Acetylcholine receptors in innervated muscles of dystrophic mdx mice degrade as after denervation

Abstract

Acetylcholine receptors (AChRs) are present at the top of the postsynaptic membrane of the neuromuscular junction (NMJ) at very high density, possibly anchored to cytoskeletal elements. The present study investigated whether AChR degradation is affected in animals lacking dystrophin, a protein that is an integral part of the cytoskeletal complex and is missing in Duchenne muscular dystrophy. The animal model for Duchenne muscular dystrophy, the mutant mdx mouse, was used to determine whether disruption of the cytoskeleton, caused by the absence of dystrophin, affects AChR degradation. Of the two populations of junctional AChRs, Rs (expressed in innervated adult muscles) and Rr (expressed in embryonic or denervated muscles), only Rs are affected in mdx animals. In innervated mdx soleus, diaphragm, and sternomastoid muscles, the AChRs have an accelerated degradation rate (t1/2 of approximately 3-5 d), similar to that acquired by Rs in control muscles after denervation. The Rs in mdx NMJs do not accelerate further when the muscles are denervated. The absence of dystrophin does not affect the degradation rate of the Rr AChRs (t1/2 of 1 d), which are expressed after denervation in mdx as in control muscles. These results suggest that dystrophin or an intact cytoskeletal complex may be required for neuronal stabilization of Rs receptors at the adult neuromuscular junctions.

Fig. 1.

In vivo degradation rates of Rs AChRs in soleus muscles are faster in mdx (○) than in control (•) muscles. AChRs were labeled with ¹²⁵I-α-BTX and counted at different days thereafter. The slope of residual radioactivity on a semi-log plot gives the rate of AChR degradation. Each data point is presented as mean ± SEM (n= ≥3).

Fig. 2.

Denervation accelerates Rs AChR degradation in control muscles (A) but not in mdxmuscles (B). Right soleus muscles of adult mice were denervated at time of AChR labeling with ¹²⁵I-α-BTX. Contralateral muscles served as innervated controls. Data points are mean ± SEM (n = ≥3).

Fig. 3.

The degradation rates of Rr AChRs are the same inmdx as in control muscles. A, Leg muscles in cell culture (3 experiments with 6 dishes each); B, long-term denervated diaphragm muscles in organ culture: control (•) (n = 4) and mdx (○) (n = 3) mice. Overall the degradation rate of Rr in denervated muscles in organ culture is slower than that in tissue-cultured muscles, but in both systems there is no difference between Rr in control and mdx muscle. Data points are mean ± SEM. In all figures, SEM is not seen when smaller than symbol.

Fig. 5.

dB-cAMP stabilizes the slow component (Rs AChR) ofmdx muscles labeled on day 6 in cell culture.A, The degradation data are best fit (least squares) by the sum (solid line) of two exponentials: 90% constitutes a fast component (Rr; t_1/2 ∼14 hr) and 10% a slow component (accelerated Rs;t_1/2 of 3.4 d); B, dB-cAMP stabilized the slow component to at_1/2 of 9.7 d without affecting the fast component or altering the ratio of the two populations. Data points are mean ± SEM (n = 6).

Fig. 4.

dB-cAMP stabilized Rs AChRs in denervatedmdx muscle. Adult innervated mdxdiaphragm muscles were labeled with ¹²⁵I-α-BTX at time of denervation to selectively label Rs. Muscles were placed in organ culture 6 d later, at a time when the control Rs had already undergone denervation-induced acceleration. dB-cAMP (500 μm) was added daily from day 3. Spontaneous fibrillation was used to verify muscle viability. Residual radioactivity, normalized to 100% on day 3 and plotted on a semi-log scale, gives the degradation rate from the slope. dBcAMP stabilized the degradation rate of the mdx Rs receptors to that of innervated controls (Table 1). Each data point is mean ± SEM (n = ≥3).

Fig. 6.

Forskolin, the activator of adenylate cyclase, stabilizes the AChRs in both control (A) andmdx (B) diaphragm muscles in organ culture. Diaphragm muscles were treated with 40 μmforskolin, starting on day 3 (n = 6). In both cultures, the half-lives of the AChRs doubled after forskolin treatment.

Fig. 7.

mdx and control muscles have the same amount of cAMP in innervated soleus muscles, and their adenylate cyclase can be activated equally with forskolin. In mdxmuscles, there was 40 ± 12 pmol/mg soluble cAMP protein (mean ± SEM; n = 3) compared with 35 ± 2.4 pmol/mg cAMP soluble protein (mean ± SEM;n = 3) in controls. Treatment with 40 μm forskolin overnight caused the cAMP level to increase to 337 ± 49 pmol/mg soluble protein (average ± range;n = 2) in mdx muscles and to 298 ± 93 pmol/mg soluble protein (average ± range;n = 2) in controls.