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Post-transcriptional regulation plays a key role in transcriptome reprogramming during cardiac pathogenesis. In previous studies, we have identified that cardiac enriched RNA-binding protein, RBFox1 plays key role in cardiac hypertrophy through mRNA alternative splicing regulation in nuclei. However, RBFox1 gene also generates a cytosolic isoform (RBFox1c), suggesting additional functions of post-transcriptional regulation in heart. In adult heart, RBFox1c mRNA constituted ~ 40% of total RBFox1 level but was significantly repressed in pressure-overloaded failing mouse heart. Using CRISPR-Cas9 technology, we have established an isoform specific RBFox1c-cKO mouse. At baseline inactivation of RBFox1c led to decreased cardiac function along with induction of cardiac fibrosis. RBFox1c-cKO mice also showed macrophages infiltration into myocardium post 7days MI. In contrast, restoration of RBFox1c expression in adult intact hearts significantly reduced cardiac fibrosis post stress. RNA-seq analyses in RBFox1c expressing cardiomyocytes showed that RBFox1c specifically suppressed the expression of pro-inflammatory genes. Secondly, CLIP-Seq analysis and targeted RNA-IP showed that RBFox1c could directly interact with inflammatory pathway mRNAs. These results suggested the inflammatory mRNAs are direct downstream targets regulated by RBFox1c. Using both in vitro cultured cardiomyocytes and intact mouse hearts, we demonstrated that expression of RBFox1c reduces pro-inflammatory mRNA expression at baseline and upon hypertrophy stimulation. Lastly, we characterized the interactome of RBFox1c through proteomic analysis and found RBFox1c specifically interacted with a component of the RNA NMD machinery-Upf1. RBFox1c interaction with Upf1 in cardiomyocytes was diminished upon hypertrophic stress. Furthermore, by inactivation of Upf1 via siRNA, we demonstrated that RBFox1c mediated repression of proinflammatory genes was Upf1 dependent.RBFox1 regulates cardiac transcriptome reprogramming in two post-transcriptional processes via distinct isoforms. While the RBFox1n regulates RNA splicing, the RBFox1c functions through targeted mRNA repression of proinflammatory genes by recruitment of Upf1 mediated RNA degradation.

Background: RBFox1 is known to be an RNA splicing regulator with enriched expression in cardiac muscle. Loss of RBFox1 expression is a molecular hallmark associated with heart failure in mouse and human. Genetic manipulation of RBFox1 reveals a major function of RBFox1 in the pathogenesis of cardiac hypertrophy, fibrosis and dysfunction under pathological stresses. However, much of our current knowledge about RBFox1 focuses on the nuclear RBFox1 with a major impact on global alternative splicing changes. Yet, RBFox1 gene also generates a cytosolic isoform-RBFox1c through alternative splicing, and the specific function of RBFox1c has not been characterized. Goal and Methods: This study investigated the functional impact and the underlying mechanism of the RBFox1c in cardiac pathological remodeling and targeted gene regulation. Results: RBFox1c expression is significantly repressed in stressed cardiomyocytes in vivo and in vitro. RNA-Seq combined with IPA analyses from cardiomyocytes revealed RBFox1c but not the nucleus RBFox1 specifically suppressed pro-inflammatory genes. In the cardiac specific RBFox1 knockout mice, enhanced cardiac fibrosis is observed following I/R injury associated with elevated expression of pro-inflammatory genes. In contrast, cardiac specific expression of RBFox1c reduced cardiac fibrosis and inflammatory gene expression following pressure-overload and myocardial infarct injury associated with improved ejection fraction. Motif enrichment analysis identified significant enrichment of the RBFox1 binding motif in the 3’UTR of the RBFox1c regulated genes. We performed CLIP analysis followed by RT-PCR and observed RBFox1c interacted with inflammatory gene 3’UTR. Lastly, we explored the interactome of RBFox1c and found RBFox1c specifically interacted with a component of RNA decay machinery-Upf1. Indeed, while expression of RBFox1c repressed inflammatory gene expression, inactivation of Upf1 abolished this effect in cardiomyocytes. Conclusion: RBFox1 regulates cardiac transcriptome reprogramming at two post-transcriptional steps. The RBFox1 nuclei isoform regulates RNA splicing reprogramming, and the RBFox1c represses proinflammatory genes via recruitment of Upf1 mediated RNA degradation.

