/h30558320846873-88 873 Glycine protects cardiomyocytes against lethal reoxygenation injury by inhibiting mitochondrial permeability transition Marisol Ruiz-Meana,Pilar Pina,David Giarcia-Dorado.Antonio Rodriguez-Sinovas,Ignasi Barba, Elisabet Mir6-Casas,Maribel Mirabet and Jordi Soler-Soler Unidad de icnHesiral Utinertarin Vl d'r,Pg VaNl donW5 Brrerm,Spaiu Post-ischaemic reperfusion may precipitate cardiomyocyte death upon correction of intra- cellularacidosis due in part to mitochondrial permeability transition.Weinvestigated whether glycine,an amino acid with poorly understood cytoprotective properties,may interfere with this mechanism.In cardiomyocyte cultures,addition of glycine during re-energization following 1 h of simulated ischaemia (NaCN/2-deoxyglucose,pH 6.4)completely prevented necrotic cell death associated with pH normalization.Glycine also protected against cell death associated with pH normalization in reoxygenated rat hearts.Glycine prevented cyclosporin-sensitive swelling and calcein release associated with re-energization in rat heart mitochondria submitted to simulated ischaemia or to Caf stress under normoxia.NMR spectroscopy revealed a marked glycine depletion in re-energized cardiomyocytes that was reversed by exposure to 3 mM glycine.These results suggest that intracellular glycine exerts a previously unrecognized inhibition on mitochondrial permcability transition in cardiac myo- cytes,and thatintracellular glycine depletion duringmyocardialhypoxia/reoxygenation makes the cell more vulnerable to necrotic death. (Receined 18 May 2004;acrrpled after revisice 17 June 2004;fev puhlihed online 24 June 2004] Corresponding author D.Gercia-Dorodo:Unidod de Imestigacion B.Hospital Laiversiano Vall d'Hebroa,Fg Vall dHebcon 119-129,08035 Barceln,Spain.Email dadordovhebooa.net Mitochondria are increasingly recognized as central by preventing the development of structural cell fragility regulators of cell death and survival in cardiac myocytes (Iausenloy et al.2003:Javadov et aL 2003). exposed to ischaemia-reperfusion (Duchen,1999).A The amino acid glycine (Gly)has been shown to exert prominent player in the mitochondrial death pathway is protective effect against cell death after diverse insults the mitochondrial permeability transition pore,a largely including ischaemia-reperfusion injury in different cell unselective megachannel connecting the mitochondrial types other than cardiomyocytes,but the mechanism matrix with the cytosol and allowing the passige of this effect remains elusive (Nishimura etal.1998; of molecules up to 1.5kDa.During reperfusion. Schemmer et al.1999.Nishimura Lemasters,2001;Dong mitochondrial permeability transition (MPT)may be et al.2001;Zhang er al.2003).In this study we investigated triggered by elevated Ca concentrations,radical oxygen the effect of Gly on MPT and reoxygenation-induced species and normalization of intracellular pll,and it cell death in cultured IL-I cardixc myocytes,intact rat is followed by the collapse of mitochondrial membrane myocardium and rat heart mitochondria.The results potential,interruption of ATP synthesis,release of show that Ghy.an endogenous amino acid,may play a pro-apoptotic molecules into the cytosol (Petronilli er cardioprotective effect in reaxygenated cardiomyocytes by 2001:Di Lisa et ai.2001)and profound ionic disturbances preventing MPT associated with pH normalization. that muay result in necrotic or apoptotic death (Lemasters et ml 1998:Kim eral 2003).It has been recently proposed that inhibition of MPT may have a strong protective effect Methods This study was performed on cultured HL!cardiac myocytes,Langendorf-perfuased hearts from Sprague. M.Ruie-Memna und P.Piu uuultibutodl cyinily to the pupet Dawley rats,and isolated mitochondria.The experimental he Phrynlog★lry0M nGt in 111uphyeel 7004 0son
J Physiol 558.3 (2004) pp 873–882 873 Glycine protects cardiomyocytes against lethal reoxygenation injury by inhibiting mitochondrial permeability transition Marisol Ruiz-Meana, Pilar Pina, David Garcia-Dorado, Antonio Rodr´ıguez-Sinovas, Ignasi Barba, Elisabet Miro-Casas, Maribel Mirabet and Jordi Soler-Soler ´ Unidad de Investigacion B, Hospital Universitario Vall d’Hebron, Pg. Vall d’Hebron 119–129, 08035 Barcelona, Spain Post-ischaemic reperfusion may precipitate cardiomyocyte death upon correction of intracellular acidosis due in part to mitochondrial permeability transition. We investigated whether glycine, an amino acid with poorly understood cytoprotective properties, may interfere with this mechanism. In cardiomyocyte cultures, addition of glycine during re-energization following 1 h of simulated ischaemia (NaCN/2-deoxyglucose, pH 6.4) completely prevented necrotic cell death associated with pH normalization. Glycine also protected against cell death associated with pH normalization in reoxygenated rat hearts. Glycine prevented cyclosporin-sensitive swelling and calcein release associated with re-energization in rat heart mitochondria submitted to simulated ischaemia or to Ca2+ stress under normoxia. NMR spectroscopy revealed a marked glycine depletion in re-energized cardiomyocytes that was reversed by exposure to 3 mM glycine. These results suggest that intracellular glycine exerts a previously unrecognized inhibition on mitochondrial permeability transition in cardiac myocytes, and that intracellular glycine depletion during myocardial hypoxia/reoxygenation makes the cell more vulnerable to necrotic death. (Received 18 May 2004; accepted after revision 17 June 2004; first published online 24 June 2004) Corresponding author D. Garcia-Dorado: Unidad de Investigacion B, Hospital Universitario Vall d’Hebron, Pg. Vall d’Hebron 119–129, 08035 Barcelona, Spain. Email: dgdorado@vhebron.net Mitochondria are increasingly recognized as central regulators of cell death and survival in cardiac myocytes exposed to ischaemia–reperfusion (Duchen, 1999). A prominent player in the mitochondrial death pathway is the mitochondrial permeability transition pore, a largely unselective megachannel connecting the mitochondrial matrix with the cytosol and allowing the passage of molecules up to 1.5 kDa. During reperfusion, mitochondrial permeability transition (MPT) may be triggered by elevated Ca2+ concentrations, radical oxygen species and normalization of intracellular pH, and it is followed by the collapse of mitochondrial membrane potential, interruption of ATP synthesis, release of pro-apoptotic molecules into the cytosol (Petronilli et al. 2001; Di Lisa et al. 2001) and profound ionic disturbances that may result in necrotic or apoptotic death (Lemasters et al. 1998; Kim et al. 2003). It has been recently proposed that inhibition of MPT may have a strong protective effect M. Ruiz-Meana and P. Pina contributed equally to the paper by preventing the development of structural cell fragility (Hausenloy et al. 2003; Javadov et al. 2003). The amino acid glycine (Gly) has been shown to exert a protective effect against cell death after diverse insults including ischaemia–reperfusion injury in different cell types other than cardiomyocytes, but the mechanism of this effect remains elusive (Nishimura et al. 1998; Schemmeret al. 1999; Nishimura & Lemasters, 2001; Dong et al. 2001; Zhang et al. 2003). In this study we investigated the effect of Gly on MPT and reoxygenation-induced cell death in cultured HL-1 cardiac myocytes, intact rat myocardium and rat heart mitochondria. The results show that Gly, an endogenous amino acid, may play a cardioprotective effect in reoxygenated cardiomyocytes by preventing MPT associated with pH normalization. Methods This study was performed on cultured HL-1 cardiac myocytes, Langendorf-perfused hearts from SpragueDawley rats, and isolated mitochondria. The experimental C The Physiological Society 2004 DOI: 10.1113/jphysiol.2004.068320
