Mineralocorticoid receptor antagonists attenuate exaggerated exercise pressor reflex responses in hypertensive rats

Exaggerated cardiovascular responses to exercise in hypertensive patients are linked with overactive exercise pressor reflexes (EPRs). Administration of low-dose mineralocorticoid receptor antagonists (spironolactone or eplerenone) effectively ameliorates abnormal EPR function in hypertension. Effective treatment of EPR overactivity may reduce the cardiovascular risks associated with physical activity in hypertension.

Abstract

Exaggerated heart rate (HR) and blood pressure responses to exercise in hypertension are mediated, in part, by overactivity of the exercise pressor reflex (EPR). The mechanisms underlying this EPR dysfunction have not been fully elucidated. Previous studies have shown that stimulation of mineralocorticoid receptors (MRs) with exogenous administration of aldosterone in normal, healthy rats reproduces the EPR overactivity characteristic of hypertensive animals. Conversely, the purpose of this study was to examine whether antagonizing MR with spironolactone (SPIR) or eplerenone (EPL) in decerebrated hypertensive rats ameliorates abnormal EPR function. Changes in mean arterial pressure (MAP) and HR induced by EPR or muscle mechanoreflex (a component of EPR) activation were assessed in normotensive Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs) fed normal chow (NC) or a customized diet containing either SPIR or EPL for 3 wk. SHRs treated with SPIR or EPL had significantly attenuated MAP responses to EPR (NC: 45 ± 7 mmHg, SPIR: 26 ± 4 mmHg, and EPL: 24 ± 5 mmHg, P = 0.02) and mechanoreflex (NC: 34 ± 9 mmHg, SPIR: 17 ± 3 mmHg, and EPL: 15 ± 3 mmHg, P = 0.03) activation. SHRs treated with SPIR or EPL also showed significantly attenuated HR responses to EPR (NC: 17 ± 3 beats/min, SPIR: 9 ± 1 beats/min, and EPL: 9 ± 2 beats/min, P = 0.01) and mechanoreflex (NC: 15 ± 3 beats/min, SPIR: 6 ± 1 beats/min, and EPL: 7 ± 1 beats/min, P = 0.01) activation. Wistar-Kyoto rats treated with SPIR did not demonstrate significant differences in MAP or HR responses to EPR or mechanoreflex activation. The data suggest that antagonizing MRs may be an effective strategy for the treatment of EPR overactivity in hypertension.

NEW & NOTEWORTHY Exaggerated cardiovascular responses to exercise in hypertensive patients are linked with overactive exercise pressor reflexes (EPRs). Administration of low-dose mineralocorticoid receptor antagonists (spironolactone or eplerenone) effectively ameliorates abnormal EPR function in hypertension. Effective treatment of EPR overactivity may reduce the cardiovascular risks associated with physical activity in hypertension.

the cardiovascular response to exercise in hypertensive patients is abnormally exaggerated (34, 54). These augmented increases in arterial blood pressure (BP) and heart rate (HR) have been shown to be strongly associated with elevated risks for myocardial ischemia, myocardial infarction, cardiac arrest, stroke, and possibly death during and after physical activity (19, 27, 35, 49). Clearly, identifying the mechanisms responsible for this hyperexcitability is clinically relevant and timely given the present growth rate of hypertension cases, which are expected to increase 60% worldwide in the next 10 yr (24).

During physical activity, afferent information from working skeletal muscle is transmitted to the brain stem and contributes significantly to sympathetic regulation of the cardiovascular system (31). These contraction-induced sensory signals are generated by stimulation of group III (primarily mechanically sensitive Aδ-fibers associated with the muscle mechanoreflex) and group IV (predominantly chemically sensitive C-fibers associated with the muscle metaboreflex) afferent fibers, which collectively constitute the skeletal muscle exercise pressor reflex (EPR) (31, 33). It has been shown in several animal models of human hypertension that an overactive EPR contributes significantly to the exaggerated increases in BP and HR during physical activity (26, 38, 49). However, the mechanisms underlying this EPR dysfunction in hypertension have yet to be fully elucidated.

