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J Appl Physiol 102: 2288-2293, 2007. First published March 22, 2007; doi:10.1152/japplphysiol.00161.2007
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Effect of muscle interstitial pH on P2X and TRPV1 receptor-mediated pressor response

Zhaohui Gao, Jialiu D. Li, Lawrence I. Sinoway, and Jianhua Li

Heart and Vascular Institute and Department of Medicine, Penn State College of Medicine and Milton S. Hershey Medical Center, Hershey, Pennsylvania

Submitted 8 February 2007 ; accepted in final form 21 March 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of purinergic P2X receptors and transient receptor potential vanilloid type 1 (TRPV1) on muscle afferent nerve evokes the pressor response. Because P2X and TRPV1 receptors are sensitive to changes in pH, the aim of this study was to examine the effects of muscle acidification on those receptor-mediated cardiovascular responses. In decerebrate rats, the pH in the hindlimb muscle was adjusted by infusing acidic Ringer solutions into the femoral artery. Dialysate was then collected using microdialysis probes inserted into the muscles, and pH was measured. The interstitial pH was 7.53 ± 0.01, 7.22 ± 0.02, 6.94 ± 0.04, and 6.59 ± 0.03 in response to arterial infusion of the Ringer solution at pH 7.4, 6.5, 5.5, and 4.5, respectively. Femoral arterial injection of {alpha},beta-methylene-ATP (P2X receptor agonist) in the concentration of 0.25 mM (volume, 0.15–0.25 ml; injection duration, 1 min) at the infused pH of 7.4, 6.5, and 5.5 increased mean arterial pressure (MAP) by 29 ± 2, 24 ± 3, and 21 ± 3 mmHg, respectively (P < 0.05, pH 5.5 vs. pH 7.4). When pH levels in the infused solution were 7.4, 6.5, 5.5, and 4.5, capsaicin (1 µg/kg), a TRPV1 agonist, was injected into the artery. This elevated MAP by 29 ± 4, 33 ± 2, 35 ± 3, and 40 ± 3 mmHg, respectively (P < 0.05, pH 4.5 vs. pH 7.4). Furthermore, blocking acid-sensing ion channel (ASIC) blunted pH effects on TRPV1 response. Our data indicate that 1) muscle acidosis attenuates P2X-mediated pressor response but enhances TRPV1 response; 2) exaggerated TRPV1 response may require lower pH in muscle, and the effect is likely to be mediated via ASIC mechanisms. This study provides evidence that muscle pH may be important in modulating P2X and TRPV1 responsiveness in exercising muscle.

P2X receptor; transient receptor potential vanilloid type 1; muscle pH; exercise; blood pressure

DURING EXERCISE, contracting skeletal muscles produce metabolic by-products (such as H+, lactic acid, ATP, and phosphate) (13, 30, 36, 49), which stimulate thin-fiber muscle afferent nerve endings (receptors) located in the muscle interstitium (11, 22, 36, 44, 46, 56). This, in turn, leads to reflex increases in the sympathetic nerve activities (6, 36, 37, 56) in contribution to increases in arterial blood pressure, heart rate (HR), and peripheral vasoconstriction (2, 38–40, 45). The reflex rise in blood pressure with exercise has been termed the "exercise pressor reflex" (2, 38, 39). The afferent arm of this reflex is known to be composed of thinly myelinated and unmyelinated groups III and IV muscle afferents, respectively (17), which respond to mechanical deformation of the muscle afferent receptive fields (19, 26), as well as to metabolic stimulation (20, 44, 46, 48). Group III afferents are more mechanically sensitive than group IV afferents (19), but both are stimulated or sensitized by muscle metabolites produced in contracting skeletal muscle (11, 22, 44, 46).

It has been reported that activation of purinergic P2X receptors and transient receptor potential vanilloid type 1 (TRPV1) on thin muscle afferent nerves induces reflex pressor response (9, 10, 12, 33, 34). P2X and TRPV1 receptors are reported to be sensitive to changes in pH (24, 41, 50, 55). Furthermore, muscle interstitial pH decreases in exercising and ischemic muscles (14, 47, 51). Thus we first examined the effects of decreasing muscle pH on the pressor response induced by stimulation of P2X and TRPV1 receptors.

