Parkinson-Susceptibility Gene DJ-1/PARK7 Protects the Murine Heart from Oxidative Damage in Vivo

Parkinson-susceptibility gene DJ-1/PARK7 protects the murine heart from oxidative damage in vivo

Filio Billiaa,b, Ludger Haucka, Daniela Grothea, Filip Konecnyc, Vivek Raod, Raymond H. Kima, and Tak W. Maka,1

aCampbell Family Cancer Research Institute, Princess Margaret Hospital, Toronto, ON, Canada M5G 2M9; bDivision of Cardiology, University Health Network, Toronto, ON, Canada M5G 2C4; cToronto Medical Discovery Tower, University of Toronto, ON, Canada M5G 2C4; and dDivision of Cardiovascular Surgery, University Health Network, Toronto, ON, Canada M5G 2C4

Contributed by Tak W. Mak, February 27, 2013 (sent for review January 23, 2013)

Oxidative stress is caused by an imbalance between the production glutathione synthesis (11–13). In contrast, oxidized DJ-1 is of reactive oxygen species (ROS) and the ability of an organism to thought to dissociate from these transcripts, allowing their eliminate these toxic intermediates. Although the Parkinson-sus- translation (13, 14). ceptibility gene, Parkinson protein 7/DJ-1 (DJ-1), has been linked to The heart’s high energy demand necessitates an immense the regulation of oxidative stress, the exact mechanism by which mitochondrial content, and therefore an increased vulnerability this occurs and its in vivo relevance have remained elusive. In the to oxidative damage. We reasoned that if the primary function heart, oxidative stress is a major contributor to the development of of DJ-1 is to inhibit the action of ROS, then its inactivation heart failure (HF). Therefore, we hypothesized that DJ-1 inhibits should result in enhanced ROS production, increased oxidative the pathological consequences of ROS production in the heart, damage, and deterioration in cardiac performance. Here we the organ with the highest oxidative burden. We report that DJ-1 is show that DJ-1 is indeed highly expressed in the normal heart, whereas it is dramatically down-regulated in end-stage human highly expressed in normal heart tissue but is markedly reduced in HF. During oxidative stress, DJ-1-deficient mice develop ex- end-stage human HF. DJ-1-deficient mice subjected to oxidative aggerated cardiac hypertrophy and susceptibility to the de- stress by transaortic banding exhibited exaggerated cardiac hyper- velopment of HF. Our study provides substantial evidence in trophy and susceptibility to developing HF. This was accompanied vivo that the Parkinson-susceptibility gene, DJ-1, functions as by a Trp53 (p53)-dependent decrease in capillary density, an exces- a unique and nonredundant cardiac antioxidant participating in sive oxidation of DNA, and increased cardiomyocyte apoptosis, the cellular defense against ROS. key events in the development of HF. Impaired mitochondrial MEDICAL SCIENCES biogenesis and progressive respiratory chain deficiency were also Results evident in cardiomyocytes lacking DJ-1. Our results provide com- DJ-1 Protein Expression Is Reduced in End-stage Human HF. Because pelling in vivo evidence that DJ-1 is a unique and nonredundant enhanced oxidative stress has been well-documented in patients antioxidant that functions independent of other antioxidative with HF (14–17), we reasoned that DJ-1 protein expression pathways in the cellular defense against ROS. would be reduced in this patient population. Therefore, we analyzed left ventricular (LV) tissue obtained from patients with cardiomyopathy | angiotensin II | JC-1 end-stage HF and normal healthy controls by Western blotting (Fig. 1A and Table 1) and quantitative real-time PCR (qRT- xidative stress is created by the imbalance between reactive PCR; Fig. 1B). There was a substantial decrease in total DJ-1 Ooxygen species (ROS) production and its elimination by protein levels in LV samples from patients with end-stage HF antioxidant systems. Because of the heart’s high metabolic rate compared with normal control patients. In contrast, the vari- and limited capacity for regeneration, it is particularly sensitive ability in DJ-1 mRNA levels in these patient samples was not to oxidative stress. On exposure