Vascular K_ATP channels mitigate severe muscle O₂ delivery-utilization mismatch during contractions in chronic heart failure rats

Abstract

The vascular ATP-sensitive K+ (K_ATP) channel is a mediator of skeletal muscle microvascular oxygenation (PO₂mv) during contractions in health. We tested the hypothesis that K_ATP channel function is preserved in chronic heart failure (CHF) and therefore its inhibition would reduce PO₂mv and exacerbate the time taken to reach the PO₂mv steady-state during contractions of the spinotrapezius muscle. Moreover, we hypothesized that subsequent K_ATP channel activation would oppose the effects of this inhibition. Muscle PO₂mv (phosphorescence quenching) was measured during 180 s of 1-Hz twitch contractions (~6 V) under control, glibenclamide (GLI, K_ATP channel antagonist; 5 mg/kg) and pinacidil (PIN, K_ATP channel agonist; 5 mg/kg) conditions in 16 male Sprague-Dawley rats with CHF induced via myocardial infarction (coronary artery ligation, left ventricular end-diastolic pressure: 18±1 mmHg). GLI reduced baseline PO₂mv (control: 28.3±0.9, GLI: 24.8±1.0 mmHg, p<0.05), lowered mean PO₂mv (average PO₂mv during the overall time taken to reach the steady-state; control: 20.6±0.6, GLI: 17.6±0.3 mmHg, p<0.05), and slowed the attainment of steady-state PO₂mv (overall mean response time; control: 66.1±10.2, GLI: 93.6±7.8 s, p<0.05). PIN opposed these effects on the baseline PO₂mv, mean PO₂mv and time to reach the steady-state PO₂mv (p<0.05 for all vs. GLI). Inhibition of K_ATP channels exacerbates the transient mismatch between muscle O₂ delivery and utilization in heart failure rats and this effect is opposed by PIN. These data reveal that the K_ATP channel constitutes one of the select few well-preserved mechanisms of skeletal muscle microvascular oxygenation control in CHF.

1. Introduction

Oxygen transfer to peripheral tissues via both convective and diffusive transport routes is routinely interrupted as part of the inexorable progression of chronic, congestive heart failure (Hirai et al., 2015; Poole et al., 2012). Decrements in skeletal muscle blood flow and vascular conductance combined with derangement of capillary structure, geometry and hemodynamics lower the muscle microvascular O₂ pressure (PO₂mv) and impair blood-myocyte O₂ flux thereby reducing work capacity commensurate with the severity of chronic heart failure (CHF) (Miyazaki et al., 2007; Piepoli et al., 2010a, 2010b). Muscle PO₂mv is a powerful measurement as it represents the final pressure head driving blood-myocyte O₂ flux. More importantly, because PO₂mv is set by the ratio of O₂-delivery to O₂-utilization, and occurs at the terminal cell-to-cell interface of the O₂ transport chain, it is impacted by the collective upstream cardiopulmonary perturbations. This makes the PO₂mv sensitive to both the spatial and temporal aspects of vascular function and therefore invaluable in revealing the O₂ transport depredations of diseases such as CHF (Diederich et al., 2002) as well as the role of vasoactive pathways including nitric oxide (NO) function (Ferreira et al., 2006).

Maintaining adequate muscle PO₂mv requires tight matching of O₂-supply to O₂-demand given that even transient mismatch can deplete muscle phosphocreatine stores and exacerbate the accumulation of muscle metabolites associated with fatigue such as ADP, H+, K+ and P_i (Hogan et al., 1992; Richardson et al., 1995; Wilson et al., 1977). Potassium channels that are sensitive to ATP and ADP (K_ATP) constitute a potential coupling between tissue metabolism and vasomotor control (i.e. O₂ supply) via K⁺ efflux-induced hyperpolarization. Accordingly, recent studies in isolated skeletal muscle arterioles demonstrate that the K_ATP channel opener levcromakalim evokes both local and conducted vasodilation (Dora, 2017). K_ATP channel inhibition in vivo exaggerates baroreflex-mediated vasoconstriction (Keller et al., 2004) whereas its activation attenuates α-adrenergic vasoconstriction (Nakai and Ichihara, 1994; Tateishi and Faber, 1995). However, it remains unclear under which circumstances this channel is obligatory for adequate skeletal muscle O₂ transport in health and disease.

