Effects of non-euphoric plant cannabinoids on muscle quality and performance of dystrophic mdx mice

Abstract

Background and purpose: Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, results in chronic inflammation and irreversible skeletal muscle degeneration. Moreover, the associated impairment of autophagy greatly contributes to the aggravation of muscle damage. We explored the possibility of using non-euphoric compounds present in Cannabis sativa, cannabidiol (CBD), cannabidivarin (CBDV) and tetrahydrocannabidivarin (THCV), to reduce inflammation, restore functional autophagy and positively enhance muscle function in vivo.

Experimental approach: Using quantitative PCR, western blots and [Ca2+ ]i measurements, we explored the effects of CBD and CBDV on the differentiation of both murine and human skeletal muscle cells as well as their potential interaction with TRP channels. Male dystrophic mdx mice were injected i.p. with CBD or CBDV at different stages of the disease. After treatment, locomotor tests and biochemical analyses were used to evaluate their effects on inflammation and autophagy.

Key results: CBD and CBDV promoted the differentiation of murine C2C12 myoblast cells into myotubes by increasing [Ca2+ ]i mostly via TRPV1 activation, an effect that undergoes rapid desensitization. In primary satellite cells and myoblasts isolated from healthy and/or DMD donors, not only CBD and CBDV but also THCV promoted myotube formation, in this case, mostly via TRPA1 activation. In mdx mice, CBD (60 mg·kg-1 ) and CBDV (60 mg·kg-1 ) prevented the loss of locomotor activity, reduced inflammation and restored autophagy.

Conclusion and implications: We provide new insights into plant cannabinoid interactions with TRP channels in skeletal muscle, highlighting a potential opportunity for novel co-adjuvant therapies to prevent muscle degeneration in DMD patients.

Linked articles: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Conflict of interest statement

F.A.I. and V.D. are supported by GW Pharmaceuticals, UK, for research on phytocannabinoids and skeletal muscle degenerative disorders (grant number GWCRI15107 to F.A.I.). V.D. has a Consultancy Agreement with GW Pharmaceuticals.

© 2018 The British Pharmacological Society.

Figures

Figure 1

Measurement of skeletal muscle differentiation markers in C2C12 cells following short or prolonged exposure to CBD and CBDV. Transcript levels of myogenin (Myog) and Tnnt‐1 in murine C2C12 cells exposed to DM following acute (5, 15 min and 3 h) or prolonged (24 or 72 h) exposure to CBD 1 μM (A, B) or CBDV 1 μM (C, D). The quantification of mRNA levels for myogenin and Tnnt‐1 was performed by quantitative real‐time PCR. Data represent the mean ± SEM of six separate determinations. Data are expressed as 2‐ΔΔct relative to S16, as described in Methods. (E, F) Morphological analysis of myotube formation in C2C12 cells exposed to DM for 72 h in the presence of vehicle (DMSO ≤ 1%, control; n = 5), CBD 1 μM (n = 5) or CBDV 3 μM (n = 5). MyHC (red) and DAPI (blue) (Scale bar, 10 μm). The fusion index was calculated in both vehicle‐ and CBD‐ or CBDV‐treated cells. Data sets were compared by use of one‐way ANOVA followed by Bonferroni's test. Differences were considered statistically significant when P was ≤ 0.05. The asterisk (*) denotes a P value of ≤ 0.05 versus vehicle (DMSO) control group.

Figure 2

CBD and CBDV increase the concentration of intracellular Ca2+ levels and promote the differentiation in C2C12 cells in a TRPV1, but not TRPV2, dependent manner. Concentration–response curve showing the effect of palvanil (A) and CBD or CBDV (B) in C2C12 cells. (C, D) Effect of CBD 3 μM or CBDV 3 μM evaluated in the presence of IRTX 0.6 μM (a selective TRPV1 antagonist) or tranilast 100 μM (a non‐selective TRPV2 antagonist) used alone or in combination. (E, F) mRNA expression levels of myogenin (Myog) and Tnnt‐1 in C2C12 cells exposed to DM in the presence of CBD (1 μM) plus IRTX (0.6 μM). Data represent the mean ± SEM of six separate determinations. Data sets were compared by use of one‐way ANOVA followed by Bonferroni's test. Differences were considered statistically significant when *P was ≤ 0.05.

