Introduction

Nonmuscle myosin (NM) II plays important roles in various cellular processes, including cell migration, cell morphology, cytokinesis, and cell–cell adhesion.¹ Three different NMII heavy chains (NMHCs) are expressed and encoded by 3 different genes: Myh9^2,3 Myh10,² and Myh14.^4,5 The protein products are referred to as NMHCII-A, NMHCII-B, and NMHCII-C, respectively, and mutations in NMHCII-A^6,7 and NMHCII-C^8,9 cause several human syndromes. To study how a mutation in NMII-B could affect pathophysiological processes in vivo, we mutated Arg709 to Cys in the motor domain of NMHCII-B in mice (B^R709C/B^R709C). This amino acid and the surrounding residues are conserved in all myosin II family members, including skeletal, cardiac, and smooth muscle myosin.

Clinical Perspective on p 265

To understand the effect of R709C mutation on NMII-B activity, we previously characterized a baculovirus-expressed heavy meromyosin (HMM) fragment, R709C-HMMII-B, which contains the NMII enzymatic and actin-binding domains.¹⁰ R709C-HMMII-B showed 2 important changes in biological properties compared with wild-type HMMII-B: a 70% decrease in its actin-activated MgATPase activity and a complete loss in its ability to propel actin filaments in an in vitro motility assay. Furthermore, R709C-HMMII-B displayed an increased affinity for actin and spent a prolonged period bound to actin filaments during cross-bridge cycling.¹¹

As a part of generating B^R709C/B^R709C mice using homologous recombination, we inserted the neomycin cassette for selection of the mutant embryonic stem cells into the Myh10 intron, 5′ of exon 16, thus initially producing hypomorphic mice (B^R709CN/B^R709CN) that expressed a decreased (20%) amount of the mutant NMII-B. These mice developed cardiac and brain abnormalities similar to NMII-B null (B⁻/B⁻) mice, although the onset of the abnormalities was delayed compared with the knockouts.^12,13 Somewhat surprisingly, when we removed the cassette encoding neomycin resistance thereby increasing the expression of mutant NMII to wild-type levels, the hydrocephalus and defects in myocyte cytokinesis were rescued, although the abnormalities in neuronal cell migration were not.^11,14 We interpreted these results as showing that NMII has 2 distinct functions in vivo: a structural–scaffolding function that is important for cell–cell adhesion and relies on the ability of NMII to form bipolar filaments that cross-link actin. This would explain the ability of the mutant NMII-B and other isoforms to rescue the defect in cell adhesion in the neuroepithelial cells lining the spinal canal, which causes the hydrocephalus.¹⁴ In contrast, the inability of the mutant NMII-B to rescue the neuronal migration defects was thought to reflect the defect in the mutant NMII-B motor activity as measured by the decreased MgATPase activity and the loss of in vitro motility, a property unique to each isoform.

In the present report, we characterize the novel abnormalities found in B^R709C/B^R709C and B⁺/B^R709C mice, which differ significantly from B⁻/B⁻ and hypomorphic mice. These include a major defect in midline fusion resulting in a cleft palate (homozygotes only), ectopia cordis (homozygotes only), and an omphalocele containing the liver and intestines, and diaphragmatic herniation and structural cardiac abnormalities (homozygotes only), defects similar to those first described in humans by Cantrell et al.¹⁵

Materials and Methods

NMHCII-B Mutant Mice

B⁻/B⁻, B^R709CN/B^R709CN, and B^R709C/B^R709C, B^a*/B^a* mice were generated as previously described^12,16,17 and are available through the Mutant Mouse Regional Resource Centers (Nos. 16991, 16142, 15983, and 16998). B⁻/B⁻, B^R709CN/B^R709CN, and B^a*/B^a* mice are maintained in a mixed background of 129/Sv and C57BL/6. All procedures were conducted using an approved animal protocol in accordance with National Heart, Lung, and Blood Institute Animal Care and Use Committee.