to ROS, the heart undergoes significantly different, implying that DJ-1 is regulated at the hypertrophic growth, a process that involves cell enlargement, posttranscriptional level. Our findings demonstrate that malad- myofibrillar disarray, and reexpression of fetal genes (1). Al- aptive changes leading to the development of end-stage HF in though cardiac hypertrophy is considered an initial adaptive re- humans is associated with impaired DJ-1 expression. sponse, prolonged hypertrophy is ultimately detrimental and leads to progressive heart failure (HF). DJ-1 Protects CMs Against Acute Oxidative Stress Both in Vitro and in HF, the leading cause of morbidity and mortality in North Vivo. In isolated neonatal murine CMs, DJ-1 localizes to the nu- America (2–4), is associated with poor quality of life and prog- cleus and cytoplasm, whereas it is absent from mitochondria (Fig. nosis. Our existing armamentarium of conventional pharmaco- 1C). To determine the consequences of DJ-1 loss on cellular via- logic therapy can only slow the progression of the disease, rather bility, we examined the integrity of mitochondrial transmembrane than target the disease process itself. Alternatives to medical potential after acute oxidative stress in isolated primary CMs. CMs therapy are limited to transplantation or mechanical assist devices, were labeled with the fluorochrome JC-1 (18) (3 μM) and then approaches that are highly invasive and are themselves associated exposed to antimycin (100 μM), which enhances ROS production. fi Antimycin induced a rapid decrease in the red/green ratio of JC-1 with signi cant morbidity and mortality. As such, it is exceedingly −/− important to find alternate strategies that will effectively restrict fluorescence in DJ-1 CMs (Fig. S1 A and B), indicative of compromised cell viability. This change occurred significantly later cardiomyocyte (CM) loss and hypertrophy. Thus, providing a bet- +/+ ter understanding of the mechanisms driving the heart’sresponse in the DJ-1 CM. Therefore, we conclude that DJ-1 preserves to oxidative stress may help in the development of new and ef- mitochondrial function, thus preventing cell death. fective treatments for HF. Parkinson protein 7 (PARK7)/DJ-1 (DJ-1) was first identified as a gene mutated in a recessively inherited form of early-onset Author contributions: F.B., L.H., and T.W.M. designed research; F.B., L.H., and D.G. per- Parkinson disease (5, 6). Subsequent work demonstrated that formed research; F.K., V.R., and R.H.K. contributed new reagents/analytic tools; F.B. and DJ-1 deficiency leads to an increased vulnerability to oxidative L.H. analyzed data; and F.B. and L.H. wrote the paper. stress in vitro (7–10), implying that DJ-1 plays a role in the de- The authors declare no conflict of interest. fense against ROS. In the absence of oxidative stress, DJ-1 binds Freely available online through the PNAS open access option. to and represses the translation of mRNAs encoding factors 1To whom correspondence should be addressed. E-mail: [email protected]. involved in contending with oxidative stress, such as tumor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. protein 53 (p53), superoxide dismutase, and proteins involved in 1073/pnas.1303444110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1303444110 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Therefore, our results exclude the possibility that increased apoptosis, in general, was responsible and substantiate the speci- ﬁcity of DJ-1 in regulating ROS-dependent events. − − Next, we analyzed whether DJ-1 / CMs are susceptible to cell death after myocardial infarction (MI), as would be predicted if DJ-1 reduces cardiac ROS. At 72 h after MI, we observed a signiﬁcant increase in the number of TUNEL-positive CM − − nuclei in the peri-infarct area in DJ-1 / mice compared with wild-type animals (Fig. 1D). We also observed larger scar size − − and elevated collagen deposition (Fig. 1E) in DJ-1 / mice + + + − compared with sham-operated DJ-1 / or DJ / animals. Thus, DJ-1 protects CMs against MI-induced apoptosis and is essential in the remodeling process postinfarct.

