Genes of the cGMP-PKG-Ca2+ signaling pathway are alternatively spliced in cardiomyopathy: Role of RBFOX2 - PubMed (original) (raw)

Genes of the cGMP-PKG-Ca2+ signaling pathway are alternatively spliced in cardiomyopathy: Role of RBFOX2

Xianxiu Wan et al. Biochim Biophys Acta Mol Basis Dis. 2020.

Abstract

Aberrations in the cGMP-PKG-Ca2+ pathway are implicated in cardiovascular complications of diverse etiologies, though involved molecular mechanisms are not understood. We performed RNA-Seq analysis to profile global changes in gene expression and exon splicing in Chagas disease (ChD) murine myocardium. Ingenuity-Pathway-Analysis of transcriptome dataset identified 26 differentially expressed genes associated with increased mobilization and cellular levels of Ca2+ in ChD hearts. Mixture-of-isoforms and Enrichr KEGG pathway analyses of the RNA-Seq datasets from ChD (this study) and diabetic (previous study) murine hearts identified alternative splicing (AS) in eleven genes (Arhgef10, Atp2b1, Atp2a3, Cacna1c, Itpr1, Mef2a, Mef2d, Pde2a, Plcb1, Plcb4, and Ppp1r12a) of the cGMP-PKG-Ca2+ pathway in diseased hearts. AS of these genes was validated by an exon exclusion-inclusion assay. Further, Arhgef10, Atp2b1, Mef2a, Mef2d, Plcb1, and Ppp1r12a genes consisted RBFOX2 (RNA-binding protein) binding-site clusters, determined by analyzing the RBFOX2 CLIP-Seq dataset. H9c2 rat heart cells transfected with Rbfox2 (vs. scrambled) siRNA confirmed that expression of Rbfox2 is essential for proper exon splicing of genes of the cGMP-PKG-Ca2+ pathway. We conclude that changes in gene expression may influence the Ca2+ mobilization pathway in ChD, and AS impacts the genes involved in cGMP/PKG/Ca2+ signaling pathway in ChD and diabetes. Our findings suggest that ChD patients with diabetes may be at increased risk of cardiomyopathy and heart failure and provide novel ways to restore cGMP-PKG regulated signaling networks via correcting splicing patterns of key factors using oligonucleotide-based therapies for the treatment of cardiovascular complications.

Keywords: Alternative splicing; Calcium homeostasis; Cardiomyopathy; Chagas; Diabetes; Protein kinase G; RNA-binding protein RBFOX2.

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Conflict of interest statement

Competing interests: All authors declare that they have no financial or other competing interests.

Figures

Figure 1:. Global changes in gene expression and alternative splicing in chronic Chagas disease mouse hearts.

C57BL/6 female mice were infected with Trypanosoma cruzi (10,000 parasites per mouse). Mice were euthanized at 150 days’ post-infection corresponding to chronic Chagas disease (ChD) phase. Total RNA was isolated from heart tissues of normal and ChD mice (n ≥ 3 mice per group) and high throughput sequencing was performed. After normalizing, datasets were analyzed to identify the global changes in gene expression and alternative splicing (AS) of genes as described in Materials and Methods. (A) Venn diagram shows global changes in gene expression in ChD (vs. normal controls) mice (fold change |≥ 1.2|, p value ≤ 0.01, up regulated: 753 genes, down regulated: 30 genes. The transcriptome data are presented in Table S2. (B) Ingenuity Pathway Analysis of the differential transcriptome dataset was performed to develop the network of Ca2+ mobilization and cellular homeostasis in ChD (vs. control) murine hearts. In the network, the intensity of pink/red and green colors show the extent of increase and decrease in gene expression, respectively, in ChD vs. control murine hearts. Brownish orange node/lines show predicted activation of the pathway. Gray and yellow lines are used when putative effect is not completely understood. (C) Venn diagram shows the distribution of cassette exon exclusion (74/210, 35%) and exon inclusion (136/210, 65%) in ChD (vs. control) mouse hearts (p<0.01). The list of genes that undergo AS in ChD (vs. control) hearts is presented in Table S3. (D) Top significant gene ontology and pathway analysis of alternatively spliced genes in the myocardium of ChD (vs. control) mice. Enrichr server and KEGG 2019 mouse pathway analysis were used to categorize AS genes based on their roles in signaling pathways.

