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Postural tachycardia syndrome (POTS) is characterized by excessive orthostatic tachycardia and significant functional disability. We have previously found that POTS patients had increases in plasma angiotensin II (Ang II) twice as high as normal subjects despite normal blood pressures. In this study we assess systemic and renal hemodynamic and functional responses to Ang II infusion in patients with POTS compared with healthy controls.
Following a 3 day sodium controlled diet, we infused Ang II (3 ng/kg/min) for 1 hour in POTS patients (n=15) and healthy controls (n=13) in the supine position. All study subjects were females with normal blood pressure (BP). Ages were similar for POTS and control subjects (30±2 [mean±SEM] vs. 26±1 years; P=0.11). We measured the changes from baseline mean arterial pressure (MAP), renal plasma flow (RPF), plasma renin activity (PRA), aldosterone, urine sodium and baroreflex sensitivity in both groups.
In response to Ang II infusion, POTS patients had a blunted increase compared with control subjects in MAP (10±1 mmHg vs. 14±1 mmHg; P=0.01), and diastolic BP (9±1 mmHg vs. 13±1 mmHg; P=0.01), but not systolic BP (13±2 mmHg vs. 15±2 mmHg; P=0.40). Renal plasma flow (RPF) decreased similarly with Ang II infusion in POTS patients and controls (−166±20 vs. −181±17 mL/min/1.73 kg/m2; P=0.58). Post-infusion, the decrease in PRA (−0.9±0.2 vs. −0.6±0.2 ng/mL/h; P=0.43) and the increase in aldosterone (17±1 vs. 15±2 pg/ml; P=0.34) were similar in POTS and controls. The decrease in urine sodium excretion was similar in both POTS and controls (−49±12 vs. −60±16 mEq/g Cr; P=0.55). The spontaneous baroreflex sensitivity at baseline was significantly lower in POTS compared to healthy controls (10.1±1.2 vs. 16.8±1.5 ms/mmHg, P=0.003) and it was further reduced with Ang II infusion.
Patients with POTS have blunted vasopressor response to Ang II and impaired baroreflex function. This impaired vasoconstrictive response might be exaggerated with upright posture, and may contribute to the subsequent orthostatic tachycardia that is the hallmark of this disorder.
Postural tachycardia syndrome (POTS) is a chronic disorder characterized by a marked increase in heart rate on upright posture, in the absence of orthostatic hypotension. It is estimated that more than 500,000 patients are affected in the United States1. This disorder predominantly affects young women of reproductive age2;3. Patients often suffer from a myriad of orthostatic symptoms that include palpitations, lightheadedness, and mental clouding4, and POTS is associated with significant functional disability and diminished quality of life5.
Various mechanisms may contribute to the orthostatic tachycardia and orthostatic intolerance in POTS, but the exact pathophysiology of POTS is still uncertain. The proposed mechanisms include increased sympathetic activity2;6, partial autonomic neuropathy7, venous blood pooling and low blood volume8. Abnormal regulation of the renin-angiotensin-aldosterone system (RAAS) has been implicated in the pathogenesis of POTS. We previously reported that many patients with POTS have inappropriately low levels of plasma renin activity (PRA) and aldosterone in response to both standing and to their supine low blood volume9. In addition to the blunted renin and aldosterone response, we and others demonstrated that there were two fold increases in the level of circulating angiotensin II (Ang II)10;11. The functional consequence of this high Ang II on POTS pathophysiology has not been fully elucidated. Furthermore, despite the high Ang II in POTS, the hemodynamic effects that would be expected with elevated Ang II (such as high blood pressure and fluid retention) were absent in POTS; instead a major portion of POTS patients have low blood volume in the supine position8. These data suggest that Ang II type-1 receptors might be hyporesponsive to the effects of Ang II in POTS.
Given the abnormal blood volume regulation that has already been reported in POTS, the abnormal profile of the RAAS in POTS, which includes high levels of Ang II and inappropriately low levels of plasma renin activity and aldosterone, we evaluated the responsiveness of different target tissue to Ang II infusion. We tested the hypothesis that patients with POTS have a state of decreased responsiveness to the action of Ang II specifically involving the adrenal gland. The adrenal response was assessed by the Ang II-induced aldosterone secretion. Blood pressure (BP) and renal plasma flow were both measured, which reflect the systemic and renal vascular response to Ang II.
