Co-Exposure of Cardiomyocytes to IFN-γ and TNF-α Induces Mitochondrial Dysfunction and Nitro-Oxidative Stress: Implications for the Pathogenesis of Chronic Chagas Disease Cardiomyopathy

Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes’ mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.

Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-g and TNF-a by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-g and TNF-a have been described to affect mitochondrial function, we hypothesized that IFN-g and TNF-a are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-g/TNF-a-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-g/TNF-a treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential

INTRODUCTION
Heart failure (HF) is an important worldwide public health problem. Available therapies are insufficient and do not fully address molecular abnormalities that occur in cardiomyocytes (1). Chagas disease cardiomyopathy (CCC) accounts for 25% of HF cases and is a major cause of death in Latin America (2,3). CCC is a severe inflammatory dilated cardiomyopathy caused by persistent infection by the protozoan Trypanosoma cruzi. While 60% of Chagas Disease (CD) patients are mostly asymptomatic in the so-called "indeterminate" form (IF) and do not develop heart disease, CCC patients (30%, roughly 8 million people) display HF, arrhythmia, and disability (4,5). CCC patients have 50% shorter survival rate and worse prognosis compared to patients with cardiomyopathies of non-inflammatory etiologies, such as ischemic, idiopathic and hypertensive cardiomyopathies (2).
The pathogenesis of CCC is still to be completely understood (6). Low-grade chronic T. cruzi persistence, which drives continued production of IFN-g and TNF-a by Th1 T cells immune cells, plays a central pathogenic role in CCC (2,4,5). Indeed, omics and immunohistochemistry studies revealed higher levels of IFN-g and TNF-a in heart tissue from CCC patients compared to other cardiomyopathies (7)(8)(9)(10)(11). Activation of the NF-kB/NOS2 axis by the continued production of IFN-g/ TNF-a can promote increased levels of intracellular reactive nitrogen species (RNS) which are important for parasite control (12,13). However, long-term sustained activation of this axis may promote damaging accumulation of reactive oxygen species (ROS) through induction of NADP oxidases and overproduction of mitochondrial reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ) and superoxide anion (O − 2 ) and RNS, molecules widely known to induce mitochondrial dysfunction and disturbances of heart function (14,15).
The heart is the most metabolically active organ in the body and has the highest content of mitochondria of any tissue (16). This is needed to meet the ever-demanding energy requirement for contraction and relaxation and about 95% of cellular ATP is utilized to support the contraction-relaxation cycle within the myocardium (16). Indeed, mitochondria contribute to the proper function of cardiomyocytes by multiple mechanisms (17). Beyond contraction, mitochondria also fine-tune cellular calcium homeostasis, apoptosis and oxidative stress and it has become increasingly clear that mitochondrial dysfunction is the source of heart energy deprivation in cardiomyopathies and heart failure of diverse etiologies (16).
Several myocardial mitochondrial enzymes were found to be selectively depressed in CCC as compared to other cardiomyopathies, including ATP synthase a, multiple fatty acid b-oxidation enzymes and creatine kinase activity [ (18)(19)(20) and our unpublished observations] additionally, in vivo myocardial ATP production was shown to be reduced in CCC as determined by 31 P-NMR spectroscopy (21). Taken together, results suggest that mitochondrial dysfunction may contribute to the worse prognosis of CCC. Since inflammatory cytokines IFN-g and TNF-a are abundantly produced and are top upstream regulators of gene expression changes in in CCC myocardium (9), and the sustained ROS and RNS production are known inducers of cardiomyocyte damage and mitochondrial dysfunction (22,23), we hypothesized that such cytokines contribute to mitochondrial dysfunction observed in CCC (5).
Thus, this study aimed to investigate 1) whether CCC myocardial tissue display increased levels of mitochondrial dysfunction and oxidative/nitrosative stress markers compared to non-chagasic dilated cardiomyopathy biopsies (DCM); 2) whether IFN-g and TNF-a promote mitochondrial dysfunction and nitrooxidative stress in the human cardiomyocyte cell line AC- 16; 3) whether the mitochondrial dysfunction in cardiomyocytes is triggered by the cytokines through the STAT1/NF-kB/NOS2 signaling pathway; 4) whether agonists of mitochondrial protection pathways, such as AMPK, NRF2 and SIRT1 ameliorate mitochondrial dysfunction driven by the IFN-g and TNF-a.

Ethics Statement
Tissue collection was approved by the Institutional Review Board of the University of São Paulo, School of Medicine (CAPPesq approval number 852/05) and written informed consent was obtained from the patients. All experimental methods comply with the Helsinki declaration.

Patients and Sample Collection
Human left ventricular free wall heart tissue was collected from end-stage heart failure CCC patients (n=40) and non-chagasic dilated cardiomyopathy (DCM, n=31) patients (NYHA class 3 and 4) at the moment of heart transplantation. CCC patients presented positive T. cruzi serology and typical heart conduction abnormalities (right bundle branch block and/or left anterior division hemiblock) and had a histopathological diagnosis of myocarditis, fibrosis and hypertrophy. All heart tissue samples were cleared from pericardium and fat and quickly frozen in liquid nitrogen and stored at -80°C. The list of patients is described in Table 1.

