TYPE Original Research PUBLISHED 23 September 2022 DOI 10.3389/fphar.2022.1002755 DJ-1 administration exerts OPEN ACCESS cardioprotection in a mouse EDITED BY Mahmood Mozaffari, Augusta University, United States model of acute myocardial REVIEWED BY Nehal Mohsen Elsherbiny, infarction Mansoura University, Egypt Xichun Pan, Army Medical University, China Alex Gallinat  1,2‡, Guiomar Mendieta  1,3‡, *CORRESPONDENCE Gemma Vilahur  1,4, Teresa Padró  1,4 and Lina Badimon, Lina Badimon  1,4,5* lbadimon@santpau.cat 1 ‡ Cardiovascular Program-ICCC, IR-Hospital Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain, These authors have contributed equally 2 Universitat Autònoma de Barcelona (UAB), Barcelona, Spain, 3Centro Nacional de Investigaciones to this work and share first authorship Cardiovasculares Carlos III (CNIC), Madrid, Spain, 4CIBERCV-Instituto de Salud Carlos III, Madrid, Spain, 5 SPECIALTY SECTION Cardiovascular Research Chair, UAB, Barcelona, Spain This article was submitted to Cardiovascular and Smooth Muscle Pharmacology, a section of the journal Frontiers in Pharmacology Cardiovascular diseases, and particularly acute myocardial infarction (MI), are RECEIVED 25 July 2022 the most common causes of death worldwide. Infarct size is the major predictor ACCEPTED 09 September 2022 of clinical outcomes in MI. The Parkinson’s disease associated protein, DJ-1 PUBLISHED 23 September 2022 (also known as PARK7), is a multifunctional protein with chaperone, redox CITATION sensing and mitochondrial homeostasis activities. Previously, we provided the Gallinat A, Mendieta G, Vilahur G, Padró T and Badimon L (2022), DJ-1 evidence for a central role of endogenous DJ-1 in the cardioprotection of post- administration exerts cardioprotection conditioning. In the present study, we tested the hypothesis that systemic in a mouse model of acute myocardial infarction. administration of recombinant DJ-1 exerts cardioprotective effects in a mouse Front. Pharmacol. 13:1002755. model of MI and also explored the associated transcriptional response. We doi: 10.3389/fphar.2022.1002755 report a significant treatment-induced reduction in infarct size, leukocyte COPYRIGHT infiltration, apoptosis and oxidative stress. Effects potentially mediated by © 2022 Gallinat, Mendieta, Vilahur, Padró and Badimon. This is an open- G-protein-coupled receptor signaling and modulation of the immune access article distributed under the response. Collectively, our results indicate a protective role for the terms of the Creative Commons exogenously administrated DJ-1 upon MI, and provide the first line of Attribution License (CC BY). The use, distribution or reproduction in other evidence for an extracellular activity of DJ-1 regulating cardiac injury in vivo. forums is permitted, provided the original author(s) and the copyright KEYWORDS owner(s) are credited and that the original publication in this journal is cardioprotection, DJ-1, PARK7, ischemia, reperfusion, myocardial infarction, ischemia/ cited, in accordance with accepted reperfusion injury academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Introduction Cardiovascular diseases are the leading cause of mortality worldwide. Among them, ischemic heart disease is the most common pathology (Benjamin et al., 2017). Myocardial infarction (MI) is defined as ischemia of the myocardial tissue, commonly caused by the occlusion of the coronary artery. Consequently, necrosis develops within the myocardium at risk (Reimer et al., 1979). Therefore, both the duration and the severity of the ischemic insult are major determinants of the final infarct size. Due to the limited regenerative capacity of the adult human heart, there is little replacement of lost functional tissue following MI. Rather, nearby fibroblasts are Frontiers in Pharmacology 01 frontiersin.org Gallinat et al. 10.3389/fphar.2022.1002755 activated to replace the damaged myocardial tissue with fibrous under ischemia and I/R, and evidenced its effects on tissue (Frangogiannis, 2006; Aix et al., 2018). Since the scar endothelial cell function at reperfusion, thereby suggesting lacks contractile function, it decreases cardiac output, a role in regulating cardiac injury (Gallinat and Badimon, eventually leading to heart failure and free wall rupture 2022). In this study, we tested the hypothesis that systemic (Kutty