Adult premenopausal normotensive women between 18 and 50 years of age were studied. Fifteen patients referred to the Vanderbilt University Autonomic Dysfunction Center with POTS between January 2009 and July 2010 and 13 healthy control subjects were included in this study. Due to the effect of the sex hormones on the renin-angiotensin system12, subjects were all studied in the first 1–5 days of the menstrual cycle (early follicular phase); this information was obtained by a menstrual cycle history (counting days). Patients with POTS met the conventional criteria9;13. Briefly, patients developed symptoms of orthostatic intolerance accompanied by a heart rate (HR) rise ≥30 bpm that occurred within the first 10 minutes of standing up, without any evidence of orthostatic hypotension (fall in BP of ≥20/10 mmHg). Patients had at least a 6-month history of symptoms, in the absence of another chronic debilitating disorder or prolonged bed rest. Healthy control subjects (26±1 years) were similar in age to the POTS patients (30±2 [mean±SEM] years; P=0.11). All subjects underwent a detailed history and physical examination, including assessment of blood chemistry and complete blood count. Blood pressure, heart rate and electrocardiogram were assessed in the supine and upright position. None of the control subjects had an increase in HR ≥30 bpm on standing or symptoms of orthostatic intolerance. Due to the strong female predominance in POTS, only female control subjects were recruited. POTS patients and control subjects were free of medications that could impact autonomic tone for at least 5 half-lives and did not take fludrocortisone for at least 5 days before testing. The Ang II levels of the subjects were not known at the time of study enrollment. The Vanderbilt University Investigational Review Board approved this study, and written informed consent was obtained from each subject before the study began. The protocols reported here were parts of a study entitled “The Renin-Aldosterone Axis in Postural Tachycardia Syndrome” (ClinicalTrials.gov NCT00962949).
Study investigations were performed on the Elliot V. Newman Clinical Research Center at Vanderbilt University. For at least 3 days before testing, study subjects consumed a standardized methylxanthine-free diet that provided 150 mEq/day of sodium and 100 mEq/day of potassium.
Twenty-four hour (7:00 AM to 7:00 AM) urine for assessment of urinary sodium and creatinine excretion was collected to determine compliance with the diet. Subjects reported to the unit the night before the study day. The study started at 8:00 AM after the subjects had been supine and fasting after mid-night. An 18-gauge indwelling catheter was placed at least 2 hours prior to the start of the study in both arms; one was used for infusion of Ang II and p-aminohippurate (PAH) and the other was used for obtaining blood samples.
Each subject voided immediately before the start of the study. Prior to the Ang II infusion, baseline BP, HR and renal plasma flow were obtained for 1 hour. Renal plasma flow was measured using PAH. A loading dose of PAH (8 mg/kg; Merck) was given intravenously, followed by a continuous infusion of 12 mg/min to determine effective renal plasma flow, as we have previously described13;14. Renal blood flow was calculated by dividing the effective renal plasma flow by (1- hematocrit). Renal vascular resistance was derived by dividing mean arterial pressure (MAP) by the renal blood flow (expressed as mmHg/L/min). Automated oscillometric BP (Dinamap, Critikon) was measured every 10 minutes during the first hour (baseline). Blood was drawn for plasma renin activity (PRA), aldosterone, cortisol, sodium, potassium, creatinine, Ang II and PAH (mean of two measurements). Subjects voided again at the end of the first hour, and urine was collected for electrolytes and creatinine.
Following 1 hour of baseline, as we have previously described14, subjects received a continuous intravenous infusion of Ang II (Bachem, Switzerland) at initial dose of (1 ng/kg/min) for 10 min followed by (3 ng/kg/min) for 60 min (second hour). BP was measured every 5 min during the second hour (Ang II infusion). At the end of the Ang II infusion, all pre-infusion blood and urine samples were repeated.
Plasma renin activity was assayed by conversion of angiotensinogen to Ang I by a radioimmunoassay technique (antibodies from IgG Corporation) and reported in nanograms of Ang I per milliliter per hour. Blood for aldosterone was collected in chilled vacuum tubes without preservative, and the serum was extracted and sent to the laboratory on ice. Serum aldosterone was measured by radioimmunoassay (DPC Coat-a-Count, Diagnostic Products Corp, Nashville, TN).
