Using a novel high-throughput proteomics platform, we examined 92 cardiovascular plasma biomarkers in patients diagnosed with postural orthostatic tachycardia syndrome and controls with normal orthostatic response. We identified higher plasma levels of growth hormone and lower plasma levels of myoglobin in patients with POTS, whereas other biomarkers did not significantly differ between the two groups. We also documented sex-specific patterns of significance where lower MB level in men and higher GH level in women were independently associated with both binary POTS status and changes in heart rate during head-up tilt test, even after adjustment for age and BMI.
Growth hormone
Higher levels of growth hormone in patients with postural orthostatic tachycardia syndrome, notably in females, was an unexpected finding that deserves detailed commentary. GH is an anabolic neuropeptide regulating carbohydrate and lipid metabolism via complex interactions with insulin and insulin-like growth factor-1 (IGF-1). The secretion of GH from the anterior pituitary gland is stimulated by growth hormone releasing hormone (GHRH) and inhibited by somatostatin and a negative feedback loop of IGF-1 [
25]. The hormone is released in a pulsatile manner with significant circadian rhythm and peak discharge occurring at night-time, approximately one hour after sleep onset [
26]. Previous studies reported higher incidence of CV events related to increased levels of GH [
26]. Since long-term prognosis in POTS patients is still unknown, the effects of increased GH levels on CV outcome in this patient population remain to be explored.
There is a number of possible mechanistic explanations for higher levels of GH observed in POTS patients. Firstly, an increase in plasma GH concentration could be the result of proinflammatory cytokines acting as negative regulatory signals fine-tuning the action of hormones and growth factors. Tumour necrosis factor alpha and interleukin-1 beta are believed to cause IGF-1 resistance by weakening downstream signalling in myoblasts and this could eventually cause increased release of GH due to negative feedback [
27]. This hypothesis could be tested in future studies comparing the levels of inflammatory mediators in POTS with healthy subjects. The current body of knowledge is very sparse and with only IL-6 reported to be elevated in POTS [
28].
Secondly, it has been shown that POTS patients present with autoantibodies against alpha- and beta-receptors, which belong to the G-protein coupled receptor (GPCR) family of rhodopsin type [
3]. Receptors for GHRH (GHRHr) are distributed on the anterior pituitary gland, which also belong to GPCR family, though of slightly different secretin type. Hypothetically, higher levels of GH might be the result of abnormal stimulation of GHRHr by circulating anti-GPCR antibodies. Nevertheless, the presence of such specific and functionally active anti-GHRHr autoantibodies has not been yet demonstrated in POTS patients.
Thirdly, patients with POTS usually have a lean body type with lower BMI which could be related to increased lipolysis due to higher GH [
29]. Unfortunately, plasma insulin levels or HbA1c were not tested in this group of patients.
Fourthly, octreotide - a somatostatin analogue classically used to control hypersomatotropism in acromegaly through the inhibition of GH action and GH secretion [
30] – has been reported to be an effective treatment for POTS patients by reducing upright tachycardia and symptoms of orthostatic intolerance due to its splanchnic vasoconstrictor effect [
31]. Splanchnic blood flow has been shown indeed to be increased in the supine posture and to progressively increase during incremental tilt in POTS patients [
32]. In the absence of peripheral sympathetic denervation, locally mediated vasodilation - involving vasoactive autacoids such as vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, and nitric oxide - has been proposed as a possible explanation for splanchnic pooling. Interestingly, insulin-like growth factor-1 (IGF-1), that is synthesized in the liver, is secreted into the blood under the control of GH, and is known to induce peripheral vasodilation via NO synthase and/or potassium channel activity [
33]. It may, therefore, provide a possible mechanistic link among the observed high levels of GH, splanchnic pooling and octreotide efficacy in reducing symptoms in POTS patients.
Finally, the relative syndrome-dependent inactivity among POTS patients might hypothetically lead to a reversal or gross disturbance in circadian patterns of GH release. However, deconditioning as an underlying pathophysiology of POTS and to possibly related syndromes such as chronic fatigue syndrome has not been supported by recent studies, and other mechanisms such as low ventricular filling have been proposed [
34,
35]. It remains to be demonstrated if physical training may reverse GH level abnormality.
