Cholinoceptive and cholinergic properties of cardiomyocytes involving an amplification mechanism for vagal efferent effects in sparsely innervated ventricular myocardium Yoshihiko Kakinuma 1 , Tsuyoshi Akiyama 2 and Takayuki Sato 1 1 Department of Cardiovascular Control, Kochi Medical School, Nankoku, Japan 2 Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Suita, Japan Introduction Our previous studies showed that an oral low dose of donepezil, an acetylcholinesterase inhibitor and anti- Alzheimer’s disease drug, prevented ventricular remod- eling and dysfunction after large myocardial infarction Keywords acetylcholine; cardiomyocytes; donepezil; energy metabolism; non-neuronal cholinergic system Correspondence Y. Kakinuma, Department of Cardiovascular Control, Kochi Medical School, Nankoku, Kochi 783-8505, Japan Fax: +81 88 880 2310 Tel: +81 88 880 2587 E-mail: kakinuma@kochi-u.ac.jp (Received 13 May 2009, revised 8 July 2009, accepted 10 July 2009) doi:10.1111/j.1742-4658.2009.07208.x Our recent studies have shown that, as indicated by vagal stimulation, an acetylcholinesterase inhibitor donepezil, an anti-Alzheimer’s disease drug, prevents progression of heart failure in rats with myocardial infarction, and activates a common cell survival signal shared by acetylcholine (ACh) in vitro. On the basis of this and evidence that vagal innervation is extremely poor in the left ventricle, we assessed the hypothesis that ACh is produced by cardiomyocytes, which promotes its synthesis via a positive feedback mechanism. Rat cardiomyocytes expressed choline acetyltransferase (ChAT) in the cytoplasm and vesicular acetylcholine transporter with the vesicular structure identified by immunogold electron microscopy, suggesting that cardiomyocytes possess components for ACh synthesis. Intracellular ACh in rat cardiomyocytes was identified with physostigmine or donepezil. How- ever, with atropine, the basal ACh content was reduced. In response to exog- enous ACh or pilocarpine, cardiomyocytes increased the transcriptional activity of the ChAT gene through a muscarinic receptor and ChAT protein expression, and, finally, the intracellular ACh level was upregulated by pilo- carpine. Knockdown of ChAT by small interfering RNA accelerated cellular energy metabolism, which is suppressed by ACh. Although physostigmine had a minimal effect on the ChAT promoter activity by inhibiting acetylcho- linesterase, donepezil resulted in elevation of the activity, protein expression and intracellular ACh level even in the presence of sufficient physostigmine. Orally administered donepezil in mice increased the ChAT promoter activity in a reporter gene-transferred quadriceps femoris muscle and the amount of cardiac ChAT protein. These findings suggest that cardiomyocytes possess an ACh synthesis system, which is positively modulated by cholinergic stim- uli. Such an amplification system in cardiomyocytes may contribute to the beneficial effects of vagal stimulation on the ventricles. Abbreviations ACh, acetylcholine; ChAT, choline acetyltransferase; ChAT KO, cell transfected with the choline acetyltransferase sequence-specific microRNA expression vectors; DAPI, 4¢,6-diamidino-2-phenylindole; HIF, hypoxia-inducible factor; miRNA, microRNA; IPHC, isopropylhomocholine chloride; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; siRNA, small interfering RNA; VAChT, vesicular acetylcholine transporter. FEBS Journal 276 (2009) 5111–5125 ª 2009 The Authors Journal compilation ª 2009 FEBS 5111 in rats without a bradycardiac effect, and that vagal nerve stimulation protected the heart against ischemia- induced ventricular tachyarrhythmias independently of a heart rate-slowing mechanism [1–3]. We also showed that pretreatment with acetylcholine (ACh) salvaged in vitro cardiomyocytes from prolonged hypoxia- induced cell death by preserving mitochondrial function and activating cell survival signals, including phospho- inositide-3-kinase ⁄ Akt ⁄ hypoxia-inducible factor (HIF)- 1a ⁄ vascular endothelial growth factor [4], and that ACh prevented hypoxia-induced loss of gap-junction chan- nels, maintaining cell-to-cell communication and electri- cal stability [4,5]. These results initially prompted us to speculate that such beneficial effects were achieved by direct cellular actions of ACh, which appeared to be released from the vagal nerve endings at ventricular walls and to be rapidly broken down by