Conditional Ablation of Nonmuscle Myosin II-B Delineates Heart Defects in Adult Mice
Rationale: Germline ablation of the cytoskeletal protein nonmuscle myosin II (NMII)-B results in embryonic lethality, with defects in both the brain and heart. Tissue-specific ablation of NMII-B by a Cre recombinase strategy should prevent embryonic lethality and permit study of the function of NMII-B in adult hearts.
Objective: We sought to understand the function of NMII-B in adult mouse hearts and to see whether the brain defects found in germline-ablated mice influence cardiac development.
Methods and Results: We used a loxP/Cre recombinase strategy to specifically ablate NMII-B in the brains or hearts of mice. Mice ablated for NMII-B in neural tissues die between postnatal day 12 and 22 without showing cardiac defects. Mice deficient in NMII-B only in cardiac myocytes (BαMHC/BαMHC mice) do not show brain defects. However, BαMHC/BαMHC mice display novel cardiac defects not seen in NMII-B germline-ablated mice. Most of the BαMHC/BαMHC mice are born with enlarged cardiac myocytes, some of which are multinucleated, reflecting a defect in cytokinesis. Between 6 to 10 months, they develop a cardiomyopathy that includes interstitial fibrosis and infiltration of the myocardium and pericardium with inflammatory cells. Four of 5 BαMHC/BαMHC hearts develop marked widening of intercalated discs.
Conclusions: By avoiding the embryonic lethality found in germline-ablated mice, we were able to study the function of NMII-B in adult mice and show that absence of NMII-B in cardiac myocytes results in cardiomyopathy in the adult heart. We also define a role for NMII-B in maintaining the integrity of intercalated discs.
Nonmuscle myosin II (NMII) plays an important role in maintaining the integrity of the actin–myosin cytoskeleton, which, in turn, helps to determine cell shape and functions in cell migration, cell polarity, and cytokinesis. Like conventional myosin IIs, such as skeletal, cardiac, and smooth muscle myosins, NMII consists of a pair of heavy chains and 2 pairs of light chains. Three isoforms of the nonmuscle myosin heavy chain (NMHC), II-A, II-B, and II-C, which are encoded by 3 genes, Myh9, -10, and -14, respectively, and are located on different chromosomes, have been identified in humans and mice.1–3 Although there is some overlap in the localization of these 3 isoforms, growing evidence suggests that they perform distinct functions during cell migration and embryonic development.4–6 Ablation of NMII-A in mice results in lethality at embryonic day (E)6.5 because of the lack of a normal functioning visceral endoderm that results in a markedly abnormal body pattern. These embryos fail to undergo gastrulation.7 In contrast, ablation of NMII-B in mice results in embryonic lethality between E14.5 and birth, with defects in the brain and heart,8,9 suggesting that NMII-B is critical for the development of both. Unfortunately, the embryonic lethality in NMII-B–null mice has impeded further efforts to understand the physiological roles of NMII-B in adult mice. Hypomorphic mice expressing low amounts of NMHC II-B can survive to adulthood and also display defects in both brains and hearts; however, severe NMII-B hypomorphs also die before adulthood.10 Moreover, because the physiological activities of the heart are continuously regulated by the nervous system, questions arise as to whether any of the heart defects in NMII-B–ablated or hypomorphic mice are secondary to the brain defects.
In this study, we ablated NMHC II-B in mice, either in the nervous system alone or in the cardiac myocytes alone, using a loxP/Cre recombinase strategy. We crossed the NMHC II-B floxed mice with a line of mice expressing Cre recombinase regulated by the neural cell–specific nestin promoter to ablate NMHC II-B in the nervous system.11 In separate experiments, we crossed the NMHC II-B floxed mice with a line of mice expressing Cre recombinase under control of the α-myosin heavy chain (αMHC) promoter to ablate NMII-B in cardiac myocytes.12 Below, we present results showing that NMII-B plays distinct physiological roles in the brain and heart and provide evidence that absence of NMII-B in the cardiac myocytes (and not in the nonmyocytes) results in myocyte enlargement and cardiomyopathy. Moreover, we demonstrate a role for NMII-B in the intercalated disc (ID) of adult mice.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
All experiments were conducted following animal protocols approved by Animal Care and Use Committee of the National Heart, Lung, and Blood Institute. Nestin-Cre transgenic mice were from The Jackson Laboratory (no. 003771).
