Dantrolene improves in vitro structural changes induced by serum from Trypanosoma cruzi-infected mice.

Dystrophin, an important protein of the dystrophin-glycoprotein complex, has been implicated in the pathogenesis of experimental Chagas disease. It is important for the maintenance of cell shape and contraction force transmission. Dystrophin loss has been related to end-stage cardiac myopathies and proposed as a common route for myocardial dysfunction and progression to advanced heart failure. Evidence suggests that calpains, calcium-dependent proteases, digest dystrophin when the calcium concentration is compatible with their activation. The objective of this in vitro study was to test the hypothesis that dantrolene, a calcium channel blocker, improves structural changes induced by serum from Trypanosoma cruzi-infected mice. Cultured neonatal cardiac myocytes were incubated with serum from T. cruzi-infected mice and treated with dantrolene for 24 h. Immunofluorescence and immunoblotting were performed to evaluate dystrophin and calpain-1 protein expression. The levels of dystrophin decreased 13 % and calpain increased 17 % after incubation of cultured neonatal cardiac myocytes with serum from T. cruzi-infected mice. The treatment with dantrolene restored the dystrophin and calpain levels near control levels. Our results demonstrate that alterations in calcium homeostasis in cardiac myocytes are responsible, in part, for cardiac structural changes in experimentally induced T. cruzi myocarditis and that calpain inhibitors may be beneficial in Chagasic heart disease.

Reduced expression of adherens and gap junction proteins can have a fundamental role in the development of heart failure following cardiac hypertrophy in rats.

Hypertension causes cardiac hypertrophy, cardiac dysfunction and heart failure (HF). The mechanisms implicated in the transition from compensated to decompensated cardiac hypertrophy are not fully understood. This study was aimed to investigate whether alterations in the expression of intercalated disk proteins could contribute to the transition of compensated cardiac hypertrophy to dilated heart development that culminates in HF. Male rats were submitted to abdominal aortic constriction and at 90 days post surgery (dps), three groups were observed: sham-operated animals (controls), animals with hypertrophic hearts (HH) and animals with hypertrophic + dilated hearts (HD). Blood pressure was evaluated. The hearts were collected and Western blot and immunofluorescence were performed to desmoglein-2, desmocollin-2, N-cadherin, plakoglobin, Bcatenin, and connexin-43. Cardiac systolic function was evaluated using the Vevo 2100 ultrasound system. Data were considered significant when p b 0.05. Seventy percent of the animals presented with HH and 30% were HD at 90 dps. The blood pressure increased in both groups. The amount of desmoglein-2 and desmocollin-2 expression was increased in both groups and no difference was observed in either group. The expression of N-cadherin, plakoglobin and B-catenin increased in the HHgroup and decreased in the HDgroup; and connexin-43 decreased only in theHDgroup. Therewas no difference between the ejection fraction and fractional shortening at 30 and 60 dps; however, they were decreased in the HD group at 90 dps. We found that while some proteins have increased expression accompanied by the increase in the cell volume associated with preserved systolic cardiac function in theHHgroup, these same proteins had decreased expression evenwithout significant reduction in the cell volume associated with decreased systolic cardiac function in HD group. The increased expression of desmoglein-2 and desmocollin-2 in both the HH and HD groups could work as a protective compensatory mechanism, helping tomaintain the dilated heart.We can hypothesize that inappropriate intercellular mechanical and electrical coupling associated with necrosis and/or apoptosis are important factors contributing to the transition to HF.

Increase in parasympathetic tone by pyridostigmine prevents ventricular dysfunction during the onset of heart failure.

