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. Author manuscript; available in PMC: 2016 Jun 9.
Published in final edited form as: Cell Rep. 2016 May 26;15(10):2200–2213. doi: 10.1016/j.celrep.2016.05.002

Dysregulation of RBFOX2 is an early event in cardiac pathogenesis of diabetes

Curtis A Nutter 1, Elizabeth A Jaworski 1, Sunil K Verma 1, Vaibhav Deshmukh 2, Qiongling Wang 2,3, Olga B Botvinnik 4, Mario J Lozano 5, Ismail J Abass 6, Talha Ijaz 1, Allan R Brasier 6,7, Nisha J Garg 8,9,11, Xander HT Wehrens 2,3,12,13, Gene W Yeo 4,14, Muge N Kuyumcu-Martinez 1,7,10
PMCID: PMC4899142  NIHMSID: NIHMS786895  PMID: 27239029

Abstract

Alternative splicing (AS) defects that adversely affect gene expression and function have been identified in diabetic hearts; however, the mechanisms responsible are largely unknown. Here we show that the RNA-binding protein RBFOX2 contributes to transcriptome changes under diabetic conditions. RBFOX2 controls AS of genes with important roles in heart function relevant to diabetic cardiomyopathy. RBFOX2 protein levels are elevated in diabetic hearts despite low RBFOX2 AS activity. A dominant negative (DN) isoform of RBFOX2 that blocks RBFOX2-mediated AS is generated in diabetic hearts. DN RBFOX2 interacts with wild type RBFOX2 and ectopic expression of DN RBFOX2 inhibits AS of RBFOX2 targets. Notably, DN RBFOX2 expression is specific to diabetes and occurs at early stages before cardiomyopathy symptoms appear. Importantly, DN RBFOX2 expression impairs intracellular calcium release in cardiomyocytes. Our results demonstrate that RBFOX2 dysregulation by DN RBFOX2 is an early pathogenic event in diabetic hearts.

INTRODUCTION

Diabetic patients have a two to five fold increased risk of developing cardiovascular diseases, which include atherosclerosis, hypertensive heart disease and diabetic cardiomyopathy (Trachanas et al., 2014). Cardiovascular complications are the number one cause of mortality and morbidity among diabetic patients (Ferrannini and Cushman, 2012; Harcourt et al., 2013). Diabetic cardiomyopathy is particularly difficult to manage due to its asymptomatic nature and its ability to progress into heart failure (Aneja et al., 2008; Asghar et al., 2009; Boudina and Abel, 2010). The molecular mechanisms responsible for diabetic cardiomyopathy are poorly understood, though chronic activation of protein kinase C (PKC) signaling has been implicated in the pathogenesis (Geraldes and King, 2010; Inoguchi et al., 1992; Liu et al., 2009). Inhibition of PKC activity improves cardiac complications in animal models of diabetes (Connelly et al., 2009; Liu et al., 2012) by mechanisms that are not well defined.

We have previously shown that PKC activation leads to reactivation of fetal alternative splicing (AS) programs in diabetic hearts partly via phosphorylation of the RNA binding protein RBFOX2 (Verma et al., 2013). Consistent with this finding, we have shown that RBFOX2 protein levels are elevated in diabetic mouse hearts (Verma et al., 2013), suggesting a role for RBFOX2 in the pathogenesis of diabetes. In this study, we wanted to further investigate how RBFOX2 regulation is modulated in diabetic hearts and whether changes in RBFOX2 contribute to aberrant AS in diabetes.

RBFOX2 directly binds to the consensus (U)GCAUG motif in target pre-mRNAs and regulates AS in a position dependent manner. Binding to the downstream intron induces alternative exon inclusion, but binding to the upstream intron triggers exon exclusion (Huang et al., 2012; Sun et al., 2012; Yeo et al., 2009). Genome-wide RBFOX2 target pre-mRNAs have been identified using cross-linking immunoprecipitation followed by RNA sequencing (CLIP-seq) analysis in human and mouse embryonic stem (ES) cells, and human 293T cells (Jangi et al., 2014; Lovci et al., 2013; Yeo et al., 2009). RBFOX2 controls the expression of other RNA binding proteins via AS induced nonsense mediated mRNA decay (Jangi et al., 2014; Yeo et al., 2009). Therefore, RBFOX2 affects gene expression at multiple levels, either directly or indirectly via modulating other RNA binding proteins.

RBFOX2 is implicated in several cellular processes such as apoptosis, proliferation and epithelial-mesenchymal-transition (Baraniak et al., 2006; Gehman et al., 2012; Venables et al., 2013a). RBFOX2 is necessary for the survival of ES cells (Yeo et al., 2009) and brain development (Gehman et al., 2012). It also plays an important role in differentiation of pluripotent stem cells (Venables et al., 2013b). A recent study has shown that RBFOX2 is implicated in pressure overload induced heart failure (Wei et al., 2015). However, its role in diabetes is unknown.

In this study, we show that 704 alternative exons differentially spliced in diabetic hearts are RBFOX2 targets. We find that RBFOX2 regulates AS of transcripts with validated RBFOX2 binding sites. Results indicate that AS changes in RBFOX2 targets are due to low RBFOX2 levels or activity in diabetic hearts. However, RBFOX2 protein levels are paradoxically elevated in Type 2 diabetic (T2D) human patients’ hearts. Our results reveal that the mechanism for low RBFOX2 dependent AS in diabetes is due to the increased expression of a dominant negative (DN) isoform of RBFOX2, that possesses an inhibitory AS function. DN RBFOX2 is expressed early in response to high glucose levels in diabetic mice and is specific to diabetic complications in the heart. We show that DN RBFOX2 generation in diabetic hearts is dependent on upregulation of RBFOX2 protein with intact RNA binding capability. Overexpression of DN RBFOX2 inhibits AS of RBFOX2 targets and impairs intracellular calcium handling in cardiomyocytes. Finally, our data indicate that DN RBFOX2 binds to WT RBFOX2 as a likely mechanism to repress RBFOX2 splicing function. In sum, our results demonstrate that dysregulation of RBFOX2 activity triggers AS abnormalities and contributes to diabetic complications in the heart.

