The W-Loop of Alpha-Cardiac Actin Is Critical for Heart Function and Endocardial Cushion Morphogenesis in Zebrafish

Mutations in cardiac actin (ACTC) have been associated with different cardiac abnormalities in humans, including dilated car-diomyopathy and septal defects. However, it is still poorly understood how altered ACTC structure affects cardiovascular physi-ology and results in the development of distinct congenital disorders. A zebrafishmutant (s434mutation) was identified that displays blood regurgitation in a dilated heart and lacks endocardial cushion (EC) formation.We identified the mutation as a single nucleotide change in the alpha-cardiac actin 1a gene (actc1a), resulting in a Y169S amino acid substitution. This mutation is located at theW-loop of actin, which has been implicated in nucleotide sensing. Consequently, s434mutants show loss of po-lymerized cardiac actin. An analogous mutation in yeast actin results in rapid depolymerization of F-actin into fragments that cannot reanneal. This polymerization defect can be partially rescued by phalloidin treatment, which stabilizes F-actin. In addi-tion, actc1amutants show defects in cardiac contractility and altered blood flow within the heart tube. This leads to downregula-tion or mislocalization of EC-specific gene expression and results in the absence of EC development. Our study underscores the importance of theW-loop for actin functionality and will help us to understand the structural and physiological consequences of ACTCmutations in human congenital disorders. Actin is an abundantmultifunctional protein involved inmanycellular functions, includingmuscle contraction (18, 19). The highest concentration of actin is found in striated muscles, form-ing the thin microfilament “rail ” of the sarcomere, along whic