Susceptibility of DJ-1−/− Mice to Pressure Overload–Induced Cardiac Dysfunction. At 2 mo of age, heart/body weight ratios, CM cross- sectional area, and echocardiographic parameters were com- − − + + + − parable between DJ-1 / and DJ-1 / , / mice (Fig. 2 A and C and Fig. S3 A–C). In contrast, transaortic banding (TAB) in- − − creased the heart/body weight ratios in DJ-1 / mice by 51% (Fig. 2A). The length/width ratios remained unaltered, consistent with concentric hypertrophy (Fig. S3 A–C). Increased mRNA transcript levels of atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain were reflective of pathological cardiac hypertrophy (Fig. 2B). TAB treatment of − − DJ-1 / mice also led to marked reduction in fractional short- ening (Fig. 2C) and significantly increased interstitial fibrosis (Fig. 2D and Fig. S4). Thus, DJ-1 loss increases susceptibility to Fig. 1. Down-regulation of DJ-1 protein expression in human end-stage the development of HF after TAB. HF. (A) Representative Western blot of DJ-1 in total human LV extracts (Upper) with quantification by densitometry (Lower). Lane 1 was set at Enhanced Oxidative Stress in DJ-1 Knockout Mice During Pressure 100%. (B) qRT-PCR analysis of DJ-1 mRNA levels in ventricular tissues from Overload. Levels of cytotoxic 4-hydroxyalkenals (4-HAE) and human patient specimen described in A. Lane 1 was set at 1-fold. Mean ± malondialdehyde (MDA), indicators of ROS-dependent lipid − − SEM (C) Representative Western blot of DJ-1, lamin, actin, and Cytb5 in peroxidation, were significantly higher in LV tissues from DJ-1 / fractionated neonatal CM extracts (Left) with corresponding indirect im- mice subjected to TAB (Fig. 3A) (19, 20). In addition, aconitase munofluorescent micrographs (Right). (D) Quantification of apoptosis in neonatal CMs. *P < 0.05 versus mock; #P < 0.05. (E) Quantification of infarct activity, which is exquisitely sensitive to ROS, was markedly reduced in both mitochondrial and cytosolic fractions in LV sam- sizes (white outline) 4 wk post-MI (Left) and representative micrographs −/− A (Right) of cross-sections stained for α-actinin and collagen I/III. *P < 0.05. ples from DJ-1 mice (Fig. 3 ). These results indicate that DJ-1 Sample size for each group is indicated inside the respective bar. LV, left loss does indeed lead to increased ROS production during ventricle; RV, right ventricle; S, septum. pressure overload, affecting both cytosolic and mitochondrial compartments. Oxidative stress–induced impairment of oxidative phosphory- To determine whether DJ-1 plays a protective role against lation and decline in ATP metabolism is detrimental for the ROS-induced apoptosis in vitro, CMs were treated with anti- contractile function and viability of CM (21, 22). TAB treatment μ μ −/− led to a significant decrease in ATP levels in total LV samples mycin (100 M) or H2O2 (200 M). DJ-1 CMs showed a 46% − − + + (antimycin) and 39% (H O ) increase in TUNEL-positive nuclei derived from DJ-1 / mice versus DJ-1 / animals (Fig. 3B). We + + 2 2 compared with DJ-1 / CMs (Figs. S1C and S2). This effect was then assessed mitochondrial biogenesis and function on the bais not observed in CMs exposed to either camptothecin (10 μM), of the hypothesis that both of these parameters would likely be a DNA topoisomerase I inhibitor, or staurosporine (2 μM), a affected under conditions of chronic oxidative stress (23). To protein kinase inhibitor, which are both nonoxidative insults. assess mitochondrial biogenesis, we measured the mitochondrial DNA copy number of cytochrome b (Cytb), which was normalized to levels of a nuclear-encoded single-copy gene β-actin by Table 1. Patient characteristics of human LV samples as in Fig. 1A qPCR. We noted a marked reduction (40%) in TAB-treated − − DJ-1 / hearts compared with those of littermate controls (Fig. Lane Age, y Sex Etiology of HF syndrome 3B). As a surrogate for mitochondrial function, we quantified the 1 38 Female Control level of mRNA expression of the mitochondrial cytochrome b5 2 38 Female Control (Cytb5) gene by qRT-PCR, corrected for the transcript expres- β 3 36 Male Adriamycin sion of -actin, and found that the mRNA levels of Cytb5 were −/− B 4 62 Male Idiopathic reduced in the TAB-treated DJ-1 hearts (Fig. 3 ). These results strongly support the hypothesis that the phenotype ob- 5 25 Male Valvular −/− 6 60 Male Idiopathic served after TAB in DJ-1 mice is a consequence of impaired mitochondrial functionality. 7 65 Male Idiopathic Next, we examined cellular oxidative DNA damage, using 8 51 Male Ischemic ELISA-based detection of 8-hydroxy-2′-deoxyguanosine (8- 9 61 Male Ischemic OHdG), a principal oxidative lesion that can cause base mispair- − − 10 49 Female Familial ing and mutations (24). DJ-1 / mice had significantly greater +/+ Informed consent was obtained from patients with end-stage HF who TAB-induced concentrations of 8-OHdG than their DJ-1 were to have a left ventricular assist device inserted as either a bridge to counterparts (Fig. 3B). These data suggest that DJ-1 loss increases a heart transplant or as destination therapy to end of life. Samples were the sensitivity of CMs to oxidative DNA damage. Cytosolic and obtained from the LV apex and frozen in liquid nitrogen at the time of mitochondrial superoxide dismutase, an essential cardiac antioxi- −/− procurement. dant (25), was also drastically reduced in TAB-treated DJ-1

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1303444110 Billia et al. Downloaded by guest on September 30, 2021 − − Fig. 2. Susceptibility of DJ-1 / mice to oxidative stress and cardiac function induced by pressure overload. (A) Heart/ body weight ratio (Left), Masson-stain of myocardial cross-sectional (Center), and longitudinal sections (Right) after TAB. (B) mRNA levels of hypertrophic marker genes by qRT-PCR normalized to mRNA levels of GAPDH. (C) Fractional shorten- ing (FS) after TAB. (D) Quantiﬁcation of extracellular matrix area by anticollagen I/III and anti-α-actinin immunoﬂuores- cence staining of cross-sectional LV specimen. *P < 0.05 versus sham; #P < 0.05.

