EVIDENCE FOR FUNCTIONAL ALTERATIONS IN THE SKELETAL ...

Page 1 ofArticles 40 in PresS. Am J Physiol Heart Circ Physiol (July 18, 2008). doi:10.1152/ajpheart.01365.2007

EVIDENCE FOR FUNCTIONAL ALTERATIONS IN THE SKELETAL MUSCLE MECHANOREFLEX AND METABOREFLEX IN HYPERTENSIVE RATS

Running Head – Cardiovascular control during exercise in hypertension

Anna K. Leal1, Maurice A. Williams2, Mary G. Garry3, Jere H. Mitchell2 and Scott A. Smith4

Departments of Biomedical Engineering1, Internal Medicine2 and Physical Therapy4 University of Texas Southwestern Medical Center Dallas, Texas, USA 75390-9174 AND Lillehei Heart Institute3 University of Minnesota Minneapolis, MN, USA 55455

Corresponding Author: Scott A. Smith, PhD University of Texas Southwestern Medical Center Southwestern Allied Health Sciences School Department of Physical Therapy 5323 Harry Hines Boulevard Dallas, Texas 75390-9174 214-648-3294 (office) 214-648-3566 (facsimile) [email protected]

Copyright Information Copyright © 2008 by the American Physiological Society.

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2 ABSTRACT Exercise in hypertensive individuals elicits exaggerated increases in mean arterial pressure (MAP) and heart rate (HR) that potentially enhance the risk for adverse cardiac events or stroke. Evidence suggests that exercise pressor reflex function (EPR; a reflex originating in skeletal muscle) is exaggerated in this disease and contributes significantly to the potentiated cardiovascular responsiveness. However, the mechanism of EPR overactivity in hypertension remains unclear. EPR function is mediated by the muscle mechanoreflex (activated by stimulation of mechanically sensitive afferent fibers) and metaboreflex (activated by stimulation of chemically sensitive afferent fibers). Therefore, we hypothesized the enhanced cardiovascular response mediated by the EPR in hypertension is due to functional alterations in the muscle mechanoreflex and metaboreflex. To test this hypothesis, mechanically and chemically sensitive afferent fibers were selectively activated in normotensive Wistar-Kyoto (WKY) and Spontaneously Hypertensive (SHR) decerebrate rats. Activation of mechanically sensitive fibers by passively stretching hindlimb muscle induced significantly greater increases in MAP and HR in SHR than WKY over a wide range of stimulus intensities. Activation of chemically sensitive fibers by administering capsaicin (0.01 to 1.00 @g/100 @L) into the hindlimb arterial supply induced increases in MAP that were significantly greater in SHR compared to WKY. However, HR responses to capsaicin were not different between the two groups at any dose. This data is consistent with the concept that the abnormal EPR control of MAP described previously in hypertension is mediated by both mechanoreflex and metaboreflex overactivity. In contrast, the previously reported alterations in the EPR control of HR in hypertension may be principally due to overactivity of the mechanically sensitive component of the reflex. Key Words: blood pressure, heart rate, muscle afferents

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3 INTRODUCTION The cardiovascular response to exercise is exaggerated in hypertension and is characterized by augmented increases in heart rate (HR), arterial blood pressure (ABP) and vascular resistance (3, 15, 19, 25, 38, 43). These abnormal hemodynamic responses to physical activity in hypertension are associated with an increased risk for adverse cardiovascular events during and after exercise such as myocardial ischemia or infarction, cardiac arrest and stroke (13, 21, 35, 36). Therefore, dissection of the regulatory mechanisms underlying the altered hemodynamic responses to exercise in hypertension is important and clinically relevant. The cardiovascular response to exercise is mediated by three neurophysiologic mechanisms: the exercise pressor reflex (EPR), central command and the arterial baroreflex. The EPR is a feedback peripheral neural drive originating in skeletal muscle that regulates changes in ABP and HR during physical activity predominantly via the sympathetic nervous system (1, 29, 34). Central command is a feed-forward mechanism that simultaneously activates motor areas within the cerebral cortex and cardiovascular centers within the brain stem (10, 22). During exercise, central command contributes to increases in ABP and HR via sympathetic activation and parasympathetic withdrawal. The cardiovascular responses mediated by the EPR and central command are modulated by the tonically active arterial baroreflex. Arterial baroreflex afferent fibers are stimulated by mechanically sensitive receptors located within the carotid arteries and aortic arch. These receptors respond to changes in blood pressure by altering HR, stroke volume, and vascular resistance on a beat-to-beat basis at rest and during exercise (28, 40). While each of these mechanisms could potentially mediate the exaggerated circulatory response to exercise in hypertension, recent evidence from our laboratory suggests the EPR


