Vascular ATP-sensitive K⁺ channels support maximal aerobic capacity and critical speed via convective and diffusive O₂ transport

Abstract

Vascular ATP-sensitive K⁺ (K_ATP) channels support skeletal muscle blood flow and microvascular oxygen delivery-to-utilization matching during exercise. However, oral sulphonylurea treatment for diabetes inhibits pancreatic K_ATP channels to enhance insulin release. Herein we tested the hypotheses that: i) systemic K_ATP channel inhibition via glibenclamide (GLI; 10 mg kg⁻¹ i.p.) would decrease cardiac output at rest (echocardiography), maximal aerobic capacity $(\dot{\mathrm{V}}{\text{O}}_{2}max)$ and the speed–duration relationship (i.e. lower critical speed (CS)) during treadmill running; and ii) local K_ATP channel inhibition (5 mg kg⁻¹ GLI superfusion) would decrease blood flow (15 μm microspheres), interstitial space oxygen pressures (PO₂is; phosphorescence quenching) and convective and diffusive O₂ transport ($\dot{\mathrm{Q}}{\text{O}}_2$ and DO₂, respectively; Fick Principle and Law of Diffusion) in contracting fast-twitch oxidative mixed gastrocnemius muscle (MG: 9% type I+IIa fibres). At rest, GLI slowed left ventricular relaxation (2.11 ± 0.59 vs. 1.70 ± 0.23 cm s⁻¹) and decreased heart rate (321 ± 23 vs. 304 ± 22 bpm, both P < 0.05) while cardiac output remained unaltered (219 ± 64 vs. 197 ± 39 ml min⁻¹, P > 0.05). During exercise, GLI reduced $\dot{\mathrm{V}}{\text{O}}_{2}max$ (71.5 ± 3.1 vs. 67.9 ± 4.8 ml kg⁻¹ min⁻¹) and CS (35.9 ± 2.4 vs. 31.9 ± 3.1 m min⁻¹, both P < 0.05). Local K_ATP channel inhibition decreased MG blood flow (52 ± 25 vs. 34 ± 13 ml min⁻¹ 100 g tissue⁻¹) and PO₂is_nadir (5.9 ± 0.9 vs. 4.7 ± 1.1 mmHg) during twitch contractions. Furthermore, MG $\dot{\mathrm{V}}{\mathrm{O}}_2$ was reduced via impaired $\dot{\mathrm{Q}}{\mathrm{O}}_2$ and DO₂ (P < 0.05 for each). Collectively, these data support that vascular K_ATP channels help sustain submaximal exercise tolerance in healthy rats. For patients taking sulfonylureas, K_ATP channel inhibition may exacerbate exercise intolerance.

Introduction

Sulphonylureas are the most popular second-line anti-diabetic drug prescribed to patients with Type 2 diabetes mellitus (T2DM, Montvida et al. 2018), enhancing insulin release from pancreatic beta cells by inhibition of ATP-sensitive potassium (K_ATP) channels. This is true irrespective of the increased risk of adverse cardiovascular events (Simpson et al. 2006, 2015; Abdelmoneim et al. 2016), developing heart failure (HF; McAlister et al. 2008; Kristiansen et al. 2011) and all-cause mortality (Simpson et al. 2015). K_ATP channels are metabolic sensors that are also present in neural, vascular (endothelial) and muscle (smooth, cardiac and skeletal) tissue, contributing significantly to the hyperpolarization of membrane potentials via K⁺ efflux and subsequent reductions in calcium ion influx. Attention to this category of K⁺ channels, and their physiological significance in metabolic control during exercise, has increased with the use of genetic knockout/down models (Flagg et al. 2010). However, the use of genetically altered animal models may result in unknown/unrelated systemic modifications and confound the translatability of K_ATP channel function to humans (Kane et al. 2004).

Current data from animal and human studies modulating K_ATP channel function with inhibitors (i.e. glibenclamide (GLI), tolbutamide) and activators (pinacidil) suggest that normal K_ATP channel function plays a significant role in limiting myocardial damage following ischaemic events in sedentary and exercise-trained animals (cardiac; Brown et al. 2005a,b), enhance reactive and functional hyperaemia to skeletal muscle (vascular; Banitt et al. 1996; Bijlstra et al. 1996; Saito et al. 1996; Hammer et al. 2001; Keller et al. 2004; Lu et al. 2013; Holdsworth et al. 2015; but not all, Farouque & Meredith, 2003), and reduce skeletal muscle tension between contractions (myocyte; Gong et al. 2000; Matar et al. 2000). Whereas systemic administration of GLI has been shown to decrease exercising limb blood flow (Keller et al. 2004; Holdsworth et al. 2015) and maximal aerobic capacity ($\dot{\mathrm{V}}{\mathrm{O}}_{2}max$; Lu et al. 2013), it remains unknown whether these cardiovascular impairments are mediated through reductions in cardiac function, vascular function within skeletal muscle, or both. As adequate energy production via oxidative metabolism dictates contractile function during fatiguing activity and depends on heterogeneous oxygen transport within muscle (Wilson et al. 1977; Hogan et al. 1992; Richardson et al. 1998), maximal oxygen uptake ($\dot{\mathrm{V}}{\mathrm{O}}_{2}max=\dot{\mathrm{Q}}max\times \mathrm{maximal}a-v{\text{O}}_2$ difference) relies on a prodigious increase in cardiac output $(\dot{\mathrm{Q}})$ combined with a highly effective red blood cell distribution and O₂ extraction within active skeletal muscle (arterial–venous O₂ content) (reviewed by Laughlin et al. 2012; Poole & Jones, 2012). Notwithstanding the importance of $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$, the ability to sustain high-intensity exercise and daily physical tasks are more appropriately determined via a submaximal threshold (i.e. critical speed (CS) or critical power) where oxidative metabolism meets metabolic demand below this threshold but, above this threshold, increases infast-twitch fibre recruitment, fatigue-related metabolite production, and O₂ consumption leading to $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ and task failure (Monod & Scherrer, 1965; Poole et al. 1988, 2016; Jones et al. 2008; Copp et al. 2010). Importantly, it remains unknown how vascular K_ATP channels contribute to O₂ transport within highly oxidative fast-twitch muscles and their role in supporting fatiguing exercise, especially as the proportional contribution of these channels to the overall vascular response may increase in disease (Holdsworth et al. 2017).

Therefore the current investigation was designed to assess the effect of systemic K_ATP channel inhibition via GLI on: i) resting cardiac function; ii) maximal aerobic capacity $(\dot{\mathrm{V}}{\mathrm{O}}_{2}max)$; and iii) submaximal exercise tolerance (CS). Local K_ATP channel inhibition via GLI superfusion was used to assess: iv) skeletal muscle blood flow $(\dot{\mathrm{Q}}m)$; and v) interstitial space O₂ pressures (PO₂is; established by O₂ delivery-to-utilization matching immediately proximal to myocytes) within contracting fast-twitch muscle of high oxidative capacity. Incorporating the Fick principle $(\dot{\mathrm{V}}{\text{O}}_2=\dot{\mathrm{Q}}m\times (\text{C}a{\text{O}}_2-\mathrm{C}v{\mathrm{O}}_2))$ and law of diffusion $\left(\dot{\mathrm{V}}{\text{O}}_2={\text{DO}}_2\times \mathrm{\Delta }{\text{PO}}_2\right)$ direct measurements were used to estimate convective $(\dot{\mathrm{Q}}{\mathrm{O}}_2)$ and diffusive (DO₂) O₂ conductances within microvascular and interstitial compartments where the convergence of $\dot{\mathrm{Q}}{\mathrm{O}}_2$ and DO₂ establish $\dot{\mathrm{V}}{\text{O}}_2$ (Wagner, 1992, 1996). Considering that vascular function and exercise assessments of K_ATP channels have been performed in male rats (Lu et al. 2013; Holdsworth et al. 2015, 2016, 2017) when females may be more adversely affected by sulphonylurea treatment (Brown et al. 2005b; Johnson et al. 2006), the current investigation sought to bridge the translatability of K_ATP channel function to females. It was hypothesized that K_ATP channel inhibition would impair resting cardiac output and decrease $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ and CS. It was further hypothesized that local K_ATP channel inhibition would reduce skeletal muscle blood flow and PO₂is during twitch contractions, and slow the recovery of PO₂is following contractions, effectively decreasing $\dot{\mathrm{V}}{\text{O}}_2$ by impairing O₂ conductance ($\dot{\mathrm{Q}}{\mathrm{O}}_2$ and DO₂). Data in support of these hypotheses would reveal a heretofore under-appreciated peripheral vascular role for K_ATP channels in the maintenance of O₂ delivery and contractile function.

