Tsuyoshi Akiyama

Heart‐Specific Overexpression of Choline Acetyltransferase Gene Protects Murine Heart Against Ischemia Through Hypoxia‐Inducible Factor‐1α–Related Defense Mechanisms

Background Murine and human ventricular cardiomyocytes rich in acetylcholine (Ach) receptors are poorly innervated by the vagus, compared with whole ventricular innervation by the adrenergic nerve. However, vagal nerve stimulation produces a favorable outcome even in the murine heart, despite relatively low ventricular cholinergic nerve density. Such a mismatch and missing link suggest the existence of a nonneuronal cholinergic system in ventricular myocardium. Methods and Results To examine the role of the nonneuronal cardiac cholinergic system, we generated choline acetyltransferase (ChAT)–expressing cells and heart‐specific ChAT transgenic (ChAT‐tg) mice. Compared with cardiomyocytes of wild‐type (WT) mice, those of the ChAT‐tg mice had high levels of ACh and hypoxia‐inducible factor (HIF)‐1α protein and augmented glucose uptake. These phenotypes were also reproduced by ChAT‐overexpressing cells, which utilized oxygen less. Before myocardial infarction (MI), the WT and ChAT‐tg mice showed similar hemodynamics; after MI, however, the ChAT‐tg mice had better survival than did the WT mice. In the ChAT‐tg hearts, accelerated angiogenesis at the ischemic area, and accentuated glucose utilization prevented post‐MI remodeling. The ChAT‐tg heart was more resistant to ischemia–reperfusion injury than was the WT heart. Conclusions These results suggest that the activated cardiac ACh‐HIF‐1α cascade improves survival after MI. We conclude that de novo synthesis of ACh in cardiomyocytes is a pivotal mechanism for self‐defense against ischemia.

A Non-Neuronal Cardiac Cholinergic System Plays a Protective Role in Myocardium Salvage during Ischemic Insults

Background In our previous study, we established the novel concept of a non-neuronal cardiac cholinergic system–cardiomyocytes produce ACh in an autocrine and/or paracrine manner. Subsequently, we determined the biological significance of this system–it played a critical role in modulating mitochondrial oxygen consumption. However, its detailed mechanisms and clinical implications have not been fully investigated. Aim We investigated if this non-neuronal cardiac cholinergic system was upregulated by a modality other than drugs and if the activation of the system contributes to favorable outcomes. Results Choline acetyltransferase knockout (ChAT KO) cells with the lowest cellular ACh levels consumed more oxygen and had increased MTT activity and lower cellular ATP levels compared with the control cells. Cardiac ChAT KO cells with diminished connexin 43 expression formed poor cell–cell communication, evidenced by the blunted dye transfer. Similarly, the ChAT inhibitor hemicholinium-3 decreased ATP levels and increased MTT activity in cardiomyocytes. In the presence of a hypoxia mimetic, ChAT KO viability was reduced. Norepinephrine dose-dependently caused cardiac ChAT KO cell death associated with increased ROS production. In in vivo studies, protein expression of ChAT and the choline transporter CHT1 in the hindlimb were enhanced after ischemia-reperfusion compared with the contralateral non-treated limb. This local effect also remotely influenced the heart to upregulate ChAT and CHT1 expression as well as ACh and ATP levels in the heart compared with the baseline levels, and more intact cardiomyocytes were spared by this remote effect as evidenced by reduced infarction size. In contrast, the upregulated parameters were abrogated by hemicholinium-3. Conclusion The non-neuronal cholinergic system plays a protective role in both myocardial cells and the entire heart by conserving ATP levels and inhibiting oxygen consumption. Activation of this non-neuronal cardiac cholinergic system by a physiotherapeutic modality may underlie cardioprotection through the remote effect of hindlimb ischemia-reperfusion.

Centrally administered ghrelin activates cardiac vagal nerve in anesthetized rabbits.

