Opening of the Mitochondrial Permeability Transition Pore Causes Depletion of Mitochondrial and Cytosolic NAD+and Is a Causative Event in the Death of Myocytes in Postischemic Reperfusion of the Heart

The opening of the mitochondrial permeability transition pore (PTP) has been suggested to play a key role in various forms of cell death, but direct evidence in intact tissues is still lacking. We found that in the rat heart, 92% of NAD+glycohydrolase activity is associated with mitochondria. This activity was not modified by the addition of Triton X-100, although it was abolished by mild treatment with the protease Nagarse, a condition that did not affect the energy-linked properties of mitochondria. The addition of Ca2+ to isolated rat heart mitochondria resulted in a profound decrease in their NAD+ content, which followed mitochondrial swelling. Cyclosporin A(CsA), a PTP inhibitor, completely prevented NAD+ depletion but had no effect on the glycohydrolase activity. Thus, in isolated mitochondria PTP opening makes NAD+ available for its enzymatic hydrolysis. Perfused rat hearts subjected to global ischemia for 30 min displayed a 30% decrease in tissue NAD+ content, which was not modified by extending the duration of ischemia. Reperfusion resulted in a more severe reduction of both total and mitochondrial contents of NAD+, which could be measured in the coronary effluent together with lactate dehydrogenase. The addition of 0.2 μm CsA or of its analogue MeVal-4-Cs (which does not inhibit calcineurin) maintained higher NAD+ contents, especially in mitochondria, and significantly protected the heart from reperfusion damage, as shown by the reduction in lactate dehydrogenase release. Thus, upon reperfusion after prolonged ischemia, PTP opening in the heart can be documented as a CsA-sensitive release of NAD+, which is then partly degraded by glycohydrolase and partly released when sarcolemmal integrity is compromised. These results demonstrate that PTP opening is a causative event in reperfusion damage of the heart. The opening of the mitochondrial permeability transition pore (PTP) has been suggested to play a key role in various forms of cell death, but direct evidence in intact tissues is still lacking. We found that in the rat heart, 92% of NAD+glycohydrolase activity is associated with mitochondria. This activity was not modified by the addition of Triton X-100, although it was abolished by mild treatment with the protease Nagarse, a condition that did not affect the energy-linked properties of mitochondria. The addition of Ca2+ to isolated rat heart mitochondria resulted in a profound decrease in their NAD+ content, which followed mitochondrial swelling. Cyclosporin A(CsA), a PTP inhibitor, completely prevented NAD+ depletion but had no effect on the glycohydrolase activity. Thus, in isolated mitochondria PTP opening makes NAD+ available for its enzymatic hydrolysis. Perfused rat hearts subjected to global ischemia for 30 min displayed a 30% decrease in tissue NAD+ content, which was not modified by extending the duration of ischemia. Reperfusion resulted in a more severe reduction of both total and mitochondrial contents of NAD+, which could be measured in the coronary effluent together with lactate dehydrogenase. The addition of 0.2 μm CsA or of its analogue MeVal-4-Cs (which does not inhibit calcineurin) maintained higher NAD+ contents, especially in mitochondria, and significantly protected the heart from reperfusion damage, as shown by the reduction in lactate dehydrogenase release. Thus, upon reperfusion after prolonged ischemia, PTP opening in the heart can be documented as a CsA-sensitive release of NAD+, which is then partly degraded by glycohydrolase and partly released when sarcolemmal integrity is compromised. These results demonstrate that PTP opening is a causative event in reperfusion damage of the heart. permeability transition pore cyclosporin A 1,N 6-etheno-NAD+ N-methylvaline-4-cyclosporin poly(ADP-ribose) polymerase rat heart mitochondria lactate dehydrogenase 4-morpholinepropanesulfonic acid Depending on the duration and severity of myocardial ischemia, reperfusion can result in either recovery of contractile function or rapid transition toward tissue necrosis (for review see Refs. 1Jennings R.B. Reimer K.A. Annu. Rev. Med. 1991; 42: 225-246Crossref PubMed Scopus (261) Google Scholar, 2Silverman H.S. Stern M.D. Cardiovasc. Res. 1994; 28: 581-597Crossref PubMed Scopus (181) Google Scholar, 3Di Lisa F. Menabò R. Canton M. Petronilli V. Biochim. Biophys. Acta. 1998; 1366: 69-78Crossref PubMed Scopus (116) Google Scholar). Paradoxically, both events require coupled mitochondrial respiration (4Di Lisa F. Blank P.S. Colonna R. Gambassi G. Silverman H.S. Stern M.D. Hansford R.G. J. Physiol. (Lond.). 