Methods

Ethical approval

All protocols and procedures were approved by the Institutional Animal Care and Use Committee of Kansas State University and conducted according to the guidelines and ethical standards put forth by the National Institutes of Health and Journal of Physiology (Grundy, 2015). Ten female Sprague–Dawley rats (~8 months old during terminal experiments) were maintained in animal facilities accredited by the Association for the Assessment and Accreditation of Laboratory and Animal Care on a 12:12 h light:dark cycle with food and water provided ad libitum. Vaginal lavages were conducted for a minimum of 10 days to monitor menstrual cycles (Marcondes et al. 2002; Smith et al. 2017) with all testing performed during the pro-oestrus phase. In the initial 14–21 days, while menstrual cycles were monitored, acclimation to running was conducted on a custom-built treadmill for ~5 min day⁻¹ at ~25 m min⁻¹ up a 5% incline. During the final acclimation days the treadmill speed was increased progressively in the last 2–3 min up to 50–60 m min⁻¹ to familiarize the rats with high-speed running (Copp et al. 2010; Craig et al. 2019a, Poole et al. 2020). Importantly, these brief duration acclimation runs do not elicit training adaptations (Dudley et al. 1982; Armstrong & Laughlin, 1984; Musch et al. 1992).

Drug dosing

K_ATP channel inhibition was administered via the pharmacological sulphonylurea derivative glibenclamide (GLI: 494 g mol⁻¹, 5-chloro-N-{4-[N-(cyclohexylcarbamoyl)sulfamoyl]phenthyl}−2-methoxybenzamide, Sigma-Aldrich, St. Louis, MO). For acute systemic inhibition via an intraperitoneal injection on experimental days, a 10 ml stock solution was made by GLI dissolved in 9 ml saline (0.9% NaCl), 900 μl NaOH (0.1 m), and 100 μl DMSO and briefly sonicated. The amount of GLI dissolved in solution was determined on experimental days to obtain a final 1 ml dose of 10 mg kg⁻¹ (Lu et al. 2013). For local inhibition via superfusion, the stock solution utilized distilled water in place of saline and GLI was dissolved to obtain a final 5 mg kg⁻¹ dose, with 0.5 ml GLI stock solution diluted in 2.5 ml of warmed Krebs–Hensleit bicarbonate-buffered solution equilibrated with 5% CO₂–95% N₂ (pH 7.4; in mm, 4.7 KCl, 2.0 CaCl₂, 2.4 MgSO₄, 131 NaCl and 22 NaHCO₃).

GLI injections (10 mg kg⁻¹, _i._p.) occurred ~30–60 min prior to echocardiographic assessment and treadmill exercise testing ($\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ and CS; Lu et al. 2013) to align with peak plasma concentration (i.e. ~60–85 min after oral administration of 10 mg kg⁻¹ GLI, Li et al. 2012). Thus, each rat underwent at least six GLI injections over ~7–8 weeks. During interstitial PO₂ measurements, inhibition was administered locally via GLI superfusion (5 mg kg⁻¹ in Krebs–Hensleit solution, Holdsworth et al. 2017).

Echocardiography determination of left ventricular function

Transthoracic echocardiography was performed with a commercially available system (Logiq S8; GE Health Care, Milwaukee, WI) using an 18 MHz linear transducer (L8–18i). Rats were anaesthetized initially on a 5% isoflurane–O₂ mixture and then maintained on a 1.5–2% isoflurane–O₂ mixture while positioned supine on a heating pad (42°C) to maintain core temperature. Standard two-dimensional and M-mode images were obtained from the midpapillary level with frame rates >50 frames s⁻¹. Ventricular dimensions were obtained from M-mode measurements over four consecutive cardiac cycles. Left ventricular (LV) internal dimensions were measured at end diastole (LVIDd) and end systole (LVIDs). Fractional shortening (FS) was calculated from LV chamber diameters: FS = [(LVIDd – LVIDs)/LVIDd] × 100. Left end-systolic (LVESV) and end-diastolic (LVEDV) volumes were estimated using the Teichholz formula: LV volume = [7.0/(2.4 + LV dimension)] × LV dimension³. Stroke volume (SV) was calculated as: SV = LVEDV–LVESV. Ejection fraction (EF) was calculated using LV volume measurements: EF = [(LVEDV–LVESV)/LVEDV] × 100. Rates of contraction (+V) and relaxation (−V) of the posterior LV wall were also measured in M-mode by integrating the slope from end-diastolic and end-systolic internal diameter locations used for assessing LVIDd and LVIDs. Heart rate (HR) was estimated using the average contraction and relaxation times across the four cardiac cycles: HR = 60/(contraction time + relaxation time). Cardiac output (CO) was calculated using HR and SV values: CO = HR × SV.

Determination of maximal oxygen uptake and critical speed

Maximal oxygen uptake $(\dot{\mathrm{V}}{\mathrm{O}}_{2}max)$ tests were performed in a plexiglass metabolic chamber placed on the treadmill (Musch et al. 1988) and connected to O₂ (model S-3A/I) and CO₂ (model CD-3A; AEI Technologies; Pittsburg, PA) analysers. Gas measurements were performed in real time and recorded in the final 5–10 s of each stage. Treadmill speed was initially set to 25 m min⁻¹ for 2 min, increased to 40 m min⁻¹ for an additional 2 min, and then increased progressively ~5 m min⁻¹ each minute until the rat was unable to maintain pace with the treadmill or no further increases in $\dot{\mathrm{V}}{\mathrm{O}}_2$ were recorded despite increases in speed. High reproducibility of $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ measurements has been established previously in our laboratory (Copp et al. 2009).

Following $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ testing, the speed–duration relationship was determined via the multiple constant-speed method (Copp et al. 2010; Craig et al. 2019a). Critical speed tests consisted of five runs-to-exhaustion at predetermined speeds estimated to elicit exhaustion between 2 and 20 min. Each test began with a 2 min warm-up at 20 m min⁻¹, followed by 1 min of quiet rest, and then rapid increase in treadmill speed (<10 s) toward the target speed to be maintained for the duration of the test. Timing began when the investigator adjusting treadmill speed verified the attainment of the target speed. When rats drifted toward the back of the running lane a separate investigator provided encouragement via manual bursts of air toward the hindlimbs. Tests were terminated immediately when rats were unable to keep up with the treadmill speed despite apparent exertion and encouragement. The termination of all tests was determined by the same investigators who were blinded to the overall exercise time. Successful runs-to-exhaustion were verified by the absence of a righting reflex (i.e. unwilling/unable to right themselves within 2 s of being placed on their backs). The initial run was set at 60 m min⁻¹ and subsequent speeds were selected at ~5 m min⁻¹ increments to obtain the appropriate range of run durations (i.e. 2–20 min). When successful constant-speed tests were completed, the speed–duration parameters were determined by: 1) the hyperbolic speed–time model (time = D′/(speed - CS), where the asymptote of this curve is CS and the curvature constant is D′; and 2) the linear 1/time model (speed = D′ × 1/time + CS), where speed is plotted as a function of the inverse of time (s) to exhaustion, D′ is the slope, and CS is the intercept of the regression line (Copp et al. 2010, 2013; Poole et al. 2016). To mitigate any potential influence of training (increased CS) or weight gain (decreased CS) on the speed–duration relationship, the slowest of the constant-speed runs were performed early (i.e. run 2–4) under control conditions and the final runs overall consisted of control and GLI runs at the slowest speeds. Preliminary data showed that times-to-exhaustion of the slowest speed under control conditions, and thus CS, were either maintained or decreased compared with the initial slowest run. Therefore the shorter of the two was used to model the speed–duration relationship.

