Rho-kinase inhibition improves haemodynamic responses and circulating ATP during hypoxia and moderate intensity handgrip exercise in healthy older adults

Abstract

Skeletal muscle haemodynamics and circulating adenosine triphosphate (ATP) responses during hypoxia and exercise are blunted in older (OA) vs. young (YA) adults, which may be associated with impaired red blood cell (RBC) ATP release. Rho-kinase inhibition improves deoxygenation-induced ATP release from OA isolated RBCs. We tested the hypothesis that Rho-kinase inhibition (via fasudil) in vivo would improve local haemodynamic and ATP responses during hypoxia and exercise in OA. Healthy YA (25 ± 3 years; n = 12) and OA (65 ± 5 years; n = 13) participated in a randomized, double-blind, placebo-controlled, crossover study on two days (≥5 days between visits). A forearm deep venous catheter was used to administer saline/fasudil and sample venous plasma ATP ([ATP]_V ). Forearm vascular conductance (FVC) and [ATP]_V were measured at rest, during isocapnic hypoxia (80% $S_{p{O}_2}$ ), and during graded rhythmic handgrip exercise that was similar between groups (5, 15 and 25% maximum voluntary contraction (MVC)). Isolated RBC ATP release was measured during normoxia/hypoxia. With saline, ΔFVC was lower (P < 0.05) in OA vs. YA during hypoxia (∼60%) and during 15 and 25% MVC (∼25-30%), and these impairments were abolished with fasudil. Similarly, [ATP]_V and ATP effluent responses from normoxia to hypoxia and rest to 25% MVC were lower in OA vs. YA and improved with fasudil (P < 0.05). Isolated RBC ATP release during hypoxia was impaired in OA vs. YA (∼75%; P < 0.05), which tended to improve with fasudil in OA (P = 0.082). These data suggest Rho-kinase inhibition improves haemodynamic responses to hypoxia and moderate intensity exercise in OA, which may be due in part to improved circulating ATP. KEY POINTS: Skeletal muscle blood flow responses to hypoxia and exercise are impaired with age. Blunted increases in circulating ATP, a vasodilator, in older adults may contribute to age-related impairments in haemodynamics. Red blood cells (RBCs) are a primary source of circulating ATP, and treating isolated RBCs with a Rho-kinase inhibitor improves age-related impairments in deoxygenation-induced RBC ATP release. In this study, treating healthy older adults systemically with the Rho-kinase inhibitor fasudil improved blood flow and circulating ATP responses during hypoxia and moderate intensity handgrip exercise compared to young adults, and also tended to improve isolated RBC ATP release. Improved blood flow regulation with fasudil was also associated with increased skeletal muscle oxygen delivery during hypoxia and exercise in older adults. This is the first study to demonstrate that Rho-kinase inhibition can significantly improve age-related impairments in haemodynamic and circulating ATP responses to physiological stimuli, which may have therapeutic implications.

Keywords: ATP; ageing; blood flow; exercise; fasudil; hypoxia.

Figure 1. Overall experimental design and experimental visit timeline

A, double‐blind, placebo‐controlled, randomized, crossover experimental design. After screening, eligible subjects were randomized in a double‐blind manner to receive an infusion of either saline (placebo control) or fasudil for their first experimental visit. Subjects then received the opposite treatment for their second experimental visit, with at least 5 days between visits. B, experimental visit timeline. After placement of the venous catheter and treatment infusion, experimental measures were made during systemic isocapnic hypoxia and graded‐intensity rhythmic handgrip exercise trials; order of the trials was randomized and counterbalanced between subjects, but kept the same for both visits within a subject. Timing of blood sampling is indicated by arrows (Fig. 1B), with samples for plasma [ATP] taken under steady‐state conditions at rest, during hypoxia and the end of each exercise workload.

Figure 2. Effects of age and fasudil on forearm haemodynamics and plasma ATP during systemic hypoxia

A and B, fasudil significantly increased absolute forearm blood flow (FBF) and vascular conductance (FVC) in older adults during hypoxia; differences between age groups were not significant. C and D, age‐related impairments in ΔFBF and ΔFVC responses from normoxia to hypoxia were reversed with fasudil. E and F, venous plasma ATP ([ATP]_V) and ATP effluent increased vs. normoxia in older adults with fasudil, but not saline; differences between age groups were not significant. ^* P < 0.05 vs. saline; †P < 0.05 vs. young; ‡P < 0.05 vs. normoxia.

Figure 3. Effects of age and fasudil on forearm haemodynamics and plasma ATP during rhythmic handgrip exercise

A and B, with saline, forearm blood flow (FBF) was impaired during 25% maximum voluntary contraction (MVC) and forearm vascular conductance (FVC) was impaired during 15% and 25% MVC in older adults. Fasudil significantly increased FBF and FVC at 25% MVC in older adults and decreased FVC at 25% MVC in young adults. C and D, ΔFBF and ΔFVC responses from rest to exercise were impaired in older vs. young adults at 25% MVC with saline and were improved with fasudil in older adults, but not younger. E and F, with saline, [ATP]_V and ATP effluent were significantly reduced in older vs. young adults at 5% and 25% MVC, respectively, and improved with fasudil at these same exercise intensities in older adults, but not younger. ^* P < 0.05 vs. saline; †P < 0.05 vs. young; ‡P < 0.05 vs. rest ([ATP]_V only).

Figure 4. Effects of age and fasudil on forearm oxygen delivery, extraction and consumption during hypoxia (A, C and E) and rhythmic handgrip exercise (B, D and F)

A, C and E, there were no differences in forearm O₂ delivery (A), extraction (C), or consumption (E) between young and older adults. ${\dot{V}}_{{\mathrm{O}}_2}$ tended to decrease during hypoxia in older adults with saline (E) and O₂ delivery was improved with fasudil (A). B, D and F, at 25% maximum voluntary contraction (MVC), forearm O₂ delivery (B) and ${\dot{V}}_{{\mathrm{O}}_2}$ (F) were impaired in older vs. young adults with saline, which was no longer different with fasudil. ^* P < 0.05 vs. saline; †P < 0.05 vs. young; ‡P < 0.05 vs. normoxia.

Figure 5. Effects of donor age and in vivo fasudil administration on red blood cell ATP release and intracellular ATP in normoxia and hypoxia

A, extracellular ATP (i.e. ATP release) in hypoxia only increased significantly vs. normoxia from RBCs of older adults with fasudil, whereas it increased from RBCs of young adults in both conditions. B, the mean percentage increase in extracellular ATP from normoxia to hypoxia was impaired from RBCs of older adults in the saline and fasudil conditions, but tended to improve with fasudil compared to saline in older adults (P = 0.08). C, intracellular ATP increased from normoxia to hypoxia in all conditions and there were no differences between young and older adults. ^* P < 0.05 vs. saline; †P < 0.05 vs. young; ‡P < 0.05 vs. normoxia.