Pedro W.M. Almeida

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.

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.

Swim training suppresses tumor growth in mice.

The present study was designed to determine the effects of physical training on the development of cancer induced by the injection of Ehrlich tumor cells in mice. Male Swiss mice were subjected to a swim training protocol (5 days/wk for 6 wk, 1 h at 50% of maximal capacity-trained groups) or remained sedentary in their cages (sedentary groups). The inoculation of Ehrlich tumor cells was performed at the end of the fourth week, and animals were killed after 6 wk of training. Heart and solid tumor weights were recorded, and tumor volumes were calculated. Portions of the tumors were used for the evaluation of macrophages and neutrophil accumulation or fixed in neutral 10% buffered formalin for histological analysis. The tumor volume and weight were, respectively, approximately 270% and 280% greater in sedentary mice than in trained mice. Macrophage infiltration in the tumor tissue was significantly lower in trained mice (0.65 +/- 0.16 vs. 1.78 +/- 0.43 macrophages x 10(3) in the sedentary group). Moreover, neutrophil accumulation in tumors was slightly reduced after exercise training, and the amount of tumor cells was reduced in trained mice. Exercise capacity was substantially increased in trained mice, as determined by a 440% increase in the exercise time at 50% of maximal capacity. In summary, swim training retarded the development of Ehrlich tumors in mice, accompanied by a reduction in macrophage infiltration and neutrophil accumulation. These findings provide conceptual support for clinical observations that controlled physical activities may be a therapeutically important approach to preventing cancer progression and may improve the outcome of cancer treatment.

The cardiac expression of Mas receptor is responsive to different physiological and pathological stimuli.

The Mas protooncogene encodes a G protein-coupled receptor that has been described as a functional receptor for the cardioprotective fragment of the renin-angiotensin system (RAS), Angiotensin (Ang)-(1-7). The aim of this current study was to evaluate the responsiveness of Mas expression in hearts during different physiological and pathological conditions in rats. Physical training was considered a physiological condition, while isoproterenol-induced hypertrophy, myocardial infarction and DOCA-salt model of hypertension were used as pathological models of heart injury. The expression of Mas was analyzed by western blotting. Although swim-trained rats presented significant cardiac hypertrophy, our physical training protocol was unable to induce changes in the expression of Mas. On the other hand, cardiac hypertrophy and damage elicited by isoproterenol treatment led to a reduction in Mas expression. Myocardial infarction also significantly decreased the expression of Mas after 21 days of myocardial ischemia. Additionally, Mas expression levels were increased in hearts of DOCA-salt rats. Our present data indicate that Mas expression is responsive to different pathological stimuli, thereby suggesting that Mas receptor is involved in the homeostasis of the heart, as well as in the establishment and progression of cardiac diseases.

Cholinergic signaling exerts protective effects in models of sympathetic hyperactivity-induced cardiac dysfunction.

Cholinergic control of the heart is exerted by two distinct branches; the autonomic component represented by the parasympathetic nervous system, and the recently described non-neuronal cardiomyocyte cholinergic machinery. Previous evidence has shown that reduced cholinergic function leads to deleterious effects on the myocardium. Yet, whether conditions of increased cholinergic signaling can offset the pathological remodeling induced by sympathetic hyperactivity, and its consequences for these two cholinergic axes are unknown. Here, we investigated two models of sympathetic hyperactivity: i) the chronic beta-adrenergic receptor stimulation evoked by isoproterenol (ISO), and ii) the α2A/α2C-adrenergic receptor knockout (KO) mice that lack pre-synaptic adrenergic receptors. In both models, cholinergic signaling was increased by administration of the cholinesterase inhibitor, pyridostigmine. First, we observed that isoproterenol produces an autonomic imbalance characterized by increased sympathetic and reduced parasympathetic tone. Under this condition transcripts for cholinergic proteins were upregulated in ventricular myocytes, indicating that non-neuronal cholinergic machinery is activated during adrenergic overdrive. Pyridostigmine treatment prevented the effects of ISO on autonomic function and on the ventricular cholinergic machinery, and inhibited cardiac remodeling. α2A/α2C-KO mice presented reduced ventricular contraction when compared to wild-type mice, and this dysfunction was also reversed by cholinesterase inhibition. Thus, the cardiac parasympathetic system and non-neuronal cardiomyocyte cholinergic machinery are modulated in opposite directions under conditions of increased sympathetic drive or ACh availability. Moreover, our data support the idea that pyridostigmine by restoring ACh availability is beneficial in heart disease.

