Age-Related Diaphragm Vasomotor Dysfunction: The Role of Reactive Oxygen Species

Introduction: Advancing age decreases inspiratory muscle (i.e., diaphragm) blood flow capacity and vascular endothelial function. Excess reactive oxygen species (ROS) formation plays a central role in the development of age-related endothelial dysfunction in coronary and locomotory skeletal muscle vascular beds by diminishing nitric oxide (NO) bioavailability and NO-mediated signaling. Although oxidative stress induces endothelial dysfunction, superoxide (O 2 - )-derived ROS, such as peroxynitrite (ONOO - ) and hydrogen peroxide (H 2 O 2 ), are mediators of normal endothelial-dependent vasodilatory signaling. However, the involvement of ROS signaling in diaphragm vascular function and whether the contribution of these molecules to endothelial signaling changes with old age is unknown. We hypothesized that aging would blunt diaphragm arteriolar endothelial-dependent dilation (EDD) via increased ROS formation and that ROS-mediated signaling contributes to EDD in diaphragm first-order (1A) arterioles of young and old rats. Methods: Young (Y) and Old (O) Fisher 344 rats of both sexes (n=49) were used for this investigation. 1As from the medial costal diaphragm were isolated, cannulated, and pressurized at 90 cmH 2 O and allowed to equilibrate for 60 min to develop spontaneous tone. Thereafter, EDD responses to cumulative doses of acetylcholine (ACh; 10 -9 to 10 -4 M) in the absence and presence of Tempol (TEMP; 100 µM), and polyethylene glycol labeled catalase (PEG-Cat; 100U/ml) were determined. Results: Spontaneous tone development (%) was not different between Y (33 ± 7%) and O (30 ± 7%; p>0.05). Maximal ACh-mediated relaxation (%) was significantly reduced in O (58 ± 15%) versus Y (78 ± 11%;p<0.05). TEMP decreased ACh-mediated relaxation by ~20% in Y and ~45% in O diaphragm 1As (p<0.05). TEMP+PEG-Cat had no effect in Y vessels but further decreased ACh-mediated dilation in O vessels (Tempol: 32 ± 11% vs Tempol+PEG-Cat: 22 ± 10%; p<0.05). Neither TEMP nor TEMP+PEG-Cat treatment abolished age-related differences in EDD of diaphragm 1As (p<0.05). Conclusion: These findings demonstrate that ROS are obligatory for EDD in the diaphragm vasculature of Y and O rats. Further, aging increases the reliance on O 2 - and H 2 O 2 vasodilatory signaling. In contrast to locomotory skeletal muscle, H 2 O 2 does not appear to play a role in EDD in the diaphragm vasculature of Y rats. These data extend the negative vascular effects of advancing age to the diaphragm vasculature and suggest the vascular control mechanisms in the highly specialized diaphragm are distinctly different from locomotory skeletal muscle arterioles. Ruth L. Kirschstein National Research Service Awards from the National Heart, Lung, and Blood Institute awarded to AGH (1F31HL167618-02) and KMS (1F31HL170643-02). 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.