PROJECT SUMMARY Filamin C (FLNC) is a muscle-specific actin-binding protein, which localizes to the Z-disc and intercalated disc of cardiac muscle, and interacts with β1 integrin and sarcoglycans at the costamere. Multiple mutations in FLNC are associated with human cardiomyopathies, highlighting its importance for cardiac function. Individuals with compound heterozygous variants in the FLNC gene (F106L/R991*(a null allele)) develop pediatric dilated cardiomyopathy (DCM). Interestingly, individuals carrying only one FLNC (F106L or R991*) variant do not exhibit any signs of cardiomyopathy; suggesting that F106L is a loss-of-function mutation. Little is known as to the specific role of FLNC in cardiomyocytes (CMs), or mechanisms by which the F106L mutation leads to loss of function, and how individuals with FLNC F106/null compound heterozygous alleles develop DCM. Deletion of the last 8 exons of FLNC in mice results in perinatal lethality. However, given that multiple distinct FLNC mutations lead to cardiomyopathy, the absence of a cardiac phenotype in this FLNC-deficient mouse model is puzzling. As the authors noted, a truncated FLNC protein is still expressed in this mutant mice, suggesting that the mutant FLNC allele is hypomorphic. Thus, a true null FLNC mouse model and a FLNC CM-specific knockout (KO) mouse model are essential to comprehensively understand the role of FLNC in the heart. In addition, studies in CMs and in vivo animal models are essential to understand the molecular basis underlying the DCM caused by the F106L/null mutations in FLNC. To address these questions, we have generated a floxed FLNC mouse line and used it to generate a FLNC global KO (gKO) mouse model, as well as constitutive (cKO) and inducible (icKO) FLNC CM-specific KO mice. In contrast to the reported FLNC hypomorphic mouse, our FLNC gKO mouse model is lethal at E10.5 and exhibits severe chest edema and decreased CM proliferation. Moreover, FLNC cKO mice die between E10.5-E11.5 and exhibit an essentially identical morphological phenotype as observed in FLNC gKO mice, suggesting that the primary cause of lethality in FLNC gKO mice is due to loss of FLNC in CMs. We also observed that adult FLNC icKO mice develop DCM and progressive heart failure. Accordingly, our hypothesis is that FLNC plays an essential role in maintaining CM sarcomere and costamere integrity, cardiac morphogenesis, and normal cardiac function, and that the FLNC F106L mutation is a loss-of-function mutation and impairs specific interaction between FLNC and actin. Our Specific Aims are: 1. To determine the role of FLNC in the developing and adult myocardium by analyzing cKO and icKO FLNC CM-specific KO mice for heart morphogenesis, structure and function, and the progression of cardiomyopathy; and 2. To elucidate mechanisms by which the F106L FLNC mutation leads to loss of function and how individuals with F106L/null compound heterozygous alleles develop pediatric DCM by analysis of F107L/null and F107L/F107L mutation knock-in mice and human embryonic stem cell-derived CMs containing FLNC F106L/-, FLNC F106L/F106L, or FLNC -/- mutations.

PROJECT NARRATIVE Mutations in Filamin C (FLNC) result in human cardiomyopathies, highlighting its importance for cardiac function. However, little is known as to the specific role of FLNC in cardiomyocytes, and how mutations in FLNC result in cardiomyopathy. Proposed studies are aimed at understanding the function of FLNC in cardiac muscle at the molecular, cellular, and physiological levels, and gaining insights into mechanisms by which mutations in FLNC cause cardiomyopathy.