Heart failure (HF) is characterized by elevated sympathetic activity and reduced parasympathetic control of the heart. Experimental evidence suggests that the increase in parasympathetic function can be a therapeutic alternative to slow HF evolution. The parasympathetic neurotransmission can be improved by acetylcholinesterase inhibition. We investigated the long-term (4 wk) effects of the acetylcholinesterase inhibitor pyridostigmine on sympathovagal balance, cardiac remodeling, and cardiac function in the onset of HF following myocardial infarction. Myocardial infarction was elicited in adult male Wistar rats. After 4 wk of pyridostigmine administration, per os, methylatropine and propranolol were used to evaluate the cardiac sympathovagal balance. The tachycardic response caused by methylatropine was considered to be the vagal tone, whereas the bradycardic response caused by propranolol was considered to be the sympathetic tone. In conscious HF rats, pyridostigmine reduced the basal heart rate, increased vagal, and reduced sympathetic control of heart rate. Pyridostigmine reduced the myocyte diameter and collagen density of the surviving left ventricle. Pyridostigmine also increased vascular endothelial growth factor protein in the left ventricle, suggesting myocardial angiogenesis. Cardiac function was assessed by means of the pressure-volume conductance catheter system. HF rats treated with pyridostigmine exhibited a higher stroke volume, ejection fraction, cardiac output, and contractility of the left ventricle. It was demonstrated that the long-term administration of pyridostigmine started right after coronary artery ligation augmented cardiac vagal and reduced sympathetic tone, attenuating cardiac remodeling and left ventricular dysfunction during the progression of HF in rats.

Temporal changes in cardiac matrix metalloproteinase activity, oxidative stress, and TGF-ß in renovascular hypertension-induced cardiac hypertrophy.

Cardiovascular remodeling found in later phases of two-kidney, one-clip (2K1C) hypertension may involve key mechanisms particularly including MMP-2, oxidative stress, transforming growth factor-ß (TGF-ß), and inactivation of the endogenous MMP inhibitor, the tissue inhibitor of MMP (TIMP)-4. We examined whether temporal cardiac remodeling resulting from 2K1C hypertension occurs concomitantly with alterations in cardiac collagen, MMP activity, MMP-2, TIMP-4, TGF-ß, and reactive oxygen species (ROS) levels during the development of 2K1C hypertension. Sham-operated and 2K1C hypertensive rats were studied after 15, 30, and 75 days of hypertension. Systolic blood pressure was monitored weekly. Left ventricle (LV) morphometry and fibrosis were evaluated in hematoxylin/eosin and picrosirius red-stained sections, respectively. Cardiac MMP-2 levels/activity was determined by gelatin zymography, immunofluorescence, and in situ zymography. TIMP-4 levels were determined by western blotting. Cardiac TGF-ß levels were evaluated by immunofluorescence and ROS levels were evaluated with a dihydroethidium probe. 2K1C hypertension induced LV hypertrophy associated with augmented gelatinolytic activity at an early phase of hypertension and further increased after 75 days of hypertension. These alterations were associated with increased cardiac MMP-2, TGF-ß, and ROS in hypertensive rats. Higher TIMP-4 levels were found in hypertensive rats only after 75 days after surgery. Our findings show that increased MMP-2 activity is associated with concomitant development of LV hypertrophy and increased TGF-ß and ROS levels.

Sepsis: going to the heart of the matter.

Although myocardial depression is the predominant cause of death in severe sepsis/septic shock, it remains disputed whether the functional changes are a consequence of structural alterations. If we look at myocardial dysfunction from the perspective of a critically ill patient, there are a few questions to be asked: What causes myocardial dysfunction? What is the pathophysiology of cardiac dysfunction and death? Is there something that could be done to prevent the outcome? Each of these questions is interrelated and the answers will be more easily addressed if we continue to understand the basic mechanisms that are implicated. The principal mechanisms proposed for the pathogenesis of myocardial dysfunction support a prominent role for functional rather than anatomical abnormalities. However, attempts to reduce the high mortality in septic patients by manipulating the functional alterations have provided limited success. In recent years, the concept of septic cardiomyopathy has evolved, which implies alterations in the myocardial phenotype. This review includes an overview on the activation of the immune system and therapeutic approaches in sepsis, myocardial structural changes in the human septic heart, experimental models of sepsis, and cellular, molecular and functional myocardial changes seen in a variety of experimental sepsis models. The abnormal parameters discussed may emerge as therapeutic targets, for which modulation might provide beneficial effects on cardiovascular outcome and mortality in sepsis in the future.

MicroRNA Profiling Using a PCR-Based Method.

MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the post-transcriptional regulation of specific mRNA targets, thus possibly controlling many biological processes. The miRNA profiling analysis can contribute to understanding several signaling pathways, as biomarkers for molecular diagnostic, as well as potential to be used as therapeutic targets. The miRNAs expression can be analyzed by quantitative reverse transcription PCR (RT-qPCR), microarrays, and RNA sequencing. The RT-qPCR method is sensitive and specific and has a lower cost when compared to other techniques as microarrays and RNA sequencing. Therefore, the protocol presented in this chapter describes step by step all the details to perform miRNA analysis using primer-based RT-qPCR.

Prospective analysis of myocardial strain through the evolution of Chagas disease in the hamster animal model.

Speckle tracking echocardiography (STE) enables early diagnosis of myocardial damage by evaluating myocardial strain. We aimed to study sequential changes in structural and ventricular functional parameters during Chagas disease (CD) natural history in an animal model. 37 Syrian hamsters were inoculated intraperitoneally with Trypanosoma cruzi (Chagas) and 20 with saline (Control). Echocardiography was performed before the infection (baseline), at 1 month (acute phase), 4, 6, and 8 months (chronic phase) using Vevo 2100 (Fujifilm Inc.) ultrasound system. Left ventricular end-diastolic diameter, Left ventricular end-systolic diameter (LVESD), Left ventricular ejection fraction (LVEF), Global longitudinal (GLS), circumferential (GCS) and radial (GRS) strain were evaluated. Tricuspid annular plane systolic excursion (TAPSE) was used to assess right ventricular function. At 8 months, animals were euthanized and LV myocardial samples were analyzed for quantitation of inflammation and fibrosis. LVEF decreased over time in Chagas group and a difference from Control was detected at 6 months (p-value of groups#time interaction = 0.005). There was a pronounced decrease in GLS, GCS and TAPSE in Chagas group (p-value of groups#time interaction = 0.003 for GLS, < 0.001 for GCS and < 0.009 for TAPSE vs Control) since the first month. LVESD, LVEF and GLS were significantly correlated to the number of inflammatory cells (r = 0.41, p = 0.046; r = - 0.42, p = 0.042; r = 0.41, p = 0.047) but not to fibrosis. In the Syrian hamster model of CD STE parameters (GLS and GCS) showed an early decrease. Changes in LVEF, LVESD, and GLS were correlated to myocardial inflammation but not to fibrosis.

Combining two potential causes of metalloproteinase secretion causes abdominal aortic aneurysms in rats: a new experimental model.

Progress in understanding the pathophysiology of abdominal aortic aneurysms (AAA) is dependent in part on the development and application of effective animal models that recapitulate key aspects of the disease. The objective was to produce an experimental model of AAA in rats by combining two potential causes of metalloproteinase (MMP) secretion: inflammation and turbulent blood flow. Male Wistar rats were randomly divided in four groups: Injury, Stenosis, Aneurysm and Control (40/group). The Injury group received a traumatic injury to the external aortic wall. The Stenosis group received an extrinsic stenosis at a corresponding location. The Aneurysm group received both the injury and stenosis simultaneously, and the Control group received a sham operation. Animals were euthanized at days 1, 3, 7 and 15. Aorta and/or aneurysms were collected and the fragments were fixed for morphologic, immunohistochemistry and morphometric analyses or frozen for MMP assays. AAAs had developed by day 3 in 60-70% of the animals, reaching an aortic dilatation ratio of more than 300%, exhibiting intense wall remodelling initiated at the adventitia and characterized by an obvious inflammatory infiltrate, mesenchymal proliferation, neoangiogenesis, elastin degradation and collagen deposition. Immunohistochemistry and zymography studies displayed significantly increased expressions of MMP-2 and MMP-9 in aneurysm walls compared to other groups. The haemo-dynamic alterations caused by the stenosis may have provided additional contribution to the MMPs liberation. This new model illustrated that AAA can be multifactorial and confirmed the key roles of MMP-2 and MMP-9 in this dynamic remodelling process.

Disruption of sarcolemmal dystrophin and beta-dystroglycan may be a potential mechanism for myocardial dysfunction in severe sepsis.

