PROJECT SUMMARYEffective contractile force in muscle requires the proper assembly regulation and activation of actin-containingthin filaments. Leiomodins (Lmods) are a family of actin-binding proteins that regulate assembly of actinfilaments through a single tropomyosin-binding and multiple actin-binding domains. We previously discoveredthat both knockout and overexpression of the cardiac predominant isoform (Lmod2) alters the lengths of thinfilaments in vivo and results in cardiomyopathy. Our extensive preliminary data suggest that Lmod2 impactscontractile function - independent of actin-thin filaments length regulation. With a plethora of uniqueexperimental tools in hand the goal of this proposal is to definitively determine the mechanisms of howmutations 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 describethe first known human mutation in LMOD2. This mutation leads to severe neonatal dilated cardiomyopathy. AllLMOD-linked diseases have the common underlying pathophysiology of severe muscle weakness due toreduced contractility. Most of the disease-causing mutations in the LMOD gene family are nonsense orframeshift mutations predicted to result in expression of truncated proteins. However in nearly all cases ofdisease little to no LMOD protein is expressed. Extensive preliminary data suggests that nonsense-mediatedmRNA decay underlies the loss of mutant LMOD2 protein which we can restore using LMOD2-specificantisense oligonucleotides. We hypothesize that Lmod2 is a multifunctional protein that influences cardiac contractility throughmaintaining proper thin filament lengths and positively effecting activation of the thin filament. We propose amultidisciplinary approach utilizing a unique combination of in vitro assays patient-specific induced pluripotentstem cell-derived cardiomyocytes (iPSC-CMs) and novel models of human disease to accomplish two SpecificAims focused on determining: 1) the fundamental function(s) of Lmod2 particularly how Lmod2 regulates thinfilament assembly and what role it has in cardiac contractility and 2) why human mutations in LMOD2 lead to alack of protein expression how loss of protein leads to disease and whether restoring full-length or truncatedLMOD2 can prevent (rescue) the onset of cardiomyopathy. Elucidating the in vivo function(s) of Lmod2 willprovide critical missing links in our understanding of muscle contraction. In addition these studies will have abroad impact on understanding the etiology and potential treatments of a spectrum of diseases that result frommutations in the LMOD family of genes as well as other diseases that involve nonsense-mediated mRNAdecay of essential proteins.