Amplification of endothelium-dependent vasodilatation in contracting human skeletal muscle: role of KIR channels

Abstract

Key points: In humans, the vasodilatory response to skeletal muscle contraction is mediated in part by activation of inwardly rectifying potassium (K_IR ) channels. Evidence from animal models suggest that K_IR channels serve as electrical amplifiers of endothelium-dependent hyperpolarization (EDH). We found that skeletal muscle contraction amplifies vasodilatation to the endothelium-dependent agonist ACh, whereas there was no change in the vasodilatory response to sodium nitroprusside, an endothelium-independent nitric oxide donor. Blockade of K_IR channels reduced the exercise-induced amplification of ACh-mediated vasodilatation. Conversely, pharmacological activation of K_IR channels in quiescent muscle via intra-arterial infusion of KCl independently amplified the vasodilatory response to ACh. This study is the first in humans to demonstrate that specific endothelium-dependent vasodilatory signalling is amplified in the vasculature of contracting skeletal muscle and that K_IR channels may serve as amplifiers of EDH-like vasodilatory signalling in humans.

Abstract: The local vasodilatory response to muscle contraction is due in part to the activation of inwardly rectifying potassium (K_IR ) channels. Evidence from animal models suggest that K_IR channels function as 'amplifiers' of endothelium-dependent vasodilators. We tested the hypothesis that contracting muscle selectively amplifies endothelium-dependent vasodilatation via activation of K_IR channels. We measured forearm blood flow (Doppler ultrasound) and calculated changes in vascular conductance (FVC) to local intra-arterial infusion of ACh (endothelium-dependent dilator) during resting conditions, handgrip exercise (5% maximum voluntary contraction) or sodium nitroprusside (SNP; endothelium-independent dilator) which served as a high-flow control condition (n = 7, young healthy men and women). Trials were performed before and after blockade of K_IR channels via infusion of barium chloride. Exercise augmented peak ACh-mediated vasodilatation (ΔFVC saline: 117 ± 14; exercise: 236 ± 21 ml min^-1 (100 mmHg)^-1 ; P < 0.05), whereas SNP did not impact ACh-mediated vasodilatation. Blockade of K_IR channels attenuated the exercise-induced augmentation of ACh. In eight additional subjects, SNP was administered as the experimental dilator. In contrast to ACh, exercise did not alter SNP-mediated vasodilatation (ΔFVC saline: 158 ± 35; exercise: 121 ± 22 ml min^-1 (100 mmHg)^-1 ; n.s.). Finally, in a subset of six subjects, direct pharmacological activation of K_IR channels in quiescent muscle via infusion of KCl amplified peak ACh-mediated vasodilatation (ΔFVC saline: 97 ± 15, KCl: 142 ± 16 ml min^-1 (100 mmHg)^-1 ; respectively; P < 0.05). These findings indicate that skeletal muscle contractions selectively amplify endothelium-dependent vasodilatory signalling via activation of K_IR channels, and this may be an important mechanism contributing to the normal vasodilatory response to exercise in humans.

Keywords: Inwardly rectifying potassium channels; endothelium-dependent hyperpolarization; exercise hyperaemia.

Figure 1. Experimental protocols

After catheterization of the brachial artery and subject instrumentation, the change in forearm vascular conductance (ΔFVC) in response to ACh or sodium nitroprusside (SNP) was assessed during rest, handgrip exercise at 5% maximal voluntary contraction (MVC) or a high flow control vasodilator condition. Protocol 1: the vasodilatory response to ACh was assessed at rest, during 5% MVC exercise and during a high flow control SNP infusion, before and after infusion of barium chloride (BaCl₂) to block inwardly rectifying potassium (K_IR) channels. Protocol 2: the vasodilatory response to SNP was assessed at rest, during 5% MVC exercise and during a high flow control ACh infusion. In a subset of subjects, the vasodilatory response to ACh was assessed before and after pharmacological activation of K_IR channels via infusion of potassium chloride (KCl).

Figure 2. Representative tracings of exercise‐induced amplification of ACh‐mediated vasodilatation before and after inhibition of inwardly rectifying potassium channels

Each panel depicts the mean blood velocity response to an intra‐arterial infusion of Ach (initiated at each vertical line) maintained for 3 min during various conditions (indicated below each panel). Left panels: under control conditions, 5% MVC exercise significantly amplified the peak vasodilatory response to ACh compared to resting skeletal muscle (saline; Fig. 3). This effect was not observed with infusion of the endothelium‐independent nitric oxide donor, sodium nitroprusside (SNP; Fig. 5). Middle panels: surprisingly, inhibition of inwardly rectifying potassium (K_IR) channels (BaCl₂ infusion) increased the control response to ACh in quiescent skeletal muscle. However, there was no further exercise‐induced amplification of ACh after inhibition of K_IR channels (Fig. 3). Right panels: pharmacological activation of K_IR channels via infusion of KCl significantly amplified the peak vasodilatory response to ACh, similar to handgrip exercise (Fig. 6).