Histology, Microscopy, and Immunoblotting
Hematoxylin/eosin (H&E) and immunofluorescence staining, electron microscopy, and immunoblotting were performed as described previously.8
Measurement of the Cross-Sectional Area of the Cardiac Myocytes
The size of cardiac myocytes was measured following wheat germ agglutinin staining using a Zeiss measuring tool.
Echocardiography was performed using an Acuson Sequoia 256c imaging system with the 15L8 multifrequency transducer. Quantitation was performed using M-mode with Prosolv Software version 3.0.
Three-lead electrocardiograms were recorded with a model MAC 1200 (GE Medical Systems).
Data and Statistical Analysis
The data were expressed as means±SD. Student’s t test was used to compare data between 2 groups.
Generation of Neural- and Cardiac-Specific NMHC II-B Knockout Mice
To ablate NMHC II-B specifically in the brain or in the heart, we used a loxP/Cre recombinase strategy to delete exon 2, the first coding exon of Myh10 (see Figure 1). Using the gene-targeting method, we generated a mouse line designated Bflox/Bflox in which both the neomycin resistance (Neor) expression cassette and exon 2 of Myh10 are flanked by loxP sites (Figure 1c). Bflox/Bflox mice express normal amounts of NMHC II-B protein and are indistinguishable from wild-type mice. We crossed Bflox/Bflox mice with 2 different transgenic lines expressing Cre recombinase under control of different promoters. To ablate NMHC II-B in neural tissue, we used a mouse line expressing Cre recombinase with a nestin promoter and a nervous system–specific enhancer11 (Figure 1e) to generate B+/Bnest and Bnest/Bnest mice. To ablate NMHC II-B in cardiac myocytes, we used a mouse line expressing Cre recombinase driven by the αMHC promoter12 (Figure 1f) to generate B+/BαMHC and BαMHC/BαMHC mice. The transgenic mouse lines nestin-Cre and αMHC-Cre are well characterized and demonstrate that nestin-Cre mice express functional Cre recombinase in the nervous system starting at E10.511 and that αMHC-Cre mice express functional Cre recombinase specifically in cardiac myocytes starting at E9.0 and at high levels by E11.5.13
Nestin-Cre–Specific Ablation of NMHC II-B in the Mouse Brain
Immunoblot analysis of Bnest/Bnest mice on postnatal day (P)18 shows that NMHC II-B protein is markedly reduced in the cerebellum (Figure 2A, compare lanes 1 and 2), as well as throughout the entire brain (data not shown) but not in the hearts of Bnest/Bnest mice (Figure 2A, lanes 3 and 4). Immunofluorescence staining using an antibody for NMHC II-B confirms that NMHC II-B protein is ablated in the cerebellum of these mice at P18 (Figure 2B). As shown in the figure, Purkinje cells in Bflox/Bflox mice express high levels of NMHC II-B (red; Figure 2B, a). However, the NMHC II-B protein level is significantly reduced in the cerebellar Purkinje cells of Bnest/Bnest mice (Figure 2B, compare a with e). In contrast, staining for calbindin (Purkinje cell marker) is unaltered in these cells (Figure 2B, compare b with f). Figure 2B (d and h) confirms the loss of NMHC II-B (yellow in d and green in h) in the cerebellar Purkinje cells. Both immunoblot analysis and immunofluorescence staining confirm that NMHC II-B protein is also significantly reduced in the cerebral cortex of Bnest/Bnest mice (data not shown).
All of the Bnest/Bnest mice die between P12 and P22 as a result of a severe hydrocephalus, which causes enlargement of the lateral ventricles and a paper-thin cortex, with absence of most brain cortical tissue (Figure 2C, compare b and d). Figure 2C (c) also shows an underdeveloped cerebellum (ellipse), which correlates with defects in motor activity in these mice and which was also seen in hypomorphic mice that have a point mutation in NMHC II-B.14 The arrows in Figure 2C (c) point to deformities following the decompression of the lateral ventricles and loss of cerebral–spinal fluid. Of note is our finding that, similar to hypomorphic mice with a point mutation in NMHC II-B,15 the spinal canal of Bnest/Bnest mice is completely ablated at P7 (Figure 2D). This is consistent with a role for NMII-B in cell–cell adhesion in the spinal canal.