Phosphorescence quenching determination of PO₂is

On the final day of experimentation, rats were anaesthetized initially on a 5% isoflurane–O₂ mixture and maintained on 2–2.5% isoflurane–O₂ mixture for the duration of carotid and caudal (tail) artery catheterizations and surgical exposure of hindlimb muscles. Rats were placed on a heating pad to maintain core temperature at ~37–38°C, measured via rectal thermometer. Following a midline incision of the skin covering the neck, the right carotid artery was isolated and cannulated (PE-10 connected to PE-50; Intra-Medic polyethylene tubing; BD, Franklin Lakes, NJ, USA) for continuous measurements of mean arterial pressure (MAP) and HR, and infusion of fluorescent-labelled microspheres for blood flow measurements (see Fluorescent microsphere assessment of blood flow). The caudal artery was cannulated for infusion of pentobarbital sodium anaesthesia and blood sampling (i.e. blood gases and blood flow reference sample). Arterial blood samples were collected following the final contraction protocol for determination of O₂ saturation, systemic haematocrit and plasma lactate (Nova Stat Profile M; Nova Biomedical, Waltham, MA, USA).

Following catheterization, an incision was made above the lateral malleolus of the left hindlimb and the overlaying skin and fascia reflected to expose the biceps femoris. Upon tying off the lateral great saphenous artery (6–0 silk suture) the distal portion of the biceps femoris was reflected to expose the mixed gastrocnemius (MG). The MG muscle was selected for its fast-twitch fibre composition (97% type IIA+IID/X+IIB), oxidative capacity (citrate synthase: ~25 μmol min⁻¹ g⁻¹; Armstrong & Phelps, 1984; Delp & Duan, 1996), and most importantly its recruitment at and above the fatigue threshold (i.e. CS; Copp et al. 2010). The MG was left attached to its anatomical origin and insertion while variations in muscle length were minimized throughout the experimental protocol with knee and ankle joints stabilized ~90° angles. Rats were then progressively transitioned off isoflurane and onto pentobarbital sodium anaesthesia (50 mg ml⁻¹) with the depth of anaesthesia continuously monitored via toe pinch and corneal sensitivity reflexes, and additional anaesthesia provided as necessary (0.03–0.05 ml of 50 mg ml⁻¹ diluted to 0.3 ml of heparinized saline). Platinum iridium electrodes were attached (6–0 silk suture) to the proximal (cathode) and distal (anode) regions of the muscles to produce electrically induced muscle contractions. Surrounding exposed tissue was covered with Saran Wrap (Dow Brands, Indianapolis, IN) to reduce tissue dehydration and exposure to superfused solutions. Exposed muscle was superfused regularly with warmed Krebs–Henseleit bicarbonate-buffered solution equilibrated with 5% CO₂–95% N₂.

Experimental protocol.

Two separate contraction bouts were performed on the MG under control (Krebs–Heneseleit) and K_ATP channel inhibition (5 mg kg⁻¹ GLI in Krebs–Henseleit) superfused conditions. GLI superfusion was performed second, due to the long half-life of GLI, and with >20 min of recovery between contraction bouts to prevent any potential priming effect of repeated contraction bouts on PO₂is profiles. Interstitial space PO₂ (PO₂is) was measured via phosphorescence quenching at rest and during 180 s twitch contractions (1 Hz, 7 V, 2 ms pulse duration; Grass stimulator model S88, Quincy, MA) and recorded at 2 s intervals (Craig et al. 2018, 2019a,b, Hirai et al. 2018a). Recovery PO₂is was measured for an additional 240 s to ensure that PO₂is returned and stabilized at baseline prior to subsequent GLI superfusion and contractions. With PO₂is measured continuously, GLI was superfused (3 ml total volume) onto the MG for 180 s and allowed an additional 180 s before the same contraction protocol was repeated (i.e. total of >23 min elapsed between contraction bouts).

Measurement of interstitial PO₂.

A frequency domain phosphorometer (PMOD 5000; Oxygen Enterprises, Philadelphia, PA) was used to measure PO₂is as described previously (Craig et al. 2018, 2019a,b, Hirai et al. 2018a). The Oxyphor G4 (Pd-meso-tetra-(3,5-dicarboxyphenyl)-tetrabenzoporphyrin) was injected locally (2–4 10 μl injections at 10 μm concentration) with a 29 gauge needle, with care taken to avoid any visible vasculature. Following injection, the muscle was covered in Saran Wrap and allowed >20 min to allow the G4 to thoroughly diffuse throughout the interstitial space. This oxyphor is well suited for use in biological tissues because it does not cross membranes and is stable across the physiological pH range (Esipova et al. 2011). Muscle surface temperature was measured via non-contact infrared thermometer, since this oxyphor is temperature sensitive. The exposed MG temperatures were 31.6 ± 0.2°C. Previous studies have shown that the present twitch contraction protocol does not significantly change muscle temperature (Craig et al. 2018, 2019a).

Phosphorescence quenching applies the Stern–Volmer relationship (Rumsey et al. 1988; Esipova et al. 2011) describing the quantitative O₂ dependence of the phosphorescent probe G4 via the equation PO₂is=[(τ₀/τ) – 1]/(k_Q·τ₀), where k_Q is the quenching constant and τ and τ₀ are the phosphorescence lifetimes at the ambient O₂ concentration and in the absence of O₂, respectively. For G4 in tissue at ~32°C, k_Q is ~258 mmHg/s and τ₀ is ~226 μs (Esipova et al. 2011). Because muscle temperature does not change appreciably throughout the contraction protocol used herein (Craig et al. 2018, 2019a), the phosphorescence lifetime is determined exclusively by the O₂ partial pressure. Following G4 injection, the common end of the bifurcated light guide was positioned 3–4 mm above the exposed muscle surface. All PO₂is measurements were performed in a dark room to minimize extraneous exposure to light.

Analysis of interstitial PO₂ kinetics.

Contracting PO₂is responses were analysed using 30 s of resting data and the 180 s contraction bouts using a monoexponential plus time delay model (one component) or a monoexponential plus time delay with a secondary component (two component) model when necessary,

One component

$${\text{PO}}_{2}i{s}_{\text{t}}={\text{PO}}_{2}i{s}_{\text{BL}}-{\mathrm{\Delta }}_1{\text{PO}}_2\mathit{is}\left(1-{\text{e}}^{-(t-TD)/\tau }\right)$$

Two component

$$\begin{gathered} {\text{PO}}_{2}i{s}_{\text{t}}={\text{PO}}_{2}i{s}_{\text{BL}}-{\mathrm{\Delta }}_1{\text{PO}}_{2}is\left(1-{\text{e}}^{-(\text{t}-\text{TD})/\tau }\right) \\ +{\mathrm{\Delta }}_2{\text{PO}}_2\mathit{is}\left(1-{\text{e}}^{-\left(\text{t}-{\text{TD}}_2\right)/{\tau }_2}\right) \end{gathered}$$

where PO₂is _t represents the PO₂is at any point in time, PO₂is _BL is the baseline before the onset of contractions, Δ₁PO₂is and Δ₂PO₂is are the primary and secondary amplitudes, TD and TD₂ are the time delays before the fall and secondary rise in PO₂is, and τ and τ₂ are the time constants (i.e. the time required to reach 63% of the amplitude) for the primary and secondary amplitudes. The mean response time (MRT) was calculated as the sum of the model-derived TD and τ. When the secondary component model was necessary, the primary amplitude was constrained to the nadir value in order to maximize the accuracy of the primary response kinetics (Craig et al. 2018, 2019a,b). The goodness of model fit was determined using the following criteria: 1) coefficient of determination, 2) sum of the squared residuals, and 3) visual inspection and analysis of the model fits to the data and the residuals. Because Δ₂PO₂is (i.e. undershoot of PO₂is; Δ₂PO₂is = PO₂is _end – PO₂is _nadir) was often non-exponential in nature, Δ₂PO₂is was determined manually by calculating the difference between the PO₂is at the end of contractions (PO₂is _end, average of 172–180 s) minus the nadir value of PO₂is during contractions (PO₂is _nadir = PO₂is _BL – Δ₁PO₂is). Rate of PO₂is recovery was calculated in eight rats as the time taken to reach 63% of the overall response (i.e. T63) between PO₂is _end and recovery PO₂is (average of 232–240 s).

