Introduction

Obesity is associated with numerous health complications such as diabetes, systemic hypertension, heart disease, stroke and respiratory disease; all of which contribute to an increase in mortality. Although obesity is also a risk factor for pulmonary hypertension (PH), a direct ‘causal’ relationship between obesity and PH has not been demonstrated. Instead, the onset of PH has been indirectly linked to other obesity-induced health complications, such as chronic hypoxic episodes caused by obstructive sleep apnea or obesity hypoventilation syndrome,1 rather than obesity per se.2, 3 Indeed, as obesity is almost always associated with several other comorbidities, it is difficult to confirm obesity as an independent risk factor for the onset of PH.

The literature describes an intriguing phenomena known as the ‘obesity paradox’,4, 5, 6 whereby obese subjects with cardiovascular disease appear to have a better prognosis than that of non-obese subjects with comparable cardiovascular complications.7 Although the ‘obesity paradox’ appears to contravene decades of health-related research, it has been confirmed by several studies and extensive meta-analyses.8, 9 Indeed, a recent prognostic clinical study in obese humans verified that obesity appears to paradoxically ‘protect’ against PH.10 The mechanisms that underpin the obesity paradox in cardiovascular disease remain largely unexplored.6

Within the lung, sympathetic stimulation facilitates β-adrenergic-mediated vasodilation to optimize blood flow through the pulmonary circulation. Under hypoxic conditions, sympathetic nerve activity (SNA)-mediated vasodilation becomes critically important to offset potent local vasoconstriction.11 This intrinsic hypoxic pulmonary vasoconstriction (HPV) of the pulmonary blood vessels is an important homeostatic mechanism for optimizing gas exchange within the normal lung. Interestingly, obesity has been linked to an enhanced sympathetic tone, which we hypothesized may protect against the onset of PH because of enhanced β-adrenergic-mediated vasodilation of the pulmonary vessels and, thus, underpin the mechanism governing the obesity paradox in PH.

Considering that SNA outflow to functionally different organs is quantitatively non-uniform,12 this study first aimed to determine whether pulmonary SNA (pSNA) is elevated in obese Zucker rats, compared with their lean counterparts. The obese Zucker rat, characterized by a missense mutation in the leptin receptor, is a well-established animal model for clinically relevant obesity-related research.13 Second, we aimed to identify how sympathetic hyper-excitation impacts on the modulation of pulmonary vascular tone in obesity, as well as in PH, using synchrotron radiation (SR) microangiography.

Materials and methods

Animals

All experiments were approved by the local animal ethics committee and conducted in accordance with the guidelines of the Physiological Society of Japan and the University of Otago, New Zealand. In addition, experiments conform to the NIH guidelines (guide for the care and use of laboratory animals). Experiments were conducted on 27 Zucker lean rats (body weight ~400 g) and 27 Zucker obese rats (body weight ~650 g). Both lean and obese rats were age matched (12 weeks old) and further divided into two groups. Obese and lean rats were housed either in standard normoxic conditions (obese-N, lean-N) or in a chronic hypoxic chamber (10±0.1% O2) for 2 weeks (obese-CH, lean-CH).11 All rats were on a 12-h light/dark cycle at 25±1 °C and were provided with food and water ad libitum.

General anesthesia and surgical preparation

Rats were anesthetized with urethane (1.5 g kg–1, intraperitoneal). Adequate anesthesia was confirmed regularly throughout the duration of the protocol by complete elimination of the limb withdrawal reflex. Rats were placed supine on a Perspex board and body temperature was maintained at 38 °C using a rectal thermistor coupled with a thermostatically controlled heating pad. The trachea was cannulated for mechanical ventilation, and a femoral artery and vein were cannulated for measurement of systemic arterial blood pressure (ABP) and fluid/drug administration, respectively.

Experiment 1—recording pSNA (lean-N, n=7; lean-CH, n=6; obese-N, n=7; obese-CH, n=6)

The pulmonary sympathetic nerve was identified as a branch from the stellate ganglion innervating the lung parenchyma, dissected free of surrounding connective tissue and placed on a pair of platinum recording electrodes, as previously described.14 The raw nerve signal was filtered (low-cutoff 0.1 kHz; high-cutoff 1 kHz;), amplified (x10k), rectified and then integrated (time constant delay of 0.5 s) online, so that the integrated nerve signal could be displayed in real time.