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4 significantly contributes to this heightened cardiovascular responsiveness (49). However, the mechanisms driving EPR dysfunction in hypertension remain unclear. EPR function is mediated by two reflex mechanisms: the muscle mechanoreflex and the muscle metaboreflex. The muscle mechanoreflex is activated by predominately mechanically sensitive group III afferent fibers (17, 18). Receptors associated with these fibers in skeletal muscle primarily respond to mechanical distortion of their receptive fields. The muscle metaboreflex is activated predominately by chemically sensitive group IV afferent fibers (17, 18). Receptors associated with these afferents are in close proximity to the vasculature of skeletal muscle and respond to the chemical by-products of muscle metabolism. Given that each of these inputs is essential to EPR activity (34), both the mechanoreflex and metaboreflex potentially mediate the EPR dysfunction that develops in hypertension (49). This is indeed the case in disease states closely related to hypertension (e.g. heart failure) where alterations in both mechanoreflex and metaboreflex activity have been shown to contribute to the development of EPR dysfunction (2, 26, 32, 47, 50). The purpose of this investigation was, therefore, to determine the possible contributions of the muscle mechanoreflex and metaboreflex to EPR overactivity in hypertension.

We

hypothesized that the potentiated cardiovascular response to activation of the EPR in hypertension is driven by functional alterations in both the mechanically and chemically sensitive components of the reflex.

To test this hypothesis, we preferentially stimulated

mechanically and chemically sensitive skeletal muscle afferent fibers in decerebrated normotensive Wistar-Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats. Determining which component of the EPR mediates its overactivity in hypertension is important as each arm


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5 of the reflex is activated by distinctly different stimuli.

As such, the mechanoreflex and

metaboreflex represent unique targets for the treatment of EPR dysfunction in hypertension.

MATERIALS AND METHODS Subjects Experiments were performed in 30 SHR and 35 WKY age-matched (14-20 weeks) male rats (Harlan, Indianapolis, Ind.). Animals were housed in standard rodent cages on 12 hour lightdark cycles and were given food and water ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center at Dallas. In addition, all studies were conducted in accordance with the United States Department of Health and Human Services National Institutes of Health Guide for the Care and Use of Laboratory Animals.

General Surgical Procedures Rats were anesthetized with isoflurane gas (2-3% in 100% oxygen), intubated and mechanically ventilated (Harvard Apparatus). Levels of inhalant gas were increased as indicated by a withdrawal reflex to pinching of the hindpaw and/or spontaneous increases in HR. To minimize the development of edema, dexamethasone (0.2 mg) was given intramuscularly (53). Fluid-filled catheters (PE-50, polyethylene tubing) were placed in both common carotid arteries and the right jugular vein for the measurement of blood pressure and the administration of fluids, respectively. Arterial blood gases and pH were monitored throughout the experiment (50 µL blood samples; 4 to 5 times) using an automated blood gas analyzer (Model ABL 5, Radiometer) to ensure these variables were maintained within normal ranges (arterial PO2, > 80 mmHg;


