# Calpain Mediated Proteolysis of Junctophilin-1 Produces an Aggregation Prone C-Terminal Fragment in Skeletal Muscle

## TL;DR

This study shows that calpain cleavage of junctophilin-1 in skeletal muscle produces a fragment prone to aggregation, which may contribute to muscle pathology under stress.

## Contribution

The study identifies specific calpain cleavage sites in junctophilin-1 and reveals the aggregation-prone nature of a resulting C-terminal fragment.

## Key findings

- Calpain cleavage of junctophilin-1 produces a 44-kDa C-terminal fragment (JPh44) that is intrinsically aggregation-prone.
- JPh44 aggregates localize to perinuclear regions and colocalize with HSP70 and HDAC6 under stress conditions.
- HSP70 activation promotes clearance of JPh44 aggregates and enhances nuclear translocation of the fragment.

## Abstract

Junctophilin-1 (JPh1) is an essential structural protein of the calcium release units required for excitation–contraction coupling in skeletal muscle. In myopathic conditions associated with elevated intracellular calcium, calcium-activated calpains target multiple proteins. Although JPh1 is known to be a calpain substrate, the precise molecular identity of its calpain cleavage sites and the (patho)physiological roles of the resulting proteolytic fragments remain poorly defined. Here, we combined in-silico prediction with in vitro calpain cleavage analysis of dual-tagged JPh1 to identify multiple calpain cleavage sites within JPh1. We further show that a 44-kDa C-terminal fragment of JPh1 (JPh44) is intrinsically prone to aggregation. Using a combination of biophysical, biochemical, and imaging approaches, we demonstrate that under stress conditions JPh44 progressively forms aggregates that localize predominantly to perinuclear regions. Aggregated JPh44 colocalizes with HSP70 and resides near HDAC6. Pharmacological activation or overexpression of HSP70 promotes clearance of JPh44 aggregates and enhances JPh44 nuclear translocation. Finally, we identify the transmembrane domain of JPh44 as a key determinant driving its aggregation propensity. Together, these findings reveal that stress-induced proteolysis of JPh1 generates aggregation-prone fragments whose handling by heat shock proteins and autophagy-related machinery may play an important role in skeletal muscle adaptation and pathology.