acetylcholines- terase. However, this speculation concerning the mechanism of the beneficial effects of ACh has been challenged by accumulating evidence of the sparseness of cholinergic innervation of the ventricular myo- cardium, except for the specialized conduction system [6–9]. Furthermore, our recent study unexpectedly showed that, in cultured cardiomyocytes, donepezil directly activated the same cell survival pathway as ACh did, although there were no cholinergic neurons in the culture. Consequently, these results allow us to propose a working hypothesis that donepezil directly stimulates cardiomyocytes to synthesize ACh de novo. Although ACh was first identified as a vagal efferent neurotransmitter, ‘parasympathin’, recent studies have revealed that non-neuronal cells in animals and humans synthesize, store and release ACh [10,11]. In the non-neuronal cholinergic system, ACh is constitu- tively produced to act locally as an autocrine or para- crine mediator via cellular membrane-bound receptors and intracellular signaling proteins. Therefore, we hypothesize that cardiomyocytes possess the properties of the non-neuronal cholinergic system and that the cholinergic system in cardiomyocytes is activated by donepezil. An earlier observation, which appears to support our hypothesis, was reported by Kawada et al. [12], who measured myocardial interstitial ACh con- centrations in the left ventricular free wall with an in vivo microdialysis technique. In vagotomized cats, acute myocardial ischemia induced a large increase in ACh concentration in the dialysate. Although the ischemically injured nerve ending of the vagus was reported to be the only source of ACh, we now pre- sume that the ischemic cardiomyocyte is another possi- ble source of ACh. To test our hypothesis and clarify a molecular and cellular basis for the beneficial effects of donepezil, in the present study we focused our efforts on the follow- ing points. First, we investigated intracellular pathways for ACh production in cardiomyocytes. Second, we determined the intracellular concentrations and loca- tions of ACh. Third, we examined the effects of donepezil on the intracellular ACh production path- ways and the intracellular levels of ACh. Finally, we evaluated the biological significance of cardiac non- neuronal ACh system by genetic interference in intrin- sic ACh synthesis. The present results not only support our hypothesis, but also reveal a novel mechanism for a direct action of vagally released ACh on the ventric- ular cardiomyocytes, i.e. amplification of the vagal effect via ACh-induced ACh production in cardio- myocytes. This mechanism would answer the question that was raised by Rosen and Hoffman [13] three dec- ades ago: ‘how can the vagus directly exert its benefi- cial effects on the diseased ventricle in spite of its sparse innervation of the ventricular myocardium except for the conduction system?’ Results Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) expressed in cardiomyocytes Immunoreactivity for ChAT, an essential enzyme for ACh synthesis from choline and acetyl-coenzyme A, was detected in the cytoplasm of neonatal and adult rat cardiomyocytes (Fig. 1A,B). Punctate fluorescent signals for VAChTs, indispensable intracellular trans- porters of synthesized ACh, were also distributed throughout the cytoplasm (Fig. 1C,D). In agreement with the immunocytochemical results, electron micro- scopic examination of the rat heart also confirmed that gold particles conjugated with antibodies against VAChT were located in the membrane structures between the muscle fibers (Fig. 1E). Immunoblot anal- ysis showed that both ChAT and VAChT proteins are expressed in cardiomyocytes (Fig. 1F). These results suggest that cardiomyocytes possess ACh-producing properties. Basal ACh level detected in cardiomyocytes The basal level of ACh was evaluated by HPLC, using rat cultured cardiomyocytes. During the procedures for measurement, without the use of an acetylcholinester- ase inhibitor, physostigmine, ACh was not detectable in cardiomyocytes at all (Fig. 2). However, in the pres- ence of 0.1 mmolÆL )1 physostigmine during measure- ment, which is adequate for inhibition, the basal ACh de novo ACh synthesis induced by ACh in cardiomyocytes Y. Kakinuma et al. 5112 FEBS Journal 276 (2009) 5111–5125 ª 2009 The Authors Journal compilation ª 2009 FEBS Fig. 1. Rat cardiomyocytes express crucial components of ACh