Upregulation of T-type voltage-dependent Ca²⁺ channels in activated hepatic stellate cells promotes liver fibrosis in metabolic dysfunction-associated steatohepatitis.

Activation of hepatic stellate cells (HSCs) is a pivotal event in the development of liver fibrosis, a pathological process that precedes cirrhosis and hepatocellular carcinoma and for which effective therapies remain limited. The molecular mechanisms underlying HSC activation, particularly the ion channels that regulate cytosolic Ca²⁺ signaling that promote fibrogenesis, remain incompletely understood. In this study, the expression and functional roles of Ca²⁺ channels in human and mouse HSCs were investigated using quantitative real-time PCR, Western blotting, whole-cell patch-clamp electrophysiology, Ca2+ imaging, and histological analyses. RNA sequencing of human HSCs revealed upregulation of CaV3.2 transcripts upon activation. In activated human HSCs (LX-2 cells), CaV3.2 protein was detected, and transient inward currents sensitive to the T-type voltage-dependent Ca²⁺ channel (T-VDCC) blocker Z944 were recorded. Z944 concentration-dependently reduced both resting cytosolic Ca2+ concentration ([Ca2+]cyt) and mRNA levels of activation markers (ACTA2 and COL3A1) in LX-2 cells. In mouse HSCs, CaV3.1 and CaV3.2 mRNA and protein expression increased during activation and were accompanied by Z944-sensitive inward currents. Among T-VDCC isoforms, CaV3.2 was identified as the predominant contributor to both T-type Ca²⁺ currents and resting [Ca2+]cyt. In a mouse model of metabolic dysfunction-associated steatohepatitis (MASH), CaV3.2 expression was selectively elevated in HSCs. Notably, administration of Z944 significantly attenuated MASH-associated liver fibrosis compared with controls. Collectively, these findings demonstrate that CaV3.2 expression and activity are upregulated during HSC activation, resulting in enhanced cytosolic Ca²⁺ signaling and fibrogenic responses. CaV3.2 may therefore represent a potential therapeutic target for liver fibrosis.