Diminazene Aceturate, an angiotensin converting enzyme 2 (ACE2) activator, promotes cardioprotection in ischemia/reperfusion-induced cardiac injury.

This study aimed to investigate whether the Diminazene Aceturate (DIZE), an angiotensin-converting enzyme 2 (ACE2) activator, can revert cardiac dysfunction in ischemia reperfusion-induced (I/R) injury in animals and examine the mechanism underlying this effect. Wistar rats systemically received DIZE (1 mg/kg) for thirty days. Cardiac function in isolated rat hearts was evaluated using the Langendorff technique. After I/R, ventricular non-I/R and I/R samples were used to evaluate ATP levels. Mitochondrial function was assessed using cardiac permeabilized fibers and isolated cardiac mitochondria. Cardiac cellular electrophysiology was evaluated using the patch clamp technique. DIZE protected the heart after I/R from arrhythmia and cardiac dysfunction by preserving ATP levels, independently of any change in coronary flow and heart rate. DIZE improved mitochondrial function, increasing the capacity for generating ATP and reducing proton leak without changing the specific citrate synthase activity. The activation of the ACE2 remodeled cardiac electrical profiles, shortening the cardiac action potential duration at 90 % repolarization. Additionally, cardiomyocytes from DIZE-treated animals exhibited reduced sensibility to diazoxide (KATP agonist) and a higher KATP current compared to the controls. DIZE was able to improve mitochondrial function and modulate cardiac electrical variables with a cardio-protective profile, resulting in direct myocardial cell protection from I/R injury.

Comparative Cardiotoxicity of Low Doses of Digoxin, Ouabain, and Oleandrin.

The aim of this study was to evaluate the comparative effects of CGs on heart physiology. Twenty-eight Wistar rats were distributed into four groups (n = 7), control group received NaCl 0.9% every 24 h for 21 days; treated groups received respectively 50 µg/kg of digoxin (DIG), ouabain (OUA) and oleandrin (OLE) every 24 h for 21 days. Serial ECGs were performed, as well as serum levels of creatinine kinase (CK), its MB fraction, troponin I (cTnI), calcium (Ca2+) and lactic dehydrogenase (LDH). Heart tissue was processed for histology, scanning electron microscopy and Western blot analysis for cTnI, brain natriuretic peptide (BNP), sodium potassium pump alpha-1 and alpha-2. Ventricle samples were also analyzed for thiobarbituric acid reactive substances and antioxidant enzymes (SOD, GPX, and CAT). ECGs showed decrease in QT and progressive shortening of QRS. No arrhythmias were observed. No significant differences were associated with CGs treatment and serum levels of CK, CK-MB, and cTnI. Only oleandrin increased LDH levels. Histological analysis showed degenerative changes and only oleandrin promoted moderate focal necrosis of cardiomyocytes. Scanning microscopy also confirmed the greatest effect of oleandrin, with rupture and shortening of cardiac fibers. The expression of troponin I and alpha-1 isoform were not altered, however, the protein levels of BNP and alpha-2 were higher in the groups that received oleandrin and ouabain in relation to the digoxin group. All GCs affected the production of ROS, without causing lipid peroxidation, through the activation of different antioxidant pathways. It is concluded that the administration of digoxin, ouabain, and oleandrin at 50 µg/kg for 21 days caused cardiovascular damage that represent an important limitation into its future use in heart failure and antineoplastic therapy.

