Angiotensin II–Triggered p44/42 Mitogen-Activated Protein Kinase Mediates Sympathetic Excitation in Heart Failure Rats
Angiotensin II (Ang II), acting via angiotensin type 1 receptors in the brain, activates the sympathetic nervous system in heart failure (HF). We reported recently that Ang II stimulates mitogen-activated protein kinase (MAPK) to upregulate brain angiotensin type 1 receptors in HF rats. In this study we tested the hypothesis that Ang II–activated MAPK signaling pathways contribute to sympathetic excitation in HF. Intracerebroventricular administration of PD98059 and UO126, 2 selective p44/42 MAPK inhibitors, induced significant decreases in mean arterial pressure, heart rate, and renal sympathetic nerve activity in HF rats, but had no effect on these variables in sham-operated rats. Pretreatment with losartan attenuated the effects of PD98059. Intracerebroventricular administration of the p38 MAPK inhibitor SB203580 and the c-Jun N-terminal kinase inhibitor SP600125 had no effect on mean arterial pressure, heart rate, or renal sympathetic nerve activity in HF. The phosphatidylinositol 3-kinase inhibitor LY294002 induced a small decrease in mean arterial pressure and heart rate but no change in renal sympathetic nerve activity. Immunofluorescent staining demonstrated increased p44/42 MAPK activity in neurons of the paraventricular nucleus of the hypothalamus of HF rats, colocalized with Fra-like activity (indicating chronic neuronal excitation). Intracerebroventricular PD98059 and UO126 reduced Fra-like activity in the paraventricular nucleus of the hypothalamus neurons in HF rats. In confirmatory acute studies, intracerebroventricular Ang II increased mean arterial pressure, heart rate, and renal sympathetic nerve activity in baroreceptor-denervated rats and Fra-like immunoreactivity in the paraventricular nucleus of the hypothalamus of neurally intact rats. Central administration of PD98059 markedly reduced these responses. These data demonstrate that intracellular p44/42 MAPK activity contributes to Ang II–induced neuronal excitation in the paraventricular nucleus of the hypothalamus and augmented sympathetic nerve activity in rats with HF.
- angiotensin II
- renal sympathetic nerve activity
- heart failure
- paraventricular nucleus of hypothalamus
Increased renin-angiotensin system activity and enhanced sympathetic excitation account for the predominant manifestations of chronic heart failure (HF).1,2 These mechanisms originally serve as compensation for reduced cardiac function but eventually contribute to left ventricular (LV) deterioration and the progression of HF.3,4 The sustained sympathetic excitation in HF is likely multifactorial, involving blunted inhibitory reflexes, augmented excitatory reflexes, and an excess of circulating neuroactive substances (angiotensin, aldosterone, and cytokines). Data from our laboratory5 and others6,7 suggest a prominent role for angiotensin II (Ang II). Recent work in our laboratory suggests that Ang II binding to the angiotensin type 1 receptor (AT1-R) activates intracellular mitogen-activated protein kinase (MAPK) signaling pathways to upregulate AT1-R expression in cardiovascular regions of the brain,8 setting the stage for increased Ang II–mediated sympathetic drive.
