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expression and cardiac hypertrophy in the heart with pressure overload1 Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of South Dakota, Vermillion, SD 57069, USA
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Abstract |
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(TNF) is known to be a major factor contributing to cardiac remodelling and dysfunction. Parasympathetic nervous system cholinergic function can inhibit TNF expression during systemic infection. In the present study, we tested the effects of a cholinesterase inhibitor, neostigmine, and a muscarinic cholinergic agonist, pilocarpine, on cardiac hypertrophy and TNF levels during pressure overload. Rats with transverse aortic constriction exhibited elevated TNF protein levels in the heart, increased heart weight to body weight ratios (an index of cardiac hypertrophy) and decreased left ventricular diastolic function. Two weeks of infusion with neostigmine (6 µg kg–1 day–1) or pilocarpine (0.3 mg kg–1 day–1) significantly reduced cardiac hypertrophy, reduced TNF levels and elevated interleukin-10 levels in heart tissues, and improved ventricular function in rats with transverse aortic constriction. Neither of these treatments significantly changed ventricular pressure load. Furthermore, in primary cultured neonatal cardiac cells, treatment with pilocarpine attenuated adrenergic agonist phenylephrine-induced increased TNF expression and [3H]leucine (a marker of protein synthesis) incorporation in the cells. Collectively, both cholinergic agents decreased TNF levels and attenuated cardiac hypertrophy. Since both agents potentially enhanced cholinergic function, the anti-inflammatory action may be involved in the cardioprotective effect of the treatments with these agents.
(Received 31 July 2007;
accepted after revision 10 September 2007; first published online 14 September 2007)
Corresponding author Y.-F. Li: Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069, USA. Email: yli01{at}usd.edu
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Introduction |
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(TNF), interleukin (IL)-1 and IL-6, is associated with disease progression and increased mortality (Mann, 2002). Animal studies have indicated that inflammatory cytokines induce cardiac hypertrophy (Li et al. 2000), inhibit heart contractility (Yu et al. 2003) and promote cardiomyocyte apoptosis (Nian et al. 2004). Therefore, normalizing excessive inflammatory cytokine levels and elevating the levels of protective cytokines such as IL-10 may provide beneficial action against cardiac remodelling and dysfunction.
Recent studies have shown that the parasympathetic nervous system (PSNS) plays an important role in modulating immune responses by regulating inflammatory cytokine synthesis (Tracey, 2002; Wang et al. 2004; Shepherd et al. 2005). In animal studies, stimulation of cholinergic vagal nerves induced significant anti-inflammatory effects by inhibiting the expression of TNF, IL-1 and IL-6 in systemic inflammatory states (Borovikova et al. 2000; Guarini et al. 2003; Wang et al. 2003). Moreover, PSNS control of the heart is suppressed in CHF patients (Porter et al. 1990; Girgis et al. 1998; Azevedo & Parker, 1999) and in animals with experimentally induced CHF (Bibevski & Dunlap, 1999). Clinical studies indicated that impaired PSNS function was related to a poor outcome and high mortality of patients with CHF (La Rovere et al. 1998). Conversely, augmentation of PSNS activity by stimulation of the vagus (Zamotrinsky et al. 2001; Li et al. 2004) improved cardiac function and decreased mortality in CHF. However, the mechanism underlying the beneficial effects of PSNS cholinergic action remains to be addressed.
Postganglionic cholinergic fibres of the PSNS release the neurotransmitter acetylcholine (ACh), which acts on the postsynaptic muscarinic cholinergic receptors (M-AChRs) in the heart to regulate heart rate and contractility (Dhein et al. 2001). The released ACh is then rapidly hydrolysed by cholinesterases that exist with high activity in synapses. Thus, we hypothesize that inhibition of cholinesterase or stimulation of M-AChRs may enhance cholinergic action. In this study, we tested the effect of treatments with an acetylcholinesterase inhibitor, neostigmine, or an M-AChR agonist, pilocarpine, on cardiac hypertrophy and expression of TNF and IL-10 in rats with heart pressure overload.
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Methods |
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Male Sprague–Dawley rats (Harlan, Inc., Indianapolis, IN, USA) were obtained at 6 weeks of age and maintained on commercially available normal rat chow (Harlan, Inc.) and tap water on a 12 h–12 h light–dark cycle. The protocols in the present study were approved by Institutional animal care and use commitee of the University of South Dakota, and all the procedures were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996).
