Author + information
- Received April 16, 2013
- Revision received June 19, 2013
- Accepted July 1, 2013
- Published online December 24, 2013.
- Rodrigo Del Rio, PhD,
- Noah J. Marcus, PhD and
- Harold D. Schultz, PhD∗ ()
- Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska
- ↵∗Reprint requests and correspondence:
Dr. Harold D. Schultz, Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5850.
Objectives This study sought to investigate whether selective ablation of the carotid body (CB) chemoreceptors improves cardiorespiratory control and survival during heart failure.
Background Chronic heart failure (CHF) is a recognized health problem worldwide, and novel treatments are needed to better improve life quality and decrease mortality. Enhanced carotid chemoreflex drive from the CB is thought to contribute significantly to autonomic dysfunction, abnormal breathing patterns, and increased mortality in heart failure.
Methods Chronic heart failure was induced by coronary ligation in rats. Selective CB denervation was performed to remove carotid chemoreflex drive in the CHF state (16 weeks post-myocardial infarction). Indexes of autonomic and respiratory function were assessed in CB intact and CB denervated animals. CB denervation at 2 weeks post-myocardial infarction was performed to evaluate whether early targeted CB ablation decreases the progression of left ventricular dysfunction, cardiac remodeling, and arrhythmic episodes and improves survival.
Results The CHF rats developed increased CB chemoreflex drive and chronic central pre-sympathetic neuronal activation, increased indexes of elevated sympathetic outflow, increased breathing variability and apnea incidence, and desensitization of the baroreflex. Selective CB ablation reduced the central pre-sympathetic neuronal activation by 40%, normalized indexes of sympathetic outflow and baroreflex sensitivity, and reduced the incidence of apneas in CHF animals from 16.8 ± 1.8 events/h to 8.0 ± 1.4 events/h. Remarkably, when CB ablation was performed early, cardiac remodeling, deterioration of left ventricle ejection fraction, and cardiac arrhythmias were reduced. Most importantly, the rats that underwent early CB ablation exhibited an 85% survival rate compared with 45% survival in CHF rats without the intervention.
Conclusions Carotid chemoreceptors play a seminal role in the pathogenesis of heart failure, and their targeted ablation might be of therapeutic value to reduce cardiorespiratory dysfunction and improve survival during CHF.
Elevated sympathetic outflow and breathing disorders are 2 hallmarks of chronic heart failure (CHF), and both have been strongly related to decreased quality of life, poor prognosis, and increased mortality (1,2). Enhanced sympathetic drive and breathing instability during CHF have been associated with alterations in peripheral and central neural pathways that regulate autonomic function and breathing control (3,4). Despas et al. (5) showed that CHF patients with high peripheral chemosensitivity displayed higher sympathetic outflow compared with CHF patients with normal chemosensitivity. In addition, impaired baroreflex function observed in CHF has been associated with an augmented peripheral chemosensitivity in patients with CHF (6). Previous studies from our laboratory have demonstrated an augmented afferent input from the carotid body (CB) chemoreceptors in pacing-induced CHF rabbits and myocardial-infarcted CHF rats and have shown that reducing chemoreflex afferent traffic results in reductions in the sympathetic drive in CHF animals (7–9). Also, transient inhibition of the CB chemoreflex with brief hyperoxic stimulation in CHF patients results in a decreased sympathetic tone (5) and an improvement in the baroreflex function (6).
Patients with CHF exhibit a high incidence (up to 60%) of breathing disorders characterized by apnea/hypopneas and higher breathing variability (10,11) that have been related to the progression of the disease (12). Exaggerated CB-mediated ventilatory responses to apneas or hypopneas could contribute to respiratory instability. We have previously shown, in support of this notion, that reductions in CB afferent activity during CHF decrease breathing variability and apnea/hypopnea incidence in rats (13).
Together these findings suggest that the CB chemoreflex sustains the cardiorespiratory dysfunction observed in CHF. Indeed, evidence showing higher mortality rates in CHF patients with high chemosensitivity compared with patients with normal chemosensitivity (3) suggests a crucial role for the CB chemoreflex in exacerbating the pathogenesis of CHF. Although a causal link between exaggerated CB chemoreflex drive and high mortality risk during CHF has not been proven, sympathoexcitation and the apnea-related hypoxemia exacerbated by the augmented chemoreflex could lead to an increased arrhythmogenesis and deterioration of cardiac function associated with a high mortality risk (2,14).
