Project Summary/Abstract The development of large-scale, biochemical proteomics and metabolomics approaches has led to a more sophisticated understanding of the pathways and biomarkers involved in neuromuscular plasticity in response to use, disuse, and aging in the limb musculature. The laryngeal muscle proteome and metabolome, however, remain relatively unexplored. Without direct study of the laryngeal muscles, diagnostic and therapeutic approaches for communication disorders caused by presumed laryngeal muscle dysfunction remain theoretically speculative. Our long-term goal is to understand the laryngeal neuromuscular response to increased voice use, such as vocal training and voice therapy, and decreased voice use, such as voice rest and senescence. The overall objective of this proposal is to determine how the proteome and metabolome of the thyroarytenoid muscle respond to vocalization training and aging. Our central hypothesis is that vocalization training will increase signaling pathways for mitochondrial function and oxidative stress responses and that aging will have unique proteomic and metabolomic effects in the thyroarytenoid muscle relative to the limb muscles. This work will be accomplished through two aims. In male and female rats we will characterize and identify specific biomarkers and biochemical pathways in the thyroarytenoid muscle indicative of neuromuscular adaptations under the following two conditions: (1) vocalization training and (2) senescence (aging). Specifically, we will broadly characterize muscle contractile properties, bioenergetics, and redox stress responses within the proteins and metabolites of muscle tissue of the thyroarytenoid muscle. This work will build on a previously established behavioral animal model involving training rats to increase their production of ultrasonic vocalizations. Additionally, using the same proteomic and metabolomic approaches, we will elucidate how age-related changes manifest across the lifespan by examining the thyroarytenoid muscle of young adult, older adult, and senescent male and female rats, and comparing biomarkers in this muscle to biomarkers in the gastrocnemius hindlimb. The study is innovative in its use of a behavioral animal model to investigate the effects of laryngeal muscle use and senescence on neuromuscular mechanisms and in its implementation of novel high-throughput proteomic and metabolomic approaches to examine laryngeal neuromuscular structure and function. These multi-omics approaches will complement previous studies conducted in our lab on neuromuscular junction and muscle fiber plasticity in normal and aging rodent models, thereby laying the foundation for understanding the cellular and molecular neuromuscular pathways involved in normal and aging intrinsic laryngeal muscle structure and function. This translational work will provide evidence for future therapeutic targets for clinical populations such as presbyphonia and hyperfunctional voice disorders. ...