antioxidants Article The Interplay of Mitochondrial Oxidative Stress and Endoplasmic Reticulum Stress in Cardiovascular Fibrosis in Obese Rats Francisco V. Souza-Neto 1,† , Sara Jiménez-González 1,† , Beatriz Delgado-Valero 1 , Raquel Jurado-López 1 , Marie Genty 1 , Ana Romero-Miranda 1 , Cristina Rodríguez 2,3,4 , María Luisa Nieto 4,5 , Ernesto Martínez-Martínez 1,4, *,‡ and Victoria Cachofeiro 1,4, *,‡ 1 Departamento de Fisiología, Facultad de Medicina, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Universidad Complutense de Madrid, 28040 Madrid, Spain; franvasc@ucm.es (F.V.S.-N.); saraji02@ucm.es (S.J.-G.); beadel02@ucm.es (B.D.-V.); rajurado@ucm.es (R.J.-L.); marie.genty@inserm.fr (M.G.); anarom12@ucm.es (A.R.-M.) 2 Institut de Recerca del Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain; crodriguezs@santpau.cat 3 Instituto de Investigación Biomédica Sant Pau (IB Sant Pau), 08025 Barcelona, Spain 4 Ciber de Enfermedades Cardiovasculares (CIBERCV), Instituto de Salud Carlos III, 28220 Majadahonda, Spain; ml.nieto@csic.es 5 Instituto de Biología y Genética Molecular, CSIC-Universidad de Valladolid, 47002 Valladolid, Spain * Correspondence: ernmarti@ucm.es (E.M.-M.); vcara@ucm.es (V.C.); Tel.: +34-913941483 (E.M.-M.); +34-913941489 (V.C.)   † These authors contributed equally to this study. ‡ These authors contributed equally to this study. Citation: Souza-Neto, F.V.; Jiménez-González, S.; Delgado- Abstract: We have evaluated the role of mitochondrial oxidative stress and its association with Valero, B.; Jurado-López, R.; Genty, endoplasmic reticulum (ER) stress activation in the progression of obesity-related cardiovascular M.; Romero-Miranda, A.; Rodríguez, fibrosis. MitoQ (200 µM) was orally administered for 7 weeks to male Wistar rats that were fed a C.; Nieto, M.L.; Martínez-Martínez, high-fat diet (HFD, 35% fat) or a control diet (CT, 3.5% fat). Obese animals presented cardiovascular E.; Cachofeiro, V. The Interplay of Mitochondrial Oxidative Stress and fibrosis accompanied by increased levels of extracellular matrix proteins and profibrotic mediators. Endoplasmic Reticulum Stress in These alterations were associated with ER stress activation characterized by enhanced levels (in Cardiovascular Fibrosis in Obese Rats. heart and aorta vs. CT group, respectively) of immunoglobulin binding protein (BiP; 2.1-and Antioxidants 2021, 10, 1274. https:// 2.6-fold, respectively), protein disulfide-isomerase A6 (PDIA6; 1.9-fold) and CCAAT-enhancer- doi.org/10.3390/antiox10081274 binding homologous protein (CHOP; 1.5- and 1.8-fold, respectively). MitoQ treatment was able to prevent (p < 0.05) these modifications at cardiac and aortic levels. MitoQ (5 nM) and the ER stress Academic Editor: Judith Haendeler inhibitor, 4-phenyl butyric acid (4 µM), were able to block the prooxidant and profibrotic effects of angiotensin II (Ang II, 10−6 M) in cardiac and vascular cells. Therefore, the data show a crosstalk Received: 1 July 2021 between mitochondrial oxidative stress and ER stress activation, which mediates the development of Accepted: 9 August 2021 cardiovascular fibrosis in the context of obesity and in which Ang II can play a relevant role. Published: 11 August 2021 Keywords: cardiovascular fibrosis; endoplasmic reticulum stress; mitochondrial oxidative stress; obesity Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Cardiovascular fibrosis is a common feature in the context of obesity that occurs due to the imbalance between production and degradation of extracellular matrix components Copyright: © 2021 by the authors. (ECM), mainly collagen type I [1]. This excessive ECM deposit due to an increased number Licensee MDPI, Basel, Switzerland. of myofibroblasts, the main cell responsible for fibrosis, can produce an aberrant remodeling This article is an open access article that triggers functional alterations by reducing the relaxing capability of the heart, which distributed under the terms and can in turn increase its filling pressure and contribute to diastolic dysfunction [2]. Vascular conditions of the Creative Commons fibrosis is associated with arterial stiffness, which is a main determinant of cardiovascular Attribution (CC BY) license (https:// mortality [3]. creativecommons.org/licenses/by/ A variety of factors has been suggested as potential mediators of cardiac fibrosis 4.0/). in the context of obesity, including oxidative stress [4–6]. Mitochondria are the main Antioxidants 2021, 10, 1274. https://doi.org/10.3390/antiox10081274 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 1274 2 of 16 source of reactive oxygen species (ROS) in the cell through oxidative phosphorylation (OXPHOS) during the process of ATP synthesis [7]. In normal conditions, ROS levels in the mitochondria are balanced by an effective antioxidant