RESULTS

RBFOX2 binding sites are enriched in pre-mRNAs that undergo alternative splicing changes in diabetic hearts

We have previously identified genome-wide AS changes in Type 1 diabetic (T1D) mouse hearts with a corresponding increase in RBFOX2 protein levels (Verma et al., 2013). Therefore, we hypothesized that RBFOX2 is involved in AS dysregulation in diabetic hearts. Since genome-wide RBFOX2 binding sites were determined using human ES cells by CLIP-seq (Yeo et al., 2009), we wanted to determine whether transcripts mis-spliced in diabetic mouse hearts have RBFOX2 binding sites. To this end, we mapped the human RBFOX2 binding sites to the corresponding mouse genomic coordinates. 73% of total pre-mRNAs mis-spliced in diabetic hearts displayed RBFOX2 binding clusters within alternative exons and introns (adjacent 500nt) (Fig. 1A). For controls, transcripts with similar expression levels, but that did not display splicing changes in diabetic hearts were used. The majority (1023 out of 1417) of RBFOX2 binding clusters were located within introns (Fig. 1B) consistent with the role of RBFOX2 in AS regulation. RBFOX2 binding clusters within intronic regions of mis-spliced transcripts were statistically significant when compared to binding clusters within control transcripts (Figs. 1B and S1). To determine the functions of the affected RBFOX2 target genes in diabetic hearts, we performed gene ontology analysis using the NIAID Database for Annotation, Visualization and Integrated Discovery (DAVID) server using the whole genome as a background control. RBFOX2 target genes dysregulated in diabetic hearts have important biological functions in macromolecular metabolism, apoptosis, vesicular transport, calcium homeostasis and cell cycle (Fig. 1C).

Figure 1. 73% of transcripts aberrantly spliced in diabetic mouse hearts have RBFOX2 binding sites.

Figure 1

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A) Pie chart of RBFOX2 binding clusters in 967 transcripts that are mis-spliced in Type 1 diabetic mouse (T1D) hearts. Alternative splicing (AS) events affected in diabetic hearts with at least one CLIP peak at a Bayes factor ≥ 1 was designated as “significant”, <1 as “not significant”. B) RBFOX2 binding site distribution among 1417 RBFOX2 binding clusters within 704 pre-mRNAs aberrantly spliced in diabetic hearts. (See Fig. S1) C) Top gene ontology categories for RBFOX2 regulated genes affected in diabetic hearts. DAVID server was used to categorize genes based on biological or molecular function. P values represent statistical significance when compared to the whole genome, which was used as a background control. D) Effect of diabetes induced AS changes on gene expression and function. E) Schematic of intronic RBFOX2 binding sites obtained from human ES cell RBFOX2 CLIP-seq data with CLIP clusters and consensus RBFOX2 binding site motif (U)GCAUG on target pre-mRNAs mis-spliced in diabetic hearts.

RBFOX2 regulates AS events mis-spliced in diabetic hearts

Next, we wanted to determine whether RBFOX2 regulates AS of the pre-mRNAs with RBFOX2 binding sites. We validated 22 out of 24 tested AS events identified by RNA-seq in T1D diabetic mouse hearts and 8 out of 11 tested AS events in T2D human hearts (data not shown). We further focused on a few AS events based on the criteria that: a) there is a significant AS change (>20%) in diabetic hearts, b) AS removes a functional protein domain (Fig. 1D), c) the genes play important roles in heart function and disease (Fig. 1D): MTMR3 (Lorenzo et al., 2006; Taguchi-Atarashi et al., 2010), FXR1 (Blackwell et al., 2010; Kirkpatrick et al., 1999; Mientjes et al., 2004; Whitman et al., 2011), MEF2A (Mora and Pessin, 2000; Naya et al., 2002; Wang et al., 2003; Weng et al., 2005; Zhu et al., 2005), and PBX3 (Arrington et al., 2012; Monica et al., 1991; Stankunas et al., 2008), and d) pre-mRNAs display distinct RBFOX2 binding sites in upstream or downstream intronic regions (Fig. 1E). By examining the AS of these specific exons with intronic RBFOX2 binding sites (Fig. 1E), we would be able to assess if increased RBFOX2 levels correspond to diabetes induced AS changes in the heart.

We tested AS of these four pre-mRNAs (MTMR3 exon 16, FXR1 exon 15+16, MEF2A exon 9, and PBX3 exon 7) in T2D human hearts as well as in obese and hypertensive mouse models using qRT-PCR. Obesity and hypertension are common among diabetic patients, so we wanted to determine whether AS changes are induced by one of these factors under diabetic conditions. MTMR3 exon 16, FXR1 exon 15+16, MEF2A exon 9, and PBX3 exon 7 were skipped more in T2D human patients’ hearts compared to non-diabetic control human hearts, similar to the results in T1D mice [Fig. 2A and (Verma et al., 2013)]. However, none of these AS events changed in obese, hypertensive or obese-hypertensive mice (Figs. 2B and S2). Mice infused with angiotensin II had hypertrophic hearts and obese mice were two times heavier than the controls (Figs. 2C and 2D). These data suggest that diabetes induced AS changes are independent of hypertension or obesity status. We tested these AS events in mice with advanced cardiomyopathy and heart failure in order to determine whether AS changes are due to non-specific effects from failing hearts. None of the AS events was altered in chagasic mice with chronic cardiomyopathy and heart failure (Fig. 2E), indicating that AS changes were not due to sick hearts. Importantly, the alternative exons with downstream RBFOX2 binding sites were excluded in diabetic patients suggesting low RBFOX2 levels or AS activity under diabetic conditions.