synthesis, ChAT and VAChT. Rat neonatal and adult cardiomyo- cytes expressed ChAT immunoreactivity in the cytoplasm (red). The red signals were detected throughout the cardiomyocytes. Green: F-actin. Bar: 20 lm [(A) neonatal; (B) adult]. VAChT signals were also detected in both neonatal and adult rat cardiomyocytes (red), although in a punctuate fashion (blue, DAPI). Bar: 20 lm [(C) neonatal; (D) adult]. Immunogold electron microscopy demon- strated vesicle-like structures with VAChT immunoreactivity in adult rat cardiomyo- cytes. Diameter of each gold particle: 15 nm. Bar: 50 nm (E). Western blot analy- sis showed that rat neonatal cardiomyo- cytes expressed both ChAT and VAChT with proper molecular masses (F). Y. Kakinuma et al. de novo ACh synthesis induced by ACh in cardiomyocytes FEBS Journal 276 (2009) 5111–5125 ª 2009 The Authors Journal compilation ª 2009 FEBS 5113 level was determined to be 2.93 ± 0.63 nmolÆL )1 (n = 10) in rat cardiomyocyte lysates, and further- more, even with donepezil, another acetylcholinesterase inhibitor, ACh was detected in cardiomyocytes (Fig. 2). Effects of muscarinic agonists on ChAT transcription and translation The HEK293 cells used for transfection in the reporter assay were shown to express the components required for ACh synthesis, i.e. ChAT, VAChT and muscarinic ACh receptor subtype (m)2 by immunocytochemical analysis. RT-PCR analysis also revealed that m1, m2, m5, nicotinic receptor subtype (a)3 and a4 mRNAs are expressed in HEK293 cells (Fig. 3A). A reporter assay using a reporter vector, transfected into HEK293 cells, with a regulatory promoter region of rat ChAT showed that ACh (1 mmolÆL )1 ) and pilo- carpine (1 mmolÆL )1 ) increased the transcriptional activity of ChAT (Fig. 3A,B). Within 4–8 h, the lucif- erase activity driven by the ChAT promoter was signif- icantly enhanced by ACh (862.0 ± 115.0%, P < 0.01, n = 9) and pilocarpine (961.0 ± 8.5%, P < 0.01, n = 9). Increases in ChAT transcription activities were suppressed by atropine (1 mmolÆL )1 ). In agreement with the transcriptional activation, the protein levels of ChAT in rat cardiomyocytes were also upregulated by ACh (267.8 ± 30.9%, P < 0.05, n = 6) and pilocar- pine (217.5 ± 14.6%, P < 0.01, n = 6) (Fig. 3A,B). These effects of muscarinic agonists on ChAT protein expression were subsequently blocked by atropine. Effects of muscarinic agonist on the ACh level in cardiomyocytes The basal level of ACh in rat cardiomyocytes was determined in the presence of physostigmine (Fig. 4A). Pretreatment with atropine (1 mmolÆL )1 ) significantly lowered the ACh level to 45.7 ± 7.9% (P < 0.05). By contrast, treatment with pilocarpine (1 mmolÆL )1 ) sig- nificantly increased the ACh level in rat cardiomyo- cytes to 255.0 ± 28.0% of the basal level of control (P < 0.05, n = 13) (Fig. 4B). These results suggest that cardiomyocytes constitutively produce and release ACh, and that the released ACh, in turn, stimulates cardiomyocytes to synthesize ACh via muscarinic receptors; that is, there is an autocrine–paracrine cardiac ACh synthesis system. Cellular effects of small interfering RNA (siRNA) for ChAT in HEK293 cells When compared with nontransfected or unrelated siRNA-transfected HEK293 cells, ChAT-knockdown cells (siRNA-transfected HEK293 cells) had few sig- nals for ChAT and low expression of ChAT protein, as also shown by western blot analysis (Fig. 5A). It has been shown that 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) activity is reflected by cellular energy metabolism; therefore, even with comparable cell numbers, MTT activity can decline when energy metabolism is suppressed [14,15]. MTT deposits partially overlapped with mitochondria, as indicated by DePsipher, not only in HEK293 cells but also in rat cardiomyocytes (Fig. 5B). The MTT activities in HEK293 cells were significantly suppressed by ACh (1 mmolÆL )1 ) to 79.2 ± 5.0% (P < 0.01) (Fig. 5B). The inhibitory effect of ACh on the MTT activity was also verified by a reduction of oxygen con- sumption in ACh-treated HEK293 cells. Likewise, oxy- gen consumption was reduced by ACh in rat cardiomyocytes (Fig. 5B). By contrast, the knockdown of ChAT reciprocally increased MTT activity to 149.5 ± 3.1% (P < 0.01) (Fig. 5C). Finally, to further assess the role of synthesized ACh, the amount of which seems to be very small in cell lysates, oxygen consumption was measured in real time using stable HEK293 cells transfected