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Am J Physiol Heart Circ Physiol 291: H714-H723, 2006. First published March 31, 2006; doi:10.1152/ajpheart.00823.2005
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Poly(ADP-ribose) polymerase-1 hyperactivation and impairment of mitochondrial respiratory chain complex I function in reperfused mouse hearts

Hui-Zhong Zhou,1,6 Raymond A. Swanson,2,7 Ursula Simonis,3 Xiaokui Ma,6 Gary Cecchini,4,8 and Mary O. Gray1,5,6

1Department of Medicine and 5Lung Biology Center, 2Department of Neurology, and 4Department of Biochemistry and Biophysics, University of California, San Francisco, California; 6Medical Service and Cardiology Section, 7Neurology Service, and 8Molecular Biology Division, Veterans Affairs Medical Center, San Francisco, California; and 3Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California

Submitted 3 August 2005 ; accepted in final form 4 March 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Poly(ADP-ribose) polymerase-1 (PARP-1), the most abundant member of the PARP family, is a nuclear enzyme that catalyzes ADP-ribose transfer from NAD+ to specific acceptor proteins in response to DNA damage. Excessive PARP-1 activation is an important cause of infarction and contractile dysfunction in heart tissue during interruptions of blood flow. The mechanisms by which PARP-1 inhibition and disruption dramatically improve metabolic recovery and reduce oxidative stress during cardiac reperfusion have not been fully explored. We developed a mouse heart experimental protocol to test the hypothesis that mitochondrial respiratory complex I is a downstream mediator of beneficial effects of PARP-1 inhibition or disruption. Pharmacological inhibition of PARP-1 activity produced no deterioration of hemodynamic function in C57BL/6 mouse hearts. Hearts from PARP-1 knockout mice also exhibited normal baseline contractility. Prolonged ischemia-reperfusion produced a selective defect in complex I function distal to the NADH dehydrogenase component. PARP-1 inhibition and PARP-1 gene disruption conferred equivalent protection against mitochondrial complex I injury and were strongly associated with improvement in myocardial energetics, contractility, and tissue viability. Interestingly, ischemic preconditioning abolished cardioprotection stimulated by PARP-1 gene disruption. Treatment with the antioxidant N-(2-mercaptopropionyl)-glycine or xanthine oxidase inhibitor allopurinol restored the function of preconditioned PARP-1 knockout hearts. This investigation establishes a strong association between PARP-1 hyperactivity and mitochondrial complex I dysfunction in cardiac myocytes. Our findings advance understanding of metabolic regulation in myocardium and identify potential therapeutic targets for prevention and treatment of ischemic heart disease.

oxidative stress; energetics; metabolism; glycolysis

CORONARY ARTERY DISEASE is an important cause of mortality throughout the world. Although many pathophysiological processes are triggered when myocardial blood flow is interrupted and restored, excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1), the most abundant homologue of the PARP family, may be one fundamental component of reperfusion injury (23). Zingarelli et al. (34) established that coronary artery occlusion in rats caused robust myocardial poly(ADP-ribosyl)ation, creatine kinase (CK) release, ATP depletion, and neutrophil infiltration. Conversely, pharmacological inhibition of PARP-1 activity reduced infarction size, normalized cardiac metabolism, and attenuated systemic inflammatory responses (34). In a separate study (37), those investigators determined that PARP-1 disruption reduced tissue necrosis, neutrophil infiltration, and peroxynitrate generation induced by ischemia-reperfusion. Mechanisms for the anti-inflammatory effects of PARP-1 disruption were suggested by an absence of stress-induced P-selectin and ICAM-1 upregulation in PARP-1 knockout (KO) vascular endothelial cells.

Thiemermann et al. (25) showed that pharmacological inhibition of PARP-1 activity during ex vivo ischemia-reperfusion of rabbit hearts reduced infarction and improved cardiac contractile recovery, findings that supported cardioprotective effects independent of systemic inflammatory responses. Pieper et al. (18) demonstrated an equivalent resistance to injury in hearts from mice lacking PARP-1. Those investigators also observed that PARP-1 gene disruption reduced tissue NAD+ and ATP depletion during ex vivo ischemia-reperfusion (18). Grupp et al. (6) utilized an isolated working mouse heart model to examine whether PARP-1 disruption affected functional alterations associated with hypoxia-reoxygenation injury. Their study revealed modest increases in baseline systolic intraventricular pressure and decreases in half-time of relaxation for PARP-1 KO hearts compared with wild-type hearts. After 30 min of hypoxia and 30 min of reoxygenation, PARP-1 KO hearts exhibited no alteration in the maximal rate of pressure development, time to peak pressure, or half-time of relaxation and modest reduction in the maximal rate of relaxation compared with baseline. In contrast, wild-type hearts exhibited alterations in all hemodynamic parameters under the same conditions (6). Those investigators postulated that PARP-1 disruption improved contractile function during reoxygenation by accelerating recovery of cellular NAD+ and ATP and by protecting against free radical and oxidant-induced damage.

