Isoproterenol hydrochloride

All drugs were freshly prepared immediately before experiments and dissolved in phosphate-buffered 0.9% saline (Gibco, pH 7.4). Drugs were administered intraperitoneally at their corresponding dosages: Yohimbine-hydrochloride (3 mg/kg, Merck, Germany), propranolol-hydrochloride (10 mg/kg, Merck, Germany), prazosin-hydrochloride (1 mg/kg, Merck, Germany), and clozapine (0.03 mg/kg, Merck, Germany).
For intra-hippocampal infusions of isoproterenol hydrochloride (Merck, Germany), animals were restrained and the guide cannula was inserted with an injector needle (62236, RWD Life Science, China) connected to an infusion pump (R462 Syringe Pump, RWD Life Science, China) via plastic tubing. Prior to attachment, the tubing was filled with sunflower seed oil (Merck, Germany) and vehicle (0.9% saline) or isoproterenol, separated by a small air bubble. Afterward, animals were allowed to freely roam their homecage for 2 min followed by bilateral intra-hippocampal infusions of vehicle drug or 1 μl of isoproterenol (3 μg/μl diluted in phosphate-buffered 0.9% saline) at 50 μl/min. Diffusion of vehicle and isoproterenol was allowed for another 2 min, before the animal was detached from the infusion setup and returned to its homecage.

Ca²⁺ imaging started 2–5 weeks after surgical procedures to allow for postoperative recovery and fluorescent indicator expression [3 (link),63 (link),64 (link)]. On the experimental day, mice were attached to the miniature microscope and placed in the recording chamber. Mice were allowed at least 10 min to settle in the familiar chamber to which they were previously habituated. Next, baseline image capture occurred for 5 min. Mice were then injected intraperitoneally with 5 mg/kg of the β-AR agonist (-)-isoproterenol hydrochloride (#I6504, Sigma-Aldrich) made in saline [67 (link),68 (link)]. Image capture occurred 20 min post-injection for 2 min. Mice were undisturbed during recordings.

Isoproterenol hydrochloride (Sigma Aldrich) was dissolved in dimethyl sulfoxide (DMSO) (Sigma Aldrich), and Verapamil hydrochloride (Sigma Aldrich) in methanol (VWR) to prepare 1 M stock solutions. Each drug solution was 10- fold serially diluted in RPMI/B27 + Ins media to make final concentrations between 0.0001–1 µM. As untreated controls, complete RPMI/B27 + Ins media supplemented with DMSO or methanol were used. For data collection, the 3D hOCMTs with drug-supplemented media were incubated for 10 min at 37 °C, 5% CO₂ each time the drug concentration changed, followed by live imaging with video acquisition per condition with Zeiss LSM 780 confocal microscope. Chemotherapy-induced cardiotoxicity was evaluated in 3D hOCMTs treated with RPMI/B27 + Ins supplemented with doxorubicin (Doxo) (Sigma Aldrich), at 0.1 µg/mL and 1 µg/mL concentrations, or plain RPMI/B27 + Ins as control for 6 days. Growth medium was exchanged every 3 days, and 3D hOCMT morphology and beating were monitored daily by live video recording with Zeiss LSM 780 confocal microscope for 6 days. On day 6 of drug treatment, the 3D hOCMTs were collected for evaluating viability.

All the local treatments were carried out with the help of cannulas (33G). Guide cannulae (26 gauge, Plastics One) were implanted to the skull with dental cement. Mice were then given 1 week to recover from surgery. 32-gauge stainless steel injectors attached to 5-μl Hamilton syringes were inserted into the guide cannulae to deliver the following drugs—Isoproterenol Hydrochloride (0.25 μg per side; Sigma Aldrich, India); ( ±)-Propranolol hydrochloride (0.5 μg per side; Sigma Aldrich, India), and 0.9% saline. Coordinates relative to bregma are as follows: vH (AP − 3.0, ML ± 2.6, DV − 3.2). A total volume of 200 nl was injected; the injector was left for another minute to allow diffusion into the tissue.