Histology and Immunofluorescence Staining

Staining was performed as described.¹² Primary antibodies (Table I in the Data Supplement) for immunostaining were incubated at 4°C overnight after antigen retrieval in 10 mmol/L citrate buffer (pH 6). The confocal images were collected using a Zeiss LSM 510-META. In all cases, when possible, comparison was made among littermates. For each genotype, we analyzed ≥5 mice.

TUNEL Assay

The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using the In Situ Cell Death Detection Fluorescein Kit, following the manufacturer’s instructions (Roche Applied Science).

Statistical Analyses

Data are expressed as mean±SD. The Student t test was performed to compare 2 means. A 1-way ANOVA was used to compare ≥3 means at a time. Data passed normality test for statistical analysis.

Results

Defects in Ventral Wall Closure in B^R709C/B^R709C and B⁺/B^R709C Mice

The B^R709C/B^R709C mice died during embryonic development between embryonic day (E) 14.5 and E16.5. As shown in Figure 1B, E14.5 B^R709C/B^R709C mice developed generalized edema (white arrow), indicating a major failure in cardiac function. Figure Ib in the Data Supplement shows evidence for massive congestive failure in the liver of B^R709C/B^R709C mice indicated by the presence of sinusoidal dilatation, focal hemorrhages, and congestion (compare with wild-type; Figure 1A; Figure Ia in the Data Supplement). Measurements of cardiac function using in utero echocardiography at E14.5 showed a marked decrease in fractional shortening and heart rate and an increase in the left ventricular dimension in systole in B^R709C/B^R709C embryos (Table II in the Data Supplement). B^R709C/B^R709C embryos develop an umbilical hernia (Figure 1B, orange arrow), indicating a failure in ventral body wall closure. Figure 1C and 1D shows sagittal sections of E13.5 embryos from B⁺/B^R709C and B^R709C/B^R709C embryos. In both cases, the liver is abnormally herniated outside the body (arrows). Approximately 50% of B⁺/B^R709C mice and all B^R709C/B^R709C mice develop an omphalocele. Furthermore, ≈50% of B^R709C/B^R709C mice develop ectopia cordis, with the heart protruding outside the thoracic chamber (Figure 1F, black arrow). Ectopia cordis was not seen in B⁺/B^R709C mice, suggesting that the severity of the defects in ventral body wall closure is dependent on the dosage of mutant NMII-B.

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B^R709C/B^R709C embryos also develop cleft palates. At E14.5, the left and right palatal shelves of B⁺/B⁺ mice are positioned in a horizontal plane above the tongue and are joined (Figure 1G, arrow). In contrast, the B^R709C/B^R709C palatal shelves are much shorter, positioned vertically, and flank the tongue with a major gap between them (Figure 1H, arrows). This defect does not seem to be because of a delay in the development of B^R709C/B^R709C embryos. Those few B^R709C/B^R709C embryos that survive to E16.5 still show cleft palates (Figure II in the Data Supplement).

Congenital Diaphragmatic Hernia in Heterozygous and Homozygous NMII-B Mutant Mice

