Muscle injury is often accompanied by disruption of the muscle plasma membrane, and disruption of the muscle plasma membrane leads to muscle injury. The elongated nature of skeletal myofibers, while optimized for muscle contraction, places stress and strain on the sarcolemma through direct and lateral force transmission. Mutations in genes encoding dystrophin and its associated proteins are associated with a fragile plasma membrane that is more susceptible to rupture. Loss-of-function mutations in the dysferlin gene cause muscular dystrophy through abnormal muscle membrane trafficking, which includes defective sarcolemmal repair. An improved understanding of the membrane repair machinery of muscle will not only provide useful information for developing novel approaches to treat dysferlinopathies, but also will provide insight for muscle repair in many forms of muscle injury. Muscle injury occurs with disease, intense exercise, trauma, and surgery, and enhancing muscle repair may benefit each of these needs. Dysferlin, and the highly related myoferlin, are membrane-anchored proteins with multiple C2 domains, domains known to have protein and phospholipid binding properties. We used high-resolution microscopy and real time live imaging to define distinct subcomplexes within the sarcolemma repair machinery. The first subcomplex is the repair cap, which forms within seconds after membrane disruption. The repair cap is enriched for annexins, including annexins A6, A2, and A1. Immediately beneath this cap within the cytoplasm is an annexin-free zone enriched with filamentous actin. The repair cap is surrounded by a ring of shoulder proteins that include dysferlin, EHD proteins, BIN1 and others. The shoulder region is enriched for phosphatidylserine indicating the importance of phospholipid signaling for sarcolemmal repair. The repair cap and the shoulder ring are independent structures. We propose a model in which calcium and phospholipid signaling orchestrate the assembly of the repair complex in muscle by first organizing annexin caps. In this model, phospholipid signaling drives assembly of the ring of shoulder proteins, which requires their membrane-bending capacity. We will test this model of sarcolemmal repair using laser injury and high resolution imaging in isolated muscle fibers. We will also evaluate this model in the context of in vivo muscle disease and injury.
The first aim will conduct experiments to delineate annexin assembly with focus on the role of calcium as a trigger for repair cap formation.
The second aim concentrates on defining the role of dysferlin and its interaction with BIN1 and EHD proteins in forming the shoulder ring.
The third aim outlines experiments to improve muscle membrane repair. These studies will decipher repair complex interactions with the goals of mitigating sarcolemmal injury and improving muscle repair.

Public Health Relevance

Muscle injury occurs after intense exercise, due to trauma and surgery, and muscle heals by way of repair and regeneration, and we developed a new system in which we can now visualize the repair complex as it reseals disrupted muscle membranes. Using this method, we will define how the repair subcomplexes assemble and define how these complexes function during muscle injury and in muscle diseases like muscular dystrophy. The findings from this work are not only relevant during muscle recovery but also for the treatment of chronic muscle diseases like the muscular dystrophies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS047726-15
Application #
9837493
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Nuckolls, Glen H
Project Start
2003-07-01
Project End
2022-11-30
Budget Start
2019-12-01
Budget End
2020-11-30
Support Year
15
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Northwestern University at Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Quattrocelli, Mattia; Capote, Joanna; Ohiri, Joyce C et al. (2017) Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury. PLoS Genet 13:e1007070
Quattrocelli, Mattia; Barefield, David Y; Warner, James L et al. (2017) Intermittent glucocorticoid steroid dosing enhances muscle repair without eliciting muscle atrophy. J Clin Invest 127:2418-2432
Demonbreun, Alexis R; McNally, Elizabeth M (2017) Muscle cell communication in development and repair. Curr Opin Pharmacol 34:7-14
Quattrocelli, Mattia; Salamone, Isabella M; Page, Patrick G et al. (2017) Intermittent Glucocorticoid Dosing Improves Muscle Repair and Function in Mice with Limb-Girdle Muscular Dystrophy. Am J Pathol 187:2520-2535
Lamar, Kay-Marie; Miller, Tamari; Dellefave-Castillo, Lisa et al. (2016) Genotype-Specific Interaction of Latent TGF? Binding Protein 4 with TGF?. PLoS One 11:e0150358
Demonbreun, Alexis R; Allen, Madison V; Warner, James L et al. (2016) Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption. Am J Pathol 186:1610-22
Demonbreun, Alexis R; McNally, Elizabeth M (2016) Plasma Membrane Repair in Health and Disease. Curr Top Membr 77:67-96
Demonbreun, Alexis R; Quattrocelli, Mattia; Barefield, David Y et al. (2016) An actin-dependent annexin complex mediates plasma membrane repair in muscle. J Cell Biol 213:705-18
Lenhart, Kaitlin C; O'Neill 4th, Thomas J; Cheng, Zhaokang et al. (2015) GRAF1 deficiency blunts sarcolemmal injury repair and exacerbates cardiac and skeletal muscle pathology in dystrophin-deficient mice. Skelet Muscle 5:27
Demonbreun, Alexis R; McNally, Elizabeth M (2015) DNA Electroporation, Isolation and Imaging of Myofibers. J Vis Exp :e53551

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