Insulin and Angiotensin II Regulate Titin Alternative Splicing via Rbm20

Objective This study is to determine whether titin alternative splicing is regulated by insulin and angiotensin II via a muscle‐specific splicing factor, RNA binding motif 20 (Rbm20). Methods In primary cultures, neonatal cardiomyocytes (CMs) were isolated from day 1 rats. CMs from wild type (WT), heterozygous (HT), and homozygous (HM) Rbm20 deficient rats were cultured in serum‐free medium and treated with insulin, insulin plus LY294002 (LY) and angiotensin II for 5 days. In in vivo assay, HT rats were treated with streptozotocin (STZ) for 8 weeks. Titin isoforms were detected by 1% SDS‐agarose gel. Band intensity was quantified with imageJ. Western blot and QRCR were performed for protein and gene expression. Results Insulin and angiotensin II increased the expression of smaller titin isoform (N2B) in both WT and HT CMs, but no N2B was rescued by insulin and angiotensin II in HM CMs. The phosphorylation of Akt was increased in WT, HT and HM CMs. LY can inhibit Akt phosphorylation and titin isoform transition in both WT and HT insulin treated CMs. STZ treatment decreased the expression of smaller titin N2B in HT rats. The mRNA levels of titin isoforms were consistent with protein level. Rbm20 expression was increased in insulin treated CMs. Conclusion Titin alternative splicing has been shown to be largely responsible for the diastolic properties of the heart. Rbm20 is the major regulator of the titin alternative splicing. These results indicated that insulin and angiotensin II regulated titin alternative splicing is Rbm20 dependent, and Akt phosphorylation can enhance Rbm20‐regulated titin alternative splicing.