Figure 2. Alternative splicing of RBFOX2 targets is disrupted in diabetic hearts independent of hypertension and obesity.

Figure 2

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A) Percent inclusion of MTMR3 exon 16, FXR1 exon 15+16, MEF2A exon 9 and PBX3 exon 7 as determined by quantitative RT-PCR (qRT-PCR) in non-diabetic (control) or Type 2 diabetic (T2D) human left ventricles (n≥3). B) AS analysis of Mtmr3 exon 16 in hypertensive, obese and obese-hypertensive mouse models (See also Fig. S2) (n=3). C) Body weight of mock treated control, angiotensin II treated hypertensive, obese, and angiotensin II treated obese mice. D) Heart to body weight ratio of mock treated control mice or angiotensin II treated hypertensive mice. E) AS analysis in chagasic mice with severe cardiomyopathy and heart failure. Mtmr3 exon 16, Fxr1 exon 15+16, Mef2a exon 9 and Pbx3 exon 7 AS was determined in non-infected (control) or chronically infected chagasic mice with cardiomyopathy (cardiomyopathy) (n≥3). Data represent means ± SD. Statistical significance was calculated using unpaired t-test for two group comparisons or using one-way ANOVA to compare four different groups. P-values are represented as **** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05.

To further investigate if decreased RBFOX2 levels induce the AS patterns of diabetic hearts, we performed RBFOX2 depletion studies in rat heart derived cells (H9c2) and tested endogenous AS events dysregulated in diabetic hearts. We used both siRNA and shRNA to target different regions of Rbfox2 (Fig. 3A). Alternative exons with RBFOX2 binding sites (Mtmr3 exon 16, Fxr1 exon 15+16, and Mef2a exon 9) (Fig. 1E) had less exon inclusion in RBFOX2 depleted cells (Figs. 3B and 3D), similar to that in diabetic hearts (Fig. 3D vs Fig. 2A). As an additional control, we depleted RBFOX2 using an shRNA targeting a different region in the Rbfox2 mRNA (exon 3) (Figs. 3A and 3C) to make sure that changes in AS were due to specific depletion of RBFOX2. Cells expressing Rbfox2 specific shRNA displayed AS patterns similar to those in Rbfox2 siRNA treated cells (Fig. 3E vs Fig. 3D) and in T2D human patients’ hearts (Fig. 2A) indicating that low RBFOX2 levels or activity trigger the AS patterns of diabetes. AS of Pbx3 was unaffected in Rbfox2 siRNA treated cells (Fig. 3D), consistent with its lack of RBFOX2 binding sites (Fig. 1E) but was affected in Rbfox2 shRNA treated cells (Fig. 3E). It is possible that PBX3 could be an indirect downstream target of RBFOX2 because its AS was altered only after prolonged shRNA treatment. These results show that pre-mRNAs with RBFOX2 binding sites are differentially spliced in T2D human hearts, consistent with low RBFOX2 dependent splicing.

Figure 3. RBFOX2 regulates alternative splicing events altered in diabetic hearts.

Figure 3

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A) Schematic of Rbfox2 pre-mRNA (only exons 3 to 7) regions targeted by siRNA and shRNA. Introns were not drawn to scale. B) Total protein lysates from siRNA treated H9c2 cells analyzed by Western blotting (WB) using an antibody (Ab) against RBFOX2 to determine the depletion efficiency of Rbfox2 siRNA. GAPDH WB was performed to confirm even protein loading. C) RBFOX2 protein levels determined in shRNA expressing H9c2 cells by WB using an anti-RBFOX2 Ab. GAPDH WB was used as a loading control. D) AS analysis of Mtmr3 exon 16, Fxr1 exon 15+16, Mef2a exon 9, and Pbx3 exon 7 in H9c2 cells transfected with scrambled (control) or Rbfox2 specific siRNA (targets exon 6) (n≥8). E) AS analysis in H9c2 cells transfected with scrambled (control) or Rbfox2 specific shRNA (targets exon 3) (n≥6). In AS gel figures +E# = Exon # inclusion and −E# = Exon # exclusion. Data represent the means ± SD. The unpaired t-test was used to calculate statistically significant differences between two sample groups from 3 independent experiments. P-values are indicated as **** < 0.0001, *** < 0.001.

High RBFOX2 protein levels in diabetic hearts are consistent with increased expression of a dominant negative isoform of RBFOX2

We have previously shown that RBFOX2 protein levels are elevated in T1D mouse hearts (Verma et al., 2013), which is contradictory to our new findings of low RBFOX2 AS activity in diabetic hearts. We thought that RBFOX2 might be regulated differently in T2D versus T1D. Therefore, we tested RBFOX2 protein levels in T2D human patients’ hearts. Consistent with our data in T1D mice, RBFOX2 protein levels were also elevated in T2D patients’ hearts [(Verma et al., 2013) and Fig. 4A]. To determine whether the increase in RBFOX2 protein levels was due to increased mRNA levels, we performed qRT-PCR using RBFOX2 specific primers in T2D patients’ hearts and age matched non-diabetic controls. There was no significant increase in RBFOX2 mRNA levels in diabetic hearts (Fig. 4B) that could explain high protein levels (Fig. 4A), suggesting that RBFOX2 protein levels are regulated post-transcriptionally.

Figure 4. Dominant negative RBFOX2 is generated in diabetic hearts at early stages.

