The cardiac thin filament is the essential regulator of cardiac contractility and relaxation at the molecular level.
It is comprised of five discrete proteins: cTnC, cTnI, cTnT, actin and tropomyosin that have co-evolved to
sustain efficient cardiac performance at rest, during exercise and, importantly, to respond to pathologic
stressors. Mutations in genes encoding each of these proteins have been definitively linked to the development
of a range of human genetic cardiomyopathies, including hypertrophic (HCM) and dilated (DCM) forms.
Despite 25 years of study by many groups including ours, to define the direct link(s) between the biophysical
insult and the resultant complex cardiomyopathy, many questions remain and significantly limit our ability to
use genotype to prognosticate and eventually even treat individuals with genetic cardiomyopathies. The recent
development of Mavacamten, a first-in-class, targeted myosin inhibitor is a game-changing advance that was
predicated on decades of basic research into the fundamental biology of the sarcomere. Thus, the question is
no longer “if” we can target the sarcomere, but for the thin filament the question is “what function to target” and
eventually “when to treat”. The cardiac thin filament is a highly dynamic allosteric “machine” where most of the
component proteins are comprised of a-helices connected by variably sized unstructured linkers, where
dynamic flexibility is the rule, not the exception and this has limited the availability of high resolution structure
for these regions. Most of the known pathogenic mutations in cTnI and cTnT are clustered within these highly
flexible domains, where there is likely a “distribution” of tolerance, whereby mutations impair function (enough
to cause disease) but do not break it. We thus propose that by examining the range of these dynamic
perturbations within these domains we can identify new structural and dynamic disease mechanisms that can
be functionally binned, studied and modulated. We provide proof-of-principle preliminary data in this proposal.
Over the recent funding period we expanded our structural methodologies to include Time-Resolved FRET
with a Single Donor – Dual Acceptor approach that allows us to use actin as an anchor to refine highly flexible
structures. We will next use known, highly divergent (HCM vs DCM) mutations within each flexible domain to
probe both structure and dynamics with the premise that these mutations will define the limits of “tolerability” in
either direction and use spectroscopy and measurements of Ca2+ dissociation and association kinetics coupled
to computation to define and test these hypotheses. Finally, we will “close the loop” by utilizing our existing
extensively characterized transgenic animal models based on the same mutations used to set our limits and
perform 3-timepoint RNA-Seq to discover unique early transcriptional signatures to help link these
perturbations to the resultant early remodeling cascade that eventually leads to distinct patterns of ventricular
remodeling. The long term goal is to use this coupled structural – dynamic – transcriptomic platform to identify
new targets, both primary and secondary for future therapeutics and even biomarker discovery.