with the ChAT sequence-specific microRNA (miRNA) expres- sion vectors (ChAT KOs). As shown in Fig. 5D, ChAT KOs definitely consumed more oxygen than the negative control vector transfectants, suggesting that intrinsic ACh synthesized by cells definitely plays a biologically significant role in suppressing cellular energy metabolism. Effects of donepezil on ACh levels in cardiomyocytes When compared with the basal level of ACh in control rat cardiomyocytes treated with physostigmine alone, Fig. 2. ACh is present in rat cardiomyocytes. In the presence of 0.1 mmolÆL )1 physostigmine during measurement, the basal ACh level in rat cardiomyocytes, evaluated by HPLC, was 2.93 ± 0.63 nmolÆL )1 (n = 10) (black area in the chromatogram) in comparison with 20 n M IPHC (gray area in the chromatogram). With 1 lmolÆL )1 donepezil, ACh was also detected in cardiomyocytes. de novo ACh synthesis induced by ACh in cardiomyocytes Y. Kakinuma et al. 5114 FEBS Journal 276 (2009) 5111–5125 ª 2009 The Authors Journal compilation ª 2009 FEBS at a concentration adequate for acetylcholinesterase inhibition (0.1 mmolÆL )1 ) [16], a higher level of ACh was found in donepezil-treated cardiomyocytes (384.6 ± 102.7%, P < 0.05) (Fig. 6). The effect of donepezil was blocked by atropine (1 mmolÆL )1 ), but there was still a significant difference in the ACh level between the cardiomyocytes treated with donepezil plus atropine and those treated with atropine (225.0 ± 43.0% versus 45.7 ± 7.9%, P < 0.01). The facts that donepezil elevated the ACh level in control cardiomyocytes and that the effect was only partially blocked by atropine suggested that donepezil has a mechanism of action that is independent of acetylcho- linesterase inhibition or muscarinic receptors. Fig. 3. Transcription and translation of ChAT is upregulated through a muscarinic receptor. HEK293 cells expressed immunoreactivities of ChAT, VAChT, and m2. Red: m2, ChAT, and VAChT, respectively. Blue: DAPI. Bar: 20 lm. HEK293 cells expressed mRNAs of cholinergic receptors, including m1, m2, and m5, as well as a3 and a4 (A), as evaluated by RT-PCR. Rat ChAT promoter activity, evaluated by using HEK293 cells transfected with the reporter vectors, and ChAT protein expression in rat cardiomyocytes were increased by 1 mmolÆL )1 ACh (blue bars) (A) and 1 mmolÆL )1 pilocarpine (green bars) (B). The promoter activity and the protein level induced by each agonist peaked at 8 h, however, 1 mmolÆL )1 atropine inhibited ChAT transcription and translation, resulting in a decrease in the peak level. Y. Kakinuma et al. de novo ACh synthesis induced by ACh in cardiomyocytes FEBS Journal 276 (2009) 5111–5125 ª 2009 The Authors Journal compilation ª 2009 FEBS 5115 Effects of donepezil on ChAT transcription and translation The effects of donepezil on the transcriptional activity for ChAT in HEK293 cells, transfected with the repor- ter vectors, and on its protein expression in rat cardio- myocytes are illustrated in Fig. 7. In comparison with donepezil, physostigmine did not increase the promoter activity for ChAT in the reporter vector-transfected HEK293cells (Fig. 7A). By contrast, a promoter assay revealed that donepezil gradually increased the ChAT promoter activity (267.0 ± 14.1%, P < 0.01), and that the time course was quite different from that of ACh (Fig. 7B). Similarly, ChAT protein expression was slowly upregulated by donepezil (420.0 ± 31.6%, P < 0.01) in rat cardiomyocytes (Fig. 7B). These results suggest that donepezil, unlike physostigmine, activates the ACh synthesis system. This in vitro effect of donepezil on ChAT transcription was also observed in vivo. Orally administered donepezil (1 week) also elevated ChAT protein expression in the heart, as eval- uated by immunohistochemical and western analysis (Fig. 7C). An in vivo reporter assay was conducted to investigate the effect of donepezil on murine skeletal muscle, as it is a more convenient target for a gene transfer approach with sonoporation than the heart. The effect of oral donepezil on ChAT promoter activity in quadriceps fem- oris muscles is shown in Fig. 8. When compared with the skeletal muscles of untreated mice, those of donepezil- treated mice exhibited increased promoter activity for ChAT (380.0 ± 82.0% of control, P < 0.05). Fig. 4. ACh synthesis in rat cardiomyocytes is activated by the muscarinic agonist pilocarpine. The basal ACh level in rat cardiomyocytes, standardized