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. 2009 Jun;214(6):905-15.
doi: 10.1111/j.1469-7580.2009.01079.x.

Knockdown of alpha myosin heavy chain disrupts the cytoskeleton and leads to multiple defects during chick cardiogenesis

Catrin Rutland  1 , Louise Warner, Aaran Thorpe, Aziza Alibhai, Thelma Robinson, Barry Shaw, Robert Layfield, J David Brook, Siobhan Loughna
Affiliations

Knockdown of alpha myosin heavy chain disrupts the cytoskeleton and leads to multiple defects during chick cardiogenesis

Catrin Rutland et al. J Anat. 2009 Jun.

Abstract

Atrial septal defects are a common congenital heart defect in humans. Although mutations in different genes are now frequently being described, little is known about the processes and mechanisms behind the early stages of atrial septal development. By utilizing morpholino-induced knockdown in the chick we have analysed the role of alpha myosin heavy chain during early cardiogenesis in a temporal manner. Upon knockdown of alpha myosin heavy chain, three different phenotypes of the atrial septum were observed: (1) the atrial septum failed to initiate, (2) the septum was initiated but was growth restricted, or (3) incorrect specification occurred resulting in multiple septa forming. In addition, at a lower frequency, decreased alpha myosin heavy chain was found to give rise to an abnormally looped heart or an enlarged heart. Staining of the actin cytoskeleton indicated that many of the myofibrils in the knockdown hearts were not as mature as those observed in the controls, suggesting a mechanism for the defects seen. Therefore, these data suggest a role for alpha myosin heavy chain in modelling of the early heart and the range of defects to the atrial septum suggest roles in its initiation, specification and growth during development.