Figure 4

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A) WB analysis of RBFOX2 protein in non-diabetic control or Type 2 diabetic (T2D) human left ventricles using an anti-RBFOX2 Ab. α-TUBULIN WB was used as a loading control. Fold change in RBFOX2 protein levels were determined after normalizing protein levels to α-TUBULIN, the control averages were set to 1 and patient averages were summarized below. B) Relative RBFOX2 mRNA levels in non-diabetic control and T2D human patients’ hearts, determined by qRT-PCR (n≥5). C) Schematic of dominant negative (DN) RBFOX2 generation via exon 6 exclusion (RRM: RNA recognition motif). Introns were not drawn to scale. D) DN RBFOX2 mRNA levels were determined by RBFOX2 exon 6 exclusion using qRT-PCR from left ventricles of T2D human patients or non-diabetic controls (n≥5). E) DN Rbfox2 expression in hearts from non-diabetic control mice or non-obese diabetic (NOD:T1D) mice (n≥4). F) DN Rbfox2 expression in hearts from mock injected control or streptozotocin induced T1D (STZ:T1D3wks) mice hyperglycemic for 3 weeks (n≥3), G) Cardiac function of mice before (Pre-STZ) or 3 weeks after hyperglycemia (STZ:T1D3wks) was assessed by M-mode echocardiography measuring fractional shortening (%), H) stroke volume (Vol.), I) ventricular internal diameter end at diastole (LVIDd) (mm) (n≥3). J) DN Rbfox2 expression in left ventricles from chagasic mice with severe cardiomyopathy or mock infected controls (n≥3). K) DN Rbfox2 expression pattern in left ventricles of obese, hypertensive and obese/hypertensive mouse left ventricles (n=3). Data represent the means ± SD. Statistical significance between two groups was calculated using the unpaired t-test, and between four groups using one-way ANOVA. P-values are indicated as *** < 0.001, ** < 0.01.

To better understand how RBFOX2 is dysregulated in diabetic hearts, we examined the expression of a dominant negative (DN) RBFOX2 isoform that is capable of repressing the AS activity of RBFOX family of proteins (including RBFOX1, RBFOX2 and RBFOX3) (Damianov and Black, 2010; Nakahata and Kawamoto, 2005). DN RBFOX2 is generated via exclusion of exon 6, which encodes half of the RNA Recognition Motif (RRM) resulting in low RNA binding capability and newly gained RBFOX repressor activity (Damianov and Black, 2010; Nakahata and Kawamoto, 2005) (Fig. 4C). We found that the DN RBFOX2 isoform lacking exon 6 was increased in both T1D mouse models (non-obese diabetic (NOD) and streptozotocin (STZ) induced) as well as in T2D human patients’ hearts (Figs. 4D, 4E, and 4F). To determine whether expression of DN Rbfox2 is an early event in diabetic cardiomyopathy, we examined cardiac function before and after induction of diabetes by STZ using echocardiography. Left ventricle (LV) function, stroke volume, and ventricular internal diameter end at diastole (LVIDd) were unaffected after 3 weeks of hyperglycemia (Figs. 4G, 4H, and 4I). These results show that DN Rbfox2 upregulation and changes in AS of RBFOX2 targets occurred before cardiac complications were detectable in diabetic mice; indicating that DN Rbfox2 expression is an early response to high glucose levels. We also examined DN Rbfox2 expression in other mouse models. DN Rbfox2 mRNA levels were not increased in hypertensive, obese, obese-hypertensive or mice with cardiomyopathy when compared to controls (Figs. 4J and 4K). Consistent with these mouse models lack of increase in DN Rbfox2 expression, AS patterns of RBFOX2 targets were also unaffected (Figs. 4J and 4K vs Figs. 2B, 2E, and S2). These results show that the DN version of RBFOX2 is generated in both T1D mouse and T2D diabetic patients’ hearts as an early event and the expression of DN RBFOX2 is consistent with aberrant AS of RBFOX2 targets in diabetic hearts.

Increased RBFOX2 protein levels with RNA binding capability induce DN Rbfox2 expression

We investigated how DN RBFOX2 is generated in diabetic hearts. RBFOX2 AS activity is controlled such that an increase in RBFOX2 protein levels leads to expression of the DN version via autoregulation of exon 6 exclusion (Damianov and Black, 2010). We tested if DN Rbfox2 is generated due to high levels of RBFOX2 and if RNA binding activity of RBFOX2 is required. We expressed RBFOX2 in H9c2 cells and examined endogenous DN Rbfox2 expression by qRT-PCR. As a control, an RNA binding deficient mutant of RBFOX2 (RBFOX2RRM) was expressed. RBFOX2RRM mutant lacks RNA binding activity due to mutagenesis of two critical phenylalanine residues in the RRM necessary for RNA binding (Auweter et al., 2006; Mayeda et al., 1994; Sun et al., 2012). We found that overexpression of RBFOX2WT (Fig. 5A) capable of binding to RNA increased the expression of the DN Rbfox2 isoform (Fig. 5B). However, RBFOX2RRM mutant (Fig. 5A) did not affect DN expression as robustly as the WT (Fig. 5B), demonstrating that the RNA binding function of RBFOX2 is critical for autoregulation and DN Rbfox2 generation.

Figure 5. Increased RBFOX2 protein with RNA binding capability is important for generation of DN Rbfox2.