Figure 2:. Alternative splicing of genes involved in the cGMP-PKG-Ca2+ signaling pathway(s) in ChD mice.

Shown are the representative genome browser images of AS of genes of the cGMP-PKG-Ca2+ pathway in ChD mice. Data show annotation of the findings based on RNA-Seq reads in ChD murine hearts in comparison to that noted in control mice. (A) Atp2b1 exon 5, (B) Atp2b1 exon 21, (C) Mef2a exon 9, (D) Atp2a3 exon 21, (E) Arhgef10 Exon 9, and (F) Plcb1 exon 32. In each panel, data from control mice are presented below the data from ChD murine hearts.

Figure 3:. AS pattern of genes involved in the cGMP-PKG-Ca2+ pathway in diabetic mouse heart.

Shown are representative genome browser images depicting alternative splicing of (A) Cacna1c exon 21 and 22, (B) Itpr1 exon 15, (C) ppp1r12a exon 13, (D) Pde2a exon 2, (E) Plcb4 exon 13, and (F) Mef2d exon 9 in DM murine hearts. These genes were identified to be alternatively spliced based on MISO analysis of the RNA-Seq dataset in DM mouse left ventricles (vs. controls, n ≥ 3 mice per group). The RNA-Seq dataset is available at [29].

Figure 4:. Validation of differential AS of genes of the cGMP-PKG-Ca2+ pathway in ChD mice.

Total RNA was isolated from heart tissue of normal and ChD mice. Semi-quantitative RT-PCR was performed as described in Materials and Methods to examine the inclusion and exclusion of specified exons. (A) Atp2b1 exon 21, (B) Atp2b1 exon 5, (C) Atp2a3 exon 21, (D) Mef2a exon 9, (E) Pde2a exon 2, (F) Arhgef10 exon 9, and (G) Plcb1 exon 32. The representative images of AS gels are shown in top panels (+ E = Exon inclusion and − E = Exon exclusion). The bottom panels show the percentage changes in inclusion of specified exons. Data in bar graphs are representative of two independent experiments (n = 3 mice per group per experiment), and are presented as mean value ± SD. The unpaired t test was used to calculate statistically significant differences between two sample groups, and presented as *p ≤ 0.05, **p ≤ 0.01, or ***p ≤ 0.001 (ChD vs. normal).

Figure 5.. Validation of differential alternative splicing of cGMP-PKG-Ca2+ pathway genes in diabetic mice.

Total RNA was obtained from heart tissue of DM and normal control mice. Semi-quantitative RT-PCR was performed to examine the inclusion and exclusion of specified exons. (A) exon 22 in Cacna1c, (B) exon 2 in Pde2a, (C) exon 9 in Mef2d, and (D) exon 13 in Ppp1r12a. The representative images of AS gels are shown in top panels (+ E = Exon inclusion and − E = Exon exclusion). The bottom panels show the percentage changes in inclusion of specified exons and are representative of two independent experiments (n=3 mice per group per experiment). Data are presented as mean value ± SD. The unpaired t test was used to calculate statistically significant differences between two sample groups, and plotted as *p ≤ 0.05, **p ≤ 0.01, or *** p ≤ 0.001 (diabetic vs. normal controls).

Figure 6.. Presence of RBFOX2-binding sites in genes of the cGMP-PKG-Ca2+ signaling pathway.

Publicly available human CLIP-Seq dataset were used to identify the RBFOX2-binding motifs within genes of the cGMP-PKG-Ca2+ signaling pathway. Shown are the genome browser images of genes of the cGMP-PKG-Ca2+ pathway that consisted RBFOX2-binding sites. These included (A) Arhgef10, (B & C) Atp2b1, (D) Mef2a, (E) Mef2d, (F) Plcb1, and (G) Ppp1r12a. The RBFOX2-binding clusters are shown in blue dots (positive strand) and red dots (negative strand). The specific exons that were identified to be mis-spliced in each gene are marked by a red box and exon number.