Cell Culture
Human adult ventricular cardiomyocytes cell line AC-16 was kindly provided by Dr. Mercy Davidson (Columbia University, USA) (24). The cell line was propagated using Dulbecco's modified Eagle's medium/F-12 medium with 12.5% fetal bovine serum (FBS) without antibiotics for no longer than 8 passages. AC-16 cells were screened monthly for mycoplasma contamination (MycoAlert Mycoplasma Detection Kit, Lonza)

LDH Release Assay
The release of lactate dehydrogenase (LDH) in the conditioned media is an indicator of cell cytotoxicity. AC-16 cardiomyocytes were stimulated with the cytokines for 48h and the LDH content was quantified using a LDH-Cytotoxicity Assay Kit II (Abcam) using SpectraMax ® Paradigm ® .

Assessment of 3-Nitrotyrosine
Relative quantification of nitrated protein was performed by assessing 3-nitrotyrosine (3-NT) as a mean to detect nitrosative stress. Cardiac fragments and stimulated AC-16 cells were lysed in aqueous lysis solution containing 10 mM HEPES, 0.32 M sucrose, 0.1 mM Na 2 EDTA, 1.0 mM dithiothreitol, 125 μg/ml PMSF and 1.0 μl/ml of proteinase inhibitor cocktail (Sigma), pH=7.4. Cardiac fragments were lysed in a tissue homogenizer (Precellys 24) pre-chilled 4°C using 2.8 mm ceramic beads for 3 cycles of 10s (seconds) with 15s intervals at 5500 rpm. Cells were lysed with 1.4 mm ceramic beads for 2 cycles of 30s with 10s intervals at 5000 rpm. Cardiac and cell lysates were clarified by centrifugation at 10,000 rcf for 30 min at 4°C. A total of 5 μg of proteins was added to a nitrocellulose membrane, dried at 60°C for 15 min and then blocked with 3% Blotting Grade Blocker (BioRad) + TBS-T, for 1h at RT, under stirring. Membrane was washed with TBS-T and incubated with 1:1000 primary monoclonal antibody 3-nitrotyrosine (Abcam) in 3% BSA + TBS-T overnight 4°C. Then, membrane was washed twice and incubated with 1:1000 secondary antibody for 2h at RT, under agitation and protected from light. Fluorescence was captured using a scanner (LI-COR, Odyssey) or ECL revelation. Ponceau S images were captured using ImageQuant LAS-400 (GE Healthcare) and used for protein normalization.

ATP and Nitrite Production
Nitrite (NO − 2 ) was measured in the conditioned media (phenolfree) of stimulated AC-16 cells using Griess Reagent Kit for Nitrite Determination (Molecular Probes) according to manufacturer's instructions. The cells were collected and lysed with TE buffer (100 mM Tris, 4 mM EDTA, pH=7.5) and ATP measured with a luciferase-based assay kit (ATP Determination Kit, ThermoFisher Scientific) according to manufacturer's instructions.

Bioenergetic Function Analysis
A Seahorse XFe24 Analyzer (Agilent, Les Ulis, France) was used to survey bioenergetic function by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells. A quantity of 3,500 cells/0.32cm 2 were seeded in 150 μl of specific media, incubated for 1h at room temperature for adhesion and stimulated with 350 μl of specific media containing IFN-g (10 ng/ml final) and TNF-a (5 ng/ml final) for 48h. OCR and ECAR were obtained from 90% confluent monolayer culture. All the experiments were done according to the protocol provided by the manufacturer. Briefly, the Agilent Seahorse Cell Mito Stress Test kit assesses mitochondrial function. Multiple parameters are obtained such as, basal respiration, ATP-linked respiration, maximal and reserve capacities, and non-mitochondrial respiration. The ATP Real-Time rate kit is the only assay that quantifies the ATP production from glycolysis and mitochondria simultaneously. The Agilent Seahorse Mito Fuel Flex Test kit measures the basal state of mitochondrial fuel oxidation in live cells by providing information on the relative contributions of glucose, glutamine and long-chain fatty acid oxidation to basal respiration. All analyses were performed using the software Wave 2.6.1 (Agilent, Les Ulis, France).

