PROJECT SUMMARY/ABSTRACT
Cardiomyopathies occur in ~1:200 individuals and are commonly caused by inheritance of variants in genes that
encode proteins that regulate the sarcomere, the force-producing organelle of heart cells. Due to an incomplete
understanding of variant pathogenicity and cardiomyopathy pathogenesis, physicians are currently limited in
their ability to provide diagnoses, prognoses, and therapeutic options for cardiomyopathy patients. Variants in
the TTN gene, which encodes the sarcomere protein titin, are the most frequently identified genetic lesion in
dilated cardiomyopathy (DCM), which is characterized by heart chamber dilation, reduced contractile function,
risk of sudden death, and progressive heart failure. The most frequent type of TTN variant identified in DCM is
a truncation mutation that would be predicted to shorten TTN protein length and to reduce TTN protein quantities.
Significantly, truncation variants localized to distal TTN structural domains are more pathogenic than those
localized to proximal structural domains, but the mechanistic basis for this relationship is uncertain. It remains
incompletely understood how TTN truncation variants cause DCM generally, which is compounded by our lack
of understanding of the ‘length dependence’ of TTN variant pathogenicity. These knowledge gaps limit disease
prognostication, biomarker identification, and therapeutic development for DCM patients. The central goal of
our study is to define how disruptions in TTN length and dosage by TTN variants cause DCM, and exploit this
knowledge to develop DCM therapeutics for TTN variant carriers. We hypothesize that healthy cardiac
contractile function and structure depends on the regulation of TTN length and dosage, and that varying
pathogenicity of TTN truncation can be explained by distinct structural and functional consequences associated
with the specific site of truncation. In Aim 1, we will determine the functional consequences of TTN truncations
across structural domains by harnessing 3-dimensional heart tissue models composed of human cardiomyocytes
differentiated from induced pluripotent stem cells in which variants have been introduced by CRISPR-mediated
genome editing. We will interrogate these models for tissue mechanical phenotypes (such as passive tension
and Frank-Starling behavior), TTN protein length and levels (using specialized methods), proteostasis stress
pathway responses (using immunoblotting), and mechanotransduction signaling and alternative splicing (using
expression analysis and transcriptomics, respectively). In Aim 2, we will restore TTN protein levels using the
recently developed method of CRISPR activation applied to DCM engineered heart tissue models for both
evaluating the function of TTN isoforms generally and as a DCM proof-of-concept therapeutic. Through these
Aims, we will gain critical new insights into the pathophysiology of DCM-associated TTN truncation variants,
uncover features to explain the variable pathogenicity identified in DCM patients, and develop a therapeutic to
target TTN directly. We anticipate this new knowledge will improve physicians’ capacity to diagnose, prognose,
and treat patients with DCM due to TTN variants.