The functional effect of adenoviral Na+/Ca2+ exchanger overexpression in rabbit myocytes depends on the activity of the Na+/K+-ATPase.

OBJECTIVES: The functional consequences of Na+/Ca2+ exchanger (NCX) overexpression in heart failure have been controversially discussed. NCX function strongly depends on intracellular sodium which has been shown to be increased in heart failure. METHODS AND RESULTS: We investigated the Na+/K+-ATPase (NKA) inhibitor ouabain (0.5-16 micromol/l) in electrically stimulated, isotonically contracting adult rabbit cardiocytes overexpressing NCX after adenoviral gene transfer (Ad-NCX-GFP, 48 h culture time). Myocytes transfected with adenovirus encoding for green fluorescent protein (Ad-GFP) served as a control. Contractions were analyzed by video-edge detection. In the Ad-NCX-GFP group, the maximum inotropic response was significantly reduced by 50.7% (P<0.05). This was a result of an enhanced susceptibility to contracture after exposure to the drug (median concentration (25-75%): 4 (4-8) vs. 8 (6-16) micromol/l, P<0.05). When analyzing relaxation before contracture, the maximum relaxation velocity was reduced (0.15+/-0.04 vs. 0.27+/-0.04 microm/s, P<0.05) and the time from peak shortening to 90% of relaxation was increased (298+/-39 vs. 185+/-15 ms, P<0.05). No differences in systolic and diastolic parameters were observed with the Na+ channel modulator BDF9198 (1 micromol/l). CONCLUSIONS: Inhibition of NKA by ouabain induces a combined diastolic and systolic dysfunction in NCX overexpressing rabbit myocytes. This may be the consequence of cytoplasmic Ca2+ overload due to inhibition of forward mode or induction of reverse mode Na+/Ca2+ exchange. In end-stage failing human myocardium and during digitalis treatment this mechanism may be of major importance.

Out of Asia: mitochondrial DNA evidence for an Oriental origin of tiger frogs, genus Hoplobatrachus.

Most examples of intercontinental dispersal events after the Miocene contact between Africa and Asia involve mammal lineages. Among amphibians, a number of probably related groups are known from both continents, but their phylogenies are so far largely unresolved. To test the hypothesis of Miocene dispersal against a Mesozoic vicariance scenario in the context of Gondwana fragmentation, we analyzed fragments of the mitochondrial 16S rRNA gene (572 bp) in 40 specimens of 34 species of the anuran family Ranidae. Results corroborated the monophyly of tiger frogs (genus Hoplobatrachus), a genus with representatives in Africa and Asia. The African H. occipitalis was the sister group of the Asian H. crassus, H. chinensis, and H. tigerinus. Hoplobatrachus was placed in a clade also containing the Asian genera Euphlyctis and Nannophrys. Combined analysis of sequences of 16S and 12S rRNA genes (total 903 bp) in a reduced set of taxa corroborated the monophyly of the lineage containing these three genera and identified the Asian genus Fejervarya as its possible sister group. The fact that the African H. occipitalis is nested within an otherwise exclusively Asian clade indicates its probable Oriental origin. Rough molecular clock estimates did not contradict the assumption that the dispersal event took place in the Miocene. Our data further identified a similar molecular divergence between closely related Asian and African species of Rana (belonging to the section Hylarana), indicating that Neogene intercontinental dispersal also may have taken place in this group and possibly in rhacophorid treefrogs.

Cardiac phosphodiesterase 5 (cGMP-specific) modulates beta-adrenergic signaling in vivo and is down-regulated in heart failure.

Recent studies implicate increased cGMP synthesis as a postreceptor contributor to reduced cardiac sympathetic responsiveness. Here we provide the first evidence that modulation of this interaction by cGMP-specific phosphodiesterase PDE5A is also diminished in failing hearts, providing a novel mechanism for blunted beta-adrenergic signaling in this disorder. In normal conscious dogs chronically instrumented for left ventricular pressure-dimension analysis, PDE5A inhibition by EMD82639 had modest basal effects but markedly blunted dobutamine-enhanced systolic and diastolic function. In failing hearts (tachypacing model), however, EMD82639 had negligible effects on either basal or dobutamine-stimulated function. Whole myocardium from failing hearts had 50% lower PDE5A protein expression and 30% less total and EMD92639-inhibitable cGMP-PDE activity. Although corresponding myocyte protein and enzyme activity was similar among groups, the proportion of EMD82639-inhibitable activity was significantly lower in failure cells. Immunohistochemistry confirmed PDE5A expression in both the vasculature and myocytes of normal and failing hearts, but there was loss of z-band localization in failing myocytes that suggested altered intracellular localization. Thus, PDE5A regulation of cGMP in the heart can potently modulate beta-adrenergic stimulation, and alterations in enzyme localization and reduced synthesis may blunt this pathway in cardiac failure, contributing to dampening of the beta-adrenergic response.