Individual studies of K_ATP channel inhibition are equivocal; some demonstrating robust effects on the magnitude of the hyperemic response to exercise (Banitt et al., 1996; Bank et al., 2000; Bijlstra et al., 1996; Holdsworth et al., 2015) with others finding no change (Duncker et al., 2001; Farouque and Meredith, 2003; Schrage et al., 2006). However, conditions of the local microenvironment are crucial to the role of the channel in vivo and will differ among species, disease states and degree of metabolic stress (e.g. exercise). In particular, K_ATP channel function is likely to be an essential response to ischemia and the resultant tissue hypoxia (Bijlstra et al., 1996; Nakai and Ichihara, 1994; Suzuki et al., 2002; Tateishi and Faber, 1995; Wheaton and Chandel, 2011). Since these are common attributes of skeletal muscle in CHF patients blocking vascular K_ATP channel function may exaggerate muscle O₂ transport dysfunction in this condition. This is a relevant scenario in patients with diabetes-CHF comorbidities because sulphonylureas, such as glibenclamide (GLI), are widely prescribed for the management of hyperglycemia, and despite apparent high in vitro pancreatic K_ATP channel affinity, their clinical use is associated with cardiovascular morbidity and mortality (Fadini et al., 2015; Morgan et al., 2014; Simpson et al., 2006). While the cardiovascular effects of sulphonylureas in diabetic patients are a consideration for prescription guidelines, the direct, peripheral vascular consequences of sulphonylurea use in CHF are not well described.

The purpose of this study was to test the hypothesis that vascular K_ATP channel function is preserved in CHF and, therefore, its inhibition via GLI would impair the matching of muscle O₂ delivery-to-utilization following the onset of electrically-induced contractions. This would be reflected by lowered PO₂mv and slowed kinetics (i.e. increased overall time to reach steady-state) following GLI superfusion of the spinotrapezius muscle of CHF rats. Moreover, we hypothesized that these effects of GLI would be opposed by the K_ATP channel activator pinacidil (PIN).

2. Methods

2.1. Ethical approval

All procedures were approved by the Institutional Animal Care and Use Committee of Kansas State University under the guidelines established by the National Institutes of Health. 16 adult male Sprague-Dawley rats (3-6 months old) were maintained in accredited animal facilities (Association for the Assessment and Accreditation of Laboratory Animal Care) at Kansas State University on a 12-h light/12-h dark cycle with food and water provided ad libitum.

2.2. Myocardial infarction procedures

All rats underwent induction of a myocardial infarction (MI) via ligation of the left main coronary artery which has been shown to result reliably in the development of moderate to severe CHF (Musch and Terrell, 1992). Briefly, each rat was anesthetized with a gas mixture of 5% isoflurane-O₂ and intubated for mechanical ventilation on a rodent respirator (model 680, Harvard Instruments, Holliston, USA) with subsequent maintenance on a 3% isoflurane-O₂ gas mixture for the duration of the procedure. A left thoracotomy was performed to expose the heart through the fifth intercostal space. Exteriorization of the heart provided access to the left main coronary artery which was ligated with a 6-0 silk suture ~1-2 mm distal to the edge of the left atrium. The muscles of the thorax were then closed with 4-0 gut, and the skin incision was closed with 3-0 silk followed by an administration of the analgesic agents bupivacaine (1.5 mg/kg subcutaneously) and buprenorphine (0.01–0.05 mg/kg i.m.) as well as ampicillin (50 mg/kg i.m.) to reduce the risk of infection. Upon removal from mechanical ventilation and withdrawal of anesthesia the rats were monitored ~8-12 h post-operatively for the development of arrhythmias and undue stress with care administered as needed. The recovery duration prior to the final, PO₂mv protocol was ≥21 days which is consistent with the time course for complete remodeling of necrotic myocardial tissue (Fishbein et al., 1978). During this time the rats were monitored daily (appetite, weight loss, gait/posture, etc.) in conjunction with the university veterinary staff.