Fluorescent microsphere determination of blood flow

The microsphere technique was used to determine MG blood flow as described previously (Musch et al. 1986; Van Oosterhout et al. 1998; Deveci & Egginton, 1999). Two fluorescent microspheres (blue-green (430/465 nm) and red (580/605 nm), Invitrogen FluoSpheres polystyrene microspheres, ThermoFisher Scientific) were injected in random order at the end of MG and MG GLI contractions. Following 180 s contractions, blood withdrawal from the tail catheter was initiated at 0.25 ml min⁻¹ while 0.25–0.30 × 10⁶ 15.5 μm diameter fluorescent microspheres were injected into the aortic arch via the carotid artery catheter. Muscle contractions and blood withdrawal were terminated 30 s after the microsphere injection. Following the final contraction protocol, rats were killed via pentobarbital sodium overdose (>100 mg kg⁻¹ i.a.), proper catheter placement in the aortic arch was confirmed, and tissues (left and right kidneys, left and right mixed gastrocnemei) dissected and stored (−80°C) for later analyses. For the final analyses, kidney and muscle tissues were weighed and placed directly in 15 ml screw cap polypropylene tubes with a conical base. Five ml of 2 M KOH in 99% ethanol with 0.5% Tween-80 were added to the tubes, vortexed, and placed in a dry heating block (60°C) with intermittent vortexing until tissue digestion was complete. Tubes were then centrifuged at 3000 rpm (1500 g) for 15 min. Supernatant was carefully aspirated until <500 μl remained to minimize the possibility of accidental microsphere loss. One ml of deionized H₂O was added and tubes quickly vortexed to resuspend the remaining pellet, followed by the addition of 9 ml ethanoic Tween-80, vortexing and another 15 min of centrifuging. Tubes were aspirated as previously described, before 5 ml of 100 mm phosphate buffer (pH 7.0) was added to neutralize the pellet and solution, followed by 4 ml of absolute ethanol. The tubes were further vortexed, centrifuged, and aspirated to <300 μl. To ensure complete resuspension of microspheres, the tubes were vortexed again before being placed in an oven (60°C) to evaporate to 100–150 μl. To improve solvent extraction from the microspheres, which would be less efficient in a dry pellet, the tubes were periodically removed from the oven and vortexed. To dissolve the polystyrene microspheres and release the fluorescent dye, 2 ml of solvent (di(ethylene glycol) ethyl ether acetate, 98%; Sigma-Aldrich Corporation, St. Louis, MO, USA) was added and vortexed several times over 3–5 min and left for 30 min before being sonicated (5 min) in a water bath to ensure complete dye extraction. Once the solvent was added, all remaining steps were conducted in dim lighting to prevent signal decay prior to fluorescent intensity measurements. After sonication the tubes were centrifuged once more (10 min) before supernatant was pipetted (300 μl) into 96 well plates in quadruplicates for the measurement of fluorescent intensity (SpectraMax i3 Multi-Mode Platform, Molecular Devices, San Jose, CA). Total tissue blood flows were calculated according to the reference sample method (Ishise et al. 1980; Musch & Terrell, 1992) and expressed mass specifically in ml min⁻¹ 100g tissue⁻¹. Adequate mixing of microspheres prior to infusion were determined by <20% difference in left and right kidney and/or muscle blood flows.

Muscle oxygen consumption

The Fick equation was used to calculate microvascular oxygen consumption $\left(\dot{\mathrm{V}}{\text{O}}_{2}mv\right)$ with the assumptions that microvascular PO₂ (PO₂mv) can be calculated from interstitial measurements (i.e. PO₂mv = PO₂is + transcapillary PO₂; Colburn et al. 2020a) and is an appropriate analogue for venous PO₂ (McDonough et al. 2001) and, by extension from the O₂ dissociation curve, venous blood O₂ content (Roca et al. 1992). Therefore mixed venous O₂ content (CvO₂) was calculated from PO₂mv using the rat O₂ dissociation curve (constructed using the Hill coefficient (n) of 2.6, the measured [Hb], P₅₀ (the PO₂ at which Hb is 50% saturated with O₂) of 38 mmHg, and an O₂-carrying capacity of 1.34 ml O₂ (gHb)⁻¹). Arterial O₂ content (CaO₂) was measured directly via arterial blood sample and, when combined with blood flow $(\dot{\mathrm{Q}}m)$ and CvO₂ values, was used to calculate $\dot{\mathrm{V}}{\text{O}}_{2}mv$ via the principle of mass balance using the Fick Equation (i.e. $\dot{\mathrm{V}}{\text{O}}_{2}mv=\dot{\mathrm{Q}}m\times \left(\text{C}a{\text{O}}_2-\text{C}v{\text{O}}_2\right)$). Microvascular O₂ diffusion conductance (DO₂mv) was defined as $\dot{\mathrm{V}}{\text{O}}_{2}mv/{\mathrm{PO}}_{2}mv$ which provides an index of diffusive O₂ transport per unit of driving pressure. Interstitial O₂ diffusion conductance $\left({\text{DO}}_{2}is=\dot{\mathrm{V}}{\text{O}}_{2}is/{\text{PO}}_{2}is\right)$ was assessed utilizing the present PO₂is and calculated $\dot{\mathrm{V}}{\text{O}}_{2}mv$ (i.e. $\dot{\mathrm{V}}{\text{O}}_{2}is$ was presumed to be equal to $\dot{\mathrm{V}}{\text{O}}_{2}mv$ considering the absence of storage for O₂ in the interstitial fluid and, thus, O₂ leaving the microvascular compartment must equal O₂ leaving the interstitial compartment).

Statistical analyses

The effect of systemic GLI on resting LV function, exercise parameters ($\dot{\mathrm{V}}{\mathrm{O}}_{2}max$, CS and D′), and local GLI superfusion on contracting MG measurements (MAP, HR, PO₂is kinetics parameters, $\dot{\mathrm{Q}}m$, microvascular $\dot{\mathrm{Q}}{\text{O}}_{\text{2}}$ and $\dot{\mathrm{V}}{\mathrm{O}}_2$ and microvascular and interstitial DO₂) were assessed using two-tail paired t tests. PO₂is profiles were assessed via two-way repeated measure ANOVA (Time × Drug) with Tukey’s post hoc analyses. Data are presented as means ± SD. Significance was accepted at P < 0.05.

Results

Two rats were unwilling to complete all runs needed to assess the speed–duration relationship; therefore, comparisons between control and K_ATP channel inhibition were conducted on eight rats.

Resting echocardiography

Left ventricular echocardiographic measurements are presented in Table 1. Compared with control, GLI did not alter LVEDV (0.83 ± 0.24 vs. 0.83 ± 0.18, P = 0.976) nor LVESV (0.15 ± 0.07 vs. 0.19 ± 0.09 ml, P = 0.172) and thus stroke volume remained unchanged (0.68 ± 0.19 vs. 0.65 ± 0.11 ml, P = 0.354). LV fractional shortening (47 ± 6 vs. 42 ± 5, P = 0.084) and EF (83 ± 5 vs. 78 ± 6%, P = 0.088) were also not significantly altered. However, the rate of LV relaxation (2.11 ± 0.59 vs. 1.70 ± 0.23, P = 0.048), but not contraction (2.76 ± 0.49 vs. 2.44 ± 0.43 cm s⁻¹, P = 0.079), was significantly slowed resulting in a decreased HR (321 ± 23 vs. 304 ± 22 bpm, P = 0.043) during maintained cardiac output (219 ± 64 vs. 197 ± 39 ml min⁻¹, P = 0.105).

Table 1.

