The Skeletal Muscle Molecular Clock Regulates Titin Splicing and Protein Expression

Abstract

The circadian factor BMAL1 is a fundamental transcriptional regulator of cell time keeping and expression of cell specific genes important for homeostasis. Our lab developed the inducible, skeletal muscle specific Bmal1 knockout (iMSBmal1-/-) mouse to determine the role of this protein in adult skeletal muscle. Skeletal muscles from these mice exhibit decreased specific tension and reduced unstimulated tension developed during a fatigability test. Since this change could be caused by changes in the elastic properties of the muscle, we investigated whether titin isoform expression changes in these mice. The tibialis anterior muscle of the iMSBmal1-/- mice exhibits a significant shift in expression from a short to long isoform of titin protein (38% long in iMSBmal1-/- vs. 19% in iMSBmal1+/+). RNA-Seq from these muscles indicates increased inclusion of titin exons 150-155 and 190-225, which are found within titin's PEVK region. Because this region is highly extensible and acts as a spring, we performed immunohistochemistry to test whether changes to this protein could have an effect on sarcomere length. An antibody to α-actinin was used to demarcate Z-lines in longitudinal sections of the tibialis anterior muscle and sarcomere length was measured as the distance between Z-lines. While average sarcomere length was not different between iMSBmal1+/+ and iMSBmal1-/- muscle, variation in sarcomere length increased following Bmal1 knockout. To test if increased variability of titin isoform expression similar to the iMSBmal1-/- mouse could lead to variable sarcomere lengths, we are currently transfecting C2C12 myoblasts with U7 snRNPs targeted to titin PEVK regions. Sarcomere length variability will be measured in these cells following 7 days of differentation. Current studies with this model are ongoing and will lead to increased knowledge of titin's role in regulating sarcomere length in a skeletal muscle at rest.