Protocol

pSNA, ABP and heart rate (HR) were continuously recorded during (i) basal conditions (for 15 min) and (ii) exposure to acute hypoxia (8% O2 in N2) for 5 min. In order to eliminate any SNA variability within groups, it was essential to omit the highly variable background ‘noise’ levels (that is, zero nerve activity) from the recorded electroneurogram.14, 15, 16 Therefore, at the end of each experiment, the electroneurogram was continuously recorded as the rat was killed by an intra-cardiac injection of 1M KCl. After the animal was deceased, only background noise was contributing to the overall recorded electroneurogram. Subsequently, during post-experiment analysis, this ‘noise’ was subtracted from the pre-recorded SNA.

Experiment 2—SR microangiography (lean-C, n=7; lean-PH, n=7; obese-C, n=7; obese-PH, n=7)

The pulmonary circulation was visualized using SR microangiography at the SPring-8 facility BL28B2 beam line, (Sayo-Cho, Japan), as previously described.17

Protocol

The rat was strategically positioned in front of the SR SATICON X-ray camera (Hamamatsu Photonics, Shizuoka, Japan) so that the left side of the thorax was in alignment with a 9.5 × 9.5 mm imaging field (that is, between the second and third rib). The X-ray imaging procedure began when a clinical autoinjector (Nemoto Kyorindo, Tokyo, Japan) injected a single bolus of contrast agent (Iomeron 350; Eisai Co. Ltd, Tokyo, Japan) at high-speed (0.4 ml @ 0.4 ml s–1) into the right ventricle (via the jugular vein) and pulmonary vessels. Rats were given at least 10 min between contrast injections for renal clearance of the contrast agent.

Following baseline imaging, rats were exposed to acute hypoxia (8% O2 in N2) for 5 min. Lung microangiography was performed under baseline conditions and on the hypoxic lung after the 4th min of hypoxia; before and then again after (i) selective β1-adrenergic receptor (AR) blockade (atenolol, 3 mg kg–1, intravenous) and (ii) nonspecific β-adrenergic blockade (propranolol 2 mg kg–1).

Western blot analysis of pulmonary β1 and β2 ARs

After the rat was killed, the lung was retrieved and homogenized in a lysis buffer containing 300 mM mannitol, 3 mM EGTA, 1 mM EDTA, 10 mM Hepes-Tris (pH 7.4) and protease inhibitor mix composed of 1 mM benzamidine, 2 μg ml–1 leupeptin, 2 μg ml–1 pepstatin A, 2 μg ml–1 aprotinin, 2.5 mM DTT and 0.5 mM phenylmethylsulfonyl fluoride. The homogenate was incubated on ice for 30 min, at room temperature for 30 min and then centrifuged at 10 000 g for 20 min at 4 °C. The supernatant was further clarified by centrifugation at 100 000 g for 30 min at 4 °C, and the pellet re-suspended in lysis buffer. Protein concentrations were determined using the DC protein determination kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. The re-suspended pellet was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 12% gel and the separated proteins were transferred to nitrocellulose (β1-AR) or polyvinylidene fluoride (β2-AR) membranes. Following transfer, the membranes were blocked for 1 h with tris-buffered saline (pH 7.5), containing 0.05% Tween 20 and 5% (w/v) skimmed milk. The blots were then probed with specific antibodies against β1-AR, β2-AR (Genetex, Irvine,CA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Sapphire Bioscience Pty. Ltd, Redfern, NSW, Australia). All proteins were detected using an enhanced chemiluminescence kit. Protein band densities were normalized to GAPDH as an internal control and are expressed as protein to GAPDH ratio.

