Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy

Abstract

Mortality of Duchenne Muscular Dystrophy (DMD) is a consequence of progressive wasting of skeletal and cardiac muscle, where dystrophinopathy affects not only muscle fibres but also myogenic cells. Elevated activity of P2X7 receptors and increased store-operated calcium entry have been identified in myoblasts from the mdx mouse model of DMD. Moreover, in immortalized mdx myoblasts, increased metabotropic purinergic receptor response was found. Here, to exclude any potential effects of cell immortalization, we investigated the metabotropic response in primary mdx and wild-type myoblasts. Overall, analyses of receptor transcript and protein levels, antagonist sensitivity, and cellular localization in these primary myoblasts confirmed the previous data from immortalised cells. However, we identified significant differences in the pattern of expression and activity of P2Y receptors and the levels of the "calcium signalling toolkit" proteins between mdx and wild-type myoblasts isolated from different muscles. These results not only extend the earlier findings on the phenotypic effects of dystrophinopathy in undifferentiated muscle but, importantly, also reveal that these changes are muscle type-dependent and endure in isolated cells. This muscle-specific cellular impact of DMD may not be limited to the purinergic abnormality in mice and needs to be taken into consideration in human studies.

Figure 1

P2RYs transcript levels in myoblasts derived from Tibialis anterior (TA), Gastrocnemius (GC), Soleus (SOL) and Flexor Digitorum Brevis (FDB) muscles. Transcripts encoding P2RY2, P2RY4 and P2RY6 were tested in the same sample, thus their relative expression levels can be compared quantitatively., mRNAs encoding two ADP-activated receptors (P2RY1, P2RY12) were also detected in these samples. *p < 0.05, **p < 0.01 (mdx vs. w/t).

Figure 2

Western bloting alaysis of ATP-activated metabotropic receptors in primary myoblasts from TA, SOL, GC, FDB. Each bar represents the mean value from 3 independent experiments ± SD. Representative western blots are also shown. *p < 0.05 (mdx vs. w/t).

Figure 3

Western bloting analysis of ADP-activated metabotropic receptor P2RY1 in primary myoblasts. Each plot shows Western blot data quantification for the 40 kDa band (top) and 35 kDa band (bottom) Each bar represents the mean value from 3 independent experiments ± SD. Representative western blots are also shown. *p < 0.05 (mdx vs. w/t.)

Figure 4

Western bloting analysis of ADP-activated metabotropic receptors P2RY12 and P2RY13. Each bar shows Western blot data from 3 independent experiments ± SD. Representative western blots are also shown. *p < 0.05 (mdx vs. w/t).

Figure 5

Western bloting analysis of UDP-activated metabotropic receptor P2RY6. Each bar represents the mean value from 3 independent experiments ± SD. Representative western blots are also shown. *p < 0.05 (mdx vs. w/t).

Figure 6

Fluorescent microscopy visualization of P2RY2, P2RY4, P2RY6 and P2RY1 in myoblasts isolated from TA, GC, SOL and FDB muscles of w/t and mdx mice. Representative example pictures of cells stained with the specific antibody, and secondary antibody only negative controls counterstained with DAPI are shown. Size bar = 10 µM.

Figure 7

Nucleotide-induced calcium release from ER. The representative trace (Top) is shown to illustrate the general concept of the experiment. Myoblasts were incubated with or without P2RY2 inhibitor (AR-C118925XX) and then 100 µM UTP or 500 µM ATP was added. Data collected from three independent experiments are shown. Asterisk (*) marks statistically significant differences: mdx vs. w/t and (mdx + P2RY2 antagonist) vs. (w/t + P2RY2 antagonist). Hashtag (#) marks differences: mdx vs. (mdx + P2RY2 antagonist) and w/t vs. (w/t + P2RY2 antagonist). * and # p < 0.05; ** and ## p < 0.01; *** p < 0.001.

Figure 8

Nucleotide-induced calcium release from ER. The protocol is the same as shown in Fig. 7. Myoblasts were stimulated with 1 mM ADP or 1 mM UDP. Data collected from three independent experiments are shown. Asterix(s) * marks mdx vs. w/t. *p < 0.05; **p < 0.01.

Figure 9

Random motility of primary myoblasts; effects of ATP-evoked cell stimulation. Data collected from cells isolated from 4 mice, with 30 myoblasts per muscle type being tracked. Differences in distance covered (µm) are shown *p < 0.05.

Figure 10

Western blot analysis of selected proteins belonging to the calcium toolkit involved in the generation of calcium signal analysed in myoblasts isolated from TA, GC, SOL and FDB. Bars represent mean values for three independent experiments ± SD. Below each graph data from one representative western blot experiment are shown. *p < 0.05, **p < 0.01.

Figure 11

Western blot analysis of selected proteins belonging to the calcium toolkit involved in the reduction of calcium signal analysed in myoblasts isolated from TA, GC, SOL and FDB. Mean values from three experiments ± SD are shown. Representative western blots are shown. *p < 0.05, **p < 0.01.