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CARDIOVASCULAR
Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, North Carolina
Received March 14, 2005; accepted June 1, 2005.
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Abstract |
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and high-frequency (HF) indices] and sequence method and HRV measured by frequency domain as power of RR interval (RRI) spectra (low-frequency RRI and high-frequency RRI) and by time domain as standard deviation of beat-to-beat interval and root mean square of successive differences along with increase in blood pressure variability measured as standard deviation of mean arterial pressure and power of systolic arterial pressure spectra (low-frequency systolic arterial pressure). Because elevated NEFA suppressed the vagal component of the baroreflex response (HF), we tested the hypothesis that NEFA-evoked sequestration of myocardial muscarinic receptor (M2-mAChR) contributes to the reduced BRS. High NEFA level was accompanied by increased caveolar sequestration of cardiac M2-mAChRs without changing M2-mAChR protein expression. We report the first detailed analyses of NEFA's effect on the cardiac baroreflex and show that increased caveolar sequestration of cardiac M2-mAChRs constitutes a cellular mechanism for elevated NEFA-related deleterious cardiovascular outcomes.
), and these neural and hemodynamic abnormalities are associated with life-threatening cardiac arrhythmias and sudden death (Hennersdorf and Strauer, 2001). Fluctuations in blood pressure (BP) and heart rate (HR) reflect the dynamic interplay of diverse physiological processes (Akselrod et al., 1985) and are acceptable measures for cardiovascular autonomic balance. Increased HR variability (HRV) and BP variability (BPV) and reduced HRV are predictors for mortality (Stein et al., 1994; Lombardi et al., 1996; Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology, 1996; Palatini and Julius, 1997; Malliani and Montano, 2002). Spectral analysis of arterial BP and HR has provided new insight into the autonomic vascular and cardiac regulation (Cerutti et al., 1994). Analysis of BPV and HRV by the spectral approach, as well as by time domain techniques, represents a useful tool for the study of the mechanisms involved in cardiovascular regulation in both normal and diseased conditions (Parati et al., 1995b).
Elevated plasma lipids have been linked to many cardiovascular diseases and are associated with an increase in sudden death (Jouven et al., 2001; Wyne, 2003). Patients with familial combined hyperlipidemia have elevated plasma nonesterified fatty acids (NEFA) (Carlsson et al., 2000) and higher BP (Castro Cabezas et al., 1993). Moreover, plasma NEFA measured after an overnight fast and 2 h after an oral glucose load independently predicted the development of hypertension (Fagot-Campagna et al., 1998). It is imperative to note that short-term elevation in plasma NEFA following the acute infusion of intralipid in humans caused impairment of baroreflex measured by the Oxford (phenylephrine) method (Gadegbeku et al., 2002). A comprehensive investigation of the impact of elevated plasma NEFA on autonomic control of cardiac baroreflex and indices has not been reported.
At the cellular level, a high-fat diet decreases M2 muscarinic cholinergic receptor (M2-mAChR) number and function in cardiomyocytes (Pelat et al., 1999). Notably, caveolar sequestration plays a role in M2-mAChR desensitization (Feron et al., 1997; Dessy et al., 2000). The possibility has not been investigated that short-term elevation in plasma NEFA may cause sequestration of myocardial M2-mAChR, which may explain the adverse effect of NEFA on the vagal component of the baroreflex arc. Undoubtedly, the elucidation of the temporal correlation between elevation of NEFA levels and its hemodynamic effects and providing a possible mechanism for these hemodynamic changes are clinically important.