Functional Cross-Talk Between Aldosterone and Angiotensin-(1-7) in Ventricular Myocytes

High serum levels of aldosterone have been linked to the development of cardiac disease. In contrast, angiotensin (Ang)-(1-7) was extensively shown to possess cardioprotective effects, including the attenuation of cardiac dysfunction induced by excessive mineralocorticoid activation in vivo, suggesting possible interactions between these 2 molecules. Here, we investigated whether there is cross-talk between aldosterone and Ang-(1-7) and its functional consequences for calcium (Ca2+) signaling in ventricular myocytes. Short-term effects of aldosterone on Ca2+ transient were assessed in Fluo-4/AM-loaded myocytes. Confocal images showed that Ang-(1-7) had no effect on Ca2+ transient parameters, whereas aldosterone increased the magnitude of the Ca2+ transient. Quite unexpectedly, addition of Ang-(1-7) to aldosterone-treated myocytes further enhanced the amplitude of the Ca2+ transient suggesting a synergistic effect of these molecules. Aldosterone action on Ca2+ transient amplitude was mediated by protein kinase A, and was related to an increase in Ca2+ current (ICa) density. Both changes were not altered by Ang-(1-7). When cardiomyocytes were exposed to aldosterone, increased Ca2+ spark rate was measured. Ang-(1-7) prevented this change. In addition, a NO synthase inhibitor restored the effect of aldosterone on Ca2+ spark rate in Ang-(1-7)-treated myocytes and attenuated the synergistic effect of these 2 molecules on Ca2+ transient. These results indicate that NO plays an important role in this cross-talk. Our results bring new perspectives in the understanding of how 2 prominent molecules with supposedly antagonist cardiac actions cross-talk to synergistically amplify Ca2+ signals in cardiomyocytes.

Exercise attenuates pulmonary injury in mice with bleomycin-induced pulmonary fibrosis

Human idiopathic pulmonary fibrosis (IPF) is a disease with unknown etiology and poor prognosis in which patients present a decrease in functional exercise tolerance and quality of life. At present, no treatment which can improve the prognosis of this disease is available. Many biomarkers of pulmonary fibrosis have been studied, and surfactant protein A (SP-A) expression is considered a specific marker of lung disease. This study aimed to investigate the influence of exercise training on exercise endurance capacity and murine-lung lesions induced by bleomycin (BLM). Thirty-four male Balb/c mice were subdivided into four groups: control sedentary (C-SED), bleomycin-treated sedentary (BLM-SED), control exercised (C-EXE) and bleomycin-treated exercised (BLM-EXE). Mice received 6.25 U/kg of BLM or saline via intratracheal instillation. After adaptation in a swimming pool, the animals started training one hour per day, with 60% of maximum load obtained in exercise endurance capacity assessment, five days/week for four weeks. The lungs were collected 48 h after the second endurance capacity assessment, fixed in buffered formalin and embedded in paraffin. Sections were analyzed using histochemical and immunohistochemical reactions for digital morphometry of pulmonary fibrosis, type I collagen, SP-A and type II pneumocytes (PII). The exercise endurance capacity of groups C-EXE (9.20 ± 0.81 min) and BLM-EXE (8.40 ± 0.82 min) increased significantly when compared with groups C-SED (5.84 ± 0.4 min) and BLM-SED (5.67 ± 0.60 min). The amounts of connective tissue, type I collagen, PII and SP-A increased significantly in the BLM-SED group. Exercise training significantly attenuated this response as observed in the BLM-EXE group. The present study shows that exercise training can prevent the decline of exercise endurance capacity and attenuate the progression of IPF.

