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

Titin, a giant sarcomeric protein, is largely responsible for the diastolic properties of the heart. It has two major

isoforms, N2B and N2BA due to pre-mRNA splicing regulated mainly by a splicing factor RNA binding motif 20

(RBM20). Mis-splicing of titin pre-mRNA in response to external stimuli may lead to altered ratio of N2B to

N2BA, and thus, impaired cardiac contractile function. However, little is known about titin alternative splicing

in response to external stimuli. Here, we reported the detailed mechanisms of titin alternative splicing in response

to insulin. Insulin treatment in cultured neonatal rat cardiomyocytes (NRCMs) activated the PI3K-AktmTOR

kinase axis, leading to increased N2B expression in the presence of RBM20, but not in NRCMs in the

absence of RBM20. By inhibiting this kinase axis with inhibitors, decreased N2B isoform was observed in NRCMs

and also in diabetic rat model treated with streptozotocin, but not in NRCMs and diabetic rats in the absence of

RBM20. In addition to the alteration of titin isoform ratios in response to insulin, we found that RBM20 expression

was increased in NRCMs with insulin treatment, suggesting that RBM20 levels were also regulated by

insulin-induced kinase axis. Further, knockdown of p70S6K1 with siRNA reduced both RBM20 and N2B levels,

while knockdown of 4E-BP1 elevated expression levels of RBM20 and N2B. These findings reveal a major signal

transduction pathway for insulin-induced titin alternative splicing, and place RBM20 in a central position in the

pathway, which is consistent with the reputed role of RBM20 in titin alternative splicing. Findings from this

study shed light on gene therapeutic strategies at the molecular level by correction of pre-mRNA mis-splicing.