Data analysis

Hemodynamic and SNA data

The signals for right ventricular pressure (RVP), ABP and raw SNA were continuously sampled at 500 Hz (RVP and ABP) or 4 kHz (SNA) with an 8-channel MacLab/8 s interface hardware system (AD Instruments, Dunedin, New Zealand), and recorded on an Apple iMac using Chart (v. 7.2.5, AD Instruments). HR was derived from the interval between arterial systolic peaks.

Microangiography

Image Pro-plus (ver. 4.1, Media Cybernetics, Rockville,MA, USA) was used to enhance the contrast and clarity of angiogram images. The experimenter was blinded to the identity of all angiograms. All imaged vessel branches were counted. Vessels were categorized according to internal diameter (ID, μm); 100–200, 200–300, 300–500 and >500 μm. Where possible, the diameters of 2–4 vessels of each branching generation (second to fourth generation) were measured before and after acute hypoxia, with and without β-AR blockade. A 50 μm-thick tungsten filament, placed directly across the corner of the detector’s window, appeared in all recorded images and was subsequently used as a reference for calculating vessel ID.

Statistical analysis

All statistical analyses were conducted using Prism (version 6; GraphPad Software Inc., La Jolla, CA, USA). All results are presented as means±s.e.m. Based upon the variability of the data to be collected, a group size of 6–7 animals was deemed as an appropriate population size to have sufficient statistical power to measure a ‘treatment’ effect, as previously described.14 Given that the variance of data was similar between groups, two-way analysis of variance (repeated measures) was used to test whether the responses to acute hypoxia (for example, pulmonary vasoconstriction or SNA) were significantly different (i) between groups of animals and (ii) following β-AR blockade. One-way analysis of variance (factorial) was used to test for differences in (i) variables under baseline conditions compared with acute hypoxia and (ii) baseline values between animal groups. Where statistical significance was reached, post hoc analyses were incorporated using the paired, or unpaired t-test with the Tukey’s correction for multiple comparisons. A P-value ⩽0.05 was predetermined as the level of significance for all statistical analyses.

Results

Baseline

Baseline pSNA of normoxic obese rats was approximately fivefold higher than the lean counterpart (2.4±0.4 and 0.5±0.1 μV s, respectively, P<0.001; Figure 1a). Although there was no difference between obese-N and lean-N rats for both systolic RVP (~30 mmHg) and the RV/LV+Sep ratio, obese-N rats had larger hearts, after normalizing to tibia length (Table 1a; P<0.01).

Figure 1
figure 1

(a) Examples of raw electroneurogram traces (above), and quantification (mean±s.e.m.; below) of pSNA showing the differences between lean and obese rats exposed to normoxia (lean-N, n=7; obese-N, n=7), or chronic hypoxia (lean-CH, n=6; obese-CH, n=6) for 2 weeks. (b) Quantitative changes in pSNA in response to acute hypoxia (8% O2 for 5 min) in lean-N, obese-N, lean-CH and obese-CH rats. † Significant difference between lean and obese rats (P-values presented). § Significant difference between normoxia and chronic hypoxia (P-values presented). * Significant response to acute hypoxia (P<0.05).

Full size image
Table 1A Morphological characteristics of lean and obese rats exposed to either Norm. or chronic hypoxia (10% O2 for 2 weeks=CH) to induce pulmonary hypertension
Full size table

Chronic hypoxia significantly elevated pSNA in lean-CH rats (1.7±0.6 μV s), which was considerably greater in obesity (6.8±2.2 μV s; Figure 1a). The observed CH-induced increase in systolic RVP (P<0.01) was similar for both lean-CH (42±2 mmHg) and obese-CH rats (40±3 mmHg) (Table 1b), and both groups developed right ventricular hypertrophy (Table 1a), collectively indicative of PH.

Table 1B sRVP, MABP and HR data of lean and obese rats exposed to either Norm. or chronic hypoxia (10% O2 for 2 weeks=CH) to induce pulmonary hypertension
Full size table

Using SR microangiography, we were able to assess perfusion distribution (that is, branching pattern) of the pulmonary circulation (Figure 2a). The CH-induced development of PH was associated with a decrease in the number of fourth-order branches in lean-CH rats (P<0.01), but not obese-CH rats (Figure 2b). As highlighted in Figure 2c, vessel ID decreased as the branching generation increased, similar for all groups of rats.