as 100%, was reduced to 45.7 ± 7.9% (P < 0.05) in the presence of 1 mmolÆL )1 atropine with physostigmine (atropine) (A). The ACh level, measured with physostigmine, was increased to 255.0 ± 28.0% (P < 0.05, n = 8) by 12 h of treatment with pilocarpine (1 mmolÆL )1 ) (green bar) (B). Fig. 5. ChAT negatively modulates mitochondrial respiratory activity. Knockdown of the ChAT gene by ChAT-specific siRNA verified the reduction of endogenous ChAT immunoreactivities in HEK293 cells, as compared with the levels in nontreated cells and unrelated siRNA- transfected cells. Red: ChAT. Blue: DAPI. Bar: 20 lm. Reduced expression of endogenous ChAT protein caused by the siRNA in HEK293 cells was also verified by western blot analysis (A). Red signals represented by mitochondria in HEK293 cells using DePsipher partially over- lapped with MTT deposits (black). Bar: 20 lm. ACh (1 mmolÆL )1 )-treated HEK293 cells decreased their oxygen consumption within 3 h and decreased their MTT activity to 79.2 ± 5.0% (blue bar), as compared with nontreated HEK293 cells (P < 0.01, n = 8). Likewise, ACh also reduced oxygen consumption in rat cardiomyocytes, in which several MTT deposits (black) also colocalized with DePsipher signals (red). Bar: 20 lm (B). However, ChAT knockdown in HEK293 cells conversely increased MTT activity to 149.5 ± 3.1% (P < 0.01, n = 15) (C). ChAT KOs (blue and green lines), HEK293 cells stably transfected with the miRNA expression vectors possessing specific human ChAT sequences, showed greater acceleration of oxygen consumption than negative control cells (negative control, dotted lines), as depletion of oxygen contents in the culture medium of ChAT KOs was greater than in the negative control. Representative data from two clones in each group are shown (D). de novo ACh synthesis induced by ACh in cardiomyocytes Y. Kakinuma et al. 5116 FEBS Journal 276 (2009) 5111–5125 ª 2009 The Authors Journal compilation ª 2009 FEBS Y. Kakinuma et al. de novo ACh synthesis induced by ACh in cardiomyocytes FEBS Journal 276 (2009) 5111–5125 ª 2009 The Authors Journal compilation ª 2009 FEBS 5117 Discussion The present results suggest that cardiomyocytes possess an ACh-producing pathway and synthesize a basal level of ACh constitutively, and that exogenously administered and endogenously produced ACh stimu- lates the ACh-producing pathway, mainly via musca- rinic receptors. On the basis of these results, we propose that the cholinoceptive cholinergic properties of cardiomyocytes could act as a positive feedback system for ACh synthesis in an autocrine ⁄ paracrine manner. Another important result is that, concerning the biological significance, the intracellular ACh syn- thesis system plays a role in negatively regulating energy metabolism in cardiomyocytes. In addition to these results, we showed that donepezil activates the ACh-producing pathway and elevates the ACh level in cardiomyocytes via both acetylcholinesterase-inhibitory and acetylcholinesterase-independent mechanisms. Therefore, donepezil is a potent upregulator of the ACh level in the ventricular myocardium, in spite of the sparseness of cholinergic innervation of the ventric- ular myocardium (except for the specialized conduc- tion system). Our previous studies demonstrated that vagal stimu- lation improves the survival rate of rats with heart fail- ure by inhibition of myocardial infarction-induced fatal arrhythmia and remodeling of the left ventricle [2,17], and that ACh protects cardiomyocytes from hypoxia-induced apoptosis through the phosphoinosi- tide-3-kinase ⁄ Akt ⁄ HIF-1a ⁄ vascular endothelial growth factor pathway [4]. These results suggest that ACh plays beneficial roles in both salvaging cardiomyocytes and suppressing arrhythmia. However, recent findings have demonstrated that vagal nerve endings are rarely detected in the left ventricle in humans, guinea pigs and rats, although the innervation may vary between species [6–9,18]. Consequently, there is a discrepancy between the cardioprotective effects of ACh on cardio- myocytes and the paucity of vagal nerve endings in the left ventricle. Since previous studies pioneered by Kawashima, Fujii [19,20] and Wessler [21,22], showing that ACh is synthesized in non-neuronal cells, i.e. T-lymphocytes and bronchial epithelial cells, accumulating experimen- tal evidence has revealed that ACh is widely present in both prokaryotic and eukaryotic non-neuronal cells, and is also