et al., 2013), Therefore, infarct size is the major administration of recombinant protein DJ-1 exerts a predictor of clinical outcomes following MI (Sobel et al., cardioprotective effect in a mouse model of acute MI. 1972; Kelle et al., 2009). Considering the time-dependent transition from ischemia to necrosis of the myocardium at risk, early reperfusion Materials and methods remains the definitive treatment for the ischemic heart. However, the rapid transition from ischemia to normoxia Murine model of myocardial infarction during reperfusion carries the potential to exacerbate damage in a process known as ischemia/reperfusion (I/R) The present study was performed in male CH3 mice of injury (Yellon and Hausenloy, 2007). This is in part related 8–10 weeks’ old weighing 25–30 g (n = 26; Jackson Laboratory, to the generation of reactive oxygen species (ROS) (Zweier, Bar Harbor, ME, United States). Mice were randomly given an 1988) affecting, among others, the integrity of the intraperitoneal injection of 50 μg of DJ-1 (n = 10; full length sarcoplasmic reticulum which leads to calcium overload, recombinant human DJ-1 > 95% purity, MBS143125, hyper-contracture, and the opening of the mitochondrial MyBioSource, San Diego, CA, United States); or equal PBS permeability transition pore, eventually causing cell death. volume for vehicle/controls (n = 16), 60 min prior to the Also, the changes in intracellular pH and the triggered induction of MI by 45 min ligation of the left anterior immune response compromise the viability of the descending coronary artery (LAD), as previously described myocardium upon reperfusion (Lemasters et al., 1996; Liu (Cubedo et al., 2016; Mendieta et al., 2019). Briefly, animals et al., 2016). The aforementioned detrimental effects of were anesthetized with a mixture of O2/isoflurane, intubated reperfusion occur within minutes of restoration of coronary and mechanically ventilated (rate 90 breath/min, tidal volume flow and account for a significant part of the final infarct size 0.1 ml; TOPO dual mode ventilator, Kent Scientific (Yellon and Hausenloy, 2007; Garcia-Dorado et al., 2009). A Corporation; Torrington, CT, United States). Core large number of pharmacological agents have shown to reduce temperature was continuously monitored throughout the infarct size in pre-clinical studies, but they have failed to surgery and maintained within 37–38°C using a heat pad demonstrate significant clinical benefits (Hausenloy et al., and heat lamp. An anterior thoracotomy was performed; 2013; Heusch, 2017). Thus, cardioprotection is currently an the heart was exposed and the LAD coronary artery was unmet clinical need. occluded with an intramural stitch (7–0 silk suture) for The early-onset Parkinson’s disease associated protein DJ- 45 min. The success of complete coronary ligation was 1 (also known as PARK7) is a multifunctional protein with verified by electrocardiographic visualization of the ST- cardioprotective effects against I/R injury (Dongworth et al., elevation-MI pattern that was continuously monitored and 2014; Shimizu et al., 2016; Shimizu et al., 2020) and oxidative the visualization of a pale and hypokinetic ventricular region stress (Billia et al., 2013). In a previous study, where the distal to the site of occlusion. After 45 min of ischemia, mitochondrial proteomic response to I/R and post- animals were sacrificed (Ischemia group; n = 11 vehicle and conditioning was analysed in a pre-clinical model of MI, we n = 5 DJ-1) or reperfused for 2 h (I/R group; n = 5 vehicle provided evidences for a central role of DJ-1 in the and n = 5 DJ-1). A sham-operated group (n = 4), which cardioprotection conferred by post-conditioning (Gallinat underwent the same surgical procedure without ligation of et al., 2022). The reported functions of DJ-1 include the LAD was included. Afterwards, hearts were carefully chaperone (Shendelman et al., 2004), protease (Chen et al., excised and processed for the following procedures. 2010), and deglycase (Richarme et al., 2015; Richarme and The dose of DJ-1 was chosen based on previous studies Dairou, 2017) activities, regulation of transcription (Arac et al., 2011; Cubedo et al., 2016). All analyses were (Takahashi-Niki et al., 2017), redox sensing (Wilson, 2011) performed blindly with regard to the treatment received by the and modulation of mitochondrial homeostasis (Hayashi et al., animals. 