Abstract
The Proline, Glutamate, Valine and Lysine-rich (PEVK) region of titin constitutes an entropic spring that provides passive tension to striated muscle. To study the functional and structural repercussions of a small reduction in the size of the PEVK region, we investigated skeletal muscles of a mouse with the constitutively expressed C-terminal PEVK exons 219–225 deleted, the TtnΔ219–225 model (MGI: TtnTM 2.1Mgot). Based on this deletion, passive tension in skeletal muscle was predicted to be increased by ∼17% (sarcomere length 3.0 μm). In contrast, measured passive tension (sarcomere length 3.0 μm) in both soleus and EDL muscles was increased 53 ± 11% and 62 ± 4%, respectively. This unexpected increase was due to changes in titin, not to alterations in the extracellular matrix, and is likely caused by co-expression of two titin isoforms in TtnΔ219–225 muscles: a larger isoform that represents the TtnΔ219–225 N2A titin and a smaller isoform, referred to as N2A2. N2A2 represents a splicing adaption with reduced expression of spring element exons, as determined by titin exon microarray analysis. Maximal tetanic tension was increased in TtnΔ219–225 soleus muscle (WT 240 ± 9; TtnΔ219–225 276 ± 17 mN/mm2), but was reduced in EDL muscle (WT 315 ± 9; TtnΔ219–225 280 ± 14 mN/mm2). The changes in active tension coincided with a switch toward slow fiber types and, unexpectedly, faster kinetics of tension generation and relaxation. Functional overload (FO; ablation) and hindlimb suspension (HS; unloading) experiments were also conducted. TtnΔ219–225 mice showed increases in both longitudinal hypertrophy (increased number of sarcomeres in series) and cross-sectional hypertrophy (increased number of sarcomeres in parallel) in response to FO and attenuated cross-sectional atrophy in response to HS. In summary, slow- and fast-twitch muscles in a mouse model devoid of titin’s PEVK exons 219–225 have high passive tension, due in part to alterations elsewhere in splicing of titin’s spring region, increased kinetics of tension generation and relaxation, and altered trophic responses to both functional overload and unloading. This implicates titin’s C-terminal PEVK region in regulating passive and active muscle mechanics and muscle plasticity.
Highlights
Titin is a giant protein located inside the striated muscle sarcomere where it anchors inside the Z-disk and M-band and spans the half-sarcomere
Our findings revealed that the Ttn 219−225 soleus and extensor digitorum longus (EDL) muscles have increased passive tension, partly due to the appearance of a smaller secondary titin isoform
Considering that the deleted exons 219–225 comprise a small fraction of the PEVK region of the titin gene (Figure 1A), the expected level of passive tension increase in Ttn 219−225 skeletal muscle was assessed first
Summary
Titin is a giant protein located inside the striated muscle sarcomere where it anchors inside the Z-disk and M-band and spans the half-sarcomere. This layout in the sarcomere uniquely positions titin to act as a molecular spring (Labeit and Kolmerer, 1995). The spring function of titin is primarily regulated through elastic domains in the I-band region of the sarcomere In skeletal muscle, these domains consist of serially linked immunoglobulin(Ig)-like repeats and a spring element consisting of the PEVK (Proline, Glutamate, Valine, Lysinerich) region (Watanabe et al, 2002; Nagy et al, 2005). Complete removal of titin results in loss of sarcomere structure and passive and active tension (Horowits et al, 1986; Radke et al, 2019), supporting titin’s vital role in muscle mechanics and sarcomerogenesis
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