Insulin regulates titin pre-mRNA splicing through the PI3K-Akt-mTOR kinase axis in a RBM20-dependent manner

<p>Titin, a giant sarcomeric protein, is largely responsible for the diastolic properties of the heart. It has two major</p> <p>isoforms, N2B and N2BA due to pre-mRNA splicing regulated mainly by a splicing factor RNA binding motif 20</p> <p>(RBM20). Mis-splicing of titin pre-mRNA in response to external stimuli may lead to altered ratio of N2B to</p> <p>N2BA, and thus, impaired cardiac contractile function. However, little is known about titin alternative splicing</p> <p>in response to external stimuli. Here, we reported the detailed mechanisms of titin alternative splicing in response</p> <p>to insulin. Insulin treatment in cultured neonatal rat cardiomyocytes (NRCMs) activated the PI3K-AktmTOR</p> <p>kinase axis, leading to increased N2B expression in the presence of RBM20, but not in NRCMs in the</p> <p>absence of RBM20. By inhibiting this kinase axis with inhibitors, decreased N2B isoform was observed in NRCMs</p> <p>and also in diabetic rat model treated with streptozotocin, but not in NRCMs and diabetic rats in the absence of</p> <p>RBM20. In addition to the alteration of titin isoform ratios in response to insulin, we found that RBM20 expression</p> <p>was increased in NRCMs with insulin treatment, suggesting that RBM20 levels were also regulated by</p> <p>insulin-induced kinase axis. Further, knockdown of p70S6K1 with siRNA reduced both RBM20 and N2B levels,</p> <p>while knockdown of 4E-BP1 elevated expression levels of RBM20 and N2B. These findings reveal a major signal</p> <p>transduction pathway for insulin-induced titin alternative splicing, and place RBM20 in a central position in the</p> <p>pathway, which is consistent with the reputed role of RBM20 in titin alternative splicing. Findings from this</p> <p>study shed light on gene therapeutic strategies at the molecular level by correction of pre-mRNA mis-splicing.</p>