Chagas disease (ChD) is the second most important cause of infectious cardiomyopathy worldwide [1,2], only behind COVID-19 [3]. Chagas heart disease is the most disabling and deadly manifestation of T. cruzi infection [1,2]. This is a complex condition that courses with cardiomyocytolysis, autonomic denervation, persistent inflammation, autoimmune damage, and oxidative stress determined by free radicals upregulation (e.g., OH•−, O2•−, and ONOO•⁻) and antioxidants insufficiency (e.g., enzymatic and non-enzymatic effectors) [4,5]. Although Chagas cardiomyopathy (CC) is multifactorial, redox imbalance plays a prominent role in its pathogenesis, being mediated by disturbances in mitochondrial complexes I and III in T. cruzi-infected cardiomyocytes [4,6,7], inducible nitric oxide synthase (iNOS) and NADPH oxidase II upregulation in activated leukocytes (e.g., respiratory burst) [4,6,7]. Despite the antiparasitic purpose, reactive species can also stimulate T. cruzi replication and favor cellular parasitism [8]. In addition, the nonspecific nature of these pro-oxidant reactions determines extensive microstructural and molecular cardiac damage, worsening myocarditis [8,5]. As the host cannot naturally eliminate the parasite, inflammation and oxidative stress persist and feed on each other, determining progressive morphofunctional cardiac degeneration, heart failure and eventually death [[8], [9], [10]].

Classically, it has been proposed that T. cruzi is highly sensitive to oxidative stress [11]. Accordingly, some studies suggest that pro-oxidant effectors (e.g., NO, HClO and H2O2) participate in T. cruzi elimination and replicative control by the host's cellular-based immune defenses [12,13]. However, divergent evidence indicates that the oxidative environment favors infection, becoming a stimulus for parasite growth in the acute phase, as well as for cardiomyopathy onset and progression in the chronic phase of ChD [14]. Despite this controversy, previous studies indicate that modulation of redox metabolism may be relevant to attenuating pathological outcomes associated with T. cruzi infection, including CC [15,2,9]. In this sense, antioxidant therapy with vitamins (e.g., C and E) [16], resveratrol [17] and curcumin [15] exerted marked myoprotective and/or cardioprotective effects, attenuating parasitism, molecular oxidation (e.g., lipid and protein), and myocarditis in T. cruzi-infected mice [9,17].

Currently, antioxidant therapy applied to Chagas disease is mainly focused on free radical scavengers [18,2,9]. However, the neutralization of these molecules only occurs after their production, enabling the prior activation of signaling pathways linked to cell parasitism. This characteristic could partially explain the limited therapeutic effectiveness of some antioxidants, including vitamins C [19] and E [2]. Although T. cruzi-induced mitochondrial dysfunction is the main source of pro-oxidant effectors in cardiomyocytes, direct mitochondrial inhibition of free radical biosynthesis has been overlooked. Thus, the availability of mitochondrial uncouplers represent a valuable opportunity in the search for more effective cytoprotective and cardioprotective redox modulators [20] in ChD [18,9].

2,4-dinitrophenol (DNP) is a classic mitochondrial uncoupler, which acts through the dissociation of electron transport in the respiratory chain [20]. In this process, DNP attenuates reactive oxygen-derived species (ROS) biosynthesis and prevents oxidative stress [20]. Accordingly, DNP may be more efficient in preventing oxidative stress compared to antioxidants that act in the cytosol, avoiding excessive ROS production and the exhaustion of endogenous antioxidant effectors [20,21]. Considering the mechanism of action of DNP, this drug may be relevant in modulating the oxidative stress invariably triggered by T. cruzi infection, having a potential impact on ChD severity and progression. Thus, the impact of DNP-based chemotherapy on Trypanosoma cruzi-induced acute myocarditis was investigated. In addition to ROS production, protein and lipid oxidation, immunological, parasitological and microstructural cardiac outcomes associated to DNP treatment were evaluated and compared to benznidazole (Bz)-based reference chemotherapy.