Efeitos preventivos do exercício físico sobre as proteínas ciclooxigenase-2 (COX-2) e fator de crescimento de vasos derivado do endotélio (VEGF) no músculo cardíaco em ratos tratados com dexametasona

Dexamethasone (DEXA) is a synthetic glucocorticoid widely used in the handling of several drugs, for its proven benefits in fighting inflammation and allergies. Despite their benefits, their chronic use leads to several side effects that include changes in the body in the metabolism of carbohydrates, lipids and proteins. Moreover, being an anti-inflammatory, acts on the arachidonic acid pathway, reducing the expression of the enzyme cyclooxygenase (COX-2) and growth factor derived from the endothelium of blood vessels (VEGF) in various tissues. However, its effects on the myocardium are still uncertain. The physical training (PT), in turn, promotes effects contrary to those caused by chronic use of DEXA, however, little is known about the preventive effects of TF in the side effects of Dexa in the myocardium. Therefore, the aim of this study was to determine if the TF has the ability to prevent and/or mitigate the effects of Dexa in protein expression of COX-2 and VEGF in the myocardium. Forty animals were divided into 4 groups: sedentary control (SC), sedentary treated with Dexa (SD), trained control (TC) and Trained treated with Dexa (TD) and submitted to a protocol of physical training on the treadmill for 70 days (1 h/day-5 days per week, 60% of physical capacity) or kept sedentary. Over the past 10 days, rats were treated with Dexa (Decadron, 0.5 mg/kg per day, ip) or saline. During training the animals were weighed weekly and during treatment daily. At the end of treatment was made to measure fasting glucose levels of animals. The rats were killed with excess anesthesia and cardiac muscle was removed, weighed, homogenized, centrifuged and stored at -20° C for analysis of protein expression of VEGF and COX-2 by Western blotting technique. Treatment with dexamethasone caused a weight loss of 18% in sedentary animals and 13% in trained as well as elevated levels of fasting glucose in sedentary (88%). The TF was unable to mitigate the loss in...

Identities among actin-encoding cDNAs of the Nile tilapia (Oreochromis niloticus) and other eukaryote species revealed by nucleotide and amino acid sequence analyses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Direct effects of colchicine on myocardial function: studies in hypertrophied and failing spontaneously hypertensive rats

The aging spontaneously hypertensive rat (SHR) is a model in which the transition from chronic stable left ventricular hypertrophy to overt heart failure can be observed. Although the mechanisms for impaired function in hypertrophied and failing cardiac muscle from the SHR have been studied, none accounts fully for the myocardial contractile abnormalities. The cardiac cytoskeleton has been implicated as a possible cause for myocardial dysfunction. If an increase in microtubules contributes to dysfunction, then myocardial microtubule disruption by colchicine should promote an improvement in cardiac performance. We studied the active and passive properties of isolated left ventricular papillary muscles from 18- to 24-month-old SHR with evidence of heart failure (SHR-F, n=6), age-matched SHR without heart failure (SHR-NF, n=6), and age-matched normotensive Wistar-Kyoto rats (WKY, n=5). Mechanical parameters were analyzed before and up to 90 minutes after the addition of colchicine (10(-5), 10(-4), and 10(-3) mol/L). In the baseline state, active tension (AT) developed by papillary muscles from the WKY group was greater than for SHR-NF and SHR-F groups (WKY 5.69+/-1.47 g/mm2 [mean+/-SD], SHR-NF 3.41+/-1.05, SHR-F 2.87+/-0.26; SHR-NF and SHR-F P<0.05 versus WKY rats). The passive stiffness was greater in SHR-F than in the WKY and SHR-NF groups (central segment exponential stiffness constant, Kcs: SHR-F 70+/-25, SHR-NF 44+/-17, WKY 41+/-13 [mean+/-SD]; SHR-F P<0.05 versus SHR-NF and WKY rats). AT did not improve after 10, 20, and 30 minutes of exposure to colchicine (10(-5), 10(-4), and 10(-3) mol/L) in any group. In the SHR-F group, AT and passive stiffness did not change after 30 to 90 minutes of colchicine exposure (10(-4) mol/L). In summary, the data in this study fail to demonstrate improvement of intrinsic muscle function in SHR with heart failure after colchicine. Thus, in the SHR there is no evidence that colchicine-induced cardiac microtubular depolymerization affects the active or passive properties of hypertrophied or failing left ventricular myocardium.