Non-invasive ECG recording and QT interval correction assessment in anesthetized rats and mice / Eletrocardiograma não invasivo e avaliação da correção do intervalo QT em roedores anestesiados

Rats and mice are the most common species used in experimental cardiac electrophysiology studies. Electrocardiogram (ECG) recording shows paramount importance for monitoring arrhythmias and cardiac function in several disease models, including QT syndrome. However, the lack of standardized reference values and QT correction formula for different animal species and lineages represent a challenge for ECG interpretation. The aim of this study is to provide an improved method for ECG recording, establishing reference range values and determine the QT formulas with higher correlation to heart rate (HR). A total of 10 Wistar rats, 10 Swiss mice, 10 C57BL/6 mice and 10 FVB/NJ mice were used in the study. Animals were submitted to anesthesia with isoflurane and ECG recording was performed using a six-channel non-invasive electrocardiograph. QT was corrected using the following formulas: Bazzett, Fridericia, Mitchell, Hodges, Van der Water and Framingham. Normal range values for ECG parameters were established in all animals studied. Pearsons' correlation defined Hodges formula as the most suitable for QT correction. This study demonstrated an improved method of ECG recording with reference values for Swiss, FVB/NJ, C57BL/6 mice, and Wistar rats. Hodges' formula was the most effective formula for QT correction in rodents, whereas Bazett's and Friderica formulas were ineffective for such animals. The present work contributes to arrhythmias investigation in experimental cardiology and may reduce misinterpretations in rodents' ECG.(AU)

Ratos e camundongos são as espécies mais comumente utilizadas em estudos experimentais de eletrofisiologia cardíaca. O registro do eletrocardiograma (ECG) é de suma importância para o monitoramento de arritmias e função cardíaca em vários modelos de patologias. No entanto, a falta de valores de referência padronizados e a fórmula de correção do QT para diferentes espécies e linhagens animais representam um desafio para a interpretação do ECG. O objetivo deste estudo é fornecer um método melhorado para o registro de ECG, estabelecendo valores de referência e determinar as fórmulas QT com maior correlação com a freqüência cardíaca (FC). Um total de 10 ratos Wistar, 10 camundongos Swiss, 10 camundongos C57BL/6 e 10 camundongos FVB/NJ foram utilizados no estudo. Os animais foram submetidos à anestesia com isoflurano e o registro de ECG foi realizado com eletrocardiógrafo não invasivo de seis canais. O QT foi corrigido usando as seguintes fórmulas: Bazzett, Fridericia, Mitchell, Hodges, Van der Water e Framingham. Os valores da normalidade para os parâmetros do ECG foram estabelecidos em todos os animais estudados. A correlação de Pearson definiu a fórmula de Hodges como a mais adequada para a correção do QT. Este estudo demonstra um método melhorado de registro de ECG com valores de referência para camundongos Swiss, FVB/NJ, C57BL/6 e Wistar. A fórmula de Hodges foi a mais eficaz para correção de QT em roedores, enquanto as fórmulas de Bazett e Friderica apresentaram valores mais baixos de correlação. O presente trabalho contribui para a investigação de arritmias em cardiologia experimental e pode reduzir interpretações erradas no ECG de roedores.(AU)

TNF-α mediated upregulation of NaV1.7 currents in rat dorsal root ganglion neurons is independent of CRMP2 SUMOylation.

Clinical and preclinical studies have shown that patients with Diabetic Neuropathy Pain (DNP) present with increased tumor necrosis factor alpha (TNF-α) serum concentration, whereas studies with diabetic animals have shown that TNF-α induces an increase in NaV1.7 sodium channel expression. This is expected to result in sensitization of nociceptor neuron terminals, and therefore the development of DNP. For further study of this mechanism, dissociated dorsal root ganglion (DRG) neurons were exposed to TNF-α for 6 h, at a concentration equivalent to that measured in STZ-induced diabetic rats that developed hyperalgesia. Tetrodotoxin sensitive (TTXs), resistant (TTXr) and total sodium current was studied in these DRG neurons. Total sodium current was also studied in DRG neurons expressing the collapsin response mediator protein 2 (CRMP2) SUMO-incompetent mutant protein (CRMP2-K374A), which causes a significant reduction in NaV1.7 membrane cell expression levels. Our results show that TNF-α exposure increased the density of the total, TTXs and TTXr sodium current in DRG neurons. Furthermore, TNF-α shifted the steady state activation and inactivation curves of the total and TTXs sodium current. DRG neurons expressing the CRMP2-K374A mutant also exhibited total sodium current increases after exposure to TNF-α, indicating that these effects were independent of SUMOylation of CRMP2. In conclusion, TNF-α sensitizes DRG neurons via augmentation of whole cell sodium current. This may underlie the pronociceptive effects of TNF-α and suggests a molecular mechanism responsible for pain hypersensitivity in diabetic neuropathy patients.