In addition, previous studies have shown that H+ stimulates thin muscle afferent nerves in mediating the muscle pressor reflex (44, 46). Changes in skeletal muscle pH can also alter the discharge properties of groups III and VI muscle afferents involved in the reflex pressor response (49, 52). Previous studies have further suggested that the effect of H+ and muscle pH on the muscle reflex may be mediated via acid-sensing ion channels (ASIC) on thin muscle afferent nerves (7, 32). ASIC may also play an interactive role in P2X and TRPV1 receptors’ processing of afferent inputs. Thus we further determined the effects of ASIC on the P2X and TRPV1 responses to muscle acidosis.

Evidence shows that P2X receptor activities decrease and TRPV1 receptor activities increase as pH levels fall (16, 41, 42, 57). We hypothesized that skeletal muscle acidosis would attenuate P2X receptor-mediated response but enhance TRPV1 receptor-mediated response. We further hypothesized that ASIC blockade would blunt the pH effects on P2X and TRPV1 responsiveness.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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All procedures outlined in this study were performed in compliance with the rules and regulations described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. These procedures were approved by the Animal Care Committee of this institution. Sprague-Dawley male rats (402 ± 18 g body wt) were housed in standard rat cages and regulated on a 12:12-h light-dark schedule, with food and water available ad libitum.

General Methods

Animal surgical preparation.   The rats were anesthetized by inhalation of an isoflurane-O2 mixture (2–5% isoflurane in 100% O2). An endotracheal tube was inserted and attached to a ventilator (model AWS, Hallowell EMC). Polyethylene (PE-50) catheters were inserted into an external jugular vein and the carotid arteries for the purposes of drug administration and measurement of arterial blood pressure, respectively. A continuous infusion of physiological saline (0.1 ml/h) into the jugular vein was established by using a syringe pump (Medical Industries). This maintained fluid balance and basal blood pressure. The femoral arteries were carefully isolated in both hindlimbs. An incision was made in each of the femoral arteries. PE-10 catheters were inserted into the femoral arteries so drugs could be injected into the arterial blood supply of the hindlimb muscles of each leg, as previously described (32). To adjust the muscle interstitial pH, Ringer solutions of different pH levels were perfused into the femoral arteries through the PE-10 catheters. The animals were artificially ventilated, and tidal CO2 was monitored by a respiratory gas monitor (Datex-Ohmeda, Madison, WI) and maintained within normal ranges, as previously described (32, 33). Considering P2X receptors’ sensitivity to temperature (8, 25), body temperatures were continuously monitored with a rectal thermometer (series 400, Yellow Springs Instruments) and maintained at 37.5–38.5°C by heating pads. The skeletal muscle temperature on the experimental leg was constantly maintained at 35°C throughout the experiment.

Decerebration.   The details of this procedure were described in a previous report (32). A transverse section was made anterior to the superior colliculus and extending ventrally to the mamillary bodies. The part of the brain rostral to this section was then removed. This approach afforded the opportunity to examine the effects of muscle pH on muscle afferent-mediated pressor responses without considering the confounding effects of anesthesia. Once the decerebration was complete, anesthesia was removed from the inhaled mixture. A recovery period of 60 min after decerebration was employed to allow sufficient time to eliminate the effects of anesthesia gas from early stages of preparation.

Microdialysis.   This method was used to measure pH levels in the muscle interstitium. As previously described (31), the skin directly over the triceps surae muscles of both legs was dissected, and two microdialysis probes were inserted into the gastrocnemius muscle of each leg. The probes were inserted into the muscle via a cannula in the direction parallel to the muscle fiber orientation (~2.5 mm deep from the tissue surface). After insertion, the microdialysis probes were attached to a perfusion pump and perfused with a Ringer solution (pH 7.4) at a rate of 2.5 µl/min.

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. The dialysate was continuously collected into a microcentrifuge tube, and pH levels were immediately measured using a Beetrode pH sensor (WPI).

Measurements of cardiovascular activities.   Arterial blood pressure was measured by connecting the carotid arterial catheter to a pressure transducer (model P23ID, Statham). MAP was obtained by integrating the arterial signal with a time constant of 4 s. HR was determined from the arterial pressure pulse. All measured variables were continuously recorded on an eight-channel chart recorder (model TA 4000, Gould, Valley View, OH) and stored on a PC that used the PowerLab system (ADInstruments, Castle Hill, Australia).

