Sympathetic Nervous System Modulation of Inflammation and Remodeling in the Hypertensive Heart

Abstract

Chronic activation of the sympathetic nervous system (SNS) is a key component of cardiac hypertrophy and fibrosis. However, previous studies have provided evidence to also implicate inflammatory cells, including mast cells, in the development of cardiac fibrosis. The current study investigated the potential interaction of cardiac mast cells with the SNS. Eight week old male SHR were sympathectomized to establish the effect of the SNS on cardiac mast cell density, myocardial remodeling and cytokine production in the hypertensive heart. Age-matched WKY served as controls. Cardiac fibrosis and hypertension were significantly attenuated and left ventricular mass normalized while cardiac mast cell density was markedly increased in sympathectomized SHR. Sympathectomy normalized myocardial levels of IFN-γ, IL-6 and IL-10, but had no effect on IL-4. The effect of norepinephrine and substance P on isolated cardiac mast cell activation was investigated as potential mechanisms of interaction between the two. Only substance P elicited mast cell degranulation. Substance P was also shown to induce the production of angiotensin II by a mixed population of isolated cardiac inflammatory cells, including mast cells, lymphocytes and macrophages. These results demonstrate the ability of neuropeptides to regulate inflammatory cell function, providing a potential mechanism by which the SNS and afferent nerves may interact with inflammatory cells in the hypertensive heart.

Introduction

Up-regulation of the sympathetic nervous system (SNS) is involved in numerous cardiovascular disease processes and is responsible for alterations in normal myocardial structure and function, including the induction of arrhythmias, alterations in contractile properties and cardiac remodeling. ¹ In an experimental setting, there is evidence that the SNS mediates hypertension-induced cardiac fibrosis and hypertrophy through α- and β-adrenergic receptors, respectively. ² However, concomitant with elevated SNS activity, induction of an inflammatory infiltrate into the heart also occurs, which appears to mediate the development of hypertension-induced cardiac fibrosis, but not hypertrophy. This inflammatory component includes mast cells (MC), ^3-6 macrophages ^7,8 and possibly T cells. ^9,10 MCs maintain a close anatomical association with nerves, ^11,12 with sympathetic neurons able to alter rat basophilic leukemia (RBL-2H3) MC membrane resistance. ¹³ Further, other inflammatory cells express adrenergic receptors, with modulation of cytokine production being either enhanced or repressed by stimulation of these receptors. ¹⁴ Alternatively, we have shown that cardiac MCs can also be activated by neuropeptides such as substance P, ¹⁵ which is found mainly in afferent sensory nerves. Given the prominent involvement of both the SNS and inflammation in myocardial remodeling associated with hypertension, we sought to determine if SNS-mediated regulation of inflammation is a mechanism involved in myocardial remodeling secondary to hypertension and whether afferent nerves also have the potential to regulate inflammation in the hypertensive heart.

Methods

All in vivo experiments were performed using adult male spontaneously hypertensive rats (SHR, n=18) and Wistar Kyoto rats (WKY, n=13) housed under standard environmental conditions and maintained on commercial rat chow and tap water ad libitum. SHR were chosen for this study because they have been documented to have elevated SNS activity. ¹⁶ All studies conformed to the principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Additionally, the protocol was approved by our Institution's Animal Care and Use Committee. All rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg).

Experimental Protocol and Sympathectomy Surgery

Rats were divided into four groups: 1) untreated WKY; 2) sympathectomized WKY (WKY+Symp); 3) untreated SHR; and 4) sympathectomized SHR (SHR+Symp). Data reported herein for untreated WKY and SHR have been previously published. ⁶ Bilateral sympathectomy surgery was performed at 8 weeks of age, and all rats were sacrificed at 20 weeks of age. This time-point was chosen as it represents a steady state of hypertension as well as established LV hypertrophy and fibrosis in the SHR. The sympathectomy surgery was conducted by the attending veterinarian at Charles River Laboratories using the following procedure. A ventral midline incision was made in the neck allowing the exposed osmohyoid muscle to be retracted laterally to expose the carotid artery internal and external branche region. The superior cervical ganglion lying within this bifurcation was carefully excised. This procedure was repeated on the contralateral side. A successful sympathectomy was evidenced by the occurrence of Horner's syndrome. Adrenalectomy was not performed in these animals because the aim was to examine the specific interactions between the SNS and MCs, not the role of circulating norepinephrine.