Increase in Ca2+ current by sustained cAMP levels enhances proliferation rate in GH3 cells.

AIMS: Ca2+ and cAMP are important intracellular modulators. In order to generate intracellular signals with various amplitudes, as well as different temporal and spatial properties, a tightly and precise control of these modulators in intracellular compartments is necessary. The aim of this study was to evaluate the effects of elevated and sustained cAMP levels on voltage-dependent Ca2+ currents and proliferation in pituitary tumor GH3 cells. MAIN METHODS: Effect of long-term exposure to forskolin and dibutyryl-cyclic AMP (dbcAMP) on Ca2+ current density and cell proliferation rate were determined by using the whole-cell patch-clamp technique and real time cell monitoring system. The cAMP levels were assayed, after exposing transfected GH3 cells with the EPAC-1 cAMP sensor to forskolin and dbcAMP, by FRET analysis. KEY FINDINGS: Sustained forskolin treatment (24 and 48h) induced a significant increase in total Ca2+ current density in GH3 cells. Accordingly, dibutyryl-cAMP incubation (dbcAMP) also elicited increase in Ca2+ current density. However, the maximum effect of dbcAMP occurred only after 72h incubation, whereas forskolin showed maximal effect at 48h. FRET-experiments confirmed that the time-course to elevate intracellular cAMP was distinct between forskolin and dbcAMP. Mibefradil inhibited the fast inactivating current component selectively, indicating the recruitment of T-type Ca2+ channels. A significant increase on cell proliferation rate, which could be related to the elevated and sustained intracellular levels of cAMP was observed. SIGNIFICANCE: We conclude that maintaining high levels of intracellular cAMP will cause an increase in Ca2+ current density and this phenomenon impacts proliferation rate in GH3 cells.

Probabilistic model checking analysis of palytoxin effects on cell energy reactions of the Na+/K+-ATPase.

Probabilistic model checking (PMC) is a technique used for the specification and analysis of complex systems. It can be applied directly to biological systems which present these characteristics, including cell transport systems. These systems are structures responsible for exchanging ions through the plasma membrane. Their correct behavior is essential for animal cells, since changes on those are responsible for diseases. In this work, PMC is used to model and analyze the effects of the palytoxin toxin (PTX) interactions with one of these systems. Our model suggests that ATP could inhibit PTX action. Therefore, individuals with ATP deficiencies, such as in brain disorders, may be more susceptible to the toxin. We have also used heat maps to enhance the kinetic model, which is used to describe the system reactions. The map reveals unexpected situations, such as a frequent reaction between unlikely pump states, and hot spots such as likely states and reactions. This type of analysis provides a better understanding on how transmembrane ionic transport systems behave and may lead to the discovery and development of new drugs to treat diseases associated to their incorrect behavior.

Morphine peripheral analgesia depends on activation of the PI3Kgamma/AKT/nNOS/NO/KATP signaling pathway.

Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3Kgamma/AKT protein kinase B (AKT) and culminated in increased activation of K(ATP) channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development.

Structure and activity analysis of two spider toxins that alter sodium channel inactivation kinetics.