2.3. Surgical instrumentation

On the day of the final protocol the rats were anesthetized initially with a 5% isoflurane-O₂ mixture and maintained on a 3% isoflurane-O₂ mixture for the duration of the surgical instrumentation. The carotid artery was cannulated and a two-French-catheter-tipped pressure transducer (Millar Instruments, Houston, USA) was advanced into the left ventricle (LV) for the measurement of LV end diastolic pressure (LVEDP). Subsequently, cannulation of both the carotid and caudal arteries was performed with PE-10 connected to PE-50 (Intra-Medic polyethylene tubing, Clay Adams, Spark, USA) for the measurement of mean arterial pressure (MAP) and heart rate (HR) as well as infusion of the phosphorescent probe Oxyphor G2 (Oxygen Enterprises, Philadelphia, PA, USA; carotid) and for blood sampling and administration of anesthetics (caudal). Isoflurane-O₂ inhalation was progressively removed as anesthesia was maintained with pentobarbital sodium administered via the caudal artery catheter to effect. Depth of anesthesia was monitored at frequent and regular intervals via the blink and toe-pinch reflexes as well as magnitude and frequency of ventilation. Access to the spinotrapezius was achieved by surgically reflecting the overlying skin and fascia. The spinotrapezius muscle was selected because the fiber-type composition and oxidative capacity closely resemble that of the human quadriceps muscle group which makes it a useful analog of human locomotor muscle (Delp and Duan, 1996). During both the surgical preparation and experimental protocol the muscle was superfused frequently with Krebs-Henseleit bicarbonate-buffered solution consisting of (in mM) 4.7 KCl, 2.0 CaCl₂, 2.4 MgSO4, 131 NaCl, and 22 NaHCO3 equilibrated with 5% CO₂ and 95% N₂ (pH 7.4, 37-38°C). Exposed tissue surrounding the spinotrapezius muscle was covered with Saran wrap (Dow Brands, Indianapolis, IN) to reduce dehydration and confine the exposure of GLI and PIN to the spinotrapezius muscle. Platinum iridium electrodes were sutured to the rostral (cathode) and caudal (anode) regions of the spinotrapezius muscle for electrically-induced twitch contractions. As previously reported by our laboratory these surgical procedures do not impact the microvascular integrity and responsiveness of the rat spinotrapezius muscle (Bailey et al., 2000).

2.4. Drugs

The pharmacological sulphonylurea derivative GLI (494 g/mol; 5-chloro-N-(4-[N- (cyclohexylcarbamoyl)sulfamoyl]phenethyl)-2-methoxybenzamide; Sigma-Aldrich, St Louis, MO, USA) was used to inhibit vascular K_ATP channels. Given that GLI is difficult to maintain in solution, 25 mg of GLI was dissolved in 99:1 ratio of distilled water/NaOH (1 M) during continuous sonication to produce a 2.5 mg/ml stock solution. A 5 mg/kg dose was drawn from the stock solution and diluted to ~3 ml with Krebs-Henseleit solution. PIN (245 g/mol; N-cyano-N'-pyridin-4-yl-N"-(1,2,2-trimethylpropyl)guanidine; Sigma-Aldrich, St Louis, MO, USA) is readily dissolved in distilled water and was diluted to ~3 ml with Krebs-Henseleit solution before being administered to oppose vascular K_ATP channel inhibition at the same dose as GLI.

2.5. Experimental protocol

Three separate contraction bouts were performed under control (vehicle), GLI (5 mg/kg) and PIN (5 mg/kg) conditions. Drug doses and experimental protocol were selected based on previous investigations examining the functional role of K_ATP channels on skeletal muscle blood flow and PO₂mv (Holdsworth et al., 2016, 2015) and preliminary studies in our laboratory. The vehicle was administered for control via superfusion (3 ml) of the spinotrapezius during 180 s of continuous PO₂mv recording. The recording was extended for an additional 90-180 s to ensure that baseline PO₂mv had stabilized at which time muscle contraction was evoked via electrical stimulation (1 Hz, ~6 V, 2-ms pulse duration, model s48; Grass Technologies, Quincy, MA) for 180 s. This protocol has been shown to increase spinotrapezius muscle blood flow four-to five-fold and metabolic rate six- to seven-fold without altering blood pH and is consistent with moderate intensity exercise (Behnke et al., 2002; Hirai et al., 2013). Immediately upon cessation of electrical stimulation an arterial blood sample was drawn (~0.8 ml) for the determination of blood gases, hematocrit, pH, [lactate] and [glucose]. This was followed by a brief washout with Krebs-Henseleit solution and a 20-30 min recovery period, after which the second contraction bout was performed in an identical fashion to the first after the superfusion of GLI (5 mg/kg). This was repeated for the third contraction bout with the administration of PIN. As previously reported by our laboratory, >20 min recovery elicits reproducible PO₂mv responses (Copp et al., 2009; Herspring et al., 2008). Given the long-lasting effects of GLI and the possibility that this drug may not washout completely with Krebs-Henseleit superfusion and the recovery period employed herein (Thomas et al., 1997; cf. Brayden, 2002), PIN was compared to GLI only as described in the Statistical analysis section below. At the end of the protocol, rats were euthanized with intra-arterial pentobarbital sodium overdose (>50 mg/kg) and subsequent removal of the heart. The lungs, right ventricle (RV), LV and atria were removed and weighed. Measurement of infarct size in the LV was made via planimetry as described previously (Ferreira et al., 2006).

