miR-1/133a clusters cooperatively specify the cardiomyogenic lineage by adjustment of myocardin levels during embryonic heart development

Abstract

miRNAs are small RNAs directing many developmental processes by posttranscriptional regulation of protein-coding genes. We uncovered a new role for miR-1-1/133a-2 and miR-1-2/133a-1 clusters in the specification of embryonic cardiomyocytes allowing transition from an immature state characterized by expression of smooth muscle (SM) genes to a more mature fetal phenotype. Concomitant knockout of miR-1-1/133a-2 and miR-1-2/133a-1 released suppression of the transcriptional co-activator myocardin, a major regulator of SM gene expression, but not of its binding partner SRF. Overexpression of myocardin in the embryonic heart essentially recapitulated the miR-1/133a mutant phenotype at the molecular level, arresting embryonic cardiomyocytes in an immature state. Interestingly, the majority of postulated miR-1/133a targets was not altered in double mutant mice, indicating that the ability of miR-1/133a to suppress target molecules strongly depends on the cellular context. Finally, we show that myocardin positively regulates expression of miR-1/133a, thus constituting a negative feedback loop that is essential for early cardiac development.

Figure 1. Deletion of single miR-1/133a clusters does not cause gross morphological alterations in the heart but results in decreased ejection fraction in miR-1-1/133a-2 mutants after TAC.

(A–C) No morphological abnormalities are discernable on frontal sections through hearts of miR-1-1/133a-2 and miR-1-2/133a-2 homozygous mutants. (D, E) Transverse aortic constriction led to an increase in wall thickness (D) and left ventricular mass (E) in comparison to sham-operated mice wildtype or miR-1/133a single knock-out mice as measured by MRI. (F) miR-1-1/133a-2 but not miR-1-2/133a-2 homozygous mutants showed a reduction in ejection fraction compared to wild type mice. (G) TAC-induced pressure overload resulted in a comparable increase in ANP levels in single cluster mutants and wild type controls. (H, I) qRT-PCR analysis (Taqman) of miR-1 and miR-133a expression in different single cluster mutant strains after TAC. No significant increase of miR-1 expression in miR-1-1/133a-2 and miR-1-2/133a-1 mutants after TAC compared to sham-operated mice while expression levels of miR-133a dropped slightly after TAC in both single cluster mutants.

Figure 2. miR-1/133a clusters contribute equally to miR-1/133a expression in the developing heart.

(A) Quantitative RT-PCR analysis of ca. 50% reduced expression of miR-1 and miR-133a expression in single cluster knock-out embryonic hearts and complete loss in miR-1/133 dKO embryonic hearts at E10.5 using Taqman probes. (B–H) Whole mount in situ (WISH) expression analysis of miR-1 in mutant and WT embryos using LNA oligonucleotides. (C–I) Cryosections of WISH embryos. (D, E) Deletion of the miR-1-2/133a-1 cluster uncovers expression of miR-1-1. (F, G) Deletion of the miR-1-1/133a-2 cluster uncovers expression of miR-1-2. (H, I) Deletion of both clusters confirms specificity of miR-1 signals in the heart. Residual staining in somites might be due to cross hybridization with miR-206, which is not expressed in the heart. Scale bar in (B) corresponds 1000 µm in B, D, F, H, scale bar in (C) corresponds to 200 µm in C, E, G, I. at: atrium, ht: heart, ot: outflow tract, s: somites, v. ventricle.

Figure 3. Loss of miR-1/133a leads to aberrant heart development and causes embryonic lethality.

(A, B) Morphological analysis of heart development at E10.5 and (C, D) E11.5 using H&E stained sections. Arrest of heart development at E10.5 and reduced diameter of the compact layer of the ventricular wall in miR-1/133a dKO embryos are clearly visible. (E, F) Immunofluorescence analysis of ANP up-regulation in the compact layer of miR-1/133a dKO embryonic hearts. (F) The thinned compact layer of miR-1/133a dKO hearts expresses high levels of ANP. (G–I) Quantitative evaluation of reduced proliferation of cardiomocytes in miR-1/133a dKO hearts at E9.5 and E10.5 by EdU incorporation (G, H) and pH3 staining (I). (J) Quantitative RT-PCR analysis (Taqman) of increased expression of ANP in miR-1/133a dKO hearts. The scale bar in B corresponds to 50 µm in A–D. The scale bar in F corresponds to 100 µm in E–F.

Figure 4. Deletion of miR-1/133a clusters induces up-regulation of smooth muscle-specific genes leading to multiple transcriptional changes in embryonic hearts.