Proteomic Analysis of AC-16 Cardiomyocytes
The proteomic analysis of AC-16 cells under IFN-g (10 ng/ml) and TNF-a (5 ng/ml) treatment was assessed by high-resolution mass spectrometric analysis. After 48h incubation with the combination of IFN-g and TNF-a, proteins from AC-16 were extracted using lysis buffer (12 mM sodium deoxycholate, 12 mM N-lauroylsarcosine sodium salt, 100 mM Tris-HCl, pH=9.0) supplemented with protease inhibitor cocktail (Sigma-Aldrich). Total cell lysates from control and IFN-g+TNF-a-treated cells were subject to in-solution trypsin digestion according to Phase Transfer Surfactant (PTS)-aided trypsin digestion protocol (32). Digested proteins were desalted using StageTip protocol (33) and dried by centrifugation under vacuum. Peptide samples were analyzed by RP-LC-MS/MS. For MS analysis, peptide mixtures were dissolved in 0.1% formic acid (solution A). Aliquots of 3 ml were automatically injected by a nano chromatography EASY-nLCII system (Thermo Scientific) on a 40 mm x 100 mm ID C-18 pre-column packed with Jupiter 10 mm resin (Phenomenex) and submitted to a chromatographic separation in a 100 mm x 75 mm ID C-18 column packed with Reprosil-Pur 3 mm C-18 beads (Dr. Maisch) coupled to an LTQ Orbitrap Velos (Thermo Scientific). Peptides were eluted with a linear gradient of 5-30% solution B (0.1% formic acid in acetonitrile) at 200 nl/min for 55 min. Spray voltage was set at 2.5kV and the mass spectrometer was operated in data dependent mode, in which one full MS scan was acquired in the m/z range of 400-1,800 at 60,000 resolution (at 400m/z) followed by collisional induced fragmentation (CID) and MS/MS acquisition of the ten most intense ions from MS scan. Unassigned and +1 charge states were not subjected to fragmentation. The maximum injection time and AGC target were set to 100ms (milliseconds) and 1E6 for full MS, and 10ms and 1E4 for MS/MS. The minimum signal threshold to trigger fragmentation event, isolation window and normalized collision energy (NCE) were set to, respectively, 1000 cps, 2m/z and 35%. Dynamic peak exclusion (list size of 500) was applied to avoid the same m/z of being selected for the next 30s. Mass spectrometric raw data were processed using the software

Statistical Analysis
Cell data are reported as the ratio to non-treated cells. Cell viability was calculated as the ratio of the number of live cells (NucGreen-negative) and total cells (NucGreen-negative plus NucGreen-positive cells) x100. The results were expressed as mean ± standard deviation (SD) or standard error of the mean (SEM) when noted. Statistical analysis was performed using the GraphPad Prism 9.2.0 software (GraphPad Software, Inc., CA). Data were tested for normality using the Shapiro-Wilk test before statistical tests. A nonparametric Mann-Whitney test was applied if data were not normally distributed. In all other cases, one-way ANOVA followed by Dunn's post hoc test was applied for multiple comparisons. A p-value < 0.05 was considered statistically significant.

Increased RNS and Decreased mtDNA Content in CCC Myocardial Tissue
We compared the nitro-oxidative profile of CCC and DCM myocardium. We first measured nitrite, a reactive nitrogen species (RNS) marker, by chemiluminescence with a NO Analyzer. We observed that CCC biopsies possess higher content of nitrite (132%; p < 0.001) in comparison to DCM lysates ( Figure 1A). Detection of nitrotyrosine, an additional nitro-oxidative stress marker, was performed by dot-blot. We observed a significantly higher nitrotyrosine immunoreactivity in CCC (27%; p < 0.01) than DCM myocardial lysates ( Figure 1B), indicating previous exposure to peroxynitrite. Also, mitochondrial DNA (MT-ND1) was lower in CCC (44%; p < 0.001) than DCM myocardial samples ( Figure 1C) indicating a reduction of mitochondrial mass.

IFN-g and TNF-a Impair AC-16 Cardiomyocyte Mitochondrial Membrane Potential (DYm)
To investigate the role of IFN-g and TNF-a on AC-16 cardiomyocytes, we stimulated the cells with several concentrations of IFN-g and TNF-a and we measured the mitochondrial DYm in a high content screening platform. This was performed to stablish the concentrations of the cytokines for subsequent analyses. We observed that IFN-g impaired the DYm of AC-16 48h after stimulation and this impairment was enhanced when IFN-g was combined with TNF-a; TNF-a alone failed to cause statistically significant reductions in DYm (Figures 2A, B). We then selected the concentrations of 10 ng/ml of IFN-g and 5 ng/ml of TNF-a and we detected that IFN-g and TNF-a decrease DYm of mitochondria larger than 10 μm 2 ( Figures 2C, D) and the impact is higher on larger mitochondria (Supplementary Figure 1). We performed the LDH assay of the selected doses of IFN-g (10 ng/ml) and TNF-a (5 ng/ml) and we observed no cytotoxicity ( Figure 2E).