Eletrocardiograma em cães: revisão

The electrocardiogram (ECG) is an important tool used in the diagnosis of cardiac arrhythmias, since it gives the graphic representation of depolarization and repolarization processes of the cardiac muscle. This study allows recording the electrical activity of myocardial cells and the underlying differences in electric potential: the magnitude of this difference is measured in millivolts (mV), and its duration is measured in seconds. The ECG is indicated when an irregular rhythm is detected during physical examination, such as bradycardias, tachycardias or arrhythmias that are not secondary to breathing, in animals with a history of syncope or weakness, for monitoring the effectiveness of antiarrhythmic therapy, in cases of putative pleural or pericardial effusion, and also in systemic diseases that lead to arrhythmia. For a reliable assessment, the ECG must be evaluated in conjunction with findings from the physical examination and clinical signs of each patient.

Myocardial Remodeling after Large Infarcts in Rat Converts Post Rest-Potentiation in Force Decay

Background: Post-rest contraction (PRC) of cardiac muscle provides indirect information about the intracellular calcium handling. Objective: Our aim was to study the behavior of PRC, and its underlying mechanisms, in rats with myocardial infarction. Methods: Six weeks after coronary occlusion, the contractility of papillary muscles (PM) obtained from sham-operated (C, n = 17), moderate infarcted (MMI, n = 10) and large infarcted (LMI, n = 14) rats was evaluated, following rest intervals of 10 to 60 seconds before and after incubation with lithium chloride (Li+) substituting sodium chloride or ryanodine (Ry). Protein expression of SR Ca(2+)-ATPase (SERCA2), Na+/Ca2+ exchanger (NCX), phospholamban (PLB) and phospho-Ser(16)-PLB were analyzed by Western blotting. Results: MMI exhibited reduced PRC potentiation when compared to C. Opposing the normal potentiation for C, post-rest decays of force were observed in LMI muscles. In addition, Ry blocked PRC decay or potentiation observed in LMI and C; Li+ inhibited NCX and converted PRC decay to potentiation in LMI. Although MMI and LMI presented decreased SERCA2 (72 +/- 7% and 47 +/- 9% of Control, respectively) and phospho-Ser(16)-PLB (75 +/- 5% and 46 +/- 11%, respectively) protein expression, overexpression of NCX (175 +/- 20%) was only observed in LMI muscles. Conclusion: Our results showed, for the first time ever, that myocardial remodeling after MI in rats may change the regular potentiation to post-rest decay by affecting myocyte Ca(2+) handling proteins. (Arq Bras Cardiol 2012;98(3):243-251)

Azione antiossidante del sulforafane: analisi in cellule cardiache in coltura ed in un modello animale di esercizio fisico

Oxidative stress is considered to be of major relevance for a variety of pathological processes. Thus, it is valuable to identify compounds, which might act as antioxidants, i.e. compounds that antagonize the deleterious action of reactive oxygen species (ROS) on biomolecules. The mode of action of these compounds could be either to scavenge ROS directly or to trigger protective mechanisms inside the cell, thereby resulting in improved defense against ROS. Sulforaphane (SF) (1-isothiocyanato-(4R)-(methylsulfinyl)butane) is a naturally occurring cancer chemopreventive agent found as a precursor glucosinolate in Cruciferous vegetables like broccoli. Although SF is not a direct-acting antioxidant, there is substantial evidence that SF acts indirectly to increase the antioxidant capacity of animal cells and their abilities to cope with oxidative stress. Induction of phase 2 enzymes is one means by which SF enhances the cellular antioxidant capacity. Enzymes induced by SF include Glutathione S-transferases (GST) and NAD[P]H:quinone oxidoreductase (NQO1) which can function as protectors against oxidative stress. To protect themselves from oxidative stress, cells are equipped with reducing buffer systems including the GSH and thioredoxin (Trx) reductase. GSH is an important tripeptide thiol which in addition to being the substrate for GSTs maintains the cellular oxidation– reduction balance and protects cells against free radical species. Aim of the first part of this thesis was to investigate the ability of SF to induce the expression and the activity of different phase 2 and antioxidant enzymes (such as GST, GR, GPx, NQO1, TR, SOD, CAT) in an in vitro model of rat cardiomyocytes, and also to define if SF treatment supprts cells in counteracting oxidative stress induced by H2O2 It is well known that acute exhaustive exercise causes significant reactive oxygen species generation that results in oxidative stress, which can induce negative effects on health and well being. In fact, increased oxidative stress and biomarkers (e.g., protein carbonyls, MDA, and 8- hydroxyguanosine) as well as muscle damage biomarkers (e.g. plasmatic Creatine cinase and Lactate dehydrogenase) have been observed after supramaximal sprint exercises, exhaustive longdistance cycling or running as well as resistance-type exercises, both in trained and untrained humans. Markers of oxidative stress also increase in rodents following exhaustive exercise. Moreover, antioxidant enzyme activities and expressions of antioxidant enzymes are known to increase in response to exhaustive exercise in both animal and human tissues. Aim of this project was to evaluate the effect of SF supplementation in counteracting oxidative stress induced by physical activity through its ability to induce phase 2, and antioxidant enzymes in rat muscle. The results show that SF is a nutraceutical compound able to induce the activity of different phase 2 and antioxidant enzymes in both cardiac muscle and skeletal muscle. Thanks to its actions SF is becoming a promising molecule able to prevent cardiovascular damages induced by oxidative stress and muscle damages induced by acute exhaustive exercise.

