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. 2014 Apr 22:5:3603.
doi: 10.1038/ncomms4603.

Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development

Jimena Giudice  1 , Zheng Xia  2 , Eric T Wang  3 , Marissa A Scavuzzo  1 , Amanda J Ward  4 , Auinash Kalsotra  5 , Wei Wang  6 , Xander H T Wehrens  7 , Christopher B Burge  8 , Wei Li  2 , Thomas A Cooper  9
Affiliations

Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development

Jimena Giudice et al. Nat Commun. .

Abstract

During postnatal development the heart undergoes a rapid and dramatic transition to adult function through transcriptional and post-transcriptional mechanisms, including alternative splicing (AS). Here we perform deep RNA-sequencing on RNA from cardiomyocytes and cardiac fibroblasts to conduct a high-resolution analysis of transcriptome changes during postnatal mouse heart development. We reveal extensive changes in gene expression and AS that occur primarily between postnatal days 1 and 28. Cardiomyocytes and cardiac fibroblasts show reciprocal regulation of gene expression reflecting differences in proliferative capacity, cell adhesion functions and mitochondrial metabolism. We further demonstrate that AS plays a role in vesicular trafficking and membrane organization. These AS transitions are enriched among targets of two RNA-binding proteins, Celf1 and Mbnl1, which undergo developmentally regulated changes in expression. Vesicular trafficking genes affected by AS during normal development (when Celf1 is downregulated) show a reversion to neonatal splicing patterns after Celf1 re-expression in adults. Short-term Celf1 induction in adult animals results in disrupted transverse tubule organization and calcium handling. These results identify potential roles for AS in multiple aspects of postnatal heart maturation, including vesicular trafficking and intracellular membrane dynamics.