2.6. Spinotrapezius PO₂mv measurement

PO₂mv was measured via phosphorescence quenching using a frequency domain phosphorometer (PMOD 5000; Oxygen Enterprises, Philadelphia, PA). As described previously (Behnke et al., 2001), this technique applies the Stern-Volmer relationship (Rumsey et al., 1988), which describes the quantitative O₂ dependence of the phosphorescent probe G2 via the equation:

$${\text{PO}}_2\text{mv}=[(\tau °∕\tau )-1]∕({\text{k}}_{\text{Q}}\cdot \tau °)$$

where k_Q is the quenching constant and τ and τ° are the phosphorescence lifetimes at the ambient O₂ concentration and in the absence of O₂, respectively. For the phosphorescent probe G2, k_Q is 273 mmHg⁻¹ s⁻¹ and τ° is 251 µs (Bodmer et al., 2012; Dunphy et al., 2002). These characteristics do not change over the physiological range of pH (~7.4) and temperature (~38°C) in vivo and, therefore, the phosphorescence lifetime is affected solely by the O₂ partial pressure. The Pd-porphyrin cores of phosphor probes bind to albumin; the primary macromolecule in plasma. This quality as well as the probe’s negative charge ensure restriction to the vascular compartment, of which, the capillary bed volumetrically constitutes the major intramuscular space (Poole et al., 2004). After infusion of G2 the common end of the bifurcated light guide with a >500 µm penetration depth was positioned ~2-4 mm superficial to the dorsal surface of the exposed spinotrapezius muscle in an area devoid of macro-vessels. The phosphorometer modulates sinusoidal excitation frequencies between 100 Hz and 20 kHz and allows phosphorescence lifetime measurements from 10 µs to ~2.5 ms. PO₂mv was measured continuously and recorded at 2-s intervals throughout the duration of the experimental protocol.

2.7. Analysis of spinotrapezius PO₂mv kinetics

PO₂mv time course was determined via exponential regression analysis by applying the Levenberg-Marquardt algorithm (SigmaPlot 11.0; Systat software, San Jose, CA) to the contraction-induced PO₂mv transient. PO₂mv responses were fit with either a one- or two-component model as follows.

One component:

$${\text{PO}}_{2\text{mv}\left(t\right)}={\text{PO}}_{2\text{mv}\left(\text{BL}\right)}-{\Delta }_1{\text{PO}}_{2\text{mv}}[1-{\text{e}}^{-(t-{\text{TD}}_1∕{\tau }_1)}]$$

Two component:

$${\text{PO}}_{2\text{mv}\left(t\right)}={\text{PO}}_{2\text{mv}\left(\text{BL}\right)}-{\Delta }_1{\text{PO}}_{2\text{mv}}[1-e^{-(t-{\text{TD}}_1∕{\tau }_1)}]+{\Delta }_2{\text{PO}}_{2\text{mv}}[1-{\text{e}}^{-(t-{\text{TD}}_2∕{\tau }_2)}]$$

PO₂mv_(t) is the PO₂mv at a given time t; PO₂mv_(BL) corresponds to the pre-contracting resting PO₂mv; Δ₁ and Δ₂ are the amplitudes for the first and second components, respectively; TD₁ and TD₂ are the independent time delays for each component; and τ₁ and τ₂ are the time constants (i.e. time to 63% of the response) for each component. Goodness of fit was determined using three criteria: the coefficient of determination, sum of squared residuals, and visual inspection (Behnke et al., 2002).

The mean response time (MRT₁) was used to describe the overall dynamics of the PO₂mv fall following the onset of muscle contractions via the equation:

$${\text{MRT}}_1={\text{TD}}_1+{\tau }_1$$

The MRT₁ analysis was limited to the first component of the PO₂mv response given that inclusion of an emergent second component underestimates the speed of the PO₂mv fall at the onset of contractions. In the event of a second component the MRT₂ was calculated using the following equation:

$${\text{MRT}}_2={\text{TD}}_2+{\tau }_2$$

To determine the overall time taken to reach the steady-state PO₂mv the MRT_Total was calculated via the equation:

$${\text{MRT}}_{\text{Total}}={\text{MRT}}_1+{\text{MRT}}_2$$

The MRT_Total does not equal the sum of the average MRT₁ and MRT₂ because the calculation of MRT_Total is independent of the average of MRT₂. The purpose of the second component kinetics parameters is to represent the speed with which the PO₂mv_(SS) is reached after the initial fall in PO₂mv. For this reason, the MRT₂ averages are calculated from rats with a two-component fit only. However, the calculation of MRT_Total necessarily reflects the absence of MRT₂ (i.e. entries of zero) for rats fit with a one-component model.