Although central ghrelin has cardioprotective effect through inhibiting sympathetic nerve activity, the effects of central ghrelin on cardiac vagal nerve remain unknown. We investigated the effects of centrally administered ghrelin on cardiac autonomic nerve activities using microdialysis technique. A microdialysis probe was implanted in the right atrial wall adjacent to the sinoatrial node of an anesthetized rabbit and was perfused with Ringers solution containing a cholinesterase inhibitor, eserine. After injection of ghrelin (1 nmol) into the right lateral cerebral ventricle, norepinephrine (NE) and acetylcholine (ACh) concentrations in the dialysate samples were measured as indices of NE and ACh release from nerve endings to the sinoatrial node using high-performance liquid chromatography. Heart rate was 270±4 bpm at baseline and decreased gradually after ghrelin injection to 234±9 bpm (P<0.01) at 60-80 min, followed by gradual recovery. Dialysate ACh concentration was 5.5±0.8 nM at baseline and increased gradually after ghrelin injection to 8.8±1.2 nM (P<0.01) at 60-80 min; the concentration started to decrease gradually from 100 to 120 min after injection reaching 5.6±0.8 nM at 160-180 min. Central ghrelin did not change mean arterial pressure or dialysate NE concentration. The elevated dialysate ACh concentration declined rapidly after transection of cervical vagal nerves. These results indicate that centrally administered ghrelin activates cardiac vagal nerve.

Survival benefit of ghrelin in the heart failure due to dilated cardiomyopathy.

Although ghrelin has been demonstrated to improve cardiac function in heart failure, its therapeutic efficacy on the life expectancy remains unknown. We aim to examine whether ghrelin can improve the life survival in heart failure using a mouse model of inherited dilated cardiomyopathy (DCM) caused by a deletion mutation ΔK210 in cardiac troponin T (cTnT). From 30 days of age, ghrelin (150 μg/kg) was administered subcutaneously to DCM mice once daily, control mice received saline only. The survival rates were compared between the two groups for 30 days. After 30‐day treatment, functional and morphological measurements were conducted. Ghrelin‐treated DCM mice had significantly prolonged life spans compared with saline‐treated control DCM mice. Echocardiography showed that ghrelin reduced left ventricular (LV) end‐diastolic dimensions and increased LV ejection fraction. Moreover, histoanatomical data revealed that ghrelin decreased the heart‐to‐body weight ratio, prevented cardiac remodeling and fibrosis, and markedly decreased the expression of brain natriuretic peptide. Telemetry recording and heart rate variability analysis showed that ghrelin suppressed the excessive cardiac sympathetic nerve activity (CSNA) and recovered the cardiac parasympathetic nerve activity. These results suggest that ghrelin has therapeutic benefits for survival as well as for the cardiac function and remodeling in heart failure probably through suppression of CSNA and recovery of cardiac parasympathetic nerve activity.

In vivo monitoring of acetylcholine release from cardiac vagal nerve endings in anesthetized mice

We applied a microdialysis technique to the left ventricular myocardium of anesthetized mice and tried to monitor acetylcholine (ACh) release from cardiac vagal nerves. Transection of bilateral cervical vagal nerves decreased dialysate ACh concentration. Electrical stimulation of the left cervical vagal nerve increased dialysate ACh concentration in proportion to the frequency of stimulation. Intravenous administration of hexamethonium, prevented the increase in dialysate ACh concentration during vagal nerve stimulation, indicating that ACh in the dialysate primarily reflects ACh released from post-ganglionic cardiac vagal nerves. Microdialysis permits monitoring of ACh release from post-ganglionic cardiac vagal nerves that are most likely to be innervating the left ventricle in mice.