Our prior work has shown that the exaggerated chemoreflex in CHF emanates from elevated tonic afferent nerve traffic from the CB (7–9). Recently, it has been proposed that targeting the CB by denervation of the afferent inputs might be beneficial in cardiovascular diseases exacerbated by sympathetic hyperactivity (15). The impact of carotid body denervation (CBD) on autonomic function and survival during CHF has not been studied before and could represent a novel strategy to slow the progression of cardiac deterioration and lower mortality rates in CHF. In this study, we asked whether CBD improves autonomic balance and breathing regularity during CHF and whether CBD in the early stage of cardiac dysfunction reduces cardiac remodeling and arrhythmia incidence and increases survival of myocardial-infarcted CHF rats.
Induction of heart failure
Seventy-one (2-month-old) male Sprague-Dawley rats, weighing between 430 and 560 g, were studied. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center and were carried out under the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1996). Chronic heart failure (CHF) was produced by coronary artery ligation (CAL) as previously described (13).
Selective CB denervation
At 16 weeks (Protocol 1) or 2 weeks (Protocol 2) post-CAL surgery, the rats underwent cryogenic destruction of the CBs (16). Graphic timelines of the protocols are provided in Online Figure 1. We found that this surgical approach allows the elimination of the CB chemosensory, but not the carotid baroreceptor afferents (Online Figs. 2 and 3). The CBD did not affect water consumption or daily food intake (Online Fig. 4). The sham CBD surgery performed in sham and CHF rats showed no cardiorespiratory effects (Online Figs. 5 to 11).
Cardiac function was determined by echocardiography (Vevo 770, Visualsonics, Inc., Toronto, Ontario, Canada) as previously described (13,17). M-mode tracings were recorded through the anterior and posterior left ventricular (LV) walls, and anterior and posterior wall thicknesses (end-diastolic and end-systolic) and LV internal dimensions were measured. Rats with ejection fractions (EFs) of <45% were considered to be in CHF (13,17).
Radiotelemetric monitoring of arterial blood pressure and heart rate
At 14 weeks, rats were chronically implanted with a radio-telemetry pressure transducer (TA11PA-C40, DSI, St. Paul, Minnesota) with a catheter directed into the abdominal aorta. Blood pressure (BP) and heart rate were acquired in conscious resting state.
Heart rate variability (HRV) and the low-frequency component of the systolic blood pressure variability (LF-SBPV) were assessed as indirect measures of autonomic balance (18) with power spectral analysis (19,20). Spontaneous baroreflex sensitivity was assessed by spectral calculation (21).
Irregular heartbeats were visually inspected from heart rate time series (22). Arrhythmic episodes were defined as premature or delayed beats with changes >3 SDs from the mean beat-to-beat interval duration and reported as events/h.
Evaluation of respiratory variability and ventilatory chemoreflex function
Tidal volume (Vt), respiratory frequency, and minute ventilation (Vt × respiratory frequency) were determined by whole-body plethysmography as previously described (13). Respiratory stability was assessed from resting breathing recordings by Poincaré plots and analysis of SD1 and SD2 of the interbreath interval variability (13). Apnea and hypopnea incidence (cessation or ≥50% reduction in Vt over ≥3 consecutive breaths) was counted and reported as apnea and hypopnea index (events/h). Peripheral chemoreceptors were stimulated preferentially by allowing the rats to breathe hypoxic gas (10% oxygen/balance nitrogen).
Neuronal activation in the rostral ventrolateral medulla (RVLM) was assessed by immunoblot of the fos-related antigen 1 (Fra-1) (1:100, Santa Cruz Biotechnology, Dallas, Texas) in RVLM micropunches as previously described (18). Fra-1 expression is induced during sustained neuronal activation and thus serves as a sensitive marker of chronically activated brainstem neurons (23). The relative amount of protein of interest was calculated as the ratio of intensity of the band relative to the intensity of the housekeeping gene β-actin.
Localization of Fra-1 immunoreactivity in RVLM catecholaminergic pre-sympathetic neurons was assessed in 4% paraformaldehyde-fixed coronal sections (15 μm) of the brain. The RVLM catecholaminergic pre-sympathetic neurons were identified by immunoreactivity to tyrosine hydroxylase. Sections were visualized with a confocal laser microscope.