874 M Ruiz-Veana and athers m procedures conformed with the Guide for the Care and Experimental protocol After 10 min of equilibration. Use of Laboratory Animals published by the United hearts were submitted to 2 h of hyporia at pH6.4 followed States National Institutes of Health INTH Publication no. hy I h ofreoygenation.For hypaxic perfusion,a modified 85-23.revised 1996).and were approved by the Research Krebs buffer (mM:NaCl 139.5,KC1 4.7.MgSO,1.2. Commission on Ethics of the llospital Vall d'Hlebron. CaCl,2.5,NalICO,3.5,KIT,PO 12,and sucrose Il)was bubhled with 95%N3-5%6 CO2.Reoxygenation was performed by switching the hypocic Krebs solution to a Studies in HL-1 cardiomyocytes glucose-containing orygenated (95%O-5%CO.)Krebs HL-1 cardiac myocytes were plated at 20000 cells cm-2 solution.Hearts were allocated to one of three groups: density in glass-bottomed culture dishes until a (1)control reoxygenation (=6)at pH7.4 (2)acidic 70-8046 confluence was achieved,as previausly described reoxygenation (n5)at pl16.d:(3)reoxygenation in the (Ruiz-Meana ctal 2003). presence of 10 mM Gly (n5).Two additional hearts were used to calculate cumulative concentration-response curves of developed tensson and transmembrane action Simulated ischaemia-reoxygenation.Cell pellets were potential to Gly (from 10-4 to 10-1 M,each concentration submitted to 1 h simulated ischaemia (S1)by suspending given during 15 min)under normoxia.Hearts were paced them in a buffer containing (ms):NaC 140,KC13.6. CaCk 2,MgsO,1.2,Hepes 20,NaCN 2,2-deoxyglucose from the base (2.5 ms,4V pulses at 2.5 Hz)and trans- membrane action potentials were recorded from the apex, 20,at pH6.4,37C.Reoxygenation was performed by as descrihed (Rodriguex-Sinovas etal 2003). centrifugation and resuspension of the pellet in a control normoxic huffer at pH7.4,with 5 mM glucose. Enzyme release.Lactate dehpdrogenase (LDH)release during reoxygenation was measured in samples taken from Experimental interventions.The effect of modifying the the effluent as previously described (Rodriguez-Sinovas reoxygenation buffer by lowering its pH (up to 6.4)or by al2005). adding Gly (3 mM)was investigated.The role of Ca-was studied by sequcstration of either intra-or extracellular Ca+(addition of 30 jMt BAPIA or removal of CaCl:in Studies in isolated mitochondria the presence of 2 mst EGTA) Isolation of mitochondria.Rat heart mitochondria were isolated by ditferential centrifugation,according to the Cell volume and viability.Changes in cell volume were method described by Holmuhamedov etl (1998). determined by analysis of forward scatter (FACscalibur Protein was adjusted to 1 mg ml-. flow cytometer,Becton Dickinson,USA)and cell viability by exclusion of propidium iodide (PI).Double staining with annexinV-fuorescein isothiocyanate (FITC)-P] Experimental interventions.Mitochondria were sub- was used to quantify the contribution of apoptosis mitted to either (1)simulated ischaemia (SI) (annexin-FITC+-PI-cells)to cell death reoxygenation or (2)15 min Ca overlad.To simulate ischaemia,the mitochondrial pellet was suspended in a metabolic inhibition buffer (myt:KCl 150,Nicl7. Intracellular pH and Na".Changes in cytosolic [I[+) Hepes 6.KH:PO.0.5.sucrose 50.MgCl,I.ADP0.1. and [Na']during Sl-reoxygenation were monitored CaCl,0.01 and NaCN 2),at pH6.4,for 1h at 37C. by ratio-fluorescence imaging in HL-1 cells loaded, Reoxygenation was performed by switching to a respectively.with 3 AM BCECF-AM or 5 uM SBFI NaCN-free solution containing (mss):KCI 150,NaCl7, (Molecular Probes.USA)(Ruix-Meana et aL 2003). Hepes6.KH2PO:0.5.sucrose 50,MgCl:1,ADP0.1, CaCl:0.05,ATP 0_3,suocinate 0.3.ascorbic acid 2.5, at pH7.2.The effects of acid pH (6.4).Gly (3 mM) Studies in isolated rat hearts or cyclosporin A (CsA,1 jM),as well as suhstrates Heart preparation.After an intraperitoneal injectin of (5 mM succinate,5 mM pyruvate or 5 mM L-alanine) sodium pentabarbital (100 mg kg-),the hearts from 18 on MPT were assessed during the first 30 min of Sprague-Dawley rats (280-350g)were rapidly removed reoxygenation.Also,the contribution of Cat to the MPT and perfused with an oxygenated Krebs solution at 37C, was tested by adding 2 mM EGTA or by increasing the as previously described (Rodriguez-Sinowas etal 2003). extracellular Ca+to 200 Mt in the reoxygenation buffer. O The Physiologkal Soclety 2004
874 M. Ruiz-Meana and others J Physiol 558.3 procedures conformed with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication no. 85-23, revised 1996), and were approved by the Research Commission on Ethics of the Hospital Vall d’Hebron. Studies in HL-1 cardiomyocytes HL-1 cardiac myocytes were plated at 20 000 cells cm−2 density in glass-bottomed culture dishes until a 70–80% confluence was achieved, as previously described (Ruiz-Meana et al. 2003). Simulated ischaemia–reoxygenation. Cell pellets were submitted to 1 h simulated ischaemia (SI) by suspending them in a buffer containing (mm): NaCl 140, KCl 3.6, CaCl2 2, MgSO4 1.2, Hepes 20, NaCN 2, 2-deoxyglucose 20, at pH 6.4, 37◦C. Reoxygenation was performed by centrifugation and resuspension of the pellet in a control normoxic buffer at pH 7.4, with 5 mm glucose. Experimental interventions. The effect of modifying the reoxygenation buffer by lowering its pH (up to 6.4) or by adding Gly (3 mm) was investigated. The role of Ca2+ was studied by sequestration of either intra- or extracellular Ca2+ (addition of 30 µm BAPTA or removal of CaCl2 in the presence of 2 mm EGTA). Cell volume and viability. Changes in cell volume were determined by analysis of forward scatter (FACscalibur flow cytometer, Becton Dickinson, USA) and cell viability by exclusion of propidium iodide (PI). Double staining with annexinV–fluorescein isothiocyanate (FITC)–PI was used to quantify the contribution of apoptosis (annexin–FITC+–PI− cells) to cell death. Intracellular pH and Na+. Changes in cytosolic [H+] and [Na+] during SI–reoxygenation were monitored by ratio-fluorescence imaging in HL-1 cells loaded, respectively, with 3 µm BCECF-AM or 5 µm SBFI (Molecular Probes, USA) (Ruiz-Meana et al. 2003). Studies in isolated rat hearts Heart preparation. After an intraperitoneal injection of sodium pentobarbital (100 mg kg−1), the hearts from 18 Sprague-Dawley rats (280–350 g) were rapidly removed and perfused with an oxygenated Krebs solution at 37◦C, as previously described (Rodriguez-Sinovas et al. 2003). Experimental protocol. After 40 min of equilibration, hearts were submitted to 2 h of hypoxia at pH 6.4 followed by 1 h of reoxygenation. For hypoxic perfusion, a modified Krebs buffer (mm: NaCl 139.5, KCl 4.7, MgSO4 1.2, CaCl2 2.5, NaHCO3 3.5, KH2PO4 1.2, and sucrose 11) was bubbled with 95% N2–5% CO2. Reoxygenation was performed by switching the hypoxic Krebs solution to a glucose-containing oxygenated (95% O2–5% CO2) Krebs solution. Hearts were allocated to one of three groups: (1) control reoxygenation (n = 6) at pH 7.4; (2) acidic reoxygenation (n = 5) at pH 6.4; (3) reoxygenation in the presence of 10 mm Gly (n = 5). Two additional hearts were used to calculate cumulative concentration–response curves of developed tension and transmembrane action potential to Gly (from 10−4 to 10−1 m, each concentration given during 15 min) under normoxia. Hearts were paced from the base (2.5 ms, 4 V pulses at 2.5 Hz) and transmembrane action potentials were recorded from the apex, as described (Rodriguez-Sinovas et al. 2003). Enzyme release. Lactate dehydrogenase (LDH) release during reoxygenation was measured in samples taken from the effluent as previously described (Rodriguez-Sinovas et al. 2003). Studies in isolated mitochondria Isolation of mitochondria. Rat heart mitochondria were isolated by differential centrifugation, according to the method described by Holmuhamedov et al. (1998). Protein was adjusted to 1 mg ml−1. Experimental interventions. Mitochondria were submitted to either (1) simulated ischaemia (SI)– reoxygenation or (2) 15 min Ca2+ overload. To simulate ischaemia, the mitochondrial pellet was suspended in a metabolic inhibition buffer (mm: KCl 150, NaCl 7, Hepes 6, KH2PO4 0.5, sucrose 50, MgCl2 1, ADP 0.1, CaCl2 0.01 and NaCN 2), at pH 6.4, for 1 h at 37◦C. Reoxygenation was performed by switching to a NaCN-free solution containing (mm): KCl 150, NaCl 7, Hepes 6, KH2PO4 0.5, sucrose 50, MgCl2 1, ADP 0.1, CaCl2 0.05, ATP 0.3, succinate 0.3, ascorbic acid 2.5, at pH 7.2. The effects of acid pH (6.4), Gly (3 mm) or cyclosporin A (CsA, 1 µm), as well as substrates (5 mm succinate, 5 mm pyruvate or 5 mm l-alanine) on MPT were assessed during the first 30 min of reoxygenation. Also, the contribution of Ca2+ to the MPT was tested by adding 2 mm EGTA or by increasing the extracellular Ca2+ to 200 µm in the reoxygenation buffer. C The Physiological Society 2004