Aldosterone is known to contribute to the development of hypertension. It also has been shown to centrally stimulate the sympathetic nervous system (8, 17). Infusion of aldosterone directly into the cerebral ventricles induces sustained increases in BP and renal sympathetic nerve activity (SNA) in rats and dogs (9, 15, 16, 21, 55). We recently demonstrated that exogenous administration of aldosterone generates EPR overactivity in otherwise healthy, normal rats (36). In consideration of these effects, aldosterone may provide a potential pathway for inducing overactivity in the EPR. This appears to be mediated primarily through activation of mineralocorticoid receptors (MRs), which are widely expressed in the brain (10, 42), kidney (11, 12), heart (30, 43), circulatory system (53), and other tissues. Previous studies have demonstrated that blockade of MRs alters BP regulation and kidney function (46). With regard to hypertension, blockade of the actions of aldosterone using MR antagonists has been linked to tissue protection and regeneration (47) and prevention of myocardial fibrosis (2). Given these positive effects in combination with the finding that aldosterone induces EPR overactivity, it is logical to suggest that antagonizing MRs may improve EPR function in hypertension.

Using this background as a basis, the present study was designed to test the hypothesis that MR antagonists attenuate exaggerated cardiovascular responses to EPR activation in hypertension. To test this, we examined BP and HR responses to activation of the EPR, as well as one of its functional components (the skeletal muscle mechanoreflex), in normotensive and hypertensive rats treated with normal chow or a customized diet containing an MR antagonist, either spironolactone (SPIR) or eplerenone (EPL).

MATERIALS AND METHODS

Animal Models

Experiments were performed in 27 spontaneously hypertensive rats (SHRs) and 9 normotensive Wistar-Kyoto (WKY) age-matched (15 wk) male rats (Harlan Laboratories). For 3 wk, SHRs were fed either normal chow (NC; n = 8) or a customized diet containing either the MR antagonist SPIR (50 mg·kg⁻¹·day⁻¹, n = 10) or EPL (100 mg·kg⁻¹·day⁻¹, n = 9). WKY rats were fed either NC (n = 5) or SPIR (n = 4). These doses for SPIR and EPL were chosen based on previous studies in hypertensive rats that have demonstrated evidence of cardiovascular protection with minimal effects on resting BP (1, 39). Doses received via oral administration were monitored by daily measurement of food consumption. Animals were housed individually in standard rodent cages on 12:12-h light-dark cycles and were given food and water ad libitum. Experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Texas at Southwestern Medical Center and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (4).

Experimental Protocols

Acute general surgical procedures.

Rats were anesthetized with 1–4% isoflurane in oxygen and mechanically ventilated after intubation. Arterial BP was continuously measured via a pressure transducer connected to a common carotid arterial catheter. ECG recordings were collected using needle electrodes placed in the back of each rat. HR was calculated from the R-R interval in the ECG. Animals were placed in a stereotaxic frame and rendered insentient through a precollicular decerebration. Isoflurane anesthesia was discontinued immediately after the decerebration.

Surgical procedures for activating skeletal muscle reflexes.

The lower lumbar of the spinal cord (L2–L6) was exposed through laminectomy (36, 48). The L4 and L5 ventral roots were isolated, and the cut peripheral ends were placed on bipolar electrodes. The triceps surae (gastrocnemius, plantaris, and soleus muscles) of the right hindlimb were isolated. The right hindlimb calcaneal bone was cut, and muscle tension was measured by connecting the Achilles tendon to a force transducer. To evoke contraction, constant current stimulation was used at three times motor threshold with a pulse duration of 0.1 ms at 40 Hz. This procedure concomitantly stimulates mechanically and chemically sensitive skeletal muscle afferent fibers (i.e. both functional components of the EPR) (31, 33).

Stimulation of the mechanically sensitive component of the EPR.

The right hindlimb triceps surae was passively stretched for 30 s using a rack-and-pinion system to evoke mechanical stimuli similar to those elicited during muscle contraction. Stretches were carefully conducted to manually simulate the pattern and magnitude of muscle tension developed during static contractions. This technique preferentially activates the mechanically sensitive afferent fibers associated with mechanoreflex (23, 50).

End-experiment procedures.

Insentient, decerebrated animals were humanely killed through intravenous injection of saturated potassium chloride (4 M, 2 ml/kg). The heart and lungs were removed and weighed. The tibia was also collected and measured.

