Muscle derived BMP4 regulates morphology and function of endplates on extrafusal and intrafusal muscle fibers in adult mice

Abstract

Understanding factors contributing to neuromuscular junction (NMJ) stability post-development will shed light on how this stability is lost during aging and in neuromuscular diseases. Previous work in Drosophila suggests that morphogens within the bone morphogenetic protein (BMP) family are potential candidates because the BMP homolog, gbb, along with its receptor, wit, have key roles in NMJ structure, stability, and function. Whether BMPs have similar roles at vertebrate NMJs is currently unknown. To examine this question, we generated doxycycline-inducible, muscle specific BMP4 null mice, referred to here as HSA^CreBMP4^fl/fl mice. Motor behavior tasks were examined pre- and post-induction while electrophysiological and morphological characteristics were examined 4 months later in mice of both sexes. Soleus muscles from HSA^CreBMP4^fl/fl mice had significantly reduced contractile force compared to wild-type (WT) littermates. Cross-sectional areas of type I, but not type IIa, muscle fibers were reduced. NMJs were also larger in HSA^CreBMP4^fl/fl muscles compared to controls due to a significant increase in acetylcholine receptor fragment number and distribution. HSA^CreBMP4^fl/fl NMJs displayed reduced amplitude and frequency of miniature endplate potentials (mEPPs), evoked EPP amplitude, quantal content, and had increased failure rates when stimulating at high frequencies. Behaviorally, HSA^CreBMP4^fl/fl mice performed increasingly worse over time on the rotarod after doxycycline administration compared to their WT littermates. Finally, muscle spindle structure and proprioceptive function were significantly compromised in HSA^CreBMP4^fl/fl mice. These results indicate that muscle derived BMP4 regulates morphological and electrophysiological attributes of the NMJ in adult mice as well as the structure and function of muscle spindles.

Significance statement Understanding the cellular mechanisms underlying neuromuscular junction (NMJ) stability is critically important in understanding why it is compromised during aging and in motoneuron diseases. Studies in Drosophila larvae have shown that gbb and wit, a ligand and receptor in the BMP signaling pathway, are critical for the stability and function of the NMJ. This paper uses a novel doxycycline-inducible, muscle-specific BMP4 knockdown approach to eliminate muscular BMP4 expression in adult mice. When BMP4 was excised in the adult, we found that muscle strength and neurotransmission were attenuated, endplates fragmented, and mice had locomotor deficits. Furthermore, muscle spindle innervation and proprioceptive function were impaired. Therefore, as in Drosophila larvae, BMP4 is required for normal function and morphology of adult vertebrate NMJs.