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)

Testosterone Induces Vascular Smooth Muscle Cell Migration by NADPH Oxidase and c-Src-Dependent Pathways

Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). The mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10(-7) mol/L, 0-120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day] pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase-derived ROS and c-Src-dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs. (Hypertension. 2012; 59: 1263-1271.). Online Data Supplement

Intrauterine food restriction alters the expression of uncoupling proteins in brown adipose tissue of rat newborns

A previous study from our laboratory showed that maternal food restriction (MFR) delays thermoregulation in newborn rats. In neonates brown adipose tissue (BAT) is essential for thermogenesis due to the presence of uncoupling proteins (UCPs). The aim of this study was to evaluate the influence of MFR on the UCPs mRNA and protein expression in BAT and skeletal muscle (SM) of the newborn rat. Female Wistar EPM-1 control rats (CON) received chow ad libitum during pregnancy, whereas food-restricted dams (RES) received 50% of the amount ingested by CON. Fifteen hours after birth, the litters were weighed and sacrificed. Blood was collected for hormonal analysis. BAT and SM were used for determination of UCPs mRNA and protein expression, and Ca2+-ATPase sarcoplasmic reticulum (SERCA1). RES pups showed a significant reduction in body weight and fat content at birth. MFR caused a significant increase in the expression of UCP1 and UCP2 in BAT, without changes in UCP3 and SERCA1 expression in BAT and SM. No differences between groups were found for leptin, T4 and glucose levels. RES pups showed increased insulin and decreased T3 levels. The delay in development of thermoregulation previously described in RES animals appears not to result from impairment in thermogenesis, but from an increase in heat loss, since MFR caused low birth weight in pups, leading to greater surface/volume ratio. The higher expression of UCP1 and UCP2 in BAT suggests a compensatory mechanism to increased thermogenesis. (C) 2011 Elsevier Ltd. All rights reserved.

The beta 2-Adrenoceptor Agonist Formoterol Improves Structural and Functional Regenerative Capacity of Skeletal Muscles From Aged Rat at the Early Stages of Postinjury

Skeletal muscles from old rats fail to completely regenerate following injury. This study investigated whether pharmacological stimulation of beta 2-adrenoceptors in aged muscles following injury could improve their regenerative capacity, focusing on myofiber size recovery. Young and aged rats were treated with a subcutaneous injection of beta 2-adrenergic agonist formoterol (2 mu g/kg/d) up to 10 and 21 days after soleus muscle injury. Formoterol-treated muscles from old rats evaluated at 10 and 21 days postinjury showed reduced inflammation and connective tissue but a similar number of regenerating myofibers of greater caliber when compared with their injured controls. Formoterol minimized the decrease in tetanic force and increased protein synthesis and mammalian target of rapamycin phosphorylation in old muscles at 10 days postinjury. Our results suggest that formoterol improves structural and functional regenerative capacity of regenerating skeletal muscles from aged rats by increasing protein synthesis via mammalian target of rapamycin activation. Furthermore, formoterol may have therapeutic benefits in recovery following muscle damage in senescent individuals.

The role of reactive oxygen species in the modulation of the contraction induced by angiotensin II in carotid artery from diabetic rat

