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

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.