In vivo electroporation mediated gene delivery to the beating heart

Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. In vivo electroporation, a physical method of gene transfer, has recently evolved as an efficient method for gene transfer. In the current study, we investigated the efficiency and safety of a protocol involving in vivo electroporation for gene transfer to the beating heart. Adult male rats were anesthetised and the heart exposed through a left thoracotomy. Naked plasmid DNA was injected retrograde into the transiently occluded coronary sinus before the electric pulses were applied. Animals were sacrificed at specific time points and gene expression was detected. Results were compared to the group of animals where no electric pulses were applied. No post-procedure arrhythmia was observed. Left ventricular function was temporarily altered only in the group were high pulses were applied; CK-MB (Creatine kinase) and TNT (Troponin T) were also altered only in this group. Histology showed no signs of toxicity. Gene expression was highest at day one. Our results provide evidence that in vivo electroporation with an optimized protocol is a safe and effective tool for nonviral gene delivery to the beating heart. This method may be promising for clinical settings especially for perioperative gene delivery.

Agonist specific L-type Ca(2+)-current stimulation in ventricular myocytes by a novel steroid-like compound

In cardiac muscle the amplitude of Ca(2+) transients can be increased by enhancing Ca(2+) influx. Among the processes leading to increased Ca(2+) influx, agonists of the L-type Ca(2+)-channel can play an important role. Known pharmacological Ca(2+)-channel agonists act on different binding sites on the channel protein, which may lead not only to enhanced peak currents, but also to distinct changes in other biophysical characteristics of the current. In this study, membrane currents were recorded with the patch-clamp technique in the whole-cell configuration in guinea pig isolated ventricular myocytes in combination with confocal fluorescence Ca(2+) imaging techniques and a variety of pharmacological tools. Testing a new positive inotropic steroid-like compound, we found that it increased the L-type Ca(2+)-current by 2.5-fold by shifting the voltage-dependence of activation by 20.2 mV towards negative potentials. The dose-response relationship revealed two vastly different affinities (EC(50(high-affinity))=4.5+/-1.7 nM, EC(50(low-affinity))=8.0+/-1.1 microM) exhibiting differential pharmacological interactions with three classes of Ca(2+)-current antagonists, suggesting more than one binding site on the channel protein. Therefore, we identified and characterized a novel positive inotropic compound (F90927) as a member of a new class of Ca(2+)-channel agonists exhibiting unique features, which set it apart from other presently known L-type Ca(2+)-channel agonists.

The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.

Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways

AIMS: Cardiac myopathies are the second leading cause of death in patients with Duchenne and Becker muscular dystrophy, the two most common and severe forms of a disabling striated muscle disease. Although the genetic defect has been identified as mutations of the dystrophin gene, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Here, we examined cellular mechanisms linking the initial membrane damage to the dysfunction of dystrophic heart. METHODS AND RESULTS: Cardiac ventricular myocytes were enzymatically isolated from 5- to 9-month-old dystrophic mdx and wild-type (WT) mice. Cells were exposed to mechanical stress, applied as osmotic shock. Stress-induced cytosolic and mitochondrial Ca(2+) signals, production of reactive oxygen species (ROS), and mitochondrial membrane potential were monitored with confocal microscopy and fluorescent indicators. Pharmacological tools were used to scavenge ROS and to identify their possible sources. Osmotic shock triggered excessive cytosolic Ca(2+) signals, often lasting for several minutes, in 82% of mdx cells. In contrast, only 47% of the WT cardiomyocytes responded with transient and moderate intracellular Ca(2+) signals. On average, the reaction was 6-fold larger in mdx cells. Removal of extracellular Ca(2+) abolished these responses, implicating Ca(2+) influx as a trigger for abnormal Ca(2+) signalling. Our further experiments revealed that osmotic stress in mdx cells produced an increase in ROS production and mitochondrial Ca(2+) overload. The latter was followed by collapse of the mitochondrial membrane potential, an early sign of cell death. CONCLUSION: Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.