Hemodynamic Parameters

At the experimental end-point, animals were anesthetized with sodium pentobarbital (50 mg/kg) and measurements of systolic, diastolic and mean arterial pressures (SBP, DBP, MAP) were made using a high fidelity Millar catheter inserted into the carotid artery and advanced into the aortic arch. Catheter data were analyzed using PVAN 3.5 software.

Histological Analysis

MC number and density were determined from pinacynol erythrosinate stained LV sections as previously described (please see http://hyper.ahajournals.org). ^6,17 LV interstitial collagen volume fraction was determined by analysis of picrosirius red stained sections as previously described (please see http://hyper.ahajournals.org).⁶

Cytokine Characterization

IFN-γ, IL-4, IL-6 and IL-10 were quantified from frozen LV tissue (please see http://hyper.ahajournals.org) to determine the effects of sympathectomy, using commercial ELISA kits (Alpco Diagnostics, Salem, NH).

Cardiac Inflammatory Cell Isolation Procedure

Cardiac inflammatory cells were isolated as previously described. ¹⁵ Briefly, a thoracotomy exposed the intact pericardial sac. The tip of a teflon catheter sleeve, attached to a sterile syringe, was then inserted into the pericardial sac and the sac filled with Hanks balanced salt solution (HBSS, 7.4 pH) composed of: 1) Hanks calcium and magnesium free salt solution; 2) HEPES (13 mM); 3) 607 units/mL of deoxyribonuclease I (Sigma Chemical Co., St. Louis, MO); and 4) an antibiotic-antimycotic mixture of penicillin G sodium, 10,000 mg/mL of streptomycin sulfate, and 25 mg/mL of amphotericin B (Gibco-BRL, Life Technologies, Grand Island, NY). The buffer was then aspirated into another sterile syringe. This was repeated several times resulting in the collection of predominantly T cells, MCs and monocyte/macrophages (Figure S1; please see http://hyper.ahajournals.org). The cells were pelleted and resuspended in Hyclone buffer (HBSSMC; HBSS containing magnesium sulphate (1.1 mM), calcium chloride (1.3 mM), and phenol red; Hyclone, Logan, UT) before undergoing treatment.

Isolated Cardiac Mast Cell Studies

Cardiac MCs were obtained during the cardiac inflammatory cell isolation procedure described above. Cardiac MC responses were examined by incubating 4 × 10³ MCs per treatment tube with norepinephrine or substance P at specific concentrations: 0, 1×10^-12, 1×10^-9, 1×10^-6 and 1×10^-4 M. The samples were incubated at 37 °C for 20 min and the post-treatment supernatants and pellets separated for subsequent analysis of histamine as a marker of MC degranulation. Percent histamine release was determined by dividing the histamine value from the supernatant by total histamine (supernatant plus pellet). Histamine concentration was measured using a Neogen^® Veratox Histamine ELISA (Lexington, KY).

Substance P-mediated Production of Angiotensin II by Isolated Cardiac Inflammatory Cells

Isolated cardiac inflammatory cells (2 ×10⁵/well) were maintained in DMEM containing 10% FBS and treated with substance P (1×10^-4 M) for 20 hours. The media was collected and assayed for angiotensin II using a commercial enzyme immunoassay kit (SPI-BIO), which has cross-reactivities of 4, 36 and 33% with angiotensin I, angiotensin III and angiotensin 3-8, respectively.

Immunofluorescent Staining of α-Adrenergic Receptors

Please see http://hyper.ahajournals.org.

Statistical Analysis

All grouped data were expressed as mean ± standard deviation (SD) or standard error of the mean (SEM) as appropriate. Group comparisons were made by one-way ANOVA using SPSS 11.5 software (SPSS Inc., Chicago, IL). When a significant F test (p≤0.05) was obtained, inter-group comparisons were analyzed using Fisher's protected least significant difference post-hoc testing.

Results

Biometrics

Table 1 displays grouped biometric data. While no differences in body weight were observed between WKY, SHR and WKY+Symp, a significant increase was observed in sympathectomized SHR. Table 1 also includes LV weight and LV weight indexed to body weight as measures of hypertrophy. SHR developed significant LV hypertrophy in comparison to WKY by both measures, with sympathectomy preventing this hypertrophic response. There were no weight differences between WKY and WKY+Symp. Lung weights showed no significant differences. SHR showed marked increases in SBP, DBP and MAP compared to WKY. Sympathectomy in the SHR caused a significant attenuation of the blood pressure parameters, whereas sympathectomy had no effect on blood pressure in the WKY group.

Table 1.

Biometric data.