Cytokine-Stimulated AC-16 Cells Display Increased RNS and Decreased ATP and mtDNA
We investigated whether IFN-g and TNF-a prompt nitrosative and oxidative stress in AC-16 cardiomyocytes. We used the Griess reaction to measure nitrite in the supernatant of 48h IFNg and TNF-a-stimulated AC-16 cells. Nitrite accumulation in the supernatant of AC-16 was increased by TNF-a alone and not by IFN-g or their combination after 48h of incubation ( Figure 3A). Detection of 3-NT was performed by dot-blot. We observed that although IFN-g or the cytokine combination induced a median increase of 25-72% in protein nitration, this was not statistically significant, most likely due to the high dispersion ( Figure 3B). Cytokine treatment of AC-16 cardiomyocytes with IFN-g, TNFa and combined for 48h reduced the amount of MT-ND1 as compared to non-stimulated cells ( Figure 3C). We also measured reactive oxygen species (ROS) production in AC-16 stimulated cells stained with the fluorogenic dye H 2 DCFDA and we found a 43% increase in ROS after stimulation with IFN-g and TNF-a and 23% with TNF-a ( Figure 3D). In addition, luminescence assay also showed that the TNF-a alone or combined with IFN-g decreased 50% and 58%, respectively, the ATP content in AC-16 cardiomyocytes ( Figure 3E).

Molecular Pathways Analysis of Cytokine-Induced Dym Reduction
In order to better understand the mechanisms by which IFN-g and TNF-a diminish AC-16 DYm, cells were treated with several compounds that activate or inhibit specific pathways. Each drug was titrated and the concentration of each compound was selected based on the highest restorative effect on DYm and no more than 10% loss on cell number (Supplementary Figures 2,  3). Concurrent treatment showed that agonists of signaling pathways related to response to stress and mitochondrial protection, such as AMPK (metformin, AICAR, resveratrol), NRF2 (Resveratrol and Protoporphyrin XI) and SIRT1 (SRT1720) restore DYm (Figures 4A-C). Additionally, inhibition of IFN-g downstream molecules with selected antagonists such as fludarabine (STAT1 inhibitor), emodin (NF-kB activation) JSH23 (NF-kB nuclear translocation), IKK16 (IkB kinase) and NOS2 (1400W) also restores AC-16 DYm (Figures 4D-G). Finally, inhibition of TNF-a-downstream molecules such as MEK (PD98059), JNK (SP600125) and MAPK (SB202190) also restores AC-16 DYm ( Figures 4H-J respectively). The effect of NF-kB inhibitors on NF-kB nuclear translocation was validated by additional experiments. Immunocytochemistry shows that IFN-g and TNF-a promote NF-kB nuclear translocation ( Figure 4K), which was antagonized with emodin and JSH23 and simultaneously restored DYm in AC-16 cardiomyocytes (Figures 4L-M). In addition, Figure 4N shows that NOS inhibitor 1400W significantly decreased nitrite accumulation caused by TNF-a (p < 0.001). A schematic representation of the signaling pathways and the targets of the compounds are detailed in Supplementary Figure 4.

IFN-g+TNF-a Costimulation on the AC-16 Cell Line Induces a Significant Decrease of Total, Glycolysis and Mitochondrial ATP Production
The effect of IFN-g+TNF-a stimulation on AC-16 was investigated by measuring the two major production pathways in mammalian cells (glycolysis and oxidative phosphorylation). Twenty-two independent measurements were done on non-stimulated AC-16 cells and 16 independent measurements were done on IFN-g+TNFa stimulated AC-16 cells. The OCR, ECAR and PER values were used to calculate glycolytic and mitochondrial ATP (Figures 5A-D). IFN-g+TNF-a induced a significant decrease of total ATP production (33%; p=0.0001) ( Figure 5E), glycolysis ATP production (30%; p<0.0001) ( Figure 5F), and the mitochondrial ATP production (41%; p=0.02) ( Figure 5G) was in line with the previous finding ( Figure 3E). The percentage of oxidative phosphorylation was unchanged ( Figure 5H).

Nunes et al. Mitochondrial Dysfunction in Chagasic Cardiomyopathy
Frontiers in Immunology | www.frontiersin.org November 2021 | Volume 12 | Article 755862 palmitoyltransferase-1 (CPT-1, fatty acid oxidation). We then calculated the dependency, which is the cell's reliance on a particular fuel pathway to maintain basal respiration, and capacity, i.e. the ability of mitochondria to oxidize a fuel when other fuel pathways are inhibited. We found that 48h stimulation with IFN-g+TNF-a decreased 62% (p=0.0104 n=9) the reliance of AC-16 cells on glutamine ( Figures 6A, D) and 87% dependency of fatty acid oxidation (p=0.0411 n≥9) ( Figures 6B, E), with no effect on glucose dependency ( Figures 6C, F). In addition, cells' capacity to oxidize fatty acid and glucose increased 46% (p=0.0037 n≥10 Figures 6H, K) and 105% (p<0.0001 n≥11 Figures 6I, L) respectively after stimulation with the cytokines. No changes were detected in glutamine capacity (Figures 6G, J). A summary of the global fuel oxidation of not-stimulated and cytokinestimulated cells is shown in Figure 6M.  Table 1). Among downregulated proteins, 6 belonged to the TCA cycle and OXPHOS (ACO2, DLAT, FH, CS, NDUFV2, NDUFS8). We found reduced expression of two mitochondrial protein import enzymes (TIMM23, TOMM22) two linked to protein synthesis (RARS, GRSF1) and 2 to maintenance of mitochondrial membrane function and polarization (TRAP1, IMMT) and reduced levels of the ion carriers SLC25A40, SLC25A11, SLC25A3. We found downregulated proteins belonging to the lipid beta oxidation (ACOT13, HADH, FASN, ACAT1) and involved in ATP metabolism, ATP synthase regulation and ATP transport to the cytoplasm (ATPIF1, USMG5, PPIF and SLC25A6). PRDX3 and PRDX5 were downregulated and SOD2 was upregulated, consistent with oxidative stress. Reactome analysis of differentially expressed mitochondrial proteins disclosed repressed mitochondrial betaoxidation and TCA cycle, increased glycolysis, increased mitochondrial protein import, ketone body metabolism and detoxification of reactive oxygen species. IPA canonical pathways analysis indicated mitochondrial dysfunction, reduced sirtuin-1 signaling, reduced TCA cycle, downregulated ketolysis, upregulation of superoxide radical degradation. A comparison of DEPs both in the cytokine-stimulated AC-16 cardiomyocytes and CCC myocardium (data not shown; submitted for publication) identified 19 matching proteins. Twelve out of the 19 were mitochondrial or related to energy metabolism, and 8 of them were similarly modulated in both proteome datasets. Among matching and differentially expressed mitochondrial proteins, we observed reduced expression of proteins including HADH and ACAT1 (fatty acid beta-oxidation), NDUFV2 (OXPHOS), and FH (TCA), with increased expression of PKM (glycolysis) and LAP3 (apoptosis).