1995; 486: 1-13Crossref Scopus (235) Google Scholar). Indeed, cyanide (5Ganote C.E. Worstell J. Kaltenbach J.P. Am. J. Pathol. 1976; 84: 327-350PubMed Google Scholar) or 2,4-dinitrophenol (6Ganote C.E. McGarr J. Liu S.Y. Kaltenbach J.P. J. Mol. Cell. Cardiol. 1980; 12: 387-408Abstract Full Text PDF PubMed Scopus (32) Google Scholar) largely reduce the release of intracellular enzymes, the marker of cell death induced by postischemic reperfusion. However, after more than 25 years, the specific mechanisms underlying these phenomenological observations have yet to be elucidated. A large body of experimental evidence suggests that a suboptimal mitochondrial function could produce low levels of ATP, which in the presence of even a modest rise in Ca2+i might cause hypercontracture in isolated cardiomyocytes (7Miyata H. Lakatta E.G. Stern M.D. Silverman H.S. Circ. Res. 1992; 71: 605-613Crossref PubMed Scopus (166) Google Scholar) and sarcolemma rupture in intact hearts (8Ganote C.E. Armstrong S.C. Cardiovasc. Res. 1993; 27: 1387-1403Crossref PubMed Scopus (129) Google Scholar, 9Jennings R.B. Murry C.E. Steenbergen C. Reimer K.A. Circulation. 1990; 82: 2-12Google Scholar). Such a sequence of events could be set in motion by the opening of the mitochondrial PTP,1 a high conductance channel located in the inner mitochondrial membrane (10Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1319) Google Scholar). The open probability of this channel is regulated by several factors including mitochondrial membrane potential difference (Δψm), Ca2+, matrix pH, and CsA, a high affinity inhibitor (11Bernardi P. Broekemeier K.M. Pfeiffer D.R. J. Bioenerg. Biomembr. 1994; 26: 509-517Crossref PubMed Scopus (526) Google Scholar). PTP opening causes a Ca2+-dependent increase of mitochondrial permeability to ions and solutes with molecular masses of up to 1500 Da, matrix swelling, and mitochondrial deenergization. Several studies performed on isolated cardiomyocytes (12Nazareth W. Yafei N. Crompton M. J. Mol. Cell. Cardiol. 1991; 23: 1351-1354Abstract Full Text PDF PubMed Scopus (226) Google Scholar, 13Duchen M.R. McGuinness O. Brown L.A. Crompton M. Cardiovasc. Res. 1993; 27: 1790-1794Crossref PubMed Scopus (261) Google Scholar, 14Minezaki K.K. Suleiman M.S. Chapman R.A. J. Physiol. (Lond.). 1994; 476: 459-471Crossref Scopus (57) Google Scholar) and perfused hearts (15Griffiths E.J. Halestrap A.P. J. Mol. Cell. Cardiol. 1993; 25: 1461-1469Abstract Full Text PDF PubMed Scopus (500) Google Scholar, 16Griffiths E.J. Halestrap A.P. Biochem. J. 1995; 307: 93-98Crossref PubMed Scopus (711) Google Scholar) support the idea that PTP opening might be pivotal in determining the transition of the ischemic damage to the irreversible phase. However, the role of PTP is not yet defined due to the difficulty of assaying its opening in situ. PTP opening is likely to alter several metabolic pathways linked to energy metabolism, and results from a classic study suggest that NAD+ catabolism may be one of them (17Vinogradov A. Scarpa A. Chance B. Arch. Biochem. Biophys. 1972; 152: 646-654Crossref PubMed Scopus (103) Google Scholar). Indeed, the content of mitochondrial pyridine nucleotides was drastically reduced upon Ca2+ addition, a condition that could have induced PTP opening. Here we show that in RHM opening of the PTP causes the release of mitochondrial NAD+ followed by its hydrolysis by a CsA-insensitive NAD+ glycohydrolase localized outside the matrix space. Furthermore, we document that in the intact heart during postischemic reperfusion mitochondrial NAD+ content is severely decreased in a process that is largely reduced by PTP inhibitors, suggesting that the hydrolysis of mitochondrial NAD+ directly reflects PTP opening in situ. The maintenance of mitochondrial NAD+ is thus associated with a significant protection from myocyte death, indicating that the PTP plays a key role in this process. All aspects of animal care and experimentation were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85–23, revised in 1996), and the national laws of Italy concerning the care and use of laboratory animals and were approved by the Ethical Committee of the University of Padova. Hearts excised from male Wistar rats (weighing 180–200 g) were perfused by the nonrecirculating Langendorff technique as previously described (18Di Lisa F. Menabò R. Barbato R. Siliprandi N. Am. J. Physiol. 