mice (Fig. 3C), as were reduced glutathione/oxidized glutathione Angiotensin II Increases Oxidative Damage in DJ-1 Knockout Mice. To ratios (Fig. 3C). Combined, these data confirm that DJ-1 loss demonstrate the effect of DJ-1 on hypertrophic growth, in the increases CM sensitivity to oxidative DNA damage as a result of presence or absence of oxidative stress, we chronically exposed −1 the impairment of ROS detoxification. mice to low-dose (LD) angiotensin II (Ang II; 0.15 mg ·kg body −1· – weight d), which did not affect mean arterial pressure (under MEDICAL SCIENCES It is well accepted that extracellular signal regulated kinase − − anesthesia of 2% isoflurane, 54 ± 16 mm Hg DJ-1 / vs. 56 ± (Erk) exerts a prohypertrophic function in response to ROS (26– + + 6mmHgDJ-1 / ; P = 0.81) and intracellular ROS levels (Fig. 4A), 30). Therefore, it was of interest to determine whether the − − or to medium-dose (MD) Ang II (0.5 mg 1·kg body weight 1·d), antioxidant activity of DJ-1 could affect the regulation of this − − which increased mean arterial pressure (85 ± 7 mm Hg DJ-1 / important signaling pathway. We observed that LV samples from ± +/+ P = −/− fi vs. 91 8 mm Hg DJ-1 ; 0.19) and the generation of ROS. DJ-1 mice had signi cantly increased levels of phosphorylated fi +/+ D As early as 1 wk after Ang II infusion, we observed signi cantly Erk1/2 compared with DJ-1 mice 4 d after TAB (Fig. 3 ), elevated heart/body weight ratios, worse cardiac function (Fig. 4 substantiating the hypothesis that DJ-1 negatively modulates A–C), and impaired antioxidant activities (Fig. 4 B and C)in − − + + ROS-dependent Erk activation. MD-Ang II-treated DJ-1 / mice compared with DJ-1 / litter- − − Next, we examined the effect of DJ-1 on CM apoptosis after mates or LD-Ang II-infused DJ-1 / mice. When Ang II was TAB, expecting that oxidative stress, in the absence of DJ-1, administered as a bolus injection (20 mg/kg body weight), we would lead to increased rates of apoptosis. We observed signif- observed a higher and prolonged increase in phospho-Erk1/2 in icantly increased numbers of TUNEL-positive CM nuclei and an −/− +/+ D − − DJ-1 mice compared with DJ-1 mice (Fig. 4 ). There were induction of caspase 3 proteolytic activity in TAB-treated DJ-1 / higher levels of phosphorylated Erk1/2 observed with chronic − − + + hearts (Fig. 3E and Fig. S5), demonstrating that DJ-1 protects exposure to MD-Ang II in DJ-1 / mice compared with DJ-1 / CMs against ROS-induced apoptosis. litters (Fig. 4E) or LD-Ang II. Collectively, these observations

Fig. 3. Pressure overload enhances oxidative stress − − in DJ-1 / mice. (A) 4-HAE and MDA levels (Left)and aconitase activities (Right) in total LV extracts after TAB. (B) 8-OhdG levels after TAB (Upper Left)intotal LV samples. ATP concentrations in total LV extracts after TAB (Lower Left). Assessment of mitochondrial biogenesis and function after TAB (Right). (C)Mito- chondrial and cytoplasmic superoxide dismutase activities in fractionated LV extracts (Left). Reduced glutathione and oxidized glutathione levels in total LV extracts after TAB (Right). (D) Western blot of Erk1/2 in total LV extracts after TAB (Lower)with corresponding densitometry quantiﬁcation and cor- rection for total Erk1/2 expression (Upper). Lane 2 set at 1-fold induction. (E) Degree of apoptosis in LV specimens at 1 wk after TAB (Upper). Caspase 3 activity in total LV samples at 1 wk after TAB (Bottom). *P < 0.05 versus sham; #P < 0.05. Sample size for each group as indicated.