Figure 5

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A) WB analysis of RBFOX2 in H9c2 cells expressing empty vector (Control), RBFOX2WT, or RBFOX2RRM (RNA binding deficient mutant). B) Endogenous DN Rbfox2 expression by exclusion of exon 6 in H9c2 cells expressing control, RBFOX2WT or RBFOX2RRM determined by qRT-PCR (n=4 from 3 independent experiments). Data represent the means ± SD. C) RBFOX2 WB in H9c2 cells expressing GFP or increasing levels of GFP-tagged RBFOX2 using anti-GFP or anti-RBFOX2 Ab. Ponceau S staining was used to assess even protein loading. D) AS of Mef2a, Mtmr3 and E) Mbnl2 and expression levels of DN Rbfox2 in H9c2 cells expressing increasing levels of RBFOX2WT-GFP (See Fig. S3). To equalize the amount of DNA per plate to 2 μg, different concentrations of vector DNA were transfected together with RBFOX2WT-GFP. Quantifications were performed using samples from at least four independent experiments (n≥4). Data represent the means ± SE. Statistical significance was calculated using one-way ANOVA to compare three or more different groups. P-values are indicated as **** < 0.0001, *** < 0.001, ** < 0.01 compared to the GFP control (0 μg RBFOX2 DNA). P-values indicated as #### < 0.0001 represents comparisons to the lowest RBFOX2WT-GFP level (0.2 μg RBFOX2 DNA).

Next, we examined if there is a threshold for RBFOX2 protein levels to have an inhibitory splicing effect by generating DN RBFOX2. To test this, we performed a dose response experiment by expressing increasing concentrations of GFP-RBFOX2 but keeping total transfected DNA concentration the same and tested AS of RBFOX2 targets. Expression levels of endogenous DN Rbfox2 were also monitored. As a negative control, we used GFP. Figure 5C shows an exponential increase in GFP-RBFOX2 protein levels determined by WB using both GFP and RBFOX2 antibodies. At the lowest dose of WT RBFOX2, all four RBFOX2 target exons were affected such that Mef2a, Mtmr3, Fxr1 and alternative exons were more included (Fig. 5D and S3). However, as the RBFOX2 dose increased, Mef2a exon 6 inclusion was inhibited (Fig. 5D), exon inclusions of Mtmr3 and Fxr1 were not further increased coincident with upregulation of endogenous DN Rbfox2 (Fig. 5E). Since the endogenous Rbfox2 pre-mRNA level is the limiting factor in generation of DN RBFOX2 in response to high levels of WT RBFOX2, it is possible that only exons very sensitive to changes in RBFOX2 were inhibited and others were prevented at high WT RBFOX2 doses. On the contrary, exon 6 exclusion of Rbfox2 was not inhibited at any concentration of WT RBFOX2 (Fig. 5E) that adversely affected exon inclusions (Figs. 5D and S3). Indeed, Rbfox2 exon 6 was excluded continuously from 92% to 36% as the WT RBFOX2 concentrations increased suggesting that WT RBFOX2 was not effective in inhibiting exon 6 exclusion; thus DN Rbfox2 generation (Fig. 5E). To test if WT RBFOX2 overexpression has any effect on other exon exclusion events; we analyzed Mbnl2 exon 8. At low WT RBFOX2 dose, Mbnl2 exon 8 inclusion decreased from 67% to 42% (Fig. 5E). At higher doses of WT RBFOX2, Mbnl2 exon 8 was 10% further excluded down to 32% (Fig. 5E). While Mbnl2 and Rbfox2 exons continued to be excluded as WT RBFOX2 concentration increased, exon inclusion events were either inhibited or prevented. These results suggest that overexpression of WT RBFOX2 may be ineffective in inhibiting exon exclusion. Overall, aberrant upregulation of RBFOX2 above a threshold becomes ineffective or even inhibitory in regulating RBFOX2 dependent exon inclusion.

Increased RBFOX2 protein levels during myocardial differentiation correlates with upregulation of dominant negative Rbfox2

RBFOX2 protein levels are dynamically regulated during cardiomyocyte differentiation (Verma et al., 2013). To further test if high RBFOX2 protein levels are responsible for DN Rbfox2 induction, we examined DN Rbfox2 expression during cardiomyocyte differentiation. Successful differentiation was confirmed by upregulation of cardiac troponin T (cTNT), using WB (Fig. S4A). In differentiated cells, RBFOX2 protein levels were elevated by 3.6 fold, corresponding to a 40% increase in DN Rbfox2 expression when compared to undifferentiated cells (Figs. S4A and S4B). These results support our findings that when RBFOX2 levels increase over a threshold, it induces DN expression (Figs. 4, 5 and S4).

Dominant negative RBFOX2 interacts with wild type RBFOX2 and inhibits AS of RBFOX2 targets

The next important question was to determine if ectopic expression of DN RBFOX2 inhibits AS similar to that observed in diabetic hearts. For that reason, we expressed FLAG-DN RBFOX2 in H9c2 cells (Fig. 6A) and tested AS of RBFOX2 responsive targets. FLAG-DN RBFOX2 expression induced exon exclusion of targets (Fig. 6B) resembling AS patterns in diabetic hearts (Figs. 6B vs 2A). This result indicates that the DN RBFOX2 upregulation in diabetic hearts impairs RBFOX2 dependent splicing.

Figure 6. Dominant negative RBFOX2 interacts with wild type RBFOX2 and inhibits AS of RBFOX2 regulated splicing.

Figure 6

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A) WB analysis of DN RBFOX2 protein in H9c2 cells expressing empty vector (Control) or FLAG-RBFOX2DN. B) AS analysis of Mtmr3, Fxr1, and Mef2a in H9c2 cells transfected with empty vector (Control) or FLAG-RBFOX2DN (n≥4). C) Interactions of FLAG tagged RBFOX2WT, RBFOX2DN, and RBFOX2RRM (RNA binding deficient mutant) with U1C protein in COS M6 cells determined by FLAG IP followed by WB using FLAG and U1C-specific Abs. D) Interactions of FLAG-RBFOX2DN with GFP-RBFOX2WT in H9c2 cells. Pulled down proteins were analyzed by WB using HRP conjugated anti-FLAG and anti-GFP Abs. Control lysates expressing empty vector was used as a negative control for non-specific protein binding to the FLAG-antibody coated beads (FLAG-IP control). Input lysates represents 1/20 of total protein loaded onto the FLAG-beads. Results are representative of three independent experiments.