B^R709C/B^R709C and B⁺/B^R709C mice show abnormal development of the diaphragm, which leads to herniation of the liver into the thoracic cavity. Figure 2A, 2B, and 2C shows sagittal sections of the developing diaphragm of B⁺/B⁺, B⁺/B^R709C, and B^R709C/B^R709C mice at E13.5 stained with antibodies to NMHCII-A (green) and striated muscle myosin (MF20, red). The skeletal muscle cells of B⁺/B⁺ mice are uniformly distributed throughout the entire dorsal area of the diaphragm (A, red; yellow and white boxes, enlarged in D and G). However in both B⁺/B^R709C (B) and B^R709C/B^R709C (C) mice, skeletal muscle cells accumulate abnormally in the central region (white boxes in B and C, enlarged in H and I). This results in significantly fewer muscles cells at the most lateral region of the diaphragm (yellow boxes in B and C, enlarged in E and F) consistent with a defect in migration of the skeletal muscle cells. To quantify the distribution of muscle cells, we divided the diaphragm into 3 equal segments—ventral, middle, and lateral—and calculated the percentages of muscle cells for each segment from 3 mice of each genotype. These values are 38.5±1.3%, 30.3±1.8%, and 31.1±1.2% for ventral, middle, and lateral segments, respectively, in the B⁺/B⁺ diaphragm; 56.2±1.3%, 38.1± 0.5%, and 5.8±1.7%, respectively, in the B⁺/B^R709C diaphragm; and 57.9±1.5%, 41.7±1.2%, and 0.43±0.3%, respectively, in the B^R709C/B^R709C diaphragm. Statistical analysis shows a significant increase in muscle cells in the ventral segments and a significant reduction in the lateral segments of B⁺/B^R709C and B^R709C/B^R709C compared with the B⁺/B⁺ diaphragm (ANOVA, P<0.05). The absence of muscle cells makes this part of the diaphragm vulnerable to herniation. Because the ventral body wall is wide open in B^R709C/B^R709C mice, the diaphragmatic hernia is more easily observed in B⁺/B^R709C mice (Figure IIIb and IIIc in the Data Supplement). The amuscular lateral region of the diaphragm of an E19.5 B⁺/B^R709C mouse becomes abnormally thin permitting the liver to protrude into the thoracic cavity (Figure IIIb in the Data Supplement, black arrow). Figure IIIa in the Data Supplement shows an equivalent section from a control E19.5 B⁺/B⁺ embryo. Figure IIIc in the Data Supplement shows a 2-month-old B⁺/B^R709C mouse, which did not develop an obvious omphalocele but still developed a severe diaphragmatic hernia that permitted the intestines to protrude into the thoracic cavity. The lack of muscle cells in the lateral regions of the mutant diaphragms is not associated with increased apoptosis, because no obvious apoptotic cells were observed in the developing diaphragms from the 3 genotypes.

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Importantly, none of the defects described above with respect to midline fusion are seen in B⁻/B⁻ or B^R709CN/B^R709CN mice.^12,16 The midline fusion defects are unlikely to be because of background strain differences in these mouse lines. Both B⁺/B^R709C and B⁻/B^R709C, but not B⁺/B⁻ offspring from B⁺/B⁻ and B⁺/B^R709C crosses, developed defects in ventral body wall closure. We next studied the cellular mechanisms underlying these defects.

