Electrocontractile remodeling of isolated cardiomyocytes induced during early-stage hypercholesterolemia.

Hypercholesterolemia is one of the most important risk factors for cardiovascular diseases. However, it is mostly associated with vascular dysfunction and atherosclerotic lesions, while evidence of direct effects of hypercholesterolemia on cardiomyocytes and heart function is still incomplete and controversial. In this study, we assessed the direct effects of hypercholesterolemia on heart function and the electro-contractile properties of isolated cardiomyocytes. After 5 weeks, male Swiss mice fed with AIN-93 diet added with 1.25% cholesterol (CHO), developed an increase in total serum cholesterol levels and cardiomyocytes cholesterol content. These changes led to altered electrocardiographic records, with a shortening of the QT interval. Isolated cardiomyocytes displayed a shortening of the action potential duration with increased rate of depolarization, which was explained by increased IK, reduced ICa.L and altered INa voltage-dependent inactivation. Also, reduced diastolic [Ca2+]i was found with preserved adrenergic response and cellular contraction function. However, contraction of isolated hearts is impaired in isolated CHO hearts, before and after ischemia/reperfusion, although CHO heart was less susceptible to arrhythmic contractions. Overall, our results demonstrate that early hypercholesterolemia-driven increase in cellular cholesterol content is associated with direct modulation of the heart and cardiomyocytes' excitability, Ca2+ handling, and contraction.

Inhibition of calcium/calmodulin (Ca2+ /CaM)-Calcium/calmodulin-dependent protein kinase II (CaMKII) axis reduces in vitro and ex vivo arrhythmias in experimental Chagas disease.

Chagasic cardiomyopathy (CCC) is one of the main causes of heart failure and sudden death in Latin America. To date, there is no available medication to prevent or reverse the onset of cardiac symptoms. CCC occurs in a scenario of disrupted calcium dynamics and enhanced oxidative stress, which combined, may favor the hyper activation of calcium/calmodulin (Ca2+ /CaM)-calcium/calmodulin-dependent protein kinase II (CaMKII) (Ca2+ /CaM-CaMKII) pathway, which is fundamental for heart physiology and it is implicated in other cardiac diseases. Here, we evaluated the association between Ca2+ /CaM-CaMKII in the electro-mechanical (dys)function of the heart in the early stage of chronic experimental Trypanosoma cruzi infection. We observed that in vitro and ex vivo inhibition of Ca2+ /CaM-CaMKII reversed the arrhythmic profile of isolated hearts and isolated left-ventricles cardiomyocytes. The benefits of the limited Ca2+ /CaM-CaMKII activation to cardiomyocytes' electrical properties are partially related to the restoration of Ca2+ dynamics in a damaged cellular environment created after T. cruzi infection. Moreover, Ca2+ /CaM-CaMKII inhibition prevented the onset of arrhythmic contractions on isolated heart preparations of chagasic mice and restored the responsiveness to the increase in the left-ventricle pre-load. Taken together, our data provide the first experimental evidence for the potential of targeting Ca2+ /CaM-CaMKII pathway as a novel therapeutic target to treat CCC.

Diminazene aceturate (DIZE) has cellular and in vivo antiarrhythmic effects.

Diminazene aceturate (DIZE) is an anti-protozoan compound that has been previously reported to increase the activity of the angiotensin-converting enzyme 2 (ACE2) and thus increase Angiotensin-(1-7) production, leading to cardioprotection against post-myocardial infarction dysfunction and structural remodelling. Moreover, DIZE is able to ameliorate morpho-functional changes after myocardial infarction by enhancing ACE2 activity, thus increasing Angiotensin-(1-7) production (a benefic peptide of the renin-angiotensin system). However, despite the improvement in cardiac function/structure, little is known about DIZE effects on arrhythmia suppression, contraction/excitable aspects of the heart and importantly its mechanisms of action. Thus, our aim was to test the acute effect of DIZE cardioprotection at the specific level of potential antiarrhythmic effects and modulation in excitation-contraction coupling. For this, we performed in vitro and in vivo techniques for arrhythmia induction followed by an acute administration of DIZE. For the first time, we described that DIZE can reduce arrhythmias which is explained by modulation of cardiomyocyte contraction and excitability. Such effects were independent of Mas receptor and nitric oxide release. Development of a new DIZE-based approach to ameliorate myocardial contractile and electrophysiological dysfunction requires further investigation; however, DIZE may provide the basis for a future beneficial therapy to post-myocardial infarction patients.

