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Division of Cardiology, Department of Medicine, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania
Submitted 22 March 2006 ; accepted in final form 22 September 2006
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ABSTRACT |
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sympathetic nervous system; exercise; blood flow
It is known that skeletal muscle interstitial K+ rises with exercise (21). Increased K+ can then activate the group III and IV muscle afferent nerves and lead to an increase in blood pressure (38). This activation is a part of the exercise pressor reflex (27).
Na+-K+ pump function is a major regulator of interstitial K+ and contributes to alterations in K+ concentrations (4). Previous studies have shown that the number of Na+-K+ pumps was reduced in rats with myocardial infarction (MI) (31, 35). However, conclusive data on whether muscle interstitial K+ concentration ([K+]o) accumulates to a greater degree in MI rats than in control rats have not been collected. Thus the purpose of the present study was to determine the following: 1) [K+]o in resting muscle and K+ responses during muscle contraction in healthy controls and in MI rats and 2) the role of the Na+-K+ pump activity on K+ responses to muscle contraction in control rats and rats with heart failure (HF). We hypothesized that an increase in [K+]o during muscle contraction in rats with MI would be greater than that seen in control animals. To examine pump activity, we added ouabain to the muscle perfusate. We hypothesized that the greater rise in K+ with contraction in HF was due to attenuated Na+-K+ pump activity in HF.
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METHODS |
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All procedures outlined in this study were approved by the Animal Care Committee of this institution. Sprague-Dawley male rats (150–180 g) were anesthetized by inhalation of isoflurane-oxygen mixture, intubated, and artificially ventilated. A left thoracotomy between the fourth and fifth ribs was performed, exposing the left ventricular wall. The left coronary artery was ligated. Microdialysis experiments were performed 8–14 wk after coronary ligation. Age- and body weight-matched rats served as controls.
Transthoracic echocardiography was performed 1 to 2 wk before the microdialysis experiments. The rats were anesthetized by inhalation of isoflurane-oxygen mixture. The transducer was positioned on the left anterior chest, and left ventricular dimensions were measured.
Experimental Preparation
The rats were anesthetized by inhalation of isoflurane-oxygen mixture. An endotracheal tube was inserted into the trachea and attached to a ventilator. Polyethylene catheters (PE-50) were inserted into the common carotid artery and external jugular vein for measurement of arterial blood pressure and for drug administration, respectively. The sciatic nerve of each leg was isolated and then placed on a stimulating electrode. The animals were ventilated, and respiratory parameters were monitored and maintained within normal ranges as previously described (17, 18). Body temperature was maintained between 37.5–38.5°C by a heating pad and external heat lamps, and fluid balance was stabilized by a continuous infusion of saline.
Decerebration was performed as previously described (17, 18). A transverse section was made anterior to the superior colliculus. Once this procedure was completed, anesthesia was removed from the inhaled mixture. The triceps surae muscle was isolated, and the calcaneal bone of the hindlimb was cut. The Achilles tendon was connected to a force transducer for the measurement of muscle tension during electrically induced muscle contraction. The pelvis was stabilized in a spinal unit, and the knee joints were secured by clamping the patellar tendon to a spinal unit.
On completion of each experiment, a 2-Fr microMillar pressure transducer catheter (Millar Instruments) was inserted into the right carotid artery and threaded into the left ventricle for measurement of left ventricular end-diastolic pressure. The heart was exercised after intravenous injection of an overdose of pentobarbital sodium (120 mg/kg body wt), followed by 2 ml of a saturated solution of KCl. MI size was then estimated by examination of cardiac tissue as described previously (3). Briefly, the left ventricle was pressed flat. The circumference of the entire flat left ventricle and visualized infarcted area were outlined on a transparent paper sheet. The difference in weight between the two marked areas on the sheet was used to determine the size of MI that was expressed as the percentage of left ventricle surface area. The data collected from rats with MI size of >35% of the left ventricle were included in this report.
Microdialysis
The skin directly over the triceps surae muscles of both legs was dissected away, and four microdialysis probes were inserted into the gastrocnemius muscle of each leg. Briefly, the probes were inserted into the muscle (two probes in each side of the muscle at 
2.5 mm deep from the tissue surface) via a cannula in the direction parallel to muscle fiber orientation. We did the same procedure on all animals used in this study. After insertion, the microdialysis probes were attached to a perfusion pump and perfused at a rate of 2.5 µl/min with physiological saline solution.