1h5限3 Gldne prevents mitochondrial permeabrlity transition 875 In the control group,mitochondria were submitted to Statistical analysis the same experimental manipulations in a NaCN-free, low Ca'+(0.1 AM)buffer containing (msr):KCl 150, Statistical analysis was performed using commercially available software (SPSS for Windows 8.0).Comparisons NaCl 7.Iepes 6.KI,PO 0.5,sucrose 50.MgCl,1. ADP 0.1.ATP 0.3,succinate 5,ascorbic acid 2.5.at pH 7.2. hetween two independent groups were performed by The inhibitory effect of acid pH and Gly on MPT was Student's i test.The effect of 5H-reoxygenation with or also assessed in normoxic mitochondria suhmitted to without Gly supplementation on intracellular Gly content was assessed by means of a t test for paired samples. 200 u CaCl for 15 min. Evaluation of unhomogeneity between multiple groups Swelling assay.Changes in mitochondrial volume was performed by analysis of variance (ANOVA).Curves were assayed spectropbotometrically in mitochondrial of IDH release were compared hy repeated measures suspensions at 25C using either 96-well-plates (Garlid ANOVA.Data are expressed as the mcan +s.E.M.The significanoe level was set at 0.05. erl 1996)or 500 ul cuvettes.Light absorbance was determined at520 nm under basal conditions,after 60 min of SI,and at the end of reoxygenation.Changes in light Results aboorbance were normalized with respect to basal values. In normoxic Ca-stressed mitochondria light absorbance Cell death during S-reoxygenation was monitored throughout time following addition of SI did not result indetoctablecell death (Fig 1A).However, 200 Ca+to the mitochandrial suspension.MPT was reoxygenation after 1h of SI was associated with a dentifed as a CsA-sensitive abrupt decrease in light marked increase in the number of PI+cardiomyocytes, absorbance,reflecting pussive matrix swelling. reflecting sarcolemmal disruption (Fig.1A).Sarcolemmal rupture occurred carly upon reoxygenation,was fully Assessment of calcein release.Calcein release was observable at 30 min,and did not further increase when monitored in mitochondria using a 96-well fluoro- the duration of reoxygenation was prolonged to 2 h (data metry assay.The mitochondrial pellet was loaded with not shown).Apopcosis seemed not to contribute to cell I uM calcein-AM at 37C (Molecular Probes,USA). death:the proportion of annexin-PI-cells after 30 min washed and submitted to 200 uM CaCl.At the end of reoxygenation was low and remained unchanged after of the experiment,mitochondria were centrifuged and 2h of reoxygenation (data not shown).Furthermore,this calcein fluorescence was determined (Excitation Wave- proportion was not significantly modified by either SI or length:485 nm.Emission Wavelength:538 nm)in the reoxygenation (Fig.1B). pellet (intramitocbondrial compartment)and in the Cell death associated with reaxygenation was closely supernatant (extramitochondrial compartment).MPT related to pH normalieation and could be abolished was identified as a CsA-sensitive loss of calcein from when pH remained at 6.4 during reoxygenation (Fig.1A). the intramitocbondrial compartment.This effect was Addition of 3 mM Gly to the reoxygenation buffer nvariably associated with an increase in the caloein at the completely prevented pH-dependent cell death (Fig.1A). extramitochondrial compartment. Chelation of Ca+from the extracellulr medium with EGTA had no effect on reaxygenation-induced cell NMR spectroscopy death(15.l±0.4%andl6.3±0.26%in control and EGTA-treated cells,respectively,n.s).Similarly,intra- Cell pellets ((3-5)x 10 cells per sample)were extracted cellular Ca+sequestration with BAPTA.did not reduce with 1.2 ml of chloroform-methanol (1:2)at room cell death during,reoxygenation (Fig.IC). temperature for 1 h.After addition of D.5 ml chloroform and 0.5 ml of water,two phases were separated by centrifugation (1500 g.5 min).The upper phase Changes in cell volume,intracellular pH (methanal-water)was separated and dried under N.. and Nat during Sl-reoxygenation Dried extracts were dissolved with 400al of D.O SI induced a significant volume increase foell swelling), containing I mst 3-(trimethylsilyl)propionic acid(TSP)as followed by cell size correction upon restoration of chemical shift and concentration reference.Spectroscopy energy and normalization of pH.Interestingly,the was performed at 27C with a 400 MHz Bruker ARX protective effect of acid pH and Gly on cell death was spectrometer.Peak areas were measured by integration in closely associated with an impaired capacity of cells to fully relaxed spectra. recover their volume (Fig.2A).In hoth interventions, The Physiologkcal Society 2004
J Physiol 558.3 Glycine prevents mitochondrial permeability transition 875 In the control group, mitochondria were submitted to the same experimental manipulations in a NaCN-free, low Ca2+ (0.1 µm) buffer containing (mm): KCl 150, NaCl 7, Hepes 6, KH2PO4 0.5, sucrose 50, MgCl2 1, ADP 0.1, ATP 0.3, succinate 5, ascorbic acid 2.5, at pH 7.2. The inhibitory effect of acid pH and Gly on MPT was also assessed in normoxic mitochondria submitted to 200 µm CaCl2 for 15 min. Swelling assay. Changes in mitochondrial volume were assayed spectrophotometrically in mitochondrial suspensions at 25◦C using either 96-well-plates (Garlid et al. 1996) or 500 µl cuvettes. Light absorbance was determined at 520 nm under basal conditions, after 60 min of SI, and at the end of reoxygenation. Changes in light absorbance were normalized with respect to basal values. In normoxic Ca2+-stressed mitochondria light absorbance was monitored throughout time following addition of 200 µm Ca2+ to the mitochondrial suspension. MPT was identified as a CsA-sensitive abrupt decrease in light absorbance, reflecting passive matrix swelling. Assessment of calcein release. Calcein release was monitored in mitochondria using a 96-well fluorometry assay. The mitochondrial pellet was loaded with 1 µm calcein-AM at 37◦C (Molecular Probes, USA), washed and submitted to 200 µm CaCl2. At the end of the experiment, mitochondria were centrifuged and calcein fluorescence was determined (Excitation Wavelength: 485 nm, Emission Wavelength: 538 nm) in the pellet (intramitochondrial compartment) and in the supernatant (extramitochondrial compartment). MPT was identified as a CsA-sensitive loss of calcein from the intramitochondrial compartment. This effect was invariably associated with an increase in the calcein at the extramitochondrial compartment. NMR spectroscopy Cell pellets ((3–5) × 106 cells per sample) were extracted with 1.2 ml of chloroform–methanol (1 : 2) at room temperature for 1 h. After addition of 0.5 ml chloroform and 0.5 ml of water, two phases were separated by centrifugation (1500 g, 5 min). The upper phase (methanol–water) was separated and dried under N2. Dried extracts were dissolved with 400 µl of D2O containing 1 mm3-(trimethylsilyl) propionic acid (TSP) as chemical shift and concentration reference. Spectroscopy was performed at 27◦ C with a 400 MHz Bruker ARX spectrometer. Peak areas were measured by integration in fully relaxed spectra. Statistical analysis Statistical analysis was performed using commercially available software (SPSS for Windows 8.0). Comparisons between two independent groups were performed by Student’s t test. The effect of SI–reoxygenation with or without Gly supplementation on intracellular Gly content was assessed by means of a t test for paired samples. Evaluation of unhomogeneity between multiple groups was performed by analysis of variance (ANOVA). Curves of LDH release were compared by repeated measures ANOVA. Data are expressed as the mean ± s.e.m. The significance level was set at 0.05. Results Cell death during SI–reoxygenation SI did not result in detectable cell death (Fig. 1A). However, reoxygenation after 1 h of SI was associated with a marked increase in the number of PI+ cardiomyocytes, reflecting sarcolemmal disruption (Fig. 1A). Sarcolemmal rupture occurred early upon reoxygenation, was fully observable at 30 min, and did not further increase when the duration of reoxygenation was prolonged to 2 h (data not shown). Apoptosis seemed not to contribute to cell death: the proportion of annexin+–PI− cells after 30 min of reoxygenation was low and remained unchanged after 2 h of reoxygenation (data not shown). Furthermore, this proportion was not significantly modified by either SI or reoxygenation (Fig. 1B). Cell death associated with reoxygenation was closely related to pH normalization and could be abolished when pH remained at 6.4 during reoxygenation (Fig. 1A). Addition of 3 mm Gly to the reoxygenation buffer completely prevented pH-dependent cell death (Fig. 1A). Chelation of Ca2+ from the extracellular medium with EGTA had no effect on reoxygenation-induced cell death (15.1 ± 0.4% and 16.3 ± 0.26% in control and EGTA-treated cells, respectively, n.s.). Similarly, intracellular Ca2+ sequestration with BAPTA, did not reduce cell death during reoxygenation (Fig. 1C). Changes in cell volume, intracellular pH and Na+ during SI–reoxygenation SI induced a significant volume increase (cell swelling), followed by cell size correction upon restoration of energy and normalization of pH. Interestingly, the protective effect of acid pH and Gly on cell death was closely associated with an impaired capacity of cells to recover their volume (Fig. 2A). In both interventions, C The Physiological Society 2004
