Mechanisms of Dysregulated Alternative Splicing in Cardiac and Skeletal Muscle under Diabetic Conditions

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Under diabetic conditions, cardiac and skeletal muscle undergoes changes that can lead to cardiomyopathy and myopathy respectively. Diabetes patients suffer from skeletal muscle weakness and atrophy. Diabetic cardiomyopathy can progress into heart failure. Currently, no treatments are available for diabetic cardiomyopathy or skeletal muscle myopathy. Protein kinase C (PKC) signaling is a central pathogenic factor in diabetes, although downstream effectors are not well known. Our lab determined a novel role for PKC in alternative splicing (AS) regulation during heart development. We found that PKC phosphorylates and controls RNA-binding proteins (RBPs) CELF1 and RBFOX2 that are involved in developmentally regulated AS in the heart. Since PKC is chronically activated in diabetes, we hypothesized that aberrant PKC activity alters CELF1 and RBFOX2 and disrupts AS. To test this hypothesis, we performed RNA-sequencing to determine genome-wide AS changes in diabetic mouse hearts. We identified 967 AS changes and discovered a subset of embryonic AS patterns that were re-activated in diabetic hearts. Some of these AS patterns were also activated in diabetic skeletal muscle; demonstrating that disruption of developmentally regulated AS is a hallmark of diabetic cardiac and skeletal muscle. In diabetic hearts, we found that the majority (73%) of mis-spliced transcripts harbored RBFOX2 binding sites. RBFOX2 controlled AS of target RNAs. We found that RBFOX2 protein levels were upregulated consistent with high PKC activity in diabetic hearts. However, our computational and experimental analyses indicated that upregulation of RBFOX2 in diabetic hearts did not correlate with AS of target RNAs. Our results showed that RBFOX2 pre-mRNA was alternatively spliced in diabetes leading to increased expression of a dominant negative (DN) isoform. Our analysis of DN RBFOX2 suggested that DN interacted with WT RBFOX2 and inhibited AS of WT targets. Notably, dysregulation of RBFOX2 by DN RBFOX2 adversely affected AS of genes involved in muscle contraction, contributing to cardiomyopathy. A further analysis of genome wide mRNA expression changes in the diabetic heart identified targets of CELF1 and PTBP1 that are mis-spliced. Interestingly we found evidence that diabetes induced alterations in RBFOX2, CELF1, and PTBP1 coordinately regulate AS changes. In skeletal muscle similar pathogenic changes in AS occur and correlate with an increase in CELF1 protein levels. In conclusion, we a) identified global AS changes in diabetic hearts, b) determined RBPs involved in aberrant AS in diabetic muscle, and c) elucidated mechanisms by which RBPs are dysregulated in diabetes.

Diabetes, RNA, Splicing, Heart, Cardiac, Skeletal, Muscle, RNA binding protein, Cardiomyopathy, Myopathy, Developmental, RBFOX2, PTBP1, CELF1:PKC