Bitter taste receptor (TAS2R) 46 in human skeletal muscle: expression and activity

Abstract

Bitter taste receptors are involved not only in taste perception but in various physiological functions as their anatomical location is not restricted to the gustatory system. We previously demonstrated expression and activity of the subtype hTAS2R46 in human airway smooth muscle and broncho-epithelial cells, and here we show its expression and functionality in human skeletal muscle cells. Three different cellular models were used: micro-dissected human skeletal tissues, human myoblasts/myotubes and human skeletal muscle cells differentiated from urine stem cells of healthy donors. We used qPCR, immunohistochemistry and immunofluorescence analysis to evaluate gene and protein hTAS2R46 expression. In order to explore receptor activity, cells were incubated with the specific bitter ligands absinthin and 3ß-hydroxydihydrocostunolide, and calcium oscillation and relaxation were evaluated by calcium imaging and collagen assay, respectively, after a cholinergic stimulus. We show, for the first time, experimentally the presence and functionality of a type 2 bitter receptor in human skeletal muscle cells. Given the tendentially protective role of the bitter receptors starting from the oral cavity and following also in the other ectopic sites, and given its expression already at the myoblast level, we hypothesize that the bitter receptor can play an important role in the development, maintenance and in the protection of muscle tissue functions.

Keywords: absinthin; bitter taste receptor; calcium imaging; skeletal muscle; urine stem cell-derived skeletal muscle cells; urine stem cells.

FIGURE 1

Expression of hTAS2Rs in human skeletal muscle biopsies. (A) qPCR analysis of hTAS2R subtypes expression in human biopsies (n = 4) of skeletal muscles (oral cavity and locomotor system). Nicotinic acetylcholine receptor subunits (nACHRa4 and nACHRa9) were used as positive control, cytokeratin 10 (CK10) as negative control. Data are expressed as 2-DCt and are means ± SEM. (B, C) Immunohistochemistry analysis of TAS2R46 expression on sections of locomotor system (B) and tongue (C) skeletal muscle. Magnification ×100. Positive (stained with both primary and secondary antibody, (D) and negative (stained with secondary antibody only, (E) control of immunohistochemistry analysis of section of sternoclaidomastoid skeletal muscle.

FIGURE 2

Expression of hTAS2R46 in human myoblast/myotube. (A–D) Immunofluorescence analysis of TAS2R46 expression in myoblasts (A) and myotubes (B) isolated from biopsies and primary SkMCs before (C) and after (D) last step of differentiation. Green: TAS2R46; Blue: Nuclei; Red. Magnification ×400. (E) Real time analysis of TAS2R46 expression on myoblasts and myotubes from biopsies and a primary cell line of SkMCs. Data are expressed as 2^−DCt and are means ± SEM of at least three independent experiments.

FIGURE 3

Expression of hTAS2R46 in USC-SkMCs. (A–B) Immunofluorescence analysis of TAS2R46 expression USC-SkMCs. Green: TAS2R46; Blue: Nuclei; Red: Phalloidin. Magnification ×100 and 400x. (C) Real-time PCR analysis of TAS2R46 gene transcript expression in USCs and USC-SkMCs. Data are expressed as 2^−DCt and are means ± SEM of five independent experiments. **p < 0.01 vs. USCs.

FIGURE 4

Membrane potential modulation by absinthin and acetylcholine. Human primary myotubes were charged with FluoVolt membrane dye and the variations in the fluorescence intensity were evaluated at the cytofluorimeter. Data are illustrated in histograms representing the mean ± SEM of the mean fluorescence intensity variations (ΔMFI) before and after cells stimulation with acetylcholine (100 μM) and absinthin (10 μM) alone or combined (n = 5).

FIGURE 5

Absinthin reduces acetylcholine-induced Ca²⁺ transients in a TAS2R46-dependent manner. Data are illustrated in representative traces as well as in histogram expressing the mean ± SEM of maximum peak of cytosolic Ca2+ release of at least 32 cells in three independent experiments. (A) Fura-2a.m.–loaded USC-SkMCs were stimulated with acetylcholine (Ach) 100 μm and absinthin (Abs) 10 μm alone or combined. ****p < 0.001 vs. Ach. (B) USC-SkMCs were stimulated with acetylcholine (Ach) 100 μm, absinthin (Abs) 10 μm and increasing concentrations of receptor antagonist 3HDC (1, 10, 100 μm). ****p < 0.0001 vs. Ach; **** p < 0.0001 vs. Ach + Abs. (C) Fura-2a.m.-loaded USC-SkMCs silenced for hTAS2R46 expression (referred as shRNA) were stimulated with acetylcholine (Ach) 100 μm, absinthin (Abs) 10 μm alone ore combined.

FIGURE 6

Absinthin reduces acetylcholine-induced Ca²⁺ transients via cAMP in USC-SkMCs. Data are illustrated in representative traces as well as in histogram expressing the mean ± SEM of maximum peak of cytosolic Ca²⁺ release of at least 54 cells in three independent experiments. Fura-2a.m.–loaded USC-SkMCs were stimulated with acetylcholine (Ach) 100 μm, absinthin (Abs) 1 μM in presence of the PKA inhibitor H-89 (10 μM), or a pan-EPAC inhibitor (ESI-09, 10 μm) or MCU inhibitor KB-R7943 (10 μm). ****p < 0.0001 vs. Ach; *** p < 0.001**** p < 0.0001 vs. Ach + Abs.

FIGURE 7

Absinthin increased mithocondrial Ca²⁺ entry. Data are illustrated in representative traces as well as in histogram expressing the mean ± SEM of maximum peak of cytosolic Ca²⁺ release of at least 43 cells in three independent experiments. Rhod-2a.m.–loaded USC-SkMCs were stimulated with acetylcholine (Ach) 100 μm, absinthin (Abs) 1 μM in presence of the antagonist 3HDC (10 μm), or the PKA inhibitor H-89 (10 μM), or the pan-EPAC inhibitor (ESI-09, 10 μm) or MCU inhibitor KB-R7943 (10 μm). **p < 0.01 vs. Ach; **** p < 0.0001 vs. Ach + Abs.

FIGURE 8

Absinthin counteracted acetylcholine-induced USC-SkMCs contraction. Cells were plated on collagen disc and treated with absinthin 10 μM, acetylcholine 100 µM. (A) The collagen area was measured at the indicated time point; (B) zoom of the time between the second and the fourth hours. (C) Histograms represent the mean ± SEM of collagen area (cm2) at several time points (n = 4) ****p < 0.0001; *p < 0.05 vs CTL (no stimuli); ° p < 0.05 vs Ach. (D) SkMCs were stimulated with absinthin 10 µM and acetylcholine 100 μM, also in presence of the PKA inhibitor (H89, 10 µM) and EPAC inhibitor (ESI-09); in (E) zoom of the time between the second and the fourth hours.