Thiamine Deficiency Increases Ca2+ Current and CaV1.2 L-type Ca2+ Channel Levels in Cerebellum Granular Neurons.

Thiamine (vitamin B1) is co-factor for three pivotal enzymes for glycolytic metabolism: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase. Thiamine deficiency leads to neurodegeneration of several brain regions, especially the cerebellum. In addition, several neurodegenerative diseases are associated with impairments of glycolytic metabolism, including Alzheimer's disease. Therefore, understanding the link between dysfunction of the glycolytic pathway and neuronal death will be an important step to comprehend the mechanism and progression of neuronal degeneration as well as the development of new treatment for neurodegenerative states. Here, using an in vitro model to study the effects of thiamine deficiency on cerebellum granule neurons, we show an increase in Ca2+ current density and CaV1.2 expression. These results indicate a link between alterations in glycolytic metabolism and changes to Ca2+ dynamics, two factors that have been implicated in neurodegeneration.

Basal and ß-adrenergic cardiomyocytes contractility dysfunction induced by dietary protein restriction is associated with downregulation of SERCA2a expression and disturbance of endoplasmic reticulum Ca2+ regulation in rats.

BACKGROUND: The mechanisms responsible for the cardiac dysfunction associated with dietary protein restriction (PR) are poorly understood. Thus, this study was designed to evaluate the effects of PR on calcium kinetics, basal and ß-adrenergic contractility in murine ventricular cardiomyocytes. METHODS: After breastfeeding male Fisher rats were distributed into a control group (CG, n = 20) and a protein-restricted group (PRG, n = 20), receiving isocaloric diets for 35 days containing 15% and 6% protein, respectively. Biometric and hemodynamic variables were measured. After euthanasia left ventricles (LV) were collected for histopathological evaluation, SERCA2a expression, cardiomyocytes contractility and Ca(2+)sparks analysis. RESULTS: PRG animals showed reduced general growth, increased heart rate and arterial pressure. These animals presented extracellular matrix expansion and disorganization, cardiomyocytes hypotrophy, reduced amplitudes of shortening and maximum velocity of contraction and relaxation at baseline and after ß-adrenergic stimulation. Reduced SERCA2a expression as well as higher frequency and lower amplitude of Ca(2+)sparks were observed in PRG cardiomyocytes. CONCLUSION: The observations reveal that protein restriction induces marked myocardial morphofunctional damage. The pathological changes of cardiomyocyte mechanics suggest the potential involvement of the ß-adrenergic system, which is possibly associated with changes in SERCA2a expression and disturbances in Ca(2+) intracellular kinetics.

Warifteine, a bisbenzylisoquinoline alkaloid, induces relaxation by activating potassium channels in vascular myocytes.

The present study used functional and electrophysiological approaches to investigate the mechanisms by which warifteine, a bisbenzylisoquinoline alkaloid isolated from Cissampelos sympodialis Eichl., causes vasorelaxation of the rat thoracic aorta. Warifteine (1 pmol/L-10 µmol/L) induced concentration-dependent relaxation (pD(2) = 9.40 ± 0.06; n = 5) of endothelium-intact aortic rings precontracted with noradrenaline (10-100 µmol/L). The relaxation effects were not attenuated by removal of the endothelium. Warifteine also induced the relaxation of prostaglandin F(2α) (1-10 mmol/L)-precontracted rings (pD(2) = 9.2 ± 0.2; n = 8). In contrast, the relaxant activity of warifteine was nearly abolished in high K(+) (80 mmol/L)-precontracted aortic rings. In preparations incubated with 20 mmol/L KCl or with the K(+) channel blockers tetraethylammonium (1, 3 and 5 mmol/L), iberiotoxin (20 nmol/L), 4-aminopyridine (1 mmol/L) or glibenclamide (10 µmol/L), the vasorelaxant activity of warifteine was markedly reduced. However, BaCl(2) (1 mmol/L) had no effect on the relaxant effects of warifteine. In vascular myocytes, warifteine (100 nmol/L) significantly increased whole-cell K(+) currents (at 70 mV). Under nominally Ca(2+) -free conditions, warifteine did not reduce extracellular Ca(2+) -induced contractions in rings precontracted with high K(+) or noradrenaline (100 µmol/L). Together, the results of the present study indicate that warifteine induces potent concentration-dependent relaxation in the rat aorta via an endothelium-independent mechanism that involves the activation of K(+) channels.