(A) DNA microarray-based transcriptional analysis of miR-1/133a dKO mutant hearts at E10.5. Genes associated with heart development showing significant (red: p-values) expression changes are shown (up-regulated genes: red, down-regulated genes: green). Putative miR-1/133a target genes are indicated. (B–F) qRT-PCR analysis (Taqman) of increased expression of ANP, myocardin, smooth muscle actin, transgelin, myh11 and BMP-10 in miR-1/133a dKO hearts.

Figure 5. Myocardin is a primary target of miR-1 in the embryonic heart.

(A, A′) Western blot analysis of increased myocardin expression in miR-1/133a dKO embryonic hearts at E10.5 compared to WT. (B, B′) No increase of SRF protein expression in miR-1/133a dKO embryonic hearts at E10.5 compared to WT. (C, D) Putative miR-1 (C) and mir-133a (D) WT and mutant binding sites located in the 3′ UTRs of myocardin and Kcnmb1 mRNAs were cloned into luciferase reporter vectors. (E, F) miR-1 (E) and miR-133a (F) mediated suppression of luciferase activity via WT but not mutant miRNA binding sites located in myocardin (E) and (F) Kcnmb1 mRNAs. Embryonic cardiomyocytes were isolated from embryonic hearts (E11.5-13.5) (G, H, H′). Transfection of miR-1 or of scrambled control (scr) into embryonic cardiomyocytes confirms miR-1 mediated repression of endogenous myocardin transcripts (qRT-PCR; G) and of myocardin protein (Western Blot; H, H′). (I, J, J′) Transfection of miR-133a into embryonic cardiomyocytes confirms miR-133a mediated repression of Kcnmb1 mRNA (qRT-PCR; I) and Kcnmb1 protein (Western Blot; J, J′).

Figure 6. Expression of the miR-1 target myocardin induces smooth muscle cell-like morphology in NIH3T3 cells.

(A) An immunofluorescence staining for myocardin is shown. Transfected cells are labeled by EGFP-fluorescence. The scale bar corresponds to 50 µm. (B–E) Quantitative RT-PCR expression analysis of myocardin (B), smooth muscle actin (C), Kcnmb1 (D), and ANP (E) in transfected NIH3T3 cells using Taqman probes.

Figure 7. Transgenic overexpression of myocardin in the embryonic heart recapitulates the miR-1/133a phenotype.

(A, B) Immunofluorescence analysis of myocardin expression in myocardin transgenic and control embryonic hearts at E10.5. (C, D) Morphological analysis of myocardin transgenic and control embryonic hearts at E10.5 using H&E stained sections. The reduced diameter of the compact layer of myocardin trangenic embryos is clearly visible. (E, F) Immunofluorescence staining for the cardiomyocyte marker MyHC and the proliferation marker phospho-histone 3 (pH3) in myocardin transgenic and WT hearts. (G, H) Immunofluorescence staining of increased ANP expression in myocardin trangenic hearts. (I, J) Immunofluorescence staining of increased smooth muscle actin expression in myocardin transgenic hearts. The scale bar in (B) corresponds to 100 µm in (B, F, H, J), the scale bar in (D) corresponds to 50 µm. (K) Quantitative evaluation of proliferating cardiomyocytes in myocardin transgenic and WT hearts. (L–N) Quantitative RT-PCR analysis of increased smooth muscle actin (Acta2) (L), Myh11 (M), and Erbb4 (N) in myocardin transgenic compared to WT hearts. (O) Quantitative RT-PCR analysis of increased expression of miR-1 and miR-133a in myocardin transgenic E10.5 embryonic hearts. (P) Model illustrating the negative feedback loop controlling expression of miR-1/133a, myocardin and Kcnmb1.

Figure 8. Increased expression of pri-miR-1/133a in Myocardin overexpressing embryos and analysis of the interaction of myocardin with SRF-binding sequences in miR-1-1/133a-2 and miR-1-2/133a-1 promoters.

(A) qRT-PCR expression analysis of pri-miR1-1, pri-miR1-2, pri-miR133-a1 and pri-miR133a-2 in Myocardin overexpressing embryonic hearts. Overexpression of myocardin leads to up-regulation of miR-1 and miR-133a. Taqman probes specific for individual pri-miRNAs representing primary unprocessed transcripts of either the miR-1-1/133a-2 or the miR-1-2/133a-1 gene were used for amplification. (B) Schematic representation of the location of SRF-binding sites and control sequences in miR-1-1/133a-2 and miR-1-2/133a-1 clusters. (C) Chromatin immunoprecipitation using anti-myocardin antibodies demonstrates binding of myocardin to an SRF-site (bs) 5′ of the miR-1-2/133a-1 cluster but not to a SRF-site in the miR-1-1/133a-2 cluster. Sequences within respective clusters not carrying CArG motifs (con) were used as controls. (D) Myocardin ChIP qRT-PCR products analyzed by gel electrophoresis. Results of PCRs for input, myocardin ChIP and IgG control ChIP are shown.