Transverse aortic constriction-induced cardiac hypertrophy
The rats (body weight 300–350 g) were pre- anaesthetized using ketamine (50 mg kg–1, I.P.) and xylazine (5 mg kg–1, I.P.). The trachea was intubated and the lungs ventilated and anaesthesia was maintained using isoflurane gas (1 to 3%) in oxygen via ventilation during the period of surgery. Under sterile conditions, the transverse aorta was located through a surgical incision at the second left intercostal space. Transverse aortic constriction (TAC) was induced by tying a 4–0 (1.5 metric) nylon suture against a 19 gauge needle. Then the needle was quickly removed to yield a consistent aortic narrowing. The internal incision was closed using absorbable suture and external incision closed using surgical glue. Prior to applying surgical glue, air was removed from the chest cavity using PE 20 tubing attached to a 27 gauge needle and syringe. Sham operations consisted of similar procedures except that the aorta was not tied. Postoperatively, pain relief Buprenex (Reckitt & Colman, Hull, UK) (0.05 to 0.1 mg kg–1, sc) was given to the animals for two days. During the first two days, food and water intake and behaviour were carefully monitored.
Chronic administration of cholinergic agents
An osmotic minipump (Alzet, Palo Alto, CA, USA) was filled with test agents or saline. The minipumps were placed intraperitoneally in rats at the time of TAC surgery. The peripheral cholinesterase inhibitor neostigmine (3 or 6 µg kg–1 day–1) or the M-AChR agonist pilocarpine (0.15 or 0.3 mg kg–1 day–1) were infused by the minipump for two weeks after surgery. At the end of the experiments, the animals were killed by cardiac excision under anaethesia with Inactin (Thiobutabarbital sodium, 150 mg kg–1; Sigma, St. Louis, MO, USA) the minipumps were recovered and the remaining drug volume was measured to validate success of the infusion.
Measurement of left ventricular function
After 2 weeks of TAC and treatments, the rats were anaesthetized with Inactin (100–150 mg kg–1). The left ventricle was catheterized through the right common carotid artery with a Millar P-V catheter (2 French, Model SPR-838 Millar Inc., Huston, TX, USA). After a stabilization period of 20 min, left ventricular function parameters (end-diastolic pressure, end-systolic pressure and rate of change of pressures, dP/dt) were measured using Millar P-V software and PowerLab A/D data acquisition system (AD instruments, Pty Ltd, Colorado springs, CO, USA).
Primary culture of neonatal cardiac cells
One to 3-day-old neonatal Sprague–Dawley rat pups were decapitated and vertricular tissues were obtained. The tissues were chopped into small pieces and digested using trypsin and then collagenase. The isolated cells were cultured in 24-well plates in Dulbecco's modifiied Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin (100 U ml–1) and streptomycin (100 mg ml–1) (Cambrex Bio Science, Walkersville, MD, USA). Before the treatments, the medium was changed to FBS-free DMEM containing 1% penicillin–streptomycin and the cells were cultured for 24 h. The cells were then treated with phenylephrine (PE; 20 and 10 µM) in the presence or absence of the cholinergic agonist pilocarpine (1 µM) for 24 h and 0.5 ml of medium was collected from each well for measurement of TNF concentrations by enzyme-linked immunosorbent assay (ELISA). Then [3H]leucine (1 µCi ml–1) was loaded into each well and incubated for an additional 8 h. After removal of the medium and washing with phosphate-buffered saline (PBS), the cells were solubilized, and [3H]leucine incorporation was detected using a scintillation counter (Beckman Coulter Inc., Fullerton, CA, USA).
Assessments of TNF and IL-10 levels
Extracted protein samples from the hearts were subjected to standard Western blot procedures as previously described (Li et al. 2003). Fluorescence-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) were used, and the fluorescent signals were scanned and quantified by a LI-COR imaging system (LI-COR, Lincoln, NE, USA). The primary antibodies against TNF or IL-10 (Bio-resource, Cotati, CA, USA) were diluted in 5% dry milk in Tris buffered saline (1:500). An ELISA method was also used to detect the TNF levels in media and within some heart samples using a commercial ELISA kit (R&D Inc., Minneapolis, MN, USA) following the manufacturer's instructions.
Statistical analysis
The results are expressed as means ± S.D. Comparison of data for physiological variables and biochemical measurements among multiple groups were conducted using one-way analysis of variance followed by Student–Newman–Keuls test. Significance was accepted when P value was less than 0.05. Some data were subjected to correlation analysis. The statistical analysis was carried out using software StatView 3.0 (SAS Inc., CA, USA).