Mechanism of the antihypertensive and vasorelaxant effects of the flavonoid tiliroside in resistance arteries.

Hypertension is a leading cause of death and disability globally, and its prevalence continues to accelerate. The cardiovascular effects of the flavonoid tiliroside have never been reported. In this work, using complementary in vivo and in vitro approaches, we describe the antihypertensive effect of tiliroside and the underlying mechanisms involved in the reduction of blood pressure. Tiliroside (1, 5 or 10 mg/kg) induced a dose-dependent long-lasting decrease in blood pressure in conscious DOCA-salt hypertensive rats that was accompanied by an increased heart rate. Tiliroside also induced a concentration-dependent vasodilation of mesenteric resistance arteries precontracted with phenylephrine. Removal of the endothelium or pretreatment of the preparation with L-NAME or indomethacin did not modify the vasodilator response for tiliroside. When vessels were precontracted with a high K⁺ (50 mM) solution, tiliroside exhibited a vasodilator effect similar to that observed in vessels precontracted with phenylephrine. Experiments carried out in nominally Ca²âº-free solution showed that tiliroside antagonized CaCl2-induced contractions. Moreover, tiliroside reduced the rise in intracellular Ca²âº concentration induced by membrane depolarization in vascular smooth muscle cells. Finally, tiliroside decreased the voltage-activated peak amplitude of the L-type Ca²âº channel current in freshly dissociated vascular smooth muscle cells from mesenteric arteries. Altogether, our results point to an antihypertensive effect of tiliroside due to a reduction in peripheral resistance through blockage of voltage-activated peak amplitude of the L-type Ca²âº channel in smooth muscle cells.

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.

Chronic exercise partially restores the transmural heterogeneity of action potential duration in left ventricular myocytes of spontaneous hypertensive rats.

Hypertension leads to electrophysiological changes in the heart. Chronic exercise induced by a treadmill-running programme (TRP) is considered a potential non-pharmacological treatment for hypertension and may have implications in heart remodelling. However, it is not known whether the TRP is able to improve the electrophysiological properties of the heart in spontaneously hypertensive rats (SHR). In the present study, we investigated whether TRP affects the electrical properties of left ventricular (LV) myocytes isolated from different layers of the LV wall of SHR. Male SHR were divided into exercised (chronic treadmill running for 8 weeks; CEX-SHR) and sedentary (SED-SHR) groups. Age-matched normotensive Wistar male rats served as controls. Action potentials (AP) and transient outward potassium current (I(to) ) were recorded in subepicardial (EPI) and subendocardial (ENDO) LV myocytes. In normotensive controls, AP duration (APD) was longer in ENDO cells than in EPI cells. This sort of transmural heterogeneity in the LV was not observed in sedentary SHR and was partially restored in SHR subject to chronic exercise. This partial recovery was associated with an increase in I(to) density in EPI cells but not in ENDO cells. The electrophysiological changes observed in the CEX-SHR group were not accompanied by either amelioration of systolic blood pressure or a reduction in heart hypertrophy. These findings imply that a TRP is able to improve the electrophysiological parameters of isolated cardiac myocytes in SHR. This sort of adaptation contributes to the overall improvement of heart physiology in this model.

Impact of IFN-γ Deficiency on the Cardiomyocyte Function in the First Stage of Experimental Chagas Disease.

Chagas disease (CD) is caused by the parasitic protozoan T. cruzi. The progression of CD in ~30% of patients results in Chagasic Cardiomyopathy (CCM). Currently, it is known that the inflammatory system plays a significant role in the CCM. Interferon-gamma (IFN-γ) is the major cytokine involved in parasitemia control but has also been linked to CCM. The L-type calcium current (ICa,L) is crucial in the excitation/contraction coupling in cardiomyocytes. Thus, we compared ICa,L and the mechanical properties of cardiomyocytes isolated from infected wild type (WT) and IFN-γ(-/-) mice in the first stage of T. cruzi infection. Using the patch clamp technique, we demonstrated that the infection attenuated ICa,L in isolated cardiomyocytes from the right and left ventricles of WT mice at 15 days post-infection (dpi), which was not observed in the IFN-γ(-/-) cardiomyocytes. However, ICa,L was attenuated between 26 and 30 dpi in both experimental groups. Interestingly, the same profile was observed in the context of the mechanical properties of isolated cardiomyocytes from both experimental groups. Simultaneously, we tracked the mortality and MCP-1, TNF-α, IL-12, IL-6, and IL-10 serum levels in the infected groups. Importantly, the IFN-γ(-/-) and WT mice presented similar parasitemia and serum inflammatory markers at 10 dpi, indicating that the modifications in the cardiomyocyte functions observed at 15 dpi were directly associated with IFN-γ(-/-) deficiency. Thus, we showed that IFN-γ plays a crucial role in the electromechanical remodeling of cardiomyocytes during experimental T. cruzi infection in mice.

