Akt/eNOS signaling and PLN S-sulfhydration are involved in H2S-dependent cardiac effects in frog and rat

Hydrogen sulfide (H2S) has recently emerged as an important mediator of mammalian cardiovascular homeostasis. In nonmammalian vertebrates, little is known about the cardiac effects of H2S. This study aimed to evaluate, in the avascular heart of the frog, Rana esculenta, whether and to what extent H2S affects the cardiac performance, and what is the mechanism of action responsible for the observed effects. Results were analyzed in relation to those obtained in the rat heart, used as the mammalian model. Isolated and perfused (working and Langendorff) hearts, Western blot analysis, and modified biotin switch (S-sulfhydration) assay were used. In the frog heart, NaHS (used as H2S donor, 10−12/10−7 M) dose-dependently decreased inotropism. This effect was reduced by glibenclamide (KATP channels blocker), N G-monomethyl-l-arginine (NOS inhibitor), 1H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (guanylyl cyclase inhibitor), KT5823 (PKG inhibitor), and it was blocked by Akt1/2 (Akt inhibitor) and by detergent Triton X-100. In the rat, in addition to the classic negative inotropic effect, NaHS (10−12/10−7 M) exhibited negative lusitropism. In both frog and rat hearts, NaHS treatment induced Akt and eNOS phosphorylation and an increased cardiac protein S-sulfhydration that, in the rat heart, includes phospholamban. Our data suggest that H2S represents a phylogenetically conserved cardioactive molecule. Results obtained on the rat heart extend the role of H2S also to cardiac relaxation. H2S effects involve KATP channels, the Akt/NOS-cGMP/PKG pathway, and S-sulfhydration of cardiac proteins.

Effect of perphenazine on electrogram of rat isolated heart

The aim of this study was to clarify whether administration of antipsychotic perphenazine contributes to the electrophysiological changes of the isolated heart. Fourteen adult male Wistar rats were divided into two groups. Langendorff hearts were perfused with Krebs-Henseleit solution at constant pressure (85 mmHg) and 37 °C (CaCl2, 1.2 mM). The electrogram was recorded by touch-free method. The hearts of the first group were exposed to 3.10-5 M perphenazine, the hearts of the second group to 3.10-8 M perphenazine. The incidence of arrhythmias and the heart rate and QT interval changes were studied on electrogram during 30 min periods of control, the first perphenazine administration, washout, and the second drug administration. Perphenazine administration significantly prolonged QT (p < 0.001) and QTc (p < 0.05) in group 1. In group 2, QT and QTc prolongation was less pronounced (p < 0.05). A number of arrhythmias appeared, from single premature ventricular complexes to more severe ones in both groups. The heart rate changes were non-significant. We conclude that although phenothiazines are still medicaments of choice in certain psychoses, their cardiovascular side effects should be always taken in consideration.

Abstract 1355: Determining Wavefront Size during Ventricular Fibrillation: Optical Mapping Study from Human Langendorff Hearts

Background: We had recently established a model for the study of VF in humans using an optical mapping technique in Langendorff perfused human hearts. The optical mapping technique allows for the unequivocal evaluation of wavefront identification, the origin of wavefronts and their directional spread. Objective: The objective of this study was to measure wavefront sizes from different regions during human VF to determine the minimal spatial resolution required to study human VF in-vitro. Methods: The hearts were explanted from seven cardiomyopathic patients who underwent transplantation. The hearts were Langendorff perfused with Tyrode’s solution (95%O 2 +5%CO 2 ) and the potentiometric dye (0.2 mg Di-4-ANEPPS dissolved in 20mg of perfusate) was injected as a bolus into the coronary circulation. The imaged region was physically immobilized to minimize motion. The fluorescence was excited at 530 nm with a HL 150 W halogen lamp, the emission signal was longpass filtered at 610 nm and recorded with a CCD and CMOS camera (MiCAM02, Ultima, BrainVision Inc., Japan). The spatial resolution depending on the mapping window was 0.65–0.85 mm and the temporal resolution was 1 kHz. The heart was paced at a cycle length of 600 ms and the VF was induced by briefly touching the heart with the two poles of a 9V battery. The changes in dye signal were recorded over 1887 ms and analyzed using BV-analyzer software (BrainVision Inc, Japan). Wavefronts were identified using high-pass filter (0.01 Hz) on the fluorescence data. Recordings were performed at 4 sites: LV Septum (LVS), Right Ventricle Endocardium (RV), Left Ventricle Endocardium (LVendo) and Left Ventricle Epicardium (LVepi). Results: 16 episodes of VF were recorded and a total of 54 wavefronts were identified. Average wavefront size was 23.6 mm. By region, the average wavefront sizes were: LVS = 26.3 mm; RV = 22.4 mm; LVendo = 29.7 mm; LVepi = 19.3 mm. Conclusion: These results show that different regions on the human ventricle during VF show different wavefront size suggesting that propensity for wavebreak maybe spatially differentially distributed. To study human VF with multi-electrode mapping tools using inter-electrode spacing of less than 1–2 cm should provide sufficient resolution.