To investigate if DN RBFOX2 inhibits splicing via sequestering spliceosome components known to interact with RBFOX2, we overexpressed either FLAG-tagged WT or DN RBFOX2 in COS M6 cells and performed FLAG immunoprecipitation (IP) to assess the binding of RBFOX2 to U1 snRNP. It was previously shown that RBFOX2 binds to U1C to assemble the U1 snRNP complex and promote splicing (Huang et al., 2012). We also tested whether the RNA binding deficient mutant of RBFOX2 (RBFOX2RRM) can interact with U1C. To determine if there is non-specific protein binding to the FLAG-antibody coated beads, we used protein lysates from cells expressing empty vector (control). Loading control shows equal amounts of lysates (input) loaded onto beads coated with similar amounts of FLAG-antibody (IgG) (Figs. 6C and 6D). As expected, WT RBFOX2 pulled down endogenous U1C protein (Fig. 6C). However, neither RBFOX2RRM nor DN RBFOX2 was able to pull down U1C (Fig. 6C), indicating that RBFOX2 mutants lacking RNA binding activity do not interact with U1C. Dominant negative proteins are known to bind to the wild type protein counterpart to inhibit activity. To test whether DN RBFOX2 interacts with WT RBFOX2 to inhibit its splicing activity, we co-expressed GFP-WT RBFOX2 with FLAG-DN RBFOX2 and performed FLAG-IP. GFP-WT RBFOX2 was pulled down together with FLAG-DN RBFOX2 indicating that DN and WT RBFOX2 proteins interact (Fig. 6D). These results suggest that DN RBFOX2 inhibits AS via interacting with WT RBFOX2 not by sequestering U1C.

Dysregulation of RBFOX2 adversely affects cytoskeleton associated gene function and intracellular calcium handling

To determine the consequences of RBFOX2 induced AS defects, we focused on the downstream targets of RBFOX2. Ablation of the RBFOX2 target gene Fxr1 in animal models causes cardiomyopathy, partly by upregulation of the TALIN2 protein, which functions in the assembly of the actin cytoskeleton (Whitman et al., 2011). FXR1 exons 15 and 16, necessary for striated muscle specific translational regulation (Kirkpatrick et al., 1999), are almost completely excluded in diabetic human hearts (Fig. 2A) and in RBFOX2 depleted cells (Figs. 3D and 3E). When we checked TALIN2 protein levels in RBFOX2 depleted cells, TALIN2 protein levels were significantly elevated (Fig. 7A). TALIN2 protein levels were also upregulated in T1D mouse hearts (Fig. 7B). This result shows that low RBFOX2 levels/activity correlates with increased TALIN2 protein levels in diabetic mouse hearts.

Figure 7. Consequences of aberrant AS of RBFOX2 targets.

Figure 7

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A) WB analysis of TALIN2 (TLN2), RBFOX2, and GAPDH in H9c2 cells treated with scrambled siRNA (control) or Rbfox2 specific siRNA. Fold changes in TLN2 protein levels were determined after normalizing to GAPDH protein levels (n≥9 from 3 independent experiments). B) Representative WB analysis of TLN2 protein levels in STZ:T1D mice. α-TUBULIN WB was used as a loading control. Fold changes in TLN2 levels were determined after quantifying protein levels and normalizing to α-TUBULIN levels. TLN2 levels in control mice were set as 1. Average values were represented as means ± SD (n=4). C) Representative tracing of calcium transients from human primary cardiomyocytes infected with AAV9-CMV-Null (Control) and AAV9-CMV-DN RBFOX2 (DN RBFOX2). Quantification of D) time to peak and E) amplitude of calcium transients. The numbers in the bar graphs represent the number of cardiomyocytes used for quantification from 3 independent experiments. Statistical significance was calculated using the unpaired t-test to compare 2 groups. P-values are indicated as **** < 0.0001, ** < 0.01, * < 0.05.

Next, we tested whether DN RBFOX2 expression affects cardiomyocyte function. It has been previously shown that ablation of Rbfox2 in adult mouse hearts impairs excitation:coupling (Wei et al., 2015) and our results show that RBFOX2 targets affected in diabetic hearts have functions in calcium homeostasis (Fig. 1C). Thus, we examined intracellular calcium handling in human cardiomyocytes expressing DN RBFOX2. DN RBFOX2 expression induced a delayed upstroke of the calcium transients in cardiomyocytes (Figs. 7C and 7D) without a change in calcium transient amplitudes (Fig. 7E), indicating a defect in intracellular calcium handling, which is necessary for cardiomyocyte contraction. In sum, our results show that DN RBFOX2 may contribute to diabetes pathogenesis by altering intracellular calcium signaling.

DISCUSSION

We have previously shown that AS is dysregulated in diabetic hearts. Our goal in this study was to determine the responsible AS regulators. We found that the RNA binding protein RBFOX2 contributes to AS defects in the heart under diabetic conditions. Comparing our RNA sequencing data from diabetic mouse hearts to the RBFOX2 CLIP-seq data, we found that RBFOX2 binds to 73% of transcripts that are differentially spliced. In diabetic hearts, RBFOX2 binding clusters were significantly enriched in mis-spliced transcripts in comparison to control transcripts that were not mis-spliced but displayed similar expression levels. Importantly, we found that RBFOX2 controls AS of target genes with RBFOX2 binding sites and that AS changes impair normal gene expression patterns in the heart especially genes important for macromolecular metabolism, apoptosis, calcium homeostasis and protein trafficking. Supporting these findings, expression of DN RBFOX2 in cardiomyocytes impaired intracellular calcium handling.

