Determining the Molecular Mechanism of Titin-Based Longitudinal Hypertrophy in Skeletal Muscle

Abstract

Longitudinal muscle hypertrophy is the lengthening of muscle fibers through the addition of sarcomeres in series. The molecular mechanisms of longitudinal hypertrophy are poorly understood. Identifying the factors that cause longitudinal hypertrophy is vital to developing therapeutic interventions to prevent muscle weakness in diseases such as cerebral palsy, chronic obstructive pulmonary disorder and fascioscapulahumeral muscle dystrophy. Titin, an ~4 MDa protein, spans from the Z-disk to the M-band of the sarcomere and maintains the structure of the sarcomere during stretch. Titin forms the molecular spring of the sarcomere and is ideally situated to sense mechanical signals. It has been proposed that titin regulates longitudinal hypertrophy by sensing muscle stretch and triggering mechano-sensitive signaling pathways that induce gene expression. I hypothesize that mechanically coupled vectors of titin regulate longitudinal hypertrophy in skeletal muscle, and therefore, an increase in titin-based stiffness will promote longitudinal hypertrophy. The TtnΔ112-158 mutant mouse model has a 47-exon deletion in the PEVK region of titin, the main spring region, which results in a stiffer titin molecule. By passively stretching muscles from WT and TtnΔ112-158 mice, the mechanisms by which titin promotes longitudinal hypertrophy was tested. This TtnΔ112-158 mouse model presented an ~34% increase in serial sarcomeres in skeletal muscle compared to control mice, making this an ideal model to study longitudinal hypertrophy. A series of in vivo and ex vivo experiments were performed, and proteomic techniques were used to identify changes in protein expression and post-translational modifications of skeletal muscle to uncover signaling proteins involved in longitudinal hypertrophy. The findings from these studies identified the PI3-AKT pathway involved in longitudinal hypertrophy. SPEG and synemin proteins were recognized as consistently being upregulated in TtnΔ112-158 stretched muscle compared to controls. These proteins associate with the Z-disk region and the M-Band region of titin, where phosphorylation activity is increased during muscle stretch. These studies further the understanding of muscle growth pathways and how skeletal muscle responds to mechanical signals providing insight into possible therapeutic targets for combating and treating muscle weakness in disease.