Calpain-4 Knockdown Modulates Cholesterol Metabolism and LXRα Nuclear Localization in Alcohol-Related Liver Disease

Background & Aims: Ethanol affects lipid metabolism through multiple pathways, leading to fatty liver development in most ALD patients. Recent studies have highlighted the role of calpain, a calcium-dependent protease, in liver inflammation and fibrosis. Calpain activity is regulated by its essential subunit, Capns1, (calpain-4, Capn4), which stabilizes and modulates the activity of its catalytic isoforms, calpain-1 and calpain-2. This study investigated calpain's impact on lipid metabolism in ALD. Approach & Results: Six-week-old C57Bl6/J mice were injected with rAAV8 vectors encoding Capn4 shRNA or control vectors. After four weeks, mice underwent a 10-day period of ad libitum ethanol consumption, followed by a single gavaged ethanol administration on day 11. Following Capn4 knockdown, microvesicular steatosis was attenuated. While triglycerides and free fatty acids levels showed no significant changes, cholesterol levels were significantly reduced in the ethanol (EtOH) group with Capn4 knockdown. Cpt1a expression increased significantly in the EtOH group with Capn4 knockdown. Western blot analysis revealed increased Cleaved-HMGCR to Pro-HMGCR ratio in Capn4 knockdown mice, suggesting reduced HMGCR activity and suppressed cholesterol biosynthesis. LXRα expression was mainly increased in the cytoplasm in the EtOH group, and following Capn4 knockdown, it was relocalized to the nucleus via its activation. In addition, RNA sequencing analysis suggests that Capn4 knockdown contributes to the reprogramming of ethanol-induced disruptions in metabolic and homeostatic pathways, primarily those involving cholesterol metabolism. Conclusions: Further investigation into the relationship between Capn4 and cholesterol biosynthesis proteins may provide insights into using calpain inhibitors as a therapeutic approach for alcohol-related hepatitis.