A0937

cEND and MyEND were treated with a physiological and supraphysiological CAT mix—corresponding concentration c1–c3—as determined from TTS patient serum, comp. Table 1, Epinephrine (Sigma-Aldrich, E4250), norEpinephrine (Sigma-Aldrich, A0937), and dopamine (Sigma-Aldrich, H8502), the respective cell culture media, for 24 h. Epinephrine and norEpinephrine were dissolved in 0.5 M HCl, and culture medium was used as a solvent for dopamine.
For CAT treatment of iPSC-derived cardiomyocytes, human iPSC-derived cardiomyocytes were treated with a physiological mix of these CATs, Epinephrine (Sigma-Aldrich, E4250), norEpinephrine (Sigma-Aldrich, A0937), and dopamine (Sigma-Aldrich, H8502),—corresponding concentration c3/ TTS acute levels as determined from TTS patient serum, comp. Table 1 in RPMI medium supplemented with 1× B-27 (Gibco, 17504001) for 24 h. Additionally to the CAT mix designated c3, cells were treated with a supraphysiological concentration of 5 mM isoprenaline (Sigma-Aldrich, I6504) [42 (link)].

The HL-1 rat cardiomyocytes were cultured on the NWMEA biodevice to test the cellular electroporation and record electrical potential. In the preparation before cell culture, the NWMEA biodevice was incubated overnight with 5 µg/mL fibronectin in 0.02% gelatin solution at 4 °C to enhance cell attachment on the device surface. During the cell culture, the frozen HL-1 cardiac muscle cell line (Louisiana State University Health Science Center, USA) was thawed in a 37 °C water bath and incubated in an HL-1 expansion medium to recover the cardiomyocytes. To purify the HL-1 cardiomyocytes, they were pelleted by centrifuging the HL-1 expansion medium including the cells at 300 × g for 2–3 min and resuspended into 10 to 15 mL of fresh HL-1 expansion medium. The HL-1 cardiomyocyte suspension was transferred into the NWMEA biodevice at a density of 10⁴ cells/cm² and maintained in a standard incubator at 37 °C and 5% CO₂ for the subsequent experiments. The HL-1 expansion medium was made from a mixture of 43.5 mL Claycomb basal medium (51800 C, Sigma-Aldrich), 5 mL qualified FBS (TMS-016-B, EMD Millipore), 0.5 mL l-glutamine at 200 mM (A0937, Sigma-Aldrich), 0.5 mL norepinephrine at 10 mM (TMS-002-C, EMD Millipore) and 0.5 mL penicillin/streptomycin at 100X (TMS-AB2-C, EMD Millipore). The cardiomyocyte culture was imaged by an upright widefield microscope (Olympus BX63, Japan).

The mesenteric bed was immediately removed after animal sacrifice, and isolated mesenteric vessels with an average internal diameter of 250 μm were dissected and prepared as segments with intact PVAT and segments without PVAT. The vessels were studied using a multi-wire myograph system (Model 610 M, version 2.2; Danish Myo Technology) and analysed with ADInstruments Chart™ 5 (version 5.5.1). The vessels were bathed in physiologic saline solution (PSS; as above), maintained at 37°C and gassed with a mixture of 95% air and 5% CO₂. The vessels were normalised and their viability was checked by the addition of PSS containing 60 mM of KCl, following which concentration-response curves to norepinephrine (NE; A0937; Sigma-Aldrich) were constructed. To assess the effect of free radical scavengers on the obese PVAT, vessels from the obese cohort were incubated with superoxide dismutase (SOD; 100 U/ml; Sigma-Aldrich) and catalase (100 U/ml, Sigma-Aldrich) for 45 min, following which another NE concentration-response curve was constructed. Similarly, vessels were incubated with L-NNA (10^-4M, 45 min; Sigma-Aldrich) to assess NO bioavailability within PVAT. When required, removal of the endothelium was performed by a careful brushing technique using a 40-μm-diameter wire (the same as that used for myography) under visualisation by microscopy.