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Results |
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Transverse aortic constriction markedly increased left ventricular systolic pressure (LVSP), which caused cardiac overload. Treatment with neostigmine or pilocarpine did not significantly reduce LVSP in rats with TAC, suggesting that the effect of the treatments on cardiac hypertrophy is not due to reduction of cardiac load (Table 1).
Relative to saline, neostigmine and pilocarpine treatments did not affect heart rate in TAC and sham groups (Table 1). Infusion with neostigmine and pilocarpine for 2 weeks did not cause significant changes in body weight compared with the saline control group.
We next determined whether treatment with neostigmine or pilocarpine affects the expression of TNF in the heart. Using Western blot, we found that levels of TNF protein were significantly increased in the heart in the group of TAC with saline treatment compared with the sham with saline group. Treatment with neostigmine (6 µg kg–1 day–1) or pilocarpine (0.3 mg kg–1 day–1) significantly reduced the TAC-induced increase in TNF levels relative to those in the TAC with saline group (Fig. 2A).
We also assessed an anti-inflammatory cytokine, IL-10 protein, in the heart. As shown in Fig. 2B, TAC reduced IL-10 expression. In contrast, neostigmine and pilocarpine treatments markedly increased IL-10 protein levels in the heart.
We also used ELISA method to measure the TNF concentrations in some homogenate samples of heart tissues from the rats with TAC that had been treated with neostigmine or saline. Consistent with the Western blot (Fig. 3A), the treatments with neostigmine reduced TNF protein levels in a dose-dependent manner. There was a significant positive correlation between the TNF protein levels and HW/BW ratio (Fig. 3B).
After 2 weeks of TAC, left ventricular end-diastolic pressure (LVEDP) was elevated in the group of TAC with saline compared to that of the sham group treated with saline. The elevated LVEDP was significantly reduced by treatment with neostigmine (6 µg kg–1 day–1) or pilocarpine (0.3 mg kg–1 day–1; Fig. 4). The maximal rate of change of pressure (dP/dtmax) of the left ventricle remained at similar levels in all the groups (data not shown). However, the minimal dP/dt (dP/dtmin) in the TAC group with saline was significantly reduced compared with the sham group with saline, indicating that TAC impaired cardiac diastolic function. This impaired diastolic function was improved in the rats with TAC treated with neostigmine (6 µg kg–1 day–1) or pilocarpine (0.3 mg kg–1 day–1; Fig. 4).
We further tested whether cholinergic agonist elicits a direct effect on hypertrophic response and TNF expression in isolated and primary cultured cardiac cells. Mixed cardiomyocytes and fibroblast cells in primary culture were treated with the adrenergic agonist phenylephrine (PE; 1 and 10 µM) in the presence or absence of treatment with the muscarinic agonist pilocarpine. As shown in Fig. 5, PE treatment caused a dose-dependent increase in [3H]leucine incorporation, indicating increased protein synthesis (hypertrophic response). Phenylephrine treatment also increased TNF protein concentration in culture media by 65% compared with the untreated control cells. Cotreatment with pilocarpine (1 µM) significantly attenuated [3H]leucine incorporation induced by PE treatment. In the cells cotreated with PE (10 µM) and pilocarpine compared with the cells treated with PE alone, [3H]leucine incorporation was reduced by approximately 68% (Fig. 5A). Concomitantly, the PE-induced increase in TNF levels was also abolished by pilocarpine cotreatment (Fig. 5B). These in vitro studies suggest that cholinergic action may exert a direct anti-inflammatory effect on PE-stressed cardiac cells.
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Discussion |
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expression and concurrently increased IL-10 expression in the hearts of rats with cardiac pressure overload. The attenuation of the increase in TNF was correlated with attenuation of cardiac hypertrophy; and (iii) in vitro, the cholinergic agonist pilocarpine elicited a direct inhibition on increased TNF production and hypertrophic responses in primary cultured cardiac cells. This is the first study to indicate a cardioprotective effect of these cholinergic agents. These data may suggest a new strategy to prevent and treat cardiac remodelling and dysfunction.
Pharmacologically, neostigmine inhibits acetylcholine inactivation and thus prolongs the excitation of cholinergic synapses. Pilocarpine stimulates postsynaptic muscarinic cholinergic receptors. By different mechanisms, both agents can potentially enhance cholinergic activity. In this study, both agents reduced the levels of the pro-inflammatory cytokine TNF, increased the levels of the anti-inflammatory cytokine IL-10 and attenuated cardiac hypertrophy, suggesting that these beneficial effects may be through enhancement of cholinergic function.