Blood for determination of Ang peptides (10 ml) was poured into pre-chilled tubes that contained 0.5 ml of an inhibitor solution composed of 25 mM NH4-EDTA, 0.44 mM o-phenanthroline (Sigma, St. Louis MO), 0.12 mM pepstatin A (Sigma, St. Louis, MO) and sodium p-hydroxymercuribenzoate (Sigma, St. Louis MO). This cocktail prevents the in vitro metabolism of Ang I and Ang II during manipulation of the sample. Blood samples were centrifuged at 3000 rpm for 20 min at 4°C, and aliquots of plasma were stored at −80°C until assayed. Angiotensin samples were analyzed at the Wake Forest Hypertension Core Laboratory. Plasma was extracted using Sep-Pak columns, as previously described15;16. The sample was eluted, reconstituted and split for the three radioimmunoassays. Recoveries of radiolabeled Ang added to the sample and followed through the extraction were 92% (n = 23). Samples were corrected for recoveries. Ang II was measured using a kit produced by ALPCO Diagnostics (Windham, NH, USA) as described previously17;18. The minimum detectable level of the assay for Ang II was 0.8 pg/tube. Values at or below the minimum detectable level of the assay were arbitrarily assigned half that value for statistical analysis. The interassay coefficient of variation for Ang II was 12%. The antibody used in the Ang II kit shows cross-reactivity with Ang III-(2–8) and Ang IV-(3–8), but no cross-reactivity with Ang I. Therefore the values reported for Ang II do not distinguish between Ang II, Ang III and Ang IV.
PAH concentrations (mean of two measurements) were determined by spectrophotometry14. Serum cortisol level was measured by radioimmunoassay. Serum and urine sodium, potassium and creatinine analyses were performed in the clinical chemistry laboratory of Vanderbilt University Medical Center, and the reference ranges are those used by these laboratories.
The data were recorded using a WINDAQ data acquisition system (DI720; DATAQ, Akron, Ohio, USA; 14 Bit, 500Hz) and processed off-line using custom- written software in PV-Wave language (PV-Wave; Visual Numerics Inc., Houston, Texas, USA). Beat- to-beat values of detected R–R intervals and blood pressure values were interpolated, low-pass filtered (cutoff 2 Hz) and re-sampled at 4 Hz. Data segments of 300 s recorded at baseline and at the end of infusion step were used for spectral analysis. Linear trends were removed and power spectral density was estimated with the FFT-based Welch algorithm using three segments of 256 data points with 50% overlapping and Hanning window. The power in the frequency range of low frequencies (LF: 0.04 to < 0.15 Hz), and high frequencies (HF: 0.15 to < 0.40 Hz) was calculated following Task Force recommendations19. Variability was also expressed as a percentage of total power or as normalized units (nu) to total power minus the power in the very-low-frequency range (<0.04 Hz).
Sponteaneous BRS evaluation was based on analyzing simultaneous fluctuations in both BP and HR using cross-spectral analysis and the sequence method. Cross spectra, coherence and transfer function analysis were used to capture inter-relationships between R–R interval and systolic blood pressure. Baroreflex gain was defined as the mean magnitude value of the transfer function in the low-frequency band (LF-band) with negative phase and squared coherence value greater than 0.520. The sequence method analyzes at least three heart beats in which both SBP and pulse intervals are steadily decreasing (BRS- sequence down). Spontaneous baroreflex slope was calculated as the slope of the linear regression line between SBP and the subsequent R–R intervals using sequences with more than 0.01 mm Hg SBP per beat. Only those sequences for which changes in the two parameters had a correlation coefficient of 0.85 were analyzed20.
Data including baseline characteristics (demographics, clinical and biochemical data) are expressed as mean ± SEM (unless otherwise noted). For continuous variables, data for the POTS and control group were compared with the Student’s t test. The Mann-Whitney U test was also used to confirm all the results obtained from the Student’s t test, and the significances of the reported parameters were not different between the two tests. Paired t-test was used to compare the difference in means within the same group; while Wilcoxon-signed rank test was used to confirm paired t-test results. Relationships between two variables were assesses by bivariate correlations generating Pearson’s correlation coefficient. Statistical analyses were carried out using the statistical software SPSS for Windows version 17.0 (SPSS Inc., Chicago, IL). All of the tests were 2-sided, and P<0.05 was considered statistically significant.
