Effect Of Testosterone On CD4+ T Cell Metabolism And Cardiac Hypertrophy In An Experimental Model Of Gender-Affirming Hormone Therapy (Testo-GAHT)

Introduction: Testosterone is utilized in gender transition protocols for transmasculine individuals. It has pro-hypertrophic effects in the myocardium and enhances glycolytic pathways in cardiomyocytes that modulate cardiac remodeling. T cells are crucial for immune responses and contribute to cardiovascular disease. Effector CD4+ T cells drive adverse cardiac remodeling. Pyruvate kinase (PK) regulates glycolysis, but its PKM2 isoform is mostly inactive in monomeric/dimeric forms, requiring allosteric activation for full function. In the present study, we tested the hypothesis that testosterone, impairs cardiac function by increasing the glycolytic metabolism in CD4 + T cells in an experimental model of gender-affirming hormone therapy (testo-GAHT). Methods: Female C57/BL6 wild type (WT) mice, female Rag1 knockout (Rag1 -/- ) mice, Rag1 -/- that received WT CD4 + T cells (Rag1 -/- ¬CD4 + ), and female mice that do not express the enzyme Pkm2 in CD4 + T cells (CD4 Cre Pkm2 fl/fl ) were treated with testosterone cypionate (48 mg.kg -1 .wk -1 ) or vehicle (peanut oil), for 8 weeks. Results: Echocardiogram analysis showed that testo-GAHT in WT females promoted cardiac hypertrophy characterized by increased intraventricular septum thickness (IVSd), and decreased left ventricular internal diameter (LVIDd); and functional changes - characterized by increased E/A wave ratio as an indicator of diastolic dysfunction, along with elevated ejection fraction (EF%) and fractional shortening (FS%) as measures of contractility, and heart rate (BPM); and decreased left ventricular stroke volume (LV Vols). Testo-GAHT (for 24 weeks) also increased blood pressure in WT females. Flow cytometry analysis of ex vivo hearts showed increased CD31 + ICAM + cells and decreased CD45 + CD11b + in testo-GAHT WT mice. In the mediastinal lymph nodes (mLN), the number of T cells remained unchanged, although effector CD4 + CD44 hi CD62L lo cells increased. In the spleen, there was a decrease in CD4+ T cells, but like the mLN, the frequency of effector cells increased, while the frequency of CD4 + RORgt + cells decreased. The cardiovascular changes induced by testo were not observed in Rag1 -/- females. Transfer of CD4 + cells to Rag -/- mice (Rag1 -/- ¬CD4 + ) restored the cardiovascular effects (IVSd, EF%, FS%, BPM and CO) of testo-GAHT. Ex vivo, by extracellular flux assays (Seahorse XF96 analyzer), testo increased glycolysis, glycolytic capacity, and non-glycolytic acidification, assessed by the extracellular acidification rate (ECAR) assay, and increased gene expression of solute carrier family 2 (GLUT1), lactate dehydrogenase A (Ldha) and Pkm2, suggesting an increased metabolism in these cells. CD4 Cre Pkm2 fl/fl females on testo-GAHT showed preserved EF%, BPM and CO, parameters elevated in littermate controls under hormonal treatment. In vitro, testo-treated CD4+ T cells preincubated with a pharmacological inhibitor and activator of Pkm2 tetramerization exhibited reduced differentiation of activated CD4+ T cells toward Th17 profile, analyzed by flow cytometry. Conclusion: Despite the adverse effects on cardiac function and blood pressure, testo provides significant immunomodulatory advantages. The glycolytic pathway plays a crucial role in these benefits, modulating CD4 T cell activity and reducing the inflammatory response, revealing important targets that can be used as markers of cardiovascular risk in transmasculine individuals undergoing testo-GAHT. Approval by the Ethics Committee: CEUA-FMRP/USP (1026/2021) and IACUC-Tufts University (B2023-25). Financial support: FAPESP, CAPES, CNPq and NIH. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.