Isoproterenol

To generate a model of isoproterenol-induced hypertrophy, mice received a dose of 10 mg/kg BW per day isoproterenol (Sigma) or saline control for 10 days via a subcutaneously implanted osmotic minipump (Alzet). For minipump implantation, mice were anesthetized with 3% isoflurane. A small horizontal incision was made through the dermal layers on the dorsal surface of the mouse to create a small pocket into which minipumps were implanted. The skin was sutured shut, following which mice received analgesia (0.1 mg/kg BW buprenorphine) and were allowed to recover at 30°C before return to normal housing.

The patch-clamp experiments was performed as previously described [19 (link),22 (link)]. Briefly, 35,000 atrial iPSC-CMs were plated on glass-bottom Fluoro Dishes and incubated with either isoproterenol (50 nmol/L, Sigma-Aldrich, Taufkirchen, Germany)) or isoproterenol + PF01247324 (1 µmol/L, Sigma-Aldrich, Taufkirchen, Germany)) for 15 min before starting the measurements. The experiments were conducted at room temperature.
Action potential recordings were performed using the whole-cell patch-clamp technique. To elicit action potentials, square current pulses with amplitudes of 0.5–1 nA and durations of 1–5 ms were applied. The stimulation frequency was increased gradually from 0.5 to 2 Hz.
The late sodium current (I_NaL) was measured using the ruptured-patch whole-cell patch-clamp technique. The pipette used had a resistance ranging from 2 to 3 mega-ohms (MΩ). I_NaL recordings were performed exclusively in CMs where a seal with a resistance of over 1 giga-ohm (GΩ) was achieved, and the access resistance remained below 7 MΩ. After a stabilization period of 3 min, the iPSC-derived CMs were held at a holding potential of −120 mV and then depolarized to −35 mV for 1000 ms with 10 pulses and a basic cycle length of 2 s. The I_NaL was quantified as the integral current amplitude between 100 and 500 ms and was normalized to the membrane capacitance.

PGF pads were obtained from wild-type mice after sacrifice by cervical dislocation. Tissues were cut into small pieces (3–5 mg) and pre-equilibrated in pre-warmed Dulbecco’s Modified Eagle Medium (DMEM) (31966047; Life Technologies Thermo Fisher Scientific, Darmstadt, Germany) at 37 °C, 5% CO₂ for 30 min. Then, tissues were incubated with 200 µM L-serine, 400 µM L-serine, with or without 1 µM isoproterenol (Sigma Aldrich, Taufkirchen, Germany) or with 1 µM isoproterenol alone in DMEM for 3 h at 37 °C, 5% CO₂. After the incubation, the media were used for glycerol quantification with the free glycerol colorimetric assay kit (Biovision, Milpitas, CA, USA) and fat pads were delipidated with 2:1 chloroform/methanol solution at 37 °C with shaking for 1 h. Delipidated tissues were transferred to a 0.3 N NaOH/0.1% SDS solution and kept at 40 °C overnight with shaking for protein extraction. Protein measurement was done with the BCA protein assay kit (Thermo Fisher Scientific, Darmstadt, Germany). Lipolysis was calculated based on free glycerol content in the assay media normalized by protein content.

Adipose tissues were collected and washed in PBS before subjected to lipolysis assays. For isoproterenol stimulation, adipose tissues were cut into small tissue pieces and incubated in serum‐free DMEM–High Glucose (Sigma, D5796) with 2% fatty acid‐free BSA (Sigma, 126579) in the absence or presence of 10 μM isoproterenol (Sigma, I6504) for the indicated time. TNFα‐induced lipolysis was induced as previously described (Ju et al, 2019 (link)). In brief, small adipose tissue pieces were cultured in DMEM–High Glucose for 24 h in the absence or presence of 100 ng/ml recombinant TNFα (Peprotech, 315‐01A) and then transferred into serum‐free DMEM containing 2% fatty acid‐free BSA for 3 h. Supernatants were collected and FFA concentration normalized to adipose tissue input.

To determine the effect of E-4031 (an inhibitor of the KCNH2 channel) on beating frequency, videos were taken at the baseline (drug-free culture medium), with the drug, and following washout of the drug. Baseline videos were first acquired for EBs from all groups, in fresh CM Media after equilibration. After baseline videos were acquired, 5 μM of E-4031 (Tocris) were supplemented to fresh CM Media and the EBs were allowed to equilibrate for 20 min before taking videos. EBs were then washed with CM media three times, allowed to equilibrate for 20 min, and videos of the same EBs were taken the third time to determine the frequency of beating after the drug washout.
To determine the effect of constitutive culture with an inhibitor of KCNH2, 5 μM E-4031 was added to the media at the start of the stimulation period. Videos were taken on days 1, 3 and 7 following 20 min of equilibration to fresh media containing E-4031. At the end of the culture period, the cardiomyocytes were washed with fresh CM Media without E-4031 three times, allowed to equilibrate for 20 min and re-evaluated for frequency.
To examine the effects of isoproterenol on cardiomyocytes, videos were first taken at the baseline in fresh CM media after equilibration, and then in CM media containing 1 μM isoproterenol (Sigma).

All hearts were subjected to a stabilization period (15–20 min) before baseline measurement. After that, hearts were perfused with isoproterenol (Sigma-Aldrich, St. Louis, MO, USA) at increasing concentrations (1 nM–1 µM range). A group of aged hearts was treated with 100 μmol/L apocynin (Sigma-Aldrich, St. Louis, MO, USA), a non-selective inhibitor of NADPH oxidase, before and during the isoproterenol challenge. Another group of aged hearts was treated identically with another NADPH oxidase inhibitor, VAS2870 [36 (link)], 20 μM (Merck, Darmstadt, Germany).

We employed a qPCR-based gene expression system that measures the expression of 96 NF-κB dependent immunological genes (StellARray, Lonza, Basel, Switzerland). These genes are recognized for having both κB binding sites in the promoter and gene expression changes associated with increased NF-κB activity. The genes included in the array encode diverse proteins such as cytokines, chemokines, complement proteins, immunological receptors, and transcription factors (for the complete list, see Additional file
1: Table S1). We performed the qPCR array on three biological replicates for each of the following inductions (6 h treatments): untreated (vehicle), isoproterenol only (Sigma-Aldrich), TNF-α only, and isoproterenol + TNF-α. This initial exploration was performed with isoproterenol, known to be a mixed β₁ and β₂ agonist. However, the 1321 N1 cell-line expresses only the β₂-subtype
[19 (link)]. For confirmation with RT-qPCR and rat studies the selective β₂ agonist clenbuterol was preferred excluding effects mediated by other β-adrenergic subtypes.