Regulation of thin filament architecture by CAP2 in health and myopathy - Aberrant actin-thin filament assembly results in life-threatening muscle diseases such as dilated cardiomyopathy (DCM) and nemaline myopathy. Our long-term goal is to determine the physiological link between thin filament architecture and myopathies, with the ultimate goal of formulating novel therapeutic strategies. It is known that sequential exchange of three α-actin isoforms: α-smooth (α-SMA), α-skeletal (α-SKA) and α-cardiac (α-CAA) into thin filaments is critical for development and function of striated muscle, and perturbations in this process lead to altered contraction and myopathies. However, the mechanisms underlying regulation of this process remain largely unknown. We discovered actin-binding cyclase associated protein 2 (CAP2) is a major regulator of cardiac thin filament assembly. Specifically, we found that it mediates α-actin isoform exchange by both regulating cofilin-2’s ability to depolymerize filaments comprised of α-SMA and promoting preferential incorporation of α-CAA into thin filaments; in other words, CAP2 acts as novel “actin monomer exchanger” of thin filaments. Accordingly, we found that cardiac thin filaments from Cap2-knockout (KO) mice contain low levels of α-CAA and high levels of α-SKA and α-SMA, while normally α-SKA and α-SMA are down-regulated in hearts of wild type mice as they age. These findings are important since CAP2 mRNA levels are reduced in human cardiomyopathies and the protein is critical for life; human mutations in CAP2, as well as knockout of Cap2 in mice, lead to DCM and sudden cardiac death. Unexpectedly, we discovered that CAP2-mediated subcellular alterations, and cardiac dysfunction are significantly more prominent in male mice, which may help to explain why human idiopathic DCM is more often frequent in men, with earlier age of onset and higher mortality. Together, these data lead us to an overarching hypothesis that aberrant α-actin isoform exchange exacerbates cardiac disease progression, and restoring proper actin isoform expression in myopathic hearts will ameliorate disease development. Excitingly, in support of this hypothesis, we found that expression of GFP-α-CAA in Cap2-KO hearts via adeno-associated virus (AAV) remarkably ameliorates onset of cardiomyopathy. We propose a multidisciplinary approach utilizing a unique combination of state-of-the art in vitro biochemical, biophysical and structural assays, cellular experiments, and novel CAP2 murine models to accomplish Specific Aims to determine: the role of CAP2/cofilin-2 complex in thin filament assembly and contraction; the role of individual CAP2 domains in -actin isoform switch; the relationship between thin filament organization and sex-specific cardiomyopathy; and whether modulating α-actin expression in diseased hearts improves cardiac differentiation and contractility. This work is of broad interest since abnormal α-actin isoform switching is a hallmark of many myopathies, regardless of the underlying cause of disease. This proposal leverages our ~30 years of experience filling critical gaps in our understanding of muscle contraction in health and disease.