Mitochondrial Genes Are Differentially Expressed in Cytokine-Treated AC-16 Cardiomyocytes
The AC-16 cell line was stimulated with IFN-g and TNF-a during 1h to 48h. Gene expression analysis was performed in the various time points taking as reference the T=0h time point. We considered differentially expressed genes, genes characterized by a corrected p-value<0.05 (Benjamini Hochberg) and absolute log2 Fold Change>1.5. A total of 1443 DEGs were detected in all these comparisons (Supplementary Table 2). These DEGs include mainly protein coding genes (n=1149, 79.6%), as well as pseudogenes (n=68, 4.7%), antisense (n=89, 6.2%) and lnRNAs (n=94, 6.5%). The largest number of DEGs were detected at 6h or 12h (T=1h: n=214; T=6h: n=858; T=12h: n=847; T=24h: n=588; T=48h: n=234). Volcano plots and heatmaps are described in Supplementary Figure 5 and Supplementary Table 3. The main pathways are linked to the immune response, to inflammation, to intracellular defense mechanisms, to fibrosis and cardiac diseases. Just after 1h of cytokine stimulation AC-16 cells increased the expression of cytokines and chemokines such as CXCL1, CCL2, CXCL2 and IL8, IRF1 and IRF9, indicating an interferon and proinflammatory signaling cascade with an overexpression of TNFAIP3 and NFKBIA which act as NFKB inhibitors. After 6h of stimulation, a large number of cytokine and chemokine genes and their receptors was upregulated, including CCL2, CCL7 , CCL8, CCL13, CX3CL1, CXCL1, CXCL2, CXCL3,  CXCL5, CXCL9, CXCL10, CXCL11, IFNAR2, IFNGR2,  STAT1, TNF, IL1A, IL1B, IL4R, IL6, IL7, IL7R, IL12A, IL15,  IL15RA, IL20RB, IL21R, IL32, IL33 genes). We also performed the same experiments with IFN-g or TNF-a alone (Figure 7 and Supplementary Table 4). Overall, we found 67 mitochondrial genes to be differentially expressed across at least one of the time points in response to combined cytokine stimulation. Differentially expressed mitochondrial genes peaked at 12h (46 genes) of which 25 were upregulated and 21 down-regulated (Supplementary Table 5). Among the top 10 canonical pathways of mitochondrial DEGs at 12h stimulus, we observed inhibition of the sirtuin signaling pathway, activation of aryl hydrocarbon receptor signaling, interferon signaling, and mitochondrial dysfunction. Toxicity-function pathways analysis at 12h indicated decreased transmembrane potential of mitochondria, fatty acid metabolism, mitochondrial dysfunction, pro-apoptosis, cardiac necrosis/cell death, cardiac hypertrophy among others (Supplementary Table 6). Mitochondrial dysfunction and decreased transmembrane potential were enriched toxicity functions in all time points starting at 6h (data not shown).