Figure 7.. RBFOX2 regulates AS in genes of the cGMP-PKG-Ca2+ signaling pathway.

H9c2 cells were transfected with Rbfox2 siRNA (targets exon 6) or scrambled control siRNA as described in Materials and Methods. (A) Total RNA was isolated from transfected cells, and Rbfox2 and Gapdh (control) mRNA levels were determined by real time RT-qPCR. Bar graph shows Rbfox2 mRNA level, normalized to Gapdh mRNA level, in transfected cells. (B) Total protein lysates from siRNA-treated H9c2 cells were analyzed by Western blotting by using an antibody against RBFOX2 to determine the depletion efficiency of Rbfox2 siRNA. Western blotting for GAPDH was performed to confirm equal protein loading. (C-E) Total RNA was isolated from H9c2 cells transfected with scrambled (control) or _Rbfox2_-specific siRNAs, and exclusion/inclusion assay was performed as described in Materials and Methods to determine the RBFOX2-dependent inclusion of specific exons. Shown are representative images (top panels) for alternative splicing in (C) Atp2b1 exon 21, (D) Mef2a exon 9, and (E) Atp2a3 exons 20–21 and exons 19–20 (+ E and −E indicate the inclusion and exclusion of the specific exon, respectively). The bar graphs below the gel images show the percentage changes in inclusion of specified exons. Data in bar graphs are representative of three independent experiments (duplicate observations per experiment per sample), and presented as mean value ± SD. The unpaired t test was used to calculate statistically significant differences between two sample groups and presented as **p < 0.01 and ***p < 0.001 (Rbfox2 siRNA vs. control siRNA).

Figure 8.. The genes within the cGMP-PKG-Ca2+ signaling pathway altered via alternative splicing in the myocardium of DM and ChD mice.

Schematic representation of processes and cGMP-PKG-Ca2+ signaling cascades affected via AS in the heart under ChD and diabetic conditions are shown. The genes that were identified to be alternatively spliced and encode for proteins involved in Ca2+ flux, Ca2+ signaling and contractile activity are shown in bold/italics font. Genes of the signaling cascade identified to be affected via alternative splicing under diabetic conditions are labeled in red, in ChD hearts in blue, and common in both diseases in yellow color. Please see Table 1 for gene/protein names, molecular functional details, and summarized results. Briefly, in the heart, cGMP is activated by nitric oxide and natriuretic peptides and its downstream effects are mediated by cGMP-dependent protein kinase (PKG). PKG phosphorylates several proteins including PLC-beta and plays an essential role in regulation of Ca2+ signaling and contraction and relaxation of cardiac myocytes. For example, PKG/PLC-β influence the function of plasma membrane Ca2+ ATPase (PMCA1), L-type Ca2+ channel (CACNA1C), Inositol trisphosphate receptor (IP3R), and SERCA3 that together determine the intracellular calcium homeostasis during muscle excitation, contraction and relaxation. Genes encoding for calcium sensing Rho guanine nucleotide exchange factor 10 (Arhgef10) and myosin phosphatase (MyPT) that regulate actin-myosin interaction were also impacted by alternative splicing in ChD and/or DM heart. Phosphodiesterases (PDE2, PDE3) catabolize the cGMP/cAMP and inhibit PKG activation. We have found that AS changes in the genes of the cGMP-PKG-Ca2+ signaling pathway in ChD and DM hearts. Atp2b1, and Mef2a were similarly mis-spliced in both ChD and DM hearts and were regulated by RBFOX2. Cacna1c, Itpr, Ppp1r12a were differentially spliced in the heart of diabetic mice; and Arhgef10 and Atp2a3 were differentially spliced in the heart of ChD mice. The AS events can impact the function of the target genes [20, 21] and T. cruzi infection is linked to increased risk of DM in Chagas patients (reviewed in [44]). We propose that DM can potentially exacerbate the AS-induced dysregulation of cGMP-PKG-Ca2+ pathway and the severity of cardiomyopathy and heart failure in Chagas disease.

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Genes of the cGMP-PKG-Ca2+ signaling pathway are alternatively spliced in cardiomyopathy: Role of RBFOX2 - PubMed (original) (raw)