Mean PO₂mv was calculated as the average PO₂mv from the contractions onset through the duration of MRT_Total.

2.8. Statistical analysis

MAP and HR were compared between conditions and across time points via 2-way repeated measures ANOVA with Student Newman Keuls post-hoc tests as necessary. Kinetics parameters were compared between conditions with 1-way, repeated measures ANOVA on the basis of the a priori hypotheses. Student Newman Keuls post-hoc tests were used to compared control/GLI and GLI/PIN when necessary. Results are presented as means ± SE. The level of significance was set at p < 0.05.

3. Results

The final body weight for all CHF rats was 463 ± 12 g. Table 1 summarizes the descriptive statistics for the central indices of CHF. Arterial blood [lactate], [glucose], PO₂, PCO₂, O₂ saturation, hematocrit and pH were not different with GLI relative to control and were also not different after PIN superfusion compared to GLI (p > 0.05 for all; data not shown). The MAP (control: 110 ± 4, GLI: 114 ± 5 mmHg) and HR (control: 326 ± 10, GLI: 337 ± 14 beats/min) at the start of contractions with GLI were not different from control (p > 0.05 for both). PIN reduced MAP at the start of contractions compared to GLI (PIN: 100 ± 6, GLI: 114 ± 5 mmHg, p < 0.05), but did not change HR (PIN: 367 ± 14, GLI: 337 ± 14 beats/min, p > 0.05).

Table 1.

Morphological and hemodynamic characteristics of CHF rats

	Mean ± SEM	Range
LVEDP, mmHg	18 ± 1	10 – 27
LV dp/dt, mmHg/s	6270 ± 239	4900 – 8800
LV/body mass, mg/g	1.91 ± 0.03	1.73 – 2.07
RV/body mass, mg/g	0.59 ± 0.02	0.51 – 0.78
Lung/body mass, mg/g	3.56 ± 0.16	2.78 – 5.29
Infarct size, %	30 ± 1	18 - 42

Data are mean ± SEM. LVEDP, left ventricular end diastolic pressure; LV dp/dt, left ventricular dpressure/dtime RV, right ventricle; LV, left ventricle. n = 16.

Muscle PO₂mv responses from rest to contractions are shown in Fig. 1 and the kinetics parameters are provided in Table 2. The goodness of fit for the models was demonstrated by an r² of 0.97 ± 0.01 and the sum of squared residuals of 18.9 ± 3.0. Overall, the mean PO₂mv over the early contractions transient was significantly reduced with GLI relative to control (control: 20.6 ± 0.6, GLI: 17.6 ± 0.3 mmHg, p < 0.05). During the control condition 11 of 16 rats exhibited PO₂mv profiles that transiently fell below the contracting steady-state (termed an “undershoot”, Δ₂PO₂mv) necessitating a two-component model fit. Conversely, during GLI the two-component model was required for 15 of 16 rats and during PIN for 5 of 12 rats. Compared to control, GLI reduced the PO₂mv baseline (PO₂mv_(BL)) prior to contractions. In addition, GLI increased the amplitude of Δ₂PO₂mv and slowed the attainment of the steady-state as indicated by an increase in the total mean response time (MRT_Total).

Figure 1.

Mean absolute spinotrapezius muscle PO₂mv profiles for (A) control and GLI and (B) GLI and PIN in CHF rats. (C) Mean relative spinotrapezius PO₂mv profiles for control, GLI and PIN in CHF rats. Contractions began at time zero. Notice the divergence of the GLI profile resulting in a marked undershoot of the steady-state PO₂mv.

Table 2.

Spinotrapezius PO₂mv parameters at rest and following the onset of contractions under control, GLI and PIN conditions

	Control	GLI	PIN
PO2mv(BL), mmHg	28.3 ± 0.9	24.8 ± 1.0*	32.5 ± 2.0†
Δ1PO2mv, mmHg	11.0 ± 0.7	9.7 ± 0.6	9.7 ± 1.2
Δ2PO2mv, mmHg	3.3 ± 0.2	4.3 ± 0.4*	2.4 ± 0.4†
PO2mv(SS), mmHg	19.6 ± 0.6	19.1 ± 0.9	23.8 ± 1.4†
TD1, s	5.5 ± 0.9	4.7 ± 0.6	3.0 ± 0.7
TD2, s	39.0 ± 6.9	36.8 ± 5.2	57.0 ± 12.7
τ1, s	10.6 ± 0.7	10.3 ± 1.0	16.8 ± 3.6
τ2, s	33.6 ± 4.6	47.1 ± 5.4*	32.5 ± 7.2
MRT1, s	16.2 ± 1.1	15.0 ± 1.0	19.8 ± 3.3
MRT2, s	72.6 ± 7.9	83.9 ± 6.3	89.5 ± 12.2
MRTTotal, s	66.1 ± 10.2	93.6 ± 7.8*	57.1 ± 14.1†