In this study, we investigated the effect of short-term (15 h) elevation of NEFA levels in conscious freely moving rats by: 1) overnight fasting, and 2) overnight fasting + intralipid/heparin (I/H) infusion on the systolic arterial pressure (SAP), HR, and spontaneous baroreflex sensitivity (BRS) measured by RR interval (RRI) and SAP cross-spectral analysis [low-frequency (LF) index and HF index]. We also investigated the effect of NEFA elevation on spontaneous baroreflex measured by the time domain analysis (sequence method). We followed the effect of NEFA elevation on HRV measured by frequency domain analysis as power of beat-to-beat interval spectrum in the low-frequency range (LFRRI) and power of beat-to-beat interval spectrum in the high-frequency range (HFRRI) and by time domain analysis as standard deviation of beat-to-beat interval (SDRR) and root mean square of successive differences (rMSSD). Because the spectral analyses' findings provided evidence that elevated plasma NEFA was associated with suppressed myocardial vagal tone, we hypothesized that caveolar sequestration of M2-mAChR in cardiac myocytes might underlie the NEFA-induced attenuation of the activity of the parasympathetic limb of baroreflex arc and the associated increase in HR and attenuation of HRV in fasting and I/H-treated conscious freely moving rats. These studies were undertaken in conscious rats to circumvent the confounding effects of anesthesia on data interpretation. Notably, the rat is considered an appropriate model for generating clinically relevant data on lipid regulation and cardiovascular responses to clinically prescribed medications in which fatty acid oxidation and metabolism in rats and humans are similar (Stanley et al., 1997).
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Materials and Methods |
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). Briefly, the rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). Catheters (polyethylene 10 connected to polyethylene 50), filled with heparinized saline (100 U/ml), were placed in the abdominal aorta and vena cava via the femoral artery and vein for measurement of BP and i.v. administration of drugs, respectively. The catheters were tunneled s.c., exteriorized at the back of the neck between the scapulae, and plugged by stainless steel pins. Incisions were closed by surgical staples and swabbed with povidone-iodine solution. Each rat received buprenorphine hydrochloride (30 µg/kg s.c.) (Buprenex; Reckitt Benckiser, Slough, UK) to control pain and 50,000 U/kg of benzathine and penicillin G procaine in an aqueous suspension (i.p.) (Durapen; Vedco, Overland Park, KS) and was housed in a separate cage. Each experiment started 2 days after surgery by connecting the arterial catheter to a Gould-Statham pressure transducer (Oxnard, CA). BP and HR were recorded and analyzed using the Gamma 4 data acquisition and analysis system (Grass Instruments Division, Astro-Med, West Warwick, RI). BP and HR were also displayed on a Grass polygraph (model 7D; Grass Instruments, West Warwick, RI). The venous catheters were used to infuse I/H mixture or saline. On the experiment day, blood samples were collected for the measurement of plasma NEFA concentration by means of an enzymatic colorimetric method using a commercial kit (NEFA C; Wako Bioproducts, Richmond, VA). Experiments were performed in strict accordance with institutional animal care and use guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Frequency Domain Analysis. Spontaneous BRS was calculated by the frequency domain analysis method as in reported studies (Parati et al., 1995a) using a newly developed software designed for rats (Nevrokard SA-BRS; Medistar, Ljubljana, Slovenia). Nevrokard SA-BRS is a software package for BRS analysis in small animals. It features frequency domain analysis of up to 5 Hz and a resampling rate of 10 Hz (http://www.nevrokard.medistar.si/maini/brs.html).
Power spectral densities of SAP and RRI oscillations were computed by 512-point fast Fourier transform and integrated over the specified frequency range (LF, 0.25-0.75 Hz; HF, 0.75-5.0 Hz). Basically, the SAP and RRI files generated via the data acquisition system (Gamma 4; Grass Instrument Division, Astro-Med) at 1000 Hz were analyzed using Nevrokard SA-BRS software. A Hanning window was applied, and the spectra of SAP and RRI series and their squared-coherence modulus were computed if the coherence was greater than 0.5 in accordance with reported criteria (Parati et al., 1995a). The square root of the ratio of RRI and SAP powers were computed to calculate LF and HF indices, which reflect the BRS (Parati et al., 1995a).
Power of RRI spectra in the LF and HF range (LFRRI and HFRRI) were calculated in normalized units, and the ratio of LFRRI/HFRRI was used as a measure of sympathovagal balance (Laitinen et al., 1999). Power of SAP spectra was calculated as LFSAP as a measure of BPV.