PARP-1 is primarily a nuclear enzyme that catalyzes transfer of ADP-ribose from NAD+ to specific acceptor proteins in response to DNA strand breaks, triggering the base excision repair pathway (20). However, excessive PARP-1 activation can impair the function of other cellular organelles, including mitochondria and accelerate production of reactive oxygen species (ROS). Virág et al. (28) demonstrated that inhibition or disruption of PARP-1 preserved mitochondrial transmembrane potential in mouse thymocytes exposed to peroxynitrite and blocked secondary ROS generation. Zingarelli et al. (34) showed that PARP-1 inhibition preserved mitochondrial respiration in heart myoblasts exposed to peroxynitrite. Szabados et al. (22) found that PARP-1 inhibition during reperfusion of rat hearts reduced ROS formation, lipid peroxidation, and DNA breaks. Thus PARP-1 inhibition or disruption may contribute to cardioprotection by preserving oxidative phosphorylation and by preventing secondary release of toxic biological radicals.

We propose that NADH-ubiquinone oxidoreductase (complex I) is one important mediator of acute cardioprotection caused by PARP-1 inhibition or disruption. Several lines of evidence support essential roles for mitochondrial complex I in cardiac energetics. First, oxidation of the terminal-reduced component of complex I by endogenous ubiquinone is a rate-determining step for electron transfer from mitochondrial NADH to O2 under physiological conditions. Second, complex I is a respiratory chain site relatively vulnerable to reperfusion injury, further limiting myocardial oxidative metabolism during stress conditions (9, 17). Third, complex I is one major source of ROS generation in mitochondrial matrix (8). Finally, pharmacological agents that can modulate electron flux through complex I are known to regulate the mitochondrial permeability transition, a terminal signaling event that commits myocytes to necrotic cell death (1, 2, 4, 30).

As part of our continuing interest in cardioprotective signaling, Gray et al. (5) recently published results establishing that the beneficial effects of ischemic preconditioning (IPC) on contractility and tissue viability after reperfusion require activation of protein kinase-C{epsilon}. In the present study, we used the same mouse heart experimental protocol to establish an association between nuclear PARP-1 hyperactivation and mitochondrial complex I dysfunction and its relevance to reperfusion injury. This investigation addressed the following key questions: 1) Does inhibition of PARP-1 activity during myocardial reperfusion preserve complex I function? 2) Does PARP-1 gene disruption preserve complex I function during reperfusion? 3) Is the preservation of complex I function associated with improvements in metabolism, hemodynamic recovery, and tissue integrity? 4) Can the protective effects of IPC be induced in hearts lacking PARP-1?


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. C57BL/6 mice were purchased from Charles River Laboratories (Hollister, CA). Mice lacking PARP-1 (29) and wild-type controls from the same genetic background were a gift from Dr. Basilia Zingarelli at the Cincinnati Children’s Hospital Medical Center. Animals were fed rodent chow and water ad libitum. The study was approved by the Institutional Animal Care and Use Committee of the San Francisco Veterans Affairs Medical Center. Protocols conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Mouse heart model. Hearts from male mice were cannulated via the aorta and perfused using a Krebs-Henseleit buffer containing (in mmol/l) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 24 NaHCO3, 11 D-glucose, and 0.5 sodium EDTA as described previously (33). Isolated hearts were perfused at constant pressure of 70 mmHg and paced at 6 Hz. Left ventricular (LV) developed pressures [LVDP = LV systolic pressure – LV end-diastolic pressure (LVEDP)] were measured by using a micromanometer catheter (Millar Instruments, Houston, TX) passed into a balloon within the LV cavity (33). Pressures were monitored continuously throughout each protocol. Coronary flow was measured by collecting effluent from the right ventricular outflow tract.

Experimental protocols. C57BL/6 mouse hearts were stabilized by normoxic perfusion for 20 min and then subjected to 25 min of global ischemia followed by 30 min of reperfusion. Hearts were treated with vehicle or the PARP inhibitor 3-aminobenzamide (3-AB) at a concentration of 100 µmol/l during the first 20 min of reperfusion. Normoxic controls underwent 75 min perfusion with oxygenated buffer and were not subjected to ischemia-reperfusion. All hearts intended for immunofluorescence studies were quickly removed from the apparatus, rinsed in cold phosphate-buffered saline, cut into two pieces, immediately incubated in 4% paraformaldehyde solution for overnight fixation (4°C), and embedded in paraffin. Tissue sections (5 µm) were deparaffinized in xylene and rehydrated with serial concentrations (100%, 95%, and 70%) of ethanol. Sections were permeabilized with 0.1% Tween 20 and stained with primary antibodies against PAR (BD Biosciences, San Jose, CA) or PARP-1 (Trevigen, Gaithersberg, MD), followed by secondary antibodies conjugated with Alexa Fluor 488. Images were acquired with Leica TCS confocal laser scanning microscope systems (Leica Lasertechik, Heidelberg, Germany).

