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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - FCSM (Human myosins)

Teaser

Summary

In utero development is critical for normal skeletal and cardiac muscle function throughout life. Many diseases, such as distal arthrogryposis (affecting 1/3000 live births) and clubfoot (1/1000) in skeletal muscle and arrhythmias in cardiac muscle
(1/4000), manifest in the embryonic and foetal period. They permanently affect longevity and quality of life. Because the effects of these diseases are present at birth, the study of in utero samples is essential to understanding the diseasesâ€™
properties and effects on the developing muscle tissues. Additionally, many of these afflictions are caused by mutations in the isoforms of troponin or myosin II that are predominantly expressed during human development. Studying the native muscle is all the more important as a control in furthering research on the effects of mutations in troponin and myosin. In particular, human foetal-specific isoforms of myosin II expressed in these muscle are poorly understood and very little has been published about these isoforms. We do know from the literature that myosinâ€™s use of its substrate, ATP, varies widely between isoforms and that the myosin expression changes during times of physiological distress, such as heart failure.

Because congenital abnormalities of the heart and skeletal muscle both can originate in the foetal muscle, further investigation is needed into the myosinsâ€™ biophysical and biomechanical mechanisms. The overarching goal of this project was to improve understanding of how foetal forms of skeletal and cardiac myosins work and are regulated, using biophysical-biochemical, molecular biology and computational modelling techniques. This was accomplished by experimentally determining the kinetics of myosin-ATP and myosin-actin interactions using stopped-flow kinetic analysis, experimentally determining the rates of force development and relaxation as well as the quantity of force developed using single myofibrils, and experimentally determining a qualitative and quantitative analysis of myosin composition using mass spectrometry.

Overall we have concluded that the change in force development and rates of force development and relaxation in skeletal muscle are significantly contributed to by changes in the myosin isoform in skeletal foetal development. We have found that these changes in the performance of myosin are slightly different than the changes seen in cardiac myosin isoforms, although the overall effect on the force development appears similar. However, the rates of force development and parts of the relaxation process do seem to have slight differences in the way they change between skeletal and cardiac isoforms. These changes, and the specific of how the myosins change their interaction with both their substrate ATP and actin, help us in better understanding how mutations that appear in similar locations on the myosin molecule lead to different outcomes in the growth and performance of the muscle as it matures, and will help with developing targeted therapeutics in the future.

Work performed

Overall this project has been very productive. We have refined the myosin extraction protocol to successfully extract functional myosin from <10 mg of tissue, and used these ex vivo myosin to test sensitivity to ATP in both an actin-bound and actin-free state, as well as the sensitivity to the product ADP, which is essential for understanding the myosin speed in force generation and relaxation.
We have publicly disseminated results through several university-wide seminars, as well as 6 international conferences. In addition, work with myosin and out reach has been communicated to the public through an event at the local museum as well as \'in the classroom\' demonstrations and group projects for 6th form students (16-18 years of age).

Key results from experiments with skeletal muscle are in manuscript form, with the goal of submitting this manuscript to a peer reviewed journal in the next few months. Similarly, a manuscript with the cardiac muscle results is in progress. The key findings in these myosins are that in skeletal muscle significant changes in the myosin isoform have a strong correlation with the activation and relaxation of this muscle and change how that muscle performs as it matures. In contrast, while cardiac muscle shows great changes in its activation and relaxation throughout development, changes in the myosin isoform cannot be strongly correlated to these performance changes. This opens up new opportunities for research into other proteins which might be responsible for the changes seen in cardiac muscle, as well as research into therapeutic exploitation of the skeletal muscle myosin role in maturation of the skeletal muscle during the foetal period.

Final results

To date, developmental biology and tissue engineering studies on human muscle tissue use early-stage muscle cells derived from embryonic, foetal, or inducible pluripotent stem cell sources where the primary comparator is adult tissue. Human foetal skeletal & cardiac tissue, cell, and myosin functional research is very limited, with few publications and little knowledge of the structure and function with early stage isoforms of muscle sarcomere contractile and regulatory proteins. Until the fellowâ€™s 2013 publication on foetal tissue, there was no significant work on human muscle of similar developmental stage. Dr. Geeves, collaborating with Dr. Leinwand, was able to express & isolate the motor domain of human embryonic & perinatal myosins in mouse cell lines for detailed kinetic studies, which confirmed the results in this project regarding the speed and function of the developing skeletal muscle myosin. Our data are the first biochemical and biophysical studies to date on the whole myosin protein isolated from tissue, and thus, offer validation for studies on myosin from mouse cell lines and highlight post- translational modifications of the protein. Visits to the laboratory of Poggesi & Tesi (Florence, Italy) for studies on foetal myofibrils, allowed the fellow to examine myosin function under high-strain situations, important because myosin\'s enzymatic functions vary more than 10-fold under strain. Together this work has established a novel framework for studying foetal muscle from the molecular to
the tissue level. With our top-to bottom, molecule to muscle tissue, thorough functional assessment in developing muscles, engineered muscle tissue can be compared to human muscle of similar developmental stage and limitations in the engineered tissue better understood through such a comparison. As the tissue engineering field grows, a better understanding of the native developmental biophysical attributes offers insights into the potentials and limitations of such projects, and how to overcome barriers in function.