Skeletal muscle weakness is a known contributor to morbidity and mortality in aging and other diseases; however, the underlying mechanisms have not been well defined. It has recently been shown that disruption of circadian rhythms leads to significant weakness. Preliminary data from our lab shows that mice in which Bmal1, a core circadian gene, has been inducibly knocked-out in adult skeletal muscle (iMSBmal1-/-) express an increased amount of a longer spliceform of titin protein than their vehicle-treated counterparts (iMSBmal1+/+). These muscles also display increased variability in sarcomere length, decreases in specific tension, as well as diminished unstimulated baseline tension, a preliminary measure of passive tension. These data lead to my hypothesis that loss of Bmal1 expression in adult skeletal muscle will lead to 1) the increased inclusion of PEVK exons in titin that will contribute to sarcomere length variability and 2) changes in titin spliceform and sarcomere length will be associated with deficits in both the isometric length-tension relationship and elastic properties of this muscle. I will test my novel hypothesis through two aims.
Specific Aim 1 will define the splicing changes in titin of iMSBmal1-/- skeletal muscle. RNA-Seq will be used to determine exon inclusion/exclusion in titin of iMSBmal1-/- and iMSBmal1+/+ tibialis anterior muscle. I will also test if changes to the length of the PEVK domain in titin protein account for the increased sarcomere length variability in iMSBmal1-/- skeletal muscle using immunohistochemical techniques combined with deconvoluted confocal microscopy.
This aim will define the changes to a key sarcomeric protein, titin, following muscle-specific Bmal1 knockout and links this change to maintaining sarcomere length homogeneity.
Specific Aim 2 tests if properties of titin-based skeletal muscle active and passive tension are diminished following Bmal1 knockout. I will perform in situ mechanical experiments to test both the isometric length-tension relationship of the muscle as well as the elasticity of the TA muscle without contractile contributions. I will then relate these measurements to the titin spliceform expressed within the muscle as well as immunohistochemical measurements (i.e., fiber cross-sectional area and fibrosis). These experiments will help determine the effects a loss of Bmal1 in skeletal muscle has on altered titin expression and sarcomere length maintenance with implications for the active length-tension relationship. The findings from this project hold potential to provide insight into a novel finding that the molecular clock helps to maintain skeletal muscle's structure and basic functional properties of the muscle through its control of titin spliceform expression.
Disruption of circadian rhythms is linked to muscle weakness. This project explores a change in titin isoform expression in skeletal muscle and the downstream effects on skeletal muscle structure and function following knockout of Bmal1, a core component of the molecular clock. Understanding the links between the change in titin spliceform and altered sarcomere structure and muscle function will provide novel insight into potential targets and therapeutics for weakness.