2009; Heo et al., 2012). Also, some reports suggest extracellular activities for DJ-1. It is secreted under several pathological conditions including breast cancer (Le Naour Morphometric assessment of infarct size et al., 2001), Parkinson’s disease (Maita et al., 2008; Tsuboi et al., 2008) or stroke (Allard et al., 2005), and a protective role Hearts (n = 26) were immersed in a fixative solution (4% against ischemia (Kaneko et al., 2014) and I/R (Han et al., paraformaldehyde), embedded in optimal cutting temperature 2017). Likewise, we have reported endothelial DJ-1 secretion compound (OCT) and sequentially cross-sectioned from the Frontiers in Pharmacology 02 frontiersin.org Gallinat et al. 10.3389/fphar.2022.1002755 apex to the base (10 µm thick sections 200 µm distanced). (5 random fields per heart). All images were acquired in the same Sections were then stained with haematoxylin-eosin and conditions. infarct size analysis was morphometrically determined with the image analysis software ImageJ (Schindelin et al., 2012). Infarct size was calculated as the sum of myocardial infarct RNA extraction areas between total left ventricle wall surface, as previously reported (Takagawa et al., 2007; Cubedo et al., 2016). Frozen tissue was grinded using mortar and pestle, and Three measurements per each histological section were total RNA was extracted using a combined organic extraction determined. and silica-membrane columns method (RNesasy Mini Kit, Qiagen; Valencia, CA, United States). RNA was then quantified by spectrophotometry using the Nanodrop ND- Immunohistochemical analysis 1000 (Thermo Fisher Scientific). RNA quality was assessed with the Agilent 2100 Bioanalyzer technology (Agilent OCT-embedded frozen specimens were cut into 5 μm thick Technologies; Santa Clara, CA, United States) and the serial sections, placed on poly-L-lysine coated slides, and Agilent RNA 6000 Nano Kit (Agilent Technologies; Santa processed for immunohistochemistry. The sections were Clara, CA, United States). Only RNA samples with an RNA incubated for 2 h with rabbit polyclonal antibodies against DJ- Integrity Number (RIN) > 7 were chosen for microarray 1 (AP01249PU-N; 1:50 dilution; Acris Antibodies GmbH, experiments. Herford, Germany), neutrophil elastase (ab68672; 1: 100 dilution; Abcam, Cambridge, United Kingdom), inducible nitrogen oxide synthase (iNOS; NB300-605; 1:20 dilution; Novus Transcriptomic and in silico analysis Biologicals; Littelton, CO, United States) and cleaved caspase-3 (Asp175; 9,661; 1:200 dilution; Cell Signaling; Danvers; MA, Myocardial gene expression changes were analysed with a United States), mouse monoclonal antibody against monocyte/ GeneChip Mouse 1.0ST array approach (Affymetrix, Santa macrophages (ab33451; 1:50 dilution; Abcam, Cambridge, Clara, CA, United States). Using the Ambion WT Expression United Kingdom), or goat polyclonal antibody against 8- Kit (Ambion, Life Tecnologies, Carlsbad, CA, United States) hydroxy-deoxy-Guanine (8-OHdG; MBS536217; 1: 100 ng of total RNA (mixed with poly-A controls; Affymetrix, 300 dilution; MyBioSource, San Diego, CA, United States). Santa Clara, CA, United States) were retro-transcribed to Thereafter, sections were rinsed and incubated with the double strand DNA, in two steps, in order to obtain cRNA. appropriate biotinylated antibodies (1:200 dilution; Vector Single strand DNA was generated from 10 ug of cRNA. Then Laboratories, Burlingame, CA, United States). Endogenous 5.5 µg of single strand DNA were fragmented and labelled peroxidase activity, as well as unspecific unions were blocked with biotin using the WT Terminal Labeling Kit (Affymetrix, before incubation with primary antibodies. Finally, sections were Santa Clara, CA, United States). Hybridization controls from incubated with avidin-biotin complex (Vector Laboratories, the Hybridization, Wash and Stain Kit (Affymetrix, Santa Burlingame, CA, United States), and 3,3′-diaminobenzidine Clara, CA, United States) were added to the sample. was used as the substrate for peroxidase. Images were Thereafter, every sample was hybridized to a GeneChip acquired with a Nikon Eclipse 80i microscope (Nikon, Tokyo, Mouse Gene 1.0 ST array for 16 h at 45°C and 60 rpm, Japan), digitized by a Retiga 1300i camera (Teledyne according to manufacturer’s