Wild-type (WT) and PARP-1 KO hearts were randomly assigned to one of four experimental groups: 1) prolonged ischemia-reperfusion consisting of 25 min ischemia and 30 min reperfusion; 2) IPC with 2 min ischemia and 5 min reperfusion before prolonged ischemia-reperfusion; 3) IPC before prolonged ischemia and infusion of the free radical scavenger N-(2-mercaptopropionyl)-glycine (MPG) at 800 µmol/l during the first 20 min of reperfusion; and 4) IPC before prolonged ischemia-reperfusion and allopurinol infusion at 100 µmol/l throughout the experimental protocol to inhibit xanthine oxidase activity.

Measurement of cardiac ADP-ribosylation. C57BL/6 mouse hearts were subjected to 75 min normoxic perfusion or to 20 min normoxic perfusion, 25 min ischemia, and 30 min reperfusion. Hearts were randomly assigned to receive vehicle or 100 µmol/l 3-AB during the first 20 min of reperfusion. At the end of the experimental protocol, hearts were removed from the perfusion apparatus, washed with cold phosphate-buffered saline, and immediately frozen in liquid nitrogen before storage at –80°C. Ventricular lysates were subjected to SDS-PAGE and transferred to nitrocellulose membrane as previously described (33). The poly(ADP-ribosyl)ated proteins were detected after incubation with anti-PAR primary antibody (Trevigen) and appropriate chemiluminescence reagents (Amersham, Piscataway, NJ). Cardiac PAR immunoreactivity was quantitated using National Institutes of Health Image densitometry software (33).

Measurement of mitochondrial respiratory chain complex I function. After normoxic perfusion or ischemia-reperfusion, individual mouse hearts were employed as a source of submitochondrial particles (SMPs) as previously described (14). Standard reaction buffer containing 250 mmol/l sucrose, 50 mmol/l Tris·HCl (pH 7.8), 0.2 mmol/l potassium EDTA, 0.5 mg/ml bovine serum albumin, and 100 µmol/l NADH was maintained at a constant temperature of 25°C. NADH oxidase activity was assayed as a decrease in absorbance at 340 nm after addition of SMPs (10–25 µg) to the spectrophotometer cuvette (Uvikon 810, Kontron Instruments, San Diego, CA). This enzymatic reaction was halted by addition of 1 mmol/l KCN. NADH-Q1 reductase activity was assayed as a decrease in absorbance at 340 nm following addition of 20 µmol/l ubiquinone-1 to the same cuvette. This enzymatic reaction was halted by addition of 2 µmol/l rotenone. Under the experimental conditions described, >95% of the NADH-Q1 reductase activity was rotenone sensitive. Finally, NADH-ferricyanide reductase activity was assayed as a decrease in absorbance at 420 nm after addition of 1 mmol/l K3Fe(CN)6 to the same cuvette. Mitochondrial complex I activities were expressed as micromoles of NADH oxidized per minute per milligram of SMP protein.

Measurement of myocardial metabolites. After reperfusion, individual mouse hearts were extracted with 10% perchloric acid and neutralized with KOH. Myocardial NAD+ content was measured by using the alcohol dehydrogenase reaction (32). ATP, total adenine nucleotides, and lactate concentrations in heart extracts reconstituted with deuterium oxide were measured using 1H NMR spectroscopy. All spectra were acquired on an Avance DRX 300 MHz spectrometer (Bruker Instruments, Billerica, MA) operating at 299.1912 MHz proton frequency. The FIDs were obtained at ambient temperature using a 90° pulse, 10-s relaxation delay, spectral width of 6,032 Hz, 32 K data points, and 250–300 acquisitions. The data were apodized using a line-broadening factor of either 0.1 or 0.2 Hz before Fourier transformation and were then phase corrected to yield one-dimensional spectra. Spectra were referenced to 3-trimethylsilylproprionate resonating at 0 ppm as an internal standard. Relative peak areas were calculated by using peak integration. Baseline correction (Lorentzian and Gaussian) was applied to peaks of interest.

Measurement of cardiac tissue lipid peroxidation products. Lipid peroxidation in individual hearts was estimated by the appearance of thiobarbituric acid reactive substances, according to Ohkawa et al. (15). Myocardial malondialdehyde (MDA) concentrations were measured at an absorbance of 532 nm and expressed as nanomoles of MDA per gram of wet heart weight.

Measurement of myocardial infarction and CK release. At the completion of each experimental protocol, hearts were perfused with 1% triphenyltetrazolium chloride, fixed with 10% neutral buffered formalin, and then sectioned (5). Planimetry of viable (stained) and necrotic (unstained) tissue was performed using National Institutes of Health Image software. Infarction size was corrected for the weight of each section and expressed as a percentage of LV mass. CK activity in coronary effluent collected during reperfusion was measured using a commercial kit (Sigma, St. Louis, MO), corrected for flow rate and wet heart weight as described previously (33).