Figure 2
figure 2

(a) Typical microangiogram images showing the branching pattern of small pulmonary arteries in an obese normoxic rat. (b) The number of opaque vessels (mean±s.e.m.), and (c) the range of vessel sizes (box and whisker graph) at each of the first four branching generations of the pulmonary circulation, in lean and obese rats exposed to normoxia (lean-N, n=7; obese-N, n=7), or chronic hypoxia (lean-CH, n=7; obese-CH, n=7) for 2 weeks. (d) Microangiograms illustrating vasoconstriction of pulmonary vessels (black arrows) in response to acute hypoxia (8% O2 for 5 min) in an obese normoxic rat. Severe vasoconstriction in some vessels prevented the entry of contrast medium into the vessel, as highlighted by the red box. The tungsten wire in the bottom right corner of each angiograpm is a reference of 50 μm diameter. A full color version of this figure is available at the International Journal of Obesity journal online.

Full size image

Responses to acute hypoxia

Normoxic rats

Acute hypoxia (8% O2 for 5 min) provoked an increase in pSNA in lean-N rats (P<0.05), which was markedly amplified (P<0.001) in the obese-N counterparts (Figure 1b). Angiographic evidence (Figure 2d) revealed that acute hypoxia caused significant constriction (decrease in ID) of all vessels smaller than 300 μm, which was similar in magnitude between normoxic lean and obese rats (Figure 3a). However, constriction of the 200–300 μm vessels was amplified in the obese-N rats compared with the lean counterpart (for example, 25±3% and 16±3% decrease in ID, respectively; P<0.05). Despite the significant pulmonary vasoconstriction, there was no significant increase in systolic RVP for either lean-N or obese-N rats (~5% increase, NS; Figure 4), which may be attributable to a decrease in cardiac output, based on the ~50% decrease in mean ABP (P<0.01) and 11% decrease in HR (significant for lean rats only; P<0.05; Figure 4).

Figure 3
figure 3

The relationship between vessel size and the magnitude of HPV (HPV – % decrease in vessel diameter) in response to acute hypoxia (8% O2 for 5 min) in (a) lean and obese rats exposed to normoxia (lean-N, n=7; obese-N, n=7), or (b) chronic hypoxia (lean-CH, n=7; obese-CH, n=7) for 2 weeks, before and after; (c) and (d) β1-AR blockade (atenolol, 3 mg kg–1, intravenous (i.v.)) and; (e) and (f) β1+β2-AR blockade (propranolol, 2 mg kg–1, i.v.). *Significant vasoconstriction response to acute hypoxia (P<0.05). †Significant difference between lean and obese rats (P<0.05). §Significant difference between normoxia and chronic hypoxia (P<0.05). ‡Significant effect of either atenolol or propranolol on the magnitude of vasoconstriction (P<0.05).

Full size image
Figure 4
figure 4

Hemodynamic responses (% change) of lean and obese rats exposed to normoxia (lean-N, n=7; obese-N, n=7), or chronic hypoxia (lean-CH, n=7; obese-CH, n=7) for 2 weeks, to acute hypoxia (8% O2 for 5 min), before and after β1-AR blockade (atenolol, 3 mg kg–1, intravenous (i.v.)), followed by β1+β2-AR blockade (propranolol, 2 mg kg–1, i.v.). *Significant response to acute hypoxia (P<0.05).

Full size image

Chronic hypoxic rats

The magnitude of pSNA response to acute hypoxia was not modified by CH for either lean or obese-CH rats (Figure 1b). Consequently, the pSNA response to acute hypoxia was still greater for obese-CH rats (P<0.05), compared with the lean counterpart (Figure 1b). Interestingly, the larger pSNA response to hypoxia in obese-CH rats was matched by a reduction in the magnitude of constriction of the 200–300 μm pulmonary vessels, so that the overall HPV profile (that is, magnitude of constriction for all vessels) was now identical between lean and obese-CH rats (Figure 3b).