DISCUSSION
In this study, we investigated the mitochondrial function, nitrooxidative profile, and gene and protein expression of myocardial samples from CCC patients. We also surveyed the effect of IFN-g and TNF-a in AC-16 cardiomyocytes' mitochondria. These cytokines are known to be selectively produced by the myocardium of CCC patients and not DCM (7-11, 36, 37). We have identified that CCC myocardium displays an increased nitro-oxidative stress profile, as well as reduced mtDNA content in comparison to DCM samples and that these phenomena were also observed by IFN-g/TNF-a treatment of AC-16 cardiomyocyte cell line. We performed mechanistic studies to better understand the role of the multiple signaling pathways in the mitochondrial function of IFN-g/TNF-a-treated AC-16 cardiomyocytes. We have found that the inhibition of STAT1/ NF-kB/NOS2 axis and activation of AMPK, NRF2 and SIRT1 signaling pathways promoted protective effects in the IFN-g/ TNF-a-induced impairment of mitochondrial DY. Pathways analysis of gene and protein expression involved mitochondrial dysfunction and decreased DYm were found in cytokine-treated AC-16 cells. In addition, we observed a cytokine-induced reduction in ATP production at the expense of mitochondrial energy metabolism in AC-16 cardiomyocytes that paralleled that observed in CCC myocardial tissue. IFN-g, but not TNF-a alone, was shown to impair the mitochondrial DYm of AC-16 cells. We also observed that concurrent treatment with IFN-g and TNF-a results in a further decrease in DYm, and this reduction is higher in larger mitochondria. This finding is especially crucial since the mitochondrial DYm is the OXPHOS proton motive force that drives ATP production through the ATP synthesis complex and thus it is an essential mechanism for contraction and survival of cardiac cells (38). Several in vitro studies have found the suppressing effect of TNF-a (39-42) and IFN-g plus TNF-a (43) in mitochondrial DYm in different cell types.
IFN-g exerts its deleterious effects in the mitochondria at least partially by potentiating TNF-a-mediated NF-kB signaling (44). Activation of NF-kB triggers transcriptional activity of NOS2, which in turn produces nitric oxide (NO) and in the presence of reactive oxygen species (ROS) produces peroxynitrite (ONOO -) one on the most toxic and highly oxidative reactive species with substantial effects in mitochondria (45,46). In our observations, IFN-g and/or TNF-a can increase NF-kB nuclear translocation, nitrite and ROS production, and 3-nitrotyrosine accumulation in AC-16 cardiomyocytes. Peroxynitrite and protein nitration cause inactivation of enzymes, poly(ADP-ribosylation), mitochondrial dysfunction, impaired stress signaling and also potently inhibits myofibrillar creatine kinase (MM-CK), an important controller of heart contractility (47, 48) whose activity has been shown to be decreased in CCC (18). Increases in ROS by IFN-g and/or TNF-a was also reported in several cell lines (39,41,49,50), including cardiomyocytes (42,(51)(52)(53). Enhanced ROS production is associated with increased levels of lipid peroxidation, decreased mtDNA copy number and impaired OXPHOS capacity, affecting cardiomyocyte structure and function which triggers signaling pathways involved in myocardial remodeling and failure (54,55). Enhanced oxidative stress has been observed in CCC heart tissue as measured by the accumulation of malondialdehyde (submitted for publication). We here reported reduction of mtDNA content in CCC myocardium and IFN-g/TNF-astimulated cardiomyocytes. The mtDNA is a circular double stranded DNA located in the mitochondrial matrix and codes for 37 genes (56) and the 13 polypeptides are the essential subunits of the OXPHOS complexes I, III, IV, and V (56,57). Deficiency in mtDNA replication was shown to cause ROS accumulation and oxidative stress in murine cardiomyocytes (58,59), which is also indicative of a reduced mitochondrial mass. Thus, our data suggest that mtDNA reduction observed in the CCC myocardium might be linked to the oxidative stress observed in IFN-g and TNF-a-treated human cardiomyocytes. This is corroborated by the gene and protein expression analysis in cytokine-treated AC-16 cardiomyocytes, where decreased levels of proteins involved in ATP generation mitochondrial protein FIGURE 7 | Gene expression analysis on AC-16 cardiomyocytes. Cells were stimulated with IFN-g (10 ng/ml) or TNF-a (5 ng/ml) or IFN-g + TNF-a during 1 or 6 or 12 or 24 or 48h. On cardiomyocytes stimulated with TNF-a 1052 DEGs were detected whereas on cardiomyocytes stimulated with IFN-g 769 DEGs were detected. Finally, on cardiomyocytes stimulated 48h with IFN-g + TNF-a 1443 DEGs were detected. Venn diagram describes the DEGs shared by the various stimulations. Each stimulation was performed 4 times (4n). and ion import and mitochondrial structural maintenance proteins and upregulated expression of proteins involved in ATP catabolism and mitochondrial transition pore. In addition, we observed pathways analysis of CCC heart tissue indicative of mitochondrial dysfunction, increased oxidative stress, and cardiac necrosis, all pointing towards mitochondrial stress and reduced functional capacity. In line with these, stimulation with IFN-g and TNF-a depleted ATP production in AC-16 cells. Previous studies described the dampening of energy metabolism enzymes in CCC heart tissue, such as ATP synthase a and creatine kinase activity in patients (18)(19)(20)60) and studies with animal models correlated with this outcome (61). Decreased in vivo ATP production was also observed in the CCC myocardium (21). In addition, our group identified an accumulation of heterozygous pathogenic variations including loss-of-function and stopgain/truncation of key mitochondrial genes in CCC patients (62). The loss of function mutation in one of the studied families was dihydroorotate dehydrogenase (DHODH) R135C. DHODH is involved in the oxidative phosphorylation by donating electrons to complex III and treatment with DHODH inhibitor Brequinar in IFN-g+TNF-atreated AC-16 cardiomyocytes caused additive damage to mitochondrial DYm (62).
The inhibition of IFN-g and TNF-a downstream molecules and pathways -STAT1/NF-kB, NOS2 -was important for the restoration of AC-16 DYm and reduction of nitrite levels in AC-16 cardiomyocytes. The inflammatory milieu (IFN-g, TNF-a, and IL-1b) enhanced ROS production in T. cruzi infected cardiomyocytes (51). Also, ROS production directly signaled the nuclear translocation of RelA (p65), NF-kB activation in AC-16 cells (63), indicating a positive feedback loop of stimulation between oxidative stress and NF-kB signaling. Studies reported that long-term sustained increase in ROS and RNS promotes cardiomyocyte dysfunction and apoptosis (64) resulting in reduction of mitochondrial DYm, lipid betaoxidation (65) and ATP generation (66,67).