Figure 3

Effect of short or prolonged stimulation with palvanil in C2C12 cells. (A, B) Quantification of transcripts levels of myogenin (Myog) and Tnnt‐1, evaluated by quantitative real‐time PCR, in C2C12 cells induced to differentiate in DM following the acute (5 and 15 min) or prolonged (48 h) exposure to palvanil 1 μM. (C) Representative blot showing the chemiluminescent signal generated by the anti‐phosphoTRPV1 antibody (at the phosphorylation site Ser800 of TRPV1) evaluated by western blot analysis in C2C12 cells exposed for 48 h to DM in the presence of palvanil (3 μM) or IRTX (1 μM). (D) The graph shows the quantification of pTRPV1 signal (OD) normalized to the OD of TRPV1. Data represent the mean ± SEM of five separate determinations. Data sets were compared by use of one‐way ANOVA followed by Bonferroni's test. Differences were considered statistically significant when *P was ≤ 0.05.

Figure 4

Effect of CBD and CBDV in differentiating myoblasts isolated from DMD donors. (A, B) Grouped column scatter plot showing the mRNA expression levels of MYOG and TNNT‐1 in primary human myoblasts isolated from DMD patients and induced to differentiate in the presence of vehicle (0.001% DMSO), CBD (1 μM) or CBDV (3 μM). The quantification of transcripts was performed by quantitative real‐time PCR. (C, D) The graphs show the differences in the expression level of MYOG and TNNT‐1 between vehicle and CBD (1 μM) or CBDV (3 μM) treated myoblasts, calculated by combining the DMD patient's results together. Data are expressed as 2‐ΔΔct formula relative to S16, as described in Methods. Data represent the mean ± SEM from the seven patients, repeated in quadruplicate. Data sets were compared by one‐way ANOVA followed by Bonferroni's test. Differences were considered statistically significant when *P was ≤ 0.05.

Figure 5

Effect of CBD, CBDV and THCV in differentiating primary human satellite cells. (A) Transcript levels of myogenin (MYOG), TNNT‐1 and MyHC in human satellite cells exposed to DM in the presence of CBD 1 μM, CBDV 3 μM and THCV 3 μM for 5 days. The quantification of transcripts for MYOG and TNNT‐1 was performed by quantitative real‐time PCR. Data represent the mean ± SEM of n ≥ 5 determinations. Data are expressed as 2‐ΔΔct relative to S16, as described in Methods. (B) Morphological analysis of myotube formation in satellite cells exposed to DM for 5 days in the presence of vehicle (0.003% DMSO, control; n = 14), CBD 1 μM (n = 14), CBDV 3 μM (n = 14) or THCV 3 μM (n = 14). MyHC (red) and DAPI (blue) (Scale bar, 10 μm). The fusion index was calculated in vehicle and CBD‐, CBDV‐ or THCV‐treated cells. Data sets were compared by use of one‐way ANOVA followed by Bonferroni's test. Differences were considered statistically significant when *P was ≤ 0.05.

Figure 6

Effect of CBD, CBDV and THCV on intracellular Ca2+ levels in the presence or absence of TRPV1, TRPV2 and TRPA1 antagonists in human satellite cells. Concentration–response curves showing the effect of CBD (A), CBDV (B) and THCV (C) on satellite cells. (D, F) Effect of CBD 3 μM, CBDV 3 μM and THCV 3 μM measured in the presence of IRTX 0.6 μM, a TRPV1 antagonist, 100 μM tranilast, a non‐selective TRPV2 antagonist, or 20 and 50 μM of AP18, a selective TRPA1 antagonist. Data represent the mean ± SEM of ≥5 determinations. Data sets were compared by use of one‐way ANOVA followed by Bonferroni's test. Differences were considered statistically significant when *P was ≤ 0.05.