Impaired Apoptosis in the Fusing Sternum of B^R709C/B^R709C Mice

Ectopia cordis is usually associated with defects in sternal fusion.¹⁸ In B⁺/B⁺ mice at E14.5, the fusing halves of the lower sternum are aligned side by side (Figure 1E, green arrow). In B^R709C/B^R709C mice, they are widely separated (Figure 1F, green arrows; Figure 3D, inset). To understand the cellular mechanisms underlying this defect, we examined apoptotic activity in the fusing sternum of E14.5 B⁺/B⁺ and B^R709C/B^R709C mice. Most of the B⁺/B⁺ sternal mesenchymal cells are undergoing apoptosis manifested by nuclear condensation and fragmentation (Figure 3A, arrows). In contrast, few apoptotic cells were found in the same area in B^R709C/B^R709C mice (Figure 3B). TUNEL assays confirmed the apoptosis in B⁺/B⁺ sternums (Figure 3C, green), which was decreased in B^R709C/B^R709C mice (Figure 3D, green). The percentage of apoptotic mesenchymal cells in B⁺/B⁺ and B^R709C/B^R709C sternums was 14.4±7.7% and 1.4±0.8% (P<0.001, t test), respectively (n=5 mice for each genotype). Previous studies from cultured cells have shown that NMII is required for the final stages of apoptosis.^19–21 We next examined a step in the upstream pathway, activation of caspase-3, using immunostaining for activated caspase-3. In B⁺/B⁺ mice, a significant number of mesenchymal cells (46.2±7.2%) in the fusing sternum were positive for activated caspase-3 (Figure 3E, red), whereas few mesenchymal cells (5.4±2.3%; n=5 mice each genotype; P<0.001, t test) stained positively for activated caspase-3 in the B^R709C/B^R709C sternum (Figure 3F, red). We then examined these same cells for expression of p53, the signaling molecule that initiates apoptosis. There were no major difference in p53 expression in B^R709C/B^R709C sternal mesenchymal cells compared with B⁺/B⁺ mesenchymal cells (Figure 3E and 3F, green). The relative average fluorescence intensities of p53 staining from B⁺/B⁺ and B^R709C/B^R709C sternums were 52.8±1.5% and 61.9±5.3% (n=3 mice each; P>0.05, t test). These results indicate that NMII-B functions upstream of caspase-3 but downstream of p53 in regulating mesenchymal cell apoptosis of the fusing sternum. The requirement for enzymatic NMII activity in apoptosis has been reported in various cell types in culture. We further asked which enzymes are responsible for activation of NMII during sternal fusion. We examined the expression of myosin light chain kinase (MLCK) and Rho kinase (ROCK1) in the fusing sternum and found that ROCK1, but not MLCK, is expressed (Figure IV in the Data Supplement). Thus, ROCK1-mediated NMII activation is most likely involved in normal sternum fusion, although further investigation is required to test this hypothesis.

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Sternal Mesenchymal Cells Express both NMII-A and NMII-B But Not NMII-C

It has previously been reported that dorsal wall closure in Drosophila (corresponding to mammalian ventral wall closure) requires zipper, the sole Drosophila NMII isoform.²² However, mice and humans express 3 different isoforms of NMII. We, therefore, examined the expression patterns of NMII-A, NMII-B, and NMII-C in the developing mouse sternum. Figure 3G to 3I shows immunofluorescence confocal images for NMII-A, NMII-B, and NMII-C from an E14.5 wild-type mouse embryo. Both NMII-A (G) and II-B (H), but not II-C (I), were detected in mesenchymal cells of the developing sternums. Previous reports have demonstrated that ablation of NMII-B did not impair ventral body wall closure in mice,¹⁶ indicating that expression of NMII-A alone is sufficient to support ventral body wall closure. Importantly, despite normal expression of NMII-A, expression of R709C-NMII-B leads to defects in ventral body wall closure. Because these defects did not occur in B⁻/B⁻ mice, they are unlikely the result of loss of NMII-B function. This raises the possibility that in B^R709C/B^R709C mice, the mutant NMII-B isoform is interfering with the normal function of NMII-A during sternal fusion.

Failure in Fusion and Remodeling of the Atrioventricular Cushions in B^R709C/B^R709C Mouse Hearts

B^R709C/B^R709C mouse hearts show defects in fusion and remodeling of the atrioventricular endocardial cushions, which are not seen in B⁻/B⁻ or wild-type hearts. Figure 4A to 4I shows the developing atrioventricular valves of B⁺/B⁺, B^R709C/B^R709C, and B⁻/B⁻ mouse hearts from E11.5 to E14.5. At E11.5, before the fusion of the superior and inferior cushions, no differences in shape, size, and positioning of the cushions were found among B⁺/B⁺ (A), B^R709C/B^R709C (B) and B⁻/B⁻ (C) hearts, suggesting a normal endothelial–mesenchymal transition in developing B^R709C/B^R709C and B⁻/B⁻ hearts. At E12.5 the B⁺/B⁺ atrioventricular cushions have fused and started to elongate (D). In contrast, the B^R709C/B^R709C cushions have not fused and show no sign of elongation (E). By E14.5, B⁺/B⁺ atrioventricular cushions have developed into elongated, mature mitral, and tricuspid valves (G). The superior and inferior cushions of B^R709C/B^R709C mice are still not fused or remodeled (H), suggesting that the defects in B^R709C/B^R709C atrioventricular cushions are not simply because of a delay in development. In B⁻/B⁻ hearts, the atrioventricular cushions were fused normally at E12.5 (Figure 4F). However, the maturation into cardiac valves is delayed at E14.5 (Figure 4I) at the time B⁻/B⁻ mice start to die.