Group	BW (g)	LV (mg)	LV/BW (mg/g BW)	Lung Weights (mg)	SBP (mmHg)	DBP (mmHg)	MAP (mmHg)
WKY	328±28	786.8±54.0	2.24±0.11	1476±217	116±19	86±20	100±20
SHR	345±11	929.5±81.1*	2.70±0.24*	1445±122	229±26*	167±13*	193±17*
WKY+Symp	332±14	755.8±45.3	2.28±0.06	1303±179	123±20	98±17	109±19
SHR+Symp	421±18*†	715.8±26.9†	1.70±0.04*†	1511±154	147±16*†	116±11*†	130±13*†

All values are mean ± SD. BW, body weight; LV, left ventricle; SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure.

^*p<0.05 versus WKY;

^†p<0.05 versus SHR.

Collagen Volume Fraction

Collagen volume fraction (CVF) results are displayed in Figure 1. LV CVF in the SHR was markedly increased in comparison with that in WKY. However, this increase was significantly attenuated in the sympathectomized SHR group. CVF was not affected by sympathectomy in the WKY.

Figure 1.

Left ventricular collagen volume fraction in WKY (n=4), SHR (n=4), WKY+Symp (n=4) and SHR+Symp (n=4). All values are mean ± SEM. * = p<0.05 versus WKY; † = p<0.05 versus SHR.

Cardiac Mast Cell Number and Density

A significant increase in the number of MCs occurred within the LV of SHR (WKY, 73.3±10.3 versus SHR 105.7±14.8 cells/section; p<0.05). However, due to the larger area of the SHR LV, there was no difference when expressed as MC density (Figure 2). In contrast, SHR that had undergone sympathectomy showed a dramatic increase in both LV MC number and density (SHR+Symp, 316.3±68.6 cells/section; p<0.05 versus WKY and SHR). In contrast, sympathectomy had no effect on MC number or density in WKY rats (WKY+Symp, 84.2±12.1 cells/section).

Figure 2.

Changes in left ventricular MC density in WKY (n=4), SHR (n=6), WKY+Symp (n=6) and SHR+Symp (n=4). All values are mean ± SD. * = p<0.05 versus WKY; † = p<0.05 versus SHR.

Myocardial Cytokine Profile

Elevated levels of IFN-γ in the SHR (Figure 3A) were normalized by sympathectomy. Sympathectomy had no effect in the WKY. Although myocardial IL-4 levels were also elevated in the SHR (Figure 3B) sympathectomy did not change these levels. However, sympathectomy in the WKY did cause a significant increase in IL-4. Myocardial IL-6 (Figure 3C) was found to be markedly lower in the SHR than the WKY, but was normalized by sympathectomy. Sympathectomy did not change IL-6 levels in the WKY. IL-10 was also found to be decreased in the SHR myocardium (Figure 3D). Sympathectomy resulted in increased amounts of IL-10 in both WKY and SHR.

Figure 3.

Changes in left ventricular levels of (A) IFN-γ, (B) IL-4, (C) IL-6 and (D) IL-10 in WKY (n=4), SHR (n=4), WKY+Symp (n=4) and SHR+Symp (n=4). All values are mean ± SEM. * = p<0.05 versus WKY; † = p<0.05 versus SHR.

Effect of SNS Products on Cardiac Mast Cell Activation and Angiotensin II Production

Incubation of isolated cardiac MCs with norepinephrine at concentrations of 1×10^-12, 1×10^-9, 1×10^-6 and 1×10^-4 M, did not induce degranulation (Figure 4A). In contrast, while no effects of substance P were seen at lower concentrations, 1×10^-4 M did stimulate cardiac MC degranulation (Figure 4B). Also, this concentration of substance P stimulated the production of angiotensin II by a mixed population of isolated cardiac inflammatory cells (Figure 4C).

Figure 4.

Concentration-responses for histamine release by isolated cardiac MCs in response to norepinephrine (n=4) (A) and substance P (n=4) (B). Angiotensin II produced in response to stimulation with substance P (1 × 10^-4 M) by a mixed population of isolated cardiac inflammatory cells, including MCs, lymphocytes and macrophages (C). p<0.05 versus control.

Identification of α-Adrenergic Receptors on Cardiac Mast Cells

Isolated cardiac mast cells were found to not possess α-adrenergic receptors (Figure S2; please see http://hyper.ahajournals.org).