Evidence from our laboratory has shown alterations in myocardial structure in severe sepsis/septic shock. The morphological alterations are heralded by sarcolemmal damage, characterized by increased plasma membrane permeability caused by oxidative damage to lipids and proteins. The critical importance of the dystrophin-glycoprotein complex (DGC) in maintaining sarcolemmal stability led us to hypothesize that loss of dystrophin and associated glycoproteins could be involved in early increased sarcolemmal permeability in experimentally induced septic cardiomyopathy. Male C57Bl/6 mice were subjected to sham operation and moderate (MSI) or severe (SSI) septic injury induced by cecal ligation and puncture (CLP). Using western blot and immunofluorescence, a downregulation of dystrophin and beta-dystroglycan expression in both severe and moderate injury could be observed in septic hearts. The immunofluorescent and protein amount expressions of laminin-alpha2 were similar in SSI and sham-operated hearts. Consonantly, the evaluation of plasma membrane permeability by intracellular albumin staining provided evidence of severe injury of the sarcolemma in SSI hearts, whereas antioxidant treatment significantly attenuated the loss of sarcolemmal dystrophin expression and the increased membrane permeability. This study offers novel and mechanistic data to clarify subcellular events in the pathogenesis of cardiac dysfunction in severe sepsis. The main finding was that severe sepsis leads to a marked reduction in membrane localization of dystrophin and beta-dystroglycan in septic cardiomyocytes, a process that may constitute a structural basis of sepsis-induced cardiac depression. In addition, increased sarcolemmal permeability suggests functional impairment of the DGC complex in cardiac myofibers. In vivo observation that antioxidant treatment significantly abrogated the loss of dystrophin expression and plasma membrane increased permeability supports the hypothesis that oxidative damage may mediate the loss of dystrophin and beta-dystroglycan in septic mice. These abnormal parameters emerge as therapeutic targets and their modulation may provide beneficial effects on future cardiovascular outcomes and mortality in sepsis.

Matrix metalloproteinase inhibition improves cardiac dysfunction and remodeling in 2-kidney, 1-clip hypertension.

BACKGROUND: Enhanced cardiac matrix metalloproteinase activity (MMPs) has been associated with ventricular remodeling and cardiac dysfunction. It is unknown whether MMPs contribute to systolic/diastolic dysfunction and compensatory remodeling in 2-kidney, 1-clip (2K1C) hypertensive rats. To test this hypothesis, we used 2K1C rats after 2 weeks of surgery treated or not with a nonspecific inhibitor of MMPs (doxycycline). METHODS AND RESULTS: We found that blood pressure and +/- dP/dt increased in 2K1C rats compared with sham groups, and these parameters were attenuated by doxycycline treatment (P < .05). Doxycycline also reversed cardiac hypertrophy observed in 2K1C rats (P < .05). Hypertensive rats showed increased MMP-2 levels in zymograms and in the tissue by immunofluorescence (P < .05) compared with sham groups. Increased total gelatinolytic activity was observed in untreated 2K1C rats when compared with sham groups (P < .05). Doxycycline decreased total gelatinolytic activity in 2K1C rats to control levels (P < .05). CONCLUSION: An imbalance in gelatinolytic activity, with increased MMP-2 levels and activity underlies the development of morphological and functional alterations found in the compensatory hypertrophy observed in 2K1C hearts. Because function and structure were restored by doxycycline, the inhibition of MMPs or their modulation may provide beneficial effects for therapeutic intervention in cardiac hypertrophy.

Turbulent blood flow plays an essential localizing role in the development of atherosclerotic lesions in experimentally induced hypercholesterolaemia in rats.

Taking into account that atherosclerosis is a focal disease and high levels of plasma cholesterol are closely correlated with its pathogenesis, it is a challenge to explain how equal concentrations of cholesterol bathing the endothelium can produce local, rather than global, effects on arteries. The focal distribution of atherosclerotic lesions has been considered to be dependent, at least in part, on hydrodynamic factors. The present study was carried out to further test the hypothesis that these forces are an important localizing factor in rats feeding a hypercholesterolaemic diet and submitted to infra-diaphragmatic aortic constriction. These animals develop a normotensive prestenotic region with laminar blood flow that serves as control for a normotensive poststenotic region with turbulent blood flow. Our findings clearly demonstrated that the combination of turbulent blood flow and low wall shear stress (WSS) in the presence of hypercholesterolaemia and oxidative stress creates conditions to the formation of focally distributed incipient atherosclerotic lesions observed in the poststenotic segment. In contrast, only diffuse fatty streaks could be observed in the normotensive prestenotic segment with laminar blood flow and normal WSS in the presence of hypercholesterolaemia and oxidative stress. Although haemodynamic forces are not by themselves responsible for the pathogenesis of atherosclerosis, they prime the local vascular wall in which the lesion develop. Further studies are required to establish how haemodynamic forces are detected and transduced into chemical signalling by the cells of the artery wall and then converted into pathophysiologically relevant phenotypic changes.

