Myosin-binding protein C (MyBP-C), a thick filament associated protein of vertebrate striated muscle, is
a key modulator of muscle contractility. Multiple distinct skeletal muscle MyBP-C isoforms are encoded by two
genes in mammals (i.e., MYBPC1 (slow-type) and MYBPC2 (fast-type)) with mutations to these genes now
linked to human skeletal myopathies, such as distal arthrogryposis. In vitro reductionist approaches have
proposed mechanisms by which the MyBP-C N terminus modulates muscle contractility through its binding-
partner interactions with the actin-thin filament and the myosin head region. Specifically, MyBP-C is believed to
sensitize the thin filament to calcium, stabilize the myosin super-relaxed state, and/or act as a molecular “brake”
to slow myofilament sliding. However, since multiple MyBP-C isoforms are co-expressed in mammalian muscle,
it has been impossible to define which of these modulatory roles are linked to specific MyBP-C isoforms within
the context of an intact muscle, let alone how they may be altered by genetic mutations. Here we propose a
novel approach in zebrafish to generate ‘designer’ muscles exclusively expressing a single, transgene-encoded
MyBP-C isoform as desired. Our preliminary data indicate that MyBP-H, an MyBP-C family member, comprises
~95% of myosin binding protein in larval zebrafish swimming muscles. Therefore, Aim 1 makes use of the
recently developed CRISPR/Cas9 ‘GeneWeld’ method, to generate precise integration alleles by homology
mediated end joining (HMEJ) that will simultaneously: i) interrupt endogenous MyBP-H expression, and; ii) place
a DNA cassette, encoding one of two most functionally extreme MyBP-C isoforms, under regulatory control of
the most highly expressed endogenous MyBP gene locus. By this approach, we propose to create zebrafish with
“designer MyBP-C” muscles. Quantitative proteomics will enable us to determine whether transgenic MyBP-C
accumulates to wildtype levels, while immunofluorescence of FLAG-tagged transgenic MyBP-C will be used to
confirm proper subcellular localization. In Aim 2 we use biophysical assays previously developed in the Warshaw
lab to define the functional impact of transgenic MyBP-C isoforms across multiple scales. Specifically, native
myosin thick filaments will be used to assess MyBP-C “braking” action, while myofibrils will be used to assess
the presence of the super-relaxed myosin state. Data from these simplified muscle systems obtained from the
proposed “designer MyBP-C” zebrafish will be correlated with intact larval muscle mechanics. Thus, this project
will provide significant insight into how an individual MyBP-C isoform modulates both molecular and cellular
contractility in the context of intact muscle. This “designer MyBP-C” zebrafish model system will create a platform
for future mechanistic studies of MyBP-C mutations associated with human skeletal myopathies as a first step
to therapeutic design.
