The cardiac mouse cell line HL-1, a cell line derived from the AT-1 mouse atrial cardiomyocyte tumour lineage , was kindly provided by Dr. W.C. Claycomb (Louisiana State University, New Orleans, LA, USA). HL-1 cells were cultivated in Claycomb medium containing 10 % fetal calf serum (FCS), 100 µM norepinephrine and 4 mM l-glutamine (all from Sigma-Aldrich, Munich, Germany) on gelatine/fibronectin coated plates. H9c2 cells (ATCC CRL-1446), stably transfected with the human IR in our laboratory  (H9c2-E2) were cultivated in DMEM, low glucose containing 10 % FCS, 1 % non–essential amino acids and 600 µg/ml G418 (all from Invitrogen, Carlsbad, CA, USA). Commercially available iPS-derived human cardiomyocytes (Cor.4U®) (Axiogenesis, Cologne, Germany) were cultured in Cor.4U® Complete Medium containing 10 % FCS on fibronectin–coated 96-well E-Plate (Acea Biosciences, San Diego, CA, USA). The medium was changed twice daily. All cells were incubated at 37 °C with 5 % CO2 in a humidified incubator.
Competition binding experiments on membrane embedded and solubilised insulin receptor preparations
Isolation of insulin receptor embedded plasma membranes (M-IR) and competition binding experiments were performed as previously described . Briefly, CHO-cells overexpressing the IR were collected and re-suspended in ice-cold 2.25 STM buffer (2.25 M sucrose, 5 mM Tris–HCl pH 7.4, 5 mM MgCl2, complete protease inhibitor) and disrupted using a Dounce homogenizer followed by sonication. The homogenate was overlaid with 0.8 STM buffer (0.8 M sucrose, 5 mM Tris–HCl pH 7.4, 5 mM MgCl2, complete protease inhibitor) and ultra-centrifuged for 90 min at 100,000g. Plasma membranes at the interface were collected and washed twice with phosphate buffered saline (PBS). The final pellet was re-suspended in dilution buffer (50 mM Tric-HCl pH 7.4, 5 mM MgCl2, complete protease inhibitor) and again homogenised with a Dounce homogenizer. Competition binding experiments were performed in a binding buffer (50 mM Tris–HCl, 150 mM NaCl, 0.1 % BSA, complete protease inhibitor, adjusted to pH 7.8) in 96-well microplates. In each well 2 µg isolated membrane were incubated with 0.25 mg wheat germ agglutinin polyvinyltoluene polyethylenimine scintillation proximity assay (SPA) beads. Constant concentrations of [125I]-labelled human Ins (100 pM) and various concentrations of respective unlabelled Ins (0.001–1000 nM) were added for 12 h at room temperature (23 °C). The radioactivity was measured at equilibrium in a microplate scintillation counter (Wallac Microbeta, Freiburg, Germany).
Binding on a freshly solubilised IR preparation (S-IR) was performed as previously described  with some modifications. Aliquots of membranes were incubated at 4 °C for 30 min in a solubilisation buffer (20 mM HEPES–NaOH, 100 mM NaCl, 10 mM MgSO4, 1 % (w/v) n-Dodecyl-ß-d-maltoside (Sigma-Aldrich, Munich, Germany), adjusted to pH 7.8 and Complete TM Protease Inhibitor cocktail). Thereafter, ultra-centrifugation was performed at 100,000g for 30 min and 4 °C to remove non–solubilised debris. Protein concentration in the supernatant was adjusted to 0.15 mg/ml with binding buffer (100 mM HEPES–NaOH, 100 mM NaCl, 10 mM MgSO4, 0.025 % (v/v) Tween-20, adjusted to pH 7.8 and complete TM protease inhibitor cocktail). To streptavidin SPA beads (5 mg in 1000 ml binding buffer), 50 µl of an anti-IR alpha-antibody 83-7 (Abcam, Cambridge, UK) was added. After incubation for 30 min, SPA beads were once washed and finally re-suspended in 500 µl binding buffer. A solution of solubilised receptor (1 ml, 0.15 mg/ml) was added and incubated for further 60 min, before washing and resuspension in 1.5 ml. Subsequently, 100 µl re-suspended IR-Antibody-SPA beads (containing 10 µg total protein) were mixed with 50 µl [125I]-labelled insulin tracer (100 pM) and 50 µl non-radioactive Ins (0.001 – 1000 nM), incubated for 12 h at room temperature (23 °C) under shaking, centrifuged for 2 min and measured in the scintillation counter (Wallac Microbeta, Freiburg, Germany).
