(25) Cabotegravir Impairs Mitochondrial Functions and Mitophagy in Human Trophoblast Cells

Haijun Gao¹, Oluebubechuk E. Eze¹, Jared Hall¹, Fuller Gao¹, Nickie Andescavage², Mark Burke¹, Rachel K. Scott³.

1. Department of Physiology & Biophysics, Howard University College of Medicine, Washington DC, USA

2. Division of Neonatal-Perinatal Medicine, Children’s National Hospital, Washington DC, USA

3. Department of Women’s and Infants’ Services, MedStar Health Research Institute, Washington DC, USA

Abstract Text:

Cabotegravir (CAB), an extended-release integrase inhibitor, is the first FDA approved long-acting, injectable option for HIV pre-exposure prophylaxis and is gaining preferences in pregnant women. To date, there is limited safety and outcome data in pregnancy after CAB exposure. However, a handful of studies indicated that CAB has adverse effects on the outcomes of pregnancy and interferes with embryonic stem cell differentiation. The placenta plays a central role in establishing and maintaining pregnancy, by forming multiple trophoblast sublineages. The placenta with extensive trophoblast differentiation during early pregnancy is vulnerable to nutritional, hormonal, and oxidative stresses. Mitochondria play a critical role in maintaining cellular homeostasis. Mitochondrial homeostasis is regulated by mitochondrial biogenesis and mitophagy, while impaired mitophagy occurs in major pregnancy-related disorders and is associated with reduced mitochondrial ATP production and enhanced reactive oxygen species (ROS) production in trophoblast cells. In this study, we hypothesized that CAB impairs mitochondrial functions and mitophagy in human trophoblast cells. To test this hypothesis, we used the following methods. HTR-8/SVneo cells, models for human extravillous trophoblast cells, were cultured under optimized conditions. In CAB toxicity assay, cells were treated with CAB at doses of a wide or narrow range, and the cell death rate was determined by propidium iodide (PI) staining. To measure mitochondrial membrane potential, TMRM staining after CAB treatment (4 and 8 µg/ml) for 24 hours. To measure mitochondrial mass, mitoTracker Green staining after CAB treatment (4 and 8 µg/ml) for 24 hours. To measure mitochondrial ATP production, Seahorse cell Mito Stress Test was conducted after cells were treated with CAB (4 µg/ml) for 24 hours. To analyze mitophagy flux in response to CAB, mitochondrial fractions were extracted from cells treated with CAB (4 µg/ml), chloroquine (CLQ) and their combination, respectively, and the protein abundance of mitophagy mediators (LC3II and SQSTM1) was analyzed by Western blotting. All numerical parameters between the control (CT) and treatment group were analyzed by ANOVA (n=3-5). The main findings in this study include: 1) CAB at the dose of 4 µg/ml or lower did not cause cell death, but CAB at higher doses increased the cell death rate, therefore, the dose of 4 µg/ml was selected in further investigations to avoid significant cellular toxicities; 2) CAB at the dose of 4 and 8 µg/ml reduced mitochondrial membrane potential by 1.09- and 1.21-fold (p < 0.05), respectively, compared to CT cells; 3) Mitochondrial mass reduced by 1.14-fold (p < 0.05) by CAB at the dose of 8 µg/ml, but not at the dose of 4µg/ml; 4) Mitochondrial ATP production was lower in cells treated with CAB, with reduced basal respiration, maximal respiration, ATP-production coupled respiration, coupling efficiency and spare respiratory capacity; 5) The abundance of LC3II proteins in mitochondrial fractions was reduced by 1.45-fold (p < 0.05) by CAB but increased by 6.91-fold (p < 0.001) by the combination of CAB and CLQ, compared to that in CT cells, respectively.  Similarly, the abundance of SQSTM1 proteins followed the same pattern of changes as LC3II in response to the treatment of CAB, CLQ and their combinations. These results indicate that CAB impairs mitochondrial ATP production, reduces mitochondrial membrane potential and mitophagy in human extravillous trophoblast cells, thus interfering with placental development, functions, and consequent fetal growth. (Supported by DC CFAR Pilot Award, NIH research grants R16HD116702 and R03HD95417).