TRPV1 activation and internalization is part of the LPS-induced inflammation in human iPSC-derived cardiomyocytes

Figures

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  Figure 1. (a) Detection of TRPV1 protein in hiPSC-cardiomyocytes by western blotting. Cells were incubated with LPS for 6 h, 37 °C. Unstimulated cells served as a control (“control”). TRPV1 control protein was loaded at equal concentration. Primary antibody: rabbit polyclonal anti-TRPV1, #ACC-030, alomone labs, 1:500, 4 °C overnight. TRPV1 control: TRPV1 control peptide, #ACC-030, alomone. kDa—kiloDalton, MW—molecular weight marker. A representative experiment out of 4 independent experiments is shown. (b) TRPV1 is present in the plasma membrane of hiPSC-cardiomyocytes (troponin T-positive cells). Cells were stained by immunofluorescence for plasma membrane, TRPV1, troponin T and nuclei (upper panel: Alexa350-wheat germ agglutinate anti-plasma membrane, anti-TRPV1 primary/PE-anti-rabbit secondary antibody, Alexa647-anti troponin T, propidiumjodide. Lower panel: Alexa350-wheat germ agglutinate plasma membrane, anti-TRPV1 primary/ FITC-anti-rabbit secondary antibody, Alexa647-anti troponin T, propidiumjodide). Arrows in the merged images denote TRPV1. Magnification was 40x. Scale bars: Upper panel: 244.45 µm. Lower panel: 100.68 µm. Independent experiments, n = 3. Per experiment, images of…

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  Figure 2. The metabolic activity of hiPSC-CMs is not affected by capsaicine (CAP), but by capsazepine (CPZ). Presented is the normalized optical density (OD) at 570 nm after application of a MTT assay measuring metabolic activity via NAD(P)H-dependent cellular oxidoreductase enzymes (mean ± standard error of the mean (SEM)). Kruskal–Wallis p < 0.0001, *p < 0.001 CPZ 100 µM versus control. For better presentation of low values, the y-axis was scaled logarithmic. Unstimulated cells, n = 16, n = 3 independent experiments, n = 3–7 biological replicates per experiment. CAP 10 µM, n = 9, n = 2 independent experiments, n = 3 and 6 biological replicates per experiment. CAP 100 µM, n = 14, n = 3 independent experiments, n = 3–6 biological replicates per experiment. CPZ 10 µM, n = 4, n = 1 independent experiment, n = 4 biological replicates. CPZ 100 µM, n = 17, n = 3 independent experiments, n = 3–9 biological replicates per experiment.

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  Figure 3. (a) LPS induces the release of interleukin-6 into the cell culture medium (mean ± standard error of the mean (SEM)). Kruskal–Wallis p = 0.0136; *p < 0.01 LPS 1 µg/mL, 6 h, versus unstimulated controls. Unstimulated cells, n = 16, n = 5 independent experiments, n = 1–5 biological replicates per experiment. LPS 1 µg/mL, 6 h, n = 15, n = 5 independent experiments, n = 1–7 biological replicates per experiment. LPS 1 µg/mL, 5 or 10 min, n = 3, n = 1 independent experiment, n = 3 biological replicates. LPS 5 µg/mL, n = 5, n = 4 independent experiments, n = 1–2 biological replicates per experiment. (b) Capsaicin (CAP, 10 µM, 20 min) mediates a non-significant release of interleukin-6 which is lower than the LPS-induced interleukin-6 production (LPS, 1 µg/mL, 6 h). Capsaicin (100 µM, 5 min) does not have an effect on interleukin-6 release. Presented is the mean ± standard error of the mean (SEM). Kruskal–Wallis p = 0.037; * p < 0.05 LPS versus unstimulated controls; (*) p < 0.1 CAP 10 µM versus unstimulated controls. Unstimulated cells, n = 16, n = 5 independent experiments, n = 1–5 biological replicates per experiment. LPS 1 µg/mL, 6 h, n = 15, n = 5 independent experiments, n = 1–7…

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  Figure 4. TRPV1 is internalized upon stimulation with LPS, as determined by corrected total cellular fluorescence (CTCF) of the fluorochrome-marked TRPV1 channel. Presented is the mean ± standard error of the mean (SEM). Kruskal–Wallis p = 0.005; *p < 0.05 unstimulated controls versus LPS. Results are derived from two independent experiments. Unstimulated cells, n = 26; n = 8 and n = 18 biological replicates per experiment. LPS (1 µg/mL, 6 h), n = 36; n = 12 and n = 24 biological replicates per experiment. CAP + LPS (CAP 10 µM, 20 min, + LPS, 1 µg/mL, 6 h), n = 29; n = 13 and n = 16 biological replicates per experiment. CPZ + LPS (CPZ, 100 µM, 20 min, + LPS, 1 µg/mL, 6 h), n = 39; n = 9 and n = 30 biological replicates per experiment.

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  Figure 5. TRPV1 ion channel current is attenuated by LPS and by interleukin-6 (IL-6). (a) I–V curves of the capsazepine-sensitive TRPV1 current in absence (control, Ctr, circles) and presence of LPS or IL-6 (LPS, 1 µg/mL, 6 h: squares, IL-6, 10 ng/mL, 6 h: triangles). Vm—membrane potential. (b) Mean values of the capsazepine-sensitive TRPV1 current at − 70 mV in absence (control, Ctr) and presence of LPS or IL-6 (LPS, 1 µg/mL, 6 h. IL-6, 10 ng/mL, 6 h). *p < 0.05. Data of two independent experiments are shown. Unstimulated control cells, n = 8; n = 4 biological replicates per experiment. LPS, n = 8; n = 4 biological replicates per experiment. IL-6, n = 10; n = 5 biological replicates per experiment.