Isoproterenol induces primary loss of dystrophin in rat hearts: correlation with myocardial injury.

The mechanism of isoproterenol-induced myocardial damage is unknown, but a mismatch of oxygen supply vs. demand following coronary hypotension and myocardial hyperactivity is the best explanation for the complex morphological alterations observed. Severe alterations in the structural integrity of the sarcolemma of cardiomyocytes have been demonstrated to be caused by isoproterenol. Taking into account that the sarcolemmal integrity is stabilized by the dystrophin-glycoprotein complex (DGC) that connects actin and laminin in contractile machinery and extracellular matrix and by integrins, this study tests the hypothesis that isoproterenol affects sarcolemmal stability through changes in the DGC and integrins. We found different sensitivity of the DGC and integrin to isoproterenol subcutaneous administration. Immunofluorescent staining revealed that dystrophin is the most sensitive among the structures connecting the actin in the cardiomyocyte cytoskeleton and the extracellular matrix. The sarcomeric actin dissolution occurred after the reduction or loss of dystrophin. Subsequently, after lysis of myofilaments, gamma-sarcoglycan, beta-dystroglycan, beta1-integrin, and laminin alpha-2 expressions were reduced followed by their breakdown, as epiphenomena of the myocytolytic process. In conclusion, administration of isoproterenol to rats results in primary loss of dystrophin, the most sensitive among the structural proteins that form the DGC that connects the extracellular matrix and the cytoskeleton in cardiomyocyte. These changes, related to ischaemic injury, explain the severe alterations in the structural integrity of the sarcolemma of cardiomyocytes and hence severe and irreversible injury induced by isoproterenol.

Myocardial structural changes in long-term human severe sepsis/septic shock may be responsible for cardiac dysfunction.

The present investigation sought to determine the cellular mechanisms directly dependent on long-term severe sepsis/septic shock that could lead to myocardial structural changes in humans. Human hearts from eight cases of long-term severe sepsis/septic shock arising from infection, as defined by the ACCP/SCCM Consensus Conference; eight cases of acute necrotizing pancreatitis and acute lung injury, a noninfectious pathologic cause of systemic inflammatory response; and three cases of accidental death without thoracic injury selected from autopsies were studied. Transmural blocks of myocardial tissue were excised from the middle portion of the left ventricular free wall and were fixed in formalin or were frozen. Histochemical and immunohistochemical methods were used to evaluate the cross-striations of the myocardial cells, the number and size of interstitial macrophages, the intracardiomyocyte accumulation of lipid, the actin/myosin contractile apparatus, and the expression of iNOS, nitrotyrosine, and TNF-alpha in the myocardia of septic and control hearts. Greater interstitial cellular infiltration composed of larger and elongated macrophages and TNF-alpha protein expression in myofibers, interstitial macrophage cell types, and smooth muscle cells and endothelial cell in the vessels; intracardiomyocyte lipid accumulation; scattered foci of actin/myosin contractile apparatus disruption; and increased expression for iNOS and nitrotyrosine in myocytes and interstitial macrophage cell types could be observed in long-term human septic myocardium as compared with normal and acute pancreatitis control myocardia. These findings give support to an opinion that structural changes could be responsible for long-term sepsis-induced myocardial dysfunction. The higher number of macrophages, most of them with morphological features of "activation," and TNF-alpha protein expression could favor the reduction of cardiac function in septic hearts. The intramyocyte lipid accumulation in these hearts very likely reflects myocardium ventricular contractile dysfunction. In addition, the increased expression of iNOS and the evidence for the significant presence of peroxynitrite in cardiomyocytes and interstitial macrophage cell types suggest that oxidative damage may play a role in actin/myosin disruption in the hearts of septic patients.