Acute effects of arterial baroreflex on sympathetic nerve activity and plasma norepinephrine concentration

Arterial pressure (AP) elevates as a logarithmic function of exogenously administered dose of norepinephrine (NE). In contrast, AP is nearly linearly correlated with efferent sympathetic nerve activity (SNA) during acute baroreflex intervention. The present study aimed at quantifying the relationship between SNA and plasma NE concentration during acute baroreflex intervention. Carotid sinus regions were isolated from systemic circulation in five Wistar Kyoto rats, and carotid sinus pressure was changed among 60, 100, 120, 140, and 180 mm Hg every 2 min. Arterial blood (0.2 ml) was obtained at each pressure level for plasma NE measurement. Maximum AP and minimum AP were 153.34 ± 6.28 and 67.31 ± 4.92 mm Hg, respectively, in response to pressure perturbation. Plasma NE correlated linearly with SNA for individual animal data (slope: 0.957 ± 0.090 pg · ml(-1) · %(-1), intercept: 46.57 ± 7.22 pg/ml, r(2): ranged from 0.923 to 0.992) and also for group averaged data (NE = 0.956 × SNA + 47.97, r(2 )= 0.982). Blockade of neuronal NE uptake by intravenous desipramine (1 mg/kg) administration increased the slope (2.966 ± 0.686 pg · ml(-1) · %(-1), P < 0.05) and the intercept (168.73 ± 28.53 pg/ml, P < 0.01) of the plasma NE-SNA relationship. These results indicate that the relationship between SNA and plasma NE concentration was nearly linear within the normal physiological range of acute baroreflex control of AP. While plasma NE concentration can reflect changes in SNA, it may also overestimate the sympathetic outflow from the central nervous system when neuronal NE uptake is impaired systemically.

Sympathetic afferent stimulation inhibits central vagal activation induced by intravenous medetomidine in rats

To examine whether sympathetic afferent stimulation (SAS) inhibits central vagal activation induced by α2‐adrenergic stimulation.

Monoamine oxidase-induced hydroxyl radical production and cardiomyocyte injury during myocardial ischemia-reperfusion in rats.

ABSTRACT To elucidate the involvement of monoamine oxidase (MAO) in hydroxyl radical production and cardiomyocyte injury during ischemia as well as after reperfusion, we applied microdialysis technique to the heart of anesthetized rats. Dialysate samples were collected during 30u2009min of induced ischemia followed by 60u2009min of reperfusion. We monitored dialysate 3,4-dihydrobenzoic acid (3,4-DHBA) concentration as an index of hydroxyl radical production using a trapping agent (4-hydroxybenzoic acid), and dialysate myoglobin concentration as an index of cardiomyocyte injury in the ischemic region. The effect of local administration of a MAO inhibitor, pargyline, was investigated. Dialysate 3,4-DHBA concentration increased from 1.9u2009±u20090.5u2009nM at baseline to 3.5u2009±u20090.7u2009nM at 20–30u2009min of occlusion. After reperfusion, dialysate 3,4-DHBA concentration further increased reaching a maximum (4.5u2009±u20090.3u2009nM) at 20–30u2009min after reperfusion, and stabilized thereafter. Pargyline suppressed the averaged increase in dialysate 3,4-DHBA concentration by ∼72% during occlusion and by ∼67% during reperfusion. Dialysate myoglobin concentration increased from 235u2009±u200960u2009ng/ml at baseline to 1309u2009±u2009298u2009ng/ml at 20–30u2009min after occlusion. After reperfusion, dialysate myoglobin concentration further increased reaching a peak (5833u2009±u20091017u2009ng/ml) at 10–20u2009min after reperfusion, and then declined. Pargyline reduced the averaged dialysate myoglobin concentration by ∼56% during occlusion and by ∼41% during reperfusion. MAO plays a significant role in hydroxyl radical production and cardiomyocyte injury during ischemia as well as after reperfusion.

Effects of intravenous magnesium infusion on in vivo release of acetylcholine and catecholamine in rat adrenal medulla.