Data Acquisition

Data-acquisition software (LabChart, ADInstruments) for an analog-to-digital convertor (Powerlab 8/30, AD Instruments) was used at a 1-kHz sampling rate to collect BP, HR, ECG, and contractile force data. Data analysis used 1-s averages for MAP, HR, and hindlimb tension. Baseline values were calculated from 30 s of recorded data before muscle contraction or passive stretch. Peak responses were defined as the greatest change from baseline elicited by contraction or stretch.

Statistical Analyses

Data were analyzed using independent Student’s t-test or one-way ANOVA with an uncorrected Fisher’s least-significant-different multiple-comparison post hoc test, as appropriate, to identify differences between specific group means. The significance level was set at P < 0.05. All results are presented as means ± SE.

RESULTS

Morphometric characteristics for each experimental group are shown in Table 1. Body weight was lower in WKY rats treated with SPIR compared with those on NC but not significantly so. SHRs treated with either MR antagonist, SPIR or EPL, had significantly lower body weights than SHRs on NC. Heart weight-to-body weight, lung weight-to-body weight, and tibial length-to-body weight ratios were not significantly different between groups. There were no significant differences in daily amounts of food consumed between groups of animals. All animals consumed sufficient amounts of chow to receive the effective dose listed in the materials and methods.

Table 1.

Baseline characteristics

	Wistar-Kyoto Rats		Spontaneously Hypertensive Rats
	Normal chow	Spironolactone	Normal chow	Spironolactone	Eplerenone
n	5	4	8	10	9
Body weight, g	332 ± 6	325 ± 4	334 ± 4	321 ± 4*	320 ± 3*
Heart weight-to-body weight ratio, mg/g	3.0 ± 0.1	3.4 ± 0.4	3.5 ± 0.2	3.6 ± 0.1	3.4 ± 0.1
Heart weight-to-tibial length ratio, mg/mm	26.6 ± 1.3	28.4 ± 2.6	31.2 ± 1.8	34.1 ± 2.5	29.1 ± 0.5
Lung weight-to-body weight ratio, mg/g	7.7 ± 0.8	5.4 ± 1.3	8.1 ± 1.0	7.3 ± 0.9	10.1 ± 0.5
Mean arterial pressure, mmHg
Predecerebration	130 ± 9	122 ± 7	196 ± 8	184 ± 12	183 ± 11
Postdecerebration	70 ± 5	68 ± 6	96 ± 4	100 ± 8	97 ± 5

Values are means ± SE.

^*P > 0.05 compared with spontaneously hypertensive rats fed normal chow.

After instrumentation and surgical preparation, baseline measurements of HR and BP were taken after 1 h of recovery postdecerebration. Neither SPIR nor EPL caused a significant change in baseline hemodynamics in either WKY rats or SHRs (Table 1). As expected, baseline MAP was significantly higher in SHRs compared with WKY rats. These differences between normotensive and hypertensive animals were consistent with previous experiments and values previously reported in the literature (36, 37). Representative arterial BP and tension responses to contraction are shown in Fig. 1.

Fig. 1.

Representative arterial blood pressure (ABP) and tension tracings from spontaneously hypertensive rats (SHRs) treated with normal chow (NC; A), spironolactone (SPIR; B), or eplerenone (EPL; C).

In WKY rats, MAP and HR responses to EPR activation were largely unaffected by blockade of MRs with SPIR (Fig. 2A). In contrast, the pressor (NC: 45 ± 7 mmHg, SPIR: 26 ± 4 mmHg, and EPL: 24 ± 5 mmHg, P = 0.02) and tachycardic (NC: 17 ± 3 beats/min, SPIR: 9 ± 1 beats/min, and EPL: 9 ± 2 beats/min, P = 0.01) responses to stimulation of the EPR during static muscle contraction in SHRs were significantly attenuated by SPIR and EPL compared with animals fed NC (Fig. 2B).

Fig. 2.

Mean arterial pressure (MAP) and heart rate (HR) responses to activation of the exercise pressor reflex by electrically induced muscle contraction in Wistar-Kyoto rats (A) and spontaneously hypertensive rats (B). NC, normal chow; SPIR, spironolactone; EPL, eplerenone. *P < 0.05 compared with NC.