Effect of insulin and insulin analogues on contractility of primary adult rat ventricular cardiomyocytes
Adult rat ventricular cardiomyocytes (ARVM) were isolated from wild-type Lewis rats (Lew/Crl) as previously described . ARVM were cultivated 3 h in Medium 199 with Hanks’ balanced salts containing 5 mM creatin, 2 mM carnitine and 5 mM taurine supplemented with 10 % FCS and 1 % insulin/transferrin/selene on laminin–coated dishes (ibidi GmbH, Martinsried, Germany). Subsequently, ARVM were cultivated over-night in DMEM/F12 containing 33 µM biotin and 17 µM pantothenate (Invitrogen, Carlsbad, CA, USA). Prior to measurement, ARVM were pre-incubated for 5 min with 100 nM of Ins (porcine Ins, Cat. No.: I5523, Sigma-Aldrich, Munich, Germany), IGla, IGlaM1 or IDeg (provided by Sanofi-Aventis, Frankfurt a.M., Germany) in modified Tyrodes solution: 125 mM NaCl; 1.2 mM KH2PO4; 2.6 mM KCl; 1.2 mM MgSO4*7H2O; 1 mM CaCl2*2H2O; 10 mM Glucose; 10 mM HEPES; adjusted to pH = 7.4 prior to measurement. Furthermore, untreated ARVM or ARVM treated with 10 nM isoproterenol (Sigma-Aldrich, Munich, Germany) were immediately measured. ARVM were paced with bipolar pulses in a contractility and fluorescence system (IonOptix, Milton, MA, USA) at 15 V, 1 Hz, 0.5 ms, at 37 °C for up to 10 min and 10–14 contractions of at least 10 rod–shaped ARVM per condition were recorded. Sarcomeric shortening, shortening rate and re-lengthening rate were calculated using the IonWizard software (IonOptix, Milton, MA, USA). To determine the role of Akt for the positive inotropic effect of Ins and the analogues, ARVM were pre-treated with 10 µM of the specific Akt–inhibitor triciribine (Sigma-Aldrich, Munich, Germany) for 30 min in contraction buffer. Afterwards, ARVM were treated as described above. After 30 min treatment with 10 µM triciribine, ARVM viability was assessed by incubating the cells with 0.1 % trypan blue in PBS for 5 min. Microscopic pictures were taken randomly with at least 10 pictures per condition. As a positive control 200 µM H2O2 was utilised. For each condition at least 400 cells were counted per experiment.
ARVM and HL-1 cells were treated as indicated and lysed in buffer containing 50 mM HEPES (pH 7.4) (PromoCell, Heidelberg, Germany), 1 % Triton X-100 (Sigma-Aldrich, Munich, Germany), PhosSTOP and CompleteTM protease inhibitor cocktail (Roche, Basel, Switzerland). After incubation for 2 h at 4 °C, the suspension was centrifuged at 10,000g for 15 min. 5 μg protein sample of the total cell lysate was separated by SDS/PAGE (10 % gel) and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked in tris-buffered saline (TBS) containing 0.1 % tween 20 and 5 % (w/v) non-fat dried skimmed milk powder and incubated overnight with anti-phospho Akt(Ser473) antibody, anti-phospho Akt(Thr308), anti-GAPDH antibody (all Cell Signalling Technology, Danvers, MA, USA) or anti-tubulin antibody (Abcam, Cambridge, UK). After washing, membranes were incubated with appropriate horseradish peroxidase-coupled secondary antibody and processed for enhanced chemiluminescence (ECL) detection using Immobilion horse radish peroxidase (HRP) substrate (Millipore, Darmstadt, Germany). Signals were visualised and evaluated on a VersaDoc 4000 MP Bio-Rad Laboratories work station and analysed by Quantity One analysis software (version 4.6.7) (both Bio-Rad Laboratories, Hercules, CA, USA).
Impedance measurement in human Cor.4U® cells
Cor.4U® cardiomyocytes were seeded at a density of 30,000 cells per well. From day 3 on the cells were cultured in Iscove’s Basal Medium containing 1 % GlutaMAX supplement (both Life Technologies, Carlsbad, CA, USA) and 2 µg/ml Ciprobay (Bayer, Leverkusen, Germany). Treatment with 500 nM of the designated Ins or analogue or 100 nM isoproterenol was started at day 4 after seeding. Impedance of each well was measured with the RTCA Cardio xCELLigence Analyser  (Acea Biosciences, San Diego, CA, USA) during the whole experiment. Beating-rate and cell index were analysed by RTCA cardio software (Acea Biosciences, San Diego, CA, USA).
Glucose uptake in HL-1 cardiomyocytes
HL-1 cells were seeded at a density of 400,000 cells per well in a 12-well plate. Glucose uptake was measured in serum-starved HL-1 cells, either kept untreated or exposed for 60 min to 200 nM Ins and the analogues, respectively. Subsequently 0.12 mM deoxy–d–glucose (Sigma-Aldrich, Munich, Germany) with 0.055 mCi 2–deoxy–D–[14C]glucose (PerkinElmer, Waltham, MA, USA) was added to the cells. After 10 min incubation the uptake was terminated by repeated washing with ice cold PBS. Afterwards, the cardiomyocytes were lysed with lysis buffer containing 1 % SDS and 200 mM NaOH. Incorporated glucose was measured by scintillation counting of the cell lysates in a liquid scintillation counter (Beckman Coulter, Pasadena, CA, USA). Values were corrected for non-specific uptake as measured by incubation with L-[14C]glucose (PerkinElmer, Waltham, MA, USA).
Caspase 3/7 activity assay
To assess the anti-apoptotic effect of Ins and its analogues, H9c2-E2 cells were seeded in a density of 5000 cells per well of a 96-well plate. The next morning cells were treated with 100 nM of the respective Ins either in the presence or absence of 800 µM of H2O2 for 2 h. Caspase 3/7 activity was then measured by the caspase-Glo® 3/7 assay system (Promega, Madison, Wisconsin, USA) as described in the manual. After 2 h incubation period caspase 3/7 activity was analysed by measuring the luminescence in an Infinite 200 plate reader (Tecan, Männedorf, Switzerland).
Results are expressed as mean values ± SEM of at least three independent experiments. For statistical analysis Graphpad Prism v5.00 (Graphpad Software, San Diego, CA, USA) was used. One-way ANOVA was performed to determine significance between conditions, with level of significance chosen at p < 0.05. In case of IC50 determinations for binding, a non-parametric Kruskal–Wallis testing was performed, again with level of significance chosen at p < 0.05.