A Potential Role of Cholinergic Dysfunction on Impaired Colon Motility in Experimental Intestinal Chagas Disease.

Background/Aims: Chagasic megacolon is caused by Trypanosoma cruzi, which promotes in several cases, irreversible segmental colonic dilation. This alteration is the major anatomic-clinical disorder, characterized by the enteric nervous system and muscle wall structural damage. Herein, we investigate how T. cruzi -induced progressive colonic structural changes modulate the colonic contractile pattern activity. Methods: We developed a murine model of T. cruzi-infection that reproduced long-term modifications of the enlarged colon. We evaluated colonic and total intestinal transit time in animals. The patterns of motor response at several time intervals between the acute and chronic phases were evaluated using the organ bath assays. Enteric motor neurons were stimulated by electric field stimulation. The responses were analyzed in the presence of the nicotinic and muscarinic acetylcholine receptor antagonists. Western blot was performed to evaluate the expression of nicotinic and muscarinic receptors. The neurotransmitter expression was analyzed by real-time polymerase chain reaction. Results: In the chronic phase of infection, there was decreased intestinal motility associated with decreased amplitude and rhythmicity of intestinal contractility. Pharmacological tests suggested a defective response mediated by acetylcholine receptors. The contractile response induced by acetylcholine was decreased by atropine in the acute phase while the lack of its action in the chronic phase was associated with tissue damage, and decreased expression of choline acetyltransferase, nicotinic subunits of acetylcholine receptors, and neurotransmitters. Conclusions: T. cruzi -induced damage of smooth muscles was accompanied by motility disorders such as decreased intestinal peristalsis and cholinergic system response impairment. This study allows integration of the natural history of Chagasic megacolon motility disorders and opens new perspectives for the design of effective therapeutic.

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.

A novel substrate for arrhythmias in Chagas disease.

BACKGROUND: Chagas disease (CD) is a neglected disease that induces heart failure and arrhythmias in approximately 30% of patients during the chronic phase of the disease. Despite major efforts to understand the cellular pathophysiology of CD there are still relevant open questions to be addressed. In the present investigation we aimed to evaluate the contribution of the Na+/Ca2+ exchanger (NCX) in the electrical remodeling of isolated cardiomyocytes from an experimental murine model of chronic CD. METHODOLOGY/PRINCIPAL FINDINGS: Male C57BL/6 mice were infected with Colombian strain of Trypanosoma cruzi. Experiments were conducted in isolated left ventricular cardiomyocytes from mice 180-200 days post-infection and with age-matched controls. Whole-cell patch-clamp technique was used to measure cellular excitability and Real-time PCR for parasite detection. In current-clamp experiments, we found that action potential (AP) repolarization was prolonged in cardiomyocytes from chagasic mice paced at 0.2 and 1 Hz. After-depolarizations, both subthreshold and with spontaneous APs events, were more evident in the chronic phase of experimental CD. In voltage-clamp experiments, pause-induced spontaneous activity with the presence of diastolic transient inward current was enhanced in chagasic cardiomyocytes. AP waveform disturbances and diastolic transient inward current were largely attenuated in chagasic cardiomyocytes exposed to Ni2+ or SEA0400. CONCLUSIONS/SIGNIFICANCE: The present study is the first to describe NCX as a cellular arrhythmogenic substrate in chagasic cardiomyocytes. Our data suggest that NCX could be relevant to further understanding of arrhythmogenesis in the chronic phase of experimental CD and blocking NCX may be a new therapeutic strategy to treat arrhythmias in this condition.

Endogenous opioid and cannabinoid systems modulate the muscle pain: A pharmacological study into the peripheral site.