instructions. Hybridization, Photometrics, Tucson, AZ, United States), and imported to washing, staining, and scanning of microarrays were ImageJ (Schindelin et al., 2012). Positive signal was then performed according to Affymetrix instructions using the quantified and expressed as the percentage of total area. For Affymetrix GeneChip 3000 7G System (645 Hybridization leukocyte infiltration and cleaved caspase-3 analyses, the number Oven, 450 Fluidic Station and GeneChip 3,000 7G Scanner). of positively labelled cells per field were counted. Six random Raw data were pre-processed (background correction, fields per sample were analysed. normalization and median polish summarization of the probes) with Robust Multiarray Average (RMA) method. Microarray quality control and statistical analyses were Apoptosis assessment performed using Expression Console (Affymetrix, Santa Clara, CA, United States) and the Partek Genomics Suite Apoptosis was histologically analysed by the dUTP neck-end software (Partek Inc., St Louis, MI, United States). p-values labeling (TUNEL) assay according to manufacturer’s were adjusted for multiple testing with Benjamini and specifications (Chemicon Inc.; Pittsburgh, PA, United States). Hochberg method, and false discovery rates (FDR) were The apoptosis rate was measured in a section below the occlusion calculated. Bioinformatic analysis was performed with and expressed as the percentage of TUNEL-positive cells per field WebGestAlt (ZhangLab; http://www.webgestalt.org/) Frontiers in Pharmacology 03 frontiersin.org Gallinat et al. 10.3389/fphar.2022.1002755 FIGURE 1 Effects of systemic DJ-1 administration on infarct size in a mouse model of MI. (A). Representative images of infarcted heart sections. Infarct area is outlined in black. (B). Infarct size quantification after ischemia and ischemia/reperfusion, in the presence and the absence of a systemic administration of DJ-1. Infarct measures are expressed as percentage of left ventricle area. (C). Representative immunohistochemical acquisitions of DJ-1 in the myocardium after ischemia and ischemia/reperfusion, in the presence and the absence of a systemic administration of DJ-1. A sham-operated group was included as baseline. (D). DJ-1 myocardium content quantification across groups. Scale bar: 100 µm; *p < 0.05, Kruskal- Wallis; †p < 0.05 vs. ischemia without DJ-1, ‡p < 0.05 vs. ischemia + DJ-1, #p < 0.05 vs. I/R without DJ-1, Dunn’s test. LV, Left Ventricle; Isch., Ischemia; I/R, Ischemia/Reperfusion. following a Gene Set Enrichment Analysis (GSEA) (Xin et al., previously described (Luquero et al., 2022). On-demand TaqMan 2019) approach and defining gene ranks by log-fold change RT-PCR assays for indicated genes were employed (Thermo Fisher (logFC). Gene sets available from Wikipathways (Martens Scientific Inc., Waltham, MA, United States). et al., 2021), Molecular Signature Data Base (MSigDB) (Liberzon et al., 2015), and Panther (Thomas et al., 2003) pathway collections were considered for the analysis. Raw Statistical analysis expression data have been deposited in the NCBI’s Gene Expression Omnibus (GEO) (Edgar et al., 2002) and are Because data were not normally distributed as assessed by the accessible under the GEO Series accession number GSE66307. Shapiro-Wilk test, a non-parametric statistical analysis was employed. Non-parametric Kruskal-Wallis followed by Dunn’s test for multiple comparisons and Mann-Whitney test were used qPCR to assess differences between groups. Results are reported as median with inter-quartile range unless otherwise stated. Correlations were Validation of myocardial gene expression changes was performed determined with Spearman’s rank correlation coefficient. All in tissue samples from the I/R group. Gprc5a, Inos and Casp3 mRNA statistical analyses were performed with the statistical software levels were analysed by real-time polymerase chain reaction, as package Statview 5.0.1 (SAS Institute Inc.; Cary, NC, United States). Frontiers in Pharmacology 04 frontiersin.org Gallinat et al. 10.3389/fphar.2022.1002755 FIGURE 2 Differential gene expression profile after ischemia/reperfusion, in the presence and the absence of a systemic administration of DJ-1. (A). Heat- map representation of differentially expressed genes. (B). Top ten up- and down-regulated genes after ischemia/reperfusion