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Similar to its role in sternal formation, apoptosis also plays an important role in the development of the endocardial atrioventricular cushions. We next investigated whether apoptosis was impaired in the developing B^R709C/B^R709C endocardial cushions using TUNEL assays. At E12.5, fusion of the superior and inferior cushions in B⁺/B⁺ hearts was accompanied by significant numbers of apoptotic mesenchymal cells (Figure 4J, green; 11.2±3.0%). However, few apoptotic cells were detected in B^R709C/B^R709C cushions at E12.5, which fail in fusion (Figure 4K; 1.1±0.9%; n=5 mice each genotype; P<0.001, t test). Note that at E11.5 no obvious apoptotic cells were detected in either B⁺/B⁺ or B^R709C/B^R709C atrioventricular cushions (Figure Va and Vb in the Data Supplement). Furthermore, we did not observe obvious apoptotic cells in B^R709C/B^R709C cushions at E14.5 (Figure Vd in the Data Supplement), suggesting that the defect in apoptosis is not because of a developmental delay in the B^R709C/B^R709C mouse heart. These results emphasize the role of NMII in apoptosis. Similar to the developing sternum, mesenchymal cells in developing atrioventricular cushions express NMII-A (Figure VIa in the Data Supplement, green) and NMII-B (Figure VIb in the Data Supplement, green) but no detectable II-C (Figure VIc in the Data Supplement). These results are consistent with the hypothesis that mutant NMII-B interferes with the normal function of NMII-A in B^R709C/B^R709C cardiac atrioventricular cushion development. This is also consistent with our findings that NMII-B/II-C double-ablated mice showed no defect in ventral wall closure or atrioventricular cushion fusion.²³

Defects in Outflow Tract Myocardialization in B^R709C/B^R709C and B⁻/B⁻ Mouse Hearts

Both B⁻/B⁻¹⁶ and B^R709C/B^R709C hearts showed defects in outflow tract (OFT) alignment, with both the aorta and the pulmonary artery emanating from the right ventricle (double outlet of the right ventricle [DORV]; Figure 5A–5C; 10 of 10 B^R709C/B^R709C mice). During heart development, the OFT cardiac cushions are initially composed of cardiac jelly. After invasion by endocardial cells and cardiac neural crest cells, the cushions expand, fuse, and consequently form a mesenchymal outlet septum. The mesenchyme of the proximal region of the outlet septum is then replaced by cardiac myocytes during a process called myocardialization.²⁴ In mice, the invasion by cardiac myocytes occurs between E10.5 and E13.5. Figure 5D and 5E shows hematoxylin and eosin–stained images of developing hearts at E11.5, illustrating the invasion by the cardiac myocytes of the B⁺/B⁺ cardiac cushion (Figure 5D, arrow) but absence of invasion in B^R709C/B^R709C mice (Figure 5E, arrow). During this process, the cardiac myocytes from the outer layer of myocardium in the OFT lose their tight cell–cell adhesions, become polarized, and migrate into the adjacent cushions. Inhibition of myocardialization leads to abnormal alignment of the OFT.²⁴ Therefore, we examined myocardialization in the OFTs of wild-type and B^R709C/B^R709C mice using immunofluorescence confocal microscopy with antibodies to MF20 to delineate the cardiac myocytes and antibodies to NMHCII-B to identify both cardiac myocytes and cardiac nonmyocytes. Figure 5F shows that at E11.5, the B⁺/B⁺ myocardial cells bordering the OFT are polarized, with extended lamellipodia- and filopodia-like structures protruding into the adjacent mesenchymal cells of the cushions (Figure 5F, arrows; Figure VIIa in the Data Supplement). However, in B^R709C/B^R709C mice there is a discrete boundary between the OFT myocardium and the cushion because the cardiac myocytes remain compact with no sign of invasion (Figure 5G; Figure VIIb in the Data Supplement).