Discussion

Chronic elevation of SNS activity in the myocardium is a key component of the altered signaling pathways that accompany cardiac remodeling resulting from hypertension. While there is evidence that the SNS mediates cardiac fibrosis in hypertension via α-adrenergic receptor stimulation, ² the exact mechanisms have not been fully elucidated. However, it is likely to be more complex than direct activation of α-adrenergic receptors on cardiac fibroblasts. To this end, inflammation is also an important component in the development of hypertension-induced cardiac fibrosis. ^3-5,7,8,18 More specifically, we recently demonstrated a causal role for MCs in regulating myocardial cytokines, macrophage recruitment and development of fibrosis in the hypertensive heart. ⁶ Given the critical roles of both SNS up-regulation and MCs in hypertension-induced myocardial remodeling, the current study aimed to explore the effects of surgical sympathectomy on cardiac MCs as well as specific cytokines in the hypertensive heart.

Following sympathectomy in SHR, we observed a dramatic almost 5-fold increase in cardiac MC density. While surgical removal of the superior cervical ganglion likely does not result in complete denervation of the heart, nerve fibers from this ganglion do project to the heart ¹⁹, and this striking change in MC density clearly demonstrates an impact of its removal on the heart. This same relationship is not present in the normal heart since such a change did not occur in sympathectomized WKY. Facoetti et al. ²⁰ also found cardiac MC density to be unchanged in normal Sprague-Dawley rats following 10 weeks of chemical sympathectomy induced by 6-hydroxydopamine. These disparate effects on MC density are likely indicative of underlying differences in the local environment of normal versus hypertensive hearts. That fibrosis was attenuated in sympathectomized SHR despite MC density being elevated, suggests that either sympathectomy altered MCs to that of a non-fibrotic phenotype, or alternatively, MCs signal the SNS, but can no longer induce a pro-fibrotic response from the damaged SNS. Interestingly, in our isolated cardiac MC experiments, incubation with norepinephrine failed to induce degranulation, and we were unable to detect α-adrenergic receptors on cardiac mast cells using flow cytometry. This is consistent with a previous report that norepinephrine was not the mechanism by which contact with sympathetic neurons decreased membrane resistance in immortalized RBL-2H3 cells, a widely used model for MCs. ¹³ However, some afferent nerve fibers projecting from the heart, travel through the superior cervical ganglia, ²¹ and substance P, which is primarily located in afferent nerve fibers, has been demonstrated to be present in superior cervical ganglia. ²² Substance P-containing nerves are associated with numerous areas of the heart including the ventricle, atria, valves and connective linings. ²³ Here we show that substance P was a strong inducer of isolated cardiac MC degranulation, consistent with what we have demonstrated previously ¹⁵ and suggestive of afferent activation of cardiac mast cells.

Suzuki et al., ²⁴ using a co-culture system, showed that RBL-2H3 mast cells activated sympathetic neurons following IgE stimulation. Activation of sympathetic neurons did not occur if RBL-2H3 cells were not present. The most obvious mechanism for MC activation of neurons to occur is through release of MC tryptase acting on protease activated receptor-2 (PAR-2). Steinhoff et al. ²⁵ recently detected PAR-2 on a large number of neurons containing substance P, and activation of these receptors by tryptase in dorsal horn slices initiated the release of substance P. However, cardiac MCs are also capable of producing renin and are, assumed to be involved in the local generation of angiotensin II in the heart. ^12,26,27 Silver et al. ¹² postulated that MC generated angiotensin II could then activate nerves through angiotensin receptors. We isolated a mixed population of inflammatory cells including lymphocytes, macrophages and MCs from rat hearts and found that incubation with substance P resulted in angiotensin II production. Thus, while in sympathectomized SHR, superficially it appears that the SNS is regulating cardiac MC density, MCs may actually be upstream of SNS activation and, therefore, responding to an underlying stimulus (e.g., increased wall stress) by unsuccessfully attempting to up-regulate SNS activity. That is, the increase in MC density may be an attempt to achieve increased SNS activity that cannot be elicited because of the sympathectomy. Therefore, we speculate, that a self-perpetuating relationship may exist, where cardiac MC tryptase could activate PAR-2 on afferent nerve fibers causing the release of substance P, which in turn induces angiotensin II production by inflammatory cells, that then acts on fibroblasts and also sympathetic nerves to release norepinephrine (Figure 5). Angiotensin II has been shown to induce myocardial necrosis through stimulation of sympathetic neurons. ^28,29 In fact, a similar situation has been described in the rat knee joint, where it appears that sensory nerve activation of mast cells led to stimulation of the SNS and subsequently plasma extravasion. ³⁰ Elaboration of norepinephrine by the SNS in response to stimulation by MCs would explain how α-adrenergic receptors mediate fibrosis ² when cardiac MCs do not degranulate in response to norepinephrine in our study. This may also explain why sympathectomy does not result in increased cardiac MC density in the normal heart where there is no underlying pathologic stimulus driving MCs to up-regulate sympathetic activity.