in the presence of DJ-1 systemic administration. (C). Gprc5a transcript analysis by qPCR. (D). Gene-set enrichment analysis result. Gene-set collections from Wikipathways, Molecular Signature, and Panther databases were considered. *p < 0.05, Mann-Whitney. I/R, Ischemia/Reperfusion; logFC, log-Fold Change. Frontiers in Pharmacology 05 frontiersin.org Gallinat et al. 10.3389/fphar.2022.1002755 Results Systemic DJ-1 administration reduces myocardial leukocyte infiltration Systemic administration of recombinant following I/R DJ-1 protects the heart against I/R injury In order to functionally validate the predicted effects of DJ-1 In order to test whether the administration of DJ-1 exerts administration in modulating the immune response to I/R, we cardioprotection after MI, a group of mice were quantified the leukocyte infiltration by immunohistochemistry intraperitoneal treated with human recombinant DJ-1 (Figure 3). As a result, animals treated with DJ-1 exhibited (50 µg) 1 h before LAD coronary artery ligation, in a significantly lower infiltration of neutrophils and double-blind experimental design, and heart samples were macrophages, both after ischemia and I/R. processed for histological analysis. Computer-assisted morphometric assessment of infarct size revealed an infarct size reduction of about 75% for the animals treated with DJ-1 Anti-apoptotic effects of DJ-1 (Figure 1A,B). Interestingly, also a reduction in infarct size of administration about 20% was detected in the ischemia group, for the animals treated with DJ-1 (Figure 1A). Importantly, a 5.4-fold mean An effect of a systemic DJ-1 administration upon the increase in the DJ-1 signal was found in the myocardium of the induction of apoptosis following myocardial I/R was further treated animals (Figure 1C,D), meaning the administrated DJ- investigated. Notably, animals treated with DJ-1 exhibited a 1 reached the heart. diminished expression of Casp3 gene after I/R, as assayed by qPCR (Figure 4A). Consistently, we could detect a significant reduction of the cleaved-Casp-3 myocardial content (p = 0.04), as DJ-1 administration modifies the well as the decrease of the TUNEL-positive cells (p = 0.009) myocardial transcriptomic response to I/R following I/R for the DJ-1 treated group (Figure 4B,C). In order to outline possible mechanisms at play, we analysed the transcriptomic response of the myocardium Anti-oxidant effects of DJ-1 upon MI in the presence and the absence of a systemic administration administration of DJ-1. As depicted in the heat-map, the administration of DJ-1 before I/R induced a multi-genic Because ROS-induced damage has a central role in I/R response in the myocardium different from that of the injury, we evaluated whether a systemic administration of vehicle group (Figure 2A). Figure 2B shows the top 10 up- DJ-1 has an effect upon the oxidative damage to the and down-regulated genes in the DJ-1-treated mice compared myocardium following I/R. As a result, the administration of to the vehicle group. DJ-1 significantly reduced the expression level of Inos gene, as Genome-wide RNA expression data was then analysed well as the staining of the oxidative damage marker 8-hydroxy- following a GSEA (Subramanian et al., 2005) approach deoxy-Guanine (8-OHdG) (Figure 5). Additionally, we found a considering all gene set collections available at negative correlation between the staining signals of DJ-1 and Wikipathway (Martens et al., 2021), Molecular Signature iNOS (Figure 5E), and between DJ-1 and 8-OHdG (Figure 5F). (Liberzon et al., 2015), and Panther (Thomas et al., 2003) A positive correlation between iNOS and 8-OHdG staining was databases. As a result, different pathways were detected to be also evidenced (Figure 5G). significantly enriched (FDR < 0.05) within each analysis (Figure 2D). From them all, G protein-coupled receptors (GPCR) mediated signaling and immune response-related Discussion gene sets were the most consistently detected. Interestingly, an apoptosis-related gene set was also significantly enriched In the present study, we explored the effects of a systemic (FDR = 0.017). administration of recombinant DJ-1 upon ischemia and I/R As a surrogate of the GPCR-mediated signaling-related injury in a mouse model of acute MI. We report a significant gene set, the expression of Gprc5a gene was validated by qPCR