Sectioning of the hearts confirms that there is none of the cardiac abnormalities in the Bnest/Bnest mice that are found in B−/B− mice. Specifically, there is no evidence for a ventricular septal defect (VSD), double outlet of the right ventricle (DORV), myocyte hypertrophy, decreased myocyte number, or increased cardiac myocyte binucleation, as seen in B−/B− mice.8,16 This demonstrates that ablation of NMHC II-B in the nervous system does not affect cardiac development. B+/Bnest mice appear normal in all respects.
Pathological Changes in the Heart Following αMHC-Cre–Specific Ablation of NMHC II-B
To address the role of NMII-B in heart development and in the adult mouse, we crossed the Bflox/Bflox mice with αMHC-Cre mice (Figure 1f), so that ablation would occur specifically in the cardiac myocytes starting at midgestation or after approximately E11.5 to avoid the early lethality found in germline-ablated B−/B− mice. Similar to other investigators who have used this particular line of mice expressing Cre recombinase,17 we found no adverse effects of the enzyme on the tissues in which it was expressed (see also Figures 4 and 6⇓ and Online Figure I). The NMHC II-B protein level is significantly reduced in the hearts of BαMHC/BαMHC mice compared to Bflox/Bflox mice at P0, as demonstrated in the immunoblot in Figure 3A. However, the NMHC II-B level is not affected in the brain of BαMHC/BαMHC mice (Figure 3A). The presence of residual NMHC II-B in the heart of BαMHC/BαMHC mice can be attributed to nonmyocytes in the heart, which continue to express wild-type amounts of NMHC II-B. Immunofluorescence staining of E13.5 mouse heart sections using antibodies to NMHC II-A, II-B, and II-C helps to clarify the ablation of NMHC II-B from the myocytes alone. It also demonstrates that ablation of NMHC II-B in cardiac myocytes has no effect on NMHC II-A and II-C expression. Figure 3B (a and d) shows that NMHC II-A (green) is only present in the nonmyocytes in the heart. This is indicated by the lack of green signal coincident with desmin (red) in the cardiac myocytes, because NMHC II-A is not present at this age in these cells. In contrast, in Figure 3B (b), NMHC II-B (green) and desmin (red) costain the myocytes (yellow) and stain the nonmyocytes green, showing that in the Bflox/Bflox heart NMHC II-B is expressed in both cell types. The arrows are pointing to the nonmyocytes (green). However, in the BαMHC/BαMHC heart the cardiac myocytes now appear red because NMHC II-B has been ablated, but desmin remains. Again, the arrows point to the nonmyocytes, which stain green for II-B, indicating that NMII-B is not ablated in nonmyocytes. Figure 3B (c and f) shows that there is very little NMHC II-C in these hearts and that this does not change after II-B is ablated.
H&E staining on the day of birth (P0) (Figure 4) shows evidence of an increase in the size of the cardiac myocytes in BαMHC/BαMHC hearts (Figure 4f) compared to B+/BαMHC hearts (Figure 4c). Figure 4f also shows examples of abnormally shaped nuclei (arrows), reflecting an abnormality in cytokinesis attributable to the loss of NMHC II-B.16 A VSD in a BαMHC/BαMHC heart (Figure 4d and enlarged in 4e) is also shown. These abnormalities were not seen in B+/B+ or B+/BαMHC mice (Figure 4a and 4b). Unlike B−/B− mice, in which the heart phenotype of a membranous VSD and DORV is almost 100% penetrant,8 only 2 of 9 BαMHC/BαMHC mice examined were born with a VSD and neither displayed a DORV. We did not see any difference in the deletion of NMII-B in the BαMHC/BαMHC cardiac myocytes with or without the presence of a VSD. However, 5 of 9 newborn BαMHC/BαMHC mice examined had an obvious increase in cardiac myocyte size along with nuclear changes. The absence of a DORV and the small percentage of mice with VSDs compared to B−/B− mice are likely attributable to the timing of the loss of NMHC II-B from the cardiac myocytes.