Background: Left ventricle (LV) and right ventricle (RV) in mouse heart undergo dramatically different chamber-specific remodeling after birth, leading to rapid increase in LV vs. RV chamber size. However, the underlying regulatory mechanism mediating chamber specific remodeling process remains enigmatic. Results and Methods: In neonatal mouse heart, p38 MAP kinase activity is dynamically activated in a chamber specific manner. p38 activity is specifically elevated in RV comparing to LV at E18.5, postnatal day 3 (P3) and P7 stages whereas p38 activity is lower in both ventricles at P0 and P1. In mouse heart with cardiomyocyte specific-knockout of p38α and β (p38ab-cdKO), total p38 activity was diminished in both chambers. The p38ab-cdKO mice had significant neonatal lethality associated with RV specific chamber enlargement and significant increase in both RV wall thickness (RVW) and inner diameter of RV (RVID) as early as P3. Interestingly, p38 inactivation suppressed myocyte apoptotic activity specifically in RV while increased RV myocyte proliferation and hypertrophy during neonatal period. Unexpectedly, RNA-seq results implicated Xbp1 mediated transcriptional regulation significantly contributing to p38 dependent transcriptome reprogramming in RV. Indeed, IRE1α expression in neonatal cardiomyocyte is sufficient to induce proliferation in vitro. Furthermore, knockdown of Xbp1 blunted p38 inhibition-induced myocyte proliferation, suggesting that IRE1a/Xbp1 mediate p38 signaling in neonatal myocyte proliferation. Conclusion: Chamber-specific remodeling in neonatal heart involves temporally regulated and RV specific p38 MAP kinase activity. RV specific myocyte proliferation and hypertrophy concurrent with RV specific programmed myocyte death is orchestrated by two innate stress-response pathways, p38 and Xbp1.

Rationale: Accurate and comprehensive de novo transcriptome profiling in heart is a central issue to better understand cardiac physiology and diseases. Although significant progress has been made in genome-wide profiling for quantitative changes in cardiac gene expression, current knowledge offers limited insights to the total complexity in cardiac transcriptome at individual exon level. Objective: To develop more robust bioinformatic approaches to analyze high-throughput RNA sequencing (RNA-Seq) data, with the focus on the investigation of transcriptome complexity at individual exon and transcript levels. Methods and Results: In addition to overall gene expression analysis, the methods developed in this study were used to analyze RNA-Seq data with respect to individual transcript isoforms, novel spliced exons, novel alternative terminal exons, novel transcript clusters (ie, novel genes), and long noncoding RNA genes. We applied these approaches to RNA-Seq data obtained from mouse hearts after pressure-overload–induced by transaortic constriction. Based on experimental validations, analyses of the features of the identified exons/transcripts, and expression analyses including previously published RNA-Seq data, we demonstrate that the methods are highly effective in detecting and quantifying individual exons and transcripts. Novel insights inferred from the examined aspects of the cardiac transcriptome open ways to further experimental investigations. Conclusions: Our work provided a comprehensive set of methods to analyze mouse cardiac transcriptome complexity at individual exon and transcript levels. Applications of the methods may infer important new insights to gene regulation in normal and disease hearts in terms of exon utilization and potential involvement of novel components of cardiac transcriptome.

Background: During postnatal maturation of mammalian heart the neonatal cardiomyocytes undergo dramatic changes including complete maturation, proliferation arrest, and terminal exit from the cell cycle (CC). However, transcriptome-wide analysis of CC programs has not been performed in perinatal stages among different cardiac chambers. In particular, the contribution of alternative RNA splicing to the chamber-specific CC activities is unexplored Design/Methods: To achieve comprehensive analysis of differential expression (DE) and alternative splicing (AS) of CC-related genes in left ventricle (LV) versus right ventricle (RV) during maturation deep RNA-seq was performed on male newborn mouse LV and RV at 3 time points of perinatal transition: P0, P3 and P7. Reads were mapped to mouse Transcriptome, and to mouse Genome. Transcriptome-Wide difference in inclusion of individual exons was performed using MATS. DE genes and AS variants were defined as those with fold change ≥2, at expression level ≥3 RBKM and a false discovery rate ≤0.05. Significant gene ontology (GO) terms were determined at P-value ≤0.05. Levels of expression were validated using qRT-PCR Results: Altogether, 2116 DE genes and 1162 AS events were observed. Among them, 109 CC-related genes were further analyzed. Distinct temporal patterns of DE and GO enrichment of CC genes in LV vs. RV during maturation were identified. Chamber -specific induction of genes involved in mitosis, karyokinesis, and cytokinesis was found at P7. RNA Splicing analysis of CC genes revealed 77 AS events. Skipping exon accounted for nearly half splicing events. Among 30 spliced exon variants, significant chamber-and temporal-specific inclusion were observed. Interestingly, the majority of AS variants exhibited opposing patterns of exon usage in RV vs. LV at p7 Conclusions: Our findings suggest novel molecular basis for chamber-specific programming of cellular proliferation and maturation in neonatal heart, including potential splicing regulation of dynamic exon enrichment of cell cycle related genes. Further functional studies to decipher putative splicing regulators of CC programming in LV vs. RV during maturation will likely lead to novel chamber-specific regenerative and therapeutic targets.