Sequence Method. BRS calculated by this method is based on quantification of sequences of at least three beats (n) in which SAP consecutively increases [UP sequence (seq)] or decreases (DOWN seq), which are accompanied by changes in the same direction of the RRI of the subsequent beats (n + 1). To be included in the BRS estimate, each sequence must fulfill the following criteria (Wang et al., 2004): 1) minimal RRI change, 3 ms; 2) minimal SAP change, 1 mm Hg; 3) minimal number of beats, 3 or more, in the sequence; and 4) minimal correlation coefficient of 0.85. The software scans the RRI and SAP records, identifies sequences, and then calculates linear correlation between RRI and SAP for each sequence. If the correlation coefficient exceeds a preset critical value (0.85), the regression coefficient (slope) is calculated and accepted. The mean of all the individual regression coefficients (slopes), which is a measure of seq BRS, was then calculated. Overall, three parameters were obtained by this method (seq BRS: SAP UP, DOWN, and TOTAL).
Time Domain Analysis. Three time domain parameters were used to measure hemodynamic variability as in previous studies (Stein et al., 1994; Sgoifo et al., 1997). HRV was determined by computing the SDRR and the rMSSD in RRI duration. The standard deviation of the mean arterial pressure (SDMAP) was used as a measure for BPV.
Immunoblotting. Ventricular tissue was homogenized in a homogenization buffer [50 mM Tris, pH 7.5, 0.1 mM EGTA, 0.1 mM EDTA, 2 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1% (v/v) Nonidet P-40, 0.1% SDS, and 0.1% deoxycholate]. After centrifugation (12,000g for 5 min), protein was quantified in the supernatant using a modified Lowry assay procedure (the Bio-Rad protein assay system; Bio-Rad, Hercules, CA). For Western blot analysis of M2 and caveolin-3 (C3), 40 µg was loaded onto Novex (San Diego, CA) Bis-Tris 4 to 12% gels and run according to the manufacturer's manual for best resolution. Proteins were transferred to nitrocellulose membranes after electrophoresis. After being blocked with 5% nonfat dry milk in Tris-buffered saline (TBS), the blots were incubated with the specified primary antibody anti-M2 dilution at 1:300 (Chemicon International, Temecula, CA) and anti-C3 dilution at 1:2500 (Transduction Laboratories, Lexington, KY) in TBS buffer containing 5% nonfat dry milk overnight at 4°C. After four washes, the blots were incubated for 2 h at room temperature with secondary antibody diluted in TBS buffer containing 5% nonfat dry milk. After three additional washes in TBS buffer with 0.1% (v/v) Tween 20, the blots were detected by an enhanced chemiluminescence system (Amersham Biosciences Inc., Piscataway, NJ) and exposed to X-ray film at room temperature. Films were developed in a Kodak D-19 developer (Eastman Kodak, Rochester, NY) and analyzed on a video-based computerized system. Protein levels were quantified by measuring the optical density of the specific bands on a Macintosh computer (Apple Computer, Cupertino, CA) using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/) (Shaw et al., 1995). The protein concentration was presented as percentage from the control sample loaded in the same gel.
Immunoprecipitation. Ventricular tissue lysates were prepared using the same method described for immunoblotting. Lysates containing 1 mg of detergent-soluble protein were precleared by incubating with protein A-Sepharose for 1 h at 4°C and then transferred to a fresh tube. Five microliters of anti-M2 polyclonal antibody (10 µg/ml; Chemicon International) were added and gently mixed overnight at 4°C. Thirty microliters of 50% protein A-Sepharose slurry were added to the mixture. After an overnight incubation rotating at 4°C, immune complexes were collected by centrifugation and washed three times with 1 ml of immunoprecipitation buffer and once with wash buffer (50 mM Tris, pH 8). The complexes were disrupted by heating at 95°C for 3 min in 30 µl of immunoblotting sample buffer (100 mM Tris, 100 mM DTT, 1% SDS, pH 7.5). The supernatant was then analyzed by Novex Bis-Tris 4 to 12% gels, followed by protein immunoblotting for C3 using anti-C3 dilution of 1:2500 (Transduction Laboratories), and analyzed as described above (Dessy et al., 2000).