Mitochondrial ATP-sensitive potassium channels inhibit apoptosis induced by oxidative stress in cardiac cells.

Mitochondria can either enhance or suppress cell death. Cytochrome c release from mitochondria and depolarization of the mitochondrial membrane potential (DeltaPsi) are crucial events in triggering apoptosis. In contrast, activation of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels prevents lethal ischemic injury in vivo, implicating these channels as key players in the process of ischemic preconditioning. We probed the relationship between mitoK(ATP) channels and apoptosis in cultured neonatal rat cardiac ventricular myocytes. Incubation with 200 micromol/L hydrogen peroxide induced TUNEL positivity, cytochrome c translocation, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and dissipation of DeltaPsi. Pharmacological opening of mitoK(ATP) channels by diazoxide (100 micromol/L) preserved mitochondrial integrity and suppressed all markers of apoptosis. Diazoxide prevented DeltaPsi depolarization in a concentration-dependent manner (EC(50) approximately 40 micromol/L, with saturation by 100 micromol/L), as shown by both flow cytometry and quantitative image analysis of cells stained with fluorescent DeltaPsi indicators. These cytoprotective effects of diazoxide were reproduced by pinacidil, another mitoK(ATP) agonist, and blocked by the mitoK(ATP) channel antagonist 5-hydroxydecanoate (500 micromol/L). Our findings identify a novel mitochondrial pathway that is protective against apoptosis. The results also pinpoint mitoK(ATP) channels as logical therapeutic targets in diseases of enhanced apoptosis and oxidative stress.

Evidence against functional heteromultimerization of the KATP channel subunits Kir6.1 and Kir6.2.

K(ATP) channels consist of pore-forming potassium inward rectifier (Kir6.x) subunits and sulfonylurea receptors (SURs). Although Kir6.1 or Kir6.2 coassemble with different SUR isoforms to form heteromultimeric functional K(ATP) channels, it is not known whether Kir6.1 and Kir6.2 coassemble with each other. To define the molecular identity of K(ATP) channels, we used adenoviral gene transfer to express wild-type and dominant-negative constructs of Kir6.1 and Kir6.2 in a heterologous expression system (A549 cells) and in native cells (rabbit ventricular myocytes). Dominant-negative (DN) Kir6.2 gene transfer suppressed current through heterologously expressed SUR2A + Kir6.2 channels. Conversely, DN Kir6.1 suppressed SUR2B + Kir6.1 current but had no effect on coexpressed SUR2A + Kir6. 2. We next probed the ability of Kir6.1 and Kir6.2 to affect endogenous K(ATP) channels in adult rabbit ventricular myocytes, using adenoviral vectors to achieve efficient gene transfer. Infection with the DN Kir6.2 virus for 72 h suppressed pinacidil-inducible K(ATP) current density measured by whole-cell patch clamp. However, there was no effect of infection with the DN Kir6.1 on the K(ATP) current. Based on these functional assays, we conclude that Kir6.1 and Kir6.2 do not heteromultimerize with each other and that Kir6.2 is the sole K(ATP) pore-forming subunit in the surface membrane of heart cells.

Phylogenetic relationships in the Ranidae. Independent origin of direct development in the genera Philautus and Taylorana.

Phylogenetic relationships within a group of Ranidae were studied, particularly between the genera Philautus and Taylorana, to determine whether their original reproductive mode (direct development) appeared independently. To study these relationships, we used the DNA sequences of the 5' end of the gene coding for mitochondrial ribosome small subunit in 28 species. Parsimony analysis resulted in two equiparsimonious trees, 867 steps long (CI = 0.382; RI = 0.429). Data saturation was studied and we applied weighting (weight of 5 for transversions and 1 for transitions) to enhance the phylogenetic information. The tree we obtained (2,212 steps long, CI = 0.408; RI = 0.482) is best supported and allows us to determine clearly that direct development has appeared independently in Philautus and Taylorana. In addition, it is confirmed that the genus Rana sensu lato is heterogeneous. The genera Occidozyga and Phrynoglossus form a clade basal to all the group but this position is not supported. The inclusion of the Rhacophorinae in the Ranidae is confirmed. Finally, this study confirms that the relationships within the group Tomopterna sensu Boulenger, 1918 should be re-evaluated.

Activation of mitochondrial ATP-dependent potassium channels by nitric oxide.