reduction in infarct size, leukocyte infiltration, apoptosis, and (Figure 2C). Effects upon the immune response and apoptosis oxidative stress associated with the treatment. Also, we analysed related gene sets were functionally validated as follows. the transcriptional response of the myocardium. Frontiers in Pharmacology 06 frontiersin.org Gallinat et al. 10.3389/fphar.2022.1002755 FIGURE 3 Effects of DJ-1 systemic administration on myocardial leukocyte infiltration after ischemia and ischemia/reperfusion. (A). Representative immunohistochemical acquisitions of myocardial neutrophils (upper panel) and macrophages (lower panel) after ischemia and ischemia/reperfusion in the presence and the absence of a systemic administration of DJ-1. A sham-operated group was included as baseline. (B) and (C) quantification of infiltrated leukocytes. Scale bar: 100 µm; *p < 0.05, Kruskal-Wallis; §p < 0.05 vs. sham, †p < 0.05 vs. ischemia without DJ-1, ‡p < 0.05 vs. ischemia + DJ-1, #p < 0.05 vs. I/R without DJ-1, Dunn’s test. Isch., Ischemia; I/R, Ischemia/Reperfusion. A role for the endogenous DJ-1 in cardioprotection has (Takahashi-Niki et al., 2017), redox sensing (Wilson, 2011) already been proven by gain- and loss-of-function studies. and the modulation of mitochondrial homeostasis (Hayashi Hence, while DJ-1 deficiency do not affect cardiac et al., 2009; Heo et al., 2012). However, the exact molecular performance at baseline, animals lacking DJ-1 exhibited function of DJ-1, as well as its dynamics and regulation remain larger infarcts, increased mitochondrial fission, and worse elusive. Interestingly, some reports suggest an extracellular left ventricular function upon LAD ligation, compared to activity, as it is secreted under some pathologic conditions wild-type littermates (Dongworth et al., 2014; Kaneko et al., such as breast cancer (Le Naour et al., 2001), Parkinson’s 2014; Shimizu et al., 2016; Dong et al., 2018; Xin et al., 2019). disease (Maita et al., 2008; Tsuboi et al., 2008) and stroke Similar results were found in experimental models of stroke, (Allard et al., 2005). Also, a pro-survival role for the where the lack of DJ-1 resulted in larger infarcts in vivo, and extracellular form, has been reported in ischemia (Kaneko enhanced cell death in vitro (Aleyasin et al., 2007). In a et al., 2014) and I/R (Han et al., 2017). Likewise, we previously previous study, we have described the up-regulation of DJ-1 described the endothelial secretion of DJ-1 during ischemia following post-conditioning in a pre-clinical model of MI and I/R, and evidenced a role in regulating endothelial cell (Gallinat et al., 2022). In pressure overload animal models, function (Gallinat and Badimon, 2022). The data here mice lacking DJ-1 exhibited a higher oxidative stress level, reported support a cardioprotective role for DJ-1 in MI, exaggerated cardiac hypertrophy, and were more prone to presumably for the extracellular form. develop heart failure (Billia et al., 2013). Collectively ROS are a group of small molecules derived from the reinforcing a cardioprotective role for DJ-1. reduction of the oxygen molecule, that are continuously Over the past decades, DJ-1 have been extensively studied, produced in small amounts as by-products of cell and many functions have been reported. These include respiration and metabolism, and eliminated by the chaperone (Shendelman et al., 2004), protease (Chen et al., endogenous antioxidant systems. Within the heart, ROS 2010), and deglycase (Richarme et al., 2015; Richarme and play a role as second messengers for the excitation- Dairou, 2017) activities, regulation of transcription contraction coupling, cell differentiation, and regulation of Frontiers in Pharmacology 07 frontiersin.org Gallinat et al. 10.3389/fphar.2022.1002755 previously described functions of DJ-1 enriched in the transcriptomic analysis, which suggests a distinct role for the extracellular and intracellular forms of DJ-1. GPCR- mediated signaling and immune response were rather the most consistently detected enrichments. A body of evidences has linked GPCR signaling to cardioprotection. Endogenous GPCR ligands released during ischemia and reperfusion (i.e., autacoids) have been proposed as triggers of the cardioprotection conferred by ischemic pre- and post-conditioning (Heusch, 2015). This is the case for