Doppler echocardiographic assessment of left ventricular function during control and systemic K_ATP channel inhibition

	Control	Glibenclamide
LVIDd (cm)	0.71 ± 0.08	0.72 ± 0.05
LVIDs (cm)	0.38 ± 0.08	0.41 ± 0.06
FS (%)	47 ± 6	42 ± 5
LVEDV (ml)	0.83 ± 0.24	0.83 ± 0.18
LVESV (ml)	0.15 ± 0.07	0.19 ± 0.09
SV (ml)	0.68 ± 0.19	0.65 ± 0.11
EF (%)	83 ± 5	78 ± 6
+V (cm s−1)	2.76 ± 0.49	2.44 ± 0.43
−V (cm s−1)	2.11 ± 0.59	1.70 ± 0.23 *
HR (bpm)	321 ± 23	304 ± 22 *
CO (ml min−1)	219 ± 64	197 ± 39

LVIDd, left ventricular end-diastole internal diameter; LVIDs, left ventricular end-systole internal diameter; FS, fractional shortening; LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume; SV, stroke volume; EF, ejection fraction; +V, rate of contraction; −V, rate of relaxation; HR, heart rate; CO, cardiac output. Data are means ± SD (n = 10) and compared via two-tail paired t test.

^*P < 0.05 vs. control.

Maximal aerobic capacity and speed–duration relationship

Table 2 and Figs 1 and 2 demonstrate that GLI reduced $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ (71.5 ± 3.1 vs. 67.9 ± 4.8 ml min⁻¹ kg⁻¹, P = 0.034) and CS (35.9 ± 2.4 vs. 31.9 ± 3.1 m min⁻¹, P = 0.020) whereas D′ remained unchanged (98 ± 16 vs. 91 ± 25 m, P = 0.532).

Table 2.

Individual maximal oxygen uptake and speed–duration relationship parameters during control and systemic K_ATP channel inhibition

	Control			Glibenclamide
	$\dot{\mathrm{V}}{\mathrm{O}}_{2}max$	CS (m min−1)	D′ (m)	$\dot{\mathrm{V}}{\mathrm{O}}_{2}max$	CS (m min−1)	D′ (m)
1	67.7	39.5	84	70.0	29.3	124
2	71.3	34.2	127	61.4	32.2	79
3	75.2	34.5	93	69.5	28.3	95
4	65.2	–	–	64.8	–	–
5	70.8	34.0	92	61.1	34.1	83
6	73.3	39.8	75	65.5	35.0	79
7	74.7	35.4	101	77.2	29.7	127
8	73.1	–	–	70.8	–	–
9	71.9	34.0	111	69.9	30.1	89
10	71.9	35.4	105	69.0	36.7	52
Mean ± SD	71.5 ± 3.1	35.9 ± 2.4	98 ± 16	67.9 ± 4.8 *	31.9 ± 3.1 *	91 ± 25

$\dot{\mathrm{V}}{\mathrm{O}}_{2}max$, maximal oxygen uptake; CS, critical speed; D′, curvature constant. The speed–duration relationship parameters are presented from the hyperbolic model. Data were compared via two-tail paired t tests.

^*P < 0.05 vs. control.

Figure 1. Effect of systemic K_ATP channel inhibition on maximal and submaximal exercise.

Note the significant reduction in maximal oxygen uptake ($\dot{\mathrm{V}}{\text{O}}_{2}max$; n = 10) and critical speed (n = 8) following glibenclamide (open bars) compared with control (grey bars). Data are means ± SD with individual data plotted and compared via two-tail paired t tests.

*P < 0.05 vs. control.

Figure 2. Speed–duration relationship following systemic K_ATP channel inhibition.

The hyperbolic (A) and 1/time linear (B) speed–duration relationships are modelled under control (closed circle, continuous line) and systemic K_ATP channel inhibition (GLI; open circle, dashed line) conditions to determine critical speed (vertical lines (A) and y-intercept (B)) and D′ (inset). These mean data fits are for illustrative purposes only, with individually determined critical speed and D′ and subsequent group means presented in Table 2. Data are means ± SD and compared via two-tail paired t tests.

*P < 0.05 vs. control.

Blood sample analysis and central haemodynamics during phosphorescence quenching

Arterial pH (7.39 ± 0.03), O₂ saturation (90.7 ± 2.6%), haematocrit (34.9 ± 4.2%) and lactate concentration (1.5 ± 0.5 mm) were assessed following the GLI contraction. GLI superfusion did not alter MAP or HR (both P > 0.346); therefore, MAP (102 ± 10 and 99 ± 10 mmHg, P = 0.096) and HR (361 ± 23 and 364 ± 38 bpm, P = 0.831) were not different at the start of the control and GLI contractions, respectively.

MG blood flow and interstitial PO₂

GLI superfusion impaired MG blood flow during contractions (52 ± 25 vs. 34 ± 13 ml min⁻¹ 100 g⁻¹, P = 0.015). The effect of GLI superfusion on PO₂is during the rest–contraction transient is presented in Table 3 and Fig. 3. GLI reduced MG PO₂is _BL (−1.1 ± 1.1 mmHg, P = 0.020). At the onset of contractions, there was a shortening in TD (P = 0.005) with a statistically non-significant change in τ (14.1 ± 2.7 vs. 12.2 ± 3.0, P = 0.053) yet a faster overall PO₂is fall (MRT: 22.2 ± 4.7 vs. 17.3 ± 4.3 s, P = 0.002) to a lower PO₂is _nadir (5.9 ± 0.9 vs. 4.7 ± 1.1, P = 0.013) but not a different PO₂is _end (7.3 ± 1.5 vs. 6.1 ± 1.4 mmHg, P = 0.073) compared with control (see Fig. 3; all 2 s measurements, two-way repeated measures ANOVA with Tukey’s post hoc analyses, P < 0.062). Following contractions, GLI PO₂is recovered more slowly (T₆₃: 95 ± 19 vs. 118 ± 20 s, P = 0.047) but to a similar end recovery PO₂is (14.6 ± 4.0 vs. 15.3 ± 6.5 mmHg, P = 0.556) during the observed window.

Table 3.

Interstitial PO₂ kinetics parameters during 180 s twitch contractions and 240 s recovery during control and local K_ATP channel inhibition

	Mixed gastrocnemius
	Control	Glibenclamide
Pre-superfusion PO2is (mmHg)	–	13.8 ± 3.4
PO2isBL (mmHg)	14.1 ± 1.4	12.8 ± 3.6 *
Δ1PO2is (mmHg)	8.2 ± 1.5	8.1 ± 3.1
TD (s)	8.0 ± 4.3	5.2 ± 3.2 *
τ (s)	14.1 ± 2.7	12.2 ± 3.0
MRT (s)	22.2 ± 4.7	17.3 ± 4.3 *
PO2isnadir (mmHg)	5.9 ± 0.9	4.7 ± 1.1 *
Δ2PO2is (mmHg)	1.4 ± 0.9	1.4 ± 0.8
PO2isend (mmHg)	7.3 ± 1.5	6.1 ± 1.4
Δ1PO2is/τ (mmHg s−1)	0.60 ± 0.16	0.72 ± 0.36
Recovery T63 (s)	95 ± 19	118 ± 20 *
Recovery PO2is (mmHg)	14.6 ± 4.0	15.3 ± 6.5
Δ3PO2is/T63 (mmHg s−1)	0.07 ± 0.03	0.08 ± 0.04

PO₂is _BL, resting baseline; PO₂is; Δ₁PO₂is and Δ₂PO₂is, amplitude of the first and second components, respectively; TD, time delay; τ, time constant; MRT, mean response time; PO₂is _nadir, lowest response prior to secondary rise in PO₂is; PO₂is _end, PO₂is at the end of contractions; Δ₁PO₂is/τ, rate of PO₂is fall; T₆₃, time to reach 63% of final response; Δ₃PO₂is/T₆₃, rate of PO₂is recovery. Data are means ± SD and compared via two-tail paired t tests.

^*P < 0.05 vs. control.

Figure 3. Interstitial PO₂ of fast-twitch oxidative muscle following local K_ATP channel inhibition.

