Even breathing gets old: Impact of ageing on small arteries of the diaphragm

Breathing … it is one of the first and last things we do outside the womb. Striated skeletal muscle comprises 30–40% of total body mass and is generally considered a ‘voluntary’ muscle as its movements are largely controlled consciously through the central nervous system. An exception to this paradigm is the diaphragm, a striated muscle whose contractile activity drives inspiration by lowering intrapleural pressure, leading to expansion of the lung parenchyma and the inhalation of air. Rhythmic contractions of the diaphragm occur throughout our lifetime (12–18 per minute in resting adult humans in the absence of overriding neural signals from higher brain centres) and are driven by neuronal circuits within the brain stem that communicate to the diaphragm via phrenic motor nerves. Similar to other tissues exhibiting regular activity (e.g. heart, brain), the diaphragm requires robust and uninterrupted blood flow to maintain rhythmic contractions. Despite this, control of diaphragm blood flow is relatively understudied as a result of its location and because surgical isolation interrupts its normal function. Anatomically, the diaphragm is divided into three main sections (i.e. sternal, costal and lumbar), with the medial costal region responsible for the majority of inspiratory work. As a result, this region not only normally receives a greater share of diaphragmatic blood flow to support increased contractile load, but also may be more vulnerable to impairment with abrupt changes in tissue blood flow. Ageing is associated with a decline in contractile activity of the diaphragm, which can compromise breathing activity, increase morbidity and reduce overall quality of life in aged individuals. Although a variety of scenarios may contribute to ageing-related declines in diaphragmatic function (e.g. reduced motor nerve activity, muscular weakness, injury, etc.), Horn et al. (2025) have investigated the hypothesis that structural and/or functional alterations in the vasculature of the medial costal region may impair contractility by affecting vasoactive mechanisms that regulate local tissue perfusion (e.g. reactive hyperaemia) and dynamic blood flow redistribution to match oxygen demand with supply. Using young (4–6 months) and old (22–24 months) male and female Fisher 344 rats, Horn et al. (2025) compared endothelium-dependent, acetylcholine (ACh)-evoked dilatation in isolated, myogenically active 1A resistance arteries from the medial costal diaphragm. First order (1A) arterioles from young and old animals developed similar levels of spontaneous myogenic tone at 66 mmHg intraluminal pressure (i.e. 31–34% of maximal arterial diameter) and this was comparable between males and females. Arterioles from the older rats exhibited larger passive diameters and decreased wall:lumen ratios, reminiscent of the ageing-related structural remodelling reported in arterioles from other skeletal muscle beds. Endothelium-dependent dilatation in response to bath-applied ACh was largely nitric oxide (NO)-dependent in both females and males at each age; however, the magnitude of response was decreased in older animals without a significant change in sensitivity to ACh (i.e. reduction in Emax, similar EC50 values). These observations are thus consistent with the presence of endothelial dysfunction, a common consequence of ageing and linked to increased vascular oxidative stress. Given these relations, Horn et al. (2025) observed that exogenous treatment of arterioles with Tempol, a chemical scavenger of reactive oxygen species (ROS), + catalase, a H2O2 degrading enzyme, inhibited ACh-evoked dilatation to a greater extent in old vs. young arterioles, independent of sex. In parallel, Horn et al. (2025) demonstrated a significant increase in the protein expression of the antioxidant related enzyme superoxide dismutase 2 (SOD2) and trends for greater expression of SOD1 and catalase in medial costal muscle from older rats. Collectively, these observations suggest that old arterioles may depend more upon ROS/H2O2 signalling to maintain endothelium-mediated dilatation under conditions of decreasing NO bioavailability (i.e. endothelial dysfunction), although the cellular source(s) of ROS and their downstream signalling targets in the vascular wall were not identified. Older animals also exhibited a lower density of feed arteries in the medial costal diaphragm (i.e. arterial rarefaction) that correlated with lower (whole muscle) protein expression of the pro-angiogenic molecule vascular endothelial growth factor that was more pronounced in females. A lower density of feed arteries would presumably impact the delivery and redistribution of local blood flow in the medial costal muscle. The putative contribution of ROS signalling to endothelium-dependent dilatation in older medial costal arterioles parallels findings in coronary resistance arteries from patients with coronary artery disease, which also exhibit compromised NO action and a compensatory shift to ROS/H2O2 vasodilatory signalling (Beyer et al., 2017). ROS and H2O2 participate in cellular signal transduction and H2O2 itself may mimic NO vasodilatory action in myogenically active arteries by activating protein kinase G independent of cGMP elevation (Khavandi et al., 2016). In addition to chemical signalling pathways, endothelial calcium-activated K+ (KCa) channels also contribute to evoked vasodilatation via electrical signalling and hyperpolarization of vascular smooth muscle (Jackson, 2021). By increasing the electrical driving force for external calcium entry (Behringer & Segal, 2015; Sheng et al., 2009), these channels can potentially act upstream of endothelial NO synthase to promote its calcium-dependent activation. It may therefore be of interest to examine a potential role for these channels (i.e. KCa2.3 and KCa3.1) in small resistance arteries of the diaphragm in response to vasodilatory stimuli acting on the endothelium (e.g. hormones, shear stress). Because moderate exercise is recognized to oppose endothelial dysfunction in humans, it may be of interest to explore whether aerobic training prevents or possibly reverses ageing-related deficits in diaphragmatic blood flow and function. A key question from the structural perspective is whether the loss of vessels (viewed as blood perfused elements) represented a true structural loss or was functional in nature. Furthermore, because rarefaction is often considered a phenomenon affecting capillaries as opposed to larger arterioles and conduit vessels, it will be of interest to dissect the precise tissue and cellular mechanisms underlying the observed regression of feed arteries. For example, does this regression result from declining function of the diaphragm or downstream capillary rarefaction secondary to growth factor disturbances, or does it involve vessel loss because of changing levels of matrix degrading enzymes, ROS, inflammatory mediators and cell death? Such studies may also be informed by more extensive time course considerations, examining the progression from young to middle aged to elderly with a focus on primary/causative events vs. responses to changing conditions (e.g. when does rarefaction/regression begin and does this align with changes in function?). Because arterial wall stiffness commonly increases with age, it will be important to discern the impact of vascular stiffening on the functional and structural properties of diaphragmatic arteries, as well as how both active cellular elements and altered mechanical properties of the extracellular matrix contribute mechanistically. By highlighting the impact of ageing on arterial function and structure in the diaphragm, the work by Horn et al. (2025) should help identify potential ageing-related strategies to sustain normal tissue blood flow, diaphragmatic activity and respiratory function. Because ageing in humans is often accompanied by comorbidities (including hypertension, obesity and diabetes), which themselves contribute to vascular dysfunction, it will also be of interest to investigate how these conditions interact and impact control of diaphragm blood flow and function as we age. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. No competing interests declared. A.B and M.H were responsible for the conception or design of the work; drafting the work or revising it critically for important intellectual content; and approving the final version submitted for publication. Both authors agree to be accountable for all aspects of the work. No funding was received.