Neuromuscular junction failure in sarcopenia is linked to NaV1.4 loss and reversed by ClC-1 inhibition
W. David Arnold, Jeanette Jeppesen Morgen, Pernille Bogetofte Thomasen, Martin Broch-Lips, Leatha A. Clark, Thomas Groennebaek, Martin Skov, Jeppe Blichfeldt Winther, Abdullah Ramadan, Philippa A. Rust, Jessica H. Myers, Fereshteh B. Darvishi, Anna R. Dashtmian, Lauren A. Fish, Deepti Chugh, Jane Bold, Jorge Quiroz, John Hutchison, Hiroshi Nishimune, Ross A. Jones, Xueyong Wang, Justin R. Fallon, Thomas H. Gillingwater, Mark M. Rich, Thomas Holm Pedersen, Brian C. Clark
W. David Arnold, Jeanette Jeppesen Morgen, Pernille Bogetofte Thomasen, Martin Broch-Lips, Leatha A. Clark, Thomas Groennebaek, Martin Skov, Jeppe Blichfeldt Winther, Abdullah Ramadan, Philippa A. Rust, Jessica H. Myers, Fereshteh B. Darvishi, Anna R. Dashtmian, Lauren A. Fish, Deepti Chugh, Jane Bold, Jorge Quiroz, John Hutchison, Hiroshi Nishimune, Ross A. Jones, Xueyong Wang, Justin R. Fallon, Thomas H. Gillingwater, Mark M. Rich, Thomas Holm Pedersen, Brian C. Clark
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Research Article Aging Neuroscience

Neuromuscular junction failure in sarcopenia is linked to NaV1.4 loss and reversed by ClC-1 inhibition

Abstract

Sarcopenia is the age-related loss of muscle strength and size that leads to mobility limitations and loss of independence in older adults. The underlying cellular mechanisms remain unclear, and treatments are limited. As the critical interface between the nervous system and muscle, the neuromuscular junction (NMJ) is essential for muscle activation and force production. Here, we demonstrate that weak older individuals exhibited NMJ transmission failure that correlated with muscle weakness severity. Preclinical experiments showed similar NMJ transmission failure in aged rodents that was associated with localized loss of muscle fiber excitability at the NMJ. This excitability defect, distinct from potential synaptic cholinergic transmission abnormalities, represents a disease mechanism of sarcopenia. Across species, immunohistochemistry identified a localized reduction in the voltage-gated sodium channel specific for skeletal muscle (NaV1.4) at the postsynaptic NMJ membrane. Acute NaV1.4 inhibition with μ-conotoxin GIIIB in adult rats reproduced findings of NMJ transmission failure observed in aged rodents and humans. Finally, ClC-1 chloride ion channel inhibition enhanced muscle excitability and improved NMJ transmission and muscle function in old rodents. Together, these findings demonstrate that NMJ transmission deficits are a key, reversible driver of sarcopenia and reveal a therapeutic target for addressing muscle weakness in aging.

Authors

W. David Arnold, Jeanette Jeppesen Morgen, Pernille Bogetofte Thomasen, Martin Broch-Lips, Leatha A. Clark, Thomas Groennebaek, Martin Skov, Jeppe Blichfeldt Winther, Abdullah Ramadan, Philippa A. Rust, Jessica H. Myers, Fereshteh B. Darvishi, Anna R. Dashtmian, Lauren A. Fish, Deepti Chugh, Jane Bold, Jorge Quiroz, John Hutchison, Hiroshi Nishimune, Ross A. Jones, Xueyong Wang, Justin R. Fallon, Thomas H. Gillingwater, Mark M. Rich, Thomas Holm Pedersen, Brian C. Clark

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Figure 2

Age-related decline in localized postsynaptic excitability and reduced NaV1.4 channel distribution at the NMJ.

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Age-related decline in localized postsynaptic excitability and reduced N...
(A and B) Excitability was assessed in adult and old mice using a 2-electrode approach in which electrodes E1 and E2 were positioned together near the NMJ (junctional, within 1,000 μm) and then repositioned farther from the NMJ (extrajunctional, >1,000 μm). Older mice exhibited significantly fewer fibers with localized enhanced excitability at the NMJ (gain) compared with adult mice. Gain was defined by a higher stimulation requirement in extrajunctional regions than at the NMJ to trigger an action potential (AP). Total fibers measured: adult, 81; old, 69. (C and D) Similar reductions in excitability gain were observed in old rats (23–24 months, n = 7) compared with adult rats (6–7 months, n = 5) using a 3-electrode approach. The centralstimulating electrode (Es) was positioned near the NMJ (junctional within 50 μm) and then farther from the NMJ (extrajunctional >800 μm), and APs were elicited by current injection through Es. Total fibers measured: adult, 36; old, 45. (E and F) Representative confocal images of sternomastoid NMJs from adult (3–4 months, n = 4) and old (26–28 months, n = 4) mice. AChRs were labeled with α-bungarotoxin (green) and NaV1.4 with specific antibodies (red). (G–I) Quantitative analysis in mouse NMJs: (G) AChR area, (H) NaV1.4 area, and (I) NaV1.4 area normalized to AChR area. Compared with adults, aged mice showed significant reductions across all measures. (J and K) Representative confocal images of lumbrical NMJs from adult (5.5–7 months, n = 5) and old (21–23 months, n = 5) rats. (L–N) Quantitative analysis in rat NMJs: (L) AChR area, (M) NaV1.4 area, and (N) NaV1.4 area normalized to AChR area. Old rats showed significant reductions in NaV1.4 area from adult rats. (G–I and L–N) Each data point represents mean values per animal from 25–27 NMJs per mouse and 23–25 NMJs per rat. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01 using unpaired t tests. Scale bars: 10 μm.