HEPES, 10 BDM (pH=7.4 with NaOH) for murine ventricular cardiomyocytes or .... stem cell model of catecholaminergic polymorphic ventricular tachycardia.
Online Supplementary Material Detailed Material and Methods Isolation of ventricular cardiomyocytes. Heterozygous knock-in RyR2R4496C/WT mice (1) and their wild-type littermates of either sex aged 8–16 weeks were used for the isolation of ventricular cardiomyocytes. Ventricular cardiomyocytes were isolated by retrograde perfusion through the aorta using a standardized enzymatic digestion protocol (2) with minor modifications. After cervical dislocation, the heart was quickly excised, arrested in icecold Tyrode’s solution containing (in mM) 135 NaCl, 4.7 KCl, 0.6 KH 2PO4, 0.6 Na2HPO4, 1.2 MgSO4, 10 HEPES, 30 taurine, 10 2,3-butanedione 2-monoxime (BDM), 10 glucose, pH=7.46 with NaOH) supplemented with 10 IU/ml heparin, and perfused retrogradely with Tyrode’s solution through the aorta. The heart was then placed on a Langendorff mode perfusion system and perfused at 37°C with oxygenated Tyrode’s solution containing 20 mM creatine for 5 minutes before being digested by perfusion with Liberase™ (Roche Diagnostics, Penzberg, Germany) dissolved in creatine-containing Tyrode’s solution at a final concentration of 37.5 ng/ml for 0.7 s/mg heart weight. The ventricles were transferred to modified Tyrode’s solution containing 12.5 μM CaCl2 and 10% FCS to stop digestion and cut into small pieces. After mechanical separation by trituration, the suspension was filtered through a 200 µm nylon mesh and myocytes were allowed to sediment by gravity for 10 minutes. Cells were then transferred into Tyrode’s solution with 1.5 mM CaCl 2 to reintroduce physiological Ca2+ concentrations. Only excitable, rod-shaped, quiescent cells were used for experiments. Human iPSC cardiomyocytes. iPS cells carrying RyR2S406L/WT mutation were obtained from a 24-year old caucasian female CPVT patient and have been described elsewhere (3). Cells obtained from a 32-year-old female caucasian without history of cardiac disease scheduled for plastic surgery served as a healthy control. Human iPSC generation via reprogramming of primary skin fibroblasts and cardiomyocyte differentiation was performed as described previously (3, 4). Spontaneously beating areas were explanted after three to five months and enzymatically dissociated into single cardiomyocytes with 1.5 mg/mL type II collagenase (Worthington, Troisdorf, Germany) for 1h at 37°C. Depending on dissociation, up to 4 further digestion steps (for a maximum of 30 min each) were performed. Afterwards, remaining cell clumps were subjected to a 10 min digestion step with Accumax (Millipore Merck KGaA, Darmstadt, Germany) in DPBS. Single cells were plated on fibronectincoated glass bottom dishes (MatTek, Ashland, MA, USA) and subjected to further experiments after incubation at 37°C and 5% CO2 on an average time of 7 days. Confocal Ca2+ imaging. Cardiomyocytes were loaded with 3 μM Fluo-4 AM (Thermo Fisher, Germany) and 0.06% (w/v) pluronic® F-127 (Sigma-Aldrich, Germany) under protection from light for 40 minutes at room temperature (RT) in external solution containing (in mM) 140 NaCl, 4 KCl, 1 MgCl 2, 1 CaCl2, 10 glucose, 5 HEPES, 10 BDM (pH=7.4 with NaOH) for murine ventricular cardiomyocytes or external solution containing (in mM) 140 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 10 glucose, 5 HEPES for human iPSC-derived cardiomyocytes respectively, followed by incubation in external solution for 20 minutes to allow de-esterification of the dye. Calcium transients in Fluo-4 AM loaded cardiomyocytes were elicited by electric field stimulation using a S48 square pulse Stimulator (Grass Technologies, Warwick, RI, USA) to apply 5 ms test pulses at 0.5 Hz with 30 V/cm electrode distance (80 V/cm for iPSC derived cardiomyocytes) and visualized on an inverted confocal microscope TCS SP5 (Leica, Germany) equipped with a HCX PL APO CS 63x/1.4 oil immersion objective. Fluo-4 AM was excited with the 488 nm line of an Argon laser and emission was collected between 498 and 627 nm. Line scan series were generated along the long axis of a myocyte at 400 Hz. Ca 2+ transient kinetics were further analyzed using pClamp 10.4 software (Molecular Devices LLC, CA, USA). The amplitude of the Ca 2+ transient is expressed as ∆F/F0, the activation time as time to peak and Ca 2+ removal from the cytosol was analysed by calculating the inactivation constant Tau of a monoexponential fit of the decay phase of the Ca 2+ transient. Examination of spontaneous Ca 2+ sparks was carried out using the SparkMaster plugin in ImageJ (5). Electrophysiology. Electrophysiological properties were examined in the current clamp mode of the perforated patch-clamp technique using an EPC10 amplifier (HEKA Elektronik Dr. Schulze GmbH, Lambrecht, Germany). Cardiomyocytes were bathed in the recording chamber filled with Tyrode´s solution composed of (in mM) 135
NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, (pH=7.4 with NaOH). A temperature-controlled superfusion was used to maintain the temperature of the bath solution at 35 ± 2°C. Cells were viewed on an inverted Olympus IX70 microscope (Olympus, Germany). Patch pipettes had a resistance of 2.5-10 MΩ when filled with the pipette solution composed of (in mM) 30 KCl, 110 K-aspartate, 1 MgCl 2, 10 HEPES, 0.1 EGTA, (pH=7.2 with KOH). An average of 5 min exposure to 900 µg/µL Amphotericin B (Sigma-Aldrich) was sufficient to achieve perforated patch clamp configuration. Only cardiomyocytes with a membrane potential lower than -60 mV and with series resistance of