Protocols and Experimental Groups. Fifteen conscious Sprague-Dawley male rats were used 48 h after implantation of femoral catheters. The rats had free access to food and water. Following a stabilization period of at least 30 min after connecting the arterial and venous lines to the pressure transducer and an i.v. delivery system, respectively, an arterial blood sample (0.25 ml) was drawn for the measurement of basal NEFA level. MAP, HR, BRS (as LF, HF, seq BRS-SAP UP, seq BRS-SAP DOWN, and seq BRS-SAP TOTAL), HRV (LFRRI,HFRRI, SDRR, and rMSSD), LFRRI/HFRRI ratio, and BPV as LFSAP and SDMAP were measured over a 2-h period as detailed earlier. The rats were then subjected to overnight fasting (water ad libitum) and randomly divided into two groups: 1) control (n = 6), and 2) I/H-treated (n = 9). I/H was infused as a 20% I/H mixture (Sigma Chemical, St. Louis, MO) at dose of 1.2 ml/kg over 10 min (Widmaier et al., 1992; Fabris et al., 2001; Gadegbeku et al., 2002); the same volume of heparinized saline was infused in the control group. Plasma NEFA and the hemodynamic parameters were measured at 30, 60, and 180 min after I/H or saline infusion, which was followed by euthanasia and tissue collection.
In a separate group of seven conscious male Sprague-Dawley rats, catheterized as detailed above for BP and HR measurement, BRS was measured by the Oxford (phenylephrine) method daily for 5 days as in our previous studies (El-Mas and Abdel-Rahman, 1997; Shaltout and Abdel-Rahman, 2003). Briefly, four or five randomized bolus doses of phenylephrine (1-16 µg/kg) were injected at 5-min intervals. In each rat, the peak changes in MAP and HR, obtained following phenylephrine injections, were used for the construction of the baroreflex curve, and the slope of the regression line represented the BRS. In the same rats, BRS was measured by Nevrokard SA-BRS software as detailed above.
Drugs. Phenylephrine hydrochloride (Sigma Chemical), Buprenex (buprenorphine hydrochloride; Reckitt Benckiser), Durapen (penicillin G benzathine and penicillin G procaine; Vedco), heparin (Elkins-Sinn Inc., Cherry Hill, NJ), intralipid 20% (phospholipid stabilized soybean oil, which contains 20.0 g of soybean oil, 1.2 g of phospholipids, glycerin, and 2.25 g of USP; Sigma-Aldrich, St. Louis, MO), pentobarbital sodium (Sigma-Aldrich), sterile saline (B. Braun Medical Inc., Bethlehem, PA), and NuPAGE Novex Bis-Tris Gels (Invitrogen, Carlsbad, CA) were purchased from commercial vendors.
Statistical Analysis. Values are expressed as mean ± S.E.M. GraphPad Software Inc. (San Diego, CA) Instat statistical analysis software (analysis of variance with repeated measures, followed by Bonferroni post hoc analysis) was used for multiple comparisons. Probability levels less than 0.05 were considered significant.
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Results |
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Effect of Fasting and I/H on BP and Heart Period. As shown in Fig. 1A, overnight fasting caused a slight increase in SAP. I/H infusion caused a statistically significant increase in SAP, which started during the infusion, peaked (13%) at 1 h, and remained significantly higher than the corresponding control value at 3 h. Beat-to-beat interval (RRI) was slightly but significantly reduced by overnight fasting (
4%), and I/H infusion caused a further reduction, which reached its nadir at 30 min and remained significantly lower than the control values over the 3-h observation period (Fig. 1B).
Effect of Fasting and I/H on BRS. Overnight fasting significantly reduced BRS measured by the spectral analysis (Fig. 2, A and B) and sequence method (Fig. 2, C, D, and E) in rats that subsequently received I/H infusion or heparinized saline (control). LF (Fig. 2A) and HF (Fig. 2B) were reduced 25 to 30% following overnight fast. I/H infusion caused a statistically significant reduction in total BRS, which reached its nadir at 30 min and tended to recover but was still significantly lower than the control value at 3 h (Fig. 2, A, B, and E). The reduction caused by I/H in BRS was more evident with the HF (Fig. 2B) than the LF (Fig. 2A) component (spectral method) and with SAP UP (Fig. 2C) than SAP DOWN (Fig. 2D) (sequence method).
