In this study, we have positionally cloned a zebrafish mutant (s434 mutation) that displays defects in EC morphogenesis and blood circulation. Our results show that actc1as434 mutants have a Y169S amino acid substitution in the W-loop of alpha cardiac actin (actc1a), which results in fragile actin filaments. Yeast cultures demonstrated that F169S mutant actin forms filaments, which then spontaneously disintegrate and are unable to reanneal and form F-actin. The phenotype in both zebrafish and yeast can be partially or fully rescued by treatment with the actin-stabilizing drug phalloidin, respectively. Mutant embryos display altered blood flow within the heart tube, which results in misexpression of genes associated with AVC morphogenesis and leads to the absence of EC formation.
In addition to ACTC
, other actins, including skeletal actin (acta1a
) are expressed in mouse cardiac tissue during embryogenesis and can provide redundant function (32
). While expression patterns for all of the zebrafish actin isoforms are not currently known, we have shown that acta1b
is present in the heart, as previous studies have shown expression of one zebrafish sarcomeric actin homolog (acta1a
) as well as smooth muscle aorta actin (acta2
) in the zebrafish heart (1
). However, these other isoforms of actin present in the heart (acta1a
, and acta2
) apparently cannot compensate for the lack of actc1a
. It is likely that there is partial redundancy between actc1a
because embryos heterozygous for both actc1as434
show similar defects in cardiac function. A likely explanation is that the overall amount of actin in these double heterozygous embryos is reduced below the critical threshold, similar to actc1as434−/−
Our in vitro and in vivo studies suggest that the zebrafish Y169S and analogous yeast F169S mutations have an adverse effect on actin polymerization per se. Yeast in vitro data show that the mutation causes a severe inherent filament destabilization leading to disassembly of the F-actin into small annealing-incompetent filament fragments. This behavior was not due to ATP depletion. Further, it was not due to postpolymerization denaturation of the actin since addition of phalloidin allowed the fragments to reanneal and form normal-appearing actin filaments. Yeast viability requires that stable actin filaments can form, and the phalloidin results suggest that in the cell, an actin-stabilizing protein such as tropomyosin or fimbrin, both present in yeast, might play this role. We also showed that substoichiometric WT actin levels could provide enough normalization of monomer-monomer contacts to restore filament stability. This property, coupled with the presence of filament-stabilizing proteins, could explain why a zebrafish heart producing both mutant and WT actins survives for an extended time and can still partially function. Similar to yeast, the zebrafish mutant phenotype could be partially rescued by treatment with phalloidin, which helped to improve cardiac functionality and restored circulation to a significant fraction of actc1as434 mutants. These results argue that Y169S mutation in s434 mutants affects actin polymerization, which is the primary reason for the observed cardiac defects.
alleles that have been previously associated with IDC and atrial septal defects in humans are thought to have a dominant phenotype, since heterozygous carriers are affected (24
). However, the actc1as434
mutant phenotype is inherited in a Mendelian recessive manner. We have not observed any developmental defects in heterozygous embryos; therefore, this mutation is unlikely to have a dominant-negative effect. It is possible that heterozygous zebrafish adults may have reduced cardiac performance, which is difficult to detect in the absence of appropriate assays. Because MO knockdown of actc1a
phenocopies the actc1as434
mutant phenotype and polymerized actin is greatly reduced in these mutants, this mutation likely renders actin protein function either null or close to null.
Interestingly, a structural mutation in the cardiac actin results in defective EC formation, which is the earliest stage of valvulogenesis. Hemodynamics and oscillatory blood flow have been previously implicated in restricting expression of genes associated with EC morphogenesis, including klf2a
, to the AVC (41
). Our results show that the blood flow pattern in the heart tube is altered in actc1as434
mutants, and this leads to mislocalization of klf2a
and other AVC marker expression as well as an increased RFF and VSF values. Previous studies have also demonstrated that skeletal actin mutants such as the cardiofunk mutant have defective valve formation (1
). Similar EC defects are also observed in other mutants that display regurgitant blood flow such as jekyll
- and klf2a
-MO-injected embryos (41
). These studies argue that both proper oscillatory blood flow at the AVC and myocardial function are required for EC formation and valve morphogenesis.
In summary, the actc1as434 mutant provides a tool for understanding the critical role for actin in early heart development. This mutant also demonstrates the essential function of the W-loop of ACTC in a vertebrate model. Understanding the physiological and developmental consequences of different types of actin mutations will provide better insights into and help to develop novel treatments for cardiovascular congenital diseases.