Age-Related Impairments in ATP Release by Red Blood Cells as an Important Contributor to Declines in Skeletal Muscle Blood Flow in Older Adults

Introduction

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality in developed countries and advancing age is the strongest independent risk factor for CVD. As such, the majority of deaths from CVD occur in adults over 60 years of age. Regular aerobic exercise is considered the first-line strategy for reducing CVD risk. However, exercise capacity is known to decline with aging and most older adults in the U.S. do not meet recommended physical activity guidelines. These limitations may be in part mediated by age-related impairments in skeletal muscle blood flow and oxygen delivery.

Regulation of skeletal muscle blood flow occurs through the release of vasoactive molecules by the vascular endothelium, the autonomic (sympathetic) nervous system, circulating humoral factors, and other mechanisms. The execution and coordination of these mechanisms to regulate skeletal muscle blood flow become impaired with aging. One emerging mechanism involved in the control of blood flow that may contribute to age-related declines in skeletal muscle blood flow is red blood cell (RBC) release of adenosine triphosphate (ATP), a vasodilatory factor that binds to purinergic receptors on the surface of endothelial cells to stimulate arterial dilation and blood flow, in response to RBC deoxygenation.

The investigators’ previous work elegantly showed that ATP release by RBCs is impaired in older adults and contributes to declines in skeletal muscle blood flow during exercise (Kirby et al. 2012). More recently (Racine & Dinenno et al. 2019), the investigators showed a mechanism underlying reduced ATP release by RBCs may be decreased RBC deformability, which refers to the ability of RBCs to change shape and allows them to pass through the microcirculation to transport and deliver oxygen to surrounding tissues. This previous work (Racine & Dinenno et al. 2019) isolated erythrocytes from older adults and modulated deformability ex vivo by inhibiting Rho-kinase, an enzyme involved in regulating RBC deformability by modulating actin cytoskeletal stiffness. These findings show that reduced RBC deformability secondary to increased Rho-kinase activation is an important factor in impaired ATP release with aging, but whether these mechanisms contribute to reductions in blood flow in vivo was not determined in these studies.

To advance these previous findings to an in vivo clinical setting and determine the role of age-related reductions in RBC deformability and ATP release in mediating age-related decreases in skeletal muscle blood flow, the investigators assessed whether systemic administration of a Rho-kinase inhibitor could ameliorate impairments in skeletal muscle blood flow observed in older adults (Racine et al. 2022).

Methods

Employing a randomized double-blind, placebo-controlled, cross-over design, 12 young (25 ± 3 years) and 13 older (65 ± 5 years) healthy men and women underwent a 60-minute intravenous infusion of a Rho-kinase inhibitor (fasudil) or volume-matched saline (placebo). Following infusions, subjects were exposed to hypoxia or performed dynamic handgrip exercise (order of trials randomized and counterbalanced), and outcome measures were assessed. Briefly, subjects were exposed to either normoxia (control; oxygen saturation ~98%) or systemic isocapnic hypoxia using a partial rebreathe system to achieve 3 minutes of steady-state hypoxia and elicit an oxygen saturation of 80%. The graded-intensity rhythmic handgrip exercise was performed by determining the subject’s maximum voluntary contraction (MVC) using maximal squeezes of a handgrip dynamometer, then performing 4 minutes of exercise at 5%, 15%, and 25% of MVC. A linear-array ultrasound probe was used to determine brachial artery mean blood velocity and diameter to calculate forearm blood flow (FBF) and forearm vascular conductance (FVC). Blood samples of venous plasma [ATP] and oxygen (O₂) were taken 15 minutes after the infusion and at the end of rest, hypoxia, and each handgrip exercise intensity. Arterial O₂ content, to calculate forearm O₂ consumption, was estimated using methods described previously by the laboratory. The authors also conducted ex vivo experiments to determine whether the effects of fausdil could be attributed to direct effects on RBCs. RBCs were isolated from subjects following saline and fasudil infusions, incubated for 30 minutes in normoxia, and then deoxygenated through exposure to hypoxia to replicate in vivo conditions during exercise. Intracellular and extracellular ATP release by RBCs was quantified during normoxia and hypoxia.

Results

The authors observed an age-related increase in mean arterial pressure (MAP) at rest and during systemic hypoxia and handgrip exercise during saline infusion. However, following fasudil, MAP was significantly reduced in older adults, abolishing age-related differences. Additionally, during normoxia and rest before handgrip exercise, FBF and FVC were not different between young and older adults during both saline and fasudil infusion. However, when exposed to systemic hypoxia and handgrip exercise, older adults exhibited an age-associated impairment in FBF and FVC response during saline that was rescued with fasudil infusion. Similarly, ATP effluent during normoxia and resting conditions during saline and fasudil infusion was not different between young and older adults. However, following exposure to systemic hypoxia, the older adults did not exhibit a significant response compared to normoxia under control conditions as was observed in young adults, yet this response was evident following treatment with fasudil. Further, under saline infusion during 25% MVC, older adults exhibited an impairment in ATP effluent compared to young adults that was rescued back to young levels with fasudil.