The participation of the peripheral opioid and cannabinoid endogenous systems in modulating muscle pain and inflammation has not been fully explored. Thus, the aim of this study was to investigate the involvement of these endogenous systems during muscular-tissue hyperalgesia induced by inflammation. Hyperalgesia was induced by carrageenan injection into the tibialis anterior muscles of male Wistar rats. We padronized an available Randal-Sellito test adaptation to evaluate nociceptive behavior elicited by mechanical insult in muscles. Western blot analysis was performed to evaluate the expression levels of opioid and cannabinoid receptors in the dorsal root ganglia. The non-selective opioid peptide receptor antagonist (naloxone) and the selective mu opioid receptor MOP (clocinnamox) and kappa opioid receptor KOP (nor-binaltorphimine) antagonists were able to intensify carrageenan-induced muscular hyperalgesia. On the other hand, the selective delta opioid receptor (DOP) antagonist (naltrindole) did not present any effect on nociceptive behavior. Moreover, the selective inhibitor of aminopeptidases (Bestatin) provoked considerable dose-dependent analgesia when intramuscularly injected into the hyperalgesic muscle. The CB1 receptor antagonist (AM251), but not the CB2 receptor antagonist (AM630), intensified muscle hyperalgesia. All irreversible inhibitors of anandamide hydrolase (MAFP), the inhibitor for monoacylglycerol lipase (JZL184) and the anandamide reuptake inhibitor (VDM11) decreased carrageenan-induced hyperalgesia in muscular tissue. Lastly, MOP, KOP and CB1 expression levels in DRG were baseline even after muscular injection with carrageenan. The endogenous opioid and cannabinoid systems participate in peripheral muscle pain control through the activation of MOP, KOP and CB1 receptors.

Cardiac effect induced by Crotalus durissus cascavella venom: Morphofunctional evidence and mechanism of action.

Envenoming, resulting from snake bites, is a global public health problem. The present study was undertaken to investigate the influence of Crotalus durissus cascavella (Cdcas) venom on cardiac activity and the mechanisms of action underlying its effect. To investigate the inotropic and chronotropic effects induced by Cdcas, studies were performed on the left and right atria. A series of tests were conducted to investigate whether the negative inotropic effect, induced by Cdcas, was related to cardiac damage. Cdcas venom (0.1-30 µg/mL) elicited a significant negative inotropic effect. The addition of Cdcas crude venom (7.5, 15 and 30 µg/mL) did not induce significant alterations in cell proliferation, nor in the enzymatic activity of total-CK and CKMB. Ultrastructural evaluation demonstrated that cardiac cells from isoproterenol and Cdcas groups revealed discreet swelling and displaced intermyofibrillar mitochondria with disorganization of the cristae. No change was observed in cardiac electrical activity in perfused isolated rat hearts with Cdcas. In addition, Cdcas reduced contractility in isolated cardiomyocytes from the rat left ventricle. The negative inotropic effect of Cdcas was reduced by l-NAME (100 µM), PTIO (100 µM), ODQ (10 µM) and KT5823 (1 µM), suggesting the participation of NO/cGMP/PKG pathway due to Cdcas. In non-anesthetized rats, Cdcas induced hypotension followed by bradycardia, the latter was also observed by ECG (anesthetized animals). Our results suggest that the negative inotropic effect induced by Cdcas venom is unrelated to cardiac toxicity, at least, at the concentrations tested; and occurs through of NO/cGMP/PKG pathway, likely leading to hypotension and bradycardia when administered in vivo.

Cardiac oxidative stress is involved in heart failure induced by thiamine deprivation in rats.

Thiamine is an important cofactor of metabolic enzymes, and its deficiency leads to cardiovascular dysfunction. First, we characterized the metabolic status measuring resting oxygen consumption rate and lactate blood concentration after 35 days of thiamine deficiency (TD). The results pointed to a decrease in resting oxygen consumption and a twofold increase in blood lactate. Confocal microscopy showed that intracellular superoxide (approximately 40%) and H(2)O(2) (2.5 times) contents had been increased. In addition, biochemical activities and protein expression of SOD, glutathione peroxidase, and catalase were evaluated in hearts isolated from rats submitted to thiamine deprivation. No difference in SOD activity was detected, but protein levels were found to be increased. Catalase activity increased 2.1 times in TD hearts. The observed gain in activity was attended by an increased catalase protein level. However, a marked decrease in glutathione peroxidase activity (control 435.3 + or - 28.6 vs. TD 199.4 + or - 30.2 nmol NADPH x min(-1) x ml(-1)) was paralleled by a diminution in the protein levels. Compared with control hearts, we did observe a greater proportion of apoptotic myocytes by TdT-mediated dUTP nick end labeling (TUNEL) and caspase-3 reactivity techniques. These results indicate that during TD, reactive oxygen species (ROS) production may be enhanced as a consequence of the installed acidosis. The perturbation in the cardiac myocytes redox balance was responsible for the increase in apoptosis.