bradykinin (Goto et al., 1995; Oldenburg et al., 2004), opioids (Schultz et al., 1995; Zatta et al., 2008), acetylcholine (Yao and Gross, 1993), catecholamines (Tsuchida et al., 1994), angiotensin II (Liu et al., 1995), and endothelin-1 (Wang et al., 1996). Also, a number of GPCR agonists have proven to protect the heart against I/R injury. GPCRs couple to a membrane-anchored heterotrimeric Gαβγ protein. Upon activation, GPCRs undergo a conformational change that allows the exchange of a GDP molecule, bound to the Gα subunit, for a GTP molecule, activating the Gα subunit. Thereafter, Gα dissociates from FIGURE 4 the remaining Gβγ subunits and triggers downstream Effects of DJ-1 systemic administration on myocardial apoptosis after ischemia/reperfusion. (A) Casp3 transcript analysis signaling. GPCRs can be classified depending on the by qPCR. (B) Representative acquisitions of myocardial cleaved downstream effects of the activated Gα subunit into: Gαs, Casp-3 immunohistochemistry (upper panel) and TUNEL which stimulate the adenylate cyclase, Gαi/o, which inhibit assay (lower panel) after ischemia/reperfusion. (C) myocardial cleaved Casp-3 immunohistochemistry and TUNEL positive cells adenylate cyclase, Gαq/11, which activate phospholipase C, and quantification. Scale bar: 100 µm; *p < 0.05, Mann-Whitney. I/R, Gα12/13 which regulate Rho GTPase activity. Most of the Ischemia/Reperfusion. GPCRs that play a role in cardioprotection couple to Gαi/o or Gαq/11 proteins (Heusch, 2015). Furthermore, GPCR kinases, and β-arrestins, that are also involved in GPCR- mediated signaling (Gurevich and Gurevich, 2019), play a blood flow (Forman et al., 2004; Burgoyne et al., 2012). role in regulating cardiac injury upon MI (Brinks et al., However, the accumulation of ROS or the unbalance 2010; Wang et al., 2017). The systemic administration of between ROS production and the antioxidant mechanisms DJ-1 induced the up-regulation of several GPCRs following (termed, oxidative stress) is detrimental and cause several MI, some of them belonging to the odorant GPCRs family. macromolecular modifications, such as, lipid peroxidation, Although still poorly understood, olfactory receptors have protein misfolding, and DNA damage. Indeed, oxidative been reported to be expressed in the heart and to play a stress is involved in the aetiology of a number of role in regulating cardiac function and angiogenesis (Kim pathologies and cellular insults. At the onset of reperfusion, et al., 2015; Jovancevic et al., 2017). the reintroduction of O2 cause a burst of ROS within the From the onset of ischemia to reperfusion and infarct mitochondria (Jassem et al., 2002), which challenges the healing, the immune response has a central role (Vilahur and antioxidant mechanisms and impairs the mitochondrial Badimon, 2014; Ong et al., 2018). The delicate balance electron transport chain. This compromise the between pro-inflammatory and healing signals exerts a mitochondrial function, the ATP production and cell great impact on the outcome. As a matter of fact, several viability (Arduini et al., 1988). The significant decrease in cardioprotective strategies have focused on the modulation of the 8-OHdG staining signal following I/R for the treated the immune response (Grilo et al., 2017; Andreadou et al., animals, indicates that the administration of DJ-1 mitigates 2019; Zuurbier et al., 2019). Importantly, both pro- and anti- the oxidative damage to the myocardium. This observation inflammatory signals are needed for the optimal resolution of was reinforced by the significant negative correlation found MI (Yap et al., 2019; Sun et al., 2021). For the treated animals, between DJ-1 and 8-OHdG staining signals. we could detect several pathways related to the immune In order to highlight possible mechanisms at play, we response enriched in the myocardium after MI. We further performed a whole-transcriptome analysis of the report, a diminished leukocyte infiltration following I/R for myocardium. Unexpectedly, we could not detect any of the the treated animals. Amongst the immune response related Frontiers in Pharmacology 08 frontiersin.org Gallinat et al. 10.3389/fphar.2022.1002755 FIGURE 5 Systemic DJ-1 administration effects on oxidative stress after ischemia and ischemia/reperfusion. (A) Inos transcript analysis by qPCR. (B) Representative