Protein Restriction after Weaning Modifies the Calcium Kinetics and Induces Cardiomyocyte Contractile Dysfunction in Rats

Protein restriction (PR) is associated with cardiovascular diseases. The purpose of this study was to investigate the effects on single ventricular cardiomyocyte contractile function of a short-term PR after weaning. Male Fischer rats that were 28 days old were randomly divided into a control group (CG, n = 16) and a protein-restricted group (PRG, n = 16). After weaning, CG and PRG animals received isocaloric diets containing 15 and 6% protein, respectively, for 35 days. Biometric parameters were then measured, and the hearts were removed for the analysis of contractile function and calcium transient in isolated cardiomyocytes of the left ventricule (LV), and the quantification of calcium and collagen fibers in LV myocardium. PRG animals had lower body weight (BW) and LV weight (LVW), an increased LVW to BW ratio and a higher proportion of collagen fibers than CG animals. PRG animals exhibited reduced tissue levels of calcium, reduced the length, width and volume of cardiomyocytes and their sarcomere length compared to CG animals. Cardiomyocytes from PRG animals had a lower amplitude of shortening, a slower time to the peak of shortening and a longer time to half-relaxation than those from the CG. Cardiomyocytes from PRG animals also presented a lower peak of calcium transient and a longer calcium transient decay time than CG animals. Taken together, the results indicate that short-term PR after weaning induces a marked structural remodeling of the myocardium parenchyma and stroma that coexists with contractile dysfunctions in single LV cardiomyocytes of rats, which is probably associated with pathological changes of the intracellular calcium kinetics, rather than inadequate available amounts of this mineral in cardiac tissue.

Disorder creation and annealing in Hg implanted CdTe

Abstract Mercury has been implanted in CdTe single crystals at room temperature and the damaging process studied by Rutherford backscattering (RBS) of He particles in channeling conditions for increasing fluences. Although the energy deposited in nuclear collisions is large due to the heavy mass of the implanted ion, the disordering mechanism is similar to the one observed during light ion implantation. Hence, disordering is mainly governed by CdTe characteristics rather than by nuclear collision parameters. The furnace annealing behavior at different temperatures has been followed. For T = 400°C during 2 hours a total damage recovery as characterized by RBS is reached.

Exercise Training Protects Cardiomyocytes from Deleterious Effects of Palmitate

We investigated the effects of palmitate, a high saturated fat, on Ca2+, action potential and reactive oxygen species dynamics in cardiomyocytes from untrained and trained mice. Male mice were subjected to moderate intensity exercise training on a treadmill. Cardiomyocytes of untrained and trained mice were isolated, treated for 30 min with palmitate and intracellular calcium transient and action potential duration were recorded. Additionally, we assessed reactive oxygen species generation. Treatment of cardiomyocytes from untrained mice with palmitate induced a significant decrease in Ca2+ transient magnitude by 34%. Exercise training did not change cardiomyocyte Ca2+ dynamics in the control group. However, trained cardiomyocytes were protected from deleterious effects of palmitate. Action potential duration was not altered by palmitate in either untrained or trained cardiomyocytes. Moreover, palmitate treatment increased reactive oxygen species generation in both untrained and trained cardiomyocytes. Nevertheless, the levels of reactive oxygen species in trained cardiomyocytes treated with palmitate were still 27% lower than those seen at basal conditions in untrained cardiomyocytes. Taken together, these findings indicate that exercise training protects cardiomyocytes from deleterious effects of palmitate possibly by inhibiting exacerbated ROS production.