We found that RBFOX2 autoregulation is disrupted in diabetic hearts at early stages before cardiac complications are evident. RBFOX2 mediated splicing function was low in diabetic hearts due to the expression of the DN RBFOX2. A recent study has shown that heart specific deletion of Rbfox2 in mice induces dilated cardiomyopathy (Wei et al., 2015). Our results demonstrate that expression of DN RBFOX2, which inhibits RBFOX2 dependent splicing, adversely affects calcium handling in human cardiomyocytes. These results support the conclusion that increased DN RBFOX2 expression in diabetic hearts contributes to cardiac complications.

In this study, we discovered that a DN isoform of RBFOX2 is upregulated in hearts of both human diabetic patients and mouse models via skipping of exon 6 induced by an increase in RBFOX2 protein levels. This isoform was identified as an inhibitor of RBFOX AS activity in cultured cells (Damianov and Black, 2010; Nakahata and Kawamoto, 2005). RBFOX2 has consensus binding sites in the flanking introns of exon 6, which is skipped in the DN isoform (Damianov and Black, 2010). Here, we showed that increased RBFOX2 protein in diabetes is important for DN RBFOX2 production in diabetic hearts and found that RBFOX2 RNA-binding capability is critical for generation of DN RBFOX2 via AS. Therefore, it is likely that RBFOX2 binds to its own pre-mRNA to promote exon 6 exclusion and DN expression.

An important part of our finding is that the dysregulation of AS in diabetic hearts is in agreement with the upregulation of DN RBFOX2. Notably, RBFOX2 target AS events were unaffected in obese and hypertensive mice that do not upregulate DN Rbfox2. We also found that RBFOX2 regulated AS events remained unchanged in mouse hearts with chagasic cardiomyopathy in which DN Rbfox2 expression is not induced. These results indicate that the expression of DN RBFOX2 is stimulated under diabetic conditions independent of hypertension and obesity. In addition, DN RBFOX2 overexpression impaired AS of RBFOX2 targets. Here, we provide strong evidence for mode of inhibitory function of DN RBFOX2 on RBFOX2 dependent splicing by finding that DN RBFOX2 interacts with WT RBFOX2 but does not interact with the U1snRNP-associated U1C.

In our study, FXR1 exons 15 and 16 were almost completely skipped in diabetic human hearts. FXR1 regulates mRNA translation of TALIN2, an important regulator of actin assembly (Whitman et al., 2011). FXR1 knockout mice develop cardiomyopathy in part due to increased translation and accumulation of TALIN2 (Whitman et al., 2011). Our data indicate that RBFOX2 knockdown results in exclusion of Fxr1 exon 15 and 16 and in turn high TALIN2 levels similar to the levels observed in diabetic hearts. These results suggest a role for RBFOX2 in regulating cytoskeleton interacting protein contributing to diabetic cardiomyopathy.

In summary, we find that 704 alternative exons mis-spliced in diabetic hearts are RBFOX2 targets. RBFOX2 protein levels go up in diabetic hearts inducing an AS change in RBFOX2 pre-mRNA that generates a DN isoform with inhibitory AS function, contributing to AS changes with adverse effects on gene expression and function. Importantly, DN RBFOX2 is generated specifically in diabetic hearts at early stages making it a potential diagnostic marker for diabetic cardiomyopathy. Identifying RBFOX2 as an important contributor to AS changes and diabetic complications and determining how it is dysregulated may allow us to develop novel tools to diagnose, prevent, or treat diabetic cardiomyopathy in the future.

EXPERIMENTAL PROCEDURES

Animal Experiments

Animal models used for this study are as follows: streptozotocin (STZ) induced T1D and non-obese diabetic (NOD), obese, hypertensive, obese hypertensive and Chagasic mice. Details about these mouse models are found in Supplemental Experimental Procedures.

All animal experiments were conducted in accordance with the NIH Guidelines and approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee (Protocol # 1101001).

Echocardiography

Cardiac function was assessed using M-mode echocardiography with a Visual Sonics VeVo 2100 ultrasound and a 30MHz probe at the UTMB Biomedical Imaging Network. Mice were anesthetized (1.5% isoflurane mixed with 95% oxygen) and maintained on a heated platform with ECG electrodes to measure body heat and heart rate before and during the imaging. Mice with a heart rate of 450±50 and body temperature of at least 37°C were used for all analyses. Fractional shortening, stroke volume and ventricular internal diameter end at diastole (LVIDd) were calculated as described previously (Liu et al., 2012) using Visual Sonics software at the Baylor College of Medicine Mouse Phenotyping Core.

Human tissue

Human heart tissues (diabetic and non-diabetic) were obtained from the National Disease Research Interchange. Two additional non-diabetic human heart tissues were obtained from Dr. Nisha Garg at the University of Texas Medical Branch. All human heart tissues used in this study were preexisting and deidentified. Thus, human tissue use was exempt from Institute Review Board regulations (protocol #11-087).

Cell Culture, differentiation, and transfections

Cells were cultured and transfected using standard procedures as previously described (Verma et al., 2013). H9c2 cells were differentiated into cardiomyocyte-like cells as described previously (Verma et al., 2013). Human Prenatal Primary Cardiomyocytes (DV Biologics) were cultured according to the manufacturer’s protocols and infected with AAV9 virus (Vector Biolabs) to express either empty vector or DN RBFOX2. Details are found in the Supplemental Experimental Procedures.