We studied 15 patients with POTS and 13 age-matched (all females) control subjects. Baseline characteristics were similar between the two groups and are summarized in (Table 1).
All baseline measurements were done in the supine position over 1 hour. POTS patients had a higher baseline heart rate than control subjects (72±2 bpm vs. 59±2 bpm; P=<0.001). The systolic BP (SBP), diastolic BP (DBP) and mean BP (MBP) were all similar between POTS and control subjects (SBP: 97±2 mmHg vs. 99±1 mmHg, P=0.51; DBP: 63±2 mmHg vs. 63±1 mmHg, P=0.91; MBP: 76±2 mmHg vs. 76±1 mmHg; P=0.88). Baseline effective renal plasma flow was not different between the two groups (641±30 ml/min per 1.73m2 vs. 639±39 mmHg, P=0.97). The renal blood flow and renal vascular resistance were also similar at baseline (Table 1).
Infusion of Ang II increased blood pressure in both POTS and control subjects. Compared to controls, POTS patients had a diminished response to Ang II infusion (Figure 1), with a blunted increase in MAP (10±1 mmHg vs. 14±1 mmHg; P=0.01; Figure 1C), and in DBP (9±1 mmHg vs. 13±1 mmHg; P=0.01; Figure 1B), but not SBP (13±2 mmHg vs. 15±2 mmHg; P=0.40; Figure 1A). The heart rate change in response to Ang II infusion was minimal in both POTS and control subjects (2±0.7 bpm vs. −1±0.9 bpm; P=0.002). Post Ang II infusion, renal plasma flow decreased to a similar extent in POTS patients and controls (−166±20 vs. −181±17 mL/min/1.73 kg/m2; P=0.58; Figure 2A). Renal blood flow and renal vascular resistance were not different between the two groups following Ang II infusion (data not shown).
Baseline PRA was similar (Table 1), and it was suppressed to a similar extent in the two groups following infusion (−0.9±0.2 vs. − 0.6±0.2 ng/mL/h; P=0.43; Figure 3A). Baseline aldosterone level was similar in POTS vs. controls (Table 1), and the increment in response to Ang II infusion was similar between the two groups (17±1 vs. 15±2 pg/ml; P=0.34; Figure 3B). Cortisol decreased similarly in both POTS and control groups (−0.7±0.6 vs. −1.4±0.6 ng/mL/h; P=0.43).
Consistent with our previous findings, baseline plasma Ang II levels were 2 fold higher in POTS compared to control (Figure 4A). With Ang II infusion, the plasma Ang II level increased equally in both groups (Figure 4B).
One hour of Ang II infusion decreased urine sodium from baseline to a similar extent in both POTS and controls (Figure 2B), these experiments were carried out at the same time of the day to avoid diurnal variation in sodium excretion.
The spontaneous baroreflex sensitivity (BRS) calculated by the sequence technique for down-slopes of SBP (BRS-sequence down) at baseline was significantly lower in POTS compared to healthy controls (13.7±3.5 vs. 26.0±2.6 ms/mmHg, P=0.01). Ang II infusion decreased BRS in POTS (from 13.7±3.5 to 10.4±2.3 ms/mmHg, P=0.04) and in controls (from 26.0±2.6 to 18.9±2.2 ms/mmHg, P=0.005) (Figure 5). BRS calculated as mean value of the transfer function between SBP and pulse intervals in the LF band demonstrated similar results. Baseline BRS-LF was lower in POTS vs. controls (10.1±1.2 vs. 16.8±1.5 ms/mmHg, P=0.003). Ang II infusion decreased BRS in POTS (from 10.1±1.2 to 8.7±1.3 ms/mmHg, P=0.07) and in controls (from 16.8±1.5 to 13.0±1.3 ms/mmHg, P=0.01). The mean reduction in BRS between the two groups with both techniques was not statistically significant.