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Figures

Fig. 1
Fig. 1
Morpholino knockdown and expression of alpha myosin heavy chain (αMHC). (A) Light (a and c) and dark (b and d) field microscopy of embryos that had 250 µm of fluorescein-tagged standard control morpholino (StC) applied. Upon application of the morpholino at Hamburger and Hamilton (HH) stage 16 with harvesting after 30 min (HH16/30 min; a and b), fluorescein uptake can clearly be seen throughout the embryo (b). When the morpholino was applied at HH14 and left until HH24 (HH14/24), the fluorescence was found to persist (d). Arrow denotes heart. Scale bars: a, 1000 µm (same in b); c, 500 µm (same in d). (B) Lissamine-tagged standard control was applied at HH14 and harvested at HH19 (HH14/19). The heart was then scanned by confocal microscopy with images captured at 15 µm intervals. Fluorescence can be seen to both the atrial (At) and ventricular (V) chambers. Scale bar: 300 µm. (C) Staining of αMHC was observed throughout the wall of the tubular heart (H) at HH12 (a). By HH15 (b) staining was found in the myocardial walls of the atrium (At) and in the small atrial septum that can be observed emerging from the roof of the atrial chamber. By HH19 (c), the atrial wall was clearly stained as was the septum as it extended towards the endocardial cushions (EC), which it fused with by HH24 (d). However, staining of the endocardial cushions and ventricular myocardium was not detected. Arrow, atrial septum; asterisk, trabeculae; L, liver; Untreated, untreated control embryo. Scale bars: a and b, 200 µm; c and d, 500 µm. (D) Graphical representation of densitometry data obtained from four immunoblots using S58 (specific αMHC) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; loading control) antibodies. On average a 63% reduction in αMHC protein levels was observed in the αMHC morpholino knockdown (αMHC kd) hearts in comparison to age-matched controls. Western blot control tissue consisted of hearts taken from both untreated and standard control embryos. Densitometry analysis showed no statistical differences between the two control groups; therefore the results were combined and compared against knockdown heart data. anova was used to assess statistical significance between the control and αMHC knockdown hearts (P= 0.034). n = 4 separate blots, with each sample on each blot containing three hearts pooled. OD, optical density.
Fig. 2
Fig. 2
Knockdown of alpha myosin heavy chain (αMHC) can lead to abnormal looping or an enlarged heart in the chick. In order to reduce αMHC expression, morpholino was applied at either Hamburger and Hamilton (HH) stage 14 (C–E) or HH16 (F) and appropriate untreated (A) and standard control morpholino (B) embryos were analysed using light microscopy. All embryos were harvested at HH19. In comparison to the controls (A and B) the αMHC knockdown (αMHC kd) embryos were either phenotypically normal (C), had an abnormally looped heart (D and E) or the heart was enlarged (F). The asterisk denotes the sharp bend between the outflow tract (OT) and ventricle (V) that was seen in some of the αMHC knockdown embryos (D–F) but not in the controls (A and B). H, head; StC, standard control; Scale bars: in A, 500 µm (same for all panels).
Fig. 3
Fig. 3
Abnormal atrial septal development upon knockdown of alpha myosin heavy chain (αMHC). (A) Untreated (a and b), standard control (StC) (c and d) and αMHC knockdown (αMHC kd) (e–h) embryos were sectioned, stained with the nuclear marker haemalum and phenotypically analysed using light microscopy. Morpholino was applied at Hamburger and Hamilton (HH) stage 16 and chicks were harvested at HH19 (HH16/19; c–h). Micrographs b, d, f and h are high magnifications of the atrial chambers seen in a, c, e and g, respectively. In the untreated and StC embryos, normal atrial septal development can be seen, with a large septum having grown from the dorso-cranial atrial wall (arrows in a–d denote atrial septum). In contrast, upon knockdown with αMHC an atrial septum was often observed but was reduced in comparison to control septa (arrows in e and f compared with controls a–d). Alternatively, an atrial septum was not detected but a thickening of the dorso-cranial wall was seen (arrows in g and h). At, atrium; OFT, outflow tract; V, ventricle. Scale bars: a, c, e and g, 500 µm; b, d, f and h, 100 µm. (B) A photomicrograph of an αMHC embryo that was knocked down at HH14 and harvested at HH19 (HH14/19), and stained with an antibody against αMHC (kindly provided by Professor Moorman) to specifically detect the atrial chamber and counterstained with haemalum. Micrographs b, d and f are high magnifications of the atrial septa seen in a, c and e, respectively. Analysis of serial sections taken throughout the atrial chamber (At) revealed abnormal atrial septation. Initially three septa were detected emerging from the dorso-cranial wall of the atrial chamber (a and b), of which two subsequently dominated (c and d). Further sectioning through the atrium showed these two septa fusing at their inferior end leaving a large opening between them (e and f). Arrows denote atrial septa. Scale bars: 100 µm.
Fig. 4
Fig. 4
Reduction of embryonic myosin heavy chain leads to a disrupted actin cytoskeleton in the atrium. Untreated control embryos were harvested at Hamburger and Hamilton (HH) stage 19 (A–C), whereas HH14 embryos initially had alpha myosin heavy chain (αMHC) morpholino applied prior to harvesting at HH19 (HH14/19; D–F). Embryos were subsequently stained with phalloidin to label the actin filaments and counterstained with propidium iodide (PI) (A, B and D–F) or Hoechst (C) to label the nuclei. Upon confocal microscopy, a distinct septum (arrow in A and C) could be detected within the atrial chamber (At) of the untreated control embryos. In contrast, the αMHC knockdown (αMHC kd) embryos had a slight outgrowth from the atrial dorso-cranial wall (arrow in D), with a definitive septum not observed. Upon higher magnification, phalloidin staining can be clearly seen to label distinct actin fibrils in both the untreated control and the knockdown embryos (arrows in B and E). However, the fibrils in the αMHC kd atrial wall appear thinner and punctuate in comparison to the untreated fibrils. Boxed areas in A and D are the regions shown in B and E, respectively. A more intense staining was observed in four out of five chicks at the leading edge of the septum in control hearts (C) but this feature was not observed in the αMHC kd chicks (D). An αMHC knockdown embryo is also shown with two atrial septa emerging from the roof of the atrial chamber (F). Scale bars: A, 25 µm (same in C, D and F); B, 5 µm (same in E).
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