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25%) and were further increased by I/H infusion (50%) at 30 min and remained significantly higher than the control until the end of the recording period (Fig. 4, A and B).
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, was highly correlated with plasma NEFA levels (Fig. 6A). The data included in Fig. 6A were collected from rats when they were fed ad libitum (before any treatment) and following overnight fasting in both treated and control groups. The data show a highly significant inverse relationship between BRS and NEFA.
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Discussion |
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The main findings in this study are 1) I/H infusion significantly elevated plasma NEFA levels; 2) elevation of plasma NEFA by overnight fasting followed by I/H infusion significantly increased systolic arterial BP and its variability; 3) I/H infusion significantly increased HR and reduced its variability; 4) elevation of NEFA significantly increased the power of RRI at the LF range and reduced it in the HF range, causing a significant increase in the LFRRI/HFRRI ratio, a measure for sympathovagal balance; and 5) NEFA elevation significantly increased the association between M2-mAChR and C3 without changing myocardial M2-mAChR or C3 protein level.
We show, for the first time in conscious rats, that acute elevation of NEFA caused baroreflex dysfunction over a 3-h period after intralipid infusion, which agrees with the finding that acute elevation of plasma NEFA reduces baroreflex measured by phenylephrine infusion in humans (Gadegbeku et al., 2002).
We found that elevation of NEFA with overnight fasting significantly attenuated the LF coefficient and HF calculated by cross-spectral analysis and also attenuated the time domain measures of the baroreflex (seq BRS: SAP UP, DOWN, and TOTAL). We used two nonpharmacological methods for BRS measurement. The reduction in BRS caused by elevated NEFA measured by the spectral analysis and time domain methods in the present study fully agrees with the clinical findings using the phenylephrine method (Gadegbeku et al., 2002). However, the methods used in the present study offered the opportunity to obtain a detailed analysis of the BRS and other cardiac indices on a beat-by-beat basis. Equally important, we circumvented the phenylephrine-evoked increase in BP, which if applied could have confounded the data interpretation. The phenylephrine method is based on the open-loop model, in which the increases in RRI and BP are related according to a linear model, whereas the methods used in our study provide a closed-loop estimation of BRS, in which BP oscillations induce changes of RRI that in turn are able to modify BP (Pitzalis et al., 1998). Notably, phenylephrine at 10 µg/kg, which falls within the doses used to construct the BRS curve by the Oxford method in reported studies, including ours (Gadegbeku et al., 2002; Shaltout and Abdel-Rahman, 2003), reduced the plasma NEFA level (Imura et al., 1971). Furthermore, the sequence BRS is an accepted measure for tonic parasympathetic cardiac control (Wang et al., 2004). Undoubtedly, the present findings with two different methods bolster our conclusion about the status of the baroreflex following evoked increases in plasma NEFA.
I/H infusion in overnight-fasting rats caused a further attenuation in HF and seq BRS-SAP (UP, DOWN, and TOTAL) that showed a recovery tendency but remained significantly lower (HF and seq BRS TOTAL) than the control group at the end of the study (3 h). Two findings suggest a strong link between increased NEFA and the reduced vagal component of the cardiac baroreflex. First, we showed that the increase in NEFA preceded the BRS dysfunction. Second, we showed for the first time a significant inverse correlation between plasma NEFA and BRS (Fig. 6A). Our conclusion is bolstered by the immunoprecipitation findings and that seq BRS-SAP UP (index of parasympathetic control) was more affected by overnight fasting than seq BRS-SAP DOWN (index of sympathetic control) (Parati et al., 2001).