An advantage of these cardiac myocyte–specific NMHC II-B knockout mice is that most of them survive to adulthood permitting analysis of the role of NMII-B in the adult heart. H&E-stained sections of BαMHC/BαMHC hearts examined at 6 months, similar to hearts examined at P0 (Figure 5a and 5e), show evidence for myocyte hypertrophy (Figure 5b and 5f). This hypertrophy is even more evident by 10 months (Figure 5c and 5g). Figure 6 shows wheat germ agglutinin staining to more easily visualize and quantify the increase in cardiac myocytes size in BαMHC/BαMHC hearts at 4 and 6 months of age. As shown in Figure 6f, there is a progressive increase in the size of the cardiac myocytes.
In addition to cardiac myocyte hypertrophy, BαMHC/BαMHC mice at 10 months also display additional pathological changes including interstitial fibrosis (Figure 5d and 5h). There is infiltration with inflammatory cells, such as lymphocytes, plasma cells, and macrophages in the interstitium of the myocardium and the pericardium of BαMHC/BαMHC mouse hearts. Some of these changes can be seen as early as 6 months of age in BαMHC/BαMHC mice (Figure 5f) but are much more prominent at 10 months of age (Figure 5d and 5h; n=4). We also observed vacuolation in cardiac myocytes, suggesting that the myocytes have undergone marked degeneration (Figure 5h, arrow). The presence of vacuolated cells in the heart prompted us to examine them for evidence of an increase in apoptosis. Online Figure II shows the results of a TUNEL assay comparing BαMHC/BαMHC and Bflox/Bflox mouse hearts. There is a small increase in the number of cells undergoing apoptosis in the mutant heart, as indicated by the arrows in the figure.
The presence of the inflammatory response in these hearts raises the possibility of a viral myocarditis. Of note, however, we did not observe inflammation in the hearts of Bflox/Bflox littermates. Analyses of both control and BαMHC/BαMHC mice for the presence of viruses associated with myocarditis were negative for enterovirus (Coxsackie viruses and echo virus; data not shown). In 2 of 8 BαMHC/BαMHC mice, thrombi were seen in the H&E-stained sections of the left atrium of the heart at 10 months of age, consistent with severe pathological changes in the heart and compromised cardiac function (see Online Figure III).
We also addressed the question of whether the fetal cardiac program was reactivated in the hearts of these mice by performing both immunoblot analyses and immunofluorescence microscopy on the NMII-B–ablated and normal hearts. In contrast to our previous findings for mice that were rendered hypomorphic for NMII-B, in which there is a 40-fold increase in the expression of β-cardiac myosin,10 we found only a 2- to 3-fold increase in the expression of βMHC in the 6-month-old BαMHC/BαMHC mouse heart. Microscopy confirmed this and showed that the increase was only detected in relatively few cardiac myocytes and did not correlate with the extent of myocyte hypertrophy (data not shown).
BαMHC/BαMHC Mice Develop a Cardiomyopathy With Abnormalities in the ID
To obtain information about the cardiac function of BαMHC/BαMHC mice, we carried out echocardiography at 4, 6, and 10 months of age. The Table shows that although there are no significant differences between Bflox/Bflox and BαMHC/BαMHC mice at 4 and 6 months, there are differences at 10 months of age. These include an increase in left ventricular internal diameter at the end of systole and a marked decrease in the percentage of fractional shortening from 44±8% (n=7) for Bflox/Bflox to 29±9% (n=12) for BαMHC/BαMHC mice. These results confirm that cardiac function is significantly compromised in the BαMHC/BαMHC mice at 10 months and are consistent with a cardiomyopathy. We also performed ECGs (3 standard leads) to determine whether abnormalities could be detected in BαMHC/BαMHC mice at this age. The electric axis ranged from +30° to +90° for the control animals. However, 4 of 5 of the BαMHC/BαMHC mice displayed an abnormal right axis deviation, ranging from +90° to more than +210° and in 3 of them, the severity of the deviation (more than 120°) is consistent with an abnormality in cardiac conduction (Online Figure IV).
