During the past century, physiologists have made remarkable progress in elucidating the molecular mechanisms of muscle contraction. Despite this progress, the goal of predicting how muscle force changes during natural movements has remained elusive. This project will advance the understanding of muscle contraction by (1) testing the hypothesis that the length and stiffness of the giant, elastic titin protein changes when calcium is released from the sarcoplasmic reticulum upon muscle activation and (2) developing a computer model of muscle based on this hypothesis. A mutant mouse that carries a deletion in the titin gene will be used to test the hypothesis. This research will involve: (1) gel electrophoresis to estimate molecular weights of titin in mutant genotypes, (2) force-lever experiments to characterize activation-dependent elastic properties of titin in myofibrils, single fibers and whole muscles, (3) development of a muscle model that incorporates calcium activation of titin, and (4) behavioral studies to compare kinematics and energetics of locomotion across mutant genotypes. The broader impacts of this work include: (1) interdisciplinary training of undergraduate, graduate, and post-doctoral scholars in neuroscience, engineering, and computer science, (2) participation of under-represented students, and (3) public outreach including participation in the research project by local High School teachers and students from a public science and technology magnet school. Results will be disseminated through publication in diverse media and participation in interdisciplinary conferences in the areas of computer science, engineering, and biology. This research has the potential to transform our understanding of the process of muscle activation, improve neuro-musculoskeletal simulations, inform causes and potential cures for neuromuscular diseases, and inspire the design of actuators and prostheses that function more like animal muscles.

Project Report

Intellectual Merit: During the past century, physiologists made remarkable progress in elucidating the molecular mechanisms of muscle contraction. However, despite this progress, the goal of predicting how muscle forces change during natural movements remains elusive. Muscle models based on the sliding filament - swinging cross bridge theory fail to predict changes in muscle force at strain rates or frequencies that occur during natural movements. The goals of this project were: (1) to perform experiments to test the hypothesis that, in addition to the cross-bridges, the giant titin protein is activated by calcium ions in skeletal muscle; (2) to incorporate titin activation into a muscle model based on experimental results, and to use the new model to simulate non-linear muscle properties; (3) to use experiments in intact muscles to characterize the behavior of muscles from genotypes of mdm mice, which carry a deletion in N2A titin, and to evaluate the winding ratchet model by comparing results from simulations and experiments; (4) to compare locomotion and energetics of locomotion among genotypes of mdm mice; and (5) to synthesize results from myofibrils, single fibers, and intact muscles with studies of locomotion and energetics in intact mice across mdm genotypes as a first step toward understanding the effects of variation in passive and active titin viscoelasticity from sarcomere to organism. We tested the hypothesis that activation of muscle results in binding of titin to the thin filaments by comparing active and passive load clamp tests between wild type and mdm soleus muscles. In wild-type soleus, activation shortened the titin spring by 15% and increased muscle stiffness 2.7-fold compared to passively stretched muscles. In mdm soleus, the neither spring length nor stiffness changed with activation. These results are consistent with the hypothesis that upon Ca2+-activation of muscle, an epitope of titin that is absent in the mdm mutants binds to the thin filaments. We also developed techniques for stretching myofibrils from mdm and wild-type mouse psoas fibers beyond overlap of the thick and thin filaments. Several myofibrils from wild-type psoas were stretched actively and passively beyond filament overlap. The results show that activation increases the stiffness of wild-type psoas myofibrils, as found for rabbit psoas. Our results demonstrate that 15% of titin’s enhanced state can be attributed to direct calcium effects on the protein. We suggest that the remaining unexplained 85% of this extra force results from titin binding to the thin filaments. A set of experiments performed on myofibrils from mdm psoas showed no change in stiffness with activation, suggesting that the site in titin that binds to actin upon muscle activation is absent or altered in the mutants. We developed and published a model that predicts force enhancement of muscle based on activation of titin via binding of the N2A region to the thin filaments, and winding of titin on the thin filaments due to rotation. The simulations accurately reproduce the observed pattern of variation in force enhancement with sarcomere length. Jumping performance was reduced in mdm mice compared to wild-type mice, primarily because the limbs fail to extend completely during jumping, similar to the differences in joint-angle excursions observed during walking. Passive exhalation is faster in mutants, consistent with the higher passive stiffness of their diaphragm muscles. The tremor frequency during shivering thermogenesis was significantly lower mutants, consistent with the lower titin-based stiffness of active muscles. During the past three years of this award, we have collected data from a variety of experiments on myofibrils, intact muscles, and behaving mice that are entirely consistent with our transformative new hypothesis. Not a single experimental observation from this work was in conflict with the hypothesis, and no result was ambiguous. The work that we accomplished under support for this project provides strong support for our hypothesis of calcium-dependent activation of titin, and establishes the mdm mutant as a model for understanding this process. Broader Impacts: The results of this research led to a successful application for a grant from the W. M. Keck Foundation to investigate the winding filament hypothesis using atomic force microscopy, electron tomography and holography, and fluorescent biosensors in a transgenic mouse. The project provided significant interdisciplinary training for four postdoctoral associates, 11 graduate students (one computer scientist, two chemists, and one mechanical engineer), 19 undergraduate students from biology, physics, mechanical engineering, and electrical engineering (including two Native American students and one Hispanic student), as well as research experiences for two high-school science teachers and two high school students. The winding filament hypothesis has implications for mechanical engineering, electrical engineering, prosthetics and robotics for algorithms and actuators that emulating biological actuation in human engineered devices.

National Science Foundation (NSF)
Division of Integrative Organismal Systems (IOS)
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Steven Ellis
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Northern Arizona University
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