Figure 5.

Schematic depicting a possible series of events in the development of fibrosis in the hypertensive heart. Cardiac mast cell tryptase may activate protease activated receptor-2 (PAR-2) on afferent nerve fibers resulting in the release of substance P, which in turn acts on the neurokinin-1 (NK-1) receptor to induce production of angiotensin II by inflammatory cells. Angiotensin II, in addition to activating fibroblasts, may also stimulate efferent nerves to produce norepinephrine, in turn further stimulating fibroblasts to produce collagen.

With this clear SNS-MC interaction, we sought to determine the effect of sympathectomy on selected myocardial cytokines that we have previously shown to be regulated, either directly or indirectly, by MCs in the hypertensive heart. ⁶ Both IFN-γ and IL-4 were elevated in the SHR, with sympathectomy normalizing IFN-γ, but not IL-4. IFN-γ is a marker of TH₁ T cell phenotype ³¹ and its elevation in hypertension is consistent with the supposition advanced by Yu et al. ⁹ that TH₁ cells may be pro-fibrotic. Interestingly, while sympathectomy had no effect on IFN-γ in the WKY, sympathectomy increased IL-4 levels in the WKY. Combined with our previous studies, ⁶ it would appear that, in the normal heart, the SNS has a role in regulating IL-4, but that role is shifted to MCs in the hypertensive heart. Although the levels of IL-6 and IL-10 were lower in the SHR myocardium compared to the WKY, sympathectomy returned them to normal. What is intriguing about these results is that, with the exception of IL-4, these changes are almost identical to what we have observed previously in the SHR following MC stabilization. ⁶ This similarity, together with the close spatial relationship between the nervous system and MCs, ^11,12 is more evidence of an interplay between the SNS and MCs in the regulation of the inflammatory process in the hypertensive heart.

In addition to attenuating cardiac fibrosis, sympathectomy in the SHR also prevented myocardial hypertrophy. However, our previous finding that MC stabilization did not prevent hypertrophy in the hypertensive heart indicates that MCs are not involved in this modulation of hypertrophy.⁶ Instead, rather than acting via inflammatory cells to induce hypertrophy, the SNS most likely has direct effects on cardiomyocytes via β-adrenergic receptors. While it cannot be ruled out that the changes in these parameters and the assayed cytokines were the result of the sympathectomy-related attenuation of blood pressure, we believe the following considerations support the argument that the changes in cytokines and fibrosis were due to direct interactions between the SNS and MCs. Firstly, attenuation of hypertrophy by sympathectomy has been shown to be independent of systolic blood pressure, ^2,32 and Perlini et al. ² further demonstrated that chemical sympathectomy in hypertensive rats prevented cardiac fibrosis independent of blood pressure. Secondly, cardiac MC density was dramatically with sympathectomy in the SHR but not in the normal non-hypertensive heart. Thirdly, our in vitro studies demonstrate that substance P, found in afferent nerves, can stimulate cardiac MC activation and angiotensin II production from a mixed population of cardiac inflammatory cells. Finally, the cytokine profile reported herein following sympathectomy in the SHR is almost identical to that following MC stabilization in the SHR where blood pressure was unaffected. ⁶

Perspectives

The results of this study reiterate the importance of the SNS in the development of myocardial remodeling and provide evidence of a link between the neurohormonal and inflammatory paradigms of hypertension, by demonstrating the importance of SNS interactions with cardiac MCs and cytokine production. Furthermore, even though a number of inflammatory cell types have been implicated in the development of fibrosis, ^7,9,10 our findings are consistent with the hypothesis that MCs not only trigger and orchestrate inflammatory responses, ³³ but are also responsible for cardiac fibrosis. It is important to note that not all of the cardiac remodeling effects of the SNS are mediated via mast cells. Clearly hypertrophy does not appear to involve SNS interactions with mast cells, but instead is likely the result of direct stimulation of cardiomyocyte β-adrenergic receptors. The findings herein indicate the need for further investigation regarding the mechanisms mediating SNS, inflammatory cell and afferent nerve interactions in the hypertensive heart, with particular focus on the role of substance P.