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Next, we looked for the cause of the failure of migration in the B^R709C/B^R709C myocytes. Because phosphorylation of the regulatory MLC (MLC20) is required for activation of NMII, we used antibodies to the 2 most likely kinases that are known to phosphorylate MLC20, ROCK1 and MLCK, to see whether they were present in the OFT and whether their pattern of expression was altered in the B^R709C/B^R709C OFT. Figure VIIa and VIIb in the Data Supplement shows that ROCK1 is present in the cardiac myocytes in the OFT of both B⁺/B⁺ and B^R709C/B^R709C mice. This kinase is not detected in the ventricular myocytes (Figure VIIc and VIId in the Data Supplement). In contrast, MLCK was detected in the OFT and ventricular myocytes of both the normal and mutant mice (Figure VIII in the Data Supplement). Figure VIIe and VIIf in the Data Supplement shows that MLC20 is phosphorylated in both wild-type and B^R709C/B^R709C cardiac myocytes. This makes it unlikely that the failure in migration is because of a lack of MLC20 phosphorylation or alteration in the ROCK1 or MLCK expression and suggests that the defect in myocyte migration entails an intrinsic kinetic property of the mutant NMII-B.

We sought a mechanism related to the kinetic properties of the mutant and wild-type NMII to explain the defect in migration in B^R709C/B^R709C cardiac myocytes. Previous work has shown that the disassembly of cell–cell adhesion junctions requires NMII activity.^25,26 Examination of the cell–cell adhesion boundaries in the myocytes of the OFT by immunofluorescence confocal microscopy showed marked differences between B⁺/B⁺ and B^R709C/B^R709C cardiac myocytes. Figure 5I (arrows) shows that the cell adhesion molecule N-cadherin (the only classical cadherin expressed in the myocardium) is concentrated at the cell–cell boundary of the cardiac myocytes in the B^R709C/B^R709C OFT. This indicates that the B^R709C/B^R709C cardiac myocytes retain tight cell–cell adhesions. In contrast, there is no cortical concentration of N-cadherin in the actively migrating B⁺/B⁺ cardiac myocytes at E11.5 (Figure 5H). These results suggest that the mutation R709C, which decreases NMII-B MgATPase activity and increases the time NMII-B spends bound to actin, inhibits the disassembly of cell–cell adhesions in the cardiac myocytes. This results in a failure in myocardialization, thereby contributing to the development of DORV.

The aorta of B⁻/B⁻ hearts is also abnormally localized to the right ventricle as previously reported.¹⁶Figure 6 shows that similar to the B^R709C/B^R709C OFT, the B⁻/B⁻ OFT shows defects in myocardialization (Figure 6C) and disassembly of cardiac myocyte cell–cell adhesions (Figure 6D) compared with a B⁺/B⁺ littermate (Figure 6A and 6B). Because normal alignment of the aorta is impaired in B^R709C/B^R709C and B⁻/B⁻ hearts, these findings suggest a requirement for wild-type NMII-B enzymatic motor activity in these processes during normal mouse heart development. This is also consistent with our finding that genetic replacement of NMII-B with NMII-A does not rescue the DORV¹⁷ because of the defects in OFT myocardialization (Figure IX in the Data Supplement).

