Hypoxia induces pulmonary vascular remodeling by inhibiting HectH9-mediated ubiquitination and degradation of hexokinase 2 in pulmonary artery smooth muscle cells

Objective To investigate the role and potential mechanism of hexokinase (HK) 2 and its ubiquitination in hypoxia-induced pulmonary hypertension (PH) in rat and proliferation of pulmonary artery smooth muscle cells (PASMCs).

Methods  Twenty-four male SD rats (180–200 g) were included and randomly divided into a normoxic control group, a 2-week hypoxia group (14 days of normoxia followed by 14 days of 10% O₂ hypoxia), and a 4-week hypoxia group (28 days of 10% O₂ hypoxia), with 8 rats in each group. Right ventricular systolic pressure (RVSP) of rats was measured by right heart catheterization. Right ventricular hypertrophy index (RVHI), pulmonary arteriole wall area percentage (WA%) and wall thickness percentage (WT%) were calculated. Protein levels of HK2 and the E3 ubiquitin ligase HectH9 in pulmonary artery tissues were assessed by Western-blot, and the activity of HK was assessed by colorimetric method. In vitro, PASMCs were cultured under 1% O₂ hypoxic conditions. Protein expression of HK2, HectH9, mitochondrial dynamin-related protein 1 (DRP1), mitochondrial fission factor (MFF) and mitochondrial fission 1 protein (FIS1) were detected by Western-blot. Colorimetric assays were employed to measure HK activity and glucose-6-phosphate (G6P) content. Cell proliferation levels were assessed using the cell counting kit 8 (CCK-8) assay. Mitochondrial morphology was observed via immunofluorescence. Co-immunoprecipitation was used to examine the interaction between HK2 and HectH9 and the level of HK2 ubiquitination. The effects of HectH9 silencing on HK2 ubiquitination, protein expression, activity, G6P content, and cell proliferation were further evaluated.

Results Compared with the rats in the normoxic group, rats exposed to hypoxia for 2 and 4 weeks increased in RVSP 2-week: (33.72±3.84) mmHg, 4-week: (50.54±5.30) mmHg vs normoxia: (27.34±3.77) mmHg; t=2.96, 10.41, both P＜0.01, RVHI 2-week: (38.49±2.13)%, 4-week: (42.38±2.34)% vs normoxia: (23.65±1.54)%, t=14.61, 18.65, both P＜0.01, WA% 2-week: (74.14±4.53)%, 4-week: (88.63±3.81)% vs normoxia: (55.47±5.60)%, t=7.75, 13.76, both P＜0.01, and WT% 2-week: (43.61±4.13)%, 4-week: (58.66±3.24)% vs normoxia: (27.54±4.22)%, t=8.02, 15.52, both P＜0.01, accompanied by upregulated HK2 protein expression and HK activity, and downregulated HectH9 expression in pulmonary arteries (all P＜0.05). The expression of HK2 protein, HK activity, G6P content, cell proliferation level and mitochondrial fragmentation degree in PASMCs treated with hypoxia for 48 hours were all higher than those in the normoxia controls (all P＜0.05), whereas the HectH9 expression (P＜0.05) and HK2 ubiquitination levels were lower. Overexpression of HectH9 via plasmid transfection under hypoxia resulted in higher HK2 ubiquitination levels than hypoxic PASMCs; whereas PASMCs with HectH9 knockdown showed higher HK2 protein expression, enzymatic activity , G6P content, and proliferation levels compared to normoxic controls (all P＜0.05).

Conclusion Hypoxia promotes the accumulation of HK2 by downregulating HectH9 expression and inhibiting HK2 ubiquitination and degradation, thereby disturbing DRP1/MFF-mediated mitochondrial fission homeostasis and promoting PASMCs proliferation and pulmonary vascular remodeling.