The influence of gender on serum concentrations of GH has been the object of previous investigations [
36,
37]. In many species, including rats, mice, and humans, the temporal pattern of pituitary GH secretion is sex-specific (episodic in males, more frequent in females) and leads to sex differences in downstream signaling pathways in target tissues [
38]. In our study blood samples were obtained in the morning after two-hour fasting, which ascertained stabilisation of GH concentration, but it has been also reported as a possible explanation for the observed high concentrations of GH in women, as if something in the morning - which may be regarded as very mild stress of fasting - could trigger a GH burst in almost all of the women but in very few of the men [
37]. This could be because of gender differences in the sensitivity of the pituitary or hypothalamus to the GH-releasing effects of mild stress. Furthermore, there is quite robust evidence in the literature to suggest that endogenous estrogens play a major role in increased GH secretion in women compared with men [
39]. Interestingly, in our study we observed POTS women presenting with significantly higher levels of GH than women without POTS. This could be the result of (i) complex interactions amongst sex-related and sex-unrelated immune-neuroendocrine mechanisms, (ii) sexually dimorphic patterns of GHRH secretion (iii) impaired cerebrovascular autoregulation [
40], (iv) chronic deconditioning [
41], and/or (v) hitherto unknown pathways.
Myoglobin
Myoglobin, an oxygen and iron binding protein found in muscle cells, is usually increased when muscle tissue damage occurs though small amounts are normally present in plasma. Our analysis revealed decreased plasma myoglobin in POTS patients, notably males, compared with controls. The results are difficult to interpret as the method used for protein detection in this study provides only relative values within the analyzed sample, not absolute values that could be translated to clinically useful cut-off levels. Of note, the values reported here are likely not indicative of muscle damage, since patients were free of such clinical suspicion at inclusion. More likely, the levels detected here are linked to small amounts of myoglobin found in plasma in the absence of muscle damage. Lower myoglobin levels found in POTS might be a result of immobilisation and limited physical activity in those patients, possibly a consequence of cardiovascular deconditioning or chronic fatigue [
1]. Thus, although deconditioning may not be causally related to POTS [
34], such deconditioning may be the result of reduced exercise tolerance experienced by many POTS patients [
42]. In addition, the lower myoglobin levels found in POTS may be seen to parallel reduced iron stores, which is, in turn, associated with POTS [
43]. Unfortunately, data on iron status was not available in our study population. Moreover, considering the role of myoglobin in muscle metabolism [
44], one may also hypothesize that myoglobin may, indeed, also have a role in the pathophysiology of POTS, even if such hypotheses are highly speculative at this stage.
Even though we did not measure lean body mass, POTS patients in our study did have lower BMI. It remains to be explored why this association is limited to men only.
Beyond growth hormone and myoglobin, we could observe aberrations in other proteomics biomarkers, although not achieving the adjusted significance level. However, the overall impression was that there were only slight differences in the analysed biomarkers. It may indicate that POTS is an inflammatory condition involving hitherto unknown pathophysiological mechanisms deserving further explorative and experimental studies.
Limitations
There are some limitations that must be addressed. Firstly, our control group included symptomatic individuals, who had normal hemodynamic response to tilt testing, but were referred to our center due to unexplained syncope and/or symptoms of orthostatic intolerance.
Secondly, this is a single-center experience with limited generalisability, also due to uneven sex distribution and lack of age matching; accordingly, and in the need of an external validation cohort, our findings have to be interpreted as hypothesis-generating.
Thirdly, our findings are based on one-off measurement precluding information about causality and temporal correlation of selected biomarkers with the progression of the disease, onset and burden of clinical symptoms.
Fourthly, we acknowledge the lack of information about menstrual cycle and use of hormonal contraceptives, as previous studies demonstrated that the hormonal fluctuations that occur during the normal menstrual cycle may alter autonomic regulation of blood pressure during various environmental stimuli [
46], and the intake of exogenous estrogen has been shown to increase plasma levels of GH [
37].
Fifthly, we recognize the lack of plasma GH determination on clinically validated high-sensitivity chemiluminescence sandwich immunoassay platforms for direct correlation with GH levels measured with PEA technology.
Finally, in order to rule-out false positive signals, our findings should be validated with alternative technologies enabling sensitive and robust detection and quantification of biomarkers. However, the use of a proximity extension assay technique, with the requirement for a dual binding event ensuring minimal noise signal, and the robust discovery algorithm would make a false positive result very unlikely.