Experimental Protocol

Study series 1: Muscle interstitial pH after arterial perfusion of Ringer solution at different levels of pH (n = 11).   In this experiment, pH in the rat hindlimb muscle was adjusted by infusing acidic Ringer solutions into the femoral artery. The Ringer solutions at pHs of 7.4, 6.5, 5.5, and 4.5 were infused into the femoral artery at a rate of 15 µl/min for 20 min. At the end of the infusion period, the pH level of dialysate was immediately measured. The sequence of infused Ringer solution at different levels of pH was in a random fashion. A minimum of 20 min was allowed between successive arterial infusions of the Ringer solutions.

Study series 2: Effect of interstitial pH on P2X-induced response (n = 16).   The purpose of this protocol was to examine whether the pressor response evoked by stimulation of P2X receptors was modulated by muscle interstitial pH. {alpha},beta-methylene-ATP ({alpha},beta-meATP) at 0.25 mM (dissolved in saline; Sigma) was injected into the arterial blood supply of the hindlimb muscles as the muscle interstitial pH was adjusted following the arterial infusion of the Ringer solutions with pHs of 7.4, 6.5, and 5.5. Previous reports have shown that {alpha},beta-meATP stimulates thin-fiber muscle afferent nerves and increases blood pressure via the engagement of P2X receptors (10, 11, 34). Furthermore, 0.25 mM of {alpha},beta-meATP should significantly increase MAP (by ~25 mmHg), on the basis of our previous study (9). The injection volume of {alpha},beta-meATP was 0.15–0.25 ml, depending on body weight, and the duration of the injection was 1 min. The Ringer solutions at different levels of pH were infused into the arteries in a random fashion. Finally, amiloride (6 µg/kg) was applied before {alpha},beta-meATP in another group of experiments. On the basis of previous data (7, 32), this dosage of amiloride has been shown to attenuate acid-induced pressor response. Effects of ASIC blockade on the pressor response induced by {alpha},beta-meATP were then examined as the Ringer solution with pH 5.5 was infused into the femoral arteries.

Study series 3: Effect of interstitial pH on TRPV1-induced response (n = 18).   This experiment was performed to determine whether changes in muscle pH affected TRPV1-mediated pressor response. After muscle pH was adjusted, capsaicin (1 µg/kg, Sigma) was injected into the arterial blood supply of the hindlimb muscles. This concentration of capsaicin has been reported to evoke the pressor response via a reflex mechanism (32, 33). The injection volume was 0.15–0.25 ml, and the duration of injection was 1 min. It has been previously reported that severe acidic pH can stimulate capsaicin receptors and alter its response (41). Therefore, pH values of the infused Ringer solutions were selected at 7.4, 6.5, 5.5, and 4.5 in this experiment. In another group of experiments, effects of amiloride on capsaicin-induced pressor response were examined as the Ringer solution with a pH 4.5 was infused into the femoral arteries.

Experimental Data Analysis

Control values were determined by analyzing 30 s of the data immediately before arterial injection. The peak response of each variable was determined by the peak change from the control value. Experimental data (MAP and HR) were analyzed statistically by using one-way ANOVA. As appropriate, Tukey's post hoc analyses were utilized to determine the differences between groups. Values are means ± SE. For all analyses, differences were considered significant at P < 0.05. All statistical analyses were performed using SPSS for Windows (version 11.5, SPSS, Chicago, IL).


   RESULTS
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DISCUSSION
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Muscle Interstitial pH

Figure 1 shows mean values of the dialysate pH after the femoral arterial infusion of each Ringer solution (n = 11). The muscle interstitial pH was 7.53 ± 0.01 after the Ringer solution with pH 7.4 was infused into the femoral artery. The infusion of acidic Ringer solution induced a reduction in muscle interstitial pH. With the arterial infusion of Ringer solutions at pHs of 6.5, 5.5, and 4.5, interstitial pHs were 7.22 ± 0.02, 6.94 ± 0.04, and 6.59 ± 0.03, respectively.