To examine the relationship between BRS and Ang II level, we performed correlation analysis between BRS values and Ang II levels using Pearson test. There was significant negative correlation between baseline Ang II level and baroreflex sensitivity in POTS but not in controls using the sequence method (BRS- sequence down) (r = −0.69, P= 0.009 vs. r = 0.40, P= 0.32). The decrease in BRS correlated with the increase in Ang II levels in patients with POTS. In contrast, there was no correlation in healthy controls.
The main new findings of this study are that in response to an Ang II infusion, patients with POTS have a blunted pressor response to Ang II; whereas there is normal renal plasma flow, aldosterone secretion and sodium reabsorption by the kidneys.
The rationale for this study was based on the finding that despite the fact that POTS patients exhibit high levels of plasma Ang II, the hemodynamic effects expected for such an increase are absent. This suggests that the Ang II type-1 (AT-1) receptors are probably hyporesponsive to the effect of Ang II. We hypothesized that POTS patients would display a blunted response to Ang II infusion. This might manifest as impaired renal and systemic vascular response, impaired aldosterone secretion, and impaired sodium reabsorption capacity by the kidneys.
The dose of Ang II used in this study produced a mild but immediate response in the systemic vasculature, renal vasculature and adrenal gland. The use of Ang II infusion at a physiological dose systemically provides a powerful and reproducible method of directly assessing the vascular response in vivo21. In this study, we demonstrated for the first time an attenuated systemic vascular response to Ang II infusion in POTS. This was evidenced by the significant smaller increment in mean arterial pressure in POTS patients compared to healthy controls. The impaired vascular response in POTS may be related to the elevated level of circulating plasma Ang II that we and others have previously described in this population10;11 The prolonged presence of high levels of Ang II have been shown to induce a state of relative vascular resistance to the pressor effect of Ang II22 in conditions such as Bartter syndrome, cirrhosis and pregnancy23–25. Furthermore, low sodium intake, a condition characterized by high Ang II, has been shown to reduce the pressor response to Ang II in normal subjects21.
Vasoconstriction in response to Ang II involves binding of the hormone to AT-1 receptors located in the plasma membrane of smooth muscle cells. The mechanism of decreased AT-1 receptor responsiveness in the vasculature may be as simple as receptor down-regulation in response to abundant substrate26. This might explain the inverse correlation between plasma Ang II levels and the pressor response to Ang II. Another possible explanation is that the blunted response to Ang II may reflect intravascular volume depletion in the POTS patients, although we did not measure the blood volume in this study. As an effector hormone, Ang II plays a fundamental role in the regulation of vascular tone under circumstances of sodium and volume depletion27. The dependence of the pressor effect of Ang II on the volume status is especially important in light of the clinical observation that patients with POTS feel better following acute volume expansion28. The pathophysiology of the blood volume depletion in POTS is not clear, but it is not due to the lack of Ang II mediated sodium retention, as shown in the present study. Another possibility is that patients with POTS have a state of decreased ability to constrict vascular smooth muscle in response to pressor agonists. If present, however, such an abnormality should involve other pressor agonists including norepinephrine, but the pressor response to norepinephrine has been shown to be preserved in patients with POTS29.
We observed a normal renal vasoconstrictive response to Ang II, as measured by the renal plasma flow in this study. The discordant renal vascular and systemic vascular responses to Ang II are particularly intriguing. A similar discrepancy has previously been observed in other conditions14. A major target vascular bed for the effect of Ang II is the splanchnic circulation30;31, which is of great importance as a blood reservoir. Previous studies in POTS have shown significantly increased blood pooling in the splanchnic circulation with standing32;33. It is possible that the blunted increase in vascular tone in response to Ang II may be primarily localized to the splanchnic circulation. This might contribute to the pathogenesis of blood pooling in POTS, due to the impaired translocation of blood from the splanchnic circulation to the systemic arterial system upon standing. We did not, however, study the mesenteric blood flow in the present study. Furthermore, it is well established that patients with POTS have elevated sympathetic activity1; this might be secondary to decreased sensitivity or responsiveness to other major non-adrenergic vasoconstrictor pathways, such as Ang II.