Responses to β1-receptor blockade (atenolol)

Baseline

In all groups of rats, atenolol caused a large decrease in HR (16–25% decrease; P<0.01) but did not alter systolic RVP or ABP (Table 1b). In accordance with the insignificant effect on systolic RVP, atenolol had an insignificant impact on pulmonary vessel caliber (<10% change in ID for all vessel categories in all groups of rats).

Acute hypoxia

The HPV profile of lean-N rats was not significantly altered by atenolol although the magnitude of vessel constriction appeared to be blunted, especially for the 100–200 μm vessels. (Figure 3c). In obese-N rats, the vessels larger than 300 μm were also unaffected by atenolol, but the magnitude of constriction in the 100–200 μm vessels was significantly accentuated by atenolol (16±2% vs 23±3% decrease in vessel ID before vs after atenolol, respectively; P<0.05) and, thus, was considerably larger than the lean counterpart (8±3% decrease in ID for the 100–200 μm vessels).

Intriguingly, following 2 weeks of chronic hypoxia, atenolol had no impact on the HPV profile for both lean-CH and obese-CH rats with PH (Figure 3d).

Responses to β1+β2-receptor blockade (propranolol)

Baseline

In all groups of rats, propranolol had no further effect on all hemodynamic variables on top of that previously observed for atenolol (Table 1b). Propranolol did not significantly change pulmonary vessel caliber for all vessel categories for normoxic lean-N and obese-N rats, but it did cause significant constriction of the 200–300 μm vessels in the obese-CH rats (17±5% decrease in vessel ID; P<0.05).

Acute hypoxia

The HPV profile of lean-N and lean-CH rats was not significantly altered by propranolol, and was therefore not altered by either β1- or β2-AR blockade (Figures 3e and f). In obese-N rats, the HPV was further amplified by propranolol, on top of atenolol treatment alone, because of enhanced vasoconstriction of both the 100–200 μm vessels and the 300–500 μm vessels (15±3% vs 7±2% decrease in vessel ID for propranolol compared with saline treatment, respectively; P<0.05) (Figure 3e).

In obese-CH rats, although β1-AR blockade (atenolol) did not have any effect on the HPV profile, subsequent β2-AR blockade markedly enhanced the magnitude of vasoconstriction for all vessels with ID ⩽300 μm, for example, (17±3% vs 33±4% decrease in vessel ID for the 200–300 μm vessels before and after propranolol, respectively; P<0.01; Figure 3f).

Expression of pulmonary β receptors

In spite of the clear sympathetic hyper-excitation in obesity and CH, and the enhanced contribution of β2-ARs for maintaining basal pulmonary vascular tone and HPV, the relative expression of pulmonary β2-ARs (western blots) was significantly downregulated in obese-N rats compared with lean-N rats (P<0.05). Moreover, β2-AR expression was downregulated by CH in both obese-CH (NS) and lean-CH rats (P<0.05; Figure 5). Interestingly, the relative expression of β1-receptors within the lung was similar between all four groups of rats (Figure 5).

Figure 5
figure 5

Representative images of western blots and quantitative analysis of pulmonary β1-AR and β2-AR protein from normoxia (lean-N, n=6; obese-N, n=6), or chronic hypoxia (lean-CH, n=5; obese-CH, n=5). The levels of β1-AR and β2-AR protein were normalized to GAPDH.

Full size image

Discussion

The primary results of this study demonstrate that (i) pSNA is elevated in obesity, compared with the lean counterparts, which is further amplified upon the development of PH; (ii) β-AR-mediated vasodilation has an important role in ‘restraining’ the severity of acute HPV in obesity; in normoxia rats (β1-AR) and chronic hypoxia (β2-AR); and (iii) the severity of pulmonary vessel rarefaction in PH appears to be attenuated in obesity. Collectively, these results highlight the importance of pSNA for modulating pulmonary vascular tone in obesity and PH, raising the possibility that sympathetic hyper-excitation may contribute, at least in part, to the ‘obesity paradox’ phenomenon. However, given the observation that the eventual development of PH was similar for lean and obese animals, the mechanisms that fully underpin the obesity paradox are almost certain to be multi-factorial and warrant further research.