Figure 1
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  		Fig. 1. Interstitial muscle pH after infusion of the Ringer solutions at 4 different pH levels, 7.4, 6.5, 5.5, and 4.5, into the femoral artery. The acidic solution induced a decrease in pH in the muscle interstitium. No. of animals = 11.

 
Effect of Muscle Acidosis on Cardiovascular Responses to {alpha},beta-meATP

The muscle interstitial pHs were adjusted by arterial infusion of Ringer solutions at pHs of 7.4, 6.5, and 5.5 (n = 8). At the end of each infusion, 0.25 mM of {alpha},beta-meATP was injected into the femoral artery to evoke the pressor response. There were no significant differences in baseline MAP and HR before injections of {alpha},beta-meATP (Fig. 2). Figure 2 further shows that {alpha},beta-meATP increased MAP. The arterial infusion with acidic Ringer solution at pH 5.5 significantly attenuated the MAP response to {alpha},beta-meATP compared with the solution with pH 7.4 (Fig. 3). The HR response to {alpha},beta-meATP tended to decrease with lowering muscle pHs, but there were no significant differences in the HR response among the three pH levels.


Figure 2
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  		Fig. 2. Baseline and peak mean arterial pressure (MAP) and heart rate (HR) responses induced by arterial injection of {alpha},beta-methylene-ATP ({alpha},beta-meATP) as the solutions with different pHs were infused into the femoral artery. {alpha},beta-meATP in the concentration of 0.25 mM increased MAP. *P < 0.05, vs. baseline. HR did not change significantly in response to injection of {alpha},beta-meATP at all pH levels of infused Ringer solution. No. of animals = 8.

 

Figure 3
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  		Fig. 3. Effect of muscle acidosis on MAP response induced by arterial injection of {alpha},beta-me ATP. The Ringer solution with pH 5.5 was infused into the femoral artery. This attenuated the reflex MAP response. *P < 0.05, significance vs. pH 7.4 (n = 8). The prior application of amiloride (6 µg/kg) tended to reverse attenuating effect of muscle acidosis on {alpha},beta-meATP-mediated pressor response, but there was no significant difference.

 
In addition, amiloride (6 µg/kg), an ASIC blocker, was applied before {alpha},beta-meATP as the Ringer solution of pH 5.5 was infused into the femoral artery (n = 8). Although the attenuating effect of muscle acidosis on the pressor response induced by {alpha},beta-meATP tended to be reversed by amiloride, the difference did not reach statistical significance (Fig. 3). It is noted that injection of amiloride did not significantly alter baseline MAP (96 ± 10 and 95 ± 8 mmHg before and after injection, P > 0.05) and HR (405 ± 25 and 410 ± 30 beats/min before and after injection, P > 0.05).

Effect of Muscle Acidosis on Cardiovascular Responses to Capsaicin

Muscle interstitial pH was adjusted by infusing Ringer solutions at pHs 7.4, 6.5, 5.5, and 4.5 into the femoral arteries (n = 10). The cardiovascular responses induced by arterial injections of capsaicin were then examined. There were no significant differences in baseline MAP and HR before arterial injections of capsaicin (Fig. 4). However, after injecting capsaicin (1 µg/kg), MAP response was increased (Fig. 4). With decreasing pH level, an increase in MAP response became greater. A significant enhancement in the peak MAP response evoked by capsaicin was observed as acidic Ringer solution at pH 4.5 was infused into the femoral artery compared with control of pH 7.4 (Fig. 5). There were no significant differences in HR response to the arterial injection of capsaicin at four different pH levels.


Figure 4
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  		Fig. 4. Baseline and peak MAP and HR responses induced by arterial injection of capsaicin as the solutions with different pHs were infused into the femoral artery. Capsaicin (1 µg/kg) increased MAP. *P < 0.05 vs. baseline. HR did not change significantly in response to injection of capsaicin at all pH levels of infused Ringer solution. No. of animals = 10.

 

Figure 5
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  		Fig. 5. Effect of muscle acidosis on MAP induced by arterial injection of capsaicin. The Ringer solution with pH 4.5 was infused into the femoral artery. This augmented the reflex MAP response evoked by capsaicin. The prior application of amiloride (6 µg/kg) attenuated the effect of muscle acidosis on capsaicin-induced pressor response. *P < 0.05, significance vs. pH 7.4 (n = 10). **P < 0.05, significance vs. pH 4.5 without amiloride injection (n = 8).