Three major terminal effector kinases of the MAPK family are the p44/42 MAPK (also called extracellular signal-regulated protein kinases, ERK 1/2), the stress-activated protein kinase/c-Jun NH2-terminal kinases (JNKs), and the p38 MAPK.9 Ang II–induced activation of these MAPK-dependent signaling pathways has been implicated in myocardial hypertrophy10 and inflammation11 in the periphery, as well as neurotransmitter catecholamine synthesis and release in the brain.12 Ang II also activates phosphatidylinositol 3-kinase (PI3K), which is involved in cell growth and proliferation in vascular smooth muscle cells13 and norepinephrine regulation in the brain of the spontaneously hypertensive rat.14 In addition, MAPK can be activated by other factors, such as proinflammatory cytokines15 and reactive oxygen species.16
The aim of the present study was to determine whether Ang II–triggered MAPK and PI3K signaling pathways in the brain contribute to sympathetic excitation in rats with chronic HF. We examined the effects of centrally administered selective kinase inhibitors on renal sympathetic nerve activity (RSNA) and lumbar sympathetic nerve activity (LSNA) and on Fra-like (Fra-LI) activity, a marker of neuronal excitation,17 that has been used to identify chronically activated neurons in the paraventricular nucleus of the hypothalamus (PVN).18 Additional acute studies were performed to more directly assess the role of p44/42 MAPK in Ang II–driven activation of the PVN and sympathetic nerve activity. The PVN was chosen for study because it is a well-known source of presympathetic neurons regulating sympathetic outflow.19,20
Materials and Methods
Adult male Sprague-Dawley rats, weighing 275 to 325 g, were obtained from Harlan Sprague Dawley. The animals were housed in temperature-controlled (23±2°C) rooms at the University of Iowa Animal Care Facility and exposed to a normal 12-hour light-dark cycle. They were provided with rat chow ad libitum. These studies were performed in accordance with the American Physiological Society Guiding Principles for Research Involving Animals and Human Beings.21 The experimental procedures were approved by the University of Iowa Institutional Animal Care and Use Committee.
Chronic HF Studies
Four weeks after coronary ligation to induce HF or a sham operation, rats were reanesthetized and instrumented for acute electrophysiological and hemodynamic recording studies during intracerebroventricular (ICV) drug administration. At least 30 minutes after completion of the surgical preparations, arterial pressure (AP), heart rate (HR), and RSNA or LSNA were recorded for 15 minutes at baseline and then continuously during ICV drug administration (40 μL/h for 1 hour).
RSNA, AP, and HR were recorded in sham-operated (SHAM) and HF rats treated with ICV vehicle (VEH) or the selective p44/42 MAPK inhibitors PD98059 (20 μmol/L) or UO126 (20 μmol/L). Additional HF rats were treated with the p38 MAPK inhibitor SB203580 (100 μmol/L), the JNK inhibitor SP600125 (100 μmol/L), the PI3K inhibitor LY294002 (100 μmol/L), or PD98059 (20 μmol/L) administered 15 minutes after ICV injection of the AT1-R blocker losartan (1 mmol/L). LSNA, AP, and HR were recorded only in HF rats treated with ICV PD98059 (20 μmol/L) or UO126 (20 μmol/L).
At the completion of these terminal recording sessions, some rats were transcardially perfused with 4% paraformaldehyde, and the brains were harvested for immunohistochemical and immunofluorescent studies. These included VEH-treated SHAM rats and HF rats treated with ICV VEH, PD98059, UO126, SP600125, SB203580, and LY 294002.
To determine whether p44/42 MAPK mediates Ang II–induced activation of RSNA, animals were anesthetized to record RSNA, AP, and HR during ICV injection of Ang II (100 ng/kg) before and after a 1-hour ICV infusion of the p44/42 MAPK inhibitor PD98059 (20 μmol/L, 40 μL/h). These studies were conducted in baroreceptor-denervated rats to eliminate the confounding effects of baroreceptor-mediated changes on RSNA and HR.22 The recordings were carried out ≥2 hours after completion of the surgical preparation. Thirty minutes was allowed after the initial ICV Ang II injection for all of the variables to return to baseline. The second Ang II injection was made immediately after discontinuation of the ICV infusion of PD98059.
Additional studies were performed in anesthetized neurally intact rats to determine whether p44/42 MAPK mediates Ang II–induced activation of PVN neurons. These rats received a 1-hour ICV infusion of VEH, Ang II (10 ng/μL, 40 μL/h), or PD98059 (20 μmol/L, 40 μL/h) combined with Ang II (10 ng/μL, 40 μL/h). At the completion of the experiment, the brains were fixed with 4% paraformaldehyde and harvested and processed for Fra-LI immunoreactivity in PVN by immunofluorescence.
Please see the data supplement, available online at http://hyper. ahajournal.org, for specific methods.