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6 arterial PCO2, 35-45 mmHg; pH, 7.3-7.4). Body temperature was maintained between 36.5 and 38.0°C by an isothermal pad (Deltaphase). Animals were held in a stereotaxic head unit (Kopf Instruments) and a pre-collicular decerebration was performed. A bilateral craniotomy was conducted by drilling holes into the parietal skull. The bone superior to the central saggital sinus was removed. The dura mater covering the brain was cut and the cerebrum aspirated. The animal was rendered insentient by sectioning the brain rostral to the superior colliculus and aspirating the forebrain. To minimize cerebral hemorrhage, small pieces of oxidized regenerated cellulose (Ethicon, Johnson & Johnson) were placed on the internal skull surface and the cranial cavity was packed with cotton. Immediately after the decerebration procedure was completed, gas anesthesia was discontinued. To maintain fluid balance and baseline ABP, a 1 M NaHCO3, 5% dextrose, Ringer solution was continuously infused via the jugular vein at a rate of 3-5 ml h-1 kg-1 (41).

A minimum recovery period of 1 h was employed after decerebration before

beginning any experimental protocol. This allowed sufficient time for the effects of isoflurane anaesthesia to completely dissipate and ABP to stabilize.

Mechanoreflex Testing Preparation: The additional surgical procedures subsequently described were conducted on 11 WKY and 11 SHR animals. To begin, a spinal laminectomy exposing the lower lumbar portions of the spinal cord (L2-L6) was performed. The dura of the cord was cut and reflected allowing visual identification of the L4 and L5 spinal roots. The dorsal and ventral roots were carefully separated. The ventral roots were sectioned and the cut peripheral ends positioned on insulated bipolar platinum electrodes. The exposed neural tissue was covered in a pool of mineral oil. Animals were secured in a customized spinal frame by clamps placed on rostral


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7 lumbar vertebrae. The pelvis was stabilized with steel posts within the frame and the right hindlimb was fixed in one position using clamps attached to the tibial bone. The gastrocnemius and soleus muscles were isolated and the calcaneal bone was cut. Lastly, the Achilles’ tendon was connected to a force transducer (Grass Instruments, FT10) allowing the measurement of muscle tension. Experimental Protocol: During muscle contraction, both the mechanically and chemically sensitive components of the EPR are stimulated (34). Passively stretching hindlimb skeletal muscle does not increase muscle metabolism and, thus, is commonly used to selectively engage the mechanically sensitive component of the EPR (20, 26, 51). Therefore, to preferentially activate mechanically sensitive afferent fibers associated with the muscle mechanoreflex in WKY and SHR animals, the gastrocnemius and soleus muscles of the right hindlimb were passively stretched using a calibrated 9.5 mm rack and pinion system (Harvard Apparatus). To evoke a mechanical stimulus similar to that elicited during muscle contraction, care was taken to generate the same magnitude and pattern of muscle tension developed during contraction. This was achieved by first determining the maximal force developed during static contraction of the gastrocnemius and soleus muscles. These muscles were contracted by electrically stimulating the L4 and L5 ventral roots for 30s. Constant-current electrical stimulation was used at 3 times motor threshold (defined as the minimum current required to produce a muscle twitch) with a pulse duration of 0.1 ms at 40 Hz. These stimulus parameters are known to generate maximal tension development during muscle contraction in rats (46). Once maximal tension development was established, the gastrocnemius and soleus muscles were stretched at this intensity a minimum of two times with a 15 minute recovery period between each trial. In a subset of these animals (10 WKY and 9 SHR), additional stretches were performed at


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8 randomized submaximal intensities. Prior to all maneuvers, muscles were preloaded by stretching to 70-100 g of tension.