Because we used new software for the measurement of BRS as LF and HF, we undertook a preliminary study to validate the software. In this validation study, we compared the BRS measured in the same rat by the Oxford (phenylephrine) method (open loop) and by Nevrokard-SA-BRS software (closed loop). The rats used in the validation study and in the present study were of similar age and weight. The data generated showed a highly significant correlation between the BRS values obtained by the two methods (Fig. 6B). However, there are differences between the reported open-loop gain values in rats, which varied between 1.3 and 2.5 ms/mm Hg with most of the studies reporting values of 1 to 1.5 ms/mm Hg (El-Mas and Abdel-Rahman, 1997; Mosqueda-Garcia et al., 1998; Shaltout and Abdel-Rahman, 2003). The HF value obtained in the present study (2.5 ms/mm Hg) is similar to the higher end of the reported range for the open-loop gain.
It is noteworthy that overnight fasting, a standard experimental procedure followed in most of clinical and experimental studies, per se can attenuate BRS and alter cardiovascular indices. For example, our experimental design resembled the clinical setting in which the patients fasted overnight before receiving I/H infusion (Gadegbeku et al., 2002). We found that overnight fasting slightly increased SAP and that further (16 mm Hg) increase occurred 60 min following I/H infusion in conscious rats. Our finding that acute elevation of NEFA increases SAP supports the epidemiological finding that elevated NEFA predicts the development of hypertension (Fagot-Campagna et al., 1998) and that elevation of NEFA by 3 h of intralipid infusion increases BP (Manzella et al., 2001). The increase in BP could be explained by the ability of fatty acids to impair endothelial function (Davda et al., 1995) and to enhance vascular 1-adrenoceptor signaling (Stepniakowski et al., 1996; Haastrup et al., 1998). The elevation in BP after acute elevation in NEFA could also be caused by activation of sympathetic activity inferred by the increased LFSAP and SDMAP, accepted markers of sympathetic activity (Julien et al., 1995) and baroreflex dysfunction. This increase in sympathetic activity could also be responsible for the baroreflex resetting. It is noteworthy that in the present study overnight fasting (10-12 h), as in other studies (Halliwell et al., 1996), increased free fatty acids, which may explain the modest increase in MAP. Prolonged fasting involves other metabolic changes, which may overwhelm the moderate increases in plasma NEFA and sympathetic activity and may cause reduction in BP.
Elevated HR and reduced HRV are independent predictors for cardiovascular and noncardiovascular mortality (Stein et al., 1994; Palatini and Julius, 1997; Palatini, 1999; Seccareccia et al., 2001). We found that overnight fasting and/or I/H infusion increased HR, which could be because of enhancement of the sympathetic tone and/or inhibition of the parasympathetic tone. In dogs, acute elevation of NEFA causes tachycardia mainly because of impairment of parasympathetic control (Verwaerde et al., 1999). Overnight fasting and I/H infusion increased LFRRI and reduced HFRRI and hence the LFRRI/HFRRI ratio, a measure of sympathovagal balance (Laitinen et al., 1999), which suggests an enhancement of sympathetic control and a reduction in parasympathetic tone. These findings agree with similar findings in humans obtained following 3-h intralipid infusion (Manzella et al., 2001). Both SDRR and rMSSD, which primarily reflect the vagal activity of the heart (Stein et al., 1994), were reduced by overnight fasting and further reduced by I/H infusion. This suggests that the shift toward the sympathetic dominance results from the lack of a sufficient parasympathetic counteraction to sympathetic activation (Sgoifo et al., 1999). This also may explain the tachycardia associated with elevation of NEFA levels. It is noteworthy that elevation of plasma NEFA by overnight fasting and intralipid infusion increased both BP and HR, which suggests possible involvement of the central sympathetic nervous system activation.