976 M.Ruiz-Meana and others Ja5583 dose-effect studies demonstrated a narrow inverse pH recovery (Fig,3).Intracellular Nat concentration relationship between the degree of protection against steadily rose during the whole SI period (from 1+0 reoxygenation-induced cell death and the mgnitude of to 1.350.05 a.u.of SBFI ratio tuorescence),remained correction of oell volume (Fig.2B and C). stable during the 30 min reoxyyenation (1.33+0.1 a-u) Intracellular pH decreased rapidly during SI,reaching A120 6.440.04 within 15 min,and recovered to 7.2 in the 4n-0 first 10 min of reoxygenation.Addition of Gly to the 115 reoxygenation buffer did not produce any dely in 8 110 A35 n=8 R 105 0 100 74Gy64 20 B 270 15 200 Nx Sl 7.4 54 Gly 30 Arnexn' 250 n=9-15引 20 24 230 220 210 6.054 58727.6 8.0 Nx SI 250 30 I20M日APTAAM UM BAPTA-AM n=5 区 28 240 230 220 210 PI 200 ◆F8C 100 -10 987 -5 Figure 1.Cell death in cardiomyorytes submitted to 1 h 5-30 min reoxygenation Glycine log周 A,necrotic cel death (P)when reaxpgenstion was perfarmed at Figure 2.Effect of 5l and reoxygenation on cardiomyocyte 5l1 7.4,st pll6.4 or at pll7.4 in the presente of 3 m Gly.Cell death volume and desth (P cells)under different conditions 6 assodated wth nommalza on of pH upon ecmgenadon. A,cels submitted to SI cporionoed a signiticant incease in their Aadion of Gly to the roooygenanion butter provamed coli death volume,which was corectod upon reoeygonation at pH 7.4.Col associated with pH normalizaan.8,comtribution of apoptoss volume did not ecover when Gly was present during tecygena.on or annesn+-P-ceb to total cell death was small and was not when reooygenation was performed at pH 6.4.P 0.05 versus signilicantly ineteared Ey pither Sl er recpygenation f.sequestraticn normeoir celk.8 the degvee of pmtection agairst death afferded by od intracelllar Cat with BAPTA tho ghoue the teeaygensenn period knw pH closely comelated with the magnisude ol the effect ain cel dd nct affard any sonificant protection against cdll death.P<0.05 vol me C,the degre of protection aforded by Gly was aba coscly ersus nommooc cdk. comelted with its efect cn cal wolume. 有Thp,deinciral Sor时y0M
876 M. Ruiz-Meana and others J Physiol 558.3 dose–effect studies demonstrated a narrow inverse relationship between the degree of protection against reoxygenation-induced cell death and the magnitude of correction of cell volume (Fig. 2B and C). Intracellular pH decreased rapidly during SI, reaching 6.4 ± 0.04 within 15 min, and recovered to 7.2 in the first 10 min of reoxygenation. Addition of Gly to the reoxygenation buffer did not produce any delay in Figure 1. Cell death in cardiomyocytes submitted to 1 h SI–30 min reoxygenation A, necrotic cell death (PI+) when reoxygenation was performed at pH 7.4, at pH 6.4 or at pH 7.4 in the presence of 3 mM Gly. Cell death was associated with normalization of pH upon reoxygenation. Addition of Gly to the reoxygenation buffer prevented cell death associated with pH normalization. B, contribution of apoptosis (annexin+–PI− cells) to total cell death was small and was not significantly increased by either SI or reoxygenation. C, sequestration of intracellular Ca2+ with BAPTA throughout the reoxygenation period did not afford any significant protection against cell death. P < 0.05 versus normoxic cells. pH recovery (Fig. 3). Intracellular Na+ concentration steadily rose during the whole SI period (from 1 ± 0 to 1.35 ± 0.05 a.u. of SBFI ratio fluorescence), remained stable during the 30 min reoxygenation (1.33 ± 0.1 a.u), Figure 2. Effect of SI and reoxygenation on cardiomyocyte volume and death (PI+ cells) under different conditions A, cells submitted to SI experienced a significant increase in their volume, which was corrected upon reoxygenation at pH 7.4. Cell volume did not recover when Gly was present during reoxygenation or when reoxygenation was performed at pH 6.4. P < 0.05 versus normoxic cells. B, the degree of protection against death afforded by low pH closely correlated with the magnitude of the effect on cell volume. C, the degree of protection afforded by Gly was also closely correlated with its effect on cell volume. C The Physiological Society 2004
/h,d55L1 Ghrine provents mitochondral permeablity transition 877 and was not influenced by the presence of Gly in the conduction velocity or contractile function (data not reoxygenation buffer (1.320.16 a.u..n.s.). shawn). Effect of Gly on isolated rat hearts Mitochondrial swelling during reoxygenation Reoxygenation induced an important IDH release In isolated mitochondria.reaxygenation at pH7.2 (Tig-4A).In hearts submitted to reaxygenation at after 1h SI at pH6.4 was associated with an increase pH 64,total LDH release was reduced by approximately in mitochondrial volume,as measured by a fall in 50%(33.1+6.0U(g dry tissue)-(60 min)-).Addition light absorbance.Reoxygenation at pH6.4 abolished of 10 mM Gly during reoxygenation had a similar effect mitochondrial swelling Reorygenation-induced swelling (LDH release was 37.4+7.8 U(gdrytissue)-(60 min)-. can be attributed to MPT since it was blocked by 1M Fig.4B).Normocic perfusion with a Gly-containing Krehs CsA and could be precipitated by increasing Ca to solution,from 10 to 10-M,did not induce any 200 uM during reoxygenation (Fig.5A).MPT was not change in transmemhrane action potential characteristics. dependent on substrate availability,since the increase of succinate concentration from 0.3 to 5 mM,or addition of m pyruvate did not reduce mitochondrial swelling 750 (Fig.5A).Addition of 3 mM Gly to the reoxygenation ■Contro(n-0 ▣Gy加= buffer completely prevented mitochondrial swelling 725 despite pH normalization (Fig.5B).This protective effect against MPT could not be mimicked by 5 mM L-alanine 7c0 ◆cool 675 c Gly 10mM 4 Rx pH 6.4 650 《=56 626 Baseline 601 Rx 060 Centrol 055 a50 1530 4560 Time (min] 045 040 4n-5-6) 035 60 020 45 +4一Rx+ 025 016002000300340005c00 Tme (s) 置 15 Figure 3.Changes of intracellular pH in cardiomyocytes submitted to 1 h 51-30 min reoxygenation (Rx) A intrcelular pH immadiately hetore S (hacelne),amer 1 h Sl and t the end nf recoygenatien wars nort modfied by the arriton 3 mu Contrel Gly pH 6.4 Gly during the reoxygenation perod.8,original measurements of Figure 4.Reoxygenation injury in bsolatod rat hearts intracdllar I+cuncenlralion (u.of BC[OF rslio fuorescence)in A.tecoyyensbcn-induced L[I teledse in control and G-reaed 2 cepresentative experments.Addtion of Gly did rot modify the tme hes色，nd n hearts reaeyvenated at pl6A0.ulL川ee course of pH mcowery during roooeygonation curing roooeygonation. OThe Physologkal Socier 2004
J Physiol 558.3 Glycine prevents mitochondrial permeability transition 877 and was not influenced by the presence of Gly in the reoxygenation buffer (1.32 ± 0.16 a.u., n.s.). Effect of Gly on isolated rat hearts Reoxygenation induced an important LDH release (Fig. 4A). In hearts submitted to reoxygenation at pH 6.4, total LDH release was reduced by approximately 50% (33.1 ± 6.0 U (g dry tissue)−1 (60 min)−1). Addition of 10 mm Gly during reoxygenation had a similar effect (LDH release was 37.4 ± 7.8 U (g dry tissue)−1 (60 min)−1, Fig. 4B). Normoxic perfusion with a Gly-containing Krebs solution, from 10−4 to 10−1 m, did not induce any change in transmembrane action potential characteristics, Figure 3. Changes of intracellular pH in cardiomyocytes submitted to 1 h SI–30 min reoxygenation (Rx) A, intracellular pH immediately before SI (baseline), after 1 h SI and at the end of reoxygenation was not modified by the addition of 3 mM Gly during the reoxygenation period. B, original measurements of intracellular H+ concentration (a.u. of BCECF ratio fluorescence) in 2 representative experiments. Addition of Gly did not modify the time course of pH recovery during reoxygenation conduction velocity or contractile function (data not shown). Mitochondrial swelling during reoxygenation In isolated mitochondria, reoxygenation at pH 7.2 after 1 h SI at pH 6.4 was associated with an increase in mitochondrial volume, as measured by a fall in light absorbance. Reoxygenation at pH 6.4 abolished mitochondrial swelling. Reoxygenation-induced swelling can be attributed to MPT since it was blocked by 1 µm CsA and could be precipitated by increasing Ca2+ to 200 µm during reoxygenation (Fig. 5A). MPT was not dependent on substrate availability, since the increase of succinate concentration from 0.3 to 5 mm, or addition of 5 mm pyruvate did not reduce mitochondrial swelling (Fig. 5A). Addition of 3 mm Gly to the reoxygenation buffer completely prevented mitochondrial swelling despite pH normalization (Fig. 5B). This protective effect against MPT could not be mimicked by 5 mm l-alanine Figure 4. Reoxygenation injury in isolated rat hearts A, reoxygenation-induced LDH release in control and Gly-treated hearts, and in hearts reoxygenated at pH 6.4. B, total LDH release during reoxygenation. C The Physiological Society 2004
