Enéas R.M. Gomes

Molecular Mechanisms Involved in the Angiotensin-(1-7)/Mas Signaling Pathway in Cardiomyocytes

Recently there has been growing evidence suggesting that beneficial effects of angiotensin-(1-7) [Ang-(1-7)] in the heart are mediated by its receptor Mas. However, the signaling pathways involved in these effects in cardiomyocytes are unknown. Here, we investigated the involvement of the Ang-(1-7)/Mas axis in NO generation and Ca2+ handling in adult ventricular myocytes using a combination of molecular biology, intracellular Ca2+ imaging, and confocal microscopy. Acute Ang-(1-7) treatment (10 nmol/L) leads to NO production and activates endothelial NO synthase and Akt in cardiomyocytes. Ang-(1-7)–dependent NO raise was abolished by pretreatment with A-779 (1 &mgr;mol/L). To confirm that Ang-(1-7) action is mediated by Mas, we used cardiomyocytes isolated from Mas-deficient mice. In Mas-deficient cardiomyocytes, Ang-(1-7) failed to increase NO levels. Moreover, Mas-ablation was accompanied by significant alterations in the proteins involved in the regulation of endothelial NO synthase activity, indicating that endothelial NO synthase and its binding partners are important effectors of the Mas-mediated pathway in cardiomyocytes. We then investigated the role of the Ang-(1-7)/Mas axis on Ca2+ signaling. Cardiomyocytes treated with 10 nmol/L of Ang-(1-7) did not show changes in Ca2+-transient parameters such as peak Ca2+ transients and kinetics of decay. Nevertheless, cardiomyocytes from Mas-deficient mice presented reduced peak and slower [Ca2+]i transients when compared with wild-type cardiomyocytes. Lower Ca2+ ATPase of the sarcoplasmic reticulum expression levels accompanied the reduced Ca2+ transient in Mas-deficient cardiomyocytes. Therefore, chronic Mas-deficiency leads to impaired Ca2+ handling in cardiomyocytes. Collectively, these observations reveal a key role for the Ang-(1-7)/Mas axis as a modulator of cardiomyocyte function.

Highly efficient siRNA delivery system into human and murine cells using single-wall carbon nanotubes

Development of RNA interference (RNAi) technology utilizing short interfering RNA sequences (siRNA) has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA coiling into carboxyl-functionalized single-wall carbon nanotubes (SWCNTs). The CNT-siRNA delivery system successfully demonstrates nonspecific toxicity and transfection efficiency greater than 95%. This approach offers the potential for siRNA delivery into different types of cells, including hard-to-transfect cells, such as neuronal cells and cardiomyocytes. We also tested the CNT-siRNA system in a non-metastatic human hepatocellular carcinoma cell line (SKHep1). In all types of cells used in this work the CNT-siRNA delivery system showed high efficiency and apparent no side effects for various in vitro applications.

Dysautonomia Due to Reduced Cholinergic Neurotransmission Causes Cardiac Remodeling and Heart Failure

ABSTRACT Overwhelming evidence supports the importance of the sympathetic nervous system in heart failure. In contrast, much less is known about the role of failing cholinergic neurotransmission in cardiac disease. By using a unique genetically modified mouse line with reduced expression of the vesicular acetylcholine transporter (VAChT) and consequently decreased release of acetylcholine, we investigated the consequences of altered cholinergic tone for cardiac function. M-mode echocardiography, hemodynamic experiments, analysis of isolated perfused hearts, and measurements of cardiomyocyte contraction indicated that VAChT mutant mice have decreased left ventricle function associated with altered calcium handling. Gene expression was analyzed by quantitative reverse transcriptase PCR and Western blotting, and the results indicated that VAChT mutant mice have profound cardiac remodeling and reactivation of the fetal gene program. This phenotype was attributable to reduced cholinergic tone, since administration of the cholinesterase inhibitor pyridostigmine for 2 weeks reversed the cardiac phenotype in mutant mice. Our findings provide direct evidence that decreased cholinergic neurotransmission and underlying autonomic imbalance cause plastic alterations that contribute to heart dysfunction.