Mechanically sensitive afferent neurons were preferentially activated using a passive stretch maneuver. Compared with animals fed NC, there was no appreciable difference in the HR and MAP responses to passive muscle stretch in WKY animals that received SPIR treatment (Fig. 3A). Similar to responses during EPR stimulation, treatment with both SPIR and EPL in SHRs induced a significant attenuation in the pressor (NC: 34 ± 9 mmHg, SPIR: 17 ± 3 mmHg, and EPL: 15 ± 3 mmHg, P = 0.03) and tachycardic (NC: 15 ± 3 beats/min, SPIR: 6 ± 1 beats/min, and EPL: 7 ± 1 beats/min, P = 0.01) responses evoked by passive stretch (Fig. 3B). In SHRs, the magnitude of the reduction in either HR or MAP was not different between SPIR and EPL treatments.

Fig. 3.

Mean arterial pressure (MAP) and heart rate (HR) responses to activation of the mechanoreflex via passive muscle stretch in Wistar-Kyoto rats (A) and spontaneously hypertensive rats (B). NC, normal chow; SPIR, spironolactone; EPL, eplerenone. *P < 0.05 compared with NC.

The amount of tension generated in the triceps surae was similar in all EPR and mechanoreflex experiments. To enhance comparison and remain consistent with previous reports (36, 48, 49), cardiovascular data were normalized to the amount of tension developed.

DISCUSSION

The major new findings from this investigation were as follows: 1) blockade of MRs with SPIR did not significantly alter EPR or mechanoreflex-mediated increases in HR or MAP in WKY rats, suggesting that MR agonists (e.g., aldosterone) do not play a significant role in the expression of muscle reflex activity in normal, healthy rats; 2) antagonism of MRs with SPIR or EPL did significantly reduce the cardiovascular response to EPR and mechanoreflex activation in SHRs, indicating that MR agonists, such as aldosterone, significantly contribute to muscle reflex overactivity in hypertension; and 3) the ameliorating effects of SPIR and EPL on EPR and mechanoreflex activity in SHRs were similar, suggesting that both MR antagonists may be equally effective for the treatment of muscle reflex dysfunction in hypertension.

We observed that both SPIR and EPL, the two primary MR antagonists used clinically for the treatment of hypertension, attenuated the augmented EPR in SHRs. SPIR has 100- to 1,000-fold higher binding affinities for androgen, glucocorticoid, and progesterone receptors than EPL (5). However, it is unlikely that the beneficial effect of SPIR on EPR is mediated through inhibition of these receptors because EPL, a more selective MR antagonist, produced the same effect as SPIR. The mechanisms underlying the greater effect than WKY rats of SPIR and EPL on SHRs are unknown. Intracerebroventricular infusion of MR antagonists has been also shown to reduce resting BP in rats with normal circulating levels of aldosterone, such as ANG II-hypertensive Wistar rats (14), aldosterone-induced hypertensive Sprague-Dawley rats (56), Dahl-salt sensitive rats (20), and normotensive rats (46). Furthermore, central infusion of an aldosterone synthase inhibitor has been shown to reduce resting BP in these hypertensive rats (20). Thus, it is possible that aldosterone production in the brain of hypertensive rats is enhanced compared with normotensive rats, contributing to the mitigating effects of MR antagonists in the present study.

There were no significant differences in morphometric measurements between groups, other than a significant loss of body weight in SHRs treated with SPIR or EPL compared with SHRs treated with NC. One of the initial expectations from this study was that, in SHRs, heart weight-to-body weight and heart weight-to-tibial length ratios would be significantly decreased by SPIR and EPL treatment. However, the data do not indicate any significant improvement in these measures. This may be attributable to the relatively short (3 wk) period of treatment. Furthermore, previous studies have not shown a reduction in left ventricular mass with this dose of SPIR after aortic banding in rodents (45). Thus, it is possible that these doses were below the threshold required for inducing significant ratio reductions despite the significant effects on EPR activity.