Elastase-2, an angiotensin II-generating enzyme, contributes to increased angiotensin II in resistance arteries of mice with myocardial infarction.

BACKGROUND AND PURPOSE: Angiotensin II (Ang II), whose generation largely depends on angiotensin-converting enzyme (ACE) activity, mediates most of the renin-angiotensin-system (RAS) effects. Elastase-2 (ELA-2), a chymotrypsin-serine protease elastase family member 2A, alternatively generates Ang II in rat arteries. Myocardial infarction (MI) leads to intense RAS activation, but mechanisms involved in Ang II-generation in resistance arteries are unknown. We hypothesized that ELA-2 contributes to vascular Ang II generation and cardiac damage in mice subjected to MI. EXPERIMENTAL APPROACH: Concentration-effect curves to Ang I and Ang II were performed in mesenteric resistance arteries from male wild type (WT) and ELA-2 knockout (ELA-2KO) mice subjected to left anterior descending coronary artery ligation (MI). KEY RESULTS: MI size was similar in WT and ELA-2KO mice. Ejection fraction and fractional shortening after MI similarly decreased in both strains. However, MI decreased stroke volume and cardiac output in WT, but not in ELA-2KO mice. Ang I-induced contractions increased in WT mice subjected to MI (MI-WT) compared with sham-WT mice. No differences were observed in Ang I reactivity between arteries from ELA-2KO and ELA-2KO subjected to MI (MI-ELA-2KO). Ang I contractions increased in arteries from MI-WT versus MI-ELA-2KO mice. Chymostatin attenuated Ang I-induced vascular contractions in WT mice, but did not affect Ang I responses in ELA-2KO arteries. CONCLUSIONS AND IMPLICATIONS: These results provide the first evidence that ELA-2 contributes to increased Ang II formation in resistance arteries and modulates cardiac function after MI, implicating ELA-2 as a key player in ACE-independent dysregulation of the RAS.

Early dystrophin loss is coincident with the transition of compensated cardiac hypertrophy to heart failure.

Hypertension causes cardiac hypertrophy, one of the most important risk factors for heart failure (HF). Despite the importance of cardiac hypertrophy as a risk factor for the development of HF, not all hypertrophied hearts will ultimately fail. Alterations of cytoskeletal and sarcolemma-associated proteins are considered markers cardiac remodeling during HF. Dystrophin provides mechanical stability to the plasma membrane through its interactions with the actin cytoskeleton and, indirectly, to extracellular matrix proteins. This study was undertaken to evaluate dystrophin and calpain-1 in the transition from compensated cardiac hypertrophy to HF. Wistar rats were subjected to abdominal aorta constriction and killed at 30, 60 and 90 days post surgery (dps). Cardiac function and blood pressure were evaluated. The hearts were collected and Western blotting and immunofluorescence performed for dystrophin, calpain-1, alpha-fodrin and calpastatin. Statistical analyses were performed and considered significant when p<0.05. After 90 dps, 70% of the animals showed hypertrophic hearts (HH) and 30% hypertrophic+dilated hearts (HD). Systolic and diastolic functions were preserved at 30 and 60 dps, however, decreased in the HD group. Blood pressure, cardiomyocyte diameter and collagen content were increased at all time points. Dystrophin expression was lightly increased at 30 and 60 dps and HH group. HD group showed decreased expression of dystrophin and calpastatin and increased expression of calpain-1 and alpha-fodrin fragments. The first signals of dystrophin reduction were observed as early as 60 dps. In conclusion, some hearts present a distinct molecular pattern at an early stage of the disease; this pattern could provide an opportunity to identify these failure-prone hearts during the development of the cardiac disease. We showed that decreased expression of dystrophin and increased expression of calpains are coincident and could work as possible therapeutic targets to prevent heart failure as a consequence of cardiac hypertrophy.

Elastase-2, a Tissue Alternative Pathway for Angiotensin II Generation, Plays a Role in Circulatory Sympathovagal Balance in Mice.

