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.

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Abstract. We examined the involvement of the Na + /Ca 2+ exchanger in the automaticity of the pulmonary vein myocardium with a specific inhibitor, SEA0400. Action potentials were recorded from the myocardial layer of isolated guinea-pig pulmonary vein preparations, and Ca 2+transients were recorded from the cardiomyocytes. Spontaneous electrical activity was observed in 17.7% of the preparations, which was inhibited by either SEA0400 or ryanodine. In quiescent preparations, ouabain induced electrical activity and spontaneous Ca 2+ transients, which were inhibited by SEA0400, as well as ryanodine. These results provide pharmacological evidence that the Na + /Ca 2+ exchanger underlies the automaticity of the pulmonary vein myocardium.Keywords: pulmonary vein myocardium, NaPulmonary veins are considered to be involved in the initiation and maintenance of atrial fibrillation, one of the most frequent arrhythmias in clinical practice (1). Pulmonary veins contain a myocardial layer, whose electrical activity is considered to underlie their arrhythmogenic activity (2). The pulmonary vein myocardium has different electrophysiological properties from those of the working myocardium, including lower density of I K1 and a less negative resting membrane potential (3). The precise mechanisms of the pulmonary vein electrical activity as well as its pharmacological properties are now receiving attention as the basis to develop an effective therapeutic strategy against atrial fibrillation.The Na + /Ca 2+ exchanger (NCX) is involved in the physiological and pathophysiological regulation of Ca 2+ concentration in the myocardium. It functions both in the forward (Ca 2+ extrusion) and reverse (Ca 2+ influx) modes, and its functional role may vary with the region and the condition of the myocardium (4, 5). The forward mode NCX activity (inward current) is the major pathway for Ca 2+ extrusion from the cytoplasm and is also considered to be involved in the normal pacemaking of the rabbit sinoatrial node (6). It was postulated that the Ca 2+ released from the sarcoplasmic reticulum (SR) during the diastolic period is pumped out of the cell through the forward mode NCX, which generates an inward current that contributes to the diastolic depolarization of the pacemaker. Although it is possible that such a mechanism is involved in the automaticity of other myocardial regions including the pulmonary vein myocardium, pharmacological evidence is limited because of the lack of an NCX inhibitor with sufficient specificity. SEA0400 {2-[4-[(2,5-difluorophenyl) methoxy] phenoxy]-5-ethoxyaniline} is a potent and selective inhibitor of NCX in cultured neurons, astrocytes, microglia, dog sarcolemmal vesicles, and cultured rat myocytes with negligible affinities towards other transporters, ion channels, and receptors (7). We have previously shown that SEA0400 is a specific inhibitor of NCX in the myocardium (8, 9). SEA0400 (1 μM), which inhibited the NCX current by more than 80%, had no effect on the Na + current, L-type Ca 2+ current, delayed recti...

Abstract-SarcolemmalIn a previous article, we showed that I Ks enhancement by elevation of [Ca 2ϩ ] i was inhibited by a calmodulin (CaM) inhibitor, but not by an inhibitor of CaM-dependent kinase II. 6 Although this finding suggests that the allosteric regulation of CaM is crucial for Ca 2ϩ -sensitive I Ks alterations, the detailed underlying mechanism remains unknown. Recently, we found that I Ks was enhanced by nitric oxide (NO) via a cGMP-independent mechanism. 7 Because allosteric interaction of Ca 2ϩ /CaM complex with NO synthase (NOS) is a major mechanism of NOS activation and NO release, 8,9 we tested in the present study if NO plays an important role in the Ca 2ϩ -sensitive modulation of I Ks in cardiac myocytes.

Abstract. Developmental changes in excitation-contraction mechanisms were examined in the ventricular myocardium from fetal, neonatal, and 1-, 2-, and 4-week-old mice. In isolated tissue, the negative inotropic effect of nifedipine decreased, while that of ryanodine increased with age. Action potential duration decreased with age, especially during the late fetal period. In ventricular cardiomyocytes, fluorescence imaging revealed that the sarcoplasmic reticulum increases progressively during pre-and postnatal development. t-Tubules were absent in the fetus and neonate, were observed only in the subsarcolemmal region at 1 week after birth, and were present throughout the cytoplasm at 2 and 4 weeks after birth. The amplitude of Ca 2+ transients, as well as its ryanodine-sensitive component, increased with age. In the neonate and 1-week-old mice, Ca 2+ at the cell center showed slower rise than the subsarcolemmal region, but in 2-and 4-week-old mice, Ca 2+ increased simultaneously across the entire width of the cell. These results suggest that in the mouse ventricular myocardium, the shortening of the action potential during the late fetal period and the development of t-tubule-sarcoplasmic reticulum coupling during the second postnatal week largely contribute to the developmental increase in the dependence of contraction on sarcoplasmic reticulum function.

The functional role of the sodium-calcium exchanger in mouse ventricular myocardium was evaluated with a newly developed specific inhibitor, SEA0400. Contractile force and action potential configuration were measured in isolated ventricular tissue preparations, and cell shortening and Ca2+ transients were measured in indo-1-loaded isolated ventricular cardiomyocytes. SEA0400 increased the contractile force, cell shortening and Ca2+ transient amplitude, and shortened the late plateau phase of the action potential. alpha-adrenergic stimulation by phenylephrine produced a sustained decrease in contractile force, cell shortening and Ca2+ transient amplitude, which were all inhibited by SEA0400. Increasing the contraction frequency resulted in a decrease in contractile force in the absence of drugs (negative staircase phenomenon). This frequency-dependent decrease was attenuated by SEA0400 and enhanced by phenylephrine. Phenylephrine increased the Ca2+ sensitivity of contractile proteins in isolated ventricular cardiomyocytes, while SEA0400 had no effect. These results provide the first pharmacological evidence in the mouse ventricular myocardium that inward current generated by Ca2+ extrusion through the sodium-calcium exchanger during the Ca2+ transient contributes to the action potential late plateau, that alpha-adrenoceptor-mediated negative inotropy is produced by enhanced Ca2+ extrusion through the sodium-calcium exchanger, and that the negative staircase phenomenon can be explained by increased Ca2+ extrusion through the sodium-calcium exchanger at higher contraction frequencies.

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Abstract. Fluorescence resonance energy transfer (FRET) with green fluorescent protein (GFP) variants has become widely used for biochemical research. In order to expand the choice of fluorescent range in FRET analysis, we designed various color versions of the FRET-based probes for caspase activity, in which the substrate sequence of the caspase was sandwiched by donor and acceptor fluorescent proteins, and studied the potential of these color versions as fluorescent indicators. Six color versions were constructed by a combination of cyan fluorescent protein (CFP), GFP, yellow fluorescent protein (YFP), and DsRed. Real-time monitoring in single cells revealed that all probes could detect caspase activation during tumor necrosis factor (TNF)-a-induced cell death as a fluorescent change. GFP-DsRed and YFP-DsRed were as sensitive as CFP-YFP, and CFP-DsRed also showed a large fluorescent change. By using two probes, CFP-DsRed and YFP-DsRed, we carried out simultaneous multi-FRET analysis and revealed that the initiator-and effector-caspases were activated almost simultaneously in TNFa -induced cell death. These findings may give experimental bases for the development of novel techniques to analyze multi-events simultaneously in single cells by using FRET probes in combination.