The semipermeable fibers with a molecular weight cutoff of 30,000 (0.20 mm ID, 0.22 mm OD; Spectrum Laboratories, Laguna, CA) were used to construct the microdialysis probes. Each end of a single fiber was inserted 1 cm into a hollow polyamide tube (0.25 mm ID, 0.36 mm OD) and glued. The length of the probe fibers was 2.0 cm.
Interstitial K+ Measurement
To measure interstitial K+, we employed micro-flow-thru K+ electrodes (Microelectrode, Londonderry, NH). These electrodes were modified so that the dead space in the system was <5 µl. The ends of individual microdialysis probes were attached to the electrodes via a modified manifold system, and thus the dialysate from these probes continuously flowed past the electrodes. This approach allowed continuous and online measurements of K+ throughout the experiment. Prior experiments illustrate the accuracy and reliability of the online K+ electrodes to determine dialysate K+ concentrations (20, 21).
The percent recovery rate of K+ for microdialysis probe was examined in vitro. The rates for 1.0, 2.5, 5.0, and 10 mM of K+ were 63–68%. A linear regression analysis for dialysate versus standard K+ concentrations shows that the relationship is linear (r = 0.956, P < 0.001). Thus K+ concentrations measured in the dialysate were linearly related to the interstitial K+ in the rat muscle. It has been shown that probe recovery is not altered by vasoconstrictor maneuvers or twitch contractions of muscle (21). In this report, we present dialysate concentrations without considering the recovery rate of the probes.
Experimental Protocols
Experiment 1: muscle contraction: 6 healthy control rats and 6 MI rats. After the microdialysis probes were inserted, a 60-min equilibration period was allowed. Contractions induced by electrical stimulation of the sciatic nerve were then performed. Three bouts of rhythmic contraction were conducted at frequencies of 1, 3, and 5 Hz (2.5 times motor threshold and 0.1-ms duration). Each stimulation frequency was sustained for 1 min. There was a 60-min rest period between each bout of contraction. [K+], before, during, and after each workload, was measured.
Experiment 2: electrical stimulation after muscle paralysis: 6 healthy control rats. Before 3 Hz of electrical stimulation, pancuronium bromide (200 µg/kg body wt) was injected intravenously and [K+] was measured before, during, and after stimulation. There was no muscle tension produced by stimulation after paralysis.
Experiment 3: perfusion of an inhibitor of Na+-K+ pump: 6 control rats and 4 MI rats. Ouabain (5 mM) was added to the microdialysis perfusate (19). [K+] was measured before and after ouabain treatment. In this part of the experiment, we examined the alterations in resting K+ concentrations during 30 min of ouabain perfusion.
Data Acquisition and Analyses
Arterial blood pressure and developed muscle tension during muscle contraction were recorded on an eMac computer that used PowerLab software. Interstitial [K+] was recorded on a PC-based computer. Control values were determined by averaging at least 1 min of the data immediately before the interventions. The peak change of each variable was determined by the peak response from control.
Peak change data for each variable were analyzed with a one-way ANOVA. The difference in 
[K+]o 40–60 s after stimulation between control rats and MI rats was also analyzed with a one-way ANOVA. Tukey post hoc analyses were used to determine differences between groups. All values were expressed as means ± SE. For all analyses, differences were considered significant at P < 0.05. All statistical analyses were performed using SPSS for windows version 13.0.