In vitro and ex vivo experiments indicate that elastase-2 (ELA-2), a chymotrypsin-serine protease elastase family member 2A, is an alternative pathway for angiotensin II (Ang II) generation. However, the role played by ELA-2 in vivo is unclear. We examined ELA-2 knockout (ELA-2KO) mice compared to wild-type (WT) mice and determined whether ELA-2 played a role in hemodynamics [arterial pressure (AP) and heart rate (HR)], cardiocirculatory sympathovagal balance and baroreflex sensitivity. The variability of systolic arterial pressure (SAP) and pulse interval (PI) for evaluating autonomic modulation was examined for time and frequency domains (spectral analysis), whereas a symbolic analysis was also used to evaluate PI variability. In addition, baroreflex sensitivity was examined using the sequence method. Cardiac function was evaluated echocardiographically under anesthesia. The AP was normal whereas the HR was reduced in ELA-2KO mice (425 ± 17 vs. 512 ± 13 bpm from WT). SAP variability and baroreflex sensitivity were similar in both strains. The LF power from the PI spectrum (33.6 ± 5 vs. 51.8 ± 4.8 nu from WT) and the LF/HF ratio (0.60 ± 0.1 vs. 1.45 ± 0.3 from WT) were reduced, whereas the HF power was increased (66.4 ± 5 vs. 48.2 ± 4.8 nu from WT) in ELA-2KO mice, indicating a shift toward parasympathetic modulation of HR. Echocardiographic examination showed normal fractional shortening and an ejection fraction in ELA-2KO mice; however, the cardiac output, stroke volume, and ventricular size were reduced. These findings provide the first evidence that ELA-2 acts on the sympathovagal balance of the heart, as expressed by the reduced sympathetic modulation of HR in ELA-2KO mice.

Turbulent flow/low wall shear stress and stretch differentially affect aorta remodeling in rats.

OBJECTIVE: The present investigation was carried out to evaluate the relationship between local hemodynamic forces and intimal and medial remodeling in the proximal and distal segments of the arterial walls of rats in relation to severe infradiaphragmatic stenosis of the aorta. METHODS: Young male rats were divided randomly into an operated group, animals submitted to surgical abdominal aorta stenosis, and a sham-operated group, a control group of animals submitted to sham operation to simulate abdominal aorta stenosis. RESULTS AND CONCLUSIONS: Constricted aortas showed two distinct adaptive remodeling responses to hemodynamic stimuli induced by infradiaphragmatic coarctation. The first is remodeling in the hypertensive prestenotic segment with increased circumferential wall tension (CWT), associated with normal tensile stress, laminar flow/normal wall shear stress characterized by enlarged heterogeneous endothelial cells, elongated in the direction of the blood flow, diffusely distributed neointimal plaques, appearing as discrete bulging towards the vascular lumen and medial thickening. Our findings suggest that increased CWT caused by hypertension play a pivotal role in the remodeling of the prestenotic segment through biomechanical effects on oxidative stress and increased expression of transforming growth factor beta. The second is remodeling in the normotensive poststenotic segment with turbulent flow/low wall shear stress and normal CWT and tensile stress characterized by groups of endothelial cells with phenotypic alterations and focally distributed neointimal plaques, similar but many of them larger than those found in the prestenotic segments. Further studies are needed to determine how the mechanical forces of turbulent flow/low shear stress are detected and transduced into chemical signaling by the cells of the artery walls and then converted into pathophysiologically relevant phenotypic changes.

Erectile dysfunction in heart failure rats is associated with increased neurogenic contractions in cavernous tissue and internal pudendal artery.

