Genetics and Molecular Biology of Striated Muscle Myosin

We are probing the role of the myosin motor in aging and in progressive, genetically based diseases of
skeletal and cardiac muscles using transgenic models of Drosophila melanogaster. We take advantage of
the powerful genetic tools available for Drosophila to study myosin in an integrative manner, from atomic
structural details and biochemical function through muscle ultrastructure, fiber mechanics, cardiac physiology
and locomotion. We are examining the functional significance of specific residues within the skeletal muscle
myosin motor and rod domains that are post-translationally modified during human aging. These
modifications cause indirect flight muscle atrophy and dysfunction in our transgenic lines. To understand the
basis of these defects, we are assessing myosin ATPase activity, in vitro motility, thick filament formation
and fiber mechanics. We will determine if aging-related accumulation of ubiquinated protein aggregates and
inclusion bodies is augmented in these lines, and will examine whether expression of the autophagy-
enhancer FOXO ameliorates such disrupted proteostasis. Secondly, we will define the roles of proteins that
anomalously accumulate in aggregates in our Drosophila models of inclusion body myopathy type 3 and
myosin storage myopathy. Both are progressive skeletal muscle disorders that we found to exhibit aberrant
proteostasis, yet they display phenotypically different protein aggregates. We will examine the structural and
functional defects in skeletal muscles that arise from up- or down-regulating expression of the aberrant
aggregate proteins as well as the effects of over-expressing FOXO. This will yield insights into roles of
these proteins in aging and in degenerative muscle diseases. Finally, we are examining how mutations
associated with human age-exacerbated dilated cardiomyopathy affect the structure and function of the
Drosophila heart. We will determine mutation effects on cardiac proteostasis and examine if FOXO
ameliorates mutation effects. Using a unique in vivo method to produce mutant myosins, we will gain
mechanistic insights into biochemical and structural defects through ATPase and in vitro motility assays
and by solving cardiac protein structures via crystallography. Further, we will express these mutant cardiac
myosins in skeletal muscles to understand functional effects on muscle contractile properties. Overall,
our project will test significant questions regarding basic myosin function during aging of healthy and
diseased skeletal and cardiac muscles and assess whether improving proteostasis aids in rectifying muscle
degeneration.

Grant Number: 5R37GM032443-41
NIH Institute/Center: NIH
Principal Investigator: Sanford Bernstein