Billia et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 − − Fig. 4. Ang II increases oxidative damage in DJ-1 / mice. (A) 4-HAE and MDA levels (Left) and aconitase activities (Right) in LV samples after LD- and MD- Ang II infusions. (B) Heart/body weight ratios and FS at 1 wk (Left) and 4 wk (Right) after Ang II treatment. (C) Superoxide dismutase activities in fractionated LV tissues after Ang II (Left). Reduced glutathione and oxidized glutathione levels in total LV samples (Right). (D) Western blot of phosphorylated (Pi)-Erk1/2 from total LV extracts after bolus injection of Ang II. (E) Western blot (Right)and densitometry of Pi-Erk1/2 protein/lane (Left)corrected for total Erk1/2 expression. Lane 2 was set at 1-fold induction. *P < 0.05 versus saline; #P < 0.05. Sample size for each group is indicated inside the respective bar.

suggest that DJ-1-deﬁciency impairs cardiac function by en- (Fig. S6). Therefore, these results are consistent with the concept hancing the heart’s sensitivity to the damaging effects of ROS. that loss of DJ-1 inhibits TAB-dependent angiogenesis. Hypoxia- inducible factor (Hif-1α) is required for expression of VEGF and DJ-1 Functions as a Stress-Responsive Regulator of Angiogenesis in Ang-1, essential regulators of angiogenesis during cardiac hyper- − − the Murine Heart. TAB treatment decreased microvessels/CMs in trophy (31–33). Hif-1α mRNA transcript levels in DJ-1 / mice − − + + − − DJ-1 / mice compared with in DJ-1 / animals (Fig. 5A). Of were markedly reduced in DJ-1 / mice after TAB (Fig. 5B), note, mRNA levels of angiogenic factors such as VEGF and which is in agreement with our previous results demonstrating that angiopoietin 1 (Ang-1) were also signiﬁcantly lower in TAB- DJ-1 is necessary for induction of Hif-1α in cells (11). TAB − − treated DJ-1 / mice (Fig. 5B). Similar inhibitory effects on neo- induces the expression of p53, which in turn inhibits Hif-1α and − − vascularization were seen in MD-Ang II-infused DJ-1 / mice cardiac angiogenesis (31), thereby providing an explanation for the

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1303444110 Billia et al. Downloaded by guest on September 30, 2021 − − there is an exaggerated Erk1/2 activation in the DJ-1 / mice, it is not sufficient to provide protection in ROS-mediated cell death. Several studies have demonstrated that even a small degree of CM apoptosis is causally linked to LV failure (38, 39). In vitro, − − DJ-1 / CMs displayed signs of substantial mitochondrial dysfunction on ROS induction, contributing to increased apoptosis. − − In vivo, biomechanical stress in DJ-1 / mice also produced decreased mitochondrial biogenesis, increased mitochondrial DNA depletion, and compromised oxidative phosphorylation with a marked increase in CM cell death. These findings are in − − agreement with our view that the DJ-1 / mice lack the ability to quench ROS effectively. Although we have defined a primary function for DJ-1 in the defense against ROS (i.e., detoxification), it would also be of interest to define a potential role for DJ-1 in DNA repair, a sequela of oxidative damage with significant consequences, such as was described with B lymphoma Mo-MLV insertion region 1 homolog (Bmi1) (40) and forkhead box O3 (Foxo3a) (41). Angiogenesis occurs in both physiological and pathological cardiac hypertrophy (42). The regulation of angiogenesis in response to hypoxia is mediated primarily through Hif-1α (30), Fig. 5. DJ-1 is a stress responsive regulator of angiogenesis in the myo- which is a key transcription factor for the induction of angio- cardium. (A) Quantification of the number of microvessels/CMs depicted genic factors VEGF and Ang-1 during pressure overload (31). from representative immunofluorescent micrographs of wheat germ aggluti- ROS-triggered intracellular stress during TAB induces p53, nin (WGA) and von Willebrand factor (vWF) of myocardial cross-sections. (B whichinturninhibitsHif-1α-dependent gene transcription. and C) mRNA levels of VEGF and Ang-1 (B) or p53 and Hif-1α (C) by qRT-PCR. p53-mediated elimination of Hif-1α function impairs coronary *P < 0.05 versus sham; #P < 0.05. Sample size for each group as indicated. angiogenesis, myocardial oxygen supply, and ATP depletion (31). Our data confirm that the expression of Hif-1α is also strongly dependent on DJ-1. A decreased induction of VEGF MEDICAL SCIENCES − − significantly higher p53 mRNA levels in DJ-1 / hearts after TAB and Ang-1, indicative of attenuated Hif-1α activity in DJ-1- + + compared with in DJ-1 / hearts (Fig. 5C). In summary, our study deficient