To understand a possible cause of the defect in conduction in BαMHC/BαMHC hearts, we carried out an electron microscopy study. Previous work from this laboratory has demonstrated that in adult mice NMII-B was detected in the IDs in the heart.18 Moreover, deletion and mutation of proteins associated with the IDs are often associated with defects in cardiac conduction.19,20 It was, therefore, of interest to see whether the IDs of the BαMHC/BαMHC mice were normal in structure. Figure 7A is electron micrographs showing that the IDs of BαMHC/BαMHC mice are widened and distorted (4/5 mice examined) compared to Bflox/Bflox mice. Approximately 20% of the IDs of BαMHC/BαMHC mice show this abnormality, which is not found in Bflox/Bflox mice. Careful inspection of the affected IDs shows that whereas the adhesion junctions are severely disrupted (Figure 7A, b, large arrow, and c), the desmosomes (arrowheads) and gap junctions (arrows) remain mostly intact and are less affected (Figure 7A, b and c).
To gain insight into the cause of the disruption of the ID, we carried out an immunoblot analysis of a number of proteins known to be present at the disc at 6 months. Figure 7B shows that of the proteins analyzed, including a number of adhesion molecules, only the actin-binding protein mXinα is decreased (a decrease of 78.5±4.8% compared to the wild type; n=2 mice, performed in triplicate). In contrast, expression of connexin 43 is increased, most likely because of cardiac myocyte hypertrophy. Figure 7C shows the distribution of both mXinα and connexin 43 in wild-type and BαMHC/BαMHC hearts at 10 months using confocal immunofluorescence microscopy. The staining confirms the decreased expression of mXinα at the ID. It also shows that, unlike the wild-type disc, mXinα is not uniformly associated with connexin 43 in many of the discs. We propose that the loss in NMII-B at the ID is the primary cause of the disruption of cell–cell adhesion in the NMII-B–ablated heart. Moreover, the loss (NMII-B) and decrease (mXinα) of 2 actin-binding proteins at the ID, the latter of which also binds to β-catenin21 are expected to contribute to instability at the adhesion junction (see Discussion). Of note, no defects in the brain or other organs were found in BαMHC/BαMHC mice at any age.
We have previously reported that, as early as E11.5, global ablation of NMHC II-B in mice resulted in hydrocephalus associated with defects in cell–cell adhesion of the neural epithelial cells lining the spinal canal and cerebral ventricles.9,15 In Bnest/Bnest mice, ablation of NMHC II-B was initiated at E10.5, controlled by the nestin promoter, which is consistent with the delayed onset of hydrocephalus. Of particular note, despite the death of these mice between 12 to 22 days of age, most likely resulting from severe hydrocephalus, there were no abnormalities found in the heart.
In addition to learning whether the defects we found in the B−/B− mouse hearts were related directly or indirectly to those found in the nervous system, we also wanted to study the role of NMII-B in the adult mouse heart. Most BαMHC/BαMHC mice manifested progressive cardiac abnormalities, starting with myocyte hypertrophy, which was apparent as early as P0 and increased during postnatal development to 6 and 10 months of age. At 10 months, there was also evidence of myocyte vacuolation and cell degeneration, interstitial fibrosis, and an infiltration of the cardiac tissue with inflammatory cells. We hypothesize that the cardiac phenotype in the BαMHC/BαMHC mice is initiated by abnormalities specific to the cardiac myocytes, because NMII-B is ablated in these cells but not in the nonmyocytes in these mice. This loss of NMII-B (and lack of compensation by NMII-A or II-C) results in a failure in cytokinesis, as manifested by multinucleation and the abnormal nuclei found in these cells. It most likely contributes to abnormal enlargement of the cardiac myocytes, as well as their decreased numbers at P0. We, therefore, reasoned that the interstitial fibrosis and infiltration of inflammatory cells are secondary to the primary abnormality in the cardiac myocytes, which is most likely myocyte degeneration.
The pathological changes in the hearts of cardiac-specific NMHC II-B knockout mice are in agreement with the echocardiographic and ECG studies. The marked decrease in the fractional shortening at 10 months is consistent with the compromised contractility of cardiac muscle. The abnormalities noted in ECGs (an abnormal electrical axis) could reflect the striking defects found in the IDs. Previous work has shown that in the adult heart NMII-B is localized to the Z-lines and IDs.18 The IDs are composed of adherens junctions, desmosomes, and gap junctions that form cell–cell boundaries and connections between cardiac myocytes and allow the myocardium to function in synchrony. As noted above, work from a number of laboratories has shown that NMIIs play an important role in cell–cell adhesion7,15,20,22 and that abnormalities in a number of adhesion proteins result in either loss or structural changes in the cardiac IDs.20,23,24 Figure 7A provides evidence that loss of NMII-B primarily affects the adhesion junctions rather than the gap junctions or desmosomes. Moreover, BαMHC/BαMHC hearts at 6 months show a milder defect in the adhesion junction of the IDs and no defects in the desmosomes and gap junctions (data not shown).
