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. 2009 Nov 20;105(11):1102-9.
doi: 10.1161/CIRCRESAHA.109.200303. Epub 2009 Oct 8.

Conditional ablation of nonmuscle myosin II-B delineates heart defects in adult mice

Xuefei Ma  1 , Kazuyo Takeda, Aman Singh, Zu-Xi Yu, Patricia Zerfas, Anthony Blount, Chengyu Liu, Jeffrey A Towbin, Michael D Schneider, Robert S Adelstein, Qize Wei
Affiliations

Conditional ablation of nonmuscle myosin II-B delineates heart defects in adult mice

Xuefei Ma et al. Circ Res. .

Abstract

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 (B(alphaMHC)/B(alphaMHC) mice) do not show brain defects. However, B(alphaMHC)/B(alphaMHC) mice display novel cardiac defects not seen in NMII-B germline-ablated mice. Most of the B(alphaMHC)/B(alphaMHC) 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 B(alphaMHC)/B(alphaMHC) 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.

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Figures

Figure 1
Figure 1. Schematic representation of the strategy used to generate neural- or cardiac myocyte-specific NMHC II-B knockout mice
(a) A portion of the wild-type NMHC II-B gene locus including exon2. (b) Targeting construct. Neomycin-resistance cassette (Neor) and exon2 are flanked by loxP sites. (c) Bflox allele is generated after homologous recombination. (d) NMHC II-B ablated allele (Bnest or BαMHC) lacking exon2, is generated in the presence of Cre recombinase. (e) The Cre expression cassette under the control of nestin promoter in the neural-specific Cre transgenic mice. (f) The Cre expression cassette under the control of the αMHC promoter in the cardiac myocytes-specific Cre transgenic mice. Black box in a, b and c indicates exon2 and arrowheads indicate lox P sites.
Figure 2
Figure 2. NMHC II-B expression levels and phenotype changes in Bnest/Bnest mice
(A) Immunoblot analysis of whole tissue lysates from the cerebellum and the heart of Bnest/Bnest and Bflox/Bflox mice, using antibodies specific for NMHC II-B and β-tubulin (loading control) as indicated. (B) Immunofluorescence staining of sections from the cerebellum of Bflox/Bflox (panels a–d) and Bnest/Bnest (panels e–h) mice at P18 using antibodies specific for NMHC II-B (red) and calbindin (green); DAPI (blue). Calbindin is a marker for Purkinje cells and DAPI for nuclei. G indicates the granular layer and M the molecular layer in the cerebellum. (C) (a, c) Photographs of the brain from Bflox/Bflox (a) and Bnest/Bnest (c) mice. Arrows in panel c indicate the collapse of the cerebral cortex after fixation due to hydrocephalus. Dashed ellipse encloses cerebellum. (b, d) Coronal sections of the brains from Bflox/Bflox (b) and Bnest/Bnest (d) mice following H&E staining.. (D) Panels a and b show the spinal canal at P7. The canals of Bflox/Bflox mice (a) are narrow but patent at this age (boxed areas, enlarged in insets, arrow). In b, the canal of a Bnest/Bnest mouse is completely obliterated.
Figure 3
Figure 3. NMHC II-B expression levels in BαMHC/BαMHC mice at E13.5
(A) Immunoblots of whole tissue lysates from the heart and the brain of Bflox/Bflox or BαMHC/BαMHC mice were probed using antibodies specific for NMHC II-B, actin, or β-tubulin, as indicated. Note that the NMHC II-B protein level does not significantly change in the brain of BαMHC/BαMHC mice. In contrast, the NMHC II-B protein level is significantly reduced in the heart of BαMHC/BαMHC mice. (B) Immunofluorescence staining of heart sections from Bflox/Bflox (a–c) and BαMHC/BαMHC (d–f) mice at E13.5 using antibodies specific for NMHC II-A (a,d, green), NMHC II-B (b,e, green) and II-C (c,f, green background) and desmin (all panels, red). Desmin is a marker for cardiac myocytes. Note the co-staining of NMHC II-B and desmin in the cardiac myocytes (b, yellow). NMHC II-B staining in the myocytes disappears after ablation, but persists in the non-myocytes (b,e, arrows). Panels c and f confirm the low expression of NMHC II-C in the heart at this age. Note there is no change in the staining for NMHC II-A and II-C in the BαMHC/BαMHC heart. DAPI (blue) stains the nuclei.
Figure 4
Figure 4. Cardiac abnormalities in BαMHC/BαMHC mice at P0
H&E staining of the hearts from B+/BαMHC (a–c) and BαMHC/BαMHC (d–f) mice. Note the rounded shape of the BαMHC/BαMHC heart (d) and the small VSD, enlarged in panel e. Panel f shows enlarged cardiac myocytes with bizarrely shaped nuclei (arrows) in the BαMHC/BαMHC heart.
Figure 5
Figure 5. Progression of cardiac abnormalities in BαMHC/BαMHC mice at P0, 6 months and 10 months
H&E stained sections show increasing cardiac myocyte hypertrophy starting at P0 through 10 months of age in the hearts of BαMHC/BαMHC mice (d–h) compared to Bflox/Bflox litter mates (a–c). (d, h) At 10 months BαMHC/BαMHC hearts show evidence for necrosis and interstitial fibrosis. There is an infiltration of the cardiac interstitium with inflammatory cells including lymphocytes, plasma cells and macrophages (d). Arrow in h indicates vacuolated cell.
Figure 6
Figure 6. WGA staining of the cardiac myocytes
Staining of cardiac myocytes with WGA (red) at 4 months (a,b) and at 6 months (c–e) and quantification of myocyte size (f) showing a progressive increase in myocyte size in BαMHC/BαMHC hearts. ** P<0.01 (n=3 mice). There is no significant difference in myocytes size at 6 months between the B+/BαMHC heart and the Bflox/Bflox heart. DAPI (blue) stains the nuclei.
Figure 7
Figure 7. Abnormalities in the IDs in BαMHC/BαMHC mice at 6 and 10 months
(A) Electron microscope sections of Bflox/Bflox (a) and BαMHC/BαMHC (b,c) left ventricles at 10 months. (b) shows a less affected ID than (c). Both (b, large arrow) and (c) show that the adhesion type junctions of the BαMHC/BαMHC cardiac myocytes are severely distorted while the structures of the desmosomes (arrowheads) and gap junctions (small arrows) remain intact. The structure between the white arrows shows a normal adhesion junction (a). Similar results were found for 4 other wild type and 3 other BαMHC/BαMHC mice. (B) Immunoblot analysis for proteins associated with the ID at 6 months. Samples are from 2 wild type and 2 NMII-B ablated hearts. Immunoblot analysis was repeated three times and quantified using an Odyssey Infrared Imaging System. (C) Confocal immunofluorescence microscope images stained as indicated for wild type and NMII-B ablated mouse hearts at 10 months. Arrows indicate IDs where mXinα is decreased.
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References

    1. Bresnick AR. Molecular mechanisms of nonmuscle myosin-II regulation. Curr Opin Cell Biol. 1999;11:26–33. - PubMed
    1. Conti MA, Adelstein RS. Nonmuscle myosin II moves in new directions. J Cell Sci. 2008;121:11–18. - PubMed
    1. Krendel M, Mooseker MS. Myosins: tails (and heads) of functional diversity. Physiology (Bethesda) 2005;20:239–251. - PubMed
    1. 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. - PubMed
    1. 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. - PubMed

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