PKA phosphorylates multiple molecules involved in calcium (Ca 2+ ) handling in cardiac myocytes and is considered to be the predominant regulator of β-adrenergic receptor-mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular Ca 2+ storage and the magnitude of Ca 2+ movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCε. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol-and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses.

Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression-related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression-related behaviour. The NR2A subunit of the NMDA receptor is tyrosine-phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr-1325-Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock-in mice show antidepressant-like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock-in mice, DARPP-32 phosphorylation at Thr 34, which is important for the regulation of depression-related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src-induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor-mediated downstream signalling to modulate depression-related behaviour.

The ductus arteriosus (DA), an essential vascular shunt for fetal circulation, begins to close immediately after birth. Although Ca 2؉ influx through several membrane Ca 2؉ channels is known to regulate vasoconstriction of the DA, the role of the T-type voltage-dependent Ca 2؉ channel (VDCC) in DA closure remains unclear. Here we found that the expression of ␣1G, a T-type isoform that is known to exhibit a tissue-restricted expression pattern in the rat neonatal DA, was significantly upregulated in oxygenated rat DA tissues and smooth muscle cells (SMCs). Immunohistological analysis revealed that ␣1G was localized predominantly in the central core of neonatal DA at birth. DA SMC migration was significantly increased by ␣1G overexpression. Moreover, it was decreased by adding ␣1G-specific small interfering RNAs or using R(؊)-efonidipine, a highly selective T-type VDCC blocker. Furthermore, an oxygenationmediated increase in an intracellular Ca 2؉ concentration of DA SMCs was significantly decreased by adding ␣1G-specific siRNAs or using R(؊)-efonidipine. The ductus arteriosus (DA) 2 is an essential vascular shunt between the aortic arch and the pulmonary trunk during a fetal period (1). After birth, the DA closes immediately in accordance with its smooth muscle contraction and vascular remodeling, whereas the connecting vessels such as the aorta and pulmonary arteries remain open. When the DA fails to close after birth, the condition is known as patent DA, which is a common form of congenital heart defect. Patent DA is also a frequent problem with significant morbidity and mortality in premature infants. Investigating the molecular mechanism of DA closure is important not only for vascular biology but also for clinical problems in pediatrics.Voltage-dependent Ca 2ϩ channels (VDCCs) consist of multiple subtypes, named L-, N-, P/Q-, R-, and T-type. L-type VDCCs are known to play a primary role in regulating Ca 2ϩ influx and thus vascular tone in the development of arterial smooth muscle including the DA (2-4). Our previous study demonstrated that all T-type VDCCs were expressed in the rat DA (5). ␣1G subunit, especially, was the most dominant isoform among T-type VDCCs. The abundant expression of ␣1G subunit suggests that it plays a role in the vasoconstriction and vascular remodeling of the DA. In this regard, Nakanishi

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Voltage-gated Ca 2ϩ channels (VGCCs) 5 in the plasma membrane mediate the influx of Ca 2ϩ that serves as the second messenger of electrical signals to initiate many cellular events, including muscle contraction, neurotransmitter release, and gene expression (1). The pore-forming component of VGCCs is provided by the ␣ 1 subunit, a protein of about 2000 amino acid residues (2). This subunit contains four structurally conserved domains (I-IV), each of which contains six transmembrane segments (S1-S6) and a membrane-associated loop between S5 and S6 (called the pore loop or P-loop). The voltage sensitivity of VGCCs and structurally related cation channels is conveyed by the S4 segments, which contain several positively charged residues. S2 and S3 contain conserved negative charges that are likely to interact electrostatically with the positively charged residues of S4. The S5-P-loop-S6 region forms the pore domain (see Fig. 1).Mutations in the ␣ 1 subunits resulting in structural aberrations cause hereditary diseases (called Ca 2ϩ channelopathies) in mammals, such as incomplete congenital stationary night blindness, hypokalemic periodic paralysis, episodic ataxia type 2 and familial hemiplegic migraine in humans, and ataxia and seizures in mice (3-6). Most of the mutations are predicted to produce truncated ␣ 1 subunits with no significant channel activity by introducing a premature stop codon or leading to aberrant splicing. Single missense mutations can also result in the production of complete or partial loss-of-function subunits and these mutation sites are likely to be restricted to amino acid residues at transmembrane segments or loops known to be important for channel activity (7-9).A eukaryotic model organism, the yeast Saccharomyces cerevisiae, carries only one gene (designated CCH1) coding for a protein structurally homologous to the animal ␣ 1 subunits of VGCCs. Physiologically, the Cch1 protein is necessary for mating pheromone-induced Ca 2ϩ uptake (10 -13), store-operated Ca 2ϩ entry (14), endoplasmic reticulum stress-induced Ca 2ϩ uptake (15), and a hyperosmotic stressinduced increase in cytosolic Ca 2ϩ concentration (16).

-In order to characterize human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) sheets as a model for detecting drug-induced conduction disturbance, we examined their electrophysiological and electropharmacological properties by using the multi-electrode array system with a programmed electrical stimulation protocol. At pre-drug control, the conduction speed, effective refractory period and field potential duration were 0.14 ± 0.01 m/sec, 453 ± 10 msec and 361 ± 9 msec, respectively at a cycle length of 1,000 msec (n = 18). Shortening the pacing cycle length from 1,000 to 600 msec decreased the conduction speed and field potential duration, but prolonged the effective refractory period. Disopyramide, lidocaine and flecainide decreased the conduction speed but prolonged the effective refractory period and field potential duration, whereas the reverse was true for verapamil. Thus, conduction properties of the cell sheet may largely depend on the extent of Na + channel availability as is the case in the human ventricle. Importantly, there was no relationship between the conduction delay and 1 st spike amplitude reduction after the treatment of Na + channel blockers. These findings may provide crucial guide on future application of this new technology for early phase safety pharmacological screening of new chemical entities.

Azithromycin has been reported to increase the risk of death from cardiovascular causes among patients with high baseline risk. Since the information is still limited to bridge the gap between electrophysiological properties of azithromycin in vitro and cardiac death in patients, we initially assessed its electropharmacological effects in doses of 3 and 30 mg/kg, i.v., with the halothane-anesthetized dogs (n = 4). The low dose provided 5.2 times higher than the therapeutic concentration, whereas the high dose attained 17.0 times higher. The high dose delayed the ventricular repolarization in a reverse use-dependent manner, reflecting blockade of the rapid component of delayed rectifier K(+) current, and the potency was relatively weak; namely, maximum change in QTc was +20 ms (+5.6%). The high dose also induced the negative inotropic effect possibly through Ca(2+) channel-independent pathway. In order to clarify proarrhythmic risk, 30 mg/kg, i.v., of azithromycin was examined with the chronic atrioventricular block dogs (n = 4). Azithromycin neither induced torsade de pointes nor affected beat-to-beat variability of repolarization. Thus, azithromycin can be considered to lack proarrhythmic potential, but caution has to be paid on its use for patients with left ventricular dysfunction.

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