Statistical analysis. Results are reported as means ± SE. Comparisons between groups were made by one-way ANOVA or repeated-measures ANOVA. Differences were confirmed using a Bonferroni post hoc test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PARP-1 inhibition during cardiac reperfusion preserves mitochondrial complex I function. We subjected hearts of C57BL/6 mice to prolonged ischemia-reperfusion to study targeting and and timing of PARP-1 activation during stress. As indicated in Fig. 1A, immunofluorescence staining demonstrated PARP-1 protein localization to the nuclei of cardiac myocytes. Ischemia-reperfusion stimulated robust PAR formation in myocyte nuclei but not in mitochondria. Parallel immunofluorescence staining of isolated cardiac myocytes (5) with the same primary antibodies demonstrated PARP-1 and PAR localization to nuclear compartments in unstimulated and H2O2-treated cells (data not shown). As indicated in Fig. 1B, Western analysis of heart lysates showed trace poly(ADP-ribosyl)ation after normoxic perfusion alone. Ischemia-reperfusion caused a 10-fold increase in poly(ADP-ribosyl)ated heart proteins compared with controls. 3-AB reduced poly(ADP-ribosyl)ation when administered solely at the onset of reperfusion.


Figure 1
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  		Fig. 1. Poly(ADP-ribose)polymerase-1 (PARP-1) protein and activity localize to the nuclei of adult mouse cardiac myocytes. A: confocal immunofluorescence micrographs showing nuclear localization of poly(ADP-ribose) in C57BL/6 mouse hearts. Ex vivo hearts were subjected to either 75 min of normoxic perfusion (Normoxia) or 20 min normoxic perfusion (equilibration), 25 min ischemia, 30 min reperfusion (Reperfusion). Cardiac tissue sections stained with poly(ADP-ribose) primary antibodies (PAR) demonstrated a marked increase in nuclear PAR formation following ischemia-reperfusion (4 panels on left). No changes in the intensity of PARP-1 staining were observed after prolonged ischemia-reperfusion (4 panels on right). Merged fluorescence and differential interference contrast (DIC) images of the same microscopic fields (bottom row) indicate nuclear localization of both PARP-1 protein and activity in myocytes. B: Western blot analysis indicating that ischemia-reperfusion causes a 10-fold increase in myocardial poly(ADP-ribosyl)ation compared with normoxic hearts. Tissue lysates were prepared from C57BL/6 mouse hearts subjected to 75 min normoxic perfusion (NX) or 20 min normoxic perfusion, 25 min ischemia, and 30 min reperfusion. Hearts were randomly assigned to receive vehicle (CON) or 100 µmol/l 3-aminobenzamide (3-AB) during the first 20 min of reperfusion. Poly(ADP-ribose) signal between 75 and 250 kDa was quantitated by densitometry. Treatment with 3-AB blocked reperfusion-induced cardiac poly(ADP-ribosyl)ation. Each lane represents tissue lysate prepared from an individual mouse heart. n = 3 hearts per group. *P < 0.05 vs. CON.

 
We next measured NADH oxidase, NADH-Q1 reductase, and NADH-ferricyanide reductase activities in SMPs to test the hypothesis that complex I function contributes to cardioprotection caused by PARP-1 inhibition and disruption. As indicated in Fig. 2, electron transfer was rapid in SMPs prepared from C57BL/6 hearts perfused under normoxic conditions (trace 1 in each panel), at rates equivalent to those in bovine SMPs (27). Ischemia-reperfusion (trace 2 in each panel) reduced NADH-oxidase and NADH-Q1 reductase activities by half but did not alter NADH-ferricyanide reductase activity. PARP-1 inhibition by 3-AB administration during reperfusion (trace 3 in each panel) preserved NADH oxidase and NADH-Q1 reductase activities at normoxic levels. Hearts from mice lacking PARP-1 (29) were used to investigate the relative importance of PARP-1 activation for ischemia-reperfusion injury compared with other PARP homologues. PARP-1 KO hearts exhibited normal weight and hemodynamic function (Table 1). There were no differences among the NADH oxidase, NADH Q1 reductase, and NADH-ferricyanide reductase activities of SMPs from normoxic KO hearts compared with the SMPs from normoxic hearts expressing PARP-1. Both NADH oxidase and NADH-Q1 reductase activities were preserved during prolonged ischemia-reperfusion of PARP-1 KO hearts (Fig. 2).


Figure 2
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  		Fig. 2. PARP-1 inhibition during reperfusion preserves mitochondrial complex I function. Submitochondrial particles (SMPs) were prepared from C57BL/6 mouse hearts (open bars) after normoxic perfusion (trace 1) or reperfusion in the absence (trace 2) and presence (trace 3) of 3-AB and used to measure complex I function. In parallel studies, SMPs were prepared from PARP-1 null hearts (filled bars) after normoxic perfusion or prolonged ischemia-reperfusion and used to measure complex I function. A: NADH oxidase activity was measured as a decrease in absorbance at 340 nm using NADH as an electron donor. B: NADH-Q1 reductase activity was measured as a decrease in absorbance at 340 nm using exogenous ubiquinone-1 (Q1) as electron acceptor, in the presence of KCN. C: NADH-ferricyanide reductase activity was measured as a decrease in absorbance at 420 nm using K3Fe(CN)6 as the electron acceptor, in the presence of rotenone. Ischemia-reperfusion reduced NADH oxidase and NADH-Q1 reductase activities in hearts expressing PARP-1 but did not alter NADH-ferricyanide reductase activity. Inhibition or disruption of PARP-1 prevented complex I injury. n = 6 hearts per group. *P < 0.05 vs. normoxia.

