(490.9) RBM20 regulates muscle hypertrophy through titin and calcium signaling

Poster Board Number: A106

Jeffrey Gao-Hu (UW-Madison), Wei Guo (UW-Madison)

Study objective:

RNA binding motif 20 (RBM20), an RNA binding protein, regulates alternative splicing of titin and calcium handling genes that mediate hypertrophy signaling. The objective of this study is to investigate whether RBM20 regulates muscle hypertrophy through titin and/or calcium signaling.

Methods:

Soleus (SOL) and extensor digitorum longus (EDL) were collected from wild-type (WT) and Rbm20 knockout (KO) rats. Transcriptome profiling was analyzed by RNA sequencing (RNA-seq). Hypertrophic signaling was examined using HeLa cells as well as differentiated and undifferentiated C2C12 myoblasts. RBM20 was knocked down in C2C12 myoblasts by siRNA transfection. Both immunostaining and western blotting were performed to test expression of hypertrophy associated proteins in both cell lines.

Results:

RBM20 KO promoted SOL hypertrophy and EDL atrophy in rats. Hypertrophy-associated hallmark proteins CaN, Erk and p38 were elevated in KO SOL, but not in KO EDL, while atrophy-associated hallmark protein p62 was increased in KO EDL, but not in KO SOL. RNA-seq data identified differentially expressed and spliced genes associated with hypertrophy in both EDL and SOL. Titin was switched to largest isoform as a result of alternative splicing in both KO SOL and EDL muscles. To test whether titin is the major mediator that regulatew hypertrophic signaling in RBM20 KO rats, we transfected Rbm20 gene into HeLa cells that do not express titin. The results revealed that the levels of hallmark proteins did not changed with overexpression of RBM20. To further test the role of titin in RBM20-mediated hypertrophy, Rbm20 was knocked down in undifferentiated C2C12 cells which do not express titin as well, and no change was observed for hallmark proteins. C2C12 myoblasts were then differentiated to myotubes and reduced RBM20 in differentiated C2C12 cells promoted reduced expression of hypertrophic hallmark proteins. 

Conclusion:

Our results suggest that RBM20 regulates muscle hypertrophy in a muscle type-dependent manner, and RBM20-mediated hypertrophy is regulated via titin, a hypertrophic mediator.

This study is funded by United States Department of Agriculture (USDA) and National Institutes of Health (NIH).