We have analyzed the expression of a number of ID proteins and found a significant decrease in the expression of mXinα in BαMHC/BαMHC hearts compared to the wild-type hearts. Mice ablated for mXinα also show abnormal IDs.19 Unlike the mXinα knockout hearts, NMII-B–ablated hearts show no decrease in expression levels of N-cadherin or β-catenin. Moreover, there was no change in the distribution of β-catenin. We, therefore, attribute the primary cause of the disruption of the IDs to the loss of NMII-B. We speculate that the decrease of mXinα is secondary to the loss of NMII-B, and the mechanism of this decrease is of ongoing interest. The decrease in both of these proteins, one of which (mXinα) has been demonstrated to also bind to β-catenin,21 would explain the marked disruption of the IDs.
The finding of a role for NMII-B in the cardiac ID is similar to the findings for NMII-B in the spinal canal. Our hypothesis is that NMII exerts tension and stabilizes actin filaments, which, in turn, are required for maintenance of adhesion complexes between cells or, in this case, between the cardiac myocytes. The loss of NMII-B from the adhesion complex could, therefore, result in the gradual deterioration in the cardiac adhesion complex, including the loss of mXinα, over a period of time, and this would account for our failure to observe abnormal discs in B−/B− mice, which died before birth. Interestingly, generation of mice in which NMII-A replaced NMII-B did not produce defects in the IDs.25 This is consistent with the hypothesis that in cases in which NMIIs are apparently playing a structural rather than a motor role, 1 isoform is more likely to substitute for the other in vivo, as well as in cultured cells.6 When myosin is playing more of a motor role, for example, in neural cell migration, because of significant differences in the kinetics of MgATP hydrolysis and actin-binding properties between the myosin isoforms, successful substitution is much less likely, at least in vivo.15 These findings further support the idea that disruption of the IDs in BαMHC/BαMHC mice is attributable to the loss of NMII and is secondary to the development of the cardiomyopathy.
These conditionally ablated mice demonstrate that the defects we observed in the hearts and brains of the B−/B− mice are independent of each other. The availability of NMHC II-B floxed mice will allow conditional ablation of NMHC II-B in a variety of tissues and cells and thus help to further define its role both in situ and in vivo.
We are grateful to Charles W. Birdsall (National Heart, Lung, and Blood Institute) and to the NIH Mouse Imaging Facility for help with mouse echocardiograms and ECGs. We thank Douglas R. Rosing, (National Heart, Lung, and Blood Institute) for expert advice on ECGs. We acknowledge the professional skills and advice of Christian A. Combs and Daniela Malide of the Light Microscopy Core Facility (National Heart, Lung, and Blood Institute), regarding microscopy-related experiments performed in this study. We thank the members of the Laboratory of Molecular Cardiology for suggestions and criticism, in particular, Mary Anne Conti and Sachiyo Kawamoto. We thank Stephanie Jackson for editorial assistance.
Sources of Funding
This work was supported by the Division of Intramural Research, National Heart, Lung, and Blood Institute, NIH.
Original received December 8, 2008; resubmission received May 4, 2009; revised resubmission received September 17, 2009; accepted September 24, 2009.
Conti MA, Adelstein RS. Nonmuscle myosin II moves in new directions. J Cell Sci. 2008; 121: 11–18.
Betapudi V, Licate LS, Egelhoff TT. Distinct roles of nonmuscle myosin II isoforms in the regulation of MDA-MB-231 breast cancer cell spreading and migration. Cancer Res. 2006; 66: 4725–4733.
Sandquist JC, Swenson KI, Demali KA, Burridge K, Means AR. Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration. J Biol Chem. 2006; 281: 35873–35883.
Vicente-Manzanares M, Zareno J, Whitmore L, Choi CK, Horwitz AF. Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells. J Cell Biol. 2007; 176: 573–580.
