Báo cáo khoa học: Cholinoceptive and cholinergic properties of cardiomyocytes involving an amplification mechanism for vagal efferent effects in sparsely innervated ventricular myocardium ppt

Rat cardiomyocytes expressed choline acetyltransferase ChAT in the cytoplasm and vesicular acetylcholine transporter with the vesicular structure identified by immunogold electron microsc

Cholinoceptive and cholinergic properties of

cardiomyocytes involving an amplification mechanism for vagal efferent effects in sparsely innervated ventricular myocardium

Yoshihiko Kakinuma1, Tsuyoshi Akiyama2and Takayuki Sato1

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.

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

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).

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.

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)1ACh (blue bars) (A) and 1 mmolÆL)1pilocarpine (green bars) (B) The promoter activity and the protein level induced by each agonist peaked at

8 h, however, 1 mmolÆL)1atropine inhibited ChAT transcription and translation, resulting in a decrease in the peak level.

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)1atropine 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).

Y Kakinuma et al de novo ACh synthesis induced by ACh in cardiomyocytes

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)1atropine tended to

atten-uate the ACh level induced by donepezil to 225.0 ± 43.0%

(atropine) (P < 0.05, n = 6).

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)1donepezil 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).

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 novosynthesis 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.