In vivo studies on nonmuscle myosin II expression and function in heart development

Abstract

Nonmuscle myosin II-B (NM II-B) plays an important role in cardiac development and function. Genetic ablation of NM II-B in mice results in both cellular and structural defects involving cardiac myocytes. These abnormalities include a ventricular septal defect, double outlet of the right ventricle, myocyte hypertrophy and premature onset of myocyte binucleation due to abnormalities in cytokinesis. The mice die by embryonic day (E) 14.5 due to defects in heart development. Conditional ablation of NM II-B in cardiac myocytes after E11.5 allows study of NM II-B function in adult myocytes. BaMHC/BaMHC mice are born with enlarged cardiac myocytes, some of which are multinucleated. Between 6-10 months of age they develop a cardiomyopathy. Many of these mice develop a marked widening of the intercalated discs. The loss of NM II-B from the intercalated discs primarily affects the adhesion junctions rather than the gap junctions and desmosomes. Interestingly, the loss of NM II-B results in a decrease in the actin binding protein mXin which also has been shown to cause disruption of the intercalated disc in addition to cardiac arrhythmias (Gustafson-Wagner et al. Am J Physiol Heart Circ Physiol. 2007, 293:H2680-92). Finally we review the evidence showing that ablation of NM II-C (which also localizes to the intercalated disc) in mouse hearts deficient in NM II-B expression results in destabilization of N-cadherin and beta-catenin in the intercalated disc.

Figures

Figure 1. Diagram Depicting NM II Molecule, Bipolar Filament and Regulation

(A) Diagram of a myosin molecule showing the globular head region, the α-helical coiled-coil rod and the short non-helical tail (NHT). The subfragment-1 (S-1), rod and heavy meromyosin (HMM) proteolytic domains are also indicated. (B) An example of a bipolar filament, which is formed by interaction among the rod domains. The actual number of molecules is greater than shown. (C) Regulation of myosin activity by phosphorylation of RMLC of NMII by myosin light chain kinase (MLCK) and Rho kinase. Whereas MLCK can only phosphorylate RMLC, Rho kinase can phosphorylate RMLC and also a subunit of myosin phosphatase (MYPT). Phosphorylation by Rho kinase activates myosin and inactivates MYPT. Both result in an increase in phosphorylated RMLC and activation of myosin. Reproduced from reference .

Figure 2. Expression of NM II during Early Heart Formation

Immunofluorescence confocal images of wild-type mouse hearts at E7.5 and E8.5 stained for NMHC II-A (green, A–C) and II-B (green, D–F), and co-stained for MF20 (red, a marker for cardiac myocytes). At E7.5, the early cardiac myocytes (A,D) show co-staining of NMHC II-A and MF20 (A) or NMHC II-B and MF20 (D) indicating that both NMHC II-A and II-B are expressed in cardiac myocytes at this stage. At E8.5, the cardiac myocytes in the developing outflow tract (OFT) still express both NMHC II-A (B, magnified in C) and II-B (E, magnified in F), however in the ventricular myocytes at E8.5 (arrows, C,F) only NMHC II-B (E,F), and not NMHC II-A (B,C) is detected. Modified from reference .

Figure 3. Normal Sarcomere Formation in B−C−/B−C− Cardiac Myocytes

Immunofluorescence confocal images of E13.5 cardiac myocytes stained with MF20 (red, a marker for cardiac myosin II, B,E) and desmin (green, A,D) in B+C+/B+C+ (A–C) and B−C−/B−C− (D–F) mouse hearts. No difference in sarcomere formation is found between B+C+/B+C+ and B−C−/B−C− hearts. Reproduced from reference .

Figure 4. Expression of NM II in Embryonic Mouse Hearts and NM II-C in Adult Mouse Hearts

(A) Immunofluorescence confocal images of an E13.5 mouse heart stained for NMHC II-A (a,d, green), II-B (b, green), and II-C (c, green) together with desmin (d–f, red), a marker for (cardiac) myocytes. NM II-A is only expressed in nonmyocytes (a,d, green). NM II-B is detected in both myocytes (e, red and green colocalization) and nonmyocytes (e, green) in the heart. NM II-C is detected in myocytes (f, red and green colocalization) but not in nonmyocytes. The bright green spots are autofluorescence from red blood cells in c and f. (B) Immunofluorescence confocal images of adult heart sections from C+/C+ (a, magnified in b) and C−/C− (c, magnified in d) mice. N-Cadherin is a marker for the intercalated disc (red). Nuclei are stained with DAPI (blue). Arrows in b indicates the presence of NMHC II-C (green) in the intercalated disc. NMHC II-C is absent from C−/C− intercalated discs (c and d). Reproduced from reference .

Figure 5. Abnormalities in the Intercalated Discs in BαMHC/BαMHC Mice

(A) Electron microscope sections of Bflox/Bflox (a) and BαMHC/BαMHC (b and c) left ventricles at 10 months. b shows a less-affected intercalated disc than c. Both b (large arrow) and c show that the adhesion type junctions of the BαMHC/BαMHC cardiac myocytes are severely distorted, whereas 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 intercalated discs at 6 months. Note the decrease in mXimα in BαMHC/BαMHC mouse hearts. In contrast, expression of connexin 43 is increased, most likely due to cardiac myocyte hypertrophy. Modified from reference .

Figure 6. Ablation of NM II-C Accelerates Development of Cardiomyopathy in NM II-B Hypomorphic Mice

Wheat germ agglutinin staining shows plasma membranes in heart sections from B+C+/B+C+, B+C−/B+C−, BΔB1NC+/BΔB1NC+, and BΔB1NC−/BΔB1NC− mice. The average cross-sectional area of the cardiac myocytes for each genotype was measured and is shown in each panel. Compared with B+C+/B+C+ cardiac myocytes (A), the average cross-sectional area for B+C−/B+C− myocytes remains unchanged (B), but it is doubled in BΔB1NC+/BΔB1NC+ myocytes (C) and increased to 4 times in BΔB1NC−/BΔB1NC− myocytes (D). Nuclei are stained with DAPI (blue). Modified from reference .

Figure 7. Impaired Localization of β-Catenin and N-cadherin in BΔB1NC−/BΔB1NC− Intercalated Discs

(A) Immunofluorescence confocal images of adult heart sections from B+C+/B+C+, BΔB1NC−/BΔB1NC−, BΔB1NC+/BΔB1NC+, and B+C−/B+C− mice stained for β-catenin (red, a–d), connexin43 (green, a–d) and N-cadherin (red, e,f). In contrast to B+C+/B+C+ (a,e), B+C−/B+C− (d), and BΔB1NC+/BΔB1NC+ (c) cardiac myocytes where β-catenin and N-cadherin very precisely stain the intercalated discs, BΔB1NC−/BΔB1NC− myocytes (arrows, b,f) show a diffuse β-catenin and N-cadherin staining. No difference in connexin43 staining (green, a–d) is observed among these four genotypes. Nuclei were stained by DAPI (blue). (B) Profiles of β-catenin staining at the intercalated discs for B+C+/B+C+ (red lines) and BΔB1NC−/BΔB1NC− (black lines) mouse hearts. BΔB1NC−/BΔB1NC− hearts showed a diffuse β-catenin distribution manifested by widened β-catenin staining at the intercalated discs compared to the wild-type hearts. An embedded Zeiss LSM image profile tool is used to quantify the fluorescence intensity along a line drawn vertically across the intercalated discs. Reproduced from reference .