Sustained CGRP1 receptor stimulation modulates development of EC coupling by cAMP/PKA signalling pathway in mouse skeletal myotubes

Abstract

We investigated modulation of excitation-contraction (EC) coupling by calcitonin gene-related peptide (CGRP), which is released by motorneurons during neuromuscular transmission. Mouse skeletal myotubes were cultured either under control conditions or in the presence of 100 nm CGRP ( approximately 4-72 h). T- and L-type Ca(2+) currents, immobilization resistant charge movement, and intracellular Ca(2+) transients were characterized in whole-cell patch-clamp experiments. CGRP treatment increased the amplitude of voltage-gated Ca(2+) release ((DeltaF/F)(max)) approximately 75-350% and moderately increased both maximal L-current conductance (G(max)) and charge movement (Q(max)). In contrast, CGRP treatment did not affect their corresponding voltage dependence of activation (V(1/2) and k) or T-current density. CGRP treatment enhanced voltage-gated Ca(2+) release in approximately 4 h, whereas the effect on L-channel magnitude took longer to develop ( approximately 24 h), suggesting that short-term potentiation of EC coupling may lead to subsequent long-term up-regulation of DHPR expression. CGRP treatment also drastically increased caffeine-induced Ca(2+) release in approximately 4 h ( approximately 400%). Thus, short-term potentiation of EC coupling is due to an increase in sarcoplasmic reticulum Ca(2+) content. Both application of a phosphodiesterase inhibitor (papaverine) and a membrane-permeant cAMP analogue (Db-cAMP) produced a similar potentiation of EC coupling. Conversely, this potentiation was prevented by pretreatment with either CGRP1 receptor antagonist (CGRP(8-37)) or a PKA inhibitor (H-89). Thus, CGRP acts through CGRP1 receptors and the cAMP/PKA signalling pathway to enhance voltage-gated Ca(2+) release. Effects of CGRP on both EC coupling and L-channels were attenuated at later times during myotube differentiation. Therefore, we conclude that CGRP accelerates maturation of EC coupling.

Figure 7. CGRP enhancement of voltage-gated Ca²⁺ release is mediated by a CGRP1 receptor activated cAMP/PKA signalling pathway

A, effects of CGRP and other compounds on the amplitude of voltage-gated SR Ca²⁺ release ((ΔF/F)_max). Values of (ΔF/F)_max were determined from the end of 30 ms depolarizating pulses to saturating voltages (+30 to +70 mV). Four different experimental series were carried out (Cultures 1–4). The average (ΔF/F)_max for each control group was (mean ±s.e.m.): 0.6 ± 0.2, 1.2 ± 0.3, 0.8 ± 0.2 and 0.7 ± 0.2; for Cultures 1, 2, 3 and 4, respectively. All (ΔF/F)_max values for each culture were divided by the mean value obtained from the corresponding control group (normalized (ΔF/F)_max). The number of myotubes that were investigated in each experimental condition is indicated near to the corresponding error bar. Both the CGRP1 receptor antagonist CGRP_8-37 (8–37; 3 μm) and the PKA inhibitor (H-89; 10 μm) were added 60 min prior to CGRP exposure and remained present thereafter. For Culture 4, the cAMP analogue, Db-cAMP, was used at a concentration of 0.5 mm and the phosphodiesterase inhibitor, papaverine (PAP), was used at a concentration of 10 μm. Simultaneous treatment with both Db-cAMP and PAP was accomplished by applying the two compounds at the same time (PAP + Db-cAMP). For Cultures 1, 2 and 4, treatments lasted 1 day and CGRP was used at a concentration of 100 nm. In contrast, one, two, or three aliquots (delivered every 2 h) of either 100 nm CGRP or 0.5 mm Db-cAMP (∼4 h treatment) were used for Culture 3. ^aP < 0.05 compared to CGRP. ^bP < 0.15 compared to both CGRP and Db-cAMP. ^cP < 0.10 compared to both PAP and PAP + Db-cAMP. Statistical tests (one-way ANOVA) were only performed between groups of the same culture. B, examples of Ca²⁺ transients recorded from the experimental conditions described in A.

Figure 4. Time course of CGRP-mediated increase in L-type Ca²⁺ current density

A, representative L-type Ca²⁺ currents from myotubes cultured for 0–3 days in either the absence (left) or the presence of 100 nm CGRP (right). The time after initiation of CGRP treatment (in days) is shown in italicized numbers. Current traces were elicited using 200 ms depolarizing pulses to +30 mV that were preceded by a 1 s prepulse to inactivate T-type Ca²⁺ channels. B, time course of average peak L-type Ca²⁺ current density obtained from control (black triangles) and CGRP-treated (grey triangles) myotubes. Data represent means ±s.e.m. from 12–19 myotubes for each condition. Two-way ANOVA indicates significant differences between control and CGRP-treated (100 nm) groups (P < 0.002). The P-values obtained from post hoc Holms–Sidak tests between time-matched control and CGRP-treated for 0.5, 1, 2 and 3 days of treatment were 0.700, 0.049, 0.036 and 0.069, respectively. *P < 0.05.