Electrophysiological and hemodynamic data were analyzed with Spike2 software. In studies of chronic HF or SHAM rats, responses of mean AP (MAP; millimeters of mercury), HR (bpm), RSNA, and LSNA (millivolts) sampled over 5-minute intervals during ICV infusion were compared with baseline values averaged over 5-minute intervals immediately preceding each intervention. RSNA and LSNA responses are reported as the percentage changes in integrated voltage (millivolts) from baseline. In the acute studies, variables were sampled over 1-minute intervals. All of the values are expressed as the means±SEMs. The significance of differences among groups was analyzed by 2-way, repeated-measure ANOVA followed by posthoc Fisher’s least significant difference test. Echocardiographic parameters were analyzed using 1-way ANOVA followed by Fisher’s least significant difference test. For immunohistochemical and immunofluorescent analysis, data were represented as positive cells per 104 μm2 and analyzed using 1-way ANOVA followed by Fisher’s least significant difference test. The correlation coefficient of p44/42 active neurons and Fra-LI–positive neurons was determined by regression analysis using SigmaPlot software. Differences were considered significant at P<0.05.
Chronic HF Studies
Echocardiographic, Hemodynamic, and Anatomic Assessment of HF
The HF animals assigned to each group for treatment with MAPK inhibitors and PI3K inhibitor versus VEH were well matched with regard to echocardiographically defined LV function (Table S1). The estimated size of the ischemic zone and the LV ejection fraction were not different in HF rats assigned to the different treatment groups. Compared with SHAM rats, LV ejection fraction was reduced and LV end-diastolic volume was increased in the rats with HF (Table S1).
Four weeks after myocardial infarction, LV peak systolic pressure and maximum rate of rise of LV pressure were lower and LV end-diastolic pressure was higher in VEH-treated HF rats than SHAM rats. The right ventricular weight:body weight and lung weight:body weight ratios were significantly higher in VEH-treated HF compared with SHAM rats. Neither the MAPK inhibitors PD98059, SP600125, and SB203580, nor the PI3K inhibitor LY294002 affected these variables. (Table S1).
Effects of ICV p44/42 MAPK Inhibitor PD98059 on Hemodynamics and Sympathetic Nerve Activity
In SHAM rats (n=7), ICV infusion of PD98059, a selective p44/42 MAPK inhibitor, had no effect on MAP, HR, or RSNA (Figure 1A and 1D). In HF rats (n=8), however, ICV infusion of PD98059 substantially reduced MAP, HR, and RSNA (Figure 1B and 1D). The maximum responses occurred 30 to 40 minutes after starting the ICV administration of PD98059. MAP decreased from 99.6±3.7 to 87.8± 3.1 mm Hg, HR decreased from 367.3±6.8 to 350.0±5.8 bpm, and renal RSNA decreased by 37.2±5.3%. ICV administration of PD98059 had no effect on LSNA in the HF rats (Figure S1).
Pretreatment of HF rats with ICV AT1-R antagonist losartan significantly attenuated the inhibitory effects of PD98059. In losartan-pretreated HF rats (n=7), ICV PD98059 induced a maximum response 30 to 40 minutes after injection. MAP decreased from 94.8±2.6 to 88.6±2.5 mm Hg, HR decreased from 343.7±4.3 to 335.1±4.1 bpm, and RSNA decreased by 16.8±2.5% (Figure 1C and 1D).
With both treatment protocols, MAP, HR, and RSNA remained below baseline levels 30 minutes after the PD98059 infusion was stopped.
Effects of ICV p44/42 MAPK Inhibitor UO126 on Hemodynamics and RSNA
Similar to PD98059, ICV administration of UO126, another selective p44/42 MAPK inhibitor, induced a significant reduction in these variables in HF rats (n=8). The maximum changes compared with the baseline in MAP (−10.0±2.3 mm Hg), HR (−15.4±3.2 bpm), and RSNA (−29.8±6.2%) occurred ≈40 minutes after initiating the ICV infusion (Figure 2B and 2C). MAP, HR, and RSNA remained below baseline levels 30 minutes after the infusion was stopped. In SHAM rats (n=7), ICV administration of UO126 had no obvious effect on MAP, HR, or RSNA (Figure 2A and 2C).