Values are mean ± SE. PO₂mv_(BL), resting PO₂mv; Δ ₁PO2mv, amplitude of the first component; Δ₂PO₂mv, amplitude of the second component; PO₂mv_(SS), contracting steady-state PO₂mv; TD₁, time delay of the first component; TD₂, time delay of the second component; τ ₁, time constant of the first component; τ₂, time constant of the second component; MRT₁, mean response time of the first component; MRT₂, mean response time of the second component; MRT_Total, sum of the first and second component mean response times. The two-component model was used to describe the PO₂mv kinetics in the following conditions: CON (11/16); GLI (15/16); PIN (5/12).

^*p < 0.05 vs. control.

^†p < 0.05 vs. GLI.

PIN increased the mean PO₂mv across MRT_Total compared to GLI (GLI: 17.6 ± 0.3, PIN: 25.7 ± 0.5 mmHg, p < 0.05). This is in direct opposition to the effect of GLI during the early contraction transient relative to control. PIN also increased both the baseline and steady-state PO₂mv with no difference in Δ₁PO₂mv relative to the GLI condition. The effects of PIN were such that a faster attainment of the steady-state (i.e. lower MRT_Total) was observed when compared to GLI.

4. Discussion

K_ATP channel inhibition via GLI fundamentally altered the spinotrapezius PO₂mv profile such that mean PO₂mv was lowered at rest and following the onset of contractions in CHF rats. The major effects of GLI were 1) a reduction in the baseline PO₂mv; 2) a near doubling of the PO₂mv undershoot amplitude with a 42% increase in the overall time to reach the contracting steady-state and MRT_Total; and 3) a reduced mean PO₂mv during the transition from contraction onset to the steady-state PO₂mv. These data indicate that vascular smooth muscle and/or endothelial K_ATP channel function are essential to the control of muscle PO₂mv kinetics at the onset of contractions in CHF, and the K_ATP channel may represent one of the select few well-preserved mechanisms of skeletal muscle vascular control in CHF. This K_ATP channel-conferred protection against severe O₂-delivery to O₂-utilization mismatch during contractions should raise serious concerns when sulphonylureas such as GLI are prescribed for patients with coincident diabetes and CHF.

The central indices of dysfunction (Table 1) support that moderate CHF was induced in these animals (Ferreira et al., 2006) which is consistent with New York Heart Association (NYHA) class II and III CHF in humans (Musch et al., 1988). The spectrum included animals with elevated LVEDP’s (up to 27 mmHg) and MI size’s greater than 40% (severe CHF) however the sample size was not sufficient for adequately powered statistical comparisons between moderate and severe sub-groups. Thus, the moderate CHF herein may underestimate the effect of GLI on the PO₂mv profiles during contractions. Nonetheless, moderate CHF induced via the rat model of coronary artery ligation does result in decrements to skeletal muscle blood flow, vascular conductance, PO₂mv kinetics, $\stackrel{.}{\mathrm{V}}{\text{O}}_2\text{max}$, and time-to-exhaustion during exercise akin to NYHA Class II and III CHF in humans (Hirai et al., 2015; Musch and Terrell, 1992; Poole et al., 2012; Richardson et al., 2003).