Recent epidemiological evidence suggests the presence of an ‘obesity paradox’ in PH, whereby an elevated BMI is correlated with decreased mortality.10 The physiological mechanism governing this paradox is unclear, but the presence of an obesity paradox suggests that pulmonary vascular tone is differentially regulated in obesity. Notwithstanding the above, there is a paucity of studies that have investigated the regulation of pulmonary vascular tone in obesity, particularly relating to the pathogenesis of PH.

Sympathetic hyper-excitation in obesity and chronic hypoxia (PH)

Obesity is associated with a ‘global’ increase in SNA in animal models, for example, Zucker rats18 and humans.19 Although we too observed that SNA was elevated in obesity, this is the first study to specifically assess changes in ‘pulmonary’ SNA in the context of obesity and PH. Indeed, a strength of this study is that we recorded directly from the pulmonary sympathetic nerve, which is of significance because sympathetic outflow to functionally different organs are quantitatively non-uniform.12 In accordance with the literature,20, 21 we noted that chronic hypoxia provoked an increase in SNA in all animals. Of particular interest is that, not only did obese rats have a higher basal pSNA, compared with their lean counterparts, but they were also much more sensitive to CH-induced amplification of pSNA, suggesting the homeostatic control of sympathetic outflow in obesity is augmented; by mechanistic pathways yet to be elucidated.

The underlying mechanisms for sympathetic hyper-excitation in obesity and chronic hypoxia remain to be fully elucidated. However, altered peripheral chemo- and baro-reflexes,22, 23 as well as progressive cellular and molecular adaptations centrally within the nucleus tractus solitarius,24 ventral lateral medulla25 and hypothalamic nuclei26 have also been charged with sustaining an increase in SNA. Indeed, alterations in leptin signaling within the central nervous system has also been shown to alter SNA in obesity.27, 28 Importantly, Shirai et al.29 reported that central signaling pathways, other than those directly triggered by chemo- and baro-reflexes, are more likely to have a predominant role in modulating ‘pulmonary’ SNA.

Sympathetic-mediated pulmonary vasodilation

We used the high-resolution capability of SR microangiography to assess pulmonary blood flow distribution and microvascular responses to β-AR blockade. In accordance with previous reports in the literature,11, 30 β-AR blockade had a negligible impact on the modulation of vascular tone for both the normoxic lean and obese rats. The efficacy of β-AR blockade was confirmed by large decreases in HR by atenolol. In the obese-CH rats with PH, however, targeted β2-AR blockade did cause significant pulmonary vasoconstriction (200–300 μm vessels), highlighting a potential role of sympathetic hyper-excitation for limiting the progression of PH in obesity; a proposal that is supported by observations that the chronic central inhibition of SNA31 or chronic peripheral β2-AR blockade30 significantly exacerbates the severity of PH.

The amplified sympathetic tone in obesity did not fully prevent the onset of PH, indicating that the obesity paradox culminates from an interaction of multiple complex mechanistic pathways. Interestingly, however, we did note that structural vessel rarefaction, which is normally evidence of advanced and severe PH,11 was attenuated in obesity compared with the lean counterparts. Whether sympathetic hyper-excitation aids in attenuating vessel rarefaction is an intriguing notion that warrants further research.

Hypoxia pulmonary vasoconstriction

In this study, the magnitude of HPV across the range of vessel sizes was generally similar between all groups of animals, in spite of the observation that obese rats had considerably larger pSNA responses compared with their lean counterparts. These results perhaps reflect the homeostatic importance of preserving a robust HPV, that is, maintaining optimal ventilation/perfusion matching, and that reflex increases in pSNA serve as a compensatory mechanism to ensure the HPV is maintained. Indeed, the importance of pSNA for maintaining HPV in obesity and PH became apparent following β-AR blockade.

