Characterization of testosterone and androgen receptor action in human and rhesus macaque heart muscle cells
by Yuliia Tereshchenko
Date of Examination:2024-03-13
Date of issue:2024-03-26
Advisor:Prof. Dr. Rüdiger Behr
Referee:Prof. Dr. Rüdiger Behr
Referee:Prof. Dr. Stefan Jakobs
Persistent Address:
http://dx.doi.org/10.53846/goediss-10407
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Description:Main PhD thesis
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Abstract
English
Androgens are sex steroid hormones that influence various processes in reproductive tissues and peripheral organs. Their lipophilic nature enables free diffusion through the plasma membrane. In the cytoplasm, testosterone (T) binds to its receptor, the androgen receptor (AR), and the complex then translocates to the nucleus for activation or inhibition of gene expression. Although the effects of androgens are studied greatly in healthy and diseased reproductive tissues, little is known about their cellular and molecular action in the cardiovascular system. Clinical research reported the impact of high and low T levels on the human heart and vasculature, but the data are currently conflicting. Additionally, most of the molecular and cellular processes triggered by androgens in heart muscle cells were researched using rodents. These models vastly contributed to our understanding of biological processes, though are partially limited due to differences in heart physiology to humans. This work focused on studying the impact of male physiological (25 nM) and supraphysiological (100 nM) concentrations of T on primate cardiomyocytes (CMs) of human and rhesus macaque origin. For the first time, AR was detected in CM nuclei of male rhesus macaque tissue. Notably, no signal was detected in female heart cells. For further in vitro studies we used an induced pluripotent stem cell (iPSC) model. AR was detected in human and rhesus macaque iPSCs and was induced and translocated by T. A directed cardiac differentiation protocol was optimized and used to generate CMs from male and female human and rhesus macaque cell lines. Non-treated cells showed cytoplasmic AR staining while T-treated cells displayed mostly nuclear signals. T treatment induced AR abundance in male rhesus iPSCs and iPSC-CMs. Interestingly, we detected an AR-V7 isoform with no T-binding capabilities in human iPSC-CMs and human left ventricular tissue. Since T may also exhibit AR-independent action, we generated a clonal AR knock-out (KO) human male iPSC line to investigate AR-independent effects. Further analysis of the AR KO cell line showed fewer CMs generated after differentiation, a higher proliferation rate, and a more disorganized contractile apparatus compared with wild-type (WT) cells. The analysis of differentially expressed genes in KO and WT cells further highlighted a more immature state of KO CMs. While 24 hours of T treatment resulted in a decrease in Ca2+ current density, 48 hours of exposure did not result in significant changes between treated and non- treated groups of WT cells. Overall, the data has shown a more drastic effect of the AR KO itself than T treatment. That is why we hypothesized T conversion to other androgens. LC- MS/MS analysis demonstrated low levels of T in cell culture media 48 hours after T addition confirming our assumption. Moreover, androstenedione, a weaker androgen than T, was detected in human samples, but almost none in rhesus. In conclusion, this work demonstrates relevant effects of the T / AR system on proliferation, cytoskeleton organization, electrophysiological parameters, and gene expression of primate CMs.
Keywords: Testosterone; Induced pluripotent stem cells (iPSC); Cardiomyocyte; Hormone regulation; Primates