Effects of ICV p38 MAPK, JNK, and PI3K Inhibitors on Hemodynamics and RSNA
ICV administration of the VEH (n=7), the p38 MAPK inhibitor SB203580 (n=7), and c-Jun N-terminal kinase inhibitor SP600125 (n=7) had no effect on MAP, HR, or RSNA in HF rats (Figure 3A, 3B, 3C, and 3E). ICV injection of the PI3K inhibitor LY294002 (n=8) induced a mild but significant decrease in MAP and HR 40 to 60 minutes after initiating the ICV administration but had no effect on RSNA in HF rats (Figure 3D and 3E). Unlike the responses to PD98059 and UO126, these variables had returned to baseline level 20 minutes after the injection of LY294002. Because there was no apparent effect on RSNA seen in HF rats, no SHAM rats were tested with these inhibitors.
Effects of ICV MAPK and PI3K Inhibitors on Fra-LI Activity in PVN Neurons
In VEH-treated HF rats (n=7) compared with SHAM rats (n=6), Fra-LI immunoreactivity was significantly increased in the dorsal parvocellular (PVN-dp), the ventrolateral parvocellular (PVN-vlp), and the posterior magnocellular (PVN-pm) subdivisions of PVN23 (Figure 4A and 4B). The PVN-vlp and PVN-pm had a higher density of Fra-LI immunoreactivity than PVN-dp in both SHAM and HF rats. After treatment with p44/42 inhibitors PD98059 (n=7) or UO126 (n=6), the number of Fra-LI positive neurons in PVN-dp, PVN-vlp, and PVN-pm was greatly decreased in HF rats (Figure 4A and 4B). ICV SP600125 (n=6), SB203580 (n=6), or LY294002 (n=6) also inhibited Fra-LI immunoreactivity in the PVN-dp, PVN-vlp, and PVN-pm but to a lesser degree than p44/42 MAPK inhibitors (Figure 4A and 4B).
Relationship Between Phosphorylated p44/42 MAPK and Fra-LI Activity in PVN Neurons
Confocal immunofluorescent images revealed an increase in p44/42 MAPK activity, indicated by the enhanced expression of phosphorylated p44/42 MAPK in the PVN (Figure 5A and 5B) in HF (n=6) compared with SHAM rats (n=6). The density of phosphorylated p44/42 MAPK-like immunoreactivity was higher in PVN-vlp and PVN-pm than in PVN-dp in both SHAM and HF rats. The numbers of neurons in which p44/42 MAPK colocalized with Fra-LI activity were also increased in all 3 of the subdivisions of PVN in HF compared with SHAM rats (Figure 5A and 5B). Phosphorylated p44/42 MAPK-like immunoreactivity colocalized with Fra-LI positivity in PVN neurons to a similar extent (>90%) in both SHAM and HF rats, but there were more double-labeled neurons in the PVN-vlp than in the PVN-pm or PVN-dp (Figure 5A and 5B). Regression analysis indicated that Fra-LI positivity correlated with p44/42 MAPK activity in PVN neurons in both SHAM and HF rats (Figure 5C).
Effects of PD98059 on Ang II–Induced Pressor Responses in Baroreceptor-Denervated Rats
In baroreceptor-denervated rats (n=7), ICV administration of Ang II elicited significant increases in MAP, HR, and RSNA (Figure 6A and 6C). The maximum increases in MAP (26.8±3.7 mm Hg, from baseline 101.6±4.5 mm Hg), HR (26.7±4.2 bpm, from baseline 350.1±12.8 bpm), and RSNA (22.2±3.6%) occurred 2 to 3 minutes after ICV injection of Ang II. Pretreatment with PD98059 greatly attenuated ICV Ang II–induced pressor responses in MAP, HR, and RSNA (Figure 6B and 6C).
