Role of NRF-2-Keap1 pathway modulation in endothelial cells on mitochondrial function in striated muscles

Introduction: Oxidative stress and mitochondrial dysfunction play a critical role in the development of cardiometabolic diseases. However, the exact interplay between local redox stress and mitochondrial dysfunction across various tissues is still poorly understood. Purpose: This study aims to determine whether endothelial-specific genetic modulation of the Nrf2-Keap1 signaling pathway, a central regulator of the antioxidant response, influences mitochondrial respiratory function in downstream tissues such as cardiac and skeletal muscles. METHOD: Cardiac tissues (left ventricle, n=4-10) and skeletal muscle (white gastrocnemius, n=4-13) were harvested for high-resolution respirometry from young mature C57BL/6J mice with genetic downregulation of Keap1 (Keap1 - ) or Nrf2 (Nrf2 - ), and wild type (WT). Mitochondrial respiratory function was assessed at 37°C in permeabilized muscle fibers for leak respiration, complex I-specific respiration, maximal ADP-stimulated respiration, complex II-specific respiration, electron transport system (ETS) capacity, and ETS reserve capacity. Results: In the white gastrocnemius muscle, the maximal ADP-stimulated respiration with convergent electron flow through complex I and II was not significantly different between groups (Keap1 - : 59 ± 22 pmolO 2 .s -1 .mg -1 , Nrf2 - : 59 ± 24 pmolO 2 .s -1 .mg -1 , WT: 64 ± 19 pmolO 2 .s -1 .mg -1 , p>0.05). All the other respiratory parameters were not significantly different between groups. In stark contrast, leak respiration (Nrf2 - : 102 ± 37 pmolO 2 .s -1 .mg -1 and WT: 63 ± 16 pmolO 2 .s -1 .mg -1 ; p = 0.034) and Complex I-specific respiration of the cardiac tissues were higher in Nrf2 - mice than WT (Nrf2 - : 180 ± 79 pmolO 2 .s -1 .mg -1 and WT: 88 ± 37 pmolO 2 .s -1 .mg -1 ; p = 0.026). As a result, the respiratory control ratio (RCR) in cardiac muscle was not significantly different between groups. Keap1 - mice showed a significant increase only in the ETS capacity compared with WT (Keap1 - : 199 ± 41 pmolO 2 .s -1 .mg -1 and WT: 123 ± 26 pmolO 2 .s -1 .mg -1 ; p = 0.002). Conclusions: These findings demonstrate that endothelial-specific knockdown of Keap1 and Nrf2 genes significantly modulated mitochondrial respiratory function in downstream cardiac tissues, with high metabolic turnover, but not in the predominantly fast-twitch glycolytic white gastrocnemius muscle, characterized by lower metabolic activity. The increased maximal mitochondrial respiratory rates with unchanged RCR in Nrf2 - and Keap1 - mice suggest a unique adaptation in cardiac muscle to compensate for greater mitochondrial leak caused by redox imbalance in endothelial cells. Together, these findings demonstrate the high sensitivity of cardiac tissues to redox stress from adjacent endothelial cells, which further highlights the critical role of the heart as a primary target for chronic diseases characterized by chronic oxidative stress and metabolic dysregulation. The University of Nebraska Omaha This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.