Given the clear differences in pSNA between obese and lean rats, it was not surprising that β-AR blockade exacerbated the magnitude of HPV significantly more for obese, compared with lean animals, and even more so when in conjunction with PH. Of interest, both β1-AR blockade (100–200 μm vessels) and β2-AR blockade (200–300 μm vessels) appeared to modulate the HPV in obese-N rats whereas only β2-AR blockade exacerbated HPV (100–300 μm vessels) in obese-CH animals. These results concur with previous reports showing that the HPV of rats with pulmonary sympathetic hyper-excitation (induced with chronic intermittent hypoxia)14 is accentuated by selective β2-AR blockade alone.30 Collectively, these results further confirm that an amplified pSNA in obesity serves, at least in part, to ‘protect’ the pulmonary vasculature against excessive constriction via β-AR-mediated vasodilation, particularly in the face of a hypoxic challenge,14 although further research is essential to identify the precise mechanistic pathways that underpin this interaction.

Interestingly, these results also suggest there may be a differential switch in the relative role or β-AR subtypes during the progressive development of PH, for modulating pulmonary vascular sensitivity to sympathetic input. This intriguing phenomenon is known to occur in heart failure whereby the relative expression of β1-ARs to β2-ARs can change because of either downregulation of one specific β-AR subtype,32 or redistribution/compartmentalization of various β-AR subtypes,33 thus altering the physiological role of each β-AR subtype.

β-AR expression

In spite of the amplified dependence of β-AR for constraining the magnitude of HPV in obesity and PH, we noted that the expression of β-AR within the lung was either unchanged (β1-AR) or downregulated (β2-AR) in obesity and PH. Remarkably, the relative expression of β2-AR between lean and obese animals, with or without PH, appeared to be an inverse reflection of baseline pSNA.

Hypercatecholemia or ‘agonist-induced’ downregulation of β-ARs (internalization or decreased expression) and/or desensitization (impaired signaling) has been well documented for several cardiac diseases34, 35 and pulmonary disorders30, 36 whereby an increase in SNA and/or CH have been implicated as primary contributing factors. In contrast to this study, however, Nagai et al.30 reported a preferential decrease in the expression of β1-AR in chronic intermittent hypoxic rats, with no change in β2-AR expression. The reason for the discrepancy between studies is unclear, although it is noteworthy that chronic hypoxia is fundamentally a very different stimulus to chronic ‘intermittent’ hypoxia.

Study limitations

This study is the first to identify the ‘fundamental’ changes in sympathetic and β-AR modulation of pulmonary vascular tone in obesity and PH. In doing so, this study provides the foundation for future research to ascertain specific mechanistic pathways that may underpin (i) sympathetic hyper-excitation (within the central nervous system), or (ii) changes in β-AR expression and sensitivity in obesity. Although western blots provide semiquantitative analysis concerning the overall expression of β-AR subtypes, it does not provide critical information concerning receptor state (membrane-bound or internalized), receptor affinity for agonist, receptor state (synthesis vs degradation) or receptor localization.

β1-ARs are expressed within the pulmonary vascular endothelium and smooth muscle, whereas β2-ARs are expressed only in the endothelium.30 This may be of significance because prolonged stimulation of β2-ARs (that is, increased pSNA) has been shown to switch G-protein signaling from Gs to Gi,37 which activates a β2AR-Gi–dependent signaling cascade ultimately potentiating nitric oxide-dependent vasodilation.30 These observations highlight a potential role of enhanced endothelial function as a contributing factor (in addition to SNA) in the obesity paradox phenomenon; an intriguing hypothesis that warrants further research.

Summary

The pulmonary circulation is meticulously modulated by various intrinsic and extrinsic neuro/hormonal factors. We have utilized SR microangiography and electrophysiological recordings to demonstrate that sympathetic traffic to the pulmonary circulation is greatly enhanced in obesity and, importantly, that sympathetic hyper-excitation appears to constrain the severity of PH. The mechanisms that underpin the sympathetic hyper-excitation in obesity and how this impacts on pulmonary vasculature sensitivity remain to be fully elucidated, although changes in the expression and signaling of varying β-ARs within the lung appear to have a pronounced role. These results also highlight the clinical implications of β-AR blockade as a therapeutic strategy for obese patients with PH and other confounding comorbidities.