Exploring circadian regulation of ACE2 and antioxidant regulator Nrf2: Implications for skeletal muscle biopsy

Abstract

INTRODUCTION: After the COVID-19 pandemic, ACE2 has garnered substantial attention due to its pivotal role as the primary entry receptor for SARS-CoV-2 into host cells. Our recent study highlights the abundant presence of ACE2 protein in human skeletal muscle, which varies between sexes [1]. Elevated ACE2 protein levels may enhance infection, while diminished levels have been linked to age-related muscle mass decline, potentially exacerbating inflammation and influencing disease severity. As a key antioxidant and anti-inflammatory defence mechanism, NRF2 may exhibit a complex interaction with ACE2, suggesting a role in modulating oxidative stress responses and maintaining cellular homeostasis [2]. Moreover, ACE2 protein expression has been linked to brain and muscle Arnt-like protein 1 (BMAL1), a circadian rhythm signal, indicating temporal fluctuations influenced by circadian and environmental factors [3]. Understanding protein expression dynamics across different time points in skeletal muscle could provide crucial insights and refine biopsy extraction methodologies.

METHODS: Twelve volunteers (6 males and 6 females) participated in our study. Following a 12-hour fasting period, biopsies of the vastus lateralis muscle were taken from randomly selected legs at five time points (baseline, 4h, 8h, 12h, and 24h), totalling 60 biopsies. Leg allocation for biopsy was randomized but consistent for each participant. We ensured strict adherence to biopsy time intervals. Protein expression levels were analysed using Western Blot. Temporal changes were assessed via ANOVA in SPSS, while correlations were explored using mixed linear models in JAMOVI.

RESULTS: Compared to baseline levels, ACE2 protein expression increased by 1.9 and 1.7-fold after 4 and 8 hours, respectively (p<0.05). Similarly, significant increases were observed in BMAL (1.6-fold and 1.4-fold) and Nrf2 (2 and 1.5-fold) expression at the same time points (p<0.05). Strong linear associations were found between ACE2 and NRF2 (R² marginal = 0.71, R² conditional = 0.94, p<0.01) and between ACE2 and BMAL (R² marginal = 0.73, R² conditional = 0.84, p<0.01). Both genders showed similar responses.

CONCLUSION: Our study highlights the temporal modulation of ACE2 and Nrf2 protein expression in skeletal muscle, possibly influenced by circadian rhythms. This regulatory mechanism may significantly contribute to maintaining cellular homeostasis and responding to hormonal signals. Moreover, understanding the dynamic interplay of these signals reveals intricate mechanisms governing oxidative stress responses and circadian regulation within skeletal muscle. Considering biopsy timing in skeletal muscle signalling research is crucial for accurately capturing intervention-related changes and properly interpreting results.

GRANT: PID2021-125354OB-C21 and PID2021-125354OB-C22.