Mortality and cardiac remodeling studies
In a subset of animals, we assessed the effects of early carotid body denervation (eCBD) (2 weeks after CAL surgery) on survival rate and cardiac remodeling during CHF (Protocol 2) (Online Fig. 1). Echocardiography and survival rates were then followed through the remaining 14 weeks of the protocol. At the end of the study, the heart was removed and processed for histological quantification of tissue collagen levels from both the noninfarcted LV free wall and the interventricular septum (IVS).
Data are expressed as means ± SEM. Differences among 3 or more groups were assessed with 1 or 2-way analysis of variance tests, followed by Newman-Keuls or Bonferroni post-hoc comparisons. The Student t test was employed to compare the differences between 2 groups. The log-rank test was used to compare survival rates between CHF and CHF+eCBD rats. A p value <0.05 was considered statistically significant.
Respiratory disorders and CB ablation: Protocol 1
The CHF rats displayed cardiac chamber dilation and a significant decrease in cardiac function 16 weeks post-myocardial infarction as described previously (13,18,24). Echocardiographic parameters were measured in sham and CHF rats (Online Table 1). The CHF rats exhibited reduced EF and fractional shortening (<40% of normal values) compared with noninfarcted sham rats (p < 0.05). Infarct size was 38 ± 2% of the total LV area in CHF rats (24).
Sixteen weeks after infarct surgery, rats developed a significant increase in CB chemoreflex drive as evidenced by increased normoxic ventilation (Online Table 2) and potentiation of the hypoxic ventilatory response (Figs. 1A and 1B, Online Fig. 5). The CHF rats displayed breathing disorders at rest (Figs. 2A and 2B). Breath-to-breath (SD1) and aggregate (SD2) breathing variability in CHF-rats increased 2-fold compared with the values obtained from sham rats (Figs. 2C and 2D). In addition, CHF rats displayed a greater number of apneas and hypopneas (Fig. 2E) compared with that in sham rats (16.8 ± 1.8 events/h vs. 8.0 ± 1.4 events/h, CHF vs. sham). The increased apnea index in CHF rats was not associated with post-sigh events, which remained unchanged (Online Fig. 7).
The elevated resting ventilation and the enhanced ventilatory response to hypoxia in CHF rats were markedly reduced after CBD to values similar to sham rats that underwent CBD (Fig. 1B, Online Fig. 5). Moreover, breathing variability was normalized in CHF-CBD rats (Fig. 2). After CBD, both SD1 and SD2 interbreath variability were significantly reduced (53.4% and 57.2%, respectively) compared with the values obtained before CBD in CHF rats (Figs. 2C and 2D). The CBD also significantly decreased apnea and hypopnea index in CHF rats (Fig. 2E) to the level seen in sham rats (Fig. 2E).
Cardiac autonomic control after CBD in CHF rats
Autonomic imbalance was evident in CHF rats compared with sham rats (Fig. 3). A shift in the HRV toward augmented sympathetic drive was found during CHF (Figs. 3B to 3D) as evidenced by a 2-fold increase in the low- to high-frequency ratio of the HRV compared with sham rats (Fig. 3B). A major component to this shift was a significant reduction in the parasympathetic respiratory sinus arrhythmia during CHF as evidenced by a significant decrease in the high-frequency component of the HRV (Fig. 3D).
Ablation of the CB rescued normal HRV in CHF rats (Fig. 3, Online Fig. 8). The low- to high-frequency ratio of the HRV significantly decreased from 2.0 ± 0.3 to 0.9 ± 0.2 (before and after CBD, respectively, p < 0.05) in rats with CHF. Selective CBD did not affect resting BP or heart rate in either sham+CBD or CHF+CBD rats (Online Table 2).
Carotid chemoreceptor ablation and brainstem pre-sympathetic neuronal activation during CHF
Catecholaminergic pre-sympathetic neurons (Fig. 4A) located in the RVLM represent a key locus in the central nervous system regulation of sympathetic outflow during CHF (25). After CHF, RVLM catecholaminergic neurons (tyrosine hydroxylase-positive) exhibited a 40% increase in Fra-1 immunoreactivity, a marker of chronic neural activation, compared with sham rats (Figs. 4A to 4C). By contrast, selective CBD significantly reduced Fra-1 expression in CHF rats (1.5 ± 0.1 vs. 0.9 ± 0.1 normalized unit, CHF vs. CHF+CBD) to levels comparable to the values obtained in sham rats (Fig. 4).