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Discussion

The Table summarizes the phenotypes observed from our 3 NMII-B genetically altered mouse models. The findings from these mutant mice have resulted in 2 hypotheses with respect to the mechanism underlying the NMII-B mutation. The first is that the novel gain-of-function defects found in these mice are because of the interference of R709C-NMII-B with the normal function of a second NMII, NMII-A. The second is that the mechanism underlying these defects arises from the 2 different functions of the NMII-B molecule: the motor function and the structural function.

Evidence that the mutant NMII-B is interfering with the normal function of NMII-A during ventral wall closure or endocardial cushion fusion is as follows: hypomorphic B^R709CN/B^R709CN mice, mice ablated for NMII-B or NMII-C, or mice doubly ablated for NMII-B and NMII-C show normal closure of the ventral body wall and normal endocardial cushion fusion. Therefore, expression of NMII-A alone is sufficient for ventral body wall development. In addition, these defects are also observed in the B^R709C/B^R709C homozygous mice, which do not contain any normal NMII-B, so interference with the normal isoform of NMII-B is not possible. Furthermore, the 2 affected tissues do express significant amounts of NMII-A (but not NMII-C). This raises the novel possibility that the mutant NMII-B is interfering with NMII-A. In vitro motility studies using baculovirus-expressed NMII-A HMM and mutant NMII-B HMM showed that the presence of the R709C-NMII-B HMM markedly slowed the movement of NMII-A HMM.¹⁰ Mechanistically, prolonged binding of R709C-NMII-B to actin filaments¹¹ could affect the dynamics of actomyosin stress fibers,²⁷ which, in general, contain both NMII-A and NMII-B.²⁸ It is of interest that there have been several reports implicating a mutation in NMII-A^29–31 but not NMII-C³² in the generation of a cleft palate in humans, consistent with our hypothesis that the mutant NMII-B is interfering with NMII-A. Of course, we cannot rule out interference with a NM of a different class.

Previous work has shown that NMII-A–ablated mice die at E6.5 and the heterozygous II-A mice are entirely normal,³³ so we cannot directly test our hypothesis. However, in support of this mechanism is our finding of a significant gene dose-dependent effect: All B^R709C/B^R709C mutant mice are severely affected, showing abnormalities including cleft palate, ectopia cordis (50%), and omphalocele. Approximately one half of B⁺/B^R709C mice, expressing 50% mutant NMII-B compared with wild-type mice, are born with an omphalocele only, and hypomorphic mice (B^R709CN/B^R709CN) expressing only 20% mutant myosin display neither defect.

The requirement for NMII function in ventral body wall closure is also supported by results from ROCK-ablated mice, which show a failure in body wall closure associated with a deficiency in the formation of actomyosin bundles in the umbilical ring.³⁴ In a chick model for defective ventral body wall closure, this abnormality was attributed to reduced myosin activity because of decreased ROCK expression.³⁵ In both cases, the defects are similar to our B⁺/B^R709C mice but much milder compared with B^R709C/B^R709C mice. This is most likely because of a partial inactivation of NMII function in ROCK-ablated mice because other kinases are also capable of activating NM-II.¹

Our second hypothesis is that the defects we observed in B^R709C/B^R709C mice arise from 2 distinct, although not unrelated functions of myosin: NMII can use either its enzymatic motor domain to translocate actin filaments or NMII can act more as a structural protein to cross-link actin filaments. Both of these functions require the binding of myosin to actin; however, translocation of actin requires a particular isoform-specific, actin-activated MgATPase activity and duty cycle (amount of time the myosin head stays strongly bound to actin) to perform a normal functional role in motile processes such as cell migration. These functions of NMII are more sensitive to mutations that alter kinetic properties and cannot be rescued by other NMII isoforms with different motor and kinetic properties.¹⁷ An example of this is generation of the DORV in the B^R709C/B^R709C mice in which the mutant NMII-B cannot dissociate the cell–cell adhesion complex nor can it participate in migration of the myocytes into the cardiac cushion. We postulate that the result of this inability to migrate normally into the cardiac cushion is mislocalization of the aortic root to the right ventricle. Of note is a report by Phillips et al³⁶ attributing the development of a DORV to abnormalities in NMII function. Another example of defective migration is found in the skeletal muscle cells of the developing diaphragm R709C-NMII-B mice resulting in diaphragmatic herniation. An additional consequence of the NMII-B motor mutation is the apparent failure of the cardiac and sternal mesenchymal cells to undergo a normal apoptotic program. The loss of apoptosis results in abnormal valve formation and a defect in sternal fusion, 2 novel defects not seen in NMII-B–ablated or hypomorphic mice.

