Calpain activation disrupts ER-Phagy and leads to mitochondrial damage in hearts treated with isoproterenol

The endoplasmic reticulum (ER) is a dynamic organelle whose homeostasis is maintained by ER-phagy (ERPG). Under conditions of ER stress, calcium-dependent cysteine proteases-specifically calpain 1 and calpain 2 (CPN1/2)-are activated and contribute to mitochondrial damage. Isoproterenol (ISO) administration is a widely used experimental model for inducing cardiac dysfunction. In this study, we hypothesize that impaired ERPG worsens ER stress in ISO-treated hearts, leading to mitochondrial dysfunction through the activation of CPN1/2. Male C57BL/6 mice (2-3 months old) received ISO at a dose of 100 mg/kg via daily intraperitoneal (IP) injection for five consecutive days. Cardiac function, ER stress, and mitochondrial function were assessed before ISO treatment, and at 1-, 2-, and 4- weeks following exposure. Cardiac dysfunction was evident two weeks after ISO administration. ER stress began to increase one week after ISO treatment and continued to intensify at 2 and 4 weeks. Activation of cytosolic CPN1/2 was observed at 1 week. The content of FAM134B-a key regulator of ERPG-was reduced by 2 weeks post-treatment. Incubation experiments showed that purified CPN1 cleaves FAM134B, supporting its role as a direct target. Mitochondrial respiration, including cytochrome oxidase activity, declined two weeks after ISO exposure. This decline was associated with a loss of cytochrome c and reduced levels of ferrochelatase, both contributing to impaired mitochondrial oxidative capacity. These findings suggest that reducing ER stress may be an effective therapeutic strategy to mitigate contractile dysfunction with cardiac disease.