Contractions of skeletal muscles are regulated by a process called excitation-contraction (EC) coupling and defects in EC coupling are associated with numerous human muscle diseases. Motor neurons activate skeletal muscles by releasing neurotransmitter that causes the voltage across the muscle membrane to change. EC coupling is the process by which the change in muscle voltage is converted to a release of calcium ions from a specialized intracellular organelle called the sarcoplasmic recticulum (SR) in muscles. The increase in calcium ions in turn initiates contraction by activating the contractile proteins. EC coupling occurs at triadic junctions of the transverse tubules that are infoldings of the muscle membrane and outpocketings of the SR. The two main molecular components responsible for EC coupling are the dihydropyridine receptor (DHPR), a voltage dependent protein in the triadic transverse tubule membrane, and the ryanodine receptor (RYR), a calcium ion release channel located in the triadic SR membrane. These two proteins face each other in the triad and are thought to directly interact during EC coupling. The voltage changes across the muscle membrane are detected by DHPRs that in turn directly activate RYRs to release calcium ions from the SR. EC coupling requires a complex of proteins including DHPR and RYR localized to triads. Although much is known about the role of DHPR and RYR, relatively little is known about the identities and functions of other components of the triadic molecular complex. We identified a zebrafish mutation that is deficient in motor behaviors and found that the causative gene encodes a novel muscle adaptor protein that we found is a key regulator of EC coupling. The adaptor protein localizes to triads, binds to the DHPR-RYR1 complex and is required for proper release of calcium ions by the SR and contraction by skeletal muscles. We further found that the gene encoding this adaptor protein in humans is the basis for a debilitating congenital myopathy in which 36% of individuals afflicted die by age 18. Finally our evidence suggests that mutations of this gene lead to a decrease in DHPR in muscle by improper trafficking of DHPR to triads once they are synthesized. We propose to take advantage of the identification of this novel protein as a key regulator of EC coupling and a new causative gene for congenital myopathy to analyze how this protein regulates EC coupling and how a defect in the protein leads to congenital myopathy. For this we will take advantage of the ability to readily generate transgenic zebrafish and the unique ability to examine cellular processes in living zebrafish embryos. We propose to examine how trafficking of DHPRs are affected by mutations in this gene by generating transgenic zebrafish in which DHPRs are tagged with a fluorescent protein. We further found that the adaptor protein binds a subunit of the DHPR so will identify the sequences in the adaptor protein and DHPR subunit required for binding and examine the consequences of a loss of this binding. We also generated adaptor protein gene knockout mice to extend our analysis to mammalian muscles. This knowledge should help us better understand the biology of myopathies and could potentially lead to therapeutic agents for congenital myopathies.

Public Health Relevance

The proposal seeks to understand how congenital myopathies, which are skeletal muscle disorders that appear at birth and can be debilitating and fatal, are caused by genes that regulate the processing and functional activity of proteins important for muscle function. At present there are no cures and only one therapeutic drug for congenital myopathy. A better understanding of the molecular basis for myopathies could potentially lead to the identification of new therapeutic drugs for congenital myopathies.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Skeletal Muscle Biology and Exercise Physiology Study Section (SMEP)
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Boyce, Amanda T
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University of Michigan Ann Arbor
Schools of Arts and Sciences
Ann Arbor
United States
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Hsu, I-Uen; Linsley, Jeremy W; Varineau, Jade E et al. (2018) Dstac is required for normal circadian activity rhythms in Drosophila. Chronobiol Int 35:1016-1026
Linsley, Jeremy W; Hsu, I-Uen; Wang, Wenjia et al. (2017) Transport of the alpha subunit of the voltage gated L-type calcium channel through the sarcoplasmic reticulum occurs prior to localization to triads and requires the beta subunit but not Stac3 in skeletal muscles. Traffic 18:622-632
Linsley, Jeremy W; Hsu, I-Uen; Groom, Linda et al. (2017) Congenital myopathy results from misregulation of a muscle Ca2+ channel by mutant Stac3. Proc Natl Acad Sci U S A 114:E228-E236