present in bacteria, protozoa, algae, and primitive plants. Therefore, ACh is considered to have appeared very early in the evolutionary process and to play another significant role, not as a neurotransmitter but as a trophic factor. However, there are no reports showing that ACh is synthesized in cardiomyocytes. As demonstrated in the present study, rat cardio- myocytes possessed essential components for ACh syn- thesis, ChAT and VAChT, and the content of ACh in cultured cardiomyocytes was measurable. Surprisingly, the protein expression level and transcriptional activity of ChAT were elevated by ACh or pilocarpine through a muscarinic receptor, and the ACh level in cardio- myocytes was upregulated by pilocarpine. This sug- gests that cardiomyocytes have a positive feedback mechanism in ACh synthesis, and reinforces the previ- ous finding that chronic vagal stimulation suppressed remodeling of the myocardial infarction-induced failing heart despite the extreme sparseness of vagal nerve endings in the ventricle. In other words, ACh released from vagal nerve endings sequentially might activate local ACh synthesis in neighboring cardiomyocytes in a paracrine fashion, and propagation of ACh synthesis might progress throughout the heart. As evaluated by HPLC, the ACh level was very low in cardiomyocyte cell lysates. However, according to our data, the average concentration of ACh in one car- diomyocyte was estimated to be in the millimoles per liter range. This was surprisingly comparable to the data on ACh levels in endothelial cells reported by Malo et al. [23], who demonstrated that one brain endothelial cell, engineered to express ChAT, produced as much as 7 mmolÆL )1 of ACh. This study strongly suggests that, if cells possess functional ChAT, ACh could reach an intracellular level up to 1 mmolÆL )1 . Furthermore, results obtained with stable transfectants of ChAT KOs suggest that the ACh synthesized at the cellular levels plays a pivotal role in suppressing cellu- lar energy metabolism. Fig. 6. Donepezil increases the ACh level in rat cardiomyocytes. even in the presence of physostigmine, and the effect is partially attenuated by atropine. In rat cardiomyocytes treated with 1 lmolÆL )1 donepezil for 20 h, the ACh level was further increased to 384.6 ± 102.7% (P < 0.05, n = 10), even in the presence of physo- stigmine (red bar). In contrast, 1 mmolÆL )1 atropine tended to atten- uate the ACh level induced by donepezil to 225.0 ± 43.0% (atropine) (P < 0.05, n = 6). de novo ACh synthesis induced by ACh in cardiomyocytes Y. Kakinuma et al. 5118 FEBS Journal 276 (2009) 5111–5125 ª 2009 The Authors Journal compilation ª 2009 FEBS Although a series of our experimental studies showed that chronic vagal nerve stimulation exerts its beneficial effects on ventricular myocardium via muscarinic ACh receptors, such a therapy needs an invasive surgical procedure. A noninvasive pharmacological treatment with oral donepezil could be a potential alternative to chronic vagal nerve stimulation therapy. Donepezil was originally developed as a centrally acting inhibitor of acetylcholinesterase, and is currently available for the treatment of Alzheimer’s disease [24]. However, several in vitro studies also suggest that the neuroprotective actions of donepezil could depend principally on its pharmacological properties other than acetylcholines- terase inhibition. For example, in an earlier study, Fig. 7. Donepezil activates rat ChAT pro- moter activity in HEK293 cells and ChAT protein expression in rat cardiomyocytes in vitro and in vivo. Rat ChAT promoter activity was not affected by physostigmine (A); in contrast, it was increased by 1 lmo- lÆL )1 donepezil within 20 h (267.0 ± 14.0%, P < 0.01, n = 6), and this effect was blocked by atropine. Concomitantly, the protein expression level of ChAT was also increased by donepezil in rat cardiomyo- cytes (red bar) (B). Orally administered donepezil also elevated ChAT protein expression and immunoreactivities in the heart. Representative results were shown. Blue: DAPI. Red: ChAT. Bar: 50 lm (C). Y. Kakinuma et al. de novo ACh synthesis induced by ACh in cardiomyocytes FEBS Journal 276 (2009) 5111–5125 ª 2009 The Authors Journal compilation ª 2009 FEBS 5119 Kato et al. [16] revealed that donepezil increased ChAT activity in cholinergic neuronal cells, as compared with other types of acetylcholinesterase inhibitors, and men- tioned the specific