Effects of Purified Recombinant Neural and Muscle Agrin on Skeletal Muscle Fibers in Vivo

Abstract

Aggregation of acetylcholine receptors (AChRs) in muscle fibers by nerve-derived agrin plays a key role in the formation of neuromuscular junctions. So far, the effects of agrin on muscle fibers have been studied in culture systems, transgenic animals, and in animals injected with agrin–cDNA constructs. We have applied purified recombinant chick neural and muscle agrin to rat soleus muscle in vivo and obtained the following results. Both neural and muscle agrin bind uniformly to the surface of innervated and denervated muscle fibers along their entire length. Neural agrin causes a dose-dependent appearance of AChR aggregates, which persist ≥7 wk after a single application. Muscle agrin does not cluster AChRs and at 10 times the concentration of neural agrin does not reduce binding or AChR-aggregating activity of neural agrin. Electrical muscle activity affects the stability of agrin binding and the number, size, and spatial distribution of the neural agrin–induced AChR aggregates. Injected agrin is recovered from the muscles together with laminin and both proteins coimmunoprecipitate, indicating that agrin binds to laminin in vivo. Thus, the present approach provides a novel, simple, and efficient method for studying the effects of agrin on muscle under controlled conditions in vivo.

Evaluation of C-terminal Agrin Fragment as a marker of muscle wasting in patients after acute stroke during early rehabilitation

“…Splicing isoforms containing 0 or 4 amino acid inserts at the y splicing site of the LG2 domain and 0, 8, 11, or 19 (8 + 11) amino acid inserts at the z splicing site of the LG3 domain of the C‐terminus have been investigated ( Figure  1). 23, 40 The neural agrin containing 4 and 8 amino acid inserts at the y and z sites, respectively, has a high affinity to the AChR clustering, while muscle agrin and agrin found in other non‐neuronal cells lacks inserts and fails to cluster AChRs 41. However, splicing isoforms of agrin lacking inserts have been found in NMJs (motor neurons, skeletal muscle, and Schwann cells), in the central nervous system and peripheral tissues (lung and kidney) 23.…”

Local Tissue Geometry Determines Contractile Force Generation of Engineered Muscle Networks

“…S4), further optimization of these processes in vitro will be required to allow the efficient survival and functional integration of tissue constructs in the host neuromuscular system. The use of angiogenic and/or neurotrophic factors, 7,30 electrical and mechanical stimulation, 9,16,31 and coculture with vascular and/or neuronal cells 6,32,33 are just some of the potential strategies to achieve this goal. Development of improved immunosuppression regimes 34 and cell sources with robust regenerative capacity (e.g., from human pluripotent stem cells 35 ) are some of the other important milestones toward the therapeutic use of tissue engineering to restore lost muscle function.…”

Neural Agrin Changes the Electrical Properties of Developing Human Skeletal Muscle Cells

“…Neural agrin was purified from the conditioned media of stably transfected HEK 293 cells (gift to G.F. from Dr. M.A. Ruegg, University of Basel, Basel, Switzerland) using mono Q-Sepharose Fast Flow Beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA) as previously described (Bandi et al 2008) by a modified method (Bezakova et al 2001) and added each time with the media changes.…”