Sympathovasomotor tone and baroreflex gain after CBD
Sympathovasomotor tone as estimated by the LF-SBPV increased in CHF rats, which was normalized (from 18.4 ± 4.5 mm Hg2 to 7.2 ± 2.0 mm Hg2) after CBD (Online Fig. 9A). In addition, the impaired baroreflex sensitivity displayed by CHF rats was significantly improved after CBD (Online Fig. 9B).
Progression of cardiac remodeling and arrhythmogenesis after eCBD in CHF: Protocol 2
Necrotic myocytes in the infarct region were almost completely replaced with fibrotic scar tissue 16 weeks after CAL surgery in both CHF and CHF-eCBD rats (Fig. 5A). In addition, both groups exhibited nearly identical infarct sizes (Online Fig. 11). Nevertheless, we hypothesized that restoration of autonomic control and breathing regularity by eCBD, 2 weeks after CAL, would influence remodeling in the noninfarcted regions.
Noninfarcted cardiac tissue from CHF rats displayed a significant 4.5-fold increase in the LV free wall and a 3-fold increase in the IVS collagen deposition compared with sham hearts (Fig. 5B). Notably, collagen content was reduced in noninfarcted cardiac tissue from CHF-eCBD rats (Figs. 5C and 5D), which was indistinguishable from the LV free wall and IVS collagen content obtained in sham rats. Analogously, the incidence of arrhythmias was markedly increased in CHF rats compared with sham rats (Fig. 6), and CHF-eCBD decreased the arrhythmic episodes 2.5-fold compared with the CHF group (Fig. 6B).
Both CHF and CHF+eCBD groups had comparable EFs before CBD was performed at 2 weeks after CAL (Online Table 3), but the progressive deterioration of EF in CHF-eCBD rats was blunted over the remaining 14-week period compared with CHF rats (Fig. 7A). A diminished expansion of LV systolic volume accompanied the preservation of EF in CHF-eCBD rats (52.5 ± 16.6% vs. 98.0 ± 12.0% increase, CHF-eCBD vs. CHF, p < 0.05). The increase in LV diastolic volume also tended to be smaller in CHF-eCBD rats (37 ± 12% vs. 61 ± 10% increase, CHF-eCBD vs. CHF) but was not statistically significant (p = 0.14).
Most remarkably, eCBD significantly reduced the mortality rate in CHF rats (p = 0.04) over the 14 weeks post-infarct compared with CHF rats with the CB intact (85% vs. 45% survival, CHF-eCBD vs. CHF) (Fig. 7B).
The present study unveils a major role of the CB chemoreflex in the pathophysiology of heart failure. Here, we show for the first time to our knowledge that selective CBD normalizes control of breathing, reduces pre-sympathetic neuronal activation in the RVLM in the brainstem, and restores normal autonomic and baroreflex function in CHF rats. Notably, CBD performed early after myocardial infarction results in significant reductions in aberrant ventricular remodeling and incidence of arrhythmias and reduces the progressive deterioration of LV function. The outcome is that selective CBD improves survival in CHF rats.
Cardiac deterioration and mortality during the progression of heart failure
Autonomic dysfunction, breathing disorder, myocardial remodeling, and arrhythmogenesis constitute major predictors of morbidity and mortality in CHF (2,14,26–28). We have shown that CBD reduces sympathetic activation, respiratory instability, myocardial fibrosis, arrhythmias, and mortality during CHF. Moreover, our data strongly suggest that the effects of CBD are primarily associated with a normalization of altered central neural control of cardiovascular and ventilatory function arising from elevated input from the CB.
Selective CBD and cardiac remodeling during heart failure
Although the present results suggest a seminal role of the CB in cardiac remodeling and progression of the disease, the molecular signaling pathways in the heart affected by CBD remain to be elucidated. Nevertheless, we can speculate that the effects of CBD on cardiac remodeling are likely due, at least in part, to a reduced sympathetic input and increased parasympathetic input to the heart. Indeed, it is well known that maladaptive heightened sympathetic/reduced parasympathetic outflow to the heart after myocardial infarction constitutes a major component in the progression into CHF (1,3,7,29). Moreover, it has been proposed that the mechanism underlying cardiac deterioration is associated with neurohumoral activation and increased catecholamines release by cardiac sympathetic nerve terminals during augmented sympathetic drive in CHF (30,31). Also, although CBD markedly improved cardiac systolic function and survival during CHF, it did not alter LV chamber dilation and hypertrophy (Online Table 3). Clearly, other factors continue to play an important role in cardiac tissue remodeling after myocardial infarction that are not affected by CBD.