Effects of PD98059 on Ang II–Induced Fra-LI Activity in PVN in Neurally Intact Rats
Confocal immunofluorescent images revealed that the number of Fra-LI–positive neurons in the 3 subdivisions of PVN analyzed, PVN-dp, PVN-vlp, and PVN-pm regions, was significantly enhanced in the rats treated with ICV Ang II (n=6) compared with ICV VEH (n=6; Figure 7A, 7B, and 7D). The increased Fra-LI activity in all 3 of the subdivisions of PVN induced by ICV administration of Ang II was attenuated by ICV p44/42 MAPK inhibitor PD98059 (n=6; Figure 7B, 7C, and 7D).
This study reveals an important and previously unrecognized functional role for the intracellular MAPK pathway. We found that p44/42 MAPK mediated Ang II–induced influences on sympathetic nerve activity and hemodynamics in HF. The data presented here are the first evidence of a critical role for the p44/42 MAPK signaling cascade in the maintenance of renal sympathetic excitation in HF rats.
In the present study, central administration of 2 different selective p44/42 MAPK inhibitors had the same effect in anesthetized rats with HF, a dramatic reduction in HR, AP, and RSNA. In contrast, in sham animals, neither of these inhibitors affected any of the measured variables. Thus, p44/42 MAPK has no apparent role in the maintenance of RSNA under basal conditions, even under the stress of anesthesia, but is a key factor mediating the augmented RSNA characteristic of HF. These profound differences in the responses of HF and SHAM rats are unlikely to be explained by differential effects of urethane anesthesia, but we cannot exclude that possibility.
Consistent with their effects on peripheral sympathetic nerve activity in this study, the p44/42 inhibitors also reduced neuronal excitation, as indicated by Fra-LI activity, in the PVN of HF rats. It is believed that parvocellular neurons of PVN, particularly the neurons located in the ventrolateral parvocellular subdivision that project to the spinal cord and the rostral ventrolateral medulla (RVLM),24,25 play an essential role in regulating sympathetic outflow. This study demonstrated colocalization of p44/42 MAPK in Fra-LI–positive PVN neurons, including neurons in the ventrolateral parvocellular subdivision, and a reduction in Fra-LI–positive PVN neurons in HF rats treated with the p44/42 MAPK inhibitors. These immunohistochemical data strongly support the interpretation that p44/42 MAPK signaling contributes to activation of PVN neurons and sympathetic excitation in HF. However, PVN is not the only nucleus in the brain containing presympathetic neurons. p44/42 MAPK may also mediate the effects of Ang II on presympathetic neurons in RVLM or neurons in other cardiovascular and autonomic centers, including the subfornical organ, median preoptic nucleus and organum vasculosum of the lamina terminalis,26 which impinge on presympathetic neurons.
Ang II may be the primary stressor driving the p44/42 MAPK-mediated renal sympathetic excitation in HF. In HF rats, the effect of the p44/42 MAPK inhibitor is markedly diminished after pretreatment with an AT1-R antagonist. The acute studies in normal rats support this suggestion: both the pressor responses and the Fra-LI activity elicited by ICV Ang II were greatly attenuated by the p44/42 MAPK inhibitor. This concept is consistent with the studies of others, demonstrating that p44/42 MAPK played an important role in maintaining high blood pressure in chronic angiotensin-infused rats27 and that bilateral injection of PD98059 into the RVLM abolished the pressor response to exogenous Ang II in the RVLM in spontaneously hypertensive rats and Wistar-Kyoto rats.28 It is also consistent with the previous observation that AT1-R in the RVLM seems to have little impact on sympathetic drive under normal conditions but has prominent influence on sympathetic drive under conditions of stress.29 However, Ang II may not be the only signal activating MAPK in HF. Pretreatment with ICV losartan significantly attenuated but did not completely block the inhibitory effects of the p44/42 inhibitor PD98059. A recent study demonstrated a possible reactive oxygen species–dependent intracellular MAPK pathway in RVLM neurons.30