Treatment of osteoporosis in a mouse model of Duchenne muscular dystrophy using black bear parathyroid hormone

Duchenne muscular dystrophy (DMD) is a progressive disease affecting skeletal and cardiac muscle, as well as bone. Long term disuse and glucocorticoid treatments cause progressive osteoporosis in DMD patients, leading to an increase in fracture incidence. Treatments for osteoporosis in these patients have not been widely explored. Parathyroid hormone (PTH), an anabolic treatment for post-menopausal osteoporosis, could benefit DMD patients by improving skeletal properties and reducing fracture risk. Other PTH analogues are not currently FDA approved to treat osteoporosis, but may have improved osteogenic effects compared to the human analogue. Black bear PTH is especially promising as an osteoporosis treatment for the DMD population. Black bears are unique models of bone maintenance during disuse, since during six months of inactivity (hibernation), they maintain skeletal properties, unlike other hibernators. Additionally, black bear PTH has been correlated to bone formation markers during hibernation, indicating it may be, at least in part, the mechanism by which bears maintain bone during disuse. Employing black bear PTH as a treatment for osteoporosis in DMD patients could greatly improve quality of life for these individuals, and reduce the pain and expense associated with frequent fractures.

In vivo electroporation-mediated gene delivery to the beating heart

Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. In vivo electroporation, a physical method of gene transfer, has recently evolved as an efficient method for gene transfer. Here, we describe two protocols involving in vivo electroporation for gene transfer to the beating heart.

Angiotensin II type1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload

Hypertrophy of mammalian cardiac muscle is mediated, in part, by angiotensin II through an angiotensin II type1a receptor (AT1aR)-dependent mechanism. To understand how the level of AT1aRs is altered in this pathological state, we studied the expression of an injected AT1aR promoter-luciferase reporter gene in adult rat hearts subjected to an acute pressure overload by aortic coarctation. This model was validated by demonstrating that coarctation increased expression of the α-skeletal actin promoter 1.7-fold whereas the α-myosin heavy chain promoter was unaffected. Pressure overload increased expression from the AT1aR promoter by 1.6-fold compared with controls. Mutations introduced into consensus binding sites for AP-1 or GATA transcription factors abolished the pressure overload response but had no effect on AT1aR promoter activity in control animals. In extracts from coarcted hearts, but not from control hearts, a Fos-JunB-JunD complex and GATA-4 were detected in association with the AP-1 and GATA sites, respectively. These results establish that the AT1aR promoter is active in cardiac muscle and its expression is induced by pressure overload, and suggest that this response is mediated, in part, by a functional interaction between AP-1 and GATA-4 transcription factors.

Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins

A major physiological role of insulin is the regulation of glucose uptake into skeletal and cardiac muscle and adipose tissue, mediated by an insulin-stimulated translocation of GLUT4 glucose transporters from an intracellular vesicular pool to the plasma membrane. This process is similar to the regulated docking and fusion of vesicles in neuroendocrine cells, a process that involves SNARE-complex proteins. Recently, several SNARE proteins were found in adipocytes: vesicle-associated membrane protein (VAMP-2), its related homologue cellubrevin, and syntaxin-4. In this report we show that treatment of permeabilized 3T3-L1 adipocytes with botulinum neurotoxin D, which selectively cleaves VAMP-2 and cellubrevin, inhibited the ability of insulin to stimulate translocation of GLUT4 vesicles to the plasma membrane. Furthermore, treatment of the permeabilized adipocytes with glutathione S-transferase fusion proteins encoding soluble forms of VAMP-2 or syntaxin-4 also effectively blocked insulin-regulated GLUT4 translocation. These results provide evidence of a functional role for SNARE-complex proteins in insulin-stimulated glucose uptake and suggest that adipocytes utilize a mechanism of regulating vesicle docking and fusion analogous to that found in neuroendocrine tissues.

Regulation of fatty acid homeostasis in cells: Novel role of leptin

It is proposed that an important function of leptin is to confine the storage of triglycerides (TG) to the adipocytes, while limiting TG storage in nonadipocytes, thus protecting them from lipotoxicity. The fact that TG content in nonadipocytes normally remains within a narrow range, while that of adipocytes varies enormously with food intake, is consistent with a system of TG homeostasis in normal nonadipocytes. The facts that when leptin receptors are dysfunctional, TG content in nonadipocytes such as islets can increase 100-fold, and that constitutively expressed ectopic hyperleptinemia depletes TG, suggest that leptin controls the homeostatic system for intracellular TG. The fact that the function and viability of nonadipocytes is compromised when their TG content rises above or falls below the normal range suggests that normal homeostasis of their intracellular TG is critical for optimal function and to prevent lipoapoptosis. Thus far, lipotoxic diabetes of fa/fa Zucker diabetic fatty rats is the only proven lipodegenerative disease, but the possibility of lipotoxic disease of skeletal and/or cardiac muscle may require investigation, as does the possible influence of the intracellular TG content on autoimmune and neoplastic processes.