Selective CBD and sympathetic activation in heart failure
Activation of the RVLM is a critical component in the regulation of sympathetic outflow during CHF (17). We found that CBD decreases pre-sympathetic neuronal activation in the RVLM in CHF. Thus, it is reasonable to assume that CBD resulted in a general decrease in sympathetic outflow in the CHF rats, although the extent to specific vascular beds cannot be ascertained directly. However, the improvement in HRV and reduced arrhythmogenesis after CBD are consistent with a reduction in sympathetic outflow to the heart.
Besides the direct beneficial effects of CBD to reduce sympathetic activation of the heart during CHF, the reduction in sympathetic outflow might indirectly benefit the heart by impacting vascular peripheral resistance to decrease cardiac afterload and the kidneys to improve renal function and blood volume homeostasis. In support of improved afterload, we observed a trend (but not statistically significant) toward decreased BP after CBD (Online Table 4). In addition, CBD decreased LF-SBPV, suggesting that CBD might have decreased vasomotor tone in CHF rats. We did not assess renal function in this study; however, it is well known that sympathoexcitation impairs renal function in CHF (32). The impact of CBD on hemodynamic status and renal function during the progression of CHF warrants further study.
Selective CBD and breathing in heart failure
The incidence of breathing disorders is high in patients with CHF (10–12), and the incidence of periodic breathing and Cheyne-Stokes respiration is associated with a deterioration of cardiac function (12). Remarkably, CBD reduced breathing variability and diminished the occurrence of apnea episodes in CHF rats, which supports a role for the CB in the generation of abnormal breathing patterns observed during CHF. The central neural mechanisms underlying induction of breathing instability by the CB in CHF deserve further study.
Despite the current advances in the treatment of heart diseases, mortality rates during CHF are still high (33). The progressive deterioration of cardiac function during CHF encompasses complex pathophysiological mechanisms that are resistant to treatment by a single therapeutic intervention. Breathing disorders and heightened sympathetic outflow are 2 major hallmarks of CHF, and both are closely related to increased morbidity and mortality (1,2,5,6). Furthermore, cardiac remodeling and increases in cardiac arrhythmogenesis are both recognized to contribute to the progression of CHF. Our current findings indicate that targeted ablation of the CB is a potentially valuable therapeutic strategy that can effectively reverse autonomic and respiratory dysfunction and improve survival of CHF. Excitingly, a recent case report presented by Niewiński et al. (34) showed that surgical removal of the CB from a patient with systolic heart failure significantly decreased sympathetic tone. Thus, our results showing the more extensive beneficial effects of ablation of the CB should be relevant and potentially transferrable to humans.
The present study reports a major role of the CB chemoreflex in the cardiorespiratory alterations after CHF and shows that selective ablation of the CB effectively restores normal control of breathing and autonomic and baroreflex function during CHF. Furthermore, when performed earlier during the progression of CHF, selective CB ablation improves survival. Taken together, targeted CBD shows promise as a novel therapeutic strategy to improve autonomic control of the heart, decrease cardiac remodeling, reduce the incidence of arrhythmias, and increase life span in heart failure patients. Additionally, selective CB ablation might also have important implications for the treatment of breathing abnormalities during heart failure.
The authors wish to thank Dr. Kurtis G. Cornish for his surgical assistance and Mary Ann Zink, Francisca Allendes, Johnnie F. Hackley, and Richard Robinson for their technical assistance.
This work was supported by a Program Project Grant from the Heart, Lung and Blood Institute of the National Institutes of Health (PO1-HL62222). The authors have no conflict of interest to disclosure with regard to funding or compensation from industry for this study. However, all authors collectively provided consultancy to Coridea NCI (now Cibiem, Inc.) with regard to their interests in the efficacy of this procedure in animal models of chronic heart failure.
- Abbreviations and Acronyms
- coronary artery ligation
- carotid body
- carotid body denervation
- chronic heart failure
- early carotid body denervation
- ejection fraction
- Fos-related antigen 1
- heart rate variability
- interventricular septum
- left ventricle/ventricular
- rostral ventrolateral medulla
- Received April 16, 2013.
- Revision received June 19, 2013.
- Accepted July 1, 2013.
- American College of Cardiology Foundation
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