 

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  		Table 1. Normal baseline heart weight and function in PARP-1 KO mice

 
PARP-1 inhibition during reperfusion normalizes tissue metabolite contents. We predicted that complex I preservation following PARP-1 inhibition would correlate with an improvement in metabolic status during reperfusion because of the energy demands of myocardial contraction. As indicated in Fig. 3, NAD+ concentrations in tissue extracts from reperfused C57BL/6 hearts were only 30% of those measured in normoxic controls. PARP-1 inhibition during reperfusion produced a twofold increase in tissue NAD+ content compared with vehicle-treated hearts, consistent with the attenuation of poly(ADP-ribosyl)ation shown in Fig. 1B. ATP concentrations in tissue extracts from reperfused C57BL/6 hearts were only 30% of those in normoxic controls (Fig. 3). PARP-1 inhibition during reperfusion produced a twofold increase in ATP content compared with vehicle-treated hearts. Finally, ischemia-reperfusion produced a twofold increase in tissue lactate concentrations compared with normoxic controls (Fig. 3), whereas pharmacological inhibition of PARP-1 activity by 3-AB administration during reperfusion blocked accumulation of lactate.


Figure 3
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  		Fig. 3. Inhibition of PARP-1 during reperfusion preserves myocardial metabolite content. NAD+, ATP, total adenine nucleotide (TAN), and lactate concentrations found in perchloric acid extracts of individual hearts after NX perfusion or reperfusion in the absence (CON) and presence of 3-AB were determined by using biochemical techniques and 1H NMR spectroscopy. Ischemia-reperfusion reduced NAD+ and ATP content by 70% and produced a twofold increase in lactate levels compared with NX hearts. Inhibition of PARP with 3-AB reduced reperfusion-mediated alterations in cardiac metabolite content. n = 6 hearts per group. *P < 0.05 vs. CON.

 
PARP-1 inhibition or disruption enhances cardiac contractile recovery and tissue viability. We examined whether the beneficial effects of PARP-1 inhibition or disruption on myocardial energetics correlate with improvement in contractile recovery and tissue integrity. As shown in Fig. 4A, ischemia-reperfusion produced severe reduction of LVDP in vehicle-treated C57BL/6 mouse hearts and pathological elevation of LVEDP. In contrast, pharmacological inhibition of PARP-1 activity by 3-AB administration during reperfusion improved contractile recovery. As indicated in Fig. 4B, ischemia-reperfusion severely reduced LVDP and elevated LVEDP in control hearts expressing PARP-1. In contrast, PARP-1 gene disruption improved myocardial contractile recovery during reperfusion. Pharmacological inhibition of PARP-1 activity and PARP-1 gene disruption were equally effective in blocking pathological release of CK during reperfusion.


Figure 4
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  		Fig. 4. PARP-1 inhibition or disruption improves contractility and reduces enzyme release. Left ventricular (LV) pressures were recorded throughout each experiment, and creatine kinase (CK) activity measured in coronary effluent was collected during reperfusion. Baseline contractile function (PRE) was measured at the end of the equilibration period before prolonged ischemia-reperfusion. A: ischemia-reperfusion of vehicle-treated (CON) hearts (open circles) produced a severe impairment of LV developed pressure (LVDP) and end-diastolic pressure (LVEDP) and profound CK release (open bar). Inhibition of PARP activity with 3-AB improved contractility (filled circles) and reduced release of CK (filled bar). n = 12 hearts per group. *P < 0.05 vs. CON. B: ischemia-reperfusion of wild-type (WT) hearts (open squares) produced a severe impairment of LVDP and LVEDP and profound CK release (open bar). PARP-1 disruption (knockout, KO) improved contractility (filled squares) and CK release (filled bar). n = 12 hearts per group. *P < 0.05 vs. WT.

 
IPC abolishes the cardioprotective effects of PARP-1 gene disruption. Gray et al. (5) recently published data establishing that protein kinase-C{epsilon} is necessary for improvement of cardiac contractile recovery and reduction of infarction size caused by IPC. In the present investigation, PARP-1 KO hearts were preconditioned to test the hypothesis that PARP-1 is another signaling molecule required for inducible cardioprotection. As shown in Fig. 5, IPC or PARP-1 gene disruption alone preserved NADH oxidase and NADH-Q1 reductase activities after reperfusion at normoxic levels. In contrast, NADH oxidase and NADH-Q1 reductase activities in SMPs from preconditioned PARP-1 KO hearts were only half of normoxic levels, equivalent to activities in SMPs from nonpreconditioned control hearts.