Cardiac anti‐remodelling effect of aerobic training is associated with a reduction in the calcineurin/NFAT signalling pathway in heart failure mice

Cardiomyocyte hypertrophy occurs in response to a variety of physiological and pathological stimuli. While pathological hypertrophy in heart failure is usually coupled with depressed contractile function, physiological hypertrophy associates with increased contractility. In the present study, we explored whether 8 weeks of moderate intensity exercise training would lead to a cardiac anti‐remodelling effect in an experimental model of heart failure associated with a deactivation of a pathological (calcineurin/NFAT, CaMKII/HDAC) or activation of a physiological (Akt–mTOR) hypertrophy signalling pathway. The cardiac dysfunction, exercise intolerance, left ventricle dilatation, increased heart weight and cardiomyocyte hypertrophy from mice lacking α2A and α2C adrenoceptors (α2A/α2CARKO mice) were associated with sympathetic hyperactivity induced heart failure. The relative contribution of Ca2+–calmodulin high‐affinity (calcineurin/NFAT) and low‐affinity (CaMKII/HDAC) targets to pathological hypertrophy of α2A/α2CARKO mice was verified. While nuclear calcineurin B, NFATc3 and GATA‐4 translocation were significantly increased in α2A/α2CARKO mice, no changes were observed in CaMKII/HDAC activation. As expected, cyclosporine treatment decreased nuclear translocation of calcineurin/NFAT in α2A/α2CARKO mice, which was associated with improved ventricular function and a pronounced anti‐remodelling effect. The Akt/mTOR signalling pathway was not activated in α2A/α2CARKO mice. Exercise training improved cardiac function and exercise capacity in α2A/α2CARKO mice and decreased heart weight and cardiomyocyte width paralleled by diminished nuclear NFATc3 and GATA‐4 translocation as well as GATA‐4 expression levels. When combined, these findings support the notion that deactivation of calcineurin/NFAT pathway‐induced pathological hypertrophy is a preferential mechanism by which exercise training leads to the cardiac anti‐remodelling effect in heart failure.

Succinate modulates Ca2+ transient and cardiomyocyte viability through PKA-dependent pathway

GPR91 is an orphan G-protein-coupled receptor (GPCR) that has been characterized as a receptor for succinate, a citric acid cycle intermediate, in several tissues. In the heart, the role of succinate is unknown. We now report that rat ventricular cardiomyocytes express GPR91. We found that succinate, through GPR91, increases the amplitude and the rate of decline of global Ca(2+) transient, by increasing the phosphorylation levels of ryanodine receptor and phospholamban, two well known Ca(2+) handling proteins. The effects of succinate on Ca(2+) transient were abolished by pre-treatment with adenylyl cyclase and cAMP-dependent protein kinase (PKA) inhibitors. Direct PKA activation by succinate was further confirmed using a FRET-based A-kinase activity reporter. Additionally, succinate decreases cardiomyocyte viability through a caspase-3 activation pathway, effect also prevented by PKA inhibition. Taken together, these observations show that succinate acts as a signaling molecule in cardiomyocytes, modulating global Ca(2+) transient and cell viability through a PKA-dependent pathway.

Attenuation of isoproterenol-induced cardiac fibrosis in transgenic rats harboring an angiotensin-(1-7)-producing fusion protein in the heart

Objective: It has been shown that Ang-(1-7) has cardioprotective actions. To directly investigate the effects of Ang-(1-7) specifically in the heart, we generated and characterized transgenic (TG) rats which express an Ang-(1-7)-producing fusion protein driven by the α-MHC promoter. Methods and Results: After microinjection of the transgene into fertilized rat zygotes, we obtained four different transgenic lines. Homozygous animals were analyzed with regard to the expression profile of the transgene by ribonuclease protection assay. Transgene expression was detected mainly in the heart with weak or no expression in other organs. Heterozygous TG(hA-1-7)L7301 rats presented a significant increase in cardiac Ang-(1-7) concentration compared with control rats (17.1±2.1 versus 3.9±1.4 pg/mg protein in SD rats). Radiotelemetry analysis revealed that TG rats presented no significant changes in blood pressure and heart rate compared with normal rats. Overexpression of Ang-(1-7) in the heart produced slight improvement in resting cardiac function (+ dT/dt: 81530±1305.0 versus 77470±345.5 g/s bpm in SD rats, p < 0.05), which was in keeping with the enhanced [Ca2+] handling observed in cardiomyocytes of TG rats. TG(hA-1-7)L7301 rats also showed a greater capacity to withstand stress since TG rats showed a less pronounced deposition of collagen type III and fibronectin induced by isoproterenol treatment in the subendocardial area than in corresponding controls. In addition, hearts from TG rats showed reduced incidence and duration of reperfusion arrhythmias in comparison with SD rats. Conclusion: These results indicate that Ang-(1-7) has blood pressure-independent, antifibrotic effects, acting directly in the heart.

