Muscle fatigue is a common experience, regardless of our level of fitness, and it becomes more pronounced as an individual becomes older. One source of fatigue is repeated use of a muscle group, which also results in decreased pH due to the metabolic byproducts from producing the energy necessary to fuel an active muscle. While this is a well-established principle, the relationship between fatigue and decreased pH at the molecular level is still unclear. Protein-protein interactions lie at the center of muscle contraction and proteins can undergo pH-dependent conformational changes, though this potential mechanism is relatively unexplored as a source of muscle fatigue. The muscle protein titin plays an important role in muscle since it acts as a scaffold for proper assembly of the sarcomere, the primary contractile unit of the muscle, and also plays a key role in maintaining a relatively uniform sarcomere length as muscles are stretched. Recently, our lab identified a region within the titin protein that undergoes a pH-dependent conformational changes. This region of titin is called the PEVK region and within that region there are clusters of glutamate residues, making these regions highly negatively charged. The size and distribution of these clusters varies as a function of muscle type and this variation is known to impact force generation and stiffness in muscle. Our lab has shown that the poly-E regions undergo pH-dependent conformational changes, which has led us to consider the novel mechanism that pH-based conformational changes in titin could contribute to muscle fatigue. The long-term goal of our work is to develop a comprehensive model correlating sequence variation in the PEVK region with titin function. Understanding the relationship between pH and the sequence of the PEVK is the first step toward achieving this goal. The results of these studies will facilitate future studies aimed at understanding how these conformational changes influence passive force. The central hypothesis of this application is that longer poly-E sequences with larger glutamate clusters undergo larger conformational changes. This hypothesis was generated based on studies from our lab showing that a 28-amino acid peptide derived from the human PEVK region undergoes a conformation change in the pH range of 5 to 7, switching from an elongated structure at neutral pH to a more compact structure and lower pHs. The rationale for the proposed research is that developing an understanding of the impact of pH on titin-based passive tension will provide new insights into titin function. The approach for testing our central hypothesis comprises two specific aims: 1) Determine the relationship between sequence and pH-derived conformational changes; and 2) Determine how pH influences force and stiffness in isolated PEVK fragments. This study is innovative as it focuses on a novel contributor to muscle fatigue and the insights gained in this study will improve understanding of how pH influences the function of muscle proteins. This knowledge will help with the development of new interventions for combating fatigue that may ultimately help extend the lifetime of effective muscle function to later ages.
The proposed research is relevant to public health because decreased pH is associated with loss of function in both skeletal and cardiac muscle but there are important questions about the mechanism for this loss of function that are not understood. The focus of the proposed work is developing an understanding of how the normal function of the muscle protein titin, a critical component in normal muscle function, is impacted by changes in pH associated with overuse or cardiovascular disease. As a result, this work is relevant to the part of NIH's mission pertaining to developing knowledge of basic biological mechanisms, which will facilitate development of new therapeutic strategies for treating musculoskeletal and cardiovascular disease.