Figure 5
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  		Fig. 5. Preconditioning blocks complex I preservation associated with PARP-1 disruption. SMPs prepared from WT (open bars) and PARP-1 KO (filled bars) hearts after ischemia-reperfusion were used to measure complex I activities. Hearts were pretreated with ischemic preconditioning, the free radical scavenger N-(2-mercaptopriopionyl)-glycine (MPG), or the xanthine oxidase inhibitor allopurinol as indicated. A: ischemia-reperfusion reduced NADH oxidase activity in WT SMPs but not in PARP-1 KO SMPs. Preconditioning preserved NADH oxidase activity in WT SMPs but paradoxically blocked the protective effects of PARP-1 disruption. MPG or allopurinol infusion restored NADH oxidase activity to normoxic control values in preconditioned PARP-1 KO hearts. B: ischemia-reperfusion reduced NADH-Q1 reductase activity in WT SMPs but not PARP-1 KO SMPs. Preconditioning preserved NADH-Q1 reductase activity in WT SMPs but paradoxically blocked the protective effects of PARP-1 disruption. MPG or allopurinol restored NADH-Q1 reductase activities to normoxic control values in preconditioned PARP-1 KO hearts. C: NADH-ferricyanide reductase activity was not altered by any intervention. n = 6 hearts per group. *P < 0.05 vs. normoxic controls.

 
In pursuit of the mechanisms by which IPC produces tissue toxicity in PARP-1 KO hearts, we measured the MDA content of extracts prepared after prolonged ischemia-reperfusion as one marker of pathological lipid peroxidation. We observed that transient IPC of PARP-1 KO hearts caused substantial myocardial lipid peroxidation, equivalent to that measured in nonpreconditioned control hearts. As indicated in Fig. 6, prolonged ischemia-reperfusion caused a twofold increase in the MDA content of control hearts compared with normoxic values. IPC and PARP-1 gene disruption were equally effective in reducing lipid peroxidation. In contrast, preconditioning of PARP-1 KO hearts resulted in a twofold increase in MDA content compared with normoxic values (Fig. 6).


Figure 6
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  		Fig. 6. Preconditioning blocks benefits of PARP-1 gene disruption on lipid peroxidation. Malondialdehyde (MDA) content was measured in lysates prepared from WT (open bars) and PARP-1 KO (filled bars) hearts after prolonged ischemia-reperfusion. Hearts were treated with ischemic preconditioning, MPG, or allopurinol as indicated. Ischemia-reperfusion caused pathological lipid peroxidation in WT hearts but not in PARP-1 KO hearts. Preconditioning reduced lipid peroxidation in WT hearts but increased the MDA contents of PARP-1 KO hearts. MPG or allopurinol infusion restored lipid peroxidation in preconditioned PARP-1 KO hearts to normoxic control values. n = 6 per group. *P < 0.05 vs. normoxic controls.

 
We examined whether the detrimental effects of preconditioning on complex I function in PARP-1 KO hearts are associated with impairment of cardiac contractile recovery and viability. As shown in Fig. 7A, prolonged ischemia-reperfusion substantially decreased LVDP, increased LVEDP, and produced tissue necrosis in hearts expressing PARP-1. IPC enhanced LVDP, blunted elevations of LVEDP, and reduced infarction. As shown in Fig. 7B, PARP-1 gene disruption was equally effective for improvement of cardiac contractile recovery and viability following prolonged ischemia-reperfusion. However, IPC substantially decreased LVDP in PARP-1 KO hearts, increased LVEDP, and produced tissue necrosis equivalent to levels of injury measured in nonpreconditioned control hearts (Fig. 7B).


Figure 7
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  		Fig. 7. Preconditioning blocks benefits of PARP-1 disruption on contraction and infarction. LV pressures were recorded throughout each experiment, and infarction size was measured after reperfusion. A: ischemia-reperfusion of nonpreconditioned (CON) WT hearts resulted in severe impairment of LVDP and LVEDP (open circles) and extensive myocardial infarction (open bar). Preconditioning (IPC) of WT hearts improved contractility (filled circles) and reduced infarction (filled bar). B: PARP-1 gene disruption (KO) improved cardiac contractility (open squares) and reduced infarct size (open bar) compared with nonpreconditioned WT hearts. Preconditioning of KO hearts paradoxically blocked the benefits of PARP-1 gene disruption on contractility (filled squares) and infarction (filled bar). Contractile recovery and infarct size in KO-IPC hearts were not different from values measured in WT-CON hearts. C: MPG treatment improved contractile recovery and reduced infarction size in preconditioned PARP-1 KO hearts to values measured in preconditioned WT hearts. D: pretreatment with allopurinol improved contractile recovery and reduced infarction size in preconditioned PARP-1 KO hearts to equivalent values measured in preconditioned WT hearts. n = 12 per group. *P < 0.05 vs. corresponding CON hearts.