We report for the first time that elevation of NEFA via overnight fasting followed by I/H infusion caused an enhancement of C3 protein expression and its association with M2-mAChR without significantly changing M2-mAChR expression. We reasoned that NEFA-evoked myocardial M2 association with C3 might account, at least in part, for the attenuated vagal component of the cardiac baroreflex measured by spectral analysis. Caveolae are now recognized to be plasma membrane compartments with distinct lipid and protein composition that sequester and regulate the function of cytoplasmically oriented signal transduction molecules (Okamoto et al., 1998). Notably, caveolar sequestration plays a role in desensitization and inactivation of many G protein-coupled receptors, including M2-mAChR receptors (Dessy et al., 2000; Feron and Kelly, 2001). It may be argued that the time after intralipid infusion (minutes to hours) may not be long enough to allow the translocation and association between the M2-mAChR and C3 and therefore precludes such a cellular mechanism as an explanation for the attenuated cardiac vagal component following plasma NEFA elevation. Findings from the present study and reported studies argue against this notion. In the present study, baroreflex dysfunction (attenuated vagal component) following overnight fasting was associated with increased expression of C3 and its association with myocardial M2-mAChR (Fig. 5, B and C). Translocation of M2-mAChR and its association with C3 occurs in the myocardium within minutes following the addition of M2-mAChR agonist (Feron et al., 1997). Caveolar sequestration of G protein-coupled receptors or enzymes (e.g., endothelial NO synthase) occurs even in the absence of significant change in the expression of C3 (Dessy et al., 2000; Feron and Kelly, 2001). Collectively, these findings support the view that enhancement of M2-mAChR C3 association contributes to the NEFA-evoked baroreflex dysfunction.
In summary, the current study sought evidence to characterize the role of caveolar sequestration of cardiac muscarinic receptors (M2-mAChR) in hemodynamic changes elicited by elevated NEFA levels as a result of overnight fasting and I/H infusion. This study is the first to provide experimental evidence that implicates a cellular mechanism for NEFA-induced attenuation of BRS and HRV parameters and increased HR through enhancement of caveolar sequestration of cardiac M2-mAChR, which results in M2-mAChR inactivation and attenuation of the parasympathetic control on the heart. The results of this study may explain, at least partly, the mechanism that underlies the adverse cardiovascular outcomes associated with high plasma NEFA in the general population.
Study Limitation. Because of the limited quantities of atrial tissue, we measured C3 and M2-mAChR protein levels and their association in ventricular myocytes. We acknowledge that activation by acetylcholine of the atrial M2-mAChR determines the contribution of the vagal component to HR control and to the baroreflex response.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: BP, blood pressure; HR, heart rate; HRV, heart rate variability; BPV, blood pressure variability; NEFA, nonesterified free fatty acid(s); M2-mAChR, myocardial M2 muscarinic receptor(s); I/H, intralipid/heparin; SAP, systolic arterial pressure; BRS, baroreflex sensitivity; RRI, RR interval(s); LF, low-frequency; HF, high-frequency; LFRRI, power of beat-to-beat interval spectrum in the low-frequency range; HFRRI, power of beat-to-beat interval spectrum in the high-frequency range; SDRR, standard deviation of beat-to-beat interval; rMSSD, root mean square of successive differences; seq, sequence; SDMAP, standard deviation of the mean arterial pressure; C3, caveolin-3; TBS, Tris-buffered saline.
Address correspondence to: Dr. Abdel A. Abdel-Rahman, Department of Pharmacology, Brody School of Medicine, East Carolina University, Greenville, NC 27858. E-mail: abdelrahmana{at}mail.ecu.edu
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) and overnight fasting + I/H (
) infusion on SAP (mm Hg, A), interbeat interval (RRI, ms, B) and NEFA (mEq/l, C) in conscious unrestrained rats. I/H or saline was infused over 10 min. Values are expressed as mean ± S.E. $, p < 0.05 versus baseline at day 1 (ad libitum); *, p < 0.05 versus control (fasting); and #, p < 0.05 versus I/H baseline at day 2 (0 min).
) and overnight fasting + I/H (
) infusion on M2 muscarinic cholinergic receptors expression (A), C3 expression in rat ventricles (B), and M2 C3 association (C) in the ventricles as percentage of the ad libitum control group (
). I/H or saline was infused over 10 min. Tissues are collected at 3 h after start of infusion. Values are expressed as mean ± S.E. *, p < 0.05 versus ad libitum control and #, p < 0.05 versus fasting control.