immunohistochemical acquisitions of myocardial iNOS (upper panel) and 8-OHdG (lower panel) after ischemia and ischemia/ reperfusion in the presence and the absence of a systemic administration of DJ-1. A sham-operated group was included as baseline. (C) Myocardial iNOS content quantification. (D) Myocardial 8-OHdG content quantification. (E–G) Correlation analyses between iNOS and DJ-1 (E), 8-OHdG and DJ-1 (F), and iNOS and 8-OHdG (G) staining signals. Scale bar: 100 µm; *p < 0.05, Kruskal-Wallis; §p < 0.05 vs. sham, †p < 0.05 vs. ischemia without DJ-1, ‡p < 0.05 vs. ischemia + DJ-1, #p < 0.05 vs. I/R without DJ-1, Dunn’s test; rho, Spearman rank correlation coefficient. Isch., Ischemia; I/ R, Ischemia/Reperfusion. pathways detected, the tumour necrosis factor α (TNFα) immune response. The described extracellular activities of signaling pathway was the most enriched. Interestingly, DJ-1 offer a therapeutic opportunity to limit I/R induced TNFα signaling has been shown to display cardioprotection damage, which goes beyond MI. Whilst the present study through NFκB-mediated cardiomyocyte expression of keratin- provides the first line of evidence for an extracellular DJ-1 8 and keratin-18, which preserve the integrity and function of activity in vivo, the therapeutic potential of a systemic the intercalated discs and mitochondria upon stress administration of recombinant DJ-1, requires further (Papathanasiou et al., 2015). investigation regarding possible off-target activities of DJ-1. Collectively our results illustrate a cardioprotective role for Also, long-term studies are needed to evaluate whether DJ-1 the exogenously administration of DJ-1 upon MI, potentially protective effects translate into the amelioration of adverse mediated by GPCRs signaling and the modulation of the cardiac remodeling. Frontiers in Pharmacology 09 frontiersin.org Gallinat et al. 10.3389/fphar.2022.1002755 Limitations Funding The present study provides the first line of evidence on the This work was supported by grants from: the Spanish infarct limiting effects of administering recombinant DJ-1 on a Ministry of Science and Innovation and Agencia Estatal de mouse model of acute myocardial infarction. This study needs Investigación SAF-2016-76819-R (to LB), MCIN/AEI/ further investigation to advance our understanding of DJ-1 target 10.13039/501100011033, Plan Nacional Proyectos and off-target effects as well as the tentative side effects of Investigación Desarrollo (PID 2019-107160RB-I00 to LB), and recombinant DJ-1 preparations. A major concern for the PGC 2018-094025-B-I00 (to GV); the Instituto de Salud Carlos clinical use of recombinant proteins is their potential to III: CIBER-CV and ERA-CVD JTC 2020-023/AC 209-00054 (to induce an unwanted immune response that may compromise LB) and FIS PI19/01687 (to TP). AG is a pre-doctoral fellow from safety. Furthermore, while infarct size is widely recognized as a BES-2017-081378. This article is part of AG PhD project at major predictor of clinical outcomes, long-term effects have not Universitat Autònoma de Barcelona (UAB). yet been addressed. Further investigation, in appropriated models is warranted in order to assess whether the cardioprotective effects of DJ-1 administration are maintained Acknowledgments in the long-term. We thank the Fundación de Investigación Cardiovascular–Fundación Jesús Serra for their continuous Data availability statement support. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and Conflict of interest accession number(s) can be found below: https://www.ncbi. nlm.nih.gov/geo/, GSE66307. LB received institutional research grants from AstraZeneca; consultancy fees from Sanofi, Pfizer and Novartis; speaker fees from Amarin, Lilly, Pfizer, and AstraZeneca. TP, GV and LB are Ethics statement shareholders of the academic spin-off companies GlyCardial Diagnostics SL and Ivestatin Therapeutics SL. All unrelated to The animal study was reviewed and approved by the present work. LB, GV, and TP are authors of the patents Institutional Animal Care and Use Committees (CEEA-IR EP3219326A1 and WO2017157958A1 regarding the use of DJ-1- Sant Pau) of ICCC-Hospital de la Santa Creu i Sant Pau. derived polypeptides for the treatment of ischemia/reperfusion injury. AG and GM declare no conflict of interest. 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