878 M Ruiz-Meana and others 145a3 or by sequestration of extracellular Ca with 2 mM mitocbondrial swelling induced by high Ca'in a EGTA (Fig.5B).To further assess the potential inhibitory similar manner as acidic pH (Fig.5C and D). effect of Gly on MPT.normoxic mitochundria were stressed with 200 jm CaCl,a manoeuvre known to precipitate mitochondrial permeabilization.The Calcein release in isolated mitochondria minimum concentration of Gly found to be protective Addition of 200 uM CaCl,tocalcein-laded mitochondria against SI-reoxygenation (3 ms)also reduced induced a rapid and abrupt release of the fluorachrom A SVRI B SIRx 10-2-的 0-2-8} 100 100 95 0 85 80 Ca200 D '0 100 Ca200M 95 95 90 90 p州6.4 85 65 Control Control 0 0 2 4 810 Time (min) Gly (mMI protoools A 30 min of rcoxpgenation (d after I h of sl under the following condtiors:at pH 72)wt 2 d mcrert respiratory subestrates (5 m succinone or 5 mM pyruvate)at pH 6.4,with I M CsA or with 200 M Ld2.此st4 e is expres火时as the pert5 tage vdue with n5pt物r0 nuac miochandnia.ohU rEoxygenotion (.4)and CsA prevented the reoepgenation-nduced fal in light aboorbante,ndicative of MPL “P<005ss版且3动min ofc9m0 n (Ra)atterI h a Slunh tolwing conditions意ph了2 )with 5 mu L-Alanine,with 2 m EGTA.with 3 mu Gly and with 1 pM CA.Absornance K pepressed as the percentage vale with respect to nommac mitochondna (N.Addition of Gly to the reoppenation buffer able to completdy prevent rooygenation-incucrd mio寸on寸d swcilng.a5ddCA.P÷D.0sesk C.exposure of normoox mtochancris 1a 15 mn of 200 MM Catl2 to promole ML with or wilhoi 3 mu Gy Control mikchendrid were not yubmitted o high Ca contentration Addit on of 3 m Gy sigrificantly reduced awoling in Ca-stressed mitochondria."P c005 versu:Control."P e0.05 veesus 3 mM Gly.D,changos in light ebsorbance Shroughout time in fuily energized mitochondria submitted to 200 poM Cb to promote MP1.The emows ndicate the time at wh ch Ca+wos added.Addit on of 3 mM Gly dutinga+overlosd was able to prevent mitechondrial sweling more afecticaly than acid pH The Physiclogical Society 2004
878 M. Ruiz-Meana and others J Physiol 558.3 or by sequestration of extracellular Ca2+ with 2 mm EGTA (Fig. 5B). To further assess the potential inhibitory effect of Gly on MPT, normoxic mitochondria were stressed with 200 µm CaCl2, a manoeuvre known to precipitate mitochondrial permeabilization. The minimum concentration of Gly found to be protective against SI–reoxygenation (3 mm) also reduced Figure 5. Effect of pH and Gly on mitochondrial swelling Changes in light absorbance at 520 nm in suspensions of rat heart mitochondria submitted to different experimental protocols. A, 30 min of reoxygenation (Rx) after 1 h of SI under the following conditions: at pH 7.2 (C), with 2 different respiratory substrates (5 mM succinate or 5 mM pyruvate), at pH 6.4, with 1 µM CsA or with 200 µM CaCl2. Absorbance is expressed as the percentage value with respect to normoxic mitochondria (Nx). Both acidic reoxygenation (6.4) and CsA prevented the reoxygenation-induced fall in light absorbance, indicative of MPT. ∗P < 0.05 versus Nx. B, 30 min of reoxygenation (Rx) after 1 h of SI under the following conditions: at pH 7.2 (C), with 5 mM L-alanine, with 2 mM EGTA, with 3 mM Gly and with 1 µM CsA. Absorbance is expressed as the percentage value with respect to normoxic mitochondria (Nx). Addition of Gly to the reoxygenation buffer was able to completely prevent reoxygenation-induced mitochondrial swelling, as did CsA. ∗P < 0.05 versus Nx. C, exposure of normoxic mitochondria to 15 min of 200 µM CaCl2 to promote MPT, with or without 3 mM Gly. Control mitochondria were not submitted to high Ca2+ concentration. Addition of 3 mM Gly significantly reduced swelling in Ca2+-stressed mitochondria. ∗P < 0.05 versus Control, ∗∗P < 0.05 versus 3 mM Gly. D, changes in light absorbance throughout time in fully energized mitochondria submitted to 200 µM CaCl2 to promote MPT. The arrows indicate the time at which Ca2+ was added. Addition of 3 mM Gly during Ca2+ overload was able to prevent mitochondrial swelling more efectively than acid pH. mitochondrial swelling induced by high Ca2+ in a similar manner as acidic pH (Fig. 5C and D). Calcein release in isolated mitochondria Addition of 200 µm CaCl2 to calcein-loaded mitochondria induced a rapid and abrupt release of the fluorochrom C The Physiological Society 2004
/h,ns3越i Ghcne prevents mitochondral permeablity transition 89 into the extramitochondrial space (Fig.6).When Gly was inhibit MPI in isolated mitocbondris.Altogether,these added at the concentrations used to prevent mitochondrial results indicate that Gly protects cardiomyocytes from swelling (3 mM or higher).Caitinduced calcein release reoxygenation-induced cell death by preventing MPT was completely inhibited(Fig 6).Release of calcein clearly associated with pH normalization. reflected MPT since it could be blocked by CsA(0.1-1 u). MPT contributes to cardiomyocyte death induced Intracellular Gly content by normalization of pH during reoxygenation NMR spectroscopy revealed that Sl-reoxygenation Disruption of the mitocbondrialinner membrane by MPI induced a significant reduction of intracellular Gly triggers cell death by different pathways,including the content.In contrast,a marked increase in intracellular Gly release of cytochrome c and other proupoptotic factors concentration was observed after 30 min reoxygenation that activate caspases and initiate apoptosis (Zamzami when reoxygenation buffer contained 3 mM Gly (Fig.7). Kroemer,2001;De Giorgi et al.2002).Less known,but increasingly recognized,is the contribution of MPT to Discussion acute necrotic cell death through impaired ATP synthesis and alterations in cation,in particular of Ca,homeo- The present study shows that the amino acid Gly has a stasis (Crompton,1999;Kim er ml 2003). powerful and previously unrecagnized inhibitory effect on Growing evidence indicates that MPT may contribute MPT,similar to that of low pH,in rat heart mitochondria. to myocardial reperfusion or reoxygenation injury.During We also demonstrate for the first time that addition prolonged ischaemia,the rapid and profound fall in of 3-10 mM Ghy during reoxygenation prevents acute intracellular pll exerts a powerful inhibitory effect on necrotic cell death associated with pH normalization in MPI by antagonizing Ca binding to the adenine cultured cardiac myocytes and LDH release in isolated rat nucleotide translocase (ANT:Hakstrap,1991).Upon bearts.Moreover,exposure to Gly during reaxygenation reperfusion,the protective inhibitory effect of low pll not only reverses cellular Gly depletion ohserved in is abrogated by the rapid correction of intracellular cells submitted to SI-reoxygenation,but markedly raises acidosis,while exaoerbated oxidative stress and persistent intracellular Gly content up to the range prowen to abnormalities of Ca+handling may stimulate MPT. (n=4) SI/Rx 40 Ca200uM 020 046 至0.16 0.12 30 0.侧 004 000 Control L0 3 Contrel 0310,Csh Gly (mM) Figure 7.Gly content during Si-reoxygenation (Rx] Gly (mM) Intracclular Gly contont mcoaurod by NMR in cardomyocyoes Figure 5.Effect of Gly on mitochondrial calcein release submitted so I h SI-30 min scoygenadion in the pesance (Glylor Calcen ttlease frum rat heart m tochondna submitted to 15 min of absence ly)of Gly in the reoxygenaton buffer.Contiol goup 200 M Catlz to promate MPL in the presence of Gly at Z dfferent comesponds to ce l maintained under Gly-free normooc condans concentraticns,in its ahsance,and when MPT was blockec with SHtecaygenaticn resulted in a reduction nf intracelllar Gly contem 0.1 pM CA.Contml gmup cemesponck to mitechondria nat whil addtinn of 3 m Gly to the renepgonation huttee indured a smted to Ca overkod.Gly 3 mw and 10 mw prevented calcsin significant increase in t边e introcelular Gly content P0.Q正og release.as cid CsA P<05 versus control normucc tell,"P<001 versus normooic cels. The Phyinlog★r0M
J Physiol 558.3 Glycine prevents mitochondrial permeability transition 879 into the extramitochondrial space (Fig. 6). When Gly was added at the concentrations used to prevent mitochondrial swelling (3 mm or higher), Ca2+-induced calcein release was completely inhibited (Fig. 6). Release of calcein clearly reflected MPT since it could be blocked by CsA (0.1–1 µm). Intracellular Gly content NMR spectroscopy revealed that SI–reoxygenation induced a significant reduction of intracellular Gly content. In contrast, a marked increase in intracellular Gly concentration was observed after 30 min reoxygenation when reoxygenation buffer contained 3 mm Gly (Fig. 7). Discussion The present study shows that the amino acid Gly has a powerful and previously unrecognized inhibitory effect on MPT, similar to that of low pH, in rat heart mitochondria. We also demonstrate for the first time that addition of 3–10 mm Gly during reoxygenation prevents acute necrotic cell death associated with pH normalization in cultured cardiac myocytes and LDH release in isolated rat hearts. Moreover, exposure to Gly during reoxygenation not only reverses cellular Gly depletion observed in cells submitted to SI–reoxygenation, but markedly raises intracellular Gly content up to the range proven to Figure 6. Effect of Gly on mitochondrial calcein release Calcein release from rat heart mitochondria submitted to 15 min of 200 µM CaCl2 to promote MPT, in the presence of Gly at 2 different concentrations, in its absence, and when MPT was blocked with 0.1 µM CsA. Control group corresponds to mitochondria not submitted to Ca2+ overload. Gly 3 mM and 10 mM prevented calcein