AIMS: The rates of erectile dysfunction (ED) in heart failure (HF) are extremely high. This study tested the hypothesis that rats with HF display ED and that HF leads to increased sympathetic-mediated contractile tone of the cavernous tissue and/or internal pudendal arteries (IPA) as potential mechanisms contributing to ED. MAIN METHODS: HF was induced in Wistar rats by ligation of the left anterior descending coronary artery. Changes in the ratio of intracavernosal pressure/mean arterial pressure (ICP/MAP) after electrical stimulation of major pelvic ganglion were determined in vivo. Cavernosal and IPA contractions were induced by electric field stimulation (EFS) and phenylephrine. RhoA, Rho kinase 2 (ROCK 2) and myosin phosphatase target protein 1 (MYPT-1) protein expression and phosphorylation levels were also determined. KEY FINDINGS: HF rats display impaired erectile function represented by decreased ICP/MAP responses. EFS-mediated contractions were increased by HF in cavernous tissue and IPA. Contractions induced by phenylephrine were increased in cavernous tissue of HF rats, but decreased in IPA rings. Moreover, HF decreased RhoA protein expression, but increased ROCK 2 and MYPT-1 phosphorylation levels in cavernous tissue. In conclusion, rats with HF induced by myocardial infarction display ED in vivo and increased sympathetic-mediated contractile responses in cavernous tissue and IPA. Increased sympathetic-mediated contractile responses were associated with increased ROCK 2 and MYPT-1 phosphorylation in cavernosal tissue, suggesting the involvement of ROCK signaling pathway in ED genesis. SIGNIFICANCE: Our findings suggest new mechanisms linking HF to ED, providing potential therapeutic targets for treating ED associated to HF.

Acetylcholinesterase Inhibition Attenuates the Development of Hypertension and Inflammation in Spontaneously Hypertensive Rats.

BACKGROUND: It is hypothesized that chronic increase of availability of acetylcholine, resulting from the effect of antiacetylcholinesterases, may prevent autonomic imbalance and reduce inflammation yielding benefic effects for cardiovascular disorders in hypertension. The effect of long-term administration of antiacetylcholinesterase agents with central and/or peripheral action, i.e., donepezil and pyridostigmine, were investigated on arterial pressure (AP), sympathovagal balance, plasma cytokine levels, and cardiac remodeling in spontaneously hypertensive rats (SHR). METHODS: Chronic treatment with donepezil or pyridostigmine started before the onset of hypertension. AP was measured by plethysmography every 4 weeks. At the end of 16 weeks of treatment, methylatropine was used to evaluate the cardiac vagal tone; AP and pulse interval (PI) variability were also evaluated followed by plasma and heart collection for analysis. RESULTS: Pyridostigmine, which does not cross the blood-brain barrier, increased cardiac vagal tone, and reduced cardiomyocyte diameter and collagen density, but did not affect the AP and plasma cytokine levels. Donepezil, which crosses the blood-brain barrier, attenuated the development of hypertension, increased cardiac vagal tone, and improved AP and PI variability. Likewise, donepezil reduced the plasma levels of tumor necrosis factor-α, interleukin 6, and interferon γ, besides reducing cardiomyocyte diameter and collagen density. CONCLUSIONS: Donepezil attenuated the development of hypertension in SHR probably involving antiinflammatory effects, indicating that acetylcholinesterase inhibition yields benefic effects for antihypertensive therapy.

Pyridostigmine prevents haemodynamic alterations but does not affect their nycthemeral oscillations in infarcted mice.

The increase in acetylcholine yielded by pyridostigmine (PYR), an acetylcholinesterase inhibitor, was evaluated for its effect on the haemodynamic responses-mean arterial pressure (MAP) and heart rate (HR)-and their nycthemeral oscillation in mice before and one week after myocardial infarction (MI). Mice were anesthetized (isoflurane), and a telemetry transmitter was implanted into the carotid artery. After 5 days of recovery, the MAP and HR were recorded for 48 h (10 s every 10 min). Following this procedure, mice were submitted to surgery for sham or coronary artery ligation and received drinking water (VEHICLE) with or without PYR. Five days after surgery, the haemodynamic recordings were recommenced. Sham surgery combined with VEHICLE did not affect basal MAP and HR; nevertheless, these haemodynamic parameters were higher during the night, before and after surgery. MI combined with VEHICLE displayed decreased MAP and increased HR; these haemodynamic parameters were also higher during the night, before and after surgery. Sham surgery combined with PYR displayed similar results for MAP as sham combined with VEHICLE; however, PYR produced bradycardia. Nevertheless, MI combined with PYR exhibited no change in MAP and HR, but these haemodynamic parameters were also higher during the night, before and after surgery. Therefore, MI decreased MAP and increased HR, while PYR prevented these alterations. Neither MI nor PYR affected nycthemeral oscillations of MAP and HR. These findings indicate that the increase in acetylcholine yielded by PYR protected the haemodynamic alterations caused by MI in mice, without affecting the nycthemeral haemodynamic oscillations.