Cleavage and Migration of Junctophilin 1 in Muscle of Patients with Cytosolic Leak of Stored Calcium

Abstract

The primary defect in susceptibility to malignant hyperthermia (MHS) is an increase in “leak” or stimulated release of Ca2+ from the SR via RyR1. The mechanistic links from this defect to the MHS disease phenotypes are not well understood. We found that abnormal Ca2+ homeostasis in MHS individuals induces proteolysis of junctophilin 1 (JP1), one of the 5 proteins essential for excitation-contraction coupling (Perni et al., 2017). The highest-probability calpain cleavage site located by GPS-CCD produces a 44 kDa C-terminal JP1 fragment (JP1-Ctf). Western blot, WB, of patients’ muscle fractions confirmed the presence of both full-length JP1 and JP1-Ctf. Immunoimaging demonstrated JP1 in T-SR junctions, strictly colocalized with RyR1. Instead, JP1-Ctf was found anywhere within the I band, and at high densities within nuclei -a location forbidden for JP1. WB of muscle extracts revealed (with high significance) more JP1-Ctf and less JP1 in MHS than in MHNegative subjects. Studies on muscle extracts and primary cultures demonstrated that truncation of JP1 and translocation of the fragment require calpain 1 and are calcium-dependent. Nuclear JP1-Ctf levels were positively correlated with [Ca2+]cyto in human primary myotubes. Endogenous calpain was found in T-SR junctions; tagged calpain, expressed in myotubes, was highly colocalized with JP1 and RyR. In sum, we identified a 44 kDa JP1-Ctf, cleaved by Ca2+-activated calpain 1, which moves into the nucleus. Consistent with its Ca2+-dependent generation, its intra-nuclear presence was increased in MHS patients. By weakening the junctional structure, the corresponding decrease in full-length JP1 may boost the disease severity. The effects of intranuclear JP1-Ctf, presumably by control of gene expression, may explain other features of the MHS condition, including the association with hyperglycemia of late onset and type-2 diabetes (Tammineni et al., 2020).