REGULATORY ROLE OF TITIN IN SKELETAL MUSCLE HYPERTROPHY THROUGH ALTERNATIVE SPLICING

Abstract

Titin is a giant sarcomeric protein with a theoretical full length of 38,000 amino acids with the molecular weight approximately 4.2 MDa. Although the detectable isoforms expressed in vertebrate muscle range from 27,000 to 36,000 amino acids, it still makes titin a largest known animal protein. Titin spans half the sarcomere, anchoring at the Z-disk and M-line, with two molecules overlapping tail-to-tail to extend across the full sarcomere. This arrangement allows titin to act as a scaffold for various sarcomeric filament components. Titin is divided into four main regions—Z-disk, I-band, A-band, and M-band—each with distinct mechanical properties that define titin’s structural and mechanosensitive roles in muscle functioning. The I-band region of titin is particularly notable for its elastic properties, providing the muscle with the ability to stretch and contract. This region is subject to extensive alternative splicing, which modulates the length of its spring element, thereby influencing its mechanical properties and interactions with signaling proteins. This splicing is regulated by the RNA-binding motif protein 20 (RBM20), a splicing factor extensively studied in cardiac muscle, but much less is known about its role in skeletal muscle. Previous studies have primarily focused on deletion models targeting parts of titin’s extensible region, whereas the functional consequences of expressing full-length, highly compliant titin isoforms remain largely unexplored. Given that RBM20 knockout (KO) results in the expression of a longer titin isoform in rodents, we hypothesized that this more compliant variant impairs mechano-transduction and attenuates hypertrophic signaling in skeletal muscle. To evaluate this, we used RBM20 KO rats and assessed body weight, muscle mass, muscle fiber cross-sectional area (CSA), and fiber type composition in the extensor digitorum longus (EDL), soleus, and tibialis anterior (TA) muscles—representing fast-, slow-, and mixed-twitch muscles, respectively. Our results revealed significant fiber-type-specific atrophy across all three muscle types. In particular, CSA of fast-twitch type IIA fibers was reduced in the EDL and TA, while CSA of slow-twitch type I fibers was significantly decreased in the soleus. We also observed fiber type switching in all muscles, with type I fibers switching to type IIX in the EDL and TA, and type IIA fibers switching to type I in the soleus. These findings suggest that titin splicing plays a regulatory role not only in skeletal muscle hypertrophy but also in fiber type specification. Protein expression analyses revealed altered mechano-transductive signaling in KO muscles. The telethonin–muscle lim protein (MLP) pathway was upregulated in RBM20 KO muscle, potentially leading to cytoplasmic retention of MLP and reduced transcriptional activation of growth-promoting genes. Additionally, the FHL1/2–ERK2 pathway was activated, suggesting that increased ERK2 signaling may antagonize pro-hypertrophic pathways. Markers of protein degradation, including NBR1, MuRF1, and p62/SQSTM1, were also upregulated, indicating elevated protein turnover. Experiments using primary myoblast cultures further confirmed that titin is a necessary mediator of mechanosensitive signaling, with these pathways becoming active only after titin expression following differentiation. We also identified a profound impairment in exercise-induced hypertrophy in RBM20 KO rats. Despite undergoing progressive resistance training via ladder climbing, KO rats failed to exhibit increases in muscle mass or fiber CSA, unlike their wild-type counterparts. This indicates that longer titin isoforms are unresponsive to mechanical overload and fail to initiate hypertrophic signaling. Additionally, KO rats exhibited significantly reduced grip strength, muscle contractility, and tensile strength, demonstrating that titin size is a key determinant of skeletal muscle function. Overall, this study provides a comprehensive analysis of skeletal muscle growth, signaling, and function in the context of longer titin isoform expression resulting from RBM20 deficiency. Our findings reveal a central role for titin in regulating mechanotransduction, muscle fiber size, and fiber type switching, and underscore the importance of alternative splicing in maintaining skeletal muscle plasticity and performance.