 
Administration of allopurinol enhances the recovery of preconditioned PARP-1 KO hearts. We tested the hypothesis that generation of ROS is a critical component of preconditioning-induced tissue toxicity in PARP-1 KO hearts by administering the antioxidant MPG during the first 20 min of reperfusion. As shown in Fig. 5, MPG treatment during reperfusion preserved NADH oxidase and NADH-Q1 reductase activity in SMPs from preconditioned PARP-1 KO hearts at normoxic levels. MPG treatment also reduced pathological lipid peroxidation in preconditioned PARP-1 KO hearts (Fig. 6). As shown in Fig. 7C, cardiac contractile recovery and infarction size in preconditioned PARP-1 KO hearts treated with MPG were equivalent to values measured in preconditioned control hearts.

We examined whether xanthine oxidase-derived free radicals represent a potential source of reperfusion injury in preconditioned PARP-1 KO hearts by administering the xanthine oxidase inhibitor allopurinol throughout each protocol. As indicated in Fig. 5, allopurinol preserved the NADH oxidase and NADH-Q1 reductase activities of SMPs from preconditioned PARP-1 KO hearts at normoxic levels. Allopurinol treatment also reduced pathological lipid peroxidation in preconditioned PARP-1 KO hearts (Fig. 6). As indicated in Fig. 7D, cardiac contractile recovery and infarction in preconditioned PARP-1 KO hearts treated with allopurinol were equal to values measured in preconditioned control hearts. Thus allopurinol administration and MPG treatment were equally effective strategies for promoting recovery of preconditioned PARP-1 KO hearts.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present investigation generated three important findings. First, hyperactivation of PARP-1 during cardiac reperfusion is associated with a selective defect in mitochondrial respiratory chain complex I function distal to those protein subunits that mediate electron transfer from NADH to ferricyanide. Second, the beneficial effects of PARP-1 inhibition or disruption on complex I activity correlate closely with improved myocardial energetics, contractility, and viability. Third, IPC reverses the beneficial effects of PARP-1 gene disruption on complex I activity during prolonged ischemia-reperfusion and promotes pathological cardiac lipid peroxidation.

Our finding of PARP-1 localization in the nuclei of adult mouse cardiac myocytes provides confirmatory evidence that hyperactivation regulates complex I function in heart mitochondria primarily through indirect mechanisms. These results are consistent with observations by Ikai and Ueda (10), who used immunofluorescence microscopy to demonstrate exclusive localization of poly(ADP-ribose) synthetase within the nuclei of sectioned bovine hearts. Liaudet et al. (13) used immunohistochemical techniques to demonstrate that coronary artery occlusion stimulated poly(ADP-ribose) signal only in the nuclei of sectioned rat hearts. Similarly, Halmosi et al. (7) demonstrated that although the PARP inhibitors 3-AB, nicotinamide, O-(3-piperidino-2-hydroxy-1-propyl)nicotinic amidoxime (BGP-15), and 4-hydroxyquinazoline prevented H2O2-induced cytochrome oxidase inactivation, PARP activity was not present in isolated mitochondria by autoradiography. In contrast, Du et al. (3) identified PARP-1 protein and activity in both nuclei and mitochondria of embryonic rat cortical neurons. Those investigators also showed that PARP-1 inhibition in isolated brain mitochondria preserved transmembrane potential ({Delta}{Psi}m) and respiration after oxidative stress. Differences in subcellular distribution of PARP-1 between isolated neurons and cardiac myocytes suggest that reperfusion injury in the central nervous system may involve mechanisms distinct from those in heart tissue.

We also found that PARP-1 hyperactivation is associated with selective defects in complex I activities distal to the NADH dehydrogenase component. SMPs were used for experiments to eliminate potential substrate barriers present in permeabilized mitochondria and preserve critical protein, lipid, and prosthetic group interactions disrupted during detergent-based isolation of individual respiratory chain complexes. In each experimental group, NADH oxidase activities closely tracked NADH-Q1 reductase activities, consistent with oxidation of the terminal-reduced component of complex I by ubiquinone as the rate-limiting step of the overall NADH oxidase reaction under physiological conditions and that governing oxidative metabolism during reperfusion (17). The present study advances work by Veitch et al. (26), who determined that ischemia-reperfusion can inhibit complex I distal to the NADH dehydrogenase component in isolated mitochondria without examining PARP-1 hyperactivation or cardiac energetics. Our investigation advances work by Halmosi et al. (7), who determined that PARP-1 hyperactivation damaged mitochondria without identifying sites in purified NADH:cytochrome c oxidoreductase (complex I-III) modified by stress or global effects on contractile recovery and tissue integrity.

Our investigation does not directly identify mechanisms through which PARP-1 inhibition or disruption preserves mitochondrial complex I function during cardiac reperfusion. Zingarelli et al. (36) recently demonstrated reduced DNA binding of activator protein-1 (AP-1) in the hearts of PARP-1 KO mice after coronary artery occlusion compared with hearts of wild-type littermates. Furthermore, microarray analysis of mRNA prepared from PARP-1 KO hearts revealed reduced expression of AP-1-dependent genes encoding interleukin-6, interleukin-1beta, cyclooxygenase-2, and other mediators of inflammation (36). In a separate study (35), those investigators showed reduced DNA binding of nuclear factor-{kappa}B in PARP-1 KO hearts after coronary artery occlusion. Expression of genes encoding adipsin, calpain 3, caspase 12, and other regulators of apoptosis was also reduced in PARP-1 KO hearts (35). Thus PARP-1 inhibition or disruption may preserve heart mitochondrial function by blocking these nuclear regulatory pathways.