How Ang II–driven MAPK activity elicits an increase in sympathetic drive remains a mystery. MAPK may regulate sympathetic nerve activity by modulating ion channel activity31 in presympathetic neurons. However, the time course of the change in RSNA in response to the p44/42 MAPK inhibitors suggests a longer-term modulating effect. The sustained augmentation of RSNA in HF2 is more likely related to mechanisms that regulate gene expression and proteins synthesis. These might include sodium or potassium ion channel protein subunits or angiotensin receptors. In related work,8 we found that activated p44/42 MAPK upregulated AT1-R in the forebrain PVN and subfornical organ in HF. Increased Ang II binding to AT1-R might then account for an increase in sympathetic excitation. The MAPK signaling cascade may also affect the synthesis and release of certain key neurotransmitters, such as norepinephrine and dopamine, in presympathetic regions of the brain.12 Finally, p44/42 MAPK activity is linked to reactive oxygen species32,33 and proinflammatory cytokines,34 which are also stimulated by Ang II. A growing body of evidence indicates that reactive oxygen species increase sympathetic excitation.35
Other kinases in the MAPK family, such as p38 MAPK and JNK, are activated in HF,8 but inhibiting these kinases did not affect RSNA even at a dose 5-fold higher than the p44/42 inhibitors. Similarly, blockade of PI3K activation by its inhibitor LY294002 did not significantly change RSNA in HF. These findings suggest that p44/42 MAPK has an independent and distinctive influence on renal sympathetic outflow in HF.
The p44/42 MAPK inhibitors had no effect on LSNA in the HF rats, although both HR and MAP were reduced (Figure S1). This observation suggests a relative selectivity of the p44/42 MAPK activity in mediating sympathetic discharge. A preferential amplification of renal sympathetic discharge, affecting sodium and water excretion and renin release, might be expected in HF. A similar MAPK-dependent differential regulation is found in the regional sympathetic response to insulin36: insulin-induced sympathetic activation to brown adipose tissue is mediated by MAPK, whereas the insulin-induced enhanced lumbar nervous sympathetic activity is mediated by PI3K. Neither MAPK nor PI3K was implicated in the insulin-mediated activation of renal or adrenal sympathetic nerve activity.36
The AP responses of the HF rats to the p44/42 MAPK inhibitors are very consistent with the findings from a previous study28 in spontaneously hypertensive rats, showing that bilateral microinjection of PD98059 into the RVLM markedly decreased AP. However, the results in normal control rats are conflicting. Microinjection of PD98059 in RVLM lowered AP in Wistar-Kyoto rats,28 whereas we found no effect in sham Sprague-Dawley rats. It should be noted, however, that the measurements from the Wistar-Kyoto rats28 were acquired during a 14-hour infusion of PD98059. At 15 minutes after initiating the infusion, blood pressure was not significantly decreased; the significant change in blood pressure was observed 3 hours into PD98059 administration. Our results in sham-operated Sprague-Dawley rats showed no effect of PD98059 after a 1-hour infusion, a time point not measured in that study. We observed a mild decrease in AP and HR in HF rats with the PI3K inhibitor LY294002, less dramatic than the results reported with a different PI3K inhibitor in the spontaneously hypertensive rats,28 confirming some role for PI3K in cardiovascular regulation. Differences in the results of these 2 studies may reflect differences in rat strains, experimental conditions, and protocols.
The present study demonstrates for the first time that the intracellular p44/42 MAPK signaling pathway plays a pivotal role in mediating the effects of the brain renin-angiotensin system on sympathetic nerve activity in HF. Because inhibition of p44/42 MAPK affects renal but not LSNA in HF rats, the central p44/42 MAPK signaling pathway may have a predominant influence on the regulation of renal function. These findings suggest that manipulating brain p44/42 MAPK signaling can ameliorate the adverse effects of the brain renin-angiotensin system on cardiovascular and renal function during the progression of HF.
Sources of Funding
This work was supported by a Merit Review award (to R.B.F.) from the Department of Veterans Affairs, by HL-073986 (to R.B.F.) from the National Institutes of Health, and by institutional funds provided by the University of Iowa.
- Received January 15, 2008.
- Revision received February 3, 2008.
- Accepted May 19, 2008.
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