In this work, Phoneutria nigriventer toxins PnTx2-5 and PnTx2-6 were shown to markedly delay the fast inactivation kinetics of neuronal-type sodium channels. Furthermore, our data show that they have significant differences in their interaction with the channel. PnTx2-6 has an affinity 6 times higher than that of PnTx2-5, and its effects are not reversible within 10-15 min of washing. PnTx2-6 partially (59%) competes with the scorpion alpha-toxin AaHII, but not with the scorpion beta-toxin CssIV, thus suggesting a mode of action similar to that of site 3 toxins. However, PnTx2-6 is not removed by strong depolarizing pulses, as in the known site 3 toxins. We have also established the correct PnTx2-5 amino acid sequence and confirmed the sequence of PnTx2-6, in both cases establishing that the cysteines are in their oxidized form. A structural model of each toxin is proposed. They show structures with poor alpha-helix content. The model is supported by experimental and theoretical tests. A likely binding region on PnTx2-5 and PnTx2-6 is proposed on the basis of their different affinities and sequence differences.

(S)-reticuline induces vasorelaxation through the blockade of L-type Ca(2+) channels.

In Brazil, various species of the genus Ocotea are used in folk medicine for treating several diseases. The chemical characterization of this plant showed the presence of alkaloids belonging to the benzyltetrahydroisoquinoline family, the major component of which is (S)-reticuline. The present study investigated whether (S)-reticuline exerts an inhibitory effect on smooth muscle L-type Ca(2+) channels. Tension measurements and patch clamp techniques were utilized to study the effects of (S)-reticuline. Whole-cell Ca(2+) currents were measured using the A7r5 smooth muscle cell line. (S)-reticuline antagonized CaCl(2)- and KCl-induced contractions and elicited vasorelaxation. It also reduced the voltage-activated peak amplitude of I (Ca,L) in a concentration-dependent manner. (S)-reticuline did not change the characteristics of current density vs. voltage relationship. (S)-reticuline shifted leftwards the steady-state inactivation curve of I (Ca,L). The application of dibutyryl cyclic adenosine monophosphate to the cell decreased the amplitude of Ca(2+) currents. In cells pretreated with forskolin, an adenylate cyclase activator, the addition of (S)-reticuline caused further inhibition of the Ca(2+) currents suggesting an additive effect. The results obtained show that (S)-reticuline elicits vasorelaxation probably due to the blockade of the L-type voltage-dependent Ca(2+) current in rat aorta. The reported effect may contribute to the potential cardioprotective efficacy of (S)-reticuline.

Aqueous leaf extract of Averrhoa carambola L. (Oxalidaceae) reduces both the inotropic effect of BAY K 8644 on the guinea pig atrium and the calcium current on GH3cells / Extrato aquoso das folhas de Averrhoa carambola L. (Oxalidaceae) reduz tanto o efeito inotrópico do BAY K 8644 em átrio de cobaia, quanto a corrente de cálcio em células GH3

It was previously showed that aqueous leaf extract (AqEx) of Averrhoa carambola depresses the guinea pig atrial inotropism. Therefore, experiments were carried out on guineapig left atrium and on pituitary GH3 cells in order to evaluate the effect of AqEx on the cellular calcium infl ux. The atrium was mounted in an organ chamber (5 mL, Tyrode, 27 ± 0.1 °C, 95% O2, 5 % CO2), stretched to 10 mN, and paced at 2 Hz (0.5 ms, 400 V) and GH3 cells were submitted to a whole cell voltage clamp confi guration. In the atrium, the AqEx (1500 μg/mL) shifted to the right the concentration-effect curve of the positive inotropic effect produced by (±) BAY K 8644, an L-type calcium channel agonist. The AqEx increased EC50 (concentration required to promote 50% of the maximum effect) of the inotropic effect of BAY K 8644 from 7.8 ± 0.38 to 115.1 ± 0.44 nM (N = 3; p < 0.05). In GH3 cells assayed with 500 μg/mL of AqEx, the L-type calcium inward current declined 30 % (from 282 to 190 pA). Nevertheless, the extract did not change the voltage correspondent to the peak current. These data suggest that, at least in part, the negative inotropic effect of AqEx on the guinea pig atrium is due to a reduction of the L-type calcium current.