1994; 267: H455-H461PubMed Google Scholar). Hearts were not stimulated, and the flow was maintained at 12 ml/min throughout all the perfusion protocols except during ischemia, which was induced by the complete abolition of coronary flow for periods ranging from 30 to 90 min. Left ventricular wall temperature was maintained at 36–37 °C irrespective of coronary flow by suspending the heart in a water-jacketed chamber. Mitochondria were isolated by conventional procedures of differential centrifugation (19Lindenmayer G.E. Sordahl L.A. Schwartz A. Circ. Res. 1968; 23: 439-450Crossref PubMed Scopus (125) Google Scholar). Freshly excised rat hearts were homogenized by Ultra-Turrax in a medium containing 0.18 m KCl, 10 mm EDTA, 0.5% fatty acid-poor bovine serum albumin, 10 mm Hepes, pH 7.4. To remove EDTA and albumin, mitochondrial pellets were washed twice with 0.18 m KCl, 10 mm Hepes, pH 7.4 (18Di Lisa F. Menabò R. Barbato R. Siliprandi N. Am. J. Physiol. 1994; 267: H455-H461PubMed Google Scholar). In a separate set of experiments aimed at characterizing the localization of the mitochondrial NAD+ glycohydrolase, mitochondria were isolated after the incubation of the whole tissue homogenate with Nagarse as previously described (20Di Lisa F. Fan C.Z. Gambassi G. Hogue B.A. Kudryashova I. Hansford R.G. Am. J. Physiol. 1993; 264: H2188-H2197PubMed Google Scholar). Mitochondrial swelling was monitored as the changes in absorbance at 540 nm as previously described (21Scorrano L. Petronilli V. Di Lisa F. Bernardi P. J. Biol. Chem. 1999; 274: 22581-22585Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Incubations were carried out at 25 °C with 0.25 mg of mitochondrial protein/ml in the RB medium, 0.25 m sucrose, 10 mm Tris-Mops, 0.05 mm EGTA, pH 7.4, 5 mm pyruvate, 5 mm malate, and 1 mm Pi-Tris. PTP opening was induced by the addition of 0.25 mmCa2+. NAD+ and CoASH were measured after perchloric acid extraction. To achieve this, the hearts were freeze-clamped with aluminum tongues cooled in liquid nitrogen, and 0.3 g of freeze-clamped tissue (stored at −70 °C) was ground and mixed thoroughly with 2 ml of 14% (v/v) HClO4. After thawing at 4 °C, this mixture was homogenized and centrifuged as previously described (18Di Lisa F. Menabò R. Barbato R. Siliprandi N. Am. J. Physiol. 1994; 267: H455-H461PubMed Google Scholar). In the case of isolated mitochondria, 0.1 ml of 21% (v/v) HClO4 was added to 1 mg of protein/ml suspensions. In neutralized HClO4 extracts, NAD+ was determined fluorometrically with alcohol dehydrogenase (22Klingenberg M. Bergmeyer H.U. Methods of Enzymatic Analysis. Verlag Chemie, Weinheim, Germany1985: 251-271Google Scholar), and CoASH was assayed with an enzymatic cycling method (23Veloso D. Veech R.L. Anal. Biochem. 1974; 62: 449-450Crossref PubMed Scopus (46) Google Scholar). The mitochondrial hydrolysis of endogenous FAD was measured as the increase in fluorescence (excitation and emission wavelengths at 450 and 520 nm, respectively) in the supernatant of mitochondria pelleted after the various incubation protocols (24Barile M. Brizio C. De Virgilio C. Delfine S. Quagliariello E. Passarella S. Eur. J. Biochem. 1997; 249: 777-785Crossref PubMed Scopus (52) Google Scholar). The fluorescence increase is caused by the release of the hydrolytic products, namely flavin mononucleotide and riboflavin. The activity of mitochondrial NAD+ glycohydrolase was measured by monitoring the enhancement in fluorescence emission caused by the hydrolysis of ε-NAD (25Barrio J.R. Secrist J.A. Leonard N.J. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 2039-2042Crossref PubMed Scopus (131) Google Scholar). The assay was carried out by adding RHM (0.2 mg of protein/ml of RB medium) with 200 μmε-NAD, which was found to saturate the NADase activity. Fluorescence measurements were performed using a PerkinElmer LS5 spectrofluorometer. The excitation wavelength was set to 310 nm. Fluorescence emission was followed at 410 nm. The concentration of ε-NAD was determined by the conversion of ε-NAD to ε-NADH using the alcohol dehydrogenase reaction and assuming a molar extinction coefficient for ε-NADH of 6.2 × 106 cm2/mol at 340 nm. The fluorescence changes produced by mitochondria were calibrated by using a standard curve produced by incubating ε-NAD (at concentrations ranging from 1 to 50 μm) with excess amounts of NADase from Neurospora crassa (25Barrio J.R. Secrist J.A. Leonard N.J. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 2039-2042Crossref PubMed Scopus (131) Google Scholar). During postischemic reperfusion, 1-ml samples of the effluent were collected at 1-min intervals for the first 5 min and at 5-min intervals until the end of the reperfusion protocol. LDH activity was measured by means of a classic procedure (26Bergmeyer H.U. Bernt E. Bergmeyer H.U. Methods of Enzymatic Analysis. Verlag Chemie, Weinheim, Germany1974: 607-612Crossref Google Scholar). Neutralized HClO4extracts of the effluents were used for NAD+ assay. Data are presented as cumulative values for the entire reperfusion period. Lactate dehydrogenase, NAD+, and NADH were purchased from Roche All and were purchased from and were of the available CsA and MeVal-4-Cs were of The of study was to in isolated mitochondria NAD+ hydrolysis could be to PTP opening. In the experiments of the addition of 0.2 mm Ca2+ to RHM (0.2 mg of mitochondrial × induced a rapid decrease of absorbance at 540 nm, which is of swelling. these the NAD+ content was reduced to than of values FAD and CoASH were not not 1 that the in mitochondrial NAD+ content after the of mitochondrial swelling and that both were largely prevented by with 0.2 μm CsA, suggesting that NAD+ was to PTP opening. The decrease in NAD+ content was prevented by 5 mm an inhibitor of NAD+glycohydrolase that did not affect either the or the of mitochondrial swelling the NAD+ depletion PTP opening was modified by the addition of which M. Passarella S. G. Quagliariello E. Biochem. Mol. Biol. Google Scholar). results on the PTP opening and could be in rat mitochondria not 2 a of experiments performed to the activity and the of NAD+ RHM displayed a activity of of mitochondrial a mitochondrial content of mg of of heart tissue J.A. J.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the mitochondrial NAD+ glycohydrolase activity 92% of that measured in the whole tissue the NAD+ hydrolytic activity was by and not by not we this activity to NAD+ The of ε-NAD hydrolysis was not modified by the addition of Triton to coupled RHM Furthermore, the incubation of RHM with a protease Nagarse, used to increase the of mitochondrial from heart tissues (20Di Lisa F. Fan C.Z. Gambassi G. Hogue B.A. Kudryashova I. Hansford R.G. Am. J. Physiol. 1993; 264: H2188-H2197PubMed Google Scholar, B. J. Biol. Chem. Full Text PDF PubMed Google Scholar), produced a reduction of NAD+glycohydrolase the not the integrity of the inner mitochondrial These results demonstrate that the NAD+glycohydrolase activity is not localized the mitochondrial matrix and that NAD+ hydrolysis outside this the NAD+ glycohydrolase activity was not by CsA these we that PTP opening in isolated mitochondria causes the release of NAD+, which then a for the glycohydrolase located outside the matrix space. The of tissue and mitochondrial was then in isolated rat hearts subjected to ischemia and postischemic reperfusion. and the changes of the tissue contents of NAD+ in perfused hearts and isolated mitochondria. After 30 min of ischemia, the NAD+ content was decreased by 30% and did not show changes as the ischemic was the a reperfusion of min resulted in a severe decrease of tissue NAD+ contents 4 that the mitochondrial NAD+ was suggesting that these to a large to the changes in is that the decrease of NAD+ content was largely prevented by 0.2 μm of both CsA and its analogue A of tissue and mitochondrial in hearts to the of Ca2+ in the perfusion after 10 min of perfusion in the of which is to a intracellular PubMed Scopus Google Scholar) not the decrease in tissue and mitochondrial contents associated with postischemic reperfusion. In perfused rat coronary flow was for min after 90 min of ischemia. the end of the perfusion hearts were either freeze-clamped or used for mitochondria NAD+ was measured in perchloric acid of tissue or mitochondrial samples as described and CsA or MeVal-4-Cs (0.2 μm) was added to the perfusion and throughout the perfusion protocol. are means of difference and perfusion for 90 30 min of reperfusion after 90 min of We NAD+ was released from the The experiments of 5 show that NAD+ was in the coronary effluent together with LDH and that both events were largely reduced by both CsA and Indeed, in hearts perfused with these PTP inhibitors, the of the mitochondrial NAD+ was associated with a significant decrease of LDH release in the coronary effluent The results a the opening of the PTP and hydrolysis of mitochondrial NAD+ both in isolated and intact and document the of the PTP in the of the heart produced by postischemic reperfusion. 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