Figure 7

Measurement of locomotor activity in control and mdx mice treated or not with CBD or CBDV. Muscle coordination and strength by the rotarod test (A), weight test (B) and (C) forelimb grip strength test were measured in control (C57BL/10ScSnJ) and dystrophic (C57BL/10ScSn‐DMDmdx/J) mice treated with vehicle (10% ethanol + 10% Tween + 80% NaCl; 1:1:8; n = 15), CBD (60 mg·kg−1; n = 15) or CBDV (60 mg·kg−1; n = 15) at the beginning of the treatment (5 weeks) and after 2 weeks (7 weeks, right). Control and mdx mice were treated every other day starting from week 5 to week 7 (left). Data sets were compared by one‐way ANOVA followed by Bonferroni's test. Differences were considered statistically significant when P was ≤ 0.05. *Denotes P ≤ 0.05 versus mdx control group at week 5; oP ≤ 0.05 versus mdx control group of the same age; #P ≤ 0.05 versus mdx vehicle group of the same age.

Figure 8

Measurement of muscle strength and histological analysis in mdx mice treated with CBD and CBDV. Muscle coordination and strength by the rotarod test (A) and weight test (B) were measured in control (C57BL/10ScSnJ) and dystrophic (C57BL/10ScSn‐DMDmdx/J) mice treated with vehicle (10% ethanol + 10% Tween + 80% NaCl; n = 12), CBD (60 mg·kg−1; n = 12) or CBDV (60 mg·kg−1; n = 12) at the beginning of the treatment (32 weeks) and after 2 weeks (34 weeks, right). Control and mdx mice were treated every other day starting from week 32 to week 34 (left). Data sets were compared by one‐way ANOVA followed by Bonferroni's test. Differences were considered statistically significant when P was ≤ 0.05. *Denotes P ≤ 0.05 versus mdx control group at week 5; oP ≤ 0.05 versus mdx control group of the same age; #P ≤ 0.05 versus mdx vehicle group of the same age. (C) Representative photomicrographs of H&E‐stained transverse sections of gastrocnemius muscle isolated from wild type (wt; n = 5); mdx treated with vehicle (10% ethanol + 10% Tween + 80% NaCl; n = 5) or CBD (60 mg·kg−1; n = 5) i.p. from week 32 to week 34 of age. Scale bars = 100 μm. (D) The bar graph indicates the number of centralized nuclei in the muscle fibres of control and CBD‐treated mdx mice. Data sets were compared by one‐way ANOVA followed by Bonferroni's test. **P ≤ 0.001.

Figure 9

Analysis of expression of inflammation markers in skeletal muscles of mdx mice treated with CBD and CBDV. Scatter plot graphs showing the mRNA expression levels of IL‐6 receptors (IL6R), TNFα, TGF‐β1 and iNOS in the gastrocnemius and/or diaphragm muscles of control and mdx mice of both 7 (A and B) and 34 (C and D) weeks of age that had received vehicle or CBD or CBDV for 2 weeks. Data are expressed as 2‐ΔΔct relative to S16, as described in Methods. (E) plasma levels of IL‐6 and TNFα quantified by elisa assay in control and mdx mice (34 weeks) treated with vehicle, CBD or CBDV. Data represent the mean ± SEM of ≥ 5 independent determinations. Data sets were compared by one‐way ANOVA followed by Bonferroni's test. *Denotes P ≤ 0.05 versus mdx control group at week 5; #P ≤ 0.05 versus mdx vehicle group of the same age.

Figure 10

Expression analysis of autophagy markers in skeletal muscles of mdx mice treated with CBD and CBDV. The scatter plot graphs show the mRNA expression levels of autophagy‐related 12 and 4b (ATG12 or 4b), beclin‐1 (becn1) and ULK1 genes in the gastrocnemius (A) and/or diaphragm (B) muscles of control and dystrophic mdx mice of 7 weeks that received vehicle or CBD or CBDV for the last 2 weeks. Data are expressed as 2‐ΔΔct relative to S16, as described in Methods. (C) Representative blot showing the band intensity of the autophagic marker LC3I/II in the gastrocnemius muscles of mdx mice treated with CBD. (D) Bar graph showing the quantification of LC3I/II levels normalized to GAPDH. Data represent the mean ± SEM of ≥5 separate determinations. Data sets were compared by one‐way ANOVA followed by Bonferroni's test. Differences were considered statistically significant when P ≤ 0.05. *Denotes P ≤ 0.05 versus mdx control group at week 5; #P ≤ 0.05 versus mdx vehicle group of the same age.