defense. However, an excessive production of ROS can affect mitochondrial function and could lead to functional alterations of the heart [8,9]. In this line, some data have reported the relevance of mitochondrial oxidative stress in the development not only of cardiac fibrosis but also in the metabolic alterations in diet-induced obese rats [10,11]. In our previous study [12], we observed that obese animals presented an increase in body weight, as well as in adiposity index, which was associated with adipose tissue fibrosis and metabolic alterations. At cardiac level, obese animals exhibited hypertrophy, interstitial fibrosis and an increase in superoxide anion levels, as compared to the reference group. These modifications were prevented by treatment with the mitochondrial antioxidant, MitoQ, and were in absence of alterations in cardiac function or in blood pressure [9]. However, the mechanisms through which mitochondrial ROS plays a role in the fibrotic consequences of obesity remain unclear. In the last years, it has been described that obesogenic diets have an impact on the diversity and functionality of the gut microbiota, which plays a central role in the cardiovascular alterations observed in obesity [13,14]. In this sense, alterations in gut microbiota are associated with renal disorders [15] and cardiovascular diseases such as heart failure [16], atherosclerosis [17], hypertension [18] or vascular dysfunction [19]. Systemic inflammation plays a central role in all of these pathologies [20,21]. It has been observed that changes in gut microbiota observed in obese animals are involved in adipose tissue inflammation, oxidative stress and metabolic disorders [22]. In a recent study, we have demonstrated the relevance of oxidative stress in all of these alterations, since treatment with a mitochondrial antioxidant was able to prevent insulin resistance and cardiac fibrosis observed in obese animals. These beneficial effects of the mitochondrial antioxidant were accompanied by a restoration in gut microbiota composition [23], thus showing the link between microbiota dysbiosis, oxidative stress, inflammation and cardiac alterations in the context of obesity. In this study, we would like to go deeper into the cardiac fibrosis associated with obesity, as well as the role of mitochondrial oxidative stress. The endoplasmic reticulum (ER) is a dynamic cellular organelle with different func- tions: Ca2+ buffering, lipid and carbohydrate metabolism, as well as protein synthesis, folding and processing, among others. In a physiological state, protein synthesis and folding levels follow a balance so that the folding capacity of the ER is not saturated. However, this balance is perturbed in response to different stimuli, including oxidative stress, and misfolded proteins thus accumulate in the ER lumen. This promotes ER stress and the activation of the unfolded protein response (UPR) to normalize and return the balance in which binding immunoglobulin protein (BiP) plays a central role as a chaperone. BiP is therefore considered to be a marker of ER stress activation. Different studies have demonstrated the participation of ER stress in cardiovascular damage. The contribution of ER stress to cardiac hypertrophy has been demonstrated in transgenic mice, where the genetic ablation of mediators of ER stress reduced cardiac remodeling, as well as cardiac dysfunction, after transverse aortic constriction in mice [24]. In addition, the beneficial effects of ER stress inhibition were confirmed by the pharmacological inhibition of ER stress capability to reduce cardiac hypertrophy induced by a pressure overload model [25]. Ischemic conditions are also associated with impaired protein folding and subsequent acti- vation of ER stress. In prolonged ER stress conditions, ER activates proapoptotic signaling pathways, thereby contributing to the onset of heart failure [26]. At vascular level, preclini- cal and clinical studies have elucidated the activation of ER stress in atherosclerotic plaques. The apoptosis in vascular smooth muscle cells (VSMCs) derived from prolonged ER stress activation leads to plaque rupture, oxidative stress and enhanced calcification [27,28]. In addition, the role of ER stress has been demonstrated in vascular endothelial dysfunction in hypertension [29]. Different studies have reported the potential participation of ER stress in the development of cardiac fibrosis in obese models [30,31]. Antioxidants 2021, 10, 1274 3 of 16 The mitochondria and ER can come into contact at different sites, forming the mitochondria– ER associated membranes, through which they can coordinate their functions by participat- ing in the regulation of different processes [32]. However, whether these interactions can be relevant for the development of cardiovascular fibrosis in the context of obesity is not fully established. Therefore, the aim of this study was to evaluate whether the participation of oxidative stress in the development of cardiovascular fibrosis in the context of obesity is through the activation of ER stress. To address this aim, we evaluated the effect of the mitochondria-targeted antioxidant MitoQ in cardiovascular fibrosis in obese rats. In addition, we have studied how MitoQ treatment can affect ER stress activation. We have also analyzed the crosstalk between both processes in cultured cardiac fibroblasts and VSMCs, the main cells involved in ECM synthesis. 