NovelN6-substituted adenosine 5′-N-methyluronamides with high selectivity for human adenosine A3receptors reduce ischemic myocardial injury

We recently reported the identification of a novel human adenosine A3receptor-selective agonist, (2 S,3 S,4 R,5 R)-3-amino-5-{6-[5-chloro-2-(3-methylisoxazol-5-ylmethoxy)benzylamino]purin-9-yl}-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide (CP-608,039), with 1,260-fold selectivity for the human A3versus human A1receptor (DeNinno et al., J Med Chem 46: 353–355, 2003). However, because the modest (20-fold) rabbit A3receptor selectivity of CP-608,039 precludes demonstration of A3-mediated cardioprotection in rabbit models, we identified another member of this class, (2 S,3 S,4 R,5 R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide (CP-532,903), which both retained human A3receptor selectivity (210-fold; human A3/human A1Ki: 23/4,800 nM) and had improved rabbit A3receptor selectivity (90-fold; rabbit A3/rabbit A1Ki: 23/2,000 nM). Infarct size was measured in Langendorff hearts or in vivo after 30 min of regional ischemia and 120 min of reperfusion. Five-minute perfusion with CP-532,903 before ischemia-reperfusion elicited a concentration-dependent reduction in infarct size in isolated hearts (EC50: 0.97 nM; maximum reduction in infarct size: 77%, P < 0.05 vs. control). Furthermore, administration of CP-532,903 (150 nM) at reperfusion also significantly reduced infarct size by 64% ( P < 0.05 vs. control), which was not different ( P ≥ 0.05) from the cardioprotection provided by the same concentration of drug given before ischemia. The selective rabbit A1receptor antagonist BWA1433 did not affect CP-532,903-dependent cardioprotection. In vivo, CP-532,903 (1 mg/kg) reduced infarct size by 50% in the absence of significant hemodynamic effects (mean arterial pressure, heart rate, rate-pressure product). CP-532,903 and CP-608,039 represent a novel class of human A3receptor-selective agonists that may prove suitable for investigation of the clinical cardioprotective efficacy of A3receptor activation.

Defibrillation depresses heart sarcoplasmic reticulum calcium pump: a mechanism of postshock dysfunction

Presently, the only therapy for ventricular fibrillation is delivery of high-voltage shocks. Despite “successful defibrillation,” patients may have poor cardiac contractility, the mechanisms of which are unknown. Intracellular Ca2+ handling by the sarcoplasmic reticulum (SR) plays a major role in contractility. We tested the hypothesis that defibrillation shocks interfere with Ca2+ transport function of cardiac SR. Rats anesthetized with pentobarbital sodium had bilateral electrodes implanted subcutaneously for transthoracic shocks. A series of 10 shocks, 10 s apart, at 0–250 V was delivered from a trapezoidal defibrillator. The hearts were rapidly removed, SR-enriched membrane vesicles were isolated, and ATP-dependent Ca2+ uptake and Ca2+-stimulated ATP hydrolysis were determined. There was a marked, shock-related decline in Ca2+ uptake, whereas adenosinetriphosphatase activity remained unaltered. The polypeptide compositions were similar in control and shocked SR. In Langendorff hearts, shocks also decreased contractility and slowed relaxation. These data indicate that shocks with current densities similar to defibrillation depress Ca2+-pumping function of cardiac SR because of uncoupling of ATP hydrolysis and Ca2+ transport. Shock-induced impairment of Ca2+ pump function may underlie postshock myocardial dysfunction.

Inhibition of the calcium paradox in isolated rat hearts by high perfusate sucrose concentrations

If a colloid osmotic (oncotic) pressure gradient develops across the myocyte membrane during the calcium paradox, adding an oncotic agent to the perfusate should be inhibitory. After 10-min perfusion with Ca(2+)-free Krebs-Henseleit (KH) buffer under constant flow at 34 degrees C, myoglobin release was measured from Langendorff hearts reperfused with Ca(2+)-containing KH buffer. When the Ca(2+)-free medium contained 200 mM sucrose, myoglobin release was reduced to 5% of that observed in the absence of sucrose, a change that was not seen when 200 mosM NaCl, choline chloride, LiCl, or glycerol was added. Replacement of 75 mM NaCl in the perfusate with 150 mM sucrose resulted in myoglobin release values that were 4% of the control. Plots of myoglobin release against sucrose concentration under these hypertonic and isotonic conditions yielded similar though separate curves. Sucrose also inhibited increases in wet weight-to-dry weight ratio and decreases in ATP and phosphocreatine contents. These results support the hypothesis that an oncotic pressure gradient arises during the calcium paradox at the moment of increased membrane permeability and plays a major role in its development.