Conti MA, Even-Ram S, Liu C, Yamada KM, Adelstein RS. Defects in cell adhesion and the visceral endoderm following ablation of nonmuscle myosin heavy chain II-A in mice. J Biol Chem. 2004; 279: 41263–41266.
Tullio AN, Accili D, Ferrans VJ, Yu ZX, Takeda K, Grinberg A, Westphal H, Preston YA, Adelstein RS. Nonmuscle myosin II-B is required for normal development of the mouse heart. Proc Natl Acad Sci U S A. 1997; 94: 12407–12412.
Gaussin V, Van de PT, Mishina Y, Hanks MC, Zwijsen A, Huylebroeck D, Behringer RR, Schneider MD. Endocardial cushion and myocardial defects after cardiac myocyte-specific conditional deletion of the bone morphogenetic protein receptor ALK3. Proc Natl Acad Sci U S A. 2002; 99: 2878–2883.
McFadden DG, Barbosa AC, Richardson JA, Schneider MD, Srivastava D, Olson EN. The Hand1 and Hand2 transcription factors regulate expansion of the embryonic cardiac ventricles in a gene dosage-dependent manner. Development. 2005; 132: 189–201.
Ma X, Kawamoto S, Hara Y, Adelstein RS. A point mutation in the motor domain of nonmuscle myosin II-B impairs migration of distinct groups of neurons. Mol Biol Cell. 2004; 15: 2568–2579.
Ma X, Bao J, Adelstein RS. Loss of cell adhesion causes hydrocephalus in nonmuscle myosin II-B-ablated and mutated mice. Mol Biol Cell. 2007; 18: 2305–2312.
Takeda K, Kishi H, Ma X, Yu ZX, Adelstein RS. Ablation and mutation of nonmuscle myosin heavy chain II-B results in a defect in cardiac myocyte cytokinesis. Circ Res. 2003; 93: 330–337.
Lombardi R, Dong J, Rodriguez G, Bell A, Leung TK, Schwartz RJ, Willerson JT, Brugada R, Marian AJ. Genetic fate mapping identifies second heart field progenitor cells as a source of adipocytes in arrhythmogenic right ventricular cardiomyopathy. Circ Res. 2009; 104: 1076–1084.
Gustafson-Wagner EA, Sinn HW, Chen YL, Wang DZ, Reiter RS, Lin JL, Yang B, Williamson RA, Chen J, Lin CI, Lin JJ. Loss of mXinalpha, an intercalated disk protein, results in cardiac hypertrophy and cardiomyopathy with conduction defects. Am J Physiol Heart Circ Physiol. 2007; 293: H2680–H2692.
Kostetskii I, Li J, Xiong Y, Zhou R, Ferrari VA, Patel VV, Molkentin JD, Radice GL. Induced deletion of the N-cadherin gene in the heart leads to dissolution of the intercalated disc structure. Circ Res. 2005; 96: 346–354.
Choi S, Gustafson-Wagner EA, Wang Q, Harlan SM, Sinn HW, Lin JL, Lin JJ. The intercalated disk protein, mXinalpha, is capable of interacting with beta-catenin and bundling actin filaments. J Biol Chem. 2007; 282: 36024–36036.
Shewan AM, Maddugoda M, Kraemer A, Stehbens SJ, Verma S, Kovacs EM, Yap AS. Myosin 2 is a key Rho kinase target necessary for the local concentration of E-cadherin at cell-cell contacts. Mol Biol Cell. 2005; 16: 4531–4542.
Yang Z, Bowles NE, Scherer SE, Taylor MD, Kearney DL, Ge S, Nadvoretskiy VV, DeFreitas G, Carabello B, Brandon LI, Godsel LM, Green KJ, Saffitz JE, Li H, Danieli GA, Calkins H, Marcus F, Towbin JA. Desmosomal dysfunction due to mutations in desmoplakin causes arrhythmogenic right ventricular dysplasia/cardiomyopathy. Circ Res. 2006; 99: 646–655.
Bao J, Ma X, Liu C, Adelstein RS. Replacement of nonmuscle myosin II-B with II-A rescues brain but not cardiac defects in mice. J Biol Chem. 2007; 282: 22102–22111.