2. Materials and Methods 2.1. Animal Model Male Wistar rats of 150 g (6 weeks old; Envigo, Barcelona, Spain) were fed either a high-fat diet (HFD, 35% fat; Envigo Teklad no. TD.03307, Haslett, MI, USA; n = 16) or a standard diet (CT, 3.5% fat; Envigo Teklad no.TD.2014, Haslett, MI, USA; n = 16) for a period of 7 weeks. The mitochondrial antioxidant MitoQ (200 µM) was administered to half of the animals of each group in the drinking water for the same period of time. Animals were sacrificed by decapitation. The dose of MitoQ was based on previous data [33]. MP Murphy from medical Research Council Mitochondrial Biology Unit (Biomedical Campus, Cambridge, UK) provided MitoQ. Animals were weighed once a week. All experimental procedures were approved by the Animal Care and Use Committee of Universidad Complutense de Madrid following the Spanish Policy for Animal Protection RD53/2013 and according the European Union Directive 2010/63/UE. Serum and plasma samples were collected at the end of the study. 2.2. Histological Analysis Samples of aorta and heart were dehydrated, immersed in paraffin and cut into sec- tions 4 µm thick. Collagen fibers were detected by the staining of sections with picrosirius red. Aorta and coronary artery fibrosis was quantified in the media layer as the ratio of collagen deposition or interstitial fibrosis to the total media area. Ten to fifteen fields for each sample were analyzed with an objective (40×) under microscopy of transmitted light (Leica DM 2000; Leica AG, Wetzlar, Germany). Quantification of the data was performed with an analysis system (Leica LAS 4.3; Leica AG, Wetzlar, Germany). 2.3. Cell Isolation and Cell Culture Conditions Heart and thoracic aorta of adult male Wistar rats (weighing 250–300 g) were used to isolate cardiofibroblasts and VSMCs. Differential centrifugation was performed af- ter enzymatic digestion of the hearts with a mix of collagenase-trypsin for the isola- tion of cardiac fibroblasts and aorta with a mix of collagenase-elastase for the isolation of VSMCs, as previously described [5,34]. Cells were used between passages 5 and 6. DMEM medium supplemented with 10% FBS, 10 mmol/L L-glutamine, 100 U/mL peni- cillin/streptomycin, 10 mmol/L L-pyruvate and 2 mmol/L HEPES was used for cardiac fibroblasts. VSMCs were maintained in DMEM F-12 supplemented with 10% FBS and 100 U/mL penicillin/streptomycin. Cells were grown as monolayer culture in a T-75 tissue culture flask from an initial density of 0.5 × 106 cells. Confluent cells were passaged with 0.25% trypsin in 0.01% EDTA. Cells were serum-starved for 12 h, and 6-well plates were used with 90% confluence for each experimental condition. All assays in the present study were done at temperatures of 37 ◦ C, 95% sterile air and 5% CO2 in a saturation humidified incubator. Cells were treated with angiotensin II (Ang II; 10−6 M) for 24 h in the presence or absence of the mitochondrial antioxidant, MitoQ (5 nM), or the chemical chaperone, 4-phenyl butyric acid (4-PBA; 4µM). VSMCs were treated with the ER stress inducer, tunicamycin (1–2 µg/mL), for 24 h. Antioxidants 2021, 10, 1274 4 of 16 2.4. Western Blot Total proteins from cardiac and aortic homogenates, as well as cellular extracts, were prepared as previously described [9]. Briefly, proteins were separated by SDS-PAGE on polyacrylamide CriterionTM TGXTM Precast Gels (BioRad, Hercules, CA, USA) and trans- ferred to Hybond-c Extra nitrocellulose membranes (Hybond-P; Amersham Biosciences, Piscataway, NJ, USA). Membranes were reused after stripping a maximum of 3 times. This allows for measuring the greatest number of proteins in the same membrane. Membranes were probed with primary antibodies for activating transcription factor 6-alpha (ATF6α; Santa Cruz, TX, USA; dilution 1:250), immunoglobulin binding protein (BiP; BD Biosciences, Madrid, Spain; dilution 1:1500), CCAAT-enhancer-binding homologous protein (CHOP; Cell Signaling Technology, Danvers, MA, USA; dilution 1:500), collagen I (Calbiochem, San Diego, CA, USA; dilution 1:500), connective tissue growth factor (CTGF; Sigma-Aldrich, San Louis, MO, USA; dilution 1:1000), protein disulfide-isomerase A6 (PDIA6; Abcam plc, Cambridge, UK; dilution 1:500), transforming growth factor-β (TGF-β; Abcam plc, Cambridge, UK; dilution 1:250) and α-tubulin or β-actin (Sigma-Aldrich, San Louis, MO, USA; dilution 1:5000) as loading controls. Signals were detected using the ECL system (Millipore, Burlington, MA, USA). Results are expressed as an n-fold increase over the values of the control group in densitometric arbitrary units. 