Calcium imaging

For calcium imaging, human primary cardiomyocytes (DV Biologics) were incubated at 20°C with 8 μM calcium indicator Fluo-4-AM (Life Technologies, F14201) for 10 minutes and imaged in Hank’s Balanced Salt Solution (pH 7.4) using an Olympus IX51 microscope (20X) and CelSens Standard version 1.4.1 software.

RNA sequencing, RBFOX2 binding site overlay and Statistical Analysis

An Illumina HiSeq 1000 system was used for paired end (2 × 15 cycles) RNA sequencing at the UTMB Next-Generation Sequencing Core facility (Verma et al., 2013). ~200 million reads per sample were generated. Changes in AS were determined using the mixture-of-isoforms (MISO) model (Katz et al., 2010). To determine RBFOX2 binding sites on transcripts mis-spliced in diabetic mouse hearts, human hg19 RBFOX2 CLIP data (Yeo et al., 2009) were converted to the mm9 mouse genome coordinates using liftOver (Hinrichs et al., 2006). AS events, which displayed an absolute delta Psi >=0.1 were used for this analysis. The number of events with at least one CLIP peak at a Bayes Factor (BF) cutoff of 1 was set as “significant” and BF < 1 was set as “not significant”. For RBFOX2 binding cluster analysis, AS events that did not change significantly between all individual comparisons among six samples but exhibited similar expression levels in diabetic hearts were used as controls. Statistical analysis of AS events that were differentially included or excluded in diabetes was determined by calculating a hypergeometric p-value of the enrichment of significant AS events, which contained an RBFOX2 CLIP peak in the region of interest, vs the control AS events with RBFOX2 CLIP peaks (Dale et al., 2011; Perez and Granger, 2007; Quinlan and Hall, 2010). Genes with RBFOX2 binding sites were categorized based on their biological or molecular function using the DAVID server (version 6.7) with the whole genome as a background control.

Quantitative RT-PCR and Statistical Analysis

Quantitative RT-PCR (qRT-PCR) was performed to detect inclusion/exclusion of gene specific alternative exons and relative mRNA levels. Details are included in the Supplemental Experimental Procedures. Student t-test was used to determine statistical significance between two groups and One-way Anova for comparisons more than two groups with Bonferroni correction using Prism software.

FLAG immunoprecipitation/Western Blot analysis

For immunoprecipitation (IP) assays, a FLAG immunoprecipitation kit (Sigma FLAGIPT1-1KT) was used to pull down FLAG-tagged WT, DN or RNA binding deficient mutant RBFOX2 expressed in COS M6 or H9c2 cells per the manufacturer’s protocol. Precipitated proteins were analyzed on 4-15% gradient SDS-PAGE gels followed by Western blot (WB) analysis using an anti-FLAG antibody (Ab) conjugated to HRP (Sigma A8595), anti-GFP Ab conjugated to HRP (Santa Cruz Biotechnology sc9996), or anti-U1C Ab (Bethyl labs A303-947A).

WB analyses were performed as previously described (Verma et al., 2013). Details are included in the Supplemental Experimental Procedures.

Plasmid generation

A human RBFOX2 mutant deficient in RNA binding (RBFOX2RRM) was generated by mutating two phenylalanine (F153 and F155) residues necessary for RNA binding in the RNA Recognition Motif into aspartate residues (Auweter et al., 2006; Mayeda et al., 1994; Sun et al., 2012).

Supplementary Material

1
2
NIHMS786895-supplement-2.pdf (1,015.5KB, pdf)

ACKNOWLEDGMENTS

We thank Hong Sun, Yareli Perez, and Rosario Espejo for technical support; Dr. David Konkel for editing the manuscript; Dr. Douglas Black for providing the wild-type and dominant negative RBFOX2 plasmid DNA constructs and Dr. Corey Reynolds for his help in quantification of echocardiography data. This work was supported, in part, by a March of Dimes Starter Basil O’Connor Starter Scholar Award (5FY12-21), an American Heart Association Grant (15GRNT22830010), an UTMB Institute for Human Infections and Immunity Mini Center Pilot Award, UTMB pilot grant and startup funds to M.N.K-M. This work was supported, in part, by a grant from the National Institutes of Health/National Institute of Allergy and Infectious Diseases (2RO1AI05478-08) and UTMB Institute for Human Infections and Immunity Mini Center Pilot Award to N.J.G., National Institutes of Health (UL1TR000071 UTMB CTSA) and (NIEHS P30 ES006676) grants to ARB, a National Institutes of Health/National Heart, Blood and Lung Institute to I.J.A (5R25HL09636305); National Institutes of Health Grants (HG0046590, NS075449) to G.W.Y. G.W.Y. is an Alfred P. Sloan Research Fellow. O.B.B. is a National Defense Science and Engineering Graduate Fellow and NumFOCUS John Hunter Technical Fellow. T.I. was supported by a Ruth L. Kirschstein National Research Service Award from the National Heart Lung Blood Institute (F30HL128036). X.H.T.W. was supported by National Institutes of Health grants R01-HL089598, R01-HL091947, R01-HL117641, and R41-HL129570, and American Heart Association grant 13EIA14560061.

Footnotes

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AUTHOR CONTRIBUTIONS

C.A.N., S.K.V., V.D., I.J.B, M.J.L., and E.A.J. conducted the experiments; C.A.N. and M.N.K-M designed the experiments and wrote the paper. RBFOX2 binding site data analysis was performed by O.B.B. and G.W.Y., calcium data were analyzed by Q.W. and X.H.T.W, chronic chagasic mice were generated and provided by N.J.G., hypertensive and obese mice were generated and provided by T.I. and A.R.B.

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Supplementary Materials

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NIHMS786895-supplement-1.docx (43KB, docx)
2
NIHMS786895-supplement-2.pdf (1,015.5KB, pdf)

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