Note the difference in mixed gastrocnemius PO₂is following glibenclamide (GLI, open symbols, n = 10) superfusion compared with control (closed symbols). Dashed line denotes the onset of twitch contractions at time zero. Data are means ± SD and compared via two-way (Time × Drug) repeated measure ANOVA with Tukey’s post hoc analyses.

MG muscle oxygen delivery, consumption and diffusive conductance

Compared with control, Fig. 4 illustrates that GLI decreased MG oxygen delivery ($\dot{\mathrm{Q}}{\text{O}}_{2}mv$: 6.4 ± 3.2 vs. 4.2 ± 1.7 ml O₂ min⁻¹ 100 g⁻¹, P = 0.015) and oxygen consumption from the microvascular compartment ($\dot{\mathrm{V}}{\mathrm{O}}_{2}mv$: 5.8 ± 2.9 vs. 3.9 ± 1.6 ml O₂ min⁻¹ 100 g⁻¹, P = 0.016). Consequently, microvascular (DO₂mv: 0.40 ± 0.20 vs. 0.30 ± 0.11, P = 0.023) and interstitial diffusive conductances (DO₂is: 0.80 ± 0.38 vs. 0.66 ± 0.25 ml O₂ min⁻¹ mmHg⁻¹ 100 g⁻¹, P = 0.040) were significantly reduced. Furthermore, DO₂is was significantly greater than DO₂mv (P = 0.0002 and 0.0001, control and GLI, respectively), with the reduction in DO₂mv following GLI (−22 ± 20%) trending towards being proportionally greater than the reduction in DO₂is (−15 ± 17%; P = 0.070).

Figure 4. The effect of local K_ATP channel inhibition on the convective and diffusive determinants of oxygen transport.

Graphical representation of the relationship between convective ($\dot{\mathrm{V}}{\text{O}}_2=\dot{\mathrm{Q}}\times \text{a-v}{\text{O}}_2$ difference; curved line) and diffusive ($\dot{\mathrm{V}}{\mathrm{O}}_2={\text{DO}}_2\times {\text{PO}}_2$; slope from origin) determinants of oxygen transport in the microvascular (circles) and interstitial (down triangles) compartments of the fast-twitch oxidative mixed gastrocnemius (MG, n = 10) muscle following vascular K_ATP channel inhibition via glibenclamide (GLI, open symbols). Importantly, unlike haemoglobin–O₂ transport in the microvasculature, the lack of haeme–O₂ storage in interstitial fluid dictates that interstitial $\dot{\mathrm{V}}{\text{O}}_2$ $(\dot{\mathrm{V}}{\text{O}}_{2}is)$ must equal microvascular $\dot{\mathrm{V}}{\text{O}}_2$ $(\dot{\mathrm{V}}{\text{O}}_{2}mv)$ allowing DO₂is to be assessed with the present data $\left(\dot{\mathrm{V}}{\text{O}}_{2}mv=\dot{\mathrm{V}}{\text{O}}_{2}is={\text{DO}}_{2}is\times {\text{PO}}_{2}is\right)$. Note the reductions in both convective (QO₂mv, curved lines y-intercept) and diffusive (DO₂mv and DO₂is) components of MG muscle compared with control (closed symbols). *P < 0.05 vs. control with two-tail paired t tests

Discussion

The main original finding of this investigation is that, in female rats, GLI-induced impairment of maximal (i.e. $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$) and submaximal (i.e. CS) exercise can be attributed, in part, to reductions in peripheral vascular K_ATP channel function. Systemic GLI administration did not change cardiac output at rest, assessed via Doppler echocardiography. However, the slowed LV relaxation and reduced HR at rest following GLI highlights a peripheral insult to K_ATP channels that is likely compensated, in part, by baroreflex-mediated changes in left ventricular function. Accordingly, local inhibition of vascular K_ATP channels during contractions resulted in decreased blood flow and interstitial space O₂ delivery-to-utilization matching (i.e. PO₂is) of fast-twitch oxidative skeletal muscle and slowed the recovery of PO₂is following cessation of contractions. Using the Fick principle and law of diffusion, estimations of MG convective and diffusive conductances ($\dot{\mathrm{Q}}{\mathrm{O}}_2$ and DO₂, respectively), and thus $\dot{\mathrm{V}}{\mathrm{O}}_2$, were impaired following GLI. Therefore, the exercise intolerance that is symptomatic of patient populations (i.e. diabetes, HF), which have a greater proportion of, and blood flow to, fast-twitch fibres, may be exacerbated by oral sulphonylurea medications impairing vascular K_ATP channel-mediated vasodilation.

K_ATP channel function on maximal aerobic capacity and speed–duration relationship

Consistent with our hypothesis, systemic K_ATP channel inhibition impaired maximal aerobic capacity $(\dot{\mathrm{V}}{\mathrm{O}}_{2}max)$ and submaximal exercise tolerance (CS). Lu and colleagues (2013) previously demonstrated impaired $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ (~54 to 36 ml O₂ min⁻¹ kg⁻¹, ~33%) in rats following GLI injections; however, utilizing a comparatively steeper ramp protocol, the current data exhibited higher baseline $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ and a far smaller effect of GLI (~5%, Table 2). This discrepancy likely stems directly from the slower ramp protocol wherein longer durations at submaximal speeds would be expected to enhance glycogen depletion in skeletal muscle leading to exhaustion at lower $\dot{\mathrm{V}}{\mathrm{O}}_2$ levels. Supporting the need for steeper ramp protocols, Richardson et al. (1993) demonstrated greater $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ and maximum work rate within 13–15 min of knee extension exercise compared with those previously measured using a slower, longer ramp protocol (~40–60 min; Andersen & Saltin, 1985). Accordingly, GLI-induced reductions in HR in the face of elevated MAP occurred at slower speeds, but not at 60 m min⁻¹ (Holdsworth et al. 2015). Coupled with decreased hindlimb muscle blood flows throughout all speeds, this suggests that vascular control of O₂ delivery is impaired especially at submaximal speeds while metabolite build-up at supra-CS speeds may activate group III/IV afferents to increase HR at near-maximal speed/intensity (Holdsworth et al. 2015). This notion is highlighted in the current investigation (Fig. 1) where GLI-induced reductions in CS were greater than those seen in $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ (~12 vs. ~5%, respectively).

K_ATP channel function on cardiac function

Assessed via Doppler ultrasound under anaesthesia, GLI significantly reduced the rates of LV relaxation and HR (Table 1) but not cardiac output. Decreased HR has been demonstrated in GLI-treated rats during conscious rest which occurred simultaneously with increased MAP and decreased sympathetic activity to hindlimb muscles (Colburn et al. 2020b). Although resting cardiac function is not expected to relate directly to cardiac function during high-intensity exercise (Fig. 1), we believe that the present changes in LV relaxation and HR at rest help to emphasize a peripheral insult following systemic K_ATP channel inhibition (Holdsworth et al. 2015, 2016; Colburn et al. 2020b) that manifests changes in cardiac function likely to minimize MAP increases (Colburn et al. 2020b) and/or decrease the work of the heart during elevated MAP. Additionally, as direct K_ATP channel inhibition of cardiomyocytes would enhance contractility and hinder relaxation (Flagg et al. 2010; Kane et al. 2005; Zingman et al. 2007), it is most likely that the primary effect of systemic GLI administration herein results from a peripheral, and not cardiomyocyte-mediated, alteration in K_ATP channel function (i.e. vascular K_ATP channel inhibition leading to vasoconstriction and increased MAP with a baroreceptor-mediated secondary reduction in sympathetic activity to decrease cardiac output, Colburn et al. 2020b). These adjustments in cardiac function are likely removed at near-maximal exercise intensities when sympathetic activity is enhanced and HR is not different between control and GLI conditions (Holdsworth et al. 2015). However, future experimental designs where possible should measure SV and cardiac output during high-intensity exercise to assess the primary, or secondary, effect of K_ATP channel inhibition on cardiac function. Nevertheless, reductions in $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ herein are considered to result, in part, from $\dot{\mathrm{Q}}{\text{O}}_2:\dot{\mathrm{V}}{\text{O}}_2$ mismatch at the level of skeletal muscle and impaired systemic arterial–venous O₂ difference (i.e. $\dot{\mathrm{V}}{\mathrm{O}}_2\mathit{max}=\dot{\mathrm{Q}}{\text{O}}_2\mathit{max}\times \text{maximal}a-v{\text{O}}_2$ difference).

