Involvement of the NLRP3 inflammasome in cardiac dysfunction associated with supraphysiological levels of testosterone

Author(s)

Juliano Vilela Alves

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2024-06-20

Examining board members

Rita de Cassia Aleixo Tostes Passaglia; Michele Mazzaron de Castro; Roger Lyrio dos Santos

• Advisor: Rita de Cassia Aleixo Tostes Passaglia

Abstract

Increased serum testosterone (Testo) concentration is associated with cardiovascular risk factors, including abdominal obesity and increased blood pressure, and cardiovascular diseases (CVDs). Testo modulates vascular tone, cardiac performance, and redox and inflammatory processes, including the generation of reactive oxygen species (ROS) and the release of proand anti-inflammatory cytokines. The NLRP3 inflammasome is a component of the innate immune system and an important regulator of chronic inflammation. NLRP3 activation is mediated, e.g. by mitochondrial ROS (ROSm), leading to the production of pro-inflammatory cytokines, such as IL-1β and IL-18, and contributes to cardiac hypertrophy and renal injury in hypertension models. Considering that Testo stimulates ROSm generation, the present study tested the hypothesis that supraphysiological levels of testo activate the NLRP3 inflammasome in immune cells, resulting in the production, release, and autocrine and paracrine actions of pro-inflammatory cytokines, and cardiac dysfunction. To test our hypothesis, we used wild type (WT) C57BL/6J mice and knockout (KO) mice for the NLRP3 (NLRP3-/-), ASC (ASC-/-), and Caspase-1 (Casp1-/-) proteins. Mice were treated with testosterone propionate [Testo-P (10 mg.kg-1)] or vehicle for 30 days, and cardiac function and structure, and NLRP3 inflammasome activation were determined. Bone marrow-derived macrophages (BMDMs) were isolated and stimulated with Testo [10-7 M] for 4, 6, 12, and 24 hours (h). Cellular and molecular assays were conducted to determine NLRP3 inflammasome activation and ROS generation. WT mice treated with Testo-P exhibited cardiac dysfunction, characterized by reduced ejection fraction, fractional shortening, cardiac output, and stroke volume, determined by echocardiography. Testo-P also increased the interventricular septum, posterior wall of the left ventricle, and decreased the internal diameter of the left ventricle. These effects were not observed in NLRP3-/-, ASC-/-, or Casp1-/- mice. WT mice, but not NLRP3-/- mice, treated with Testo-P showed increased cardiac expression of the NLRP3 receptor, active Caspase-1, and IL-1β levels. Furthermore, WT mice, but not NLRP3-/- mice, treated with Testo-P showed increased ROS generation and increased macrophage infiltration in the left ventricle. In in vitro experiments, BMDMs stimulated with Testo for 12 and 24 h showed increased NLRP3 receptor expression, caspase-1 activation, IL-1β levels, and increased mitochondrial ROS generation. Flutamide [(Flu), androgen receptor antagonist], carbonyl cyanide 3-chlorophenylhydrazone [(CCCP), mitochondrial uncoupler], and a selective inhibitor of the NLRP3 inflammasome (MCC950) attenuated Testo-induced NLRP3 inflammasome activation. Together, our results suggest that supraphysiological levels of Testo induce cardiac dysfunction via mitochondrial ROS generation and NLRP3 inflammasome activation. (AU)

• FAPESP's process: 19/20692-0 - Involvement of the NLRP3 inflammasome in cardiac dysfunction associated with supraphysiological levels of testosterone
• Grantee: Juliano Vilela Alves
• Support Opportunities: Scholarships in Brazil - Doctorate