The role of metabolic stress, xanthine oxidase, NOX2, and redox-related signalling in regulating ACE2 protein expression in exercising human skeletal muscle

Abstract

The renin-angiotensin system (RAS) modulates skeletal muscle vascular and metabolic function depending on the balance between the "classical" angiotensin-converting enzyme (ACE1)/angiotensin II/angiotensin II type 1 receptor (AT1R) axis and the counter-regulatory angiotensin-converting enzyme 2 (ACE2)/angiotensin 1-7/Mas Receptor (MasR) and angiotensin II type 2 receptor (AT2R) pathways. Although ACE2 has been implicated in reactive oxygen species (ROS)-related responses, no direct mechanistic evidence in human skeletal muscle has been reported. This study aimed to determine whether the protein expression of ACE2 and other components of the RAS are modulated by intense exercise and redox-sensitive signalling mechanisms in human skeletal muscle. We hypothesised that exhaustive exercise would increase ACE2 expression, and that this effect would be amplified by severe acute hypoxia and post-exercise ischaemia via redox-signalling mechanisms. Eleven active men performed incremental exercise to exhaustion in normoxia (PIO2: 143 mmHg) and severe acute hypoxia (PIO2: 73 mmHg). At exhaustion, the circulation of one leg was occluded (300 mmHg) for 60 s. Muscle biopsies (vastus lateralis) were taken before and after exercise (at 10 and 60 s). ACE2 protein expression was increased at exhaustion, independently of inspired O2, and remained elevated during the 60 s post-exercise ischaemia, while it returned to baseline in the leg that recovered with free circulation. Transmembrane protease, serine 2 (TMPRSS2) protein expression increased at exhaustion and rose further with ischaemia. AT2R protein expression decreased modestly, whereas MasR, ACE1 and AT1R remained unchanged. Phospho-p47phox Ser359 (a proxy for NADPH oxidase 2 (NOX2) activation) and xanthine oxidase (XO) protein expression increased by exercise and even further by ischaemia. ACE2 correlated positively with TMPRSS2 protein expression and with redoxsensitive signalling and antioxidant enzyme expression. In another experiment, Zynamite (R) PX (a polyphenol mixture containing mangiferin and quercetin, known to inhibit NOX2 and XO in vitro) was administered every 8 h for 48 h before incremental exercise to exhaustion. Zynamite (R) PX prevented NOX2 activation and the increase of XO protein expression induced by exercise, thereby blunting the upregulation in ACE2 observed in the non supplemented group. Our data reveal that ACE2 is a fast, exercise-responsive enzyme that elevates its expression in human skeletal muscle during intense exercise via a redox-linked mechanism, a response inhibited by antioxidant polyphenol supplementation.