The SPIR or EPL treatments used in our study did not significantly alter baseline BP after 3 wk of dietary consumption in either WKY rats or SHRs. However, results of our study are consistent with previous reports showing no change in resting BP in hypertensive rats treated with the same or even higher doses of SPIR (1) or EPL (39). Nevertheless, both SPIR or EPL were shown to induce a clear effect on the EPR response in SHRs, suggesting that inhibition of EPR is independent of resting BP. It is possible that the brain stem centers involved in autonomic cardiovascular regulation during exercise are more sensitive to MR inhibition than the brain region involved in regulation of resting BP. For one, activation of neurons within the rostral ventrolateral medulla neuron by aldosterone can be blocked by the application of EPL (41). Another important cardiovascular autonomic regulatory center, the paraventricular nucleus (PVN) (44), has been shown to express MRs (3). However, animals were decerebrated before the measurement of baseline BPs in our study. By decerebrating the animals, it is possible that the central circuits in the forebrain that could have been affected by treatment with MR antagonists (e.g., the PVN) may have been removed. It may be possible that the chronic administration of the MR antagonists SPIR or EPL induced permanent changes in the cardiovascular regulatory pathways that originate in the PVN. Furthermore, it may be possible that centrally mediated effects of chronic MR antagonism may have peripheral effects on BP regulation through the kidney (46). There may be mechanisms that exist entirely in the periphery, such as through T cell-specific MRs (52) or MRs present in the vascular smooth muscle (18). Further work is required to elucidate these and other possible mechanisms.

One additional limitation of this study is that an oral delivery system limits the ability to identify the site or sites at which the SPIR or EPL act to induce the observed effects reported here. The intake of the drugs through these customized diets does not discriminate between systemic and peripheral effects from centrally mediated effects. As has been previously documented, MRs have broad expression throughout the body (10–12, 30, 42, 43, 53). Additionally, oral and peripheral administration of SPIR and EPL have been shown to induce centrally mediated effects, providing indirect evidence that they both cross the blood-brain barrier (6, 22, 57). Because these doses of SPIR and EPL did not alter resting BP, it is unlikely that their effects are mediated by a direct vasodilating action. Because attenuation of the overactive EPR was observed despite precollicular decerebration, our study suggested a much less important role for the forebrain in mediating the sympathoinhibitory actions of SPIR and EPL. Although we are confident that MR antagonism is attenuating the overactive EPR responses in SHRs, it is possible that the SPIR and EPL may be acting at multiple sites within the EPR reflex arc, including the muscle afferents, dorsal root ganglion, or brain stem, to produce the observed effects.

The ultimate goals of research in hypertension are to either prevent the onset of the disease, arrest its progression, or successfully treat it by lowering BP. Investigations into the mechanisms responsible for the development, onset, and advancement of hypertension ultimately lead to better treatment modalities aimed at achieving these goals. Exercise has been consistently shown to have a positive effect in reducing hypertension in patients (25, 28, 32). The problem is that, with exercise in hypertensive patients, there is an increased risk of myocardial ischemia, infarction, cardiac arrest, stroke, and possibly death during and after physical activity (19, 27, 35, 49). On the basis of previous studies in our laboratory and others, understanding the mechanisms underlying the pathogenesis of EPR overactivity in hypertension is key to making inroads toward the safe prescription of exercise in this disease. The present study advances our knowledge in this area by demonstrating that low doses of SPIR or EPL can be used as effective treatments to reduce the exaggerated cardiovascular responses produced by EPR activation in hypertension. The results of this study support the original hypothesis that antagonizing MRs in hypertensive animals attenuates EPR overactivity toward a more normal phenotype. As use of MR antagonists becomes more clinically widespread (7, 13, 29, 40, 51), understanding the mechanisms underlying their beneficial effects becomes more important. As is always the case with basic science investigations using animal models, translational studies are needed before the findings of this research can be applied to the treatment of EPR dysfunction in hypertensive humans.

GRANTS

This work was supported by National Institutes of Health (NIH) Grant HL-113738 (to W. Vongpatanasin), NIH Training Grant HL-007360 (to J. Hill, RMD Trainee), the Lawson & Rogers Lacy Research Fund in Cardiovascular Disease (to J. Mitchell), and the Univ. of Texas Southwestern O’Brien Kidney Research Core Center (NIH Grant P30-DK-079328, to P. Igarashi; Clinical and Translational Core, WV Co-Principal Investigator).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

R.M.D. performed experiments; R.M.D., M.M., and S.A.S. analyzed data; R.M.D., M.M., J.H.M., W.V., and S.A.S. interpreted results of experiments; R.M.D. prepared figures; R.M.D. drafted manuscript; R.M.D., M.M., J.H.M., W.V., and S.A.S. edited and revised manuscript; R.M.D., M.M., J.H.M., W.V., and S.A.S. approved final version of manuscript; W.V. and S.A.S. conceived and designed research.