K_ATP channel manipulation of the steady-state O₂ delivery has been characterized previously for in vivo electrical stimulation (Thomas et al., 1997), reactive hyperemia (Banitt et al., 1996; Bank et al., 2000; Bijlstra et al., 1996) and exercise-induced hyperemia (Holdsworth et al., 2015). Our laboratory has also demonstrated that GLI increased the prevalence of the undershoot and prolonged the time to reach the contracting steady-state PO₂mv in the spinotrapezius muscle of healthy rats (Holdsworth et al., 2016). The available evidence is equivocal in regards to $\stackrel{.}{\mathrm{V}}{\text{O}}_2$ and mitochondrial respiration with modulation of K_ATP channel function. Some investigations indicate no appreciable change in $\stackrel{.}{\mathrm{V}}{\text{O}}_2$ during whole body exercise with GLI in diabetic patients (Larsen et al., 1999) and that mitochondrial K_ATP channel inhibition does not impact the O₂ cost of myocardial contractions (Chen et al., 2001). Other groups have found that GLI reduces diaphragmatic $\stackrel{.}{\mathrm{V}}{\text{O}}_2$ in canines (largely driven by reductions in O₂ delivery) (Vanelli et al., 1994) and that skeletal muscle mitochondrial respiration is impaired by small doses of GLI (Montoya-Pérez et al., 2010). While GLI-induced changes in O₂ delivery may constitute the primary driver for the perturbed PO₂mv profile, it is not possible to identify whether the superfusion method utilized herein affected K_ATP channels present specifically within the arteriole wall and/or myocyte (Flagg et al., 2010; Jackson, 2000). Further studies in CHF are required to establish whether manipulation of K_ATP channel function could be influenced by the mode of drug delivery and/or specific tissue of interest.

The small-muscle mass preparation used herein poses an advantage for investigations of peripheral vascular control because it does not tax the limits of cardiac output. This is a strength for investigations of vascular K_ATP channels in CHF because the channel’s role in cardiac function may obfuscate detection and/or interpretation of the impact on peripheral vascular control (Nichols et al., 1991; Noma, 1983). Although no direct assessment of K_ATP channel activity and/or expression was performed in the current study, in CHF rats it appears that the vascular K_ATP channel pathway remains functional given the marked sensitivity of the PO₂mv profile to both GLI and PIN. The PO₂mv profiles of the CHF rats herein exhibit the characteristic undershoot demonstrated previously by our laboratory (Behnke et al., 2001; Hirai et al., 2014) and permitted the statistical comparison of the second kinetics component (i.e. undershoot) between control and GLI which was not possible in our previous investigation of K_ATP function in healthy rats (Holdsworth et al., 2016). Specifically, the present investigation revealed that GLI elevated the magnitude of the PO₂mv undershoot and slowed the overall time taken to reach steady-state. In contrast to previous results in healthy rats (Holdsworth et al., 2016), baseline PO₂mv was reduced with GLI indicating a role for K_ATP-mediated function in resting O₂ delivery-utilization matching in CHF. In addition, our findings indicate that PIN opposed the major effects promoted by GLI on muscle PO₂mv thus indicating modulation of K_ATP channel function rather than some non-specific effect with the present experimental protocol.

Intrinsic impairments in specific vasodilatory mechanisms in CHF skeletal muscle such as reduced NO bioavailability (Ferreira et al., 2006) will place greater reliance on K_ATP channel function during contractions. Figure 2 demonstrates different responses to GLI on the spinotrapezius muscle PO₂mv in healthy (data from Holdsworth et al., 2016) versus the present investigation with CHF rats. That GLI induced a greater reduction in muscle PO₂mv during the early contraction transient in CHF compared to healthy animals is consistent with augmented reliance of K_ATP channel function on O₂ delivery-utilization matching in this disease. Crucially, greater O₂ delivery-utilization mismatch with GLI in CHF occurs concomitantly with the slowing of second component (phase II) $\stackrel{.}{\mathrm{V}}{\text{O}}_2$ kinetics following the onset of contractions (Behnke et al., 2002). Exacerbating the fall in skeletal muscle PO₂mv due to a sluggish increase in O₂ delivery is also likely to impact cellular metabolism and contribute to the slowing of $\stackrel{.}{\mathrm{V}}{\text{O}}_2$ kinetics and premature fatigue that is prominent in CHF patients (Ferreira et al., 2005; Hepple et al., 1999; Wheaton and Chandel, 2011; Wilson et al., 1977).

Figure 2.

Magnitude of change in spinotrapezius muscle PO₂mv with GLI during the early transient from rest to contractions in healthy (data from Holdsworth et al., 2016) versus the present investigation with CHF rats. Contractions began at time zero. GLI superfusion of the spinotrapezius muscle was utilized herein to minimize potential alterations in MAP and HR (primarily due to reduced conductance in the renal and splanchnic vascular beds) (Duncker et al., 2001; Holdsworth et al., 2015) observed previously with intra-arterial drug administration in healthy rats (Holdsworth et al., 2016). Note the different effects of GLI on muscle PO₂mv in CHF compared to healthy rats, indicating a greater reliance of K_ATP channel function on O₂ delivery-utilization matching in this disease. See text for further details. *p < 0.05 vs. healthy.