 
Finally, Fig. 5 shows that muscle acidosis augmented the reflex MAP response induced by arterial injection of capsaicin (n = 10). Further, the prior administration of amiloride (6 µg/kg) attenuated effects of muscle acidosis on capsaicin-induced pressor response as the Ringer solution with pH 4.5 was infused into the femoral artery in eight animals (shown in Fig. 5). Amiloride alone did not significantly alter baseline MAP (102 ± 16 and 105 ± 10 mmHg before and after injection, P > 0.05) and HR (422 ± 26 and 420 ± 30 beats/min before and after injection, P > 0.05).


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
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Study Findings

P2X and TRPV1 mediated-reflex muscle responses were sensitive to muscle interstitial pH. Acidosis attenuated P2X response as muscle interstitial pH was ~6.9. TRPV1 responsiveness was augmented as interstitial pH was lowered to ~6.5. Blocking ASIC blunted the effects of lowering the pH on TRPV1. It is known that muscle interstitial pH decreases in exercising and ischemic muscle (14, 47, 51, 56). Thus our data provide evidence that muscle pH may play an important role in modulating the exercise pressor reflex by altering P2X and TRPV1 responsiveness.

Muscle Interstitial pH

Previous studies have shown that the physiological norm of extracellular pH levels at rest is ~7.38 in skeletal muscle (51). Interstitial pH in skeletal muscle during dynamic exercise gradually decreases. The mean values of interstitial pHs at 30-, 50-, and 70-W exercise are ~7.27, 7.16, and 7.04, respectively (51). The pH value can drop to ~6.5 in ischemic muscles with exercise (3). In our present experiment, the Ringer solutions at pHs 6.5, 5.5, and 4.5 were infused into the arterial supply of the hindlimb muscles to mimic muscle acidosis seen in exercise. This lowered the interstitial pH to ~7.22, 6.94, and 6.59, respectively. Thus, in this report, effects of acidosis on P2X and TRPV1-mediated reflex pressor response were examined on the basis of measurements of interstitial pH.

P2X Response at Lowered Muscle pH

The results from this study are consistent with previous findings that {alpha},beta-meATP injected into the arterial supply of hindlimb muscles increases blood pressure (10, 33, 34). Furthermore, our data suggest that lowering muscle pH by infusion of the Ringer solution at pH 5.5 attenuates the reflex pressor response to {alpha},beta-meATP. It has been reported that injecting {alpha},beta-meATP into the hindlimb muscles stimulates thin-fiber muscle afferent nerves and increases blood pressure via P2X receptors (10, 34). Thus the result of this experiment suggests that muscle pH affects reflex pressor response evoked by chemical activation of P2X receptors on thin-fiber nerves.

A prior work has also demonstrated that muscle acidosis can attenuate P2X-mediated vasoconstriction (24). The mechanisms of decreased P2X receptor-mediated response in muscle acidification are not clear, but previous studies suggest that extracellular protons, at physiological concentrations, can regulate the function of P2X purinoceptors by modulating the affinity of the ATP-binding site (29, 50). Hydrogen ion may lead to a reduction in P2X ion pore permeability (50).

Finally, {alpha},beta-meATP stimulates only P2X3 and P2X1 receptor subtypes, and P2X3 receptors dominate at dorsal ganglion neurons (15, 43). Therefore, P2X3 purinoceptors were most likely activated in the present study.

TRPV1 Response at Lowered Muscle pH

TRPV1 appears preferentially on metabolite-sensitive groups III and IV sensory neurons (35) and mediates the effects of the vanilloid compound capsaicin (1). When thin-fiber muscle afferent nerves are stimulated by capsaicin, a reflex pressor response originated from muscle is initiated (4, 32, 33). For example, when capsaicin is injected into the arterial supply of a dog's hindlimb, blood pressure rises by 20%, and the effect is abolished by sectioning afferent nerves (4). Capsaicin was found to stimulate 71% of group IV and 26% of group III dog hindlimb muscle afferent fibers (18). A recent report suggests that the capsaicin-induced reflex pressor response is mediated via TRPV1 (32). Therefore, data from the present study show that muscle acidosis enhanced TRPV1-mediated pressor reflex, and a distinct enhancement was seen when muscle interstitial pH was adjusted to ~6.59. This is the first report showing the effects of muscle acidification on the cardiovascular responses induced by stimulation of TRPV1.