Figure 2. Time course of CGRP effect on voltage-gated Ca²⁺ transients

A, representative voltage-gated Ca²⁺ transients obtained from myotubes cultured for 0–3 days either under control conditions (left) or in the presence of 100 nm CGRP (right). Ca²⁺ transient amplitude was estimated as described in Fig. 1. The approximated time (in days) following initiation of treatment is indicated with italicized numbers. B, average amplitude of voltage-gated Ca²⁺ transients ((ΔF/F)_max) obtained at different times after CGRP treatment (100 nm). Results were obtained from 6 to 12 myotubes for each condition. (ΔF/F)_max was estimated by either fitting experimental data to eqn (3) or averaging peak ΔF/F values obtained at saturating voltages (+30 mV to +70 mV). Results from two-way ANOVA and post hoc Holms–Sidak test indicated significant differences between control and CGRP-treated groups (*P≤ 0.05). Specifically, the P values between CGRP-treated and time-matched controls obtained for 0.15, 1, 2, and 3 day treatments were 0.021, 0.008, 0.050, and 0.086, respectively.

Figure 1. Effects of CGRP on voltage-gated Ca²⁺ transients

A, representative voltage-gated Ca²⁺ transients obtained from control (left) and CGRP-treated (right) myotubes. Test pulses (V_m) to the indicated voltages (shown in left margin) were elicited following 1 s prepulses used to inactivate T-type Ca²⁺ currents (see Methods). B, average voltage dependence of Ca²⁺ transients elicited as in A. Ca²⁺ transient amplitude measured at the end of each test pulse is plotted as a function of V_m. Continuous lines represent fitted ΔF/F-values obtained using eqn (3) and the resulting average Boltzmann parameters reported in Table 1. C, normalized voltage dependence of Ca²⁺ transients. Absolute ΔF/F values from each myotube were normalized by their corresponding maximal value and plotted as a function of V_m. Results were obtained from 20 control (black triangles) and 21 CGRP-treated (grey triangles) myotubes. CGRP treatment was 100 nm for 1–3 days.

Figure 3. CGRP selectively increases L-type Ca²⁺ current density

A and B, representative traces of total (A) and L-type (B) Ca²⁺ currents obtained from control (top) and CGRP-treated (bottom) myotubes. Total (T-type and L-type) Ca²⁺ currents were first elicited from the holding potential (−80 mV). Subsequently, a family of L-type Ca²⁺ currents were elicited following a 1 s prepulse to −30 mV to inactivate T-type Ca²⁺ channels. Ca²⁺ current traces are shown for the following membrane potentials (mV): −20, −10, 0, +10, +20 and +30. C and D, average current–voltage relationships (I–V curves) obtained for total (C) and L-type (D) Ca²⁺ currents. Results were obtained from 31 (C) and 54 (D) control myotubes (black triangles), and 29 (C) and 40 (D) CGRP-treated myotubes (1–3 days, 100 nm; grey triangles). The continuous lines in D represent fits to the data using eqn (1) and average values of the parameters of these fits are shown in Table 1 (G–V data). A spline curve was used to generate the smooth lines through the data in C.

Figure 5. CGRP increases immobilization-resistant charge movement

A, representative non-capacitative gating currents (asymmetric charge movements) obtained from control (left) and CGRP-treated (right) myotubes elicited at different membrane potentials (left margin). B, average voltage dependence of asymmetric charge movement estimated from integrating the non-capacitative current during the onset of each test depolarization (Q_ON) plotted as a function of V_m. The continuous lines through the data were generated by fitting each data set with eqn (2). Average values of the fitted parameters are shown in Table 1 (Q–V data). C, normalized voltage dependence of charge movement. Absolute values of Q_ON obtained from each myotube were normalized by their corresponding maximum values (Q_max), averaged, and plotted as a function of V_m. Experimental results were obtained from 15 control (black triangles) and 15 CGRP-treated (1–3 days, 100 nm; grey triangles) myotubes.

Figure 6. CGRP similarly potentiates voltage- and caffeine-induced SR Ca²⁺ release

A, representative Ca²⁺ transients elicited by either voltage (left) or exposure to 30 mm caffeine (right). Transients were obtained from one representative control (top) and one representative CGRP-treated (200 nm, ∼4 h) myotube (bottom). The vertical calibration bar indicates initiation of the depolarizing pulse (30 ms to +70 from a holding potential of −80 mV). Caffeine was applied (hatched bar) ∼20 s thereafter (//). Horizontal calibration bar represents 0.2 s (left) and 0.8 s (right). B, average peak amplitude of voltage- (left) and caffeine-induced (right) Ca²⁺ transients. Results were obtained from 13 control and 10 CGRP-treated (∼4 h) myotubes. The treatment with CGRP consisted of one, two, or three aliquots (delivered every 2 h) of 100 nm CGRP. *P < 0.005, #P < 0.001; compared to control.