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. 2022 Apr 10;23(8):4179.
doi: 10.3390/ijms23084179.

Inhibition of RhoA and Cdc42 by miR-133a Modulates Retinoic Acid Signalling during Early Development of Posterior Cardiac Tube Segment

Carlos Garcia-Padilla  1   2 , Virginio Garcia-Lopez  1 , Amelia Aranega  2   3 , Diego Franco  2   3 , Virginio Garcia-Martinez  1 , Carmen Lopez-Sanchez  1
Affiliations

Inhibition of RhoA and Cdc42 by miR-133a Modulates Retinoic Acid Signalling during Early Development of Posterior Cardiac Tube Segment

Carlos Garcia-Padilla et al. Int J Mol Sci. .

Abstract

It is well known that multiple microRNAs play crucial roles in cardiovascular development, including miR-133a. Additionally, retinoic acid regulates atrial marker expression. In order to analyse the role of miR-133a as a modulator of retinoic acid signalling during the posterior segment of heart tube formation, we performed functional experiments with miR-133a and retinoic acid by means of microinjections into the posterior cardiac precursors of both primitive endocardial tubes in chick embryos. Subsequently, we subjected embryos to whole mount in situ hybridisation, immunohistochemistry and qPCR analysis. Our results demonstrate that miR-133a represses RhoA and Cdc42, as well as Raldh2/Aldh1a2, and the specific atrial markers Tbx5 and AMHC1, which play a key role during differentiation. Furthermore, we observed that miR-133a upregulates p21 and downregulates cyclin A by repressing RhoA and Cdc42, respectively, thus functioning as a cell proliferation inhibitor. Additionally, retinoic acid represses miR-133a, while it increases Raldh2, Tbx5 and AMHC1. Given that RhoA and Cdc42 are involved in Raldh2 expression and that they are modulated by miR-133a, which is influenced by retinoic acid signalling, our results suggest the presence of a negative feedback mechanism between miR-133a and retinoic acid during early development of the posterior cardiac tube segment. Despite additional unexplored factors being possible contributors to this negative feedback mechanism, miR-133a might also be considered as a potential therapeutic tool for the diagnosis, therapy and prognosis of cardiac diseases.

Keywords: Cdc42; Raldh2; RhoA; atrial differentiation; cardiac development; miR-133a; retinoic acid signalling.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Representative data of RhoA (A) and Cdc42 (B) 3′UTR luciferase assays after premiR-133a overexpression in 3T3 fibroblasts. Luciferase activity was compared to non-transfected controls. Each luciferase assay was carried out in triplicate. Student’s t-test: ** p < 0.01.
Figure 2
Figure 2
Whole-mount ISH for miR-133a and whole mount IMH for RhoA, Cdc42 and Raldh2 during early chick cardiac development, from HH8–HH11 stages, in control embryos. Note miR-133a expression pattern at the level of the anterior region of the primitive endocardial tube (PET), being observable in the atrium (A) and ventricle (V) at later stages. Note the location of RhoA, Cdc42 and Raldh2 at the level of the PET posterior region, and subsequently in the atrium and inflow tract (IFT). The scheme illustrates the complementary location in cranial-caudal trend of miR-133a (blue) in comparison with RhoA, Cdc42 and Raldh2 (orange).
Figure 3
Figure 3
Effect of miR-133a gain- and loss-of-function on posterior cardiac segment. Whole-mount IMH for RhoA, Cdc42 and Raldh2. and ISH for Tbx5 and AMHC1. Embryos microinjected with CFDA (control), premiR-133a or antimiR-133a, at the level of the posterior cardiac precursors of both primitive endocardial tubes, and visualisation of CFDA (A). Note that, at the atrium and inflow tract levels, RhoA (B), Cdc42 (C), Raldh2 (D), Tbx5 (E) and AMHC1 (F) are dramatically reduced, and an atrophic sino-atrial region in the heart tube after premiR-133a treatment is indicated by the red arrows, whereas they are markedly increased and expanded after miR-133a inhibition (blue arrows). RT-qPCR of RNA from dissected cardiac asa (left side) in embryos microinjected either with CFDA, premiR-133a or antimiR-133a. A high level of miR-133a leads to decreased RhoA, Cdc42, Raldh2, Tbx5 and AMHC1 transcripts, whereas miR-133a inhibition leads to increased transcripts. The standard deviations are from three independent experiments. Student’s t-test: * p < 0.05, ** p < 0.01, *** p < 0.005 with respect to control (CFDA) embryos.
Figure 4
Figure 4
Effect of miR-133a gain- and loss-of-function experiments on cellular proliferation during posterior differentiation of cardiac tube. RT-qPCR of RNA from dissected cardiac asa in embryos microinjected either with CFDA, premiR-133a or anti-miR-133a, at the level of the posterior cardiac precursors of both primitive endocardial tubes. Note that miR-133a treatment leads to increased p21 (A) and decreased cyclin A (B) transcripts, whereas miR-133a inhibition leads to decreased p21 (A) and increased cyclin A (B) transcripts. The standard deviations are from three independent experiments. Student’s t-test: * p < 0.05, ** p < 0.01, *** p < 0.005 with respect to control (CFDA) embryos.
Figure 5
Figure 5
Effect of miR-133a on cellular proliferation in cardiomyocytes. Immunohistochemistry for Ki67 positive cells (arrows) and staining with DAPI (blue) in control cardiomyocytes obtained from H9c2 cell culture and subjected to premiR-133a treatment (A). Note that the percentage of Ki67 positive cells is dramatically repressed in premiR-133a treated cardiomyocytes (B). Student’s t-test: ** p < 0.01.
Figure 6
Figure 6
Whole-mount ISH for miR-133a, Tbx5, AMHC1 and IMH for Raldh2. Embryos microinjected with CFDA (control), Retinoic acid (RA) or Citral, at the level of the posterior cardiac precursors into both primitive endocardial tubes, and visualisation of CFDA (A). The gain-of-function of RA leads to diminished miR-133a expression (red arrows) at the cardiac asa level (B), accompanied by increased protein levels of Raldh2 (C) and expanded expression of Tbx5 (D) and AMHC1 (E) in the heart tube and in the inflow tract (blue arrows). Note atrophic sino-atrial region with increased miR-133a expression (blue arrows) at cardiac asa level (B), and also (red arrows) diminished Raldh2 protein level (C), and decreased Tbx5 (D) and AMHC1 (E) expressions by RA synthesis inhibition. RT-qPCR of RNA from dissected cardiac asa (left side) in embryos microinjected either with CFDA, RA or Citral. The standard deviations are from three independent experiments. Student’s t-test: * p < 0.05, ** p < 0.01, *** p < 0.005 with respect to control (CFDA) embryos.
Figure 7
Figure 7
Model proposed for the interplay between miR-133a and RA during posterior heart tube formation. Our model indicates that miR-133a downregulates RhoA and Cdc42. Consequently, Raldh2 downregulation is induced by miR-133a, via these Rho GTPases. RA synthesis is Raldh2-dependent. Thus, miR-133a modulates RA signalling via Raldh2 expression. Also, RA negatively modulates miR-133a expression during the early genetic programme of the sinoatrial region. Additionally, our model indicates that miR-133a modulates cell proliferation by acting on the cell cycle regulators p21 (via RhoA) and cyclin A (via Cdc42). We hypothesise that there is a negative feedback mechanism between miR-133a and RA signalling during early development of the posterior cardiac tube segment. A: anterior segment, P: posterior segment.
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