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.

Chronic Sympathetic Hyperactivity Triggers Electrophysiological Remodeling and Disrupts Excitation-Contraction Coupling in Heart.

The sympathetic nervous system is essential for maintenance of cardiac function via activation of post-junctional adrenergic receptors. Prolonged adrenergic receptor activation, however, has deleterious long-term effects leading to hypertrophy and the development of heart failure. Here we investigate the effect of chronic adrenergic receptors activation on excitation-contraction coupling (ECC) in ventricular cardiomyocytes from a previously characterized mouse model of chronic sympathetic hyperactivity, which are genetically deficient in the adrenoceptor α2A and α2C genes (ARDKO). When compared to wild-type (WT) cardiomyocytes, ARDKO displayed reduced fractional shortening (~33%) and slower relaxation (~20%). Furthermore, ARDKO cells exhibited several electrophysiological changes such as action potential (AP) prolongation (~50%), reduced L-type calcium channel (LCC) current (~33%), reduced outward potassium (K+) currents (~30%), and increased sodium/calcium exchanger (NCX) activity (~52%). Consistent with reduced contractility and calcium (Ca2+) currents, the cytosolic Ca2+ ([Ca2+]i) transient from ARDKO animals was smaller and decayed slower. Importantly, no changes were observed in membrane resting potential, AP amplitude, or the inward K+ current. Finally, we modified our existing cardiac ECC computational model to account for changes in the ARDKO heart. Simulations suggest that cellular changes in the ARDKO heart resulted in variable and dyssynchronous Ca2+-induced Ca2+ release therefore altering [Ca2+]i transient dynamics and reducing force generation. In conclusion, chronic sympathetic hyperactivity impairs ECC by changing the density of several ionic currents (and thus AP repolarization) causing altered Ca2+ dynamics and contractile activity. This demonstrates the important role of ECC remodeling in the cardiac dysfunction secondary to chronic sympathetic activity.

Redox-Active Drug, MnTE-2-PyP5+, Prevents and Treats Cardiac Arrhythmias Preserving Heart Contractile Function.

BACKGROUND: Cardiomyopathies remain among the leading causes of death worldwide, despite all efforts and important advances in the development of cardiovascular therapeutics, demonstrating the need for new solutions. Herein, we describe the effects of the redox-active therapeutic Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, AEOL10113, BMX-010 (MnTE-2-PyP5+), on rat heart as an entry to new strategies to circumvent cardiomyopathies. METHODS: Wistar rats weighing 250-300 g were used in both in vitro and in vivo experiments, to analyze intracellular Ca2+ dynamics, L-type Ca2+ currents, Ca2+ spark frequency, intracellular reactive oxygen species (ROS) levels, and cardiomyocyte and cardiac contractility, in control and MnTE-2-PyP5+-treated cells, hearts, or animals. Cells and hearts were treated with 20 µM MnTE-2-PyP5+ and animals with 1 mg/kg, i.p. daily. Additionally, we performed electrocardiographic and echocardiographic analysis. RESULTS: Using isolated rat cardiomyocytes, we observed that MnTE-2-PyP5+ reduced intracellular Ca2+ transient amplitude, without altering cell contractility. Whereas MnTE-2-PyP5+ did not alter basal ROS levels, it was efficient in modulating cardiomyocyte redox state under stress conditions; MnTE-2-PyP5+ reduced Ca2+ spark frequency and increased sarcoplasmic reticulum (SR) Ca2+ load. Accordingly, analysis of isolated perfused rat hearts showed that MnTE-2-PyP5+ preserves cardiac function, increases SR Ca2+ load, and reduces arrhythmia index, indicating an antiarrhythmic effect. In vivo experiments showed that MnTE-2-PyP5+ treatment increased Ca2+ transient, preserved cardiac ejection fraction, and reduced arrhythmia index and duration. MnTE-2-PyP5+ was effective both to prevent and to treat cardiac arrhythmias. CONCLUSION: MnTE-2-PyP5+ prevents and treats cardiac arrhythmias in rats. In contrast to most antiarrhythmic drugs, MnTE-2-PyP5+ preserves cardiac contractile function, arising, thus, as a prospective therapeutic for improvement of cardiac arrhythmia treatment.

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.