In an electrophysiological study, reducing the pH to 6.8 did not directly activate TRPV1 channels but resulted in a subtle increase in the activation rate of capsaicin responses (41). Increasing the acidity of the solution to pH 6.0 or pH 5.0 resulted in significant increases of the activation rate of capsaicin currents. Previous studies (53) have shown that acidification of the extracellular milieu has two primary effects on TRPV1 function: First, extracellular protons increase the potency of heat and capsaicin (as TRPV1 agonists) because the protons lower the threshold for channel activation by both stimuli. Second, extracellular protons themselves can act as TRPV1 agonists because acidification (to pH 6.0) leads to TRPV1 channel opening. A study using whole cell patch-clamp technique has shown that the low pH increased conductance of the capsaicin-gated current of the dorsal root ganglion neurons (27). In addition, acidic pH was found to increase the activation rate and decrease the deactivation rate of capsaicin-activated currents, providing evidence that potency of capsaicin is enhanced under acidic conditions (41, 53).

Effect of ASIC on pH Modulating P2X and TRPV1 Responses

Our previous report has shown that H+ does not affect TRPV1 receptors directly but stimulates ASIC in evoking the reflex response (32). The studies suggest that ASIC is likely to be frequently found on afferents containing TRPV1 receptors (32) and play a coordinated and interactive role with TRPV1 in modulating cardiovascular responses (7). However, a recent work suggests that TRPV1 (VR-1) receptors play little, if any, role in the responses of groups III and IV muscle afferents to H+ production by contracting muscles (23). Since concentrations of H+ necessary to activate ASIC and TRPV1 are different (54), there is a potential explanation for the previous observations. In mild or moderate muscle acidification, H+ stimulates ASIC but not TRPV1. Thus TRPV1 may play little role in the pressor response to muscle contraction. In severe muscle acidosis, interstitial protons may lower the threshold for channel activation of TRPV1 and increase the TRPV1 response in modulating the muscle pressor reflex.

Our data from this report have shown that augmented TRPV1 response to capsaicin in muscle acidification was attenuated after blocking ASIC with amiloride. This supports the hypothesis that there is an interaction between ASIC and TRPV1 receptors when muscle acidification develops.

Study Limitations

Amiloride is a nonspecific ASIC blocker (28). ASIC subunits are expressed on thin-fiber afferent nerves (28). Unfortunately, aside from Psalmotoxin 1, which only blocks ASIC 1a (5), no specific pharmacological blockers of the family ASIC are presently available (21). Thus, until a group of ASIC-specific blockers becomes available, one must consider the possibility that the effects seen with amiloride could be due to blockade of another member of the Na+ channel/degenerin family of ion channels (21). It is noted that higher amiloride concentrations are required to block Na+ channel/degenerin channels (21). Nevertheless, a precise mechanism for the interactive role that P2X/TRPV1 and ASIC play in processing the muscle afferent responses to H+ needs to be better explored when a specific pharmacological blocker of ASIC is available.

Summary and Conclusions

In conclusion, the data from the present study suggest that 1) P2X receptor and TRPV1-mediated effects on the reflex pressor response are sensitive to muscle acidosis; and 2) lowered pH in skeletal muscle attenuates P2X receptor-induced pressor response and augments TRPV1-induced response, but exaggerated TRPV1 response requires lower pH in muscle. This study provides evidence that muscle pH may be important in modulating P2X and TRPV1 responsiveness in exercising muscles.


   GRANTS
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 ABSTRACT
 METHODS
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 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-075533 (to J. Li), R01-HL-078866 (to J. Li), and R01-HL-060800 (to L. Sinoway).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Li, Heart and Vascular Institute H047, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Dr., Hershey, PA 17033 (e-mail: jzl10{at}psu.edu)

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.


   REFERENCES
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