We found that the treatment of AC-16 cells with agonists of AMPK (resveratrol, AICAR and metformin) and NRF2 (protoporphyrin IX cobalt and resveratrol) rescued DYm. AMPK and NRF2 are involved in the cellular response to oxidative stress by countering the damaging effects of NF-kB and by promoting ATP production and regulation of important physiological processes to restore heart function, such as autophagy (68)(69)(70)(71)(72). Our data showed that these agonists ameliorate IFN-g/TNF-a-damaging effect to cardiomyocytes DYm. Similarly, activation of AMPK (metformin) was shown to inhibit the enhancing effect of IFN-g on the DOX-induced cardiotoxicity and prolonged the survival time in DOX-treated mice (65). Indeed, a recent study showed that antioxidants such as resveratrol and mitochondria-targeted antioxidants have potential benefits for the control of oxidative stress in the myocardium of mice with experimental Chagas disease cardiomyopathy (73). Our work potentially found the mechanistic link for the findings that treatment of chronically T. cruzi-infected mice with SIRT1 and/or AMPK agonists SRT1720, resveratrol and metformin or antioxidants reduced myocardial NF-kB transcriptional activity, inflammation and oxidative stress, resulting in beneficial results for restoration of cardiac function (73,74).
Our transcriptomic profiling on cytokine-stimulated AC-16 cardiomyocytes over time (0 to 48h) showed that a cardiomyocyte can respond to inflammatory stimuli by producing inflammatory cell-attracting chemokines and inflammatory cytokines on its own as early as 1h after stimulus, perpetuating inflammation. Previous studies by our group in a subset of 10 out of the 30 CCC samples studied here observed increased mRNA expression of multiple chemokines and cytokines (9,36,75) in CCC heart tissue. At each time point, several genes associated with the mitogen-activated protein kinase (MAPK) signaling pathway were differentially expressed. This pathway is also one of the main inducers of the NF-kB pathway that is activated after inflammatory stimulus, ischemia/reperfusion, in congestive heart failure, dilated cardiomyopathy, after ischemic and pharmacological preconditioning, and in hypertrophy of isolated cardiomyocytes (76). Pathway analyses of gene and protein expression in the IFN-g and TNF-a stimulated AC-16 cardiomyocytes profile were consistent with disturbances of ATP production and increased levels of reactive oxygen species. These metabolic changes affect cardiac ion channel gating, electrical conduction, intracellular calcium handling, and fibrosis formation.
As shown in the functional energy metabolism experiments, the integrity of the mitochondria is affected (proton leak). Therefore, cytokine-treated cardiomyocytes significantly increased their respiration (basal respiration, maximal respiration, spare respiratory capacity, as well as the percentage of basal respiration used to produce ATP. However, as mitochondria are less numerous (as seen with the reduced mitochondrial DNA) and their integrity is affected, the production of mitochondrial and glycolytic ATP is decreased in association with a significant decrease of the glutamine and fatty acid oxidation dependencya reduction in metabolic flexibility that is found in failing hearts (77). This is in line with the IFN-g and TNF-a induced NF-kB activation and activation of expression of NADP oxidases, and inducible nitric oxide synthase (NOS2), leading to the production of large amounts of NO and reactive nitrogen species (12,78,79) leading to synthesis of the peroxynitrite anion (ONOO − ) production, a strong oxidant arising from the reaction of NO with superoxide radical (O − 2 ) (46). This study has limitations, since we studied frozen human heart tissue samples and cytokine-treated human cardiomyocytes in vitro. Although some of the changes observed in IFN-g and TNF-a-treated AC-16 cardiomyocytes closely parallel those observed in CCC heart tissue, this convergence is not proof that the findings in tissue are induced by the same cytokines, since several other mechanisms can induce nitro-oxidative stress and mitochondrial damage in CCC myocardium and as a consequence of heart failure. Conversely, our results with cytokine-treated cardiomyocytes can bring insight not only about Chagas disease, but also in other cardiac disorders where IFN-g and TNF-a play a role, such as inflammatory cardiomyopathies of other etiology.
This study demonstrated that stimulation with IFN-g and TNFa in human cardiomyocytes causes mitochondrial damage, oxidative and nitrosative stress, paralleling events observed in the cytokine-rich CCC heart tissue. It is important to notice that the part of the CCC samples analyzed here have been previously assessed for cytokine expression and IFN-g was among the most highly expressed cytokine, while IFN-g and TNF-a were the top upstream regulators (9,36). Both CCC myocardium and stimulated cells exhibited damaging profiles in markers of cellular stress and increased ROS and RNS, and decreased ATP. Several mitochondrial-related pathways important for mitochondrial integrity, function and ATP production were dysregulated in cytokine-stimulated cells. Also, cytokine-stimulated cells exhibited impaired DYm and increased ROS and RNS and higher amounts of ROS. It is important to point out the direct involvement of the STAT1/NF-kB/NOS2 signaling pathway in the damaging effects of IFN-g and TNF-a in cardiomyocytes' DYm, as well as the restorative effects of stimulating the AMPK, NRF2 and SIRT1 pathways ( Figure 8). Our results suggest that cytokine-induced disturbances in mitochondrial function and energy metabolism might play a role in the worse prognosis of Chagas disease cardiomyopathy. Therapy targeting mitochondrial function and energy imbalance should thus in principle be beneficial to restore cardiac function in CCC and other IFN-g-dependent inflammatory heart diseases, like viral myocarditis and inflammatory cardiomyopathy of other etiologies, age-related myocardial inflammation and functional decline (80), myocardial infarction (81), and anthracycline antitumoral agent cardiotoxicity (65). cardiomyocytes with IFN-g/TNF-a increased ROS, RNS and proton leak, impaired DYm, depleted ATP production and changed the metabolic profile of the cells. Right panel: in our rescue model, we showed that pharmacological inhibition of molecules involved in the IFN-g/TNF-a/NF-kB/NOS2 pathway ameliorated the DYm and NO production. Additionally, activation of AMPK/SIRT1 and NRF2 had beneficial impact on the DYm. Thus, we hypothesize that mitochondrial dysfunction is driven by the excessive production of IFN-g/TNF-a in CCC myocardium and is an essential component for the poor prognosis of Chagas disease cardiomyopathy. Mitochondriatargeted therapies might improve CCC disease progression. Compounds in yellow are inhibitors; Compounds in light green are agonists. Connecting arrows indicate activation and flat line means inhibitory interaction. Red and green arrows indicate the changes observed before and after treatment with the compounds.
Further studies with induced pluripotent stem cells derived cardiomyocytes (iPS-CM) can be employed to investigate, in a personalized, patient-specific manner, the effect of the cytokines in mitochondrial function.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The protocol was also approved by the INSERM Internal Review Board and the Brazilian National Ethics in Research Commission (CONEP). The patients/participants provided their written informed consent to participate in this study.  . This work was founded by the Inserm Cross-Cutting Project GOLD. This project has received funding from the Excellence Initiative of Aix-Marseille University -A*Midex a French "Investissements d'Avenir programme"-Institute MarMaRa AMX-19-IET-007. JN was a recipient of a MarMaRa fellowship. ECN and JK are recipients of productivity awards by CNPq. The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