We applied microdialysis technique to the left adrenal medulla of anesthetized rats and examined the effects of intravenous Mg(2+) infusion on presynaptic acetylcholine (ACh) release and postsynaptic catecholamine release induced by electrical stimulation of splanchnic nerves. The dialysis probes were perfused with Ringers solution containing neostigmine. Low-dose MgSO4 (25 μmol/kg/min for 30 min i.v.) increased mean plasma Mg(2+) concentration to 2.5mM; the administration suppressed norepinephrine (NE) release by approximately 30% and epinephrine (Epi) release by approximately 20%, but did not affect ACh release. High-dose MgSO4 (50 μmol/kg/min for 30 min i.v.) increased mean plasma Mg(2+) concentration to 3.8mM; the administration suppressed ACh release by approximately 25%, NE release by approximately 60% and Epi release by approximately 45%. Administration of Na2SO4 (50 μmol/kg/min for 30 min i.v.) did not change the release of ACh, NE or Epi. Local administration of nifedipine (200 μM) suppressed NE release by approximately 40% and Epi release by approximately 30%, but did not affect ACh release. In the presence of nifedipine, low-dose MgSO4 did not suppress the release of ACh, or further suppress NE or Epi compared to nifedipine alone, but high-dose MgSO4 suppressed ACh release by approximately 25% and further suppressed NE release by approximately 60% and Epi release by approximately 50% compared to nifedipine alone. In conclusion, intravenous administration of Mg(2+) inhibits both presynaptic ACh release and postsynaptic catecholamine release in the adrenal medulla, but L-type Ca(2+) channel-controlled catecholamine release may be more sensitive to Mg(2+) than non-L-type Ca(2+) channel-controlled ACh release.

Myocardial interstitial levels of serotonin and its major metabolite 5-hydroxyindole acetic acid during ischemia-reperfusion

The aim of this study was to examine the accumulation of serotonin (5-HT) and degradation of 5-HT taken up into cells in the ischemic region during myocardial ischemia-reperfusion. Using microdialysis technique in anesthetized rats, we monitored myocardial interstitial levels of 5-HT and its metabolite produced by monoamine oxidase (MAO), 5-hydroxyindole acetic acid (5-HIAA), during 30-min coronary occlusion followed by 45-min reperfusion, and investigated the effects of local administration of the MAO inhibitor pargyline and the 5-HT uptake inhibitor fluoxetine. In the vehicle group, the dialysate 5-HT concentration increased from 1.3 ± 0.2 nM at baseline to 29.6 ± 2.8 nM at 22.5-30 min of occlusion, but the dialysate 5-HIAA concentration did not change from baseline (9.9 ± 1.1 nM). Upon reperfusion, the dialysate 5-HT concentration increased further to a peak (34.2 ± 4.2 nM) at 0-7.5 min and then declined. The dialysate 5-HIAA concentration increased to 31.9 ± 5.2 nM at 7.5-15 min of reperfusion and maintained this high level until 45 min. Pargyline markedly suppressed the increase in dialysate 5-HIAA concentration after reperfusion and increased the averaged dialysate 5-HT concentration during the reperfusion period. Fluoxetine suppressed the increase in dialysate 5-HT concentration during occlusion but did not change dialysate 5-HT or 5-HIAA concentration after reperfusion. During ischemia, 5-HT secreted from ischemic tissues accumulates but 5-HT degradation by MAO is suppressed. After reperfusion, degradation of 5-HT taken up into cells is enhanced and contributes to the clearance of accumulated 5-HT. This degradation following cellular uptake is dependent on MAO activity but not the fluoxetine-sensitive uptake transporter.nnnNEW & NOTEWORTHYnBy monitoring myocardial interstitial levels of 5-HT and its metabolite, 5-hydroxyindole acetic acid, we investigated 5-HT kinetics during myocardial ischemia-reperfusion. 5-HT accumulates but 5-HT degradation is suppressed during ischemia. After reperfusion, 5-HT degradation is enhanced and this degradation is dependent on monoamine oxidase activity but not fluoxetine-sensitive uptake transporter.