Calpain-2 inhibition prevents pressure overload-induced lysosomal cathepsin release and cardiac hypertrophy.

AIMS: Dysregulated calpains and cathepsins contribute to adverse cardiac remodeling following chronic pathological stress, but their interplay and the specific contributions of the classical calpain isoforms remain undefined. Here, we tested whether calpain-2 initiates a lysosome-cathepsin proteolytic axis that promotes pathological hypertrophy through mTORC1-dependent signaling. MATERIALS AND METHODS: Calpain expression was analyzed in septal myocardium from patients with aortic stenosis and mice subjected to transverse aortic constriction (TAC). Calpain activity was modulated genetically by Capns1 deletion or pharmacologically with the calpain-2 inhibitor NA-184 in TAC mice. Cathepsins were inhibited with balicatib. In vitro, Capn2 was silenced by siRNA in H9c2 cardiomyoblasts stimulated with angiotensin II. KEY FINDINGS: TAC upregulated myocardial calpain-2 expression, which correlated with hypertrophy severity in septal tissue from patients. TAC induced lysosomal membrane permeabilization, evidenced by reduced cathepsin B-lysosome colocalization, and increased cytosolic cathepsin accumulation and activity. Mechanistically, calpain-2 cleaved the lysosomal protein LAMP2, generating a ~ 55 kDa fragment. Both Capns1 deletion and NA-184 preserved LAMP2 and lysosomal integrity, reduced autophagic dysregulation and the activation of the AKT/mTOR signaling, and attenuated cardiac hypertrophy. Capn2 silencing in H9c2 reproduced the effects by reducing LAMP2 proteolysis and hypertrophy. Balicatib produced comparable anti-hypertrophic effects, supporting a sequential calpain-cathepsin signaling cascade. When treatment was extended, NA-184 limited ventricular dilation and attenuated systolic dysfunction (30%, p < 0.001). SIGNIFICANCE: These findings identify calpain-2-mediated LAMP2 proteolysis as an upstream trigger linking lysosomal dysfunction to pathological hypertrophy, and highlight selective calpain-2 inhibition as a therapeutic strategy for heart failure treatment.