Another possibility is that PARP-1 inhibition or disruption sustains carbohydrate oxidation during oxidative stress. In the present study, PARP-1 hyperactivation rendered hearts unable to maintain ATP content during reperfusion despite abundant glucose and O2. NMR spectroscopy data suggested that accumulated tissue lactate could not be metabolized after reperfusion. NAD+ depletion in cytosolic compartments has been shown to inhibit the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis as well as lactate dehydrogenase activity in numerous cells and tissues (21). Ying et al. (31) determined that incubation of mouse astrocytes with pyruvate after PARP-1 hyperactivation reduced cell death by providing energy substrates metabolized in the absence of cytosolic NAD+. In contrast, those investigators observed that glucose or lactate incubation did not reduce cellular necrosis (31). Our current experimental findings suggest that PARP-1 inhibition or disruption reduces ATP consumption needed for resynthesis of NAD+ and promotes ATP generation by preserving glucose and lactate metabolism in reperfused tissue.

Pharmacological agents that sustain carbohydrate oxidation during reperfusion may cause cardioprotection by increasing delivery of pyruvate to the mitochondrial matrix, which inhibits pyruvate dehydrogenase kinase and favors the activated state of pyruvate dehydrogenase (11). Kudej et al. (12) observed that dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase, increased glucose oxidation and reduced stunning in reperfused hearts. Raha et al. (19) found that pyruvate dehydrogenase kinase also phosphorylated a 18-kDa complex I subunit, which inhibited electron transfer and accelerated ROS production. Thus PARP-1 inhibition may be protective in part by reducing phosphorylation of complex I negative regulatory subunits and preventing oxidation of mitochondrial lipids required for electron transfer (19). Alternatively, PARP-1 inhibition may stimulate mitochondrial protection and cellular survival through signal transduction pathways involving phosphatidylinositol 3-kinase and Akt (16, 24).

Our findings regarding preconditioning effects on PARP-1 KO hearts are consistent with the work of Liaudet et al. (13), who subjected PARP-1 KO hearts to IPC (four cycles of 5 min coronary occlusion), regional ischemia (30 min coronary artery occlusion), and reperfusion (24 h after release of coronary artery occlusion). Those investigators demonstrated that in vivo preconditioning of PARP-1 KO hearts increased infarction size to levels measured in nonpreconditioned wild-type hearts. 3-AB administration before preconditioning mimicked the effects of PARP-1 disruption (13). Preconditioning of PARP-1 KO hearts did not increase myeloperoxidase activity, suggesting mechanisms independent of neutrophil infiltration. Ex vivo experiments carried out in the present study also support reversal of the cardioprotective effects of PARP-1 disruption as a process independent of systemic inflammatory responses.

Liaudet et al. (13) did not establish the mechanisms responsible for enhanced infarction after IPC of PARP-1 KO hearts but postulated that an imbalance of endogenous purines could lead to enhanced oxidant generation. In the present study, we demonstrated that IPC of PARP-1 KO hearts was associated with increased infarction size, mitochondrial injury, hemodynamic impairment, and lipid peroxidation. In contrast, treatment with either the antioxidant MPG or the xanthine oxidase inhibitor allopurinol significantly reduced tissue damage in preconditioned PARP-1 KO hearts. These data prompt speculation that xanthine oxidase-derived radicals are critical for preconditioning-induced toxicity in PARP-1 KO hearts. However, measurements of xanthine oxidase activity and formal identification of specific radicals are needed to validate this mechanism of injury.

In summary, we established that PARP-1 inhibition and PARP-1 gene disruption are equally effective strategies for preservation of complex I function during reperfusion. Our data support an association between nuclear PARP-1 hyperactivation and mitochondrial complex I injury in cardiac myocytes and suggest possible physiological roles for limited PARP-1 activation in the development of preconditioning-induced resistance to stress. This work advances understanding of metabolic regulation and identifies potential targets for prevention of ischemic heart disease.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants R01 AA-11135 and P01 HL-068738 and by the Medical Research Service of the Department of Veterans Affairs. Ubiquinone-1 was the gift of Eisai (Tokyo, Japan). Mice lacking PARP-1 and wild-type control mice were the gifts of Dr. Basilia Zingarelli at the Cincinnati Children’s Hospital Medical Center.


   ACKNOWLEDGMENTS
 
We thank Drs. William McIntire, Elena Maklashina, and Joel S. Karliner for helpful suggestions during the investigation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. O. Gray, Div. of Cardiology 5G1, San Francisco General Hospital, 1001 Potrero Ave., San Francisco, CA 94110 (e-mail: mgray{at}medsfgh.ucsf.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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