2.5. Retrotranscription and Real-Time PCR Total RNA was isolated using Trizol Reagent (Fisher Scientific, Waltham, MA, USA) and was reverse-transcribed into cDNA using the High-Capacity cDNA Reverse Transcrip- tion Kit (Thermo Fisher Scientific Inc, Waltham, MA, USA). Quantitative PCR analysis was performed with SYBR green PCR technology (Bio-Rad, Hercules, CA, USA) and specific oligonucleotides (Table S1). Quantification of mRNA levels was performed by real-time PCR using the ∆∆Ct method. Data were normalized to hypoxanthine phosphoribosyltrans- ferase (HPRT). 2.6. Measurement of Intracellular Superoxide Anion Production Cardiomyofibroblasts and VSMCs were seeded into 48-well plates at 70% confluence for experiments measuring O2 − production. Cells were exposed for 24 h with either vehicle or Ang II (10−6 M) for 24 h in the presence or absence of the mitochondrial antioxidant, MitoQ (5 nM), or the chemical chaperone, 4-PBA (4 µM). Cells were then exposed with dihydroethidium (DHE; 5 × 10−3 mmol/L) for 30 min. Cells were washed with warm PBS and analyzed with an objective lens (40×) in a fluorescent laser scanning Leica DMI 3000 microscope. A number of cells (150–200) for each condition was analyzed. The mean fluorescence densities in the nucleus were calculated. Results are expressed as an n-fold increase over the data of the control group. The analysis was performed by a single researcher unaware of the experimental groups. 2.7. Statistical Analysis Variables are expressed as mean ± SEM. Kolmogorov–Smirnov test was used to verify the normality of distributions. Differences between 2 groups were assessed with Student’s t test. Differences among several groups were evaluated using one-way ANOVA. Newman– Keuls test was used as a post hoc test for differences in means. A value of p < 0.05 was used as the cutoff for defining statistical significance. Data were analyzed using the statistical program GraphPad Prism 5 (San Diego, CA, USA). 3. Results 3.1. Mitochondrial Oxidative Stress Promotes ECM Protein Deposition and ER Stress Activation at Cardiac Level in Obesity HFD animals presented an increase in collagen type I protein levels accompanied by an increase in the profibrotic mediators CTGF and TGF-β as compared to control animals 3. Results 3.1. Mitochondrial Oxidative Stress Promotes ECM Protein Deposition and ER Stress Activation at Cardiac Level in Obesity Antioxidants 2021, 10, 1274 5 of 16 HFD animals presented an increase in collagen type I protein levels accompanied by an increase in the profibrotic mediators CTGF and TGF-β as compared to control animals (Figure 1A). The treatment with the mitochondrial antioxidant, MitoQ, was able to pre- vent the1A). (Figure increase in ECMwith The treatment components and profibrotic the mitochondrial growth antioxidant, factors MitoQ, observed was able in HFD to prevent the (Figure increase 1A). in ECM components and profibrotic growth factors observed in HFD (Figure 1A). Figure 1. Mitochondrial oxidative stress mediates the increase in extracellular matrix proteins and endoplasmic reticulum Figureat1.cardiac stress Mitochondrial oxidative level. Protein levelsstress of (A)mediates collagenthe increase type in connective I (Col I), extracellular matrix tissue proteins growth and factor endoplasmic (CTGF) reticulum and transforming stress atfactor-beta growth cardiac level. Protein (TGF-β); levels (B) of (A) collagen immunoglobulin type I (Col binding I), connective protein tissue growth (BiP); (C) protein factor disulfide (CTGF) isomerase and transforming family A member 6 growth factor-beta (PDIA6); (TGF-β); (B) immunoglobulin (D) CCAAT-enhancer-binding binding protein protein homologous (BiP); protein (C) protein (CHOP); disulfide isomerase (E) activating family transcription A member factor 6-alpha 6 (PDIA6); (D) CCAAT-enhancer-binding protein homologous protein (CHOP); (E) activating transcription factor 6-alpha (ATF6α) in heart tissue from control rats fed a normal chow (CT) and rats fed a high-fat diet (HFD) treated with vehicle or (ATF6α) in heart tissue from control rats fed a normal chow (CT) and rats fed a high-fat diet (HFD) treated with vehicle with the mitochondrial antioxidant MitoQ (MQ; 200 µM). Bars graphs represent the mean ± SEM of 6–8 animals normalized or with the mitochondrial antioxidant MitoQ (MQ; 200 µM). Bars graphs represent the mean ± SEM of 6–8 animals nor- for α-tubulin. malized * p < 0.05,* ** for α-tubulin. p