic; CypD, cyclophilin D; 3AB, 3‐aminobenzamide; PARP, poly(ADP‐ribose) polymerase; KO, knockout.

Figure 5.

Mitochondrial NAD(H) after I/R. A, NAD(H) in mitochondrial isolates following ischemia (30 minutes) followed by 60 minutes of reperfusion: sham, n=7; WT, n=6; EUK, n=6; CypD, n=7; 3AB, n=8; PARP WT, n=10; PARP KO, n=10. Values are expressed relative to sham group. B, NAD(H) in mitochondrial fractions assayed after 1 and 4 hours of reperfusion, n=6 all groups. Values are expressed relative to sham group. *P<0.05 compared with sham and # compared with WT controls. Values are means±SEMs. I/R indicates ischemia–reperfusion; AAR, area at risk; EUK, EUK134, SODII, and catalase mimetic; CypD, cyclophilin D; 3AB, 3‐aminobenzamide; PARP, poly(ADP‐ribose) polymerase; KO, knockout.

Figure 6.

ATP and total adenine nucleotide levels after I/R. Total ATP levels in the AAR of hearts following ischemia (30 minutes) followed by (A) 1.5 hours or (B) 4 hours of reperfusion. C, Total adenine nucleotide levels in the AAR of hearts following ischemia (30 minutes) followed by 4 hours of reperfusion. At 1.5 hours, sham, n=6; WT, n=5; EUK, n=6; CypD, n=6; 3AB, n=6; PARP WT, n=6; PARP KO, n=6. At 4 hours, sham, n=5; WT, n=7; EUK, n=6; CypD, n=6; 3AB, n=6; PARP WT, n=6; PARP KO, n=6. Total adenine nucleotide levels, sham, n=8; WT, n=8; 3AB, n=8. Values are expressed relative to sham group. *P<0.05 compared with sham and # compared with WT controls. Values are means±SEMs. I/R indicates ischemia–reperfusion; AAR, area at risk; WT, wild type; EUK, EUK134, SODII, and catalase mimetic; CypD, cyclophilin D; 3AB, 3‐aminobenzamide; PARP, poly(ADP‐ribose) polymerase; KO, knockout.

Figure 7.

TMRE localization, stability, and responses to CCCP ex vivo. A, Mitochondrial‐targeted GFP‐expressing hearts were loaded with TMRE in vivo, excised, and sliced. Colocalization of GFP and TMRE fluorescence was confirmed by 2‐photon microscopy. B, Hearts were loaded with TMRE, excised, sliced, and imaged during CCCP (50 or 250 μmol/L) treatment for 15 minutes. C, Hearts were loaded with TMRE, excised, sliced, and imaged while being maintained in ice‐cold PBS for 30 minutes followed by 15 minutes of treatment with 250 μmol/L CCCP, n=5 all groups. TMRE indicates tetramethylrodamine ethyl ester; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; GFP, green fluorescent protein; PBS, phosphate‐buffered saline.

Figure 8.

Mitochondrial polarization in relation to plasma membrane rupture after I/R. TMRE fluorescence and calcein‐AM staining at different times after reperfusion. TMRE was loaded into beating hearts for 15 minutes. TMRE and calcein fluorescence change in hearts subjected to ischemia (30 minutes) followed by reperfusion for 15 minutes or 4 hours, n=5 all groups. *P<0.05 compared with sham and # compared with 15‐minute time. Values are means±SEMs. I/R indicates ischemia–reperfusion; TMRE, tetramethylrodamine ethyl ester; AAR, area at risk; LV, left ventricle.

Figure 9.

Mitochondrial polarization after I/R. Hearts were loaded with TMRE at different points after reperfusion. Hearts were then retroperfused with Hoechst to delineate the AAR, sliced, and imaged. Total fluorescence of the AAR was compared with the area not at risk and normalized to sham hearts. Fluorescence changes from genetically matched WT, 3AB, PARP‐1‐KO, and CypD‐KO hearts were compared after (A) 1.5 hours or (B) 4 hours of reperfusion. Included are representative heart slices showing TMRE voids in the AAR, n=5 all groups. *P<0.05 compared with sham and # compared with WT controls. Values are means±SEMs. I/R indicates ischemia–reperfusion; TMRE, tetramethylrodamine ethyl ester; AAR, area at risk; WT, wild type; CypD, cyclophilin D; 3AB, 3‐aminobenzamide; PARP, poly(ADP‐ribose) polymerase; KO, knockout.

Figure 10.

Cardiac troponin release in relation to cytochrome c or AIF translocation after I/R. A, Cardiac troponin in circulating plasma as a measure of plasma membrane rupture after 1.5 and 4 hours of reperfusion and in EUK, CypD‐KO, 3AB, and PARP‐KO hearts at 4 hours of reperfusion. B, Mitochondrial AIF, mitochondrial cytochrome c, and cytosolic AIF and cytochrome c in cellular fractions in sham hearts or after ischemia (30 minutes) followed by reperfusion for 1 hour (IR1h), 4 hours (IR4h), 16 hours (IR16h), or ex vivo hearts allowed to autolyse for 1 hour (AL): sham, n=6; IR1.5h, n=5; IR4h, n=6; IR16h, n=6; AL, n=5. Mitochondrial values are normalized to cytochrome oxidase subunit 4 (Cox IV), and cytosolic values are normalized to GAPDH. *P<0.05 compared with sham. Values are means±SEMs. AIF indicates apoptosis‐inducing factor; I/R, ischemia–reperfusion; AAR, area at risk; WT, wild type; EUK, EUK134, SODII, and catalase mimetic; CypD, cyclophilin D; 3AB, 3‐aminobenzamide; PARP, poly(ADP‐ribose) polymerase; KO, knockout; Cox IV, cytochrome c oxidase subunit 4.

Figure 11.

Model of myocardial I/R‐induced cell death. I/R indicates ischemia–reperfusion; ROS, reactive oxygen species; mPTP, mitochondrial permeability transition pore; PARP, poly(ADP‐ribose) polymerase.