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RESULTS |
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Twitch muscle contraction significantly increased [K+]o in healthy and MI rats. Time courses of increase in [K+]o during 1 min of contraction are shown in Fig. 1. Changes in [K+] 40–60 s after beginning of stimulation were shown in Table 2. In control rats, peak responses of [K+]o at 1-, 3-, and 5-Hz stimulation were 1.19 ± 0.11, 1.36 ± 0.14, and 1.48 ± 0.26 mM from baselines of 1.04 ± 0.08, 0.94 ± 0.05, and 0.92 ± 0.04 mM, respectively. In MI rats, contractions at 1-, 3-, and 5-Hz stimulation increased [K+]o (baseline to peak) from 1.26 ± 0.12 to 1.55 ± 0.15 mM, 1.20 ± 0.12 to 1.95 ± 0.22 mM, and 1.14 ± 0.14 to 1.87 ± 0.26 mM, respectively. This increase in [K+]o in exercising muscle was augmented after MI (Fig. 2). The peak increase in [K+]o was 0.15 ± 0.02 (controls) and 0.29 ± 0.03 mM (MI rats; P < 0.05 vs. controls) by 1-Hz stimulation; 0.42 ± 0.04 (controls) and 0.75 ± 0.06 mM (MI rats; P < 0.05 vs. controls) by 3-Hz stimulation; and 0.56 ± 0.06 (controls) and 0.73 ± 0.08 mM (MI rats; P > 0.05 vs. controls) by 5-Hz stimulation. In addition, an increase in [K+]o was blocked after paralysis of muscle with intravenous injection of pancuronium bromide (Fig. 3). This suggests that increased [K+]o was due to active muscle contraction per se and not to sciatic nerve stimulation.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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We have shown that twitch contraction of muscle significantly increased [K+]o and that the peak change was greater in animals with MI (Figs. 1 and 2). The enhanced [K+]o was not due to muscle tension, because developed tensions were similar in both healthy controls and MI rats during stimulations. In addition, the interstitial K+ response was eliminated as muscles were paralyzed (Fig. 3), suggesting that the elevated muscle interstitial K+ comes from skeletal muscle cells per se. Our results further showed that [K+]o was significantly elevated after muscle Na+-K+ pumps were inhibited by perfusing ouabain, and the inhibitory effect was significantly smaller in MI animals than in controls (Fig. 4). This result indicates that muscle activity of Na+-K+ pump is attenuated after MI. Because of impaired Na+-K+ activity, as muscle is stimulated, more K+ accumulates in the muscle interstitial space and the concentration rises to a greater degree in the MI rats.
Accumulation of [K+]o in the interstitium can be affected by three related factors: the K+ leak/release from the muscle cells, the reuptake of K+ into the cells by the Na+-K+ pump, and the clearance of K+ from the interstitium due to blood flow. Therefore, the elevated [K+]o in skeletal muscle of HF rats could be the result of an increased K+ leak/release, a decreased reuptake by the Na+-K+ pump, a decreased clearance of K+ due to blood flow, or a combination of these factors. Our current results suggest that [K+]o in contracting muscle was augmented in MI rats due to an attenuation of Na+-K+ pump activity. A previous report (30) has shown that blood flow to exercising muscle was reduced in HF rats. However, the present study did not allow us to identify precise mechanisms underlying the altered K+ balance in skeletal muscle of MI animals. We have observed that there was a difference in the changes in [K+] between controls and MI rats after 1 Hz and 3 Hz, but not 5 Hz, of stimulation. It was not clear what factor(s) could cause the difference due to the stimulation, although our data have shown that Na+-K+ pump activity was attenuated in MI rats.
Physiological Significance
Skeletal muscle system. The Na+-K+ pump establishes gradients of Na+ and K+ across the plasma membranes of cells (1). Because the amount of Na+ pumped out is larger than the amount of K+ pumped in, the pump transfers the net charge across the membrane and, in this way, contributes directly to the resting membrane potential.
Change in [K+]o has an impact on skeletal muscle performance. For instance, enhanced accumulation of [K+]o leads to reduced membrane excitability and poor contractile force (2, 10). When the sarcolemmal Na+-K+ pump is unable to keep up with the K+ efflux during maximal muscle activity, contractile function decreases due to declining membrane potential and cell excitability (9). Studies have shown a direct role of K+-Na+ gradients in modulating contractile function of skeletal muscle. Overgaard et al. (34) demonstrated that an increase in [K+]o reduces M wave and, consequently, tetanic force. Furthermore, it has been suggested that the K+-Na+ gradients have a great effect on the excitability of the T-tubular system in skeletal muscle (33). Finally, it has been reported that exercise training induces an increase in Na+-K+-ATPase content, lowers levels of [K+]o during exercise, and delays fatigue in trained subjects (32).
Similar to the findings presented in this report, others have found that Na+-K+ pumps are downregulated in MI animals (31). The present study has further shown that attenuated Na+-K+ activity after MI augmented increases of [K+]o during muscle contraction. We would speculate that the heightened rise in [K+]o shown in this report may contribute to the premature fatigue seen in HF (45).