Metaboreflex Testing Preparation: The additional surgical procedures subsequently described were conducted on 19 WKY and 15 SHR animals. To administer drugs into the arterial supply of muscle within the right hindlimb, the circulation of the hindlimb was isolated. A catheter (PE-10, polyethylene tubing) was placed in the left common iliac artery and its tip advanced to the bifurcation of the abdominal aorta. This procedure allowed the injection of substances directly into the circulation of the right hindlimb via the right common iliac artery. To limit drug delivery to the hindlimb, a reversible vascular occluder was placed around the common iliac vein emptying the right hindlimb. Experimental Protocol: Selective activation of chemically sensitive afferent fibers associated with the muscle metaboreflex was achieved by administering graded concentrations of capsaicin into the arterial supply of the right hindlimb of WKY and SHR animals. The capsaicin receptor, transient receptor potential vanilloid 1 (TRPv1), is a relatively selective marker of group IV afferent neurons although the receptor is also present on a small number of Group III afferent fibers (14, 31). Therefore, stimulation of this receptor predominately activates the neuronal population within skeletal muscle known to primarily mediate metaboreflex activity and is commonly used for this purpose (16, 26, 50). Capsaicin was injected directly into the arterial supply of the right hindlimb via the right common iliac artery as described. To limit drug delivery to the hindlimb being tested, the reversible ligature placed around the right common iliac vein was pulled for 2 minutes. In each animal, saline and/or the vehicle for capsaicin was


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9 injected first followed by the administration of capsaicin at 5 different doses (0.01, 0.03, 0.10, 0.30 and 1.00 µg/100 µL). Animals underwent a recovery period of 15 minutes between each injection. Each injection was flushed by 0.2 ml saline. Two minutes before each injection, 0.15 ml of the neuromuscular blocker vecuronium bromide was given intravenously to prevent muscle twitch and/or contraction during capsaicin injection. Control Experiments: As a control, 1.00 µg/100 µL of capsaicin was injected directly into the systemic circulation via the jugular vein at the conclusion of the protocol. This maneuver was performed to evaluate the systemic response to capsaicin compared with that of its response when injected locally in the hindlimb circulation. As an additional control, a subset of WKY (n=5) and SHR (n=5) animals received injections of capsaicin in the hindlimb circulation together with capsazepine (100 µg/100 µL), a selective TRPv1 receptor antagonist. This experiment was performed to confirm the cardiovascular effects elicited by administration of capsaicin were the result of TRPv1 receptor activation. Finally, to eliminate the possibility that cutaneous afferent neurons may contribute to the cardiovascular response elicited by hindlimb injection of capsaicin, 0.3 and 1.0 µg/100 µL doses of capsaicin were injected into the arterial supply of the hindlimb before and after removing the skin from the leg in 4 WKY rats.

Corollary Experiments In order to assess blood pressure responsiveness independent of EPR activation in normotensive and hypertensive animals, changes in ABP elicited by intravenous administration of the pressor agent phenylephrine (2 mg/kg) was determined in separate groups of WKY (n=5) and SHR (n=4) animals.


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10 Morphological Measurements At the conclusion of all experiments, decerebrated animals were euthanized by intravenous injection of saturated potassium chloride (4 M, 2ml/kg). Use of this procedure adheres to the guidelines established by the Panel on Euthanasia of the American Veterinary Medical Association.

In all animals, the heart and lungs were excised and weighed.

Additionally, the tibia was harvested, weighed and measured.

Data Acquisition: A pressure transducer (Model DTX plus-DT-NN12, Ohmeda) connected to the left carotid arterial catheter was used to measure ABP.

Mean arterial pressure (MAP) was

determined by integrating the arterial pressure signal with a time constant of 1-4 seconds. HR was derived from the blood pressure pulse wave using a biotachometer (Gould Instruments). Hindlimb tension was measured by a force transducer (FT-10, Grass Instruments). Baseline values for all variables were determined by evaluating 30 seconds of recorded data before a given stretch or drug injection. The peak response of each variable was defined as the greatest change from baseline elicited by the experimental stimulus. All cardiovascular and contractile force data were acquired, recorded, and analyzed using data acquisition software (Spike 2, version 3, Cambridge Electronic Design, Ltd) for the CED micro 1401 system (Cambridge Electronic Design Ltd).

Statistical Analyses On all data sets, statistics were performed using correlation and regression analyses, Student t tests or ANOVA with Student-Newman-Keuls post hoc tests employed as appropriate.


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11 The significance level was set at P