Em estudo prévio mostrou-se que o extrato aquoso das folhas de Averrhoacarambola (ExAq) reduziu o inotropismo atrial da cobaia. Por isso, este trabalho avaliou se o ExAq interfere com o infl uxo de cálcio através da membrana celular. A investigação foi conduzidaem átrio esquerdo de cobaia, montado em cuba (5 mL, Tyrode, 27 ± 0,1 °C, 95 % O2, 5 % CO2), estirado para uma tensão de repouso de 10 mN e submetido a uma estimulação de 2 Hz (0,5 ms, 400 V). O efeito do ExAq sobre a entrada de cálcio nas células foi avaliado em átrio de cobaia e em células GH3, estas submetidas a ‘patch clamp’ na confi guração ‘whole cell’. No átrio, o ExAq (1500 μg /mL) deslocou para direita a curva concentração-efeito do (±) BAY K 8644 (agonista dos canais de cálcio tipo-L), aumentando a CE50 (concentração capaz de produzir 50 % do efeito máximo) de 7,8 ± 0,38 para 115,1 ± 0,44 nM (N = 3, p < 0,05). Em células GH3, este extrato (500 μg /mL) reduziu de 282 para 190 pA (30 %) a corrente de cálcio, sem contudo alterar a voltagem de pico da curva desta corrente. Estes resultados mostram que, pelo menos em parte, o efeito inotrópico negativo do ExAq em átrio de cobaia se deve a uma diminuição do infl uxo de cálcio pelos canais tipo-L.

CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons.

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.

Impairment of in vitro and in vivo heart function in angiotensin-(1-7) receptor MAS knockout mice.

In this study we investigated the effects of the genetic deletion of the angiotensin (Ang)-(1-7) receptor Mas on heart function. Localization of Mas in the mouse heart was evaluated by binding of rhodamine-labeled Ang-(1-7). Cardiac function was examined using isolated heart preparations. Echocardiography was used to confirm the results obtained with isolated heart studies. To elucidate the possible mechanisms involved in the cardiac phenotype observed in Mas(-/-) mice, whole-cell calcium currents in cardiomyocytes and the expression of collagen types I, III, and VI and fibronectin were analyzed. Ang-(1-7) binding showed that Mas is localized in cardiomyocytes of the mouse heart. Isolated heart techniques revealed that Mas-deficient mice present a lower systolic tension (average: 1.4+/-0.09 versus 2.1+/-0.03 g in Mas(+/+) mice), +/-dT/dt, and heart rate. A significantly higher coronary vessel resistance was also observed in Mas-deficient mice. Echocardiography revealed that hearts of Mas-deficient mice showed a significantly decreased fractional shortening, posterior wall thickness in systole and left ventricle end-diastolic dimension, and a higher left ventricle end-systolic dimension. A markedly lower global ventricular function, as defined by a higher myocardial performance index, was observed. A higher delayed time to the peak of calcium current was also observed. The changes in cardiac function could be partially explained by a marked change in collagen expression to a profibrotic profile in Mas-deficient mice. These results indicate that Ang-(1-7)-Mas axis plays a key role in the maintenance of the structure and function of the heart.

Electrophysiological characterization and molecular identification of the Phoneutria nigriventer peptide toxin PnTx2-6.

A cDNA with 403 nucleotides encoding the precursor of the toxin PnTx2-6 was cloned and sequenced. Subsequent analysis revealed that the precursor begins with a signal peptide and a glutamate-rich propeptide. The succeeding peptide confirmed the reported sequence of PnTx2-6. The purified toxin exerted complex effects on Na(+) current of frog skeletal muscle. There was a marked decrease of the inactivation kinetics, and a shift to hyperpolarizing potentials of both the Na(+) conductance and the steady-state inactivation voltage dependences, along with a reduction of the current amplitude. The concentration dependence of the modified current suggests a K(D) of 0.8 microM for the toxin-channel complex.