The modulation played by reactive oxygen species on the angiotensin II-induced contraction in type I-diabetic rat carotid was investigated. Concentration-response curves for angiotensin II were obtained in endothelium-intact or endothelium-denuded carotid from control or streptozotocin-induced diabetic rats, pre-treated with tiron (superoxide scavenger), PEG-catalase (hydrogen peroxide scavenger), dimethylthiourea (hydroxyl scavenger), apocynin [NAD(P) H oxidase inhibitor], SC560 (cyclooxygenase-1 inhibitor), SC236 (cyclooxygenase-2 inhibitor) or Y-27632 (Rho-kinase inhibitor). Reactive oxygen species were measured by flow cytometry in dihydroethidium (DHE)-loaded endothelial cells. Cyclooxygenase and AT1-receptor expression was assessed by immunohistochemistry. Diabetes increased the angiotensin II-induced contraction but reduced the agonist potency in rat carotid. Endothelium removal, tiron or apocynin restored the angiotensin II-induced contraction in diabetic rat carotid to control levels. PEG-catalase, DMTU or SC560 reduced the angiotensin II-induced contraction in diabetic rat carotid at the same extent. SC236 restored the angiotensin II potency in diabetic rat carotid. Y-27632 reduced the angiotensin II-induced contraction in endothelium-intact or -denuded diabetic rat carotid. Diabetes increased the DHE-fluorescence of carotid endothelial cells. Apocynin reduced the DHE-fluorescence of endothelial cells from diabetic rat carotid to control levels. Diabetes increased the muscular cyclooxygenase-2 expression but reduced the muscular AT1-receptor expression in rat carotid. In summary, hydroxyl radical, hydrogen peroxide and superoxide anion-derived from endothelial NAD(P) H oxidase mediate the hyperreactivity to angiotensin II in type I-diabetic rat carotid, involving the participation of cyclooxygenase-1 and Rho-kinase. Moreover, increased muscular cyclooxygenase-2 expression in type I-diabetic rat carotid seems to be related to the local reduced AT1-receptor expression and the reduced angiotensin II potency. (C) 2011 Elsevier B. V. All rights reserved.

Differential cellular FGF-2 upregulation in the rat facial nucleus following axotomy, functional electrical stimulation and corticosterone: a possible therapeutic target to Bell's palsy

Abstract Background The etiology of Bell's palsy can vary but anterograde axonal degeneration may delay spontaneous functional recovery leading the necessity of therapeutic interventions. Corticotherapy and/or complementary rehabilitation interventions have been employed. Thus the natural history of the disease reports to a neurotrophic resistance of adult facial motoneurons leading a favorable evolution however the related molecular mechanisms that might be therapeutically addressed in the resistant cases are not known. Fibroblast growth factor-2 (FGF-2) pathway signaling is a potential candidate for therapeutic development because its role on wound repair and autocrine/paracrine trophic mechanisms in the lesioned nervous system. Methods Adult rats received unilateral facial nerve crush, transection with amputation of nerve branches, or sham operation. Other group of unlesioned rats received a daily functional electrical stimulation in the levator labii superioris muscle (1 mA, 30 Hz, square wave) or systemic corticosterone (10 mgkg-1). Animals were sacrificed seven days later. Results Crush and transection lesions promoted no changes in the number of neurons but increased the neurofilament in the neuronal neuropil of axotomized facial nuclei. Axotomy also elevated the number of GFAP astrocytes (143% after crush; 277% after transection) and nuclear FGF-2 (57% after transection) in astrocytes (confirmed by two-color immunoperoxidase) in the ipsilateral facial nucleus. Image analysis reveled that a seven days functional electrical stimulation or corticosterone led to elevations of FGF-2 in the cytoplasm of neurons and in the nucleus of reactive astrocytes, respectively, without astrocytic reaction. Conclusion FGF-2 may exert paracrine/autocrine trophic actions in the facial nucleus and may be relevant as a therapeutic target to Bell's palsy.

Morphologic and biomechanical changes of thoracic and abdominal aorta in a rat model of cigarette smoke exposure

Background: Smoking is the most relevant environmental factor that affects the development of aortic aneurysm. Smokers have elevated levels of elastase activity in the arterial wall, which leads to weakening of the aorta. The aim of this study was to verify whether cigarette smoke exposure itself is capable of altering the aortic wall. Methods: Forty-eight Wistar rats were divided into 2-, 4-, and 6-month experimental periods and into 2 groups: smokers (submitted to smoke exposure at a rate of 40 cigarettes/day) and nonsmokers. At the end of the experimental periods, the aortas were removed and crosssectioned to obtain histologic specimens for light microscopic and morphometric analyses. The remaining longitudinal segments were stretched to rupture and mechanical parameters were determined. Results: A degenerative process (i.e., a reduction in elastic fibers, the loss of lamellar arrangement, and a reduction of smooth muscle cells) was observed, and this effect was proportional in intensity to the period of tobacco exposure. We observed a progressive reduction in the yield point of the thoracic aorta over time (P < 0.05). There was a decrease in stiffness (P < 0.05) and in failure load (P < 0.05) at 6 months in the abdominal aorta of rats in the smoking group. Conclusions: Chronic exposure to tobacco smoke can affect the mechanical properties of the aorta and can also provoke substantial structural changes of the arterial wall