BACKGROUND: Nitric oxide (NO) has been implicated as a mediator of "second-window" ischemic preconditioning, and mitochondrial ATP-dependent K(+) (mitoK(ATP)) channels are the likely effectors. The links between NO and mitoK(ATP) channels are unknown. METHODS AND RESULTS: We measured mitochondrial redox potential as an index of mitoK(ATP) channel opening in rabbit ventricular myocytes. The NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP, 0.1 to 1 mmol/L) oxidized the mitochondrial matrix dose-dependently without activating sarcolemmal K(ATP) channels. SNAP-induced oxidation was blocked by the selective mitoK(ATP) channel blocker 5-hydroxydecanoate and by the NO scavenger 2-(4-carboxyphenyl)-4,4', 5,5'-tetramethylimidazole-1-oxyl-3-oxide. SNAP-induced mitochondrial oxidation was detectable either by photomultiplier tube recordings of flavoprotein fluorescence or by confocal imaging. SNAP also enhanced the oxidative effects of diazoxide when both agents were applied together. Exposure to 1 mmol/L 8Br-cGMP failed to mimic the effects of SNAP. CONCLUSIONS: NO directly activates mitoK(ATP) channels and potentiates the ability of diazoxide to open these channels. These results provide novel mechanistic links between NO-induced cardioprotection and mitoK(ATP) channels.

Molecular composition of mitochondrial ATP-sensitive potassium channels probed by viral Kir gene transfer.

Heart cells contain ATP-sensitive potassium (KATP) channels in both the sarcolemma and the inner mitochondrial membrane. The sarcolemmal channels are believed to be heteromultimeric complexes of sulfonylurea receptors (SUR) and potassium inward rectifier (Kir) gene products, but the molecular identity of mitochondrial KATP (mitoKATP) channels remains unclear. To probe the molecular composition of KATP channels, we used adenoviral gene transfer to express wild-type (WT) and dominant-negative (AFA) constructs of Kir6.1 and 6.2 in rabbit ventricular myocytes. None of the Kir6.1 or 6.2 constructs affected mitoKATPchannel activity as assayed by confocal imaging of flavoprotein fluorescence, contradicting the proposal, based on subcellular antibody localization, that Kir6.1 forms part of mitoKATP channels. As previously reported, dominant-negative Kir6.2 gene transfer suppressed sarcolemmal KATP current, while Kir6.1 constructs had no effect on sarcolemmal activity. Immunohistochemistry with an anti-Kir6.1 antibody revealed expression of this protein in heart but no apparent co-localization with mitochondria. Thus, the available evidence indicates that both Kir6.1 and 6.2 are expressed in ventricular myocytes, but neither plays a discernible functional role in the mitoKATP channel.

Cardiac and hemodynamic effects of the sinus node inhibitor tedisamil dihydrochloride in patients with congestive heart failure due to dilated cardiomyopathy.

Clinical and experimental investigations have demonstrated an inverse relation between heart rate and myocardial performance in patients with congestive heart failure. Accordingly, this study was designed to investigate the hemodynamic effect of the novel bradycardic compound tedisamil in patients with heart failure. We hypothesized that tedisamil would reduce heart rate and thereby improve hemodynamic parameters of failing hearts with an inverse force-frequency relation. Tedisamil was administered intravenously in nine patients with dilated cardiomyopathy (NYHA II-III). Hemodynamic measurements by right heart catheterization were carried out at time points -30, 10, 20 min, 1, 2, 4, and 6 h. Tedisamil decreased heart rate significantly from 84 +/- 6 beats/min to 73 +/- 4 beats/min (at 10 min; p < 0.05). Stroke volume index remained unchanged, and cardiac index tended to decrease transiently. Mean blood pressure increased from 98 +/- 5 to 104 +/- 6 mm Hg (p < 0.05) because of an increase in systemic vascular resistance from 1,619 +/- 145 to 2,079 +/- 198 dyn x s x cm(-5) (at 20 min; p < 0.05). Diastolic pulmonary pressure and pulmonary vascular resistance showed similar changes. Pulmonary capillary wedge pressure increased from 12 +/- 3 to 16 +/- 4 mm Hg (at 20 min; p < 0.05). Although tedisamil resulted in a significant heart-rate reduction, this was not associated with an improvement of hemodynamics. This may be due to increased afterload of the left and right ventricle. In these patients, tedisamil increased vascular resistance, which is unwanted in the treatment of congestive heart failure.

Mechanical restitution and recirculation fraction in cardiac myocytes and left ventricular muscle of adult rats.