hearts, supports this notion. DJ-1 deficiency sensitizes demonstrates that DJ-1 regulates several key aspects of cardiac the adult heart to oxidative stress in vivo, with resultant re- remodeling, including the protection and activation of disparate duction in neovascularization. The disruption of coordinated cell types involved in the production of extracellular matrix and cardiac hypertrophy and angiogenesis contribute to the transi- angiogenesis, all of which contribute to the development and tion to HF (43), delineating a critical function for DJ-1 in the pathogenesis of HF. angiogenic response to biomechanical stress. Conceptually, these data support a model in which DJ-1 participates in a sig- Discussion naling cascade that regulates Hif-1α and other angiogenic fac- Although DJ-1-deficient mice do not develop a cardiac pheno- tors to elicit neovascularization in vivo. In this regard, it has type at baseline, we present several important observations from been demonstrated that DJ-1 interacts with p53, leading to our genetic deletion studies supporting the function of DJ-1 in inhibition of p53-dependent target gene activation (12, 44). − − the heart in the cellular defense against ROS. DJ-1 / mice This finding could mechanistically explain how DJ-1 functions exhibited exaggerated pathological cardiac hypertrophy, elevated as a stress-responsive regulator of myocardial angiogenesis. ROS production, marked elevation in the degree of CM apo- DJ-1 deficiency affected the cardiac phenotype only in response ptosis, and a susceptibility to overt HF. The functionality of cy- to substantial hemodynamic insult, suggesting a specificand toplasmic or mitochondrial antioxidant systems was substantially unique role for DJ-1 in the complex interplay between hyper- − − compromised in DJ-1 / mice subjected to TAB or MD-Ang II trophic signaling and the response to increased oxidative stress. infusion accompanied with increased DNA damage. Protein As reduction of DJ-1 levels in CM accelerated cardiac hyper- levels of DJ-1 in end-stage human HF were also diminished, trophy and the development of HF in mice, we provide unique supporting a reciprocal relationship between DJ-1 activity and experimental evidence for DJ-1 functioning in a cardioprotective disease state. This could be a result of DJ-1 functioning in manner in the cellular defense against oxidative stress. a regulatory pathway that promotes mitochondrial biogenesis via PGC-1alpha (34, 35) or, alternatively, through the inhibition of Materials and Methods ubiquitination and proteasomal degradation of nuclear related The generation of DJ-1 gene-deficient mice was previously described (25). + + + − − − factor 2 (Nrf2), a key regulator of antioxidant transcriptional re- Age-matched syngenic male DJ-1 / , / , / mice (14–16 wk, 22–26 g) sponses (36). Thus, DJ-1-dependent loss of Nrf2-mediated acti- were used in this study. All experiments used littermate controls of vation of antioxidants could well account for the DJ-1 cardiac matched age and sex and in accordance with approved institutional an- phenotype, which is only evident after stress. Combined, all these imal care guidelines of the University Health Network (AUP 1772; Cana- findings support our view that DJ-1 is critical and functions in dian Council in Animal Care). Functional analysis was done at various a nonredundant manner by regulating multiple pathways involved times after birth and included 2D echocardiographic, biochemical, his- in balancing the production and elimination of ROS to protect tologic, and immunofluorescent assessment. Details are provided in the heart against oxidative stress. SI Materials and Methods. Other investigators have shown that ROS activates ERK1/2 through Src- and Ras-dependent pathways in isolated neonatal ACKNOWLEDGMENTS. We thank M. Badiwala (University Health Network, CMs, providing protection from apoptosis (37). In our study, we Toronto, ON, Canada) for assistance in the procurement of the human LV observed elevated levels of phospho-Erk1/2 in TAB-treated samples. This work was supported by grants awarded by the Canadian In- −/− stitute of Health Research (CIHR) and the Ontario Ministry of Health and DJ-1 mice in vivo. However, despite increased Erk activity, Long Term Care (to T.W.M.). Division of Cardiology, University Health Net- CM apoptosis in this experimental group was significantly higher work, provided support in completing this research project. F.B. is the +/+ than in DJ-1 animals. These results demonstrate that although recipient of the CIHR Phase 1 Clinician-Scientist Award.