GLI clearly exaggerates many of the CHF-induced perturbations of skeletal muscle PO₂mv kinetics including elevating the undershoot amplitude and delaying attainment of the contracting steady-state PO₂mv (Ferreira et al., 2006). This effect is also well-characterized in other O₂ supply-limited conditions such as aging (Behnke et al., 2005) and diabetes (Padilla et al., 2007) which are often coincident conditions for CHF patients. The exacerbation of this dysregulation with GLI is clinically relevant given that these populations present with severe exercise intolerance (Piepoli et al., 2010a, 2010b; Sacre et al., 2015) due, in part, to a lowering of the skeletal muscle PO₂mv pressure head intrinsic to those diseases (Hirai et al., 2015; Poole et al., 2012). Both the magnitude and time course (i.e. concomitant with the rise in $\stackrel{.}{\mathrm{V}}{\text{O}}_2$ following contractions onset) of the decrements in PO₂mv suggest that degradation of the intracellular metabolic environment is likely to impair muscle work capacity and/or $\stackrel{.}{\mathrm{V}}{\text{O}}_2$ kinetics with GLI.

Impairments in contracting muscle PO₂mv in CHF have been attributed, in part, to reductions in NO bioavailability (Ferreira et al., 2006) and the results herein suggest that preserved K_ATP function protects against a more severe mismatch of O₂-delivery to O₂-utilization in this disease. Taken together, the shared contribution of NO and K_ATP channel signaling to muscle PO₂mv suggests that these pathways are not merely independent, parallel mechanisms, but rather synergistic mediators of skeletal muscle vascular and metabolic control. NO has been shown to cause arterial hyperpolarization, in part, via K_ATP channel activation (Murphy and Brayden, 1995) which may occur through the cGMP intermediary as demonstrated previously in cardiac myocytes (Chai et al., 2011) and cultured smooth muscle cells (Kubo et al., 1994). Furthermore, as a reactive nitrogen species, NO can cause direct S-nitrosylation of cysteine residues found on the SURx subunit (Kawano et al., 2009; Yoshida et al., 2006). Thus, K_ATP channel activation via NO is likely mediated through a combination of these two mechanisms. The notion that K_ATP channels can amplify vasodilatory signaling (Cole et al., 2000; Silva et al., 2008) via modulation of membrane potential deserves further investigation and may be valuable clinically (Akrouh et al., 2009).

Both the results herein and large retrospective studies of the association of sulphonylurea monotherapy with cardiovascular mortality and morbidity are cause for concern in diabetic and CHF patients (Fadini et al., 2015; Morgan et al., 2014; Simpson et al., 2006). Our findings demonstrate the additive effect of GLI on PO₂mv dysregulation in CHF which has been minimally addressed in the risk assessments of sulphonylurea therapy. That the primary muscle $\stackrel{.}{\mathrm{V}}{\text{O}}_2$ kinetics are significantly slowed in diabetic patients raises an intriguing question as to whether it is a de facto diabetes- or alternatively sulphonlyurea-induced dysfunction of the K_ATP channel that drives the $\stackrel{.}{\mathrm{V}}{\text{O}}_2$ kinetics limitation (Bauer et al., 2007; O’Connor et al., 2015; Regensteiner et al., 1998). Clearly, when CHF attenuates other pathways vascular K_ATP channels remain one of the few potential defenses protecting against a more severe mismatch of O₂-delivery to O₂-utilization during muscle contractions. The administration of sulphonylureas may pose a previously unrecognized danger in patients afflicted with both CHF and diabetes where an additional drag on O₂ delivery could severely compromise exercise tolerance. The findings herein extend previous investigations of GLI in healthy rats (Holdsworth et al., 2016) by demonstrating a robust perturbation of the PO₂mv profile at the onset of electrically-induced contractions of the spinotrapezius muscle in CHF rats. The apparent sensitivity of the vascular K_ATP channels to pharmacologic intervention suggests that, in contrast to the well-established disruption of other mechanisms of vascular control in CHF such as NO synthase uncoupling (Maier et al., 2000; Sindler et al., 2009) and autonomic imbalance (Coats et al., 1994; Wang et al., 2010), preserved vascular K_ATP channel function protects against exaggerated transient skeletal muscle hypoxia during contractions.

Highlights.

• Chronic heart failure impairs skeletal muscle microvascular oxygenation (PO₂mv) and exercise tolerance.

• Vascular ATP-sensitive K+ (K_ATP) channels are important modulators of muscle PO₂mv during contractions in health.

• Herein we demonstrate that preserved K_ATP channel function helps preserve PO₂mv during muscle contractions in chronic heart failure.