Changes in food intake, metabolic parameters and insulin resistance are induced by an isoenergetic, medium-chain fatty acid diet and are associated with modifications in insulin signalling in isolated rat pancreatic islets

LLong-chain fatty acids are capable of inducing alterations in the homoeostasis of glucose-stimulated insulin secretion (GSIS), but the effect of medium-chain fatty acids (MCFA) is poorly elucidated. In the present study, we fed a normoenergetic MCFA diet to male rats from the age of 1 month to the age of 4 months in order to analyse the effect of MCFA on body growth, insulin sensitivity and GSIS. The 45% MCFA substitution of whole fatty acids in the normoenergetic diet impaired whole body growth and resulted in increased body adiposity and hyperinsulinaemia, and reduced insulin-mediated glucose uptake in skeletal muscle. In addition, the isolated pancreatic islets from the MCFA-fed rats showed impaired GSIS and reduced protein kinase Ba (AKT1) protein expression and extracellular signal-related kinase isoforms 1 and 2 (ERK(1/2)) phosphorylation, which were accompanied by increased cellular death. Furthermore, there was a mildly increased cholinergic sensitivity to GSIS. We discuss these findings in further detail, and advocate that they might have a role in the mechanistic pathway leading to the compensatory hyperinsulinaemic status found in this animal model.

Suppressive effect of low-level laser therapy on tracheal hyperresponsiveness and lung inflammation in rat subjected to intestinal ischemia and reperfusion

Intestinal ischemia and reperfusion (i-I/R) is an insult associated with acute respiratory distress syndrome (ARDS). It is not known if pro- and anti-inflammatory mediators in ARDS induced by i-I/R can be controlled by low-level laser therapy (LLLT). This study was designed to evaluate the effect of LLLT on tracheal cholinergic reactivity dysfunction and the release of inflammatory mediators from the lung after i-I/R. Anesthetized rats were subjected to superior mesenteric artery occlusion (45 min) and killed after clamp release and preestablished periods of intestinal reperfusion (30 min, 2 or 4 h). The LLLT (660 nm, 7.5 J/cm(2)) was carried out by irradiating the rats on the skin over the right upper bronchus for 15 and 30 min after initiating reperfusion and then euthanizing them 30 min, 2, or 4 h later. Lung edema was measured by the Evans blue extravasation technique, and pulmonary neutrophils were determined by myeloperoxidase (MPO) activity. Pulmonary tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), intercellular adhesion molecule-1 (ICAM-1), and isoform of NO synthase (iNOS) mRNA expression were analyzed by real-time PCR. TNF-α, IL-10, and iNOS proteins in the lung were measured by the enzyme-linked immunoassay technique. LLLT (660 nm, 7.5 J/cm(2)) restored the tracheal hyperresponsiveness and hyporesponsiveness in all the periods after intestinal reperfusion. Although LLLT reduced edema and MPO activity, it did not do so in all the postreperfusion periods. It was also observed with the ICAM-1 expression. In addition to reducing both TNF-α and iNOS, LLLT increased IL-10 in the lungs of animals subjected to i-I/R. The results indicate that LLLT can control the lung's inflammatory response and the airway reactivity dysfunction by simultaneously reducing both TNF-α and iNOS.