Billia et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 1. Sadoshima J, Izumo S (1997) The cellular and molecular response of cardiac myocytes 23. Ide T, et al. (2001) Mitochondrial DNA damage and dysfunction associated with ox- to mechanical stress. Annu Rev Physiol 59:551–571. idative stress in failing hearts after myocardial infarction. Circ Res 88(5):529–535. 2. Braunwald E, Bristow MR (2000) Congestive heart failure: Fifty years of progress. 24. Cheng KC, Cahill DS, Kasai H, Nishimura S, Loeb LA (1992) 8-Hydroxyguanine, an Circulation 102(20, Suppl 4)IV14–IV23. abundant form of oxidative DNA damage, causes G——T and A——C substitutions. J 3. Cohn JN (1996) The management of chronic heart failure. N Engl J Med 335(7): Biol Chem 267(1):166–172. 490–498. 25. Li Y, et al. (1995) Dilated cardiomyopathy and neonatal lethality in mutant mice 4. Jessup M, Brozena S (2003) Heart failure. N Engl J Med 348(20):2007–2018. lacking manganese superoxide dismutase. Nat Genet 11(4):376–381. 5. Bonifati V, et al. (2003) DJ-1(PARK7), a novel gene for autosomal recessive, early 26. Bueno OF, et al. (2000) The MEK1-ERK1/2 signaling pathway promotes compensated – onset parkinsonism. Neurol Sci 24(3):159–160. cardiac hypertrophy in transgenic mice. EMBO J 19(23):6341 6350. 6. Bonifati V, et al. (2003) Mutations in the DJ-1 gene associated with autosomal 27. Ueyama T, et al. (2000) Requirement of activation of the extracellular signal-regu- – recessive early-onset parkinsonism. Science 299(5604):256–259. lated kinase cascade in myocardial cell hypertrophy. J Mol Cell Cardiol 32(6):947 960. 7. Yang Y, et al. (2005) Inactivation of Drosophila DJ-1 leads to impairments of oxidative 28. Nishida M, et al. (2000) G alpha(i) and G alpha(o) are target proteins of reactive ox- – stress response and phosphatidylinositol 3-kinase/Akt signaling. Proc Natl Acad Sci ygen species. Nature 408(6811):492 495. USA 102(38):13670–13675. 29. Lorenz K, Schmitt JP, Schmitteckert EM, Lohse MJ (2009) A new type of ERK1/2 au- – 8. Meulener M, et al. (2005) Drosophila DJ-1 mutants are selectively sensitive to envi- tophosphorylation causes cardiac hypertrophy. Nat Med 15(1):75 83. 30. Dorn GW, 2nd, Force T (2005) Protein kinase cascades in the regulation of cardiac ronmental toxins associated with Parkinson’s disease. Curr Biol 15(17):1572–1577. – 9. Martinat C, et al. (2004) Sensitivity to oxidative stress in DJ-1-deficient dopamine hypertrophy. J Clin Invest 115(3):527 537. 31. Sano M, et al. (2007) p53-induced inhibition of Hif-1 causes cardiac dysfunction during neurons: An ES- derived cell model of primary Parkinsonism. PLoS Biol 2(11):e327. pressure overload. Nature 446(7134):444–448. 10. Kim RH, et al. (2005) Hypersensitivity of DJ-1-deficient mice to 1-methyl-4-phenyl- 32. Pugh CW, Ratcliffe PJ (2003) Regulation of angiogenesis by hypoxia: Role of the HIF 1,2,3,6-tetrahydropyrindine (MPTP) and oxidative stress. Proc Natl Acad Sci USA system. Nat Med 9(6):677–684. 102(14):5215–5220. 33. Carmeliet P, et al. (1999) Impaired myocardial angiogenesis and ischemic cardiomy- 11. Vasseur S, et al. (2009) DJ-1/PARK7 is an important mediator of hypoxia-induced opathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and cellular responses. Proc Natl Acad Sci USA 106(4):1111–1116. VEGF188. Nat Med 5(5):495–502. 