characteristics of donepezil. In agree- ment with that study, the present results show that de novo synthesis of ACh in cardiomyocytes was signifi- cantly promoted by donepezil, even in the presence of a dose of physostigmine sufficient to completely inhibit endogenous acetylcholinesterase [25–27], suggesting that donepezil has the ability to activate the ACh syn- thesis system in cardiomyocytes. Although a precise mechanism for the intracellular signal transduction of donepezil remains to be clarified, in addition to acetyl- cholinesterase inhibition, donepezil seems to activate ChAT transcription and translation in cardiomyocytes and skeletal muscle cells. In previous studies, we showed that ACh is involved in enhanced protein stability of HIF-1a, which nega- tively regulates cellular energy metabolism [4,14]. This finding is strongly supported by other studies showing that HIF-1a is responsible for inhibition of mitochon- drial function [28–30]. As indicated in the present study, exogenous ACh decreased oxygen consumption, resulting in downregulation of energy metabolism. By contrast, ChAT knockdown by transfection of its siRNA or the stable transfectants increased MTT activities and oxygen consumption. Taken together, these results suggest that intrinsic ACh, constitutively produced even at the basal level, contributes to the negative regulation of energy metabolism, leading to suppression of the function, and indicate that the pharmacological activation of the de novo ACh pro- duction pathway results in suppression of oxygen consumption. The increased production of reactive oxygen species in mitochondria in the case of enhanced oxygen demand causes cellular damage and death, i.e., apopto- sis, if not balanced with the capacities of the specific scavengers. As already reported in chronic heart fail- ure, cardiomyocytes are often exposed to such an imbalanced condition, resulting in apoptosis. In previ- ous studies, we showed that treatment with ACh pro- tects cardiomyocytes against ischemia and hypoxia through upregulation of antiapoptotic factors and downregulation of proapoptotic factors [3,4,14]. Fur- thermore, the present study reveals that such a cholin- ergic system is not specific for the central nervous system, but is evolutionarily conserved by non-neuro- nal cells, i.e. cardiomyocytes, in terms of ‘a molecular brake’ on cellular energy metabolism. So far, therapeu- tic modalities have not been considered from this point of view. Therefore, pharmacological activation of the intrinsic ACh-producing system in the myocardium with donepezil would be a new therapeutic strategy against chronic heart failure. Limitations Quantification of the level of ACh in cells is technically difficult, owing to the rapid degradation by acetylcho- linesterase. Therefore, it always requires acetylcholines- terase inhibitors. As HPLC is the only tool available with which to quantify ACh, and a highly specific anti- body against ACh cannot be obtained at present, it is quite difficult to perform real-time analysis of changes in intracellular ACh in various conditions. Further- more, owing to a lack of knowledge of the receptor, binding partners and action mechanisms for donepezil, it is still difficult to completely differentiate the acetyl- cholinesterase inhibitory action from acetylcholin- esterase-independent action. The development or introduction of new technologies is necessary for the detailed investigation of the intracellular ACh synthesis system. Conclusions The present study indicates that cardiomyocytes have an ACh synthesis system that is activated by musca- rinic agonists, suggesting that the cholinoceptive cho- linergic properties of cardiomyocytes could act as an amplification mechanism for vagal efferent effects in the sparsely innervated ventricular myocardium. The anti-Alzheimer’s disease drug donepezil also stimulates this ACh synthesis system and increases the ACh level in cardiomyocytes by mechanisms other than acetyl- cholinesterase inhibition. Fig. 8. Donepezil activates the transcriptional activity of ChAT in quadriceps femoris muscles. Mice given oral donepezil showed an increase in ChAT promoter activity in the quadriceps femoris mus- cle (380.0 ± 82.0%, P < 0.05, n = 7) transfected by the reporter vectors, as compared with nontreated mice. de novo ACh synthesis induced by ACh in cardiomyocytes Y. Kakinuma et al. 5120 FEBS Journal 276 (2009) 5111–5125 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... Kakinuma et al Experimental procedures The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health Care and use of the animals were also in strict accordance with the guiding principles of the Physiological Society of Japan Materials and reagents ACh, the muscarinic receptor agonist pilocarpine and the muscarinic receptor antagonist... 