Non-neuronal cholinergic machinery present in cardiomyocytes offsets hypertrophic signals

Recent work has provided compelling evidence that increased levels of acetylcholine (ACh) can be protective in heart failure, whereas reduced levels of ACh secretion can cause heart malfunction. Previous data show that cardiomyocytes themselves can actively secrete ACh, raising the question of whether this cardiomyocyte derived ACh may contribute to the protective effects of ACh in the heart. To address the functionality of this non-neuronal ACh machinery, we used cholinesterase inhibitors and a siRNA targeted to AChE (acetylcholinesterase) as a way to increase the availability of ACh secreted by cardiac cells. By using nitric oxide (NO) formation as a biological sensor for released ACh, we showed that cholinesterase inhibition increased NO levels in freshly isolated ventricular myocytes and that this effect was prevented by atropine, a muscarinic receptor antagonist, and by inhibition of ACh synthesis or vesicular storage. Functionally, cholinesterase inhibition prevented the hypertrophic effect as well as molecular changes and calcium transient alterations induced by adrenergic overstimulation in cardiomyocytes. Moreover, inhibition of ACh storage or atropine blunted the anti-hypertrophic action of cholinesterase inhibition. Altogether, our results show that cardiomyocytes possess functional cholinergic machinery that offsets deleterious effects of hyperadrenergic stimulation. In addition, we show that adrenergic stimulation upregulates expression levels of cholinergic components. We propose that this cardiomyocyte cholinergic signaling could amplify the protective effects of the parasympathetic nervous system in the heart and may counteract or partially neutralize hypertrophic adrenergic effects.

Angiotensin-(1-7)-Mediated Signaling in Cardiomyocytes

The Renin-Angiotensin System (RAS) acts at multiple targets and has its synthesis machinery present in different tissues, including the heart. Actually, it is well known that besides Ang II, the RAS has other active peptides. Of particular interest is the heptapeptide Ang-(1-7) that has been shown to exert cardioprotective effects. In this way, great compilations about Ang-(1-7) actions in the heart have been presented in the literature. However, much less information is available concerning the Ang-(1-7) actions directly in cardiomyocytes. In this paper, we show the actual knowledge about Ang-(1-7)-mediated signaling in cardiac cells more specifically we provide a brief overview of ACE2/Ang-(1-7)/Mas axis; and highlight the discoveries made in cardiomyocyte physiology through the use of genetic approaches. Finally, we discuss the protective signaling induced by Ang-(1-7) in cardiomyocytes and point molecular determinants of these effects.

Role of SOCS2 in Modulating Heart Damage and Function in a Murine Model of Acute Chagas Disease

Infection with Trypanosoma cruzi induces inflammation, which limits parasite proliferation but may result in chagasic heart disease. Suppressor of cytokine signaling 2 (SOCS2) is a regulator of immune responses and may therefore participate in the pathogenesis of T. cruzi infection. SOCS2 is expressed during T. cruzi infection, and its expression is partially reduced in infected 5-lipoxygenase-deficient [knockout (KO)] mice. In SOCS2 KO mice, there was a reduction in both parasitemia and the expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-6, IL-10, SOCS1, and SOCS3 in the spleen. Expression of IFN-γ, TNF-α, SOCS1, and SOCS3 was also reduced in the hearts of infected SOCS2 KO mice. There was an increase in the generation and expansion of T regulatory (Treg) cells and a decrease in the number of memory cells in T. cruzi-infected SOCS2 KO mice. Levels of lipoxinA(4) (LXA(4)) increased in these mice. Echocardiography studies demonstrated an impairment of cardiac function in T. cruzi-infected SOCS2 KO mice. There were also changes in calcium handling and in action potential waveforms, and reduced outward potassium currents in isolated cardiac myocytes. Our data suggest that reductions of inflammation and parasitemia in infected SOCS2-deficient mice may be secondary to the increases in Treg cells and LXA(4) levels. This occurs at the cost of greater infection-associated heart dysfunction, highlighting the relevance of balanced inflammatory and immune responses in preventing severe T. cruzi-induced disease.

Cardiotoxic effects of Loxosceles intermedia spider venom and the recombinant venom toxin rLiD1

Loxosceles spider bites cause many human injuries worldwide. Injections in mice of whole Loxosceles (L.) intermedia venom or a recombinant toxin (rLiD1) produce systemic symptoms similar to those detected in envenomed humans. This animal model was used to characterize the effects of Loxosceles intermedia venom in cardiac tissues. L. intermedia antigens were detected by ELISA in kidney, heart, lung and liver of experimentally envenomed mice. In addition, rLiD1 binding to cardiomyocytes was demonstrated by immunofluorescence and confocal microscopy. Furthermore, isolated perfused heart preparations and ventricular cardiomyocytes from envenomed mice showed heart function impairment, and a significant increase of I(Ca,L) density and intracellular Ca(2+) transients, respectively. Thus, L. intermedia spider venom, as shown through the use of the recombinant toxin rLiD1, causes cardiotoxic effects and a protein from the sphingomyelinase D family plays a key role in heart dysfunction. Thus, L. intermedia spider venom and the Loxtox rLiD1 play a key role in heart dysfunction.