release, as did CsA. P < 0.05 versus control. inhibit MPT in isolated mitochondria. Altogether, these results indicate that Gly protects cardiomyocytes from reoxygenation-induced cell death by preventing MPT associated with pH normalization. MPT contributes to cardiomyocyte death induced by normalization of pH during reoxygenation Disruption of the mitochondrial inner membrane by MPT triggers cell death by different pathways, including the release of cytochrome c and other proapoptotic factors that activate caspases and initiate apoptosis (Zamzami & Kroemer, 2001; De Giorgi et al. 2002). Less known, but increasingly recognized, is the contribution of MPT to acute necrotic cell death through impaired ATP synthesis and alterations in cation, in particular of Ca2+, homeostasis (Crompton, 1999; Kim et al. 2003). Growing evidence indicates that MPT may contribute to myocardial reperfusion or reoxygenation injury. During prolonged ischaemia, the rapid and profound fall in intracellular pH exerts a powerful inhibitory effect on MPT by antagonizing Ca2+ binding to the adenine nucleotide translocase (ANT; Halestrap, 1991). Upon reperfusion, the protective inhibitory effect of low pH is abrogated by the rapid correction of intracellular acidosis, while exacerbated oxidative stress and persistent abnormalities of Ca2+ handling may stimulate MPT. Figure 7. Gly content during SI–reoxygenation (Rx) Intracellular Gly content measured by NMR in cardiomyocytes submitted to 1 h SI–30 min reoxygenation in the presence (Gly+) or absence (Gly−) of Gly in the reoxygenation buffer. Control group corresponds to cells maintained under Gly-free normoxic conditions. SI–reoxygenation resulted in a reduction of intracellular Gly content while addition of 3 mM Gly to the reoxygenation buffer induced a significant increase in the intracellular Gly content. P < 0.05 versus normoxic cells, ∗∗P < 0.01 versus normoxic cells. C The Physiological Society 2004
88D M.Ruiz-Meana and athers JPbyun!5 Experimental evidence of MPT during reperfusion has Gly prevents reoxygenation injury by been provided by fluorescence microscopy of cardio interfering with MPT myocytes re-energized after SI showing.CsA-sensitive mitochondrial depolarization (Duchen etai.1993)and The direct protective effect of Gly against necrotic and apoptotic cell death induced hy ditferent insults,including calcein loading (Petronilli eral 1999).In reperfused ischaemia-reperfusion,and a variety of other pathological myocardium.MPT has been demonstrated by the loss of mitochondrial NAD+(Di Lisa etal 2001) conditions-Le.endotoxaemic shock,alcoholic hepatitis. and mitochondrial entrapment of Ideoxyglucose hepatic fibrosis.arthritis,tumour and drug toxicity- 6-phosphate (Tavadov er al 2000),with a time course that has heen characterized in detail (Zhong eral 1996 reflects that of pH normalization. Nishimuraer al 1998;Nishimura Lemasters,2001;Dong In the present study.acidic reorygenation markedly ctal.2001:Zhang etal.2003).Gly delays sarcolemmal attenuated cell death in cultured HI-I myocytes and in rat rupture induced by hypoxia,which was initially explained hearts.The protective effoct of low pH was independent by attenuated Nat accumulation obscurely related to of the inhibitory effect of acidosis on contractility (since stimulation of the Gly-gated Cl channels,originally HL-I cells have only a rudimentary contractile machinery descrihed in the postsynaptic membranes of the spinal and do not hypercontract upon reoxygenation),was no cord (Carini eral 2000).Other authors suggested that associated with attenuatedcytosolic Na overload.and was the protective effect of Gly was independent of receptor not modified hy elimination ofextra or intracellulr Ca+ stimulation hut rather related to the closureofnon-specific The protectiveeffect of acidic pH cannot he thusexplained kaks in plasma membranes induced by bypoxia (Dong by attenuation of Ca overload or hypercontracture,but et al.1998;Frank et al.2000;Nishimura Lemasters, it rather appears to be related to its inhibitory effect on 2001).Confocal microscopy analysis of membrane permeabilization to fluorescent molecules ofdifferentsizes MPT.as ohserved in isolated mitochondria. demonstrated that Gly delays membrane permeabilization during hypaxia which could reflect the opening of large 名Ni的.61，na anionic channels ('death pores')eventually leading to 出 massive cell swelling and sarcolemmal rupture (Nishimura Lemasters,2001).Previous studies were coincident in interpreting that Gly prevented plasma membrane failure. the final event in the pathway to necrotic cell death (Frank te↓TP.fROS tal.2000). 458s In the present study Gly completely prevented cell death associated with normalization ofpH during reorygenation in cardiomyocyte cultures,and mimicked the protective effect of acidic reoxygenation on LDII release in isolated rat hearts.Since acute cell death observed during n-enrginticn reoxygenation in our model was ascribable to MPT,we hypothesized that Gly could act at the mitochondrial kevel,interfering with MPT.The experiments in isolated MPTP mitochondria demonstrated that Gly indeed prevents CsA-sensitive mitochondrial swelling and calcein release induced by re-energization after a transient SI or by exposure to high Ca+.The protective effect of Gly is not CELL related to its utilization as a respiratory substrate or as a DEATH source of NAD(P)H,since it was not mimicked by other Figure &Prapesed role of Gly during reperfusion injury suhstrates (succinate or pyruvate)and was present in fully schemu end ischaemi-telaled concitions ate assocaled wilh energized mitochondria submitted to Ca'+owerload. cytooolic derangements that fovour MPT but ako with acidoss that The inhibitory effect of Gly an MPT was equivalent prevonts it.Roooygenation mapidy comocts introcalulor acidoss,thus to that of low pH (6.4)and was ohserved at high alowing MPI,.hi女Bfa航ate时ty:pletion of ntracellular Ghy (prchably theough volume negulatary dlecreatel.Treamment with Gly at concentrations (3 ms).Intracellular Gly concentration, the time of renrygenarion innihts MPT doseite comectice at acidesi, measured by NMR spectrascopy,was reduced in HL.I cells and prevents cell death. exposed to SI-remygenation,as compared to normosic O The Physiologkal Soclety 2004
880 M. Ruiz-Meana and others J Physiol 558.3 Experimental evidence of MPT during reperfusion has been provided by fluorescence microscopy of cardiomyocytes re-energized after SI showing CsA-sensitive mitochondrial depolarization (Duchen et al. 1993) and calcein loading (Petronilli et al. 1999). In reperfused myocardium, MPT has been demonstrated by the loss of mitochondrial NAD+ (Di Lisa et al. 2001) and mitochondrial entrapment of [3H]deoxyglucose 6-phosphate (Javadov et al. 2000), with a time course that reflects that of pH normalization. In the present study, acidic reoxygenation markedly attenuated cell death in cultured HL-1 myocytes and in rat hearts. The protective effect of low pH was independent of the inhibitory effect of acidosis on contractility (since HL-1 cells have only a rudimentary contractile machinery and do not hypercontract upon reoxygenation), was not associated with attenuated cytosolic Na+ overload, and was not modified by elimination of extra or intracellular Ca2+. The protective effect of acidic pH cannot be thus explained by attenuation of Ca2+ overload or hypercontracture, but it rather appears to be related to its inhibitory effect on MPT, as observed in isolated mitochondria. Figure 8. Proposed role of Gly during reperfusion injury Ischaemia and ischaemia-related conditions are associated with cytosolic derangements that favour MPT, but also with acidosis that prevents it. Reoxygenation rapidly corrects intracellular acidosis, thus allowing MPT, which is facilitated by depletion of intracellular Gly (probably through volume regulatory decrease). Treatment with Gly at the time of reoxygenation inhibits MPT despite correction of acidosis, and prevents cell death. Gly prevents reoxygenation injury by interfering with MPT The direct protective effect of Gly against necrotic and apoptotic cell death induced by different insults, including ischaemia–reperfusion, and a variety of other pathological conditions – i.e. endotoxaemic shock, alcoholic hepatitis, hepatic fibrosis, arthritis, tumour and drug toxicity – has been characterized in detail (Zhong et al. 1996; Nishimura et al. 1998; Nishimura & Lemasters, 2001; Dong et al. 2001; Zhang et al. 2003). Gly delays sarcolemmal rupture induced by hypoxia, which was initially explained by attenuated Na+ accumulation obscurely related to stimulation of the Gly-gated Cl− channels, originally described in the postsynaptic membranes of the spinal cord (Carini et al. 2000). Other authors suggested that the protective effect of Gly was independent of receptor stimulation but rather related to the closure of non-specific leaks