19659355) from the Ministry of Education, Science, Sports, and Culture of Japan, and by Health and Labor Sciences research grants (H15-PHYSI-001, H16-NANO-005) from the Ministry of Health, Labor, and Welfare of Japan References 1 Arikawa M, Katare RG, Kakinuma Y, Handa T, Ando M, Yamasaki F & Sato T (2007) Progression of ventricular dysfunction after myocardial infarction is prevented by anti-Alzheimer’s... mixture of type II and type IV collagenases and type XIV protease (SigmaAldrich), and inoculated onto dishes precoated with a mixture of gelatin, fibronectin, and laminin [33] HEK293 cells were transfected with a reporter vector driven by a rat promoter region ()1.9 kb) for ChAT Immunocytochemistry and confocal microscopy Rat cultured cardiomyocytes from neonates and adults were fixed in 4% paraformaldehyde,... a working curve for ACh showed adequate linearity in a range of 10 fmol to 1 pmol per injection In vivo reporter assay for ChAT promoter The reporter vector for ChAT (1 lg) and its internal control vector were conjugated with 200 lL of a nonpyrogenic suspension of microspheres of human serum albumin with perflutren for contrast enhancement during ultrasound imaging (Optison; GE Healthcare, Princeton,... synthesis induced by ACh in cardiomyocytes against ChAT (Chemicon) diluted 1 : 500 and a polyclonal antibody against VAChT (Santa Cruz) diluted 1 : 500 were used in conjunction with a horseradish peroxidase-conjugated secondary antibody The predicted molecular mass for ChAT is 70 kDa; however, an antibody against VAChT can show two possible bands, at 55 and 70 kDa, depending on the cell source For in vitro... 0.1% Triton X-100, and blocked with 1% BSA; this was followed by reaction with primary antibodies, including antibody against ChAT (Chemicon International, Temecula, CA, USA) diluted 1 : 100, antibody against VAChT (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1 : 100, and antibody against m2 (Alomone Lbs Ltd, Jerusalem, Israel) diluted 1 : 100 For F-actin staining in cardiomyocytes, Alexa... type II and type IV collagenases (Worthington Biochemical Corporation, Lakewood, NJ, USA) [31,32] The purity of cardiomyocytes in a culture dish was more than 92% They were cultured on glass-bottomed dishes precoated with 1% gelatin and 50 lgÆmL)1 fibronectin, or cultured in ordinary culture dishes coated with gelatin, for assessment of ACh content in cardiomyocytes Adult cardiomyocytes of rats and mice... (Abstract) 2 Ando M, Katare RG, Kakinuma Y, Zhang D, Yamasaki F, Muramoto K & Sato T (2005) Efferent vagal nerve stimulation protects heart against ischemiainduced arrhythmias by preserving connexin43 protein.Circulation 112, 164–170 3 Katare GR, Ando M, Kakinuma Y, Arikawa M, Handa T, Yamasaki F & Sato T (2009) Vagal nerve stimulation prevents reperfusion injury through inhibition of opening of mitochondrial... were injected into the left quadriceps femoris muscles of the mice, and were introduced with a sonoporator (Sonitron 2000; Nepa Gene, Chiba, Japan) [41,42] After introduction of the vectors, donepezil was orally administered at a daily dose of 5 mgÆkg)1 body weight for 3 days, and the muscular tissue was then isolated and homogenized Homogenates were centrifuged at 10 000 g for 5 min, and the supernatant... mitochondrial permeability transition pore independent of the bradycardiac effect J Thorac Cardiovasc Surg 137, 223–231 4 Kakinuma Y, Ando M, Kuwabara M, Katare RG, Okudela K, Kobayashi M & Sato T (2005) Acetylcholine from vagal stimulation protects cardiomyocytes against ischemia and hypoxia involving additive nonhypoxic induction of HIF-1alpha FEBS Lett 579, 2111–2118 5 Zhang Y, Kakinuma Y, Ando M, Katare RG, . Cholinoceptive and cholinergic properties of cardiomyocytes involving an amplification mechanism for vagal efferent effects in sparsely innervated ventricular myocardium Yoshihiko. properties of cardiomyocytes could act as an amplification mechanism for vagal efferent effects in the sparsely innervated ventricular myocardium. The anti-Alzheimer’s