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Figures

Figure 1
Figure 1. Extensive gene expression and RNA processing changes occur during heart development
a–b. Differentially expressed genes (a) and alternative RNA processing between E17-adult (b). c. AS events categorized by pattern. d. Proportion of skipping/inclusion of the AS regions. AS: alternative splicing.
Figure 2
Figure 2. Gene expression regulation occurs primarily before PN28
a. GO analysis (KEGG-pathways) of down- and up-regulated genes between E17-adult (ventricles). b. Differentially expressed genes between E17-PN10, PN1–PN28, and PN28–PN90 (ventricles). c. Significance of KEGG-pathways (−log p) were plotted against time windows for specific categories. d. Venn diagrams with developmentally regulated genes and those regulated after CELF1 re-expression on adult mice (RNA-seq data). GO analysis of the intersected genes (summary). ECM: extracellular-matrix.
Figure 3
Figure 3. Reciprocal gene expression transitions between CM and CF
a. Isolated adult ventricular CM and CF stained with anti-vimentin or anti-alpha-actinin. Scale bars: 10 µm. b. RT-PCR assays of CF (Vim, Ddr2) and CM (Tnnt2, Nkx2.5) markers on RNA from adult ventricles, CM and CF. Experiments were repeated with at least 3 independent samples. c. CF enrichment (CFE) for CF-enriched transcripts (blue) and CM enrichment (CME) for CM-enriched transcripts (red) calculated from RNA-seq data. d. Postnatal gene expression transitions in CM and CF (PN1–3 versus adult) by RNA-seq. e. GO analysis of down- and up-regulated genes in CM and CF (Supplementary Table 2). f. Postnatal expression of three metabolic genes (RNA-seq data) showing reciprocal CM-CF regulation. bp: base pairs. FPKM: fragments per kilobase per million mapped.
Figure 4
Figure 4. Extensive postnatal AS transitions occur primarily without changes in gene expression
a. RNA-seq data (UCSC browser) and RT-PCR assays (n=2 biological replicates) for AS events developmentally regulated (+ex: exon included; −ex: exon skipped). See Supplementary Fig. 3, and Supplementary Table 3. b. Correlation between RNA-seq and RT-PCR ΔPSI values. c. Genes undergoing postnatal AS transitions (|ΔPSI|≥20%) were intersected with differentially expressed (DE) genes in ventricles (E17-adult). d. AS transitions (|ΔPSI|≥20%; E17-adult) were plotted comparing their change between E17-PN28 (red) and PN28-adult (green). Diagonal line: no difference between time points. Dots above/below the diagonal: increased/decreased inclusion. AS: alternative splicing. bp: base pairs. PSI: percent spliced in.
Figure 5
Figure 5. Postnatal gene expression and AS transitions in CM and CF
a. AS transitions (|ΔPSI|≥20%) and gene expression changes in CM and CF. b. Genes developmentally regulated by splicing (|ΔPSI|≥20%) in CM and CF were intersected with differentially expressed (DE) genes (PN1–3 versus PN28–30). c. Postnatal AS transitions (between PN1–3 and adult) specifically in CM, CF or both. d. Analysis of the 67 events regulated in CF and CM. e. Three postnatal AS transitions occurring in opposite directions in CM and CF. AS: alternative splicing. PSI: percent spliced in.
Figure 6
Figure 6. Vesicular trafficking genes are regulated by AS during development
a. GO analysis on AS genes (|ΔPSI|≥20%) in three time windows: E17-PN1, PN1–PN28, PN28-adult (ventricles). b. KEGG-pathway analysis on AS genes detected by RNA-seq during wild type CM development (|ΔPSI|≥20%), in adult CELF1-expressing hearts (CELF1 oe) and adult Mbnl1∆E3/∆E3 hearts (|ΔPSI|≥10%). c. AS transitions (|ΔPSI|≥20%) during CM development (between PN1–3 and adult): regulation by Mbnl1 and/or CELF1. d. The 57 events regulated by Mbnl1 and CELF1 were analyzed in terms of antagonistic/not-antagonistic effects (Supplementary Table 4). AS: alternative splicing.
Figure 7
Figure 7. Vesicular trafficking genes revert to neonatal splicing patterns after CELF1 re-expression in adult hearts
a. RT-PCR (ventricles) of vesicular trafficking AS events during development and in CELF1-expressing hearts. Bar graphs: mean±s.e.m (n=2 biological replicates). b. Correlation between RNA-seq and RT-PCR data in vesicular trafficking genes (PN1-adult). c. Correlation between developmental transitions in vesicular trafficking genes and reversion after CELF1 re-expression in adults (RT-PCR). d. Network of vesicular trafficking genes developmentally regulated by splicing and responsive to CELF1 (black circles). Grey circles: linking-genes. bp: base pairs. CELF1 oe: TRECUGBP1/MHC animals. MHC: control animals. PSI: percent spliced in.
Figure 8
Figure 8. T-tubule disorganization after CELF1 re-expression in adults
a. Western blot analysis from adult hearts after CELF1 induction. b. Fractional shortening was measured in MHC (n=6 animals) and TRECUGBP1/MHC mice (n=3 animals) given doxycycline (four days). Results are expressed as the mean±s.e.m. c. Confocal imaging of T-tubules on living CM from MHC (#1–2; n= 2 animals) and CELF1-expressing (#3–4, n= 2 animals) mice. Scale bars: 10 µm. Third row: fluorescence plot over the line (first row). d–g. T-tubule and calcium spark analysis: normalized T-tubule power (d) (n=13 cells for MHC animals, n=16 cells for TRCUGBP1/MHC animals), T-tubule area (e) (n=12 cells for MHC animals, n=16 cells for TRCUGBP1/MHC animals), T-tubule irregularity (f) (n=13 cells for MHC animals, n=16 cells for TRCUGBP1/MHC animals), calcium spark frequency (g) (n=11 cells for each genotype). Asterisks show statistical differences (p≤0.05). Results are expressed as the mean±s.e.m. CELF1 oe: TRECUGBP1/MHC animals. MHC: controls. FFT: Fast Fourier Transform T-tubules: transverse tubules.
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References

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