in plasma membranes induced by hypoxia (Dong et al. 1998; Frank et al. 2000; Nishimura & Lemasters, 2001). Confocal microscopy analysis of membrane permeabilization tofluorescent molecules of different sizes demonstrated that Gly delays membrane permeabilization during hypoxia, which could reflect the opening of large anionic channels (‘death pores’) eventually leading to massive cell swelling and sarcolemmal rupture (Nishimura & Lemasters, 2001). Previous studies were coincident in interpreting that Gly prevented plasma membrane failure, the final event in the pathway to necrotic cell death (Frank et al. 2000). In the present study Gly completely prevented cell death associated with normalization of pH during reoxygenation in cardiomyocyte cultures, and mimicked the protective effect of acidic reoxygenation on LDH release in isolated rat hearts. Since acute cell death observed during reoxygenation in our model was ascribable to MPT, we hypothesized that Gly could act at the mitochondrial level, interfering with MPT. The experiments in isolated mitochondria demonstrated that Gly indeed prevents CsA-sensitive mitochondrial swelling and calcein release induced by re-energization after a transient SI or by exposure to high Ca2+. The protective effect of Gly is not related to its utilization as a respiratory substrate or as a source of NAD(P)H, since it was not mimicked by other substrates (succinate or pyruvate) and was present in fully energized mitochondria submitted to Ca2+ overload. The inhibitory effect of Gly on MPT was equivalent to that of low pH (6.4) and was observed at high concentrations (3 mm). Intracellular Gly concentration, measured by NMR spectroscopy, was reduced in HL-1 cells exposed to SI–reoxygenation, as compared to normoxic C The Physiological Society 2004
1h5限3 Glcne prevents mitochondrial permeabrlity transition 31 cells.Exposure to 3mst Gly during reoygenation raised De Giorgi F,Lartigue L Bauer MK,Schubert A.Grimm S. intracellular Gly concentration up to the range prowven to Hanson GT,Remington ST,Youle RI Ichas F(2002)The inhibit MPT.Measured intracellular content corresponds permeability transition pore signals apoptcsis by directing to an estimated Gly concentration of approximately 7 mM. Bax translocation and multimerization.FASEB 16, compared to approximately I m in normoxic cells and 607-609. 0.6 mM in cells reoxygenated without Gly. Di Lisa F.Menbo R.Canton M.Barile M&Bernardi P(2001). Opening of the mitochondria permeability transitio pore causes depktion of mitochondrial and cytosolc NAD+and Hypothetical role of Gly depletion in reoxygenation isa causativeevent in the death of myocytes in postischemic reperfusion of the heart.Hia!Cem 276. The mechanism of Gly depletion in cardiomyocytes 2571-2575 submitted to Sl-rexygenation was not imvestigated in the Dong 7 Patel Y.Saiknmar P.Weinberg IM Venkatachalam present study.However,aminoacidandCl releaseplayan MA (1998).Development of porous defects in plasma important role in regulating volume decrease in swollen membranes of adenosine triphosphate-depleted cells (Rasmusson etal.1993;Song et al 1998).It should Madin-Darby canine kidney cells and its inhibition by be thus expected that the rapid reduction in cell volume lycine.Lahuratory Inwe 78,657-668. occurring during reoxygenation and pormalization of Dong:Z.Venkatachalam MA,Weinherg:IM.Saikumar P Patel intracellular pH results in Gly efflux.Although the Y(2001)Prolection uf ATP-depkted cells by impermcant contrihution of Gly efflus to volume decrease has not strychnine derivatives:implcations for ghtinc been established,its abolition by exposure to increased cytoprotection.Am Prhoi 158.1021-1028. Duchen MR (19991.Contrihutions of mirochondria lo animal Gly concent山ation in t山e extracellular medium should phyokgy:from homeastalic sensur to calcium sling reduce it and hamper cell volume correction.In this study. d cell death.JPi51,【-l7. addition of Gly to the reoxygenation buffer attenuated Duchen MR,McGuinness O,Brown LA Crumpton M volume decrease,and this effect was closely correlated (1993).On the involvement of a cydosporin A sensitive with the magnitude of the protection afforded against cell mitochondrial pore in myocardia reperfusion injury. death. Cardiovarc Res 27.1790-1794. A hypothesis that integrates the results obtained Frank A.Kauen U de (iroot H(2000).Protection by ghcine in the present study is outlined in Fig.8.Rapid pH against hypoxic injury of rat hepatocytes:inh bition of ion normalization during reoxygenation favours MPT and fluxes through nonspecific aks fepatal 32.58-66 oell death.Intracellular Gly acts as an inhibitor of MPT. Garlid KD,Paucek P,Yarov Yarovoy V.Sun X Schindler PA However,volume regulatory decrease occurring upon (1996).The mitochoodrial KAIP channel as a receptor for potassium channel openers.Ban Cheme 271,8796-8799. normalization of extracellular pll imvolves cellular Gly Halextrap AP (1991)Calcinm-dependent opening of a loss,which makes the cell more vulnerable to MPT,an non-pccific pone in The mitochundrial inner membrane is effect that can be reversed by increasing extracellular Gly inhbited at pH valucs bdlow 7.Implicalions for the concentration. protective effect of low pH against chemical and hypce The contribution of GGly to cardiamyocyte survival after d山m实khwJ27R,715-719. an ischaemic insult represents a promising therapeutic Hausenloy DI.Duchen MI Yellon DM(2003).Imhibiting approach to preventing cell death in reperfused myo- milochondrial permeability transition pore opening at cardium.Studies in volunteers have shown that plasmatic reperfusion prolexts against ischacmia-reprfusion injury Gly concentrations higher than those found to be Cardigatsc Res 60,617-625. protective in this study can be rapidly achieved by intra. Holmuhamedov EL,fovanovic S,Dzeja PP.Jowinovic A venous infusion,and are well tolerated [Nilsson etal Terzic A(1998).Mitochoodrial ATP sensitive K channels 19961. modulale cardiac mitochondrial function.Am /Physi 275, H1567-H1576. References Javadov SA.Clarke S,Das M.Griffiths EJ.Lim KH Halestrap AP (2003).Ischaemic preconditioning inhibits opening of Carini R.De Cesris MG,Splendlore R,Bagnali M.Bdlomo G mitochondrial permeability transition pores in the &Albano E(2000以Alerations of Na*hocnoodasi本in reperfused rat heart.Phynoi 549.513-524. hepatocyte renxygrmurion injury.c Bophys Aca 1500. lavadov SA.1.im KH,Kerr PM.Suleiman M5,Angrlini GD) 297-305 Halerap AP(2000)Protection of hearts from reperfusion Crumpton M(19991.The mitochondrial permeabiliy injury by propool is associated with inhibition of the transition porc and its role in cell death.Biochem 341, mitochondrial permeability transition.Cindionise Kes 45. 233-249. 36-369. The Physiological Socicty 2004
J Physiol 558.3 Glycine prevents mitochondrial permeability transition 881 cells. Exposure to 3 mm Gly during reoxygenation raised intracellular Gly concentration up to the range proven to inhibit MPT. Measured intracellular content corresponds to an estimated Gly concentration of approximately 7 mm, compared to approximately 1 mm in normoxic cells and 0.6 mm in cells reoxygenated without Gly. Hypothetical role of Gly depletion in reoxygenation The mechanism of Gly depletion in cardiomyocytes submitted to SI–reoxygenation was not investigated in the present study. However, amino acid and Cl− release play an important role in regulating volume decrease in swollen cells (Rasmusson et al. 1993; Song et al. 1998). It should be thus expected that the rapid reduction in cell volume occurring during reoxygenation and normalization of intracellular pH results in Gly efflux. Although the contribution of Gly efflux to volume decrease has not been established, its abolition by exposure to increased Gly concentration in the extracellular medium should reduce it and hamper cell volume correction. In this study, addition of Gly to the reoxygenation buffer attenuated volume decrease, and this effect was closely correlated with the magnitude of the protection afforded against cell death. A hypothesis that integrates the results obtained in the present study is outlined in Fig. 8. Rapid pH normalization during reoxygenation favours MPT and cell death. Intracellular Gly acts as an inhibitor of MPT. However, volume regulatory decrease occurring upon normalization of extracellular pH involves cellular Gly loss, which makes the cell more vulnerable to MPT, an effect that can be reversed by increasing extracellular Gly concentration. The contribution of Gly to cardiomyocyte survival after an ischaemic insult represents a promising therapeutic approach to preventing cell death in reperfused myocardium. Studies in volunteers have shown that plasmatic Gly concentrations higher than those found to be protective in this study can be rapidly achieved by intravenous infusion, and are well tolerated (Nilsson et al. 1996). References Carini R, De Cesaris MG, Splendore R, Bagnati M, Bellomo G & Albano E (2000). Alterations of Na+ homeostasis in hepatocyte reoxygenation injury. Biochim Biophys Acta 1500, 297–305. Crompton M (1999). The mitochondrial permeability transition pore and its role in cell death. Biochem J 341, 233–249. De Giorgi F, Lartigue L, Bauer MK, Schubert A, Grimm S, Hanson GT, Remington SJ, Youle RJ & Ichas F (2002). The permeability transition pore signals apoptosis by directing Bax translocation and multimerization. FASEB J 16, 607–609. Di Lisa F, Menabo R, Canton M, Barile M & Bernardi P (2001). 