Mice either ablated for NMII-B or expressing R709C-NMII-B, either in reduced (20%) or wild-type amounts, develop abnormalities in myocardialization during cardiac OFT development, leading to misalignment of the aorta with the right ventricle. Our results indicate that both the expression level and the normal enzymatic activity of NMII-B are essential for normal OFT myocardialization. MLCK and ROCK are 2 of the major kinases that activate NMII activity. Specific expression of ROCK1 in OFT cardiac myocytes during myocardialization suggests that ROCK1 is the major upstream kinase activating NMII-B. This is supported by findings from loop-tail (Lp) mice where abnormal OFT myocardialization is associated with disruption of noncanonical Wnt/planar cell polarity–mediated RhoA/ROCK1 signaling.³⁶ Decreased expression of ROCK1 in the proximal OFT cardiac myocytes was also described in connexin 43 knockout mice that developed DORV with abnormal OFT myocardialization.³⁷ Because no changes in ROCK1 expression and NMII activation (MLC20 phosphorylation) were observed in the B^R709C/B^R709C OFT, we attribute the defects in myocardialization directly to the impaired R709C-NMII-B enzymatic activity. Our results further point to the importance of NMII-B–mediated disruption of cardiac myocyte cell–cell contacts during OFT myocardialization. Cardiac myocytes lose their epithelial context and migrate into the adjacent mesenchymal cushions during myocardialization. Expression of R709C-NMII-B in mice prevents OFT cardiac myocytes from detaching from surrounding cells, and the retention of cell–cell adhesions thereby inhibits their migration into the cushion tissue. Loss of the NMII enzymatic activity and prolonged binding of the mutant NMII to actin contribute to the inability of R709C-NMII-B to disrupt cell–cell adhesions. This is consistent with the requirement for NMII activity to perturb pre-existing epithelial cell–cell adhesions in culture.^38,39 All of the above evidence is consistent with a Wnt/RhoA/ROCK1/NMII-B pathway in regulating myocardialization during OFT development. Abnormal OFT alignment is one of the most common congenital heart defects. Defects in OFT myocardialization seem to be the common end pathway leading to this abnormality.

During revision of this article, Dr Wendy Chung's group reported a patient carrying a nonsense mutation resulting in a premature stop codon in the MYH10 transcript.⁴⁰ Among various abnormalities, the patient developed a congenital diaphragmatic hernia, which is one of the defects of pentalogy of Cantrell and is seen in our NMII-B mutant mice reported here. Our present plans call for testing our hypothesis by searching for possible mutations in NMII-B and related proteins in patients with the diagnosis of pentalogy of Cantrell.

Acknowledgments

We thank Dr Mary Anne Conti for her significant contributions to this article. Dr Sachiyo Kawamoto and members of the Laboratory of Molecular Cardiology also provided critical comments on the article. We also thank Dr Kazuyo Takeda for echocardiography. Drs Chengyu Liu and Yubin Du (National Heart, Lung, and Blood Institute [NHLBI] Transgenic Core) and Drs Christian A. Combs and Daniela Malide (NHLBI Light Microscopy Core) provided outstanding service and advice. Antoine Smith and Dalton Saunders provided excellent technical assistance.

Footnotes