Measuring Cellular ROS via Fluorescence: Cellular ROS levels were assessed using fluorescence in cells plated in 96-well microtiter plates at a density of approximately 2 × 10⁴ cells per well. After a 6-hour incubation, the cells were stimulated by exposing them to 2 μM DOX for 3 hours, inducing oxidative stress and inflammatory damage. ROS production triggered by DOX was evaluated through intracellular detection of the fluorescent probe 2',7'dichlorofluorescein (DCF). Inside the cells, DCF undergoes hydrolysis by esterases, which remove its acetate group, trapping the probe within the cytoplasm. Oxidation of DCF produces fluorescence directly proportional to the total ROS generated. In this assay, HL-1 cells exposed to DOX were incubated with 5 μM DCF in DMSO for 30 minutes at 37°C in the dark. Afterward, the cells were rinsed twice with 1× PBS, and fluorescence was measured using the Promega GloMax Explorer Microplate Reader with excitation and emission wavelengths of 488 nm and 525 nm, respectively. ROS levels were quantified using a DCF standard curve prepared in DMSO (0-1 mM). For electrochemical ROS analysis, cells were seeded in P6-well plates at a density of 5.0 × 10⁵ cells per well. After reaching 70-80% confluence, the cells were serum-starved in a serum-free medium for 6 hours before being stimulated with DOX under the same conditions described above. Following 3 hours of DOX treatment, the cells were collected in 1× PBS, and ROS levels were measured using electrochemical biosensors. In vivo animal study: Eight-week-old adult male C57BL/6J mice were procured from Charles River Laboratories in Milan, Italy. The mice were assigned to experimental groups and received DOX at a dosage of 5 mg/kg via intraperitoneal (i.p.) injection once a week for 4 weeks, resulting in a total cumulative dose of 20 mg/kg per animal. At the end of the study, the mice were humanely euthanized under general anesthesia with 2.5% isoflurane inhalation, followed by cervical dislocation. Heart tissues were collected and homogenized in PBS1X to measure ROS levels using biosensors. All animal care and experimental procedures were conducted in accordance with both national and international regulations. The study received approval from the local animal care office (Centro Servizi Veterinari, University of Naples Federico II) and the Italian Ministry of Health. It was carried out in full compliance with Italian Legislative Decree No. 26 (March 4, 2014), EU Directive 2010/63/EU on animal experimentation, ARRIVE guidelines 2.0, and the Basel Declaration, emphasizing adherence to the 3Rs principle of ethical animal research.