A momentous side-effect of the treatment with anthracyclines like doxorubicin (DOX) is the cumulative, dose-dependent damage of the heart. The chronic anthracycline-induced cardiotoxicity (ACT) leads to the development of symptomatic heart failure in 6 to 9 % of all patients treated and is greatly impairing patient outcome. The search for preventive and therapeutic strategies has yielded little success so far as molecular mechanisms are not fully understood. The production of reactive oxygen species (ROS), mitochondrial dysfunction and topoisomerase 2β inhibition have been proposed as parts of the complex and
multifactorial disease model. The aim of this study was to investigate the potential role of cardiac fibroblasts in ACT development in terms of ROS production and NAPDH oxidase subunit expression.
Human cardiac fibroblasts (cFB) were isolated from fresh cardiac tissue after heart
transplantation from an ACT and a dilative cardiomyopathy (DCM) patient. Isolated cFBs show morphological characteristics, high proliferation capacity and expression of typical cardiac fibroblast markers as periostin, transcription factor 21 and α-smooth muscle actin in qRT-PCR and immunocytochemistry experiments.
To analyze the acute reaction to anthracycline treatment, cFBs were exposed to 0.1 or 0.25 μM DOX for 24 hours and analyzed regarding the expression of NADPH oxidase subunits on mRNA level via qRT-PCR and on protein level via Western blot. Subunits RAC2 and NCF4 were expressed significantly more highly in cardiac fibroblasts compared to skin fibroblasts. As an immediate response to DOX treatment, tendencies of expression changes were found for NOX2, NOX4, RAC2 and NCF4. Additionally, the expression of NADPH oxidase subunits in tissue of chronic heart failure patients was studied showing that expression levels for the subunits differed between ACT patients, DCM patients and healthy controls.
DOX-dependent production of ROS in cFBs was assessed using the Amplex Red reagent for hydrogen peroxide and DHE-HPLC for superoxide. DOX concentrations of 0.1 to 0.25 μM DOX caused a dose-dependent significant increase of ROS. Also, hydrogen peroxide levels were higher in cardiac fibroblasts compared to skin fibroblasts and NADPH-contributable superoxide production was only measurable when the cells were triggered with DOX treatment. In conclusion, this study suggests that cardiac fibroblasts contribute to ACT development and not only to the manifestation of the fibrotic phenotype following cardiomyocyte apoptosis. Also, the NADPH oxidase appears to be a promising target for further research
based on the expression changes and differences found in this study.2021-11-0
