Fast and slow-twitching muscles are differentially affected by reduced cholinergic transmission in mice deficient for VAChT: A mouse model for congenital myasthenia

Abstract

Congenital myasthenic syndromes (CMS) result from reduced cholinergic transmission at neuromuscular junctions (NMJs). While the etiology of CMS varies, the disease is characterized by muscle weakness. To date, it remains unknown if CMS causes long-term and irreversible changes to skeletal muscles. In this study, we examined skeletal muscles in a mouse line with reduced expression of Vesicular Acetylcholine Transporter (VAChT, mouse line herein called VAChT-KD). We examined this mouse line for several reasons. First, VAChT plays a central function in loading acetylcholine (ACh) into synaptic vesicles and releasing it at NMJs, in addition to other cholinergic nerve endings. Second, loss of function mutations in VAChT causes myasthenia in humans. Importantly, VAChT-KD present with reduced ACh and muscle weakness, resembling CMS. We evaluated the morphology, fiber type (myosin heavy chain isoforms), and expression of muscle-related genes in the extensor digitorum longus (EDL) and soleus muscles. This analysis revealed that while muscle fibers atrophy in the EDL, they hypertrophy in the soleus muscle of VAChT-KD mice. Along with these cellular changes, skeletal muscles exhibit altered levels of markers for myogenesis (Pax-7, Myogenin, and MyoD), oxidative metabolism (PGC1-α and MTND1), and protein degradation (Atrogin1 and MuRF1) in VAChT-KD mice. Importantly, we demonstrate that deleterious changes in skeletal muscles and motor deficits can be partially reversed following the administration of the cholinesterase inhibitor, pyridostigmine in VAChT-KD mice. These findings reveal that fast and slow type muscles differentially respond to cholinergic deficits. Additionally, this study shows that the adverse effects of cholinergic transmission, as in the case of CMS, on fast and slow type skeletal muscles are reversible.

Evaluation of the neuromuscular junction in a middle‐aged mouse model of congenital myasthenic syndrome

“…Our group previously developed a mouse model with reduced expression of VAChT (VAChT KD HOM ) and found motor, cardiac, and synaptic alterations that resemble some of the features later discovered in CMS patients with mutations in VAChT . Interestingly, adult animals already exhibit cognitive and behavioral impairment, with a 40% to 50% decrease in expression level of VAChT, suggesting that central cholinergic synapses are even more sensitive to decreased VAChT activity than peripheral synapses, in which symptoms appear only after a 70% decrease in VAChT expression .…”

“…When we performed the wire‐hang and rotarod tests, middle‐aged VAChT KD HOM performed worse when compared with WT mice (Figure A,B). However, if we compare the performance of VAChT KD HOM middle‐aged mice with 6‐month‐old mice, no further impairment was detected due to the aging process …”

“…However, further investigation is needed to determine why MEPP frequency increased in aging VAChT KD HOM mice compared with adult counterparts. It may have been due to a compensatory mechanism built up over time due to decreased ACh release in these mice …”

“…However, if we compare the performance of VAChT KD HOM middle-aged mice with 6-month-old mice, no further impairment was detected due to the aging process. 11…”

“…On the other hand, VAChT knock‐down (VAChT KD HOM ) mice, which exhibit a 70% decrease in VAChT expression, are able to survive and reach adulthood, but they are myasthenic and have cognitive deficits . At the NMJ, adult VAChT KD HOM mice show a reduction in quantal content and size, reduced miniature end‐plate potential frequency, impairment of motor performance, and severe deficits in muscle strength . Reduced expression of VAChT does not interfere with the overall morphology of the NMJ in the adult mouse; however, changes in the distribution and shape of synaptic vesicles within the nerve terminal have been observed, suggesting that vesicular ACh content may play a key role in maintaining synaptic structure.…”