Background: The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is also implicated in many human diseases including cancer. However, the global pattern of alternative mRNA splicing during cardiac development and diseases is unknown, and the regulatory mechanisms remain unexplored. Methods and Results: Using deep RNA-Sequencing, we have identified global alternative splicing changes associated with both cardiac development and pathological remodeling in mouse heart following pressure-overload induced heart failure. The alternative RNA splicing events observed in failing hearts mimics the profile in fetal hearts, suggesting a fetal-like RNA splicing program induced in diseased hearts. Using RNA-Seq database and real-time PCR analysis, we examined the expression profile of a large number of known alternative splicing regulators. Among them, we identified Fox1 as a significantly induced regulator during cardiac development in zebrafish, mouse and human, and down-regulated in both mouse and human failing hearts. Morpholino mediated Fox1 knockdown in zebrafish embryos led to lethal phenotype associated with reduced cardiac function and defects in chamber specificity. This phenotype could be rescued by re-expressing both zebrafish and mouse Fox1 gene, suggesting a highly conserved cardiac function of Fox1 for normal cardiac development and function in vertebrates. Conclusion: Our study provided the first comprehensive analysis of mRNA splicing regulation in heart during post-natal development and heart failure, and identified Fox1 as a key regulator for alternative RNA splicing in heart. This study expands our current understanding to the complexity of cardiac transcriptome, and reveals the functional importance of RNA-splicing in cardiac development and diseases.

The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is also implicated in many human diseases including cancer. However, little knowledge is available about the scope of alternative splicing at whole genome level in heart diseases and even less about the mechanisms underlying the regulation of mRNA splicing in response to pathological injury in heart. Using a genome-wide RNA-Seq analysis, we have identified global alternative splicing changes associated with both development and pathological remodeling in mouse heart. Most significantly, the alternative RNA splicing events observed in failing heart mimicked the splicing profile in fetal hearts, suggesting a fetal like RNA splicing remodeling in failing hearts. After examining the expression profiles of splicing regulators in neonatal, normal adult, and failing adult hearts, Fox-1 was identified as one to be significantly down regulated in the failing and fetal hearts. Morpholino mediated Fox-1 knock-down in zebrafish embryos led to lethal phenotype associated with impaired cardiac development and function. This phenotype could be rescued by re-expressing both zebrafish and mouse Fox1 gene. Therefore, our established functional significance of Fox1 mediated RNA alternative splicing serves as a key molecular player in transcriptome remodeling during cardiac development and pathology.

The complexity of transcriptome and proteome is contributed by alternative splicing of RNA. Altered RNA splicing is also implicated in many human diseases including cancer. However, little knowledge is available about the scope of alternative splicing at whole genome level in heart diseases and even less about the mechanisms underlying the regulation of mRNA splicing in response to pathological injury in heart. In order to investigate the key alternative splicing events associated with cardiac pathogenesis, we used high-throughput RNA-Seq to profile total transcriptome in mouse heart failure induced by pressure-overload. From preliminary data, we identified >1000 novel exons that have not been reported in any published database. Many of the novel exons are detected in genes with potential importance in cardiac gene regulation and signal transduction. In addition, we have also identified a significant number of differentially expressed exons between normal and diseased hearts, supporting the notion that differential RNA splicing is associated with the onset of heart failure. Interestingly, the differential splicing events observed in failing hearts were also detected in neonatal mouse hearts, suggesting that differential RNA splicing is a part of the “fetal” gene expression program in diseased hearts. Furthermore, we have also validated some of these changes in human heart failure samples, supporting the clinic relevance of these findings. In summary, this study suggests that development of heart failure is associated with alternative RNA splicing events at genome level. Further studies on the functional consequences of differential RNA splicing and the underlying regulatory mechanisms in heart will advance our understanding to this important disease.