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. J Biol Chem 276, 2571–2575. Dong Z, Patel Y, Saikumar P, Weinberg JM & Venkatachalam MA (1998). Development of porous defects in plasma membranes of adenosine triphosphate-depleted Madin-Darby canine kidney cells and its inhibition by glycine. Laboratory Invest 78, 657–668. Dong Z, Venkatachalam MA, Weinberg JM, Saikumar P & Patel Y (2001). Protection of ATP-depleted cells by impermeant strychnine derivatives: implications for glycine cytoprotection. Am J Pathol 158, 1021–1028. Duchen MR (1999). Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death. J Physiol 516, 1–17. Duchen MR, McGuinness O, Brown LA & Crompton M (1993). On the involvement of a cyclosporin A sensitive mitochondrial pore in myocardial reperfusion injury. Cardiovasc Res 27, 1790–1794. Frank A, Rauen U & de Groot H (2000). Protection by glycine against hypoxic injury of rat hepatocytes: inhibition of ion fluxes through nonspecific leaks. J Hepatol 32, 58–66. Garlid KD, Paucek P, Yarov-Yarovoy V, Sun X & Schindler PA (1996). The mitochondrial KATP channel as a receptor for potassium channel openers. J Biol Chem 271, 8796–8799. Halestrap AP (1991). Calcium-dependent opening of a non-specific pore in the mitochondrial inner membrane is inhibited at pH values below 7. Implications for the protective effect of low pH against chemical and hypoxic cell damage. Biochem J 278, 715–719. Hausenloy DJ, Duchen MR & Yellon DM (2003). Inhibiting mitochondrial permeability transition pore opening at reperfusion protects against ischaemia-reperfusion injury. Cardiovasc Res 60, 617–625. Holmuhamedov EL, Jovanovic S, Dzeja PP, Jovanovic A & Terzic A (1998). Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondrial function. Am J Physiol 275, H1567–H1576. Javadov SA, Clarke S, Das M, Griffiths EJ, Lim KH & Halestrap AP (2003). Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart. J Physiol 549, 513–524. Javadov SA, Lim KH, Kerr PM, Suleiman MS, Angelini GD & Halestrap AP (2000). Protection of hearts from reperfusion injury by propofol is associated with inhibition of the mitochondrial permeability transition. Cardiovasc Res 45, 360–369. C The Physiological Society 2004
882 M.Ruiz-Mpana and others h,a'551 Kim [5.He T.Lemaslerx (20031.Milo humdrial Roelrigsne-5inowas A.Gorcia-Doratk D,Paililla F,Tmsurte T. permeabaity transitioncommon pathway to necrosis and Barrabes IA,Ruiz-Meana M,Agullo L Soler-Soler J (2003). apoptosis.Biochem Bioplys Res Commun 304.4670 Pre-treatment with the Na/H exchange inhibitor Lemasierx ]I.Nieminen Al.Qian T,Trust IC,Flmore SP. cariperide delys cell-lo-cell elctrical unoupling,during Nishimura Y.Cn:RA,Cscin WF,Rradham CA,Rrenm mysarclial ischemia.Cardlintaoe Rer 58,100-117 DA Herman B(1998).The mcochondridl pernseability Ruiz-Meana M.Giarcia-Dorado D,Pina P.leserte J.Agullo L trarsition in cell death a oommon mechanism in necrosis. Soler-Soler I (2003).Cariporide preserves mitochondrial apuptosis and autophagy.Binkioe Biopins Actu 1366. prokn gradient and delys ATP depletion in cardiamyueytes 177-16 during iscbemic conditons.Am J Flysof Honrt Carc Fhrsiol Nisson A.Randmaa 1 Hahn RG [1996).Hatmodynamic 25.H999-H1036、 effects of irrigating uid studied by Dopples Schertamer P.Bradroed BU.Rose ML Bunxendahl H,Raleish uhrasonography in volunteers.Urof 77,341-546. IA.Lemasters I Thurman RG (1999).Intravenous glycine Nishirmara Y Lemasters J (2001).Glcine blodks opening of improves sarvival in rat liver transplantaton Am J Flrsii a death channel in cultured hepatic sinusoidal endothelial 76G924-G92 cells during chemical hypeoia.Cell Deah Dr8. Sang D,O'Repan MH Philli TW(1998).Amino acid relee 850&5& during volume regalation by cardiac cells:cellular Nichirmra Y.Romer 1H Lemasen (19981,Milhundrial mochaanisms.Fur J Plarmad 341,223-280, dysfunction and cytoskeltal disruption during chemical Zamomi N Kroemer G (2001).The mitochondrio hypoxa to cultured rat hepati:simsidl endothelial cells apoptosis:how Pandora's box opens.Nir Rev Mol Cell mial 2. the pH ruradox aml cyluprerdion by gluoxr.adotir nH, 62-71. and phycine.Hejvaigy 27.1039-1049. Zhang K,Wcinbeng:TM,Venkatachalam MA Dung Z (2003). Petroeilli V.Miotto G Canton M,Brini M.Colonna R, Glycine protection of PC-12 cells against injury by Bernardi P Di Lisa F(1999).Transient and locg-lsting ATP-depletion.Neurochewe Res 26,893-901. openings nfthe milochondlril permexhilily I ransilion porr Zhang,7,Jine 5 Thurman RG(19n)Gh:ine minimixx can be moanored directly鱼mtact cells by changes鱼 reperfusion injury in a low-Dlow,reflone liver perfusion mitocboodrial calcein fluoresoence.Bioplrys 76,725-734 mode in the rat.Am Plysiw 270,G332-G538. Prtnnilli V.Penx D.Senrrano L,Rernardi P Di Lia F (2001).The matochondrial permeability trarsition,release o cyochrome c and cell death.Correlation with the duration Ackmowledgements of pore openings in situ./Biai Chem76 120-12034. This wurk ws partially supported by grants from the Spunish Rsmuscon RI.Duvis DG Lidhermon M(1993).Aminn arid Ministry ofScience Technology.CICYT-SAF2002-00759,and loss during volume regalatory decrease in cultured chack Funde ck Tnwestigaciin Somilari 01/3135.A.R.-5 has a grant heart oells Am JPlysiol 264.C136-C145. from the Miniszerio de Sanidad y Consumo (99/3142). 自Them,cg*国a年0mM
882 M. Ruiz-Meana and others J Physiol 558.3 Kim JS, He L & Lemasters JJ (2003). Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun 304, 463–470. Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, Crowe RA, Cascio WE, Bradham CA, Brenner DA & Herman B (1998). The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1366, 177–196. Nilsson A, Randmaa I & Hahn RG (1996). Haemodynamic effects of irrigating fluids studied by Doppler ultrasonography in volunteers. Br J Urol 77, 541–546. Nishimura Y & Lemasters JJ (2001). Glycine blocks opening of a death channel in cultured hepatic sinusoidal endothelial cells during chemical hypoxia. Cell Death Differ 8, 850–858. Nishimura Y, Romer LH & Lemasters JJ (1998). Mitochondrial dysfunction and cytoskeletal disruption during chemical hypoxia to cultured rat hepatic sinusoidal endothelial cells: the pH paradox and cytoprotection by glucose, acidotic pH, and glycine. Hepatology 27, 1039–1049. Petronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P & Di Lisa F (1999). Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 76, 725–734. Petronilli V, Penzo D, Scorrano L, Bernardi P & Di Lisa F (2001). The mitochondrial permeability transition, release of cytochrome c and cell death. Correlation with the duration of pore openings in situ. J Biol Chem 276, 12030–12034. Rasmusson RL, Davis DG & Lieberman M (1993). Amino acid loss during volume regulatory decrease in cultured chick heart cells. Am J Physiol 264, C136–C145. Rodriguez-Sinovas A, Garcia-Dorado D, Padilla F, Inserte J, Barrabes JA, Ruiz-Meana M, Agullo L & Soler-Soler J (2003). Pre-treatment with the Na+/H+ exchange inhibitor cariporide delays cell-to-cell electrical uncoupling during myocardial ischemia. Cardiovasc Res 58, 109–117. Ruiz-Meana M, Garcia-Dorado D, Pina P, Inserte J, Agullo L & Soler-Soler J (2003). Cariporide preserves mitochondrial proton gradient and delays ATP depletion in cardiomyocytes during ischemic conditions. Am J Physiol Heart Circ Physiol 285, H999–H1006. Schemmer P, Bradford BU, Rose ML, Bunzendahl H, Raleigh JA, Lemasters JJ & Thurman RG (1999). Intravenous glycine improves survival in rat liver transplantation. Am J Physiol 276, G924–G932. Song D, O’Regan MH & Phillis JW (1998). Amino acid release during volume regulation by cardiac cells: cellular mechanisms. Eur J Pharmacol 341, 273–280. Zamzami N & Kroemer G (2001). The mitochondrion in apoptosis: how Pandora’s box opens. Nat Rev Mol Cell Biol 2, 67–71. Zhang K, Weinberg JM, Venkatachalam MA & Dong Z (2003). Glycine protection of PC-12 cells against injury by ATP-depletion. Neurochem Res 28, 893–901. Zhong Z, Jones S & Thurman RG (1996). Glycine minimizes reperfusion injury in a low-flow, reflow liver perfusion model in the rat. Am J Physiol 270, G332–G338. Acknowledgements This work was partially supported by grants from the Spanish Ministry of Science & Technology, CICYT-SAF/2002-00759, and Fondo de Investigacion Sanitaria 01/3135. A.R.-S. has a grant ´ from the Ministerio de Sanidad y Consumo (99/3142). C The Physiological Society 2004