12. Fan J, et al. (2008) DJ-1 decreases Bax expression through repressing p53 transcrip- 34. Cookson MR (2012) Parkinsonism due to mutations in PINK1, parkin, and DJ-1 and oxi- tional activity. J Biol Chem 283(7):4022–4030. dative stress and mitochondrial pathways. Cold Spring Harb Perspect Med 2(9):a009415. 13. van der Brug MP, et al. (2008) RNA binding activity of the recessive parkinsonism 35. Wu Z, et al. (1999) Mechanisms controlling mitochondrial biogenesis and respiration protein DJ-1 supports involvement in multiple cellular pathways. Proc Natl Acad Sci through the thermogenic coactivator PGC-1. Cell 98(1):115–124. USA 105(29):10244–10249. 36. Clements CM, McNally RS, Conti BJ, Mak TW, Ting JP (2006) DJ-1, a cancer- and Par- 14. McMurray J, Chopra M, Abdullah I, Smith WE, Dargie HJ (1993) Evidence of oxidative kinson’s disease-associated protein, stabilizes the antioxidant transcriptional master – stress in chronic heart failure in humans. Eur Heart J 14(11):1493 1498. regulator Nrf2. Proc Natl Acad Sci USA 103(41):15091–15096. 15. Borchi E, et al. (2010) Enhanced ROS production by NADPH oxidase is correlated to 37. Aikawa R, et al. (1997) Oxidative stress activates extracellular signal-regulated kinases changes in antioxidant enzyme activity in human heart failure. Biochim Biophys Acta through Src and Ras in cultured cardiac myocytes of neonatal rats. J Clin Invest 100(7): – 1802(3):331 338. 1813–1821. 16. Yücel D, et al. (1998) Increased oxidative stress in dilated cardiomyopathic heart 38. Narula J, et al. (1996) Apoptosis in myocytes in end-stage heart failure. N Engl J Med – failure. Clin Chem 44(1):148 154. 335(16):1182–1189. 17. Khaper N, Kaur K, Li T, Farahmand F, Singal PK (2003) Antioxidant enzyme gene 39. Olivetti G, et al. (1997) Apoptosis in the failing human heart. N Engl J Med 336(16): expression in congestive heart failure following myocardial infarction. Mol Cell 1131–1141. Biochem 251;1(1-2):9-15. 40. Liu J, et al. (2009) Bmi1 regulates mitochondrial function and the DNA damage re- 18. James AM, Cochemé HM, Murphy MP (2005) Mitochondria-targeted redox probes as sponse pathway. Nature 459(7245):387–392. tools in the study of oxidative damage and ageing. Mech Ageing Dev 126(9):982–986. 41. Kops GJ, et al. (2002) Forkhead transcription factor FOXO3a protects quiescent cells 19. Gardner PR (1997) Superoxide-driven aconitase FE-S center cycling. Biosci Rep 17(1): from oxidative stress. Nature 419(6904):316–321. 33–42. 42. Giordano FJ, et al. (2001) A cardiac myocyte vascular endothelial growth factor 20. Gardner PR, Fridovich I (1992) Inactivation-reactivation of aconitase in Escherichia paracrine pathway is required to maintain cardiac function. Proc Natl Acad Sci USA coli. A sensitive measure of superoxide radical. J Biol Chem 267(13):8757–8763. 98(10):5780–5785. 21. Singal PK, Gupta M, Randhawa AK (1991) Reduced myocardial injury due to exoge- 43. Shiojima I, et al. (2005) Disruption of coordinated cardiac hypertrophy and nous oxidants in pressure induced heart hypertrophy. Basic Res Cardiol 86(3):273–282. angiogenesis contributes to the transition to heart failure. JClinInvest115(8): 22. Maslov MY, et al. (2007) Altered high-energy phosphate metabolism predicts con- 2108–2118. tractile dysfunction and subsequent ventricular remodeling in pressure-overload hy- 44. Kato I, et al. (2013) Oxidized DJ-1 inhibits p53 by sequestering p53 from promoters in pertrophy mice. Am J Physiol Heart Circ Physiol 292(1):H387–H391. a DNA-binding affinity-dependent manner. Mol Cell Biol 33(2):340–359.

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