Figure 1.
Expression of TRPV1 and SERCA1 in skeletal muscle.
Left panel: Western blot analysis of total protein extracts (25 µg) with anti-SERCA1 (top panel) and anti-TRPV1 antibodies (low panel) from the following tissues: flexus digitorum brevis/interosseal (FDB/IO), soleus, extensor digitorum longus (EDL), red and white gastrocnemius (R Ga and W Ga) and brain. Right panel: Localization of TRPV1 in sarcotubular membrane fractions (R1, fraction enriched in light SR; R2, fraction enriched in longitudinal SR; R3, fraction containing a mixture of longitudinal SR and terminal cisternae; R4, fraction enriched in terminal cisternae): 30 µg of protein from each fraction was separated on SDS/PAGE.
Figure 2.
Localization of TRPV1, RyR1 and SERCA1 in mouse FDB fibers.
A: Schematic organization of the sarcoplasmic reticulum (SR) in a skeletal muscle fiber. Confocal images of double immunofluorescence labeling of TRPV1 (B) and RyR1 (C) or TRPV1 (F) and SERCA1 (G). E, I: average intensity profiles from the dotted rectangle region in the next corresponding images. In isolated fibers, RyR1 shows localization in the junctional part of the SR (C) while SERCA1 appears in the longitudinal part of the SR (G). In the merged images, TRPV1 does not colocalize with RyR1 (D) while colocalization with SERCA1 staining is visible in H. Results are from at least 4 independent fibers preparations (n>10).
Figure 3.
FM1–43 labeling in intact and permeabilized FDB isolated muscle fibers.
In the top panel (A; B; C) cells were incubated in relaxing solution; in the low panel (D; E; F), cells in relaxing solution containing capsaicin (100 µM). Photomicrographs in transmitted light (A, D) or FM1–43 fluorescence in cells before (B; E) or after (C; F) saponin-permeabilization. In intact cells (B; E), FM1–43 fails to enter the FDB fiber, while once the surface membrane is disrupted, the dye enters into the cytoplasm and labels SR membranes (C; F). Results are from at least 2 independent fibers preparations (n>3).
Figure 4.
Calcium release induced by pharmacological activation of TRPV1 in FDB isolated fibers.
(A) The traces show representative curve obtained after stimulation of single fibers with capsaicin alone (100 µM; black line) or in the presence of capsazepine (light grey line) or dantrolene (dark grey line). B; C: changes in fluorescent ratio F/F0 (peak– resting) induced by drugs as indicated in table above the graphs. Summary data showing fluorescent changes in cells induced by agonists, B: with or without antagonist pretreatment or else after SR depletion and C: with or without RyR inhibitors pretreatment. Capsazepine (CPZ, 100 µM), cyclopiazonic acid (CPA, 25 µM), dantrolene (DAN, 25 µM) and ryanodine (Ry, 80 µM) were added 20 min prior to agonist treatment. Resiniferatoxin and thapsigargin were used at 10 µM and 1 µM respectively. Results are expressed as means ± S.E.M. of the indicated number of experiments from at least 4 independent fibers preparations. T-tests were performed by paired samples: *p<0.05, **p<0.002 and ***p<0.0002.
Figure 5.
Calcium release induced by physiological activation of TRPV1 in FDB isolated fibers.
(A) Time course of cytosolic Ca2+ concentration in Fluo-4 loaded cells in response to heat. The temperature of external Ca2+-free solution was increased from 25°C to 45°C (as depicted by horizontal bars on the top). Experiments were performed with or without capsazepine pre-treatment (100 µM during 25 min). (B) Cumulative data of the heat effect on SR Ca2+ release in control conditions or after capsazepine pre-treatment. (C) Cumulative data of the rate of SR Ca2+ release evoked by CPA (25 µM) in control conditions and after TRPV1 inhibition by capsazepine. Results are expressed as means ± S.E.M. T-tests were performed by paired samples: **p<0.06.
Figure 6.
Schematic drawing of the possible TRPV1 contribution in the maintenance of Ca2+ muscle homeostasis.
(1) External stimuli, such as capsaicin, resiniferatoxin or high temperature, activate TRPV1 channels located at the longitudinal SR. (2) TRPV1 channels releases Ca2+ from SR to cytoplasm. (3) This low increase in [Ca2+]c activates RyR channels (4) that in return release Ca2+ into cytoplasm increasing [Ca2+]c (5). In permanence, SERCA1 pumps continuously back Ca2+ from cytoplasm to SR and could mask TRPV1 activation in resting condition.