Renal sodium reabsorption is mediated in part by Ang II both directly, via stimulation of AT-1 receptors in the proximal tubules and indirectly by decreasing renal plasma flow in addition to promoting aldosterone secretion34. Renal sodium retention was appropriate in this study in response to Ang II infusion. This essentially rules out hyporesponsiveness to Ang II as an explanation for the low plasma volume and the tendency for salt wasting in POTS patients8. Ang II serves as a major stimulus of aldosterone secretion which was similar in POTS compared to control, we controlled for other factors that might affect the aldosterone secretion, including dietary sodium and potassium intake, as well as ACTH-induced aldosterone secretion (cortisol level). The adrenal and renal responses were dissociated from the vascular response. This discrepancy might be partly due to different mechanisms of tissue interaction with Ang II. Downregulation of AT-1 receptor with administration of Ang II has been reported in vascular smooth muscle cells,35 while infusion of Ang II increases AT-1 receptor expression in the adrenal gland, but not the aorta or the kidney36;37. Contrary to our previous findings of an inappropriately low aldosterone response to high Ang II and upright posture in POTS, the adrenal response to Ang II infusion is intact in POTS9;11. The explanation for this discrepancy is unclear. It is possible that the pressor dose used in our Ang II infusion might have excessively stimulated aldosterone production with increased AT-1 expression as previously reported36, or the immediate increase in aldosterone secretion in response to standing might be mediated by other factors such as ACTH38.
Baroreflex sensitivity (BRS) is generally defined as the amount of response in heart beat interval to a change in blood pressure expressed in ms/mmHg. A blood pressure increment leads to an increment in interval within few seconds. Both POTS and healthy subjects had a comparable reduction in BRS during Ang II infusion. In POTS the baseline spontaneous BRS was significantly diminished and strongly correlated with plasma Ang II level (negative correlation between Ang II level and BRS). In fact the reduction in baroreflex sensitivity seems to parallel the baseline level of circulating Ang II. The cardiac vagal activity can be inhibited by circulating Ang II in the absence of baroreflex loading as suggested by Twonend J.N. et al39. Furthermore, chronically elevated Ang II can shift the cardiac baroreflex rest point to higher pressure by a blood-pressure independent mechanism in animal models40. These findings of blunted baroreflex sensitivity are similar to those observed in POTS patients by Farquhar W.B et. al. using the modified Oxford technique41. The relationship between the diminished spontaneous baroreflex sensitivity and impaired orthostatic tolerance has been reported before in both normal men and women following 7-day head-down bed rest42. Taken together these data indicate that patients with POTS might have impaired baroreflex control of the heart rate most likely due to the high circulating Ang II, this impaired baroreflex function results in excessive increase in heart rate on standing and contributes to the elevated baseline heart rate and sympathetic activity in POTS.
We studied our subjects while supine and not while upright (when the tachycardia would be greater). It would have been difficult to standardize standing time in each study subject given the various tolerances to standing. Blood pooling in the splanchnic circulation when upright is characteristic of POTS, and produces acute intravascular volume depletion. This volume depletion might be expected to make the Ang II- mediated vasoconstriction even more impaired, further exaggerating the differences between patients with POTS and controls subjects. It is noted that the PRA and aldosterone levels were not lower in POTS patients, in contrast to prior reports9;11. Finally, the exogenous Ang II infusion that we used allows determination of AT-1 receptors sensitivity. It is not, however, intended specifically to determine the interaction of endogenous Ang II & AT-1 receptors.
In summary, this study revealed a blunted systemic vascular and baroreflex response to Ang II in patients with POTS. The adrenal aldosterone response and renal sodium reabsorption capacity in response to Ang II are intact in POTS. These results provide a putative mechanism for POTS - an inability to adequately elevate the systemic vascular resistance in response to Ang II, which is further aggravated by the presence of a blunted baroreflex response. Both processes are critical circulatory adjustment to orthostatic stress. The mechanisms underlying the diminished vascular reactivity to Ang II remains to be elucidated.
We would like to thank our patients. Without their participation this research project could not have been performed. We would also like to recognize the highly professional care provided by the Elliot V. Newman Clinical Research Center nursing and nutrition staff.
Funding Sources: Supported by P01 HL056693 (DR), R01 HL071784 (DR), K23 RR020783 (SRR), R01 HL102387 (SRR), U54 NS065736 (DR), and UL1 RR024975 (CTSA) all from the National Institutes of Health, Bethesda MD, USA.
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