PROJECT SUMMARY Effective contractile force in muscle requires the proper assembly, regulation, and activation of actin-containing thin filaments. Leiomodins (Lmods) are a family of actin-binding proteins that regulate assembly of actin filaments through a single tropomyosin-binding and multiple actin-binding domains. We previously discovered that both knockout and overexpression of the cardiac predominant isoform (Lmod2) alters the lengths of thin filaments in vivo and results in cardiomyopathy. Our extensive preliminary data suggest that Lmod2 impacts contractile function - independent of actin-thin filaments length regulation. With a plethora of unique experimental tools in hand, the goal of this proposal is to definitively determine the mechanisms of how mutations in LMOD2 lead to heart failure. It is becoming increasingly clear that the Lmod family of proteins play a critical role in muscle function; mutations in any of the three LMOD isoforms lead to debilitating human diseases. In this proposal, we describe the first known human mutation in LMOD2. This mutation leads to severe neonatal dilated cardiomyopathy. All LMOD-linked diseases have the common underlying pathophysiology of severe muscle weakness due to reduced contractility. Most of the disease-causing mutations in the LMOD gene family are nonsense or frameshift mutations predicted to result in expression of truncated proteins. However, in nearly all cases of disease little to no LMOD protein is expressed. Extensive preliminary data suggests that nonsense-mediated mRNA decay underlies the loss of mutant LMOD2 protein, which we can restore using LMOD2-specific antisense oligonucleotides. We hypothesize that Lmod2 is a multifunctional protein that influences cardiac contractility through maintaining proper thin filament lengths and positively effecting activation of the thin filament. We propose a multidisciplinary approach utilizing a unique combination of in vitro assays, patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and novel models of human disease to accomplish two Specific Aims focused on determining: 1) the fundamental function(s) of Lmod2, particularly how Lmod2 regulates thin filament assembly and what role it has in cardiac contractility, and 2) why human mutations in LMOD2 lead to a lack of protein expression, how loss of protein leads to disease, and whether restoring full-length or truncated LMOD2 can prevent (rescue) the onset of cardiomyopathy. Elucidating the in vivo function(s) of Lmod2 will provide critical missing links in our understanding of muscle contraction. In addition, these studies will have a broad impact on understanding the etiology and potential treatments of a spectrum of diseases that result from mutations in the LMOD family of genes, as well as other diseases that involve nonsense-mediated mRNA decay of essential proteins.