K_ATP channel function on skeletal muscle blood flow and PO₂is

To assess the contribution of vascular K_ATP channels supporting skeletal muscle O₂ transport, and thus oxidative phosphorylation, the current investigation measured the interstitial space driving pressure of O₂ (PO₂is) during the rest–contractions transient during submaximal contractions, muscle blood flow $(\dot{\mathrm{Q}}m)$ at the end of contractions, and PO₂is during recovery. Following local GLI administration, PO₂is in the MG fast-twitch oxidative muscle fell faster (i.e. MRT) and to a lower PO₂is (PO₂is _nadir; Table 3, Fig. 3) when $\dot{\mathrm{Q}}m$ was reduced. Interestingly, because PO₂is _BL was also reduced following GLI, the magnitude of PO₂is fall (i.e. Δ₁PO₂is) was unaltered, possibly due to a lowering of intracellular $\dot{\mathrm{V}}{\text{O}}_2$ to preserve interstitial-myocyte PO₂ and prevent muscle damage, as proposed by Richmond et al. (1999). Therein the ‘critical PO₂’, the point at which PO₂is ceased to continue falling and the NADH fluorescence signal increased, was ~2.4–2.9 mmHg for mixed-fibre spinotrapezius muscle and may be greater in the more oxidative MG (citrate synthase activity: ~26 vs. ~14 μmol min⁻¹ g⁻¹; Delp & Duan, 1996). While the exact contribution of lowered $\dot{\mathrm{V}}{\text{O}}_2$ (Fig. 4) is unable to be separated completely between low $\dot{\mathrm{Q}}{\text{O}}_2$- (via decreased $\dot{\mathrm{Q}}m$ which lowered PO₂is and sped the fall in PO₂is) and/or myocyte-mediated $\dot{\mathrm{V}}{\text{O}}_2$ lowering, the end result is indeed lower $\dot{\mathrm{V}}{\text{O}}_2$ which would increase the reliance on glycolytic energy sources for contractions and production of fatigue-related metabolites (Wilson et al. 1977; Hogan et al. 1992; Richardson et al. 1998).

In addition, despite recovering to a similar PO₂is, GLI significantly slowed the recovery compared with control. All of these findings during local K_ATP channel inhibition provide evidence supporting the hypothesis that the reductions in exercise tolerance resulting from systemic GLI administration were due, in part, to impaired O₂ transport at the microvascular level hindering aerobic metabolism within the contracting myocyte (see Convective and diffusive determinants of O₂ transport below) and, especially for repeated bouts of physical activity, increasing the amount of time needed to re-establish muscle PO₂ and replenish muscle energy stores (Haseler et al. 1999; Kindig et al. 2005).

Convective and diffusive determinants of O₂ transport

Utilizing direct measurements of $\dot{\mathrm{Q}}m$, arterial O₂ content (CaO₂), and the O₂ pressures nearest the contracting myocytes (PO₂is) to calculate PO₂mv (PO₂mv = PO₂is + transcapillary PO₂; Colburn et al. 2020a), the authors conflated the Fick principle $(\dot{\mathrm{V}}{\text{O}}_{2}mv=\dot{\mathrm{Q}}m\times \left(\text{C}a{\text{O}}_2-\text{C}v{\text{O}}_2\right)$; i.e. convective O₂ transport) and Fick’s law of diffusion $(\dot{\mathrm{V}}{\mathrm{O}}_2\text{m}v={\text{DO}}_2\text{m}v\times {\text{PO}}_2\text{m}v$; i.e. diffusive O₂ transport) to estimate muscle convective O₂ delivery $\left(\dot{\mathrm{Q}}{\text{O}}_{2}mv\right)$, diffusive conductance for O₂ (DO₂mv), and the resulting $\dot{\mathrm{V}}{\text{O}}_2$ from the microvascular compartment. Importantly, the convergence of the convective and diffusive determinants to O₂ transport describes the rate of O₂ consumption by skeletal muscle (Wagner, 1992, 1996).

In the current investigation, impaired PO₂is was due, in part, to reductions in $\dot{\mathrm{Q}}m$. As a result, the rate of O₂ able to be consumed from the microvascular compartment was significantly reduced $\dot{\mathrm{V}}{\mathrm{O}}_{2}mv$. As demonstrated in Fig. 4, this change in $\dot{\mathrm{V}}{\mathrm{O}}_{2}mv$ was not only a consequence of impaired $\dot{\mathrm{Q}}{\text{O}}_{2}mv(\downarrow 34\%)$ but also lowered microvascular-myocyte diffusing conductance (DO₂mv ↓25%). Traditionally interpreted in the context of microvascular-myocyte O₂ transport, DO₂mv is altered via changes in capillary haematocrit, red blood cell (RBC) flux and RBC velocity (reviewed by Poole et al. 2013; Poole, 2019). More recently, with the advent of PO₂is measurements during contractions (Hirai et al. 2018a; Colburn et al. 2020a), interstitial DO₂ (DO₂is, interstitial-myocyte) can be estimated presuming that O₂ leaving the microvascular compartment $\left(\dot{\mathrm{V}}{\mathrm{O}}_{2}mv\right)$ equals O₂ leaving the interstitial compartment ($\dot{\mathrm{V}}{\mathrm{O}}_{2}is$; i.e. negligible change in storage of O₂ in interstitial fluid) during steady-state contractions and can be calculated from $\dot{\mathrm{V}}{\mathrm{O}}_{2}mv$ and the present PO₂is (i.e. ${\text{DO}}_{2}is=\dot{\mathrm{V}}{\text{O}}_{2}mv/{\text{PO}}_{2}is$ when $\dot{\mathrm{V}}{\text{O}}_{2}mv=\dot{\mathrm{V}}{\text{O}}_{2}is$). Interestingly, since $\dot{\mathrm{V}}{\text{O}}_2$ must be equivalent in both compartments, DO₂ was greater in the interstitial compartment compared with microvascular compartment and both decreased with GLI. However, with GLI, DO₂ was almost reduced to a significantly greater extent when O₂ diffused out of the microvascular compartment compared with the subsequent O₂ diffusion out of the interstitium (↓25% and 19%, DO₂mv vs. DO₂is, respectively). These disparate magnitudes of, and potential reductions in, DO₂ between compartments are likely to result from: i) divergent surface areas for O₂ flux (i.e. across capillary wall < into myocyte); and ii) fluid dynamics wherein impaired buffering of RBCs following GLI (i.e. ↓ percent capillaries flowing, RBC flux and RBC velocity, Hirai et al. 2018b) would yield greater decrements in DO₂mv than DO₂is considering interstitial fluid volume is expected to remain relatively constant during contractions and unaffected by GLI. Furthermore, since PO₂mv for control and GLI conditions were calculated using the same transcapillary PO₂ (PO₂mv = PO₂is + transcapillary PO₂), the reduction in DO₂mv following GLI is potentially underestimated as a result of increased transcapillary PO₂. Crucially, increased $\dot{\mathrm{Q}}m$ and RBC dynamics increase DO₂mv and reduce transcapillary PO₂ (PO₂mv-PO₂is) in the contracting fast-twitch oxidative $\text{MG}\text{(MG}\text{Control:}\uparrow \dot{\mathrm{V}}{\text{O}}_2=\uparrow \uparrow {\text{DO}}_2\times \downarrow \left[{\text{PO}}_{2}mv-{\text{PO}}_{2}is\right]$; Colburn et al. 2020a) whereas the GLI-induced reduction in $\dot{\mathrm{Q}}m$ herein and impaired RBC dynamics (Hirai et al. 2018b) would serve to reduce DO₂mv compared with control and therefore increasing the actual PO₂mv following GLI (MG+GLI compared with MG Control: $\downarrow \dot{\mathrm{V}}{\text{O}}_2=\downarrow \downarrow {\text{DO}}_2\times \uparrow \left[{\text{PO}}_{2}mv-{\text{PO}}_{2}is\right]$).