A cholinergic anti-inflammatory action has been reported in animal models with systemic inflammatory reactions (Borovikova et al. 2000; Tracey, 2002; Guarini et al. 2003). Whether this cholinergic action can be beneficial in cardiac diseases has been discussed (Jankowska et al. 2006), but has yet to be demonstrated. Our study shows that two different cholinergic agents induce similar anti-inflammatory and cardioprotective effects, further suggesting the beneficial effect of cholinergic anti-inflammatory action on cardiac diseases.
Sustained increases in inflammatory cytokines, such as TNF, IL-1 and IL-6, are the major pathogenic factors involved in cardiac remodelling and the progression of chronic heart failure (Deswal et al. 2001; Mann, 2002; Malave et al. 2003). Animal studies indicate that inflammatory cytokines induce cardiac hypertrophy (Li et al. 2000), inhibit heart contractility (Yu et al. 2003) and promote cardiomyocyte apoptosis (Nian et al. 2004). In our study, after the treatment with neostigmine there was a positive correlation between decreases of TNF and cardiac hypertrophy, suggesting that the cardiac protection may be attributed to the anti-inflammatory effect of the treatment. A limitation of the present study is that we did not observe the time course of the treatments, which might further suggest a cause–effect relationship between the anti-inflammatory effect and the extent of cardiac hypertrophy.
Moreover, our study showed that treatment with low-dose neostigmine or pilocarpine reduced but did not completely suppress TNF levels. Recent major clinical trials of anti-TNF therapy indicate that complete blockade of TNF using specific antibodies induces adverse events in heart failure patients (Anker & Coats, 2002; Chung et al. 2003). Tumour necrosis factor may play more complicated roles in both physiological and pathophysiological processes (Mann, 2003; Valgimigli et al. 2005). More appropriate approaches would be to regulate excessive inflammatory cytokines, instead of inducing complete blockade. Therefore, treatment with low doses of neostigmine or pilocarpine may provide suitable approaches for prevention and treatment of heart failure.
In this study, neostigmine and pilocarpine were administered systemically. It is not clear whether the beneficial effects in the in vivo study were mediated through a systemic or local action. However, we found that the treatments significantly reduced the increased TNF levels in the heart tissues induced by TAC. Moreover, our in vitro experiment in the primary cultured cardiac cells also indicated that treatment with the cholinergic agent pilocarpine directly attenuated the increased TNF levels and hypertrophic response induced by PE. These data suggest that the cholinergic action may elicit a direct anti-inflammatory effect on the cardiac cells in the heart in vivo. The local cytokines in heart tissue, generated by cardiomyocytes (Torre-Amione et al. 1995) and fibroblasts (Zhao & Eghbali-Webb, 2001), are believed to play a very important role in cardiac remodelling and dysfunction. Therefore, the local cholinergic anti-inflammatory effect of cytokines may provide an important beneficial effect in prevention of cardiac remodelling and dysfunction. This local cholinergic anti-inflammatory action may be involved in the cardiac beneficial effect of the treatments with neostigmine and pilocarpine. Moreover, treatment with low doses of neostigmine or pilocarpine did not affect left ventricular systolic pressure, suggesting that the cardioprotective effects were unlikely to result from their haemodynamic effect.
In immune cells, such as macrophages, the nicotinic cholinergic receptor 7 subunit plays a key role in the cholinergic regulation of TNF expression (Wang et al. 2003). In the heart, although nicotinic receptors exist (Dvorakova et al. 2005), their functional roles in cardiac regulation remain unclear (Deck et al. 2005). Instead, our study indicated that the muscarinic receptor agonist pilocarpine attenuated TNF expression and cardiac hypertrophy, suggesting that muscarinic cholinergic receptors may be involved in the anti-inflammatory and cardiac protective effects. However, more studies are needed to demonstrate the mechanisms responsible for muscarinic cholinergic receptor cardioprotection.
In summary, for the first time, this study reports that treatment with low doses of neostigmine or pilocarpine attenuate the increase of inflammatory cytokine TNF in the heart and improve cardiac hypertrophic responses to pressure overload. The mechanisms of the cardiac beneficial effects of these treatments need to be elucidated. The evidence that both the cholinesterase inhibitor and the muscarinic agonist elicit a beneficial effect on the stressed heart may suggest that improving cholinergic function may provide a new strategy for the prevention and treatment of heart failure.
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