Leucine and HMB differentially modulate proteasome system in skeletal muscle under different sarcopenic conditions

In the present study we have compared the effects of leucine supplementation and its metabolite β-hydroxy-β-methyl butyrate (HMB) on the ubiquitin-proteasome system and the PI3K/Akt pathway during two distinct atrophic conditions, hindlimb immobilization and dexamethasone treatment. Leucine supplementation was able to minimize the reduction in rat soleus mass driven by immobilization. On the other hand, leucine supplementation was unable to provide protection against soleus mass loss in dexamethasone treated rats. Interestingly, HMB supplementation was unable to provide protection against mass loss in all treatments. While solely fiber type I cross sectional area (CSA) was protected in immobilized soleus of leucine-supplemented rats, none of the fiber types were protected by leucine supplementation in rats under dexamethasone treatment. In addition and in line with muscle mass results, HMB treatment did not attenuate CSA decrease in all fiber types against either immobilization or dexamethasone treatment. While leucine supplementation was able to minimize increased expression of both Mafbx/Atrogin and MuRF1 in immobilized rats, leucine was only able to minimize Mafbx/Atrogin in dexamethasone treated rats. In contrast, HMB was unable to restrain the increase in those atrogenes in immobilized rats, but in dexamethasone treated rats, HMB minimized increased expression of Mafbx/Atrogin. The amount of ubiquitinated proteins, as expected, was increased in immobilized and dexamethasone treated rats and only leucine was able to block this increase in immobilized rats but not in dexamethasone treated rats. Leucine supplementation maintained soleus tetanic peak force in immobilized rats at normal level. On the other hand, HMB treatment failed to maintain tetanic peak force regardless of treatment. The present data suggested that the anti-atrophic effects of leucine are not mediated by its metabolite HMB.

Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.

Rapidly excised and cryofixed rat tissue

All preparation efforts of biological samples in electron microscopy are focused to preserve structures as close as possible to the native state. To achieve this goal with tissues, it is of advantage to have a very short time between excision and fixation. The most common approach is chemical fixation: cross-linking of the tissue samples with aldehydes followed by postfixation with osmium tetroxide. Here, the fastest approach for tissue samples is perfusion. However, the diffusion of the fixation solution from blood vessels into the depth of the tissue is still slow and does not allow an overall instant fixation of a single cell. As a result, osmotic effects become evident (swelling or shrinkage of cell organelles). Another possibility is to take a tissue sample from the experimental animal. Excision of tissue can last quite some time, which results in even more pronounced autolytic induced osmotic effects. Furthermore, the animal does not survive the procedure in most cases. Alternatively, microbiopsies are an elegant technique to rapidly excise small quantities of tissue. Some tissues, such as liver and muscle, may be obtained using a non-lethal approach. To avoid the artifacts introduced by chemical fixation, high-pressure freezing of microbiopsies (brain, liver, kidney, and muscle) is a powerful alternative to chemical fixation. Here, we describe the microbiopsy method, and high-pressure freezing/freeze-substitution (HPF/FS) as a follow-up procedure. Cryosectioning of high-pressure frozen samples is optimally preserving the ultrastructure; however, it is not considered to be a routine approach yet.

Threshold-dependent effects on peripheral nerve in vivo excitability properties in the rat

Various factors, including maturity, have been shown to influence peripheral nerve excitability measures, but little is known about differences in these properties between axons with different stimulation thresholds. Multiple nerve excitability tests were performed on the caudal motor axons of immature and mature female rats, recording from tail muscles at three target compound muscle action potential (CMAP) levels: 10%, 40% ("standard" level), and 60% of the maximum CMAP amplitude. Compared to lower target levels, axons at high target levels have the following characteristics: lower strength-duration time constant, less threshold reduction during depolarizing currents and greater threshold increase to hyperpolarizing currents, most notably to long hyperpolarizing currents in mature rats. Threshold-dependent effects on peripheral nerve excitability properties depend on the maturation stage, especially inward rectification (Ih), which becomes inversely related to threshold level. Performing nerve excitability tests at different target levels is useful in understanding the variation in membrane properties between different axons within a nerve. Because of the threshold effects on nerve excitability and the possibility of increased variability between axons and altered electric recruitment order in disease conditions, excitability parameters measured only at the "standard" target level should be interpreted with caution, especially the responses to hyperpolarizing currents.

Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes

This work was motivated by the incomplete characterization of the role of vascular endothelial growth factor-A (VEGF-A) in the stressed heart in consideration of upcoming cancer treatment options challenging the natural VEGF balance in the myocardium. We tested, if the cytotoxic cancer therapy doxorubicin (Doxo) or the anti-angiogenic therapy sunitinib alters viability and VEGF signaling in primary cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). ARVM were isolated and cultured in serum-free medium. CMEC were isolated from the left ventricle and used in the second passage. Viability was measured by LDH-release and by MTT-assay, cellular respiration by high-resolution oxymetry. VEGF-A release was measured using a rat specific VEGF-A ELISA-kit. CMEC were characterized by marker proteins including CD31, von Willebrand factor, smooth muscle actin and desmin. Both Doxo and sunitinib led to a dose-dependent reduction of cell viability. Sunitinib treatment caused a significant reduction of complex I and II-dependent respiration in cardiomyocytes and the loss of mitochondrial membrane potential in CMEC. Endothelial cells up-regulated VEGF-A release after peroxide or Doxo treatment. Doxo induced HIF-1α stabilization and upregulation at clinically relevant concentrations of the cancer therapy. VEGF-A release was abrogated by the inhibition of the Erk1/2 or the MAPKp38 pathway. ARVM did not answer to Doxo-induced stress conditions by the release of VEGF-A as observed in CMEC. VEGF receptor 2 amounts were reduced by Doxo and by sunitinib in a dose-dependent manner in both CMEC and ARVM. In conclusion, these data suggest that cancer therapy with anthracyclines modulates VEGF-A release and its cellular receptors in CMEC and ARVM, and therefore alters paracrine signaling in the myocardium.

Abolishing myofibroblast arrhythmogeneicity by pharmacological ablation of α-smooth muscle actin containing stress fibers

Rationale: Myofibroblasts typically appear in the myocardium after insults to the heart like mechanical overload and infarction. Apart from contributing to fibrotic remodeling, myofibroblasts induce arrhythmogenic slow conduction and ectopic activity in cardiomyocytes after establishment of heterocellular electrotonic coupling in vitro. So far, it is not known whether α-smooth muscle actin (α-SMA) containing stress fibers, the cytoskeletal components that set myofibroblasts apart from resident fibroblasts, are essential for myofibroblasts to develop arrhythmogenic interactions with cardiomyocytes. Objective: We investigated whether pharmacological ablation of α-SMA containing stress fibers by actin-targeting drugs affects arrhythmogenic myofibroblast–cardiomyocyte cross-talk. Methods and Results: Experiments were performed with patterned growth cell cultures of neonatal rat ventricular cardiomyocytes coated with cardiac myofibroblasts. The preparations exhibited slow conduction and ectopic activity under control conditions. Exposure to actin-targeting drugs (Cytochalasin D, Latrunculin B, Jasplakinolide) for 24 hours led to disruption of α-SMA containing stress fibers. In parallel, conduction velocities increased dose-dependently to values indistinguishable from cardiomyocyte-only preparations and ectopic activity measured continuously over 24 hours was completely suppressed. Mechanistically, antiarrhythmic effects were due to myofibroblast hyperpolarization (Cytochalasin D, Latrunculin B) and disruption of heterocellular gap junctional coupling (Jasplakinolide), which caused normalization of membrane polarization of adjacent cardiomyocytes. Conclusions: The results suggest that α-SMA containing stress fibers importantly contribute to myofibroblast arrhythmogeneicity. After ablation of this cytoskeletal component, cells lose their arrhythmic effects on cardiomyocytes, even if heterocellular electrotonic coupling is sustained. The findings identify α-SMA containing stress fibers as a potential future target of antiarrhythmic therapy in hearts undergoing structural remodeling.