Unloaded cell shortening was measured in electrically stimulated myocytes from adult rat hearts to compare the contractile response to stimulation with that in isometrically contracting left ventricular papillary muscles under similar experimental conditions, but preloaded to produce maximum twitch tension. Mechanical restitution in cells followed a biexponential function with time constants of 0.19 +/- 0.03 s and 36.4 +/- 10.2 s (7 cells from 5 hearts, n = 7/5). The time constants for papillary muscles were 0.58 +/- 0.05 s and 14.6 +/- 1.0 s (n = 6/6). In myocytes, maximum post-rest potentiation occurred after 30 to 60 s of rest. The potentiation after 60 s of rest was 2.48 +/- 0.31 times the steadystate in cells and 2.63 +/- 0.16 in papillary muscles. Recirculation fraction of C2+ as calculated from the decay of post-rest potentiation was 0.84 +/- 0.04 in single cells and 0.59 +/- 0.02 in papillary muscles (p < 0.005). Caffeine (3mM) abolished post-rest potentiation in both types of preparations. The numerical values for the time constants of mechanical restitution, potentiation factor and recirculation fraction in papillary muscles did not depend on preload. It is concluded that interval-dependent changes of contractility are preserved in single cardiac cells but the kinetics of decay of potentiation appear to have changed quantitatively.

Electrophysiology of ion channels of the heart.

The contribution of Na+, Ca2+, and various K+ currents to the shape of the cardiac action potential is outlined based on the relation between electrophysiological properties and structure of channel molecules. These currents have also been found in human ventricular myocytes, where the most prominent K+ current is a transient outward current that is not influenced by methylsulfonanilide antiarrhythmic drugs. Combined knowledge of electrophysiological and molecular properties of ion channels is likely to form the basis for rational design of future drugs.

Effects of E-4031, almokalant and tedisamil on postrest action potential duration of human papillary muscles.

The new antiarrhythmic compounds E-4031 (1-[2-(6-methyl-2-pyridyl)ethyl]-4-(4-methylsulfonyl- aminobenzoyl)piperidine), almokalant and tedisamil prolonged the action potential duration (APD) of human right ventricular papillary muscle. In order to investigate whether drug-channel interaction takes place during rest, regular stimulation (0.5 Hz) was interrupted by three 30-min periods of quiescence. Drug was added at the beginning of the second period of rest, the third period was interposed at equilibrium of drug action. Under predrug control conditions, the first action potential after rest was longer than with regular stimulation, steady state was reached again with a monotonic time course. With E-4031 the first action potential after 30 min of drug exposure during quiescence was similar to predrug control, but drug-induced prolongation of APD developed during further stimulation, indicating drug interaction with open channels. After the third period of quiescence, the first APD remained significantly increased compared to predrug values suggesting that E-4031 may be trapped within the resting channel. With almokalant, however, the first APD after wash-in was already prolonged and APD increased further with regular pacing. The effect was partially reversed during the third period of rest. These findings are compatible with open-channel block or no evidence for trapping. On the other hand, tedisamil prolonged APD but did not change the monophasic time course neither when added during quiescence nor at equilibrium of drug action. It is concluded that changes in APD after quiescence indicate differences among these drugs in their interactions with channel subtypes controlling repolarization.

Frequency-dependent effects of E-4031, almokalant, dofetilide and tedisamil on action potential duration: no evidence for "reverse use dependent" block.

Antiarrhythmic drugs with class III action are incriminated by "reverse use dependency" which implies preferential block of resting channels (Hondeghem and Snyders 1990). The purpose of the present study was to investigate the frequency dependence of the effects of four new antiarrhythmic compounds on action potential duration (APD) in guinea-pig papillary muscle and on delayed rectifier in guinea-pig ventricular myocytes in order to scrutinize the concept of reverse use dependency and to obtain evidence for drug-channel interaction. In guinea-pig papillary muscles, E-4031 (1-[2-(6-methyl-2-pyridyl)ethyl]-4- (4-methylsulfonyl-aminobenzoyl)piperidine), almokalant, dofetilide and tedisamil prolonged APD in a concentration-dependent manner. Drug-induced APD prolongation was not affected significantly by low rates of stimulation (0.2 to 0.5 Hz. In order to investigate whether drug-channel interaction takes places during rest, regular stimulation (1 Hz) was interrupted by three 30-min periods of quiescence. Drug was added at the beginning of the second period of rest, the third period was interposed at steady state of drug action. With E-4031 and dofetilide no change in shape of the first AP after the initial 30 min of drug exposure was observed as compared with pre-drug control, but regular stimulation was required for the full effect to develop. APD did not recover to pre-drug values after the third period of quiescence. With almokalant and tedisamil, however, the first APD after wash-in was already prolonged and the effects increased further with regular pacing. Only with almokalant but not with tedisamil did APD recover during rest.(ABSTRACT TRUNCATED AT 250 WORDS)