# Cartilaginous fish and mammalian connectin evolved independently from an ancestral bony fish-like structure

## TL;DR

This study shows that connectin in cartilaginous fish and mammals evolved independently from a bony fish-like ancestor, leading to differences in muscle structure.

## Contribution

The study reveals the connectin gene structure in cartilaginous fish and shows independent evolution from bony fish-like ancestors.

## Key findings

- Elephant shark connectin has six 6-Ig and one 10-Ig super-repeat, differing from human and zebrafish.
- Tissue-specific isoform distribution is conserved between cartilaginous fish and mammals.
- Ancestral jawed vertebrates likely had a bony fish-like connectin structure.

## Abstract

Connectin, also known as titin, is the largest protein that connects the Z-line to M-line of the sarcomere, functioning as a molecular spring to regulate muscle extensibility. While comparative studies have illuminated vertebrate muscle evolution, connectin structure in cartilaginous fishes remains unexplored. Here, we investigated the connectin gene and domain structure in the elephant shark (Callorhinchus milii), revealing a 238 kb gene with 300 exons, with an overall domain structure similar to that of mammalian connectin. PCR analysis revealed tissue-specific expression, with the N2BA isoform present in the heart and the N2A isoform in skeletal muscle, indicating conservation of isoform distribution between cartilaginous fishes and mammals. Molecular phylogenetic analysis showed that the elephant shark has six 6-Ig super-repeats and one 10-Ig super-repeat within the middle-Ig segment of connectin, differing from human (three 6-Ig super-repeats and three 10-Ig super-repeats) and zebrafish (three 6-Ig super-repeats and one 10-Ig super-repeat). These findings suggest that the ancestral jawed vertebrate, approximately 450 million years ago, had a connectin structure resembling that of bony fish. Cartilaginous fishes and mammals subsequently evolved independently, leading to elongation of the connectin I-band region in skeletal muscle. Our findings provide new insights into the evolutionary adaptations of locomotion and circulation.

The online version contains supplementary material available at 10.1038/s41598-025-10916-z.