The interplay between convective and diffusive O₂ delivery on muscle $\dot{\mathrm{V}}{\text{O}}_2$ has been assessed directly in healthy skeletal muscle across fibre types (Behnke et al. 2003; McDonough et al. 2005), during handgrip exercise (Rosenberry et al. 2019) and in disease populations during isolated knee extensor and cycling exercise (chronic obstructive pulmonary disease: Broxterman et al. 2020; HFrEF: Esposito et al. 2010, 2011 (exercise trained)). To our knowledge, the current investigation is the first to assess changes in convective and diffusive O₂ conductance following specific channel/enzyme inhibition and provides evidence that this approach can be utilized in future studies examining O₂ transport in health, dysfunction related to a range of cardiovascular diseases (i.e. diabetes, sickle cell anaemia, pulmonary hypertension, HF; Padilla et al. 2006, 2007; Hirai et al. 2015; Ferguson et al. 2018), and potential therapeutic interventions aimed at increasing O₂ delivery (i.e. nitrate and nitrite supplementation; Ferguson et al. 2013a,b, 2015, 2016a,b; Glean et al. 2015; Colburn et al. 2017; Craig et al. 2019b).

Experimental considerations

Glycolytic muscle fibres experience the greatest metabolic perturbations during exercise and accordingly contain a greater content of pore-forming K_ATP channel subunit Kir6.2 (type IIB > IIX > IIA > I; Banas et al. 2011) which depresses force production and resting tension and limits intracellular calcium-mediated fibre damage (Gong et al. 2003; Thabet et al. 2005; Cifelli et al. 2008). When skeletal muscle K_ATP channels are inhibited via GLI, this could potentially lead to greater myocyte contraction and $\dot{\mathrm{V}}{\text{O}}_2$. Although augmenting K_ATP channels via pinacidil impairs force production and increases the rate of skeletal muscle fatigue ex vivo, K_ATP channel inhibition via GLI does not decrease skeletal muscle fatigue or alter force production during tetanic contractions yet appears to increase resting muscle tension between contractions and could, therefore, increase $\dot{\mathrm{V}}{\text{O}}_2$ accordingly (Gong et al. 2000; Matar et al. 2000). Nonetheless, the topical application of GLI and related disturbance of $\dot{\mathrm{Q}}{\mathrm{O}}_2:\dot{\mathrm{V}}{\text{O}}_2$ matching (i.e. PO₂is) of the fast-twitch oxidative glycolytic MG are, principally, consequent to impaired blood flow (decreased herein, and also in Holdsworth et al. 2015) rather than increased metabolic demand ($\dot{\mathrm{V}}{\text{O}}_2$; see Fig. 4).

Whereas the present experimental design precluded the assessment of blood flow at rest and during treadmill running (i.e. utilizing fluorescent microspheres to assess resting/running blood flow would prevent blood flow assessment during PO₂is in the same rat), prior investigations from our laboratory have demonstrated reduced muscle blood flow following GLI at rest and a wide range of speeds (20, 40 and 60 m min⁻¹, the latter of which yields $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ on the inclined treadmill in rats, Colburn et al. 2020b and Holdsworth et al. 2015). Therefore, the authors are assured that skeletal muscle blood flow is reduced following systemic GLI administration herein and during the cardiac assessment via Doppler ultrasound.

Clinically, a primary concern of sulphonylurea use in patient populations is the potential for hypoglycaemia. Prior to exercise, hypoglycaemia following GLI-mediated insulin release and systemic glucose uptake would inherently limit exercise duration by restricting blood glucose stores available for energy production. To minimize this concern, the authors performed all exercise testing within 30–90 min of systemic GLI administration to target K_ATP channel inhibition and not incur the confounding effects on glucose availability that would result from a longer duration GLI-mediated insulin release (Li et al. 2012). Importantly, while assessing the speed–duration relationship (Table 2 and Fig. 2), the curvature constant (D′) remained unchanged following K_ATP channel inhibition. While CS is better understood and reflects the upper threshold of oxidative phosphorylation to support metabolic demand, D′ is considered to reflect principally the contributions of finite non-aerobic energy stores and fatigue-resistant muscular properties supporting exercise above CS (reviewed by Poole et al. 2016). Additionally, exercise measurements were performed every 4 days to target the pro-oestrus phase. Repeated acute doses of GLI, which has a half-life of up to 10 h, is not anticipated to have a cumulative effect across testing days. Nevertheless, if a chronic K_ATP channel inhibition effect was captured, it would mirror more directly the use of oral sulphonylureas in T2DM patients and the present investigation may actually underestimate the long-term effects of K_ATP channel inhibition that is associated with elevated risk for adverse cardiovascular events, developing HF, and all-cause mortality (Simpson et al. 2006, 2015; McAlister et al. 2008; Kristiansen et al. 2011; Abdelmoneim et al. 2016).

Conclusions

These data emphasize the important role that vascular ATP-sensitive K⁺ (K_ATP) channels have in supporting exercise tolerance. Crucially, systemic inhibition of K_ATP channels via GLI reduces $\dot{\mathrm{V}}{\mathrm{O}}_{2}max$ and submaximal exercise tolerance (CS). These impairments during treadmill running are reflected in fast-twitch oxidative glycolytic MG muscle where local inhibition of vascular K_ATP channels reduces skeletal muscle blood flow $\dot{\mathrm{Q}}m$ and O₂ delivery during twitch contractions (i.e. PO₂is). As a result, by reducing convective O₂ $\left(\dot{\mathrm{V}}{\text{O}}_2=\dot{\mathrm{Q}}m\times {\text{arterial–venous}\text{O}}_2\text{content}\right)$ and diffusive O₂ conductances $\left(\dot{\mathrm{V}}{\text{O}}_2={\text{DO}}_2\times \mathrm{\Delta }{\text{PO}}_2\right)$, K_ATP channel inhibition lowered muscle $\dot{\mathrm{V}}{\text{O}}_2$. Therefore, the exercise (in)tolerance of disease patients taking oral sulphonylurea medication may be, in part, due to pharmacologically mediated impairments in vascular O₂ transport and muscle O₂ utilization.

Key points.

• Oral sulphonylureas, widely prescribed for diabetes, inhibit pancreatic ATP-sensitive K⁺ (K_ATP) channels to increase insulin release. However, K_ATP channels are also located within vascular (endothelium and smooth muscle) and muscle (cardiac and skeletal) tissue.

• We evaluated left ventricular function at rest, maximal aerobic capacity $\left(\dot{\mathrm{V}}{\text{O}}_{2}max\right)$ and submaximal exercise tolerance (i.e. speed–duration relationship) during treadmill running in rats, before and after systemic K_ATP channel inhibition via glibenclamide.

• Glibenclamide impaired critical speed proportionally more than $\dot{\mathrm{V}}{\text{O}}_{2}max$ but did not alter resting cardiac output.

• Vascular K_ATP channel function (topical glibenclamide superfused onto hindlimb skeletal muscle) resolved a decreased blood flow and interstitial PO₂ during twitch contractions reflecting impaired O₂ delivery-to-utilization matching.

• Our findings demonstrate that systemic K_ATP channel inhibition reduces $\dot{\mathrm{V}}{\text{O}}_{2}max$ and critical speed during treadmill running in rats due, in part, to impaired convective and diffusive O₂ delivery, and thus $\dot{\mathrm{V}}{\text{O}}_2$, especially within fast-twitch oxidative skeletal muscle.

Biography

Trenton D. Colburn is a Predoctoral Fellow in the Cardiorespiratory Physiology Laboratory at Kansas State University, mentored by Drs Timothy I. Musch and David C. Poole. His research focuses on the regulation of oxygen delivery and utilization within the microvascular and interstitial compartments of skeletal muscle in health and disease, with particular interest in nitric oxide bioavailability and ATP-sensitive potassium channel function during exercise.