Of note, forearm O₂ delivery was not different between young and older adults during normoxia and rest under saline and fasudil conditions. However, during systemic hypoxia, fasudil significantly improved O₂ delivery in older adults compared to saline, and during handgrip exercise as fasudil almost eliminated the age associated reduction observed at 25% MVC compared to the saline condition (p=0.07); forearm O₂ extraction did not change across conditions in older adults. Together, these observations indicate a trend that fasudil improves (1) differences in forearm O₂ consumption (VO₂) between normoxia and hypoxia in older adults and (2) age-related declines in forearm VO₂ during handgrip exercise at 25% MVC.

Finally, in vivo infusion of fasudil tended to improve deoxygenation-induced release of extracellular ATP from isolated RBCs ex vivo in older adults relative to saline (p=0.08) (Racine et al. 2022).

Discussion and Future Directions

Overall, Racine et al. found that systemic administration of a Rho-kinase inhibitor improves impaired ATP release and reduced skeletal muscle blood flow during hypoxia and handgrip exercise with aging. This is supported by the use of fasudil, a Rho-kinase inhibitor, as a mechanistic probe to demonstrate that inhibiting Rho-kinase activity increases circulating ATP, assessed in vivo and ex vivo, and improves skeletal muscle blood flow in vivo in older adults. These results have added to our understanding of the mechanisms contributing to age-related reductions in skeletal muscle blood flow, which may have significant implications for improving exercise capacity and tolerance in older adults and, potentially, other clinical populations.

Age is an independent non-modifiable risk factor for many chronic diseases, such as diabetes, chronic kidney disease, chronic obstructive pulmonary disease, and Alzheimer’s disease and related dementias. Many of these age-related conditions are associated with reduced exercise capacity, possibly as a consequence of decreased skeletal muscle blood flow during exercise. It is possible that these declines could be partially mediated by reductions in RBC deformability leading to impairments in RBC release of ATP, making the findings by Racine biomedically relevant in these groups and justifying translation of these findings to clinical populations.

Additionally, estrogen deficient post-menopausal women are a particular group that should be studied as they are at increased risk for CVD in part due to faster declines in endothelial function, basal blood flow, and vascular conductance following the menopausal transition compared to age-matched men (Moreau et al. 2003). Declines in circulating estradiol in post-menopausal women has been shown to be a primary mechanism contributing to impaired vascular control of blood flow (Moreau et al. 2003). The current study showed that both older men and estrogen-deficient postmenopausal women exhibit age-related decrements in ATP release, leading to reduced skeletal muscle blood flow during exercise (Racine et al. 2022); a previous study by the authors indicates this may be mediated by declines in RBC deformability with aging, as assessed ex vivo (Racine & Dinenno et al. 2019). Furthermore, it has been previously shown that pre-menopausal women show increased RBC deformability, in part related to elevated estradiol levels, compared to young men (Grau et al. 2018). However, it remains unclear (1) whether this sex difference exists after the menopause transition; and (2) whether declines in estradiol directly influence RBC deformability and skeletal muscle blood flow in post-menopausal women. RBCs carry receptors for estrogens (Grau et al. 2018), suggesting that reductions in estradiol may contribute to reductions in RBC deformability and be an additional mechanism of vascular dysfunction with aging. To address these research gaps the outcomes described in Racine et al. should be assessed in a larger number of subjects to provide proper statistical power to analyze sex differences in older adults. Additionally, ex vivo experiments exposing RBCs isolated from pre- and post-menopausal women to physiologically relevant levels of estradiol should be conducted to investigate the possible role of menopause-related reductions in estradiol on mediating these effects on RBC deformability and blood flow.

Another future direction may be to determine the contribution of Rho-kinase in modulating RBC nitric oxide (NO) production, which, in turn, may stimulate arterial dilation directly and/or have effects on RBC deformability and ATP release. Although eNOS is predominantly found in the endothelium, recent data suggest eNOS may also be present in RBCs, that when activated, increases NO bioavailability to induce vasodilation (Leo et al. 2021). As mentioned by the investigators (Racine et al. 2022), fasudil is known to enhance eNOS activity in addition to improving RBC deformability. The investigators did not assess the relative contribution of fasudil-mediated improvements to endothelial-located eNOS activation in reducing age-related declines in skeletal muscle blood flow but acknowledged this as an experimental limitation that should be further explored. However, in addition to assessing fasudil’s role in endothelial-located eNOS activation, fasudil’s role on activation of RBC-located eNOS should also be assessed.

In summary, the findings by Racine et al. provide novel evidence addressing how age-related reductions in skeletal muscle blood flow are in part mediated by impaired deformability-mediated RBC release of ATP. By adding to the collective understanding of the mechanisms involved in the regulation of skeletal muscle blood flow, these findings could have important implications for improving exercise capacity in older adults. Furthermore, to translate the significance of these findings to a wider population, the mechanistic role of RBC deformability in impaired blood flow should be extended to clinical populations. These endeavors may ultimately lead to interventions aimed at improving age-related reductions in exercise tolerance and vascular function to decrease CVD risk in older adults and clinical populations.