Integration of autonomic system: role of [K+]o in afferent nerves. [K+]o also affects sympathetic nervous system activity. In skeletal muscle, group III and IV afferent nerves have been shown to be activated by K+ (38). This leads to an activation of the cardiovascular nuclei in the brainstem, an increase of sympathetic activity, and rises in blood pressure (27). As previously discussed, this is one of the basic mechanisms of the exercise pressor reflex (5, 24, 28). Therefore, an augmented [K+]o in skeletal muscle with HF could result in the exaggerated cardiovascular responses to exercise in clinical observation and animal investigation (18, 23, 25, 26, 29, 36, 42).
The results from this report have shown that the peak change in [K+] concentrations induced by 1-, 3-, and 5-Hz stimulation was 0.15 to 0.56 mM in control rats and 0.29 to 0.75 mM in MI rats, respectively. It should be noted that the K+ concentrations were presented without considering the recovery rate of the probes (60%). If we take this into account, an increase in [K+] of 1 mM is close to the minimal concentration that stimulates group III and IV muscle afferent fibers that mediate the muscle pressor reflex (38). When [K+] concentration was increased by >4.5 mM, an increase of 18 mmHg was seen (37). Thus blood pressure was not increased by muscle contraction due to the small change of [K+] in the present study.
However, it should be noted that several studies do not support the concept that [K+]o is responsible for evoking and sustaining the exercise pressor reflex (6, 20). Specifically, Daley et al. (6) examined the relationship between [K+] and muscle sympathetic nerve activity during handgrip and found that venous [K+] and muscle sympathetic nerve activity did not correlate during exercise. Another human study suggests that changes in [K+]o tracked changes in skeletal muscle blood flow but not blood pressure during exercise (20).
A previous report suggests that the number of Na+-K+ pumps in rats with HF is reduced in muscles with oxidative fibers compared with those with glycolytic fibers (31). In the present study, the microdialysis probes were inserted into the gastrocnemius muscles without considering the types of fibers (i.e., oxidative and glycolytic fibers). Thus, this may contribute to a small increase in [K+] during muscle contraction seen in our current study.
Integration of autonomic system: role of [K+]o in sympathetic nerves. It is known that the decline in exercise performance associated with HF is partly due to decreased blood flow to active skeletal muscles. Interstitial NE released from sympathetic nerves constricts vascular smooth muscle and, in the process, reduces muscle blood flow. K+ increases the exocytotic release of NE from the nerve terminals (40). Thus we speculate that, in this study, a greater increase in [K+]o with muscle contraction in MI animals induces a larger release of NE-evoking vasoconstriction and would tend to reduce muscle blood flow to muscle.
On the other hand, increased [K+]o may relax vascular smooth muscle and evoke vasodilation (7). This direct action opposes the NE effect. Future studies are needed to examine these competing factors. Of note, prior reports suggest that NE concentrations in exercising muscle are not affected by blood flow (8, 14).
Consideration of Microdialysis Probe Recovery
In the present study, the percent recovery rate of K+ for microdialysis probe was examined in vitro. The rate was 60% with 1.0–10 mM of bath K+ concentrations. This suggests that the recovery rate of the dialysis was not altered by K+ concentrations. Furthermore, it has also been shown that the recovery of the probe inserted in muscle was not altered when the muscle was contracted (21), indicating that a mechanical event is unlikely to contribute to the probe recovery. In addition, thallium-201 was used since the internal reference and microdialysis methods were performed to examine dialysate K+ before, during, and after exercise (11). It was confirmed that loss of thallium-201 was linearly related to K+ recovery. The results from this previous study (11) also showed that stable K+ concentrations were measured at the relative loss of thallium-201 by different perfusate flows, suggesting that the changes in interstitial K+ in skeletal muscle were accurately measured by using microdialysis. Thus we believe that dialysate K+ concentrations reported in the present study were not affected by the probe recovery and reflected interstitial K+ activity.
In conclusion, our data demonstrate that muscle [K+]o rises with muscle contraction. When compared with that in healthy animals, the rise in [K+]o is larger in rats with MI. Our results further suggest that the enhanced K+ response is due to a reduction of Na+-K+ pump activity within muscle after MI. We postulate that the greater rise in K+ leads to muscle fatigue as well as a greater release of NE and greater vasoconstriction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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P < 0.05, significance vs. baseline; *P < 0.05, significant difference between MI rats and healthy control rats.