Figure 3. Exercise amplifies peak ACh‐mediated vasodilatation

The change in forearm vascular conductance (FVC) in response to ACh during saline control, sodium nitroprusside (SNP) (high flow control) and mild intensity exercise (5% MVC) before and after inhibition of inwardly rectifying potassium (K_IR) channels with barium chloride (BaCl₂). A, exercise significantly augments peak ACh‐mediated ΔFVC relative to saline and high flow control (SNP) conditions. B and C, inhibition of K_IR channels significantly increased the peak vasodilatory response to ACh during saline and high flow (SNP) conditions relative to control conditions, although inhibition of K_IR channels prevented the exercise‐induced amplification of ACh‐mediated vasodilatation (P = 0.07). ^*P < 0.05 vs. saline within condition; ^† P < 0.05 vs. SNP within condition; ^‡ P < 0.05 vs. respective control condition; ^# P = 0.07 vs. 5% MVC in control conditions.

Figure 4. Amplification of ACh‐mediated vasodilatation during exercise is attenuated by blockade of inwardly rectifying potassium channels

The effect of 5% MVC exercise and sodium nitroprusside (SNP) on ACh‐mediated vasodilatation (amplification of ACh), calculated by subtracting the change in FVC observed during control infusion from the change in FVC in response to ACh during exercise or SNP (ΔFVC − ΔFVC control). A, exercise amplifies ACh‐mediated vasodilatation beyond what is observed during a passive high flow control condition (SNP). B and C, blockade of inwardly rectifying potassium (K_IR) channels with barium chloride (BaCl₂) attenuated the effect of exercise on ACh‐mediated vasodilatation with no change on the effect of SNP. ^* P < 0.05, effect of 5% MVC vs. effect of SNP; ^† P < 0.05, effect of 5% MVC vs. effect of SNP + BaCl₂; ^# P < 0.05, effect of 5% MVC vs. effect of 5% MVC + BaCl₂.

Figure 5. Exercise does not amplify SNP‐mediated vasodilatation

The change in forearm vascular conductance (FVC) in response to sodium nitroprusside (SNP) during saline control, Ach (high flow control) and mild intensity exercise (5% MVC). A, there were no differences in SNP‐mediated vasodilatation quantified as ΔFVC from baseline. B, peak ΔFVC in response to SNP was not different during exercise or ACh (high flow control) compared to control saline conditions. ^* P < 0.05, 5% MVC vs. saline; ^† P < 0.05, 5% MVC vs. ACh.

Figure 6. Infusion of KCl in quiescent skeletal muscle amplifies peak ACh‐mediated vasodilatation

The change in forearm vascular conductance (FVC) in response to ACh is presented during control saline, and infusion of potassium chloride (KCl) to activate inwardly rectifying potassium (K_IR) channels in quiescent muscle. Peak ACh‐mediated vasodilatation was amplified during KCl infusion compared to saline control. ^* P < 0.05 vs. saline.

Figure 7. Working hypothesis on K_IR channel‐mediated regulation of skeletal muscle blood flow

In resting skeletal muscle (left panel), the prevailing extracellular potassium levels (∼4–5 mM) and membrane potential (E _m) are near the activation potential of K_IR channels, resulting in mild activation of K_IR channels and a modest contribution to resting vascular tone. Application of certain endothelium‐dependent agonists may hyperpolarize membrane potential (leftward arrow) and recruit current through K_IR channels to elicit vasodilatation. Immediately upon initiation of exercise (right panel), efflux of K⁺ from skeletal muscle fibres will increase extracellular [K⁺] (∼8–10 mM) shifting the I–V relationship (solid line) rightward, resulting in activation of K_IR channels and feedforward vasodilatation. Subsequently, any increase in endothelium‐dependent signalling associated with muscle contractions will be amplified via greater conductance through K_IR channels. Thus, for the same EDH stimulus (e.g. ACh or ATP) during muscle contractions, there will be greater recruitment of K_IR channel conductance and amplification of the original stimulus, facilitating greater upstream conduction of vasodilator signalling (figure adapted from Jackson, 2017). [Color figure can be viewed at wileyonlinelibrary.com]