Supraphysiological Levels of Testosterone Induce Cardiac Dysfunction via NLRP3 Inflammasome Activation in Macrophages

Abstract

IntroductionTestosterone (Testo) modulates vascular tone and cardiac performance. Athletes who use Testo at supraphysiological doses exhibit increased blood pressure, higher inflammatory marker levels, vascular dysfunction, and cardiac hypertrophy. NLRP3 inflammasome activation, as part of the innate immune system response, contributes to proinflammatory cytokines production, leading e.g to cardiac dysfunction.HypothesisWe hypothesized that supraphysiological levels of Testo promotes cardiac dysfunction via generation of mitochondrial reactive oxygen species (mROS) and activation of the NLRP3 inflammasome.MethodsMale, 12 week‐old wild type (WT) and NLRP3 knockout (NLRP3‐/‐) mice were used. Mice were treated with testosterone propionate [Testo‐P (10 mg/kg)] or vehicle for 30 days. Cardiac function was evaluated using echocardiography. After in vivo experiments, western blot and ELISA were performed to evaluate NLRP3 inflammasome components. ROS generation was evaluated by lucigenin. Bone marrow‐derived macrophages (BMDMs) were isolated, primed with lipopolysaccharide (LPS [500 ng/mL 4 h]) and stimulated with Testo [10‐7 M], for 4, 6, 12 and 24 h. ROS generation was evaluated by DHE fluorescence and MitoSOX.ResultsEchocardiography showed cardiac dysfunction in WT mice treated with Testo‐P, characterized by reduced ejection fraction, shortening fraction, cardiac output and systolic volume. Testo‐P also increased interventricular septum, left ventricle posterior wall and decreased left ventricle internal diameter. All these effects were observed in NLRP3‐/‐mice. Furthermore, WT mice treated with Testo‐P showed increased cardiac expression of NLRP3 receptor, active Caspase‐1 and IL‐1β levels. These effects were prevented in NLRP3‐/‐. In addition, increased ROS generation was observed in the left ventricle of WT mice, but not NLRP3‐/‐ mice, treated with TP. Testo‐P‐treated WT mice showed increased macrophage infiltrate in the left ventricle, and this effect was not seen in the WT vehicle mice. In in vitro experiments, BMDMs primed with LPS and stimulated with Testo showed increased expression of the NLRP3 receptor and IL‐1β levels after 12 and 24 h. In addition, Testo‐stimulated BMDMs exhibited increased mROS generation.ConclusionSupraphysiological levels of testosterone induce cardiac dysfunction via mROS generation and NLRP3 inflammasome activation. This study was approved by the Ethics Committee on Animal Experimentation of the Ribeirao Preto Medical School (020/2021).