ACKNOWLEDGMENTS
We thank Andreía Kuramoto Takara (Laboratory of Immunology, InCor) and Victor Debbas (Laboratory of Vascular Biology, InCor) for their always helpful technical assistance.

SUPPLEMENTARY MATERIAL
The Supplementary Figure 4 | Schematic representation of the inhibitors and agonists and their targets in the IFN-g and TNF-a signaling pathways. The proinflammatory cytokine IFN-g interacts with its transmembrane receptor IFNGR1 and signals mainly through the signal transducer and activator of transcription 1 (STAT1) and Interferon Regulatory Factor 1 (IRF1) intracellular transduction pathway to achieve transcriptional activation of IFN-g-inducible genes, such as TNF-a and Nuclear Factor Kappa B (NF-kB). Activation of NF-kB leads to an upregulation of Nitric oxide synthase 2 (NOS2) gene, which in turn increases the amount of intracellular nitric oxide (NO), reacting to reactive oxygen species (ROS) to generate peroxynitrite (ONOO -). Arrows indicate activation and flat line means inhibitory interaction. While ONOOis important for pathogenic response, it causes damages to mitochondria, leading to reduction of mitochondria membrane potential and fragmentation. Proteins such AMPK and NRF2 are involved in the cellular response to oxidative stress countering the damaging effects of NF-kB. Compounds highlighted in yellow are inhibitors and green are agonists.
Supplementary Figure 5 | Transcriptomic analysis on AC-16 cardiomyocyte cell line stimulated with IFN-g and TNF-a. AC-16 cells were stimulated with IFN-g and TNF-a during 1h to 48h. Gene expression analysis was done between the various time points taking as reference the T=0h time point. At each time is provided the volcano plot and the heatmap. Each stimulation was performed 4 times.