A high-fat meal impairs muscle vasodilatation response to mental stress in humans with Glu27 beta(2)-adrenoceptor polymorphism

Background: Forearm blood flow responses during mental stress are greater in individuals homozygous for the Glu27 allele. A high-fat meal is associated with impaired endothelium-dependent dilatation. We investigated the impact of high-fat ingestion on the muscle vasodilatory responses during mental stress in individuals with the Glu27 allele and those with the Gln27 allele of the beta(2)-adrenoceptor gene. Methods: A total of 162 preselected individuals were genotyped for the Glu27Gln beta(2)-adrenoceptor polymorphism. Twenty-four individuals participated in the study. Fourteen were homozygous for the Gln27 allele (Gln27Gln, 40 +/- 2 years; 64 +/- 2 kg), and 10 were homozygous for the Glu27 allele (Glu27Glu, 40 +/- 3 years; 65 +/- 3 kg). Forearm blood flow was evaluated by venous occlusion plethysmography before and after ingestion of 62 g of fat. Results: The high-fat meal caused no changes in baseline forearm vascular conductance (FVC, 2.2 +/- 0.1 vs. 2.4 +/- 0.2; P = 0.27, respectively), but reduced FVC responses to mental stress (1.5 +/- 0.2 vs. 0.8 +/- 0.2 units; P = 0.04). When volunteers were divided according to their genotypes, baseline FVC was not different between groups (Glu27Glu = 2.4 +/- 0.1 vs. Gln27Gln = 2.1 +/- 0.1 units; P = 0.08), but it was significantly greater in Glu27Glu individuals during mental stress (1.9 +/- 0.4 vs. 1.0 +/- 0.3 units; P = 0.04). High-fat intake eliminated the difference in FVC responses between Glu27Glu and Gln27Gln individuals (FVC, 1.3 +/- 0.4 vs. 1.2 +/- 0.4; P = 0.66, respectively). Conclusion: These findings demonstrate that a high-fat meal impairs muscle vasodilatation responses to mental stress in humans. However, this reduction can be attributed to the presence of the homozygous Glu27 allele of the beta(2)-adrenoceptor gene.

Aerobic Exercise Training-Induced Left Ventricular Hypertrophy Involves Regulatory MicroRNAs, Decreased Angiotensin-Converting Enzyme-Angiotensin II, and Synergistic Regulation of Angiotensin-Converting Enzyme 2-Angiotensin (1-7)

Aerobic exercise training leads to a physiological, nonpathological left ventricular hypertrophy; however, the underlying biochemical and molecular mechanisms of physiological left ventricular hypertrophy are unknown. The role of microRNAs regulating the classic and the novel cardiac renin-angiotensin (Ang) system was studied in trained rats assigned to 3 groups: (1) sedentary; (2) swimming trained with protocol 1 (T1, moderate-volume training); and (3) protocol 2 (T2, high-volume training). Cardiac Ang I levels, Ang-converting enzyme (ACE) activity, and protein expression, as well as Ang II levels, were lower in T1 and T2; however, Ang II type 1 receptor mRNA levels (69% in T1 and 99% in T2) and protein expression (240% in T1 and 300% in T2) increased after training. Ang II type 2 receptor mRNA levels (220%) and protein expression (332%) were shown to be increased in T2. In addition, T1 and T2 were shown to increase ACE2 activity and protein expression and Ang (1-7) levels in the heart. Exercise increased microRNA-27a and 27b, targeting ACE and decreasing microRNA-143 targeting ACE2 in the heart. Left ventricular hypertrophy induced by aerobic training involves microRNA regulation and an increase in cardiac Ang II type 1 receptor without the participation of Ang II. Parallel to this, an increase in ACE2, Ang (1-7), and Ang II type 2 receptor in the heart by exercise suggests that this nonclassic cardiac renin-angiotensin system counteracts the classic cardiac renin-angiotensin system. These findings are consistent with a model in which exercise may induce left ventricular hypertrophy, at least in part, altering the expression of specific microRNAs targeting renin-angiotensin system genes. Together these effects might provide the additional aerobic capacity required by the exercised heart. (Hypertension. 2011;58:182-189.).

Impact of gender on benefits of exercise training on sympathetic nerve activity and muscle blood flow in heart failure

We compared the effects of exercise training on neurovascular control and functional capacity in men and women with chronic heart failure (HF). Forty consecutive HF outpatients from the Heart Institute, University of Sao Paulo, Brazil were divided into the following four groups matched by age: men exercise-trained (n = 12), men untrained (n = 10), women exercise-trained (n = 9), women untrained (n = 9). Maximal exercise capacity was determined from a maximal progressive exercise test on a cycle ergometer. Forearm blood flow was measured by venous occlusion plethysmography. Muscle sympathetic nerve activity (MSNA) was recorded directly using the technique of microneurography. There were no differences between groups in any baseline parameters. Exercise training produced a similar reduction in resting MSNA (P = 0.000002) and forearm vascular resistance (P = 0.0003), in men and women with HF. Peak VO(2) was similarly increased in men and women with HF (P = 0.0003) and VE/VCO(2) slope was significantly decreased in men and women with HF (P = 0.0007). There were no significant changes in left-ventricular ejection fraction in men and women with HF. The benefits of exercise training on neurovascular control and functional capacity in patients with HF are independent of gender.

Muscle sympathetic nerve activity in patients with Chagas` disease

Background: The progression of heart failure in Chagas` disease has been explained by remodeling, leading to neurohumoral activation, or by the direct parasite damage to parasympathetic neurons during acute phase, leading to early sympathetic activation and progressive heart failure. To help distinguish between these hypotheses we studied muscle sympathetic nerve activity (MSNA) at rest and during handgrip exercise (30% of maximal voluntary contraction) in patients with Chagas` disease and normal ejection fraction vs. patients with heart failure. Methods: A consecutive study of 72 eligible out-patients/subjects was conducted between July 1998 and November 2004. The participants were classified in three advanced heart failure groups (New York Heart Association Functional Classes II-III): Chagas` disease (n-15), ischemic (n=15) and idiopathic cardiomyopathy (n-15). Twelve Chagas` disease patients without heart failure and normal ejection fraction, and 15 normal controls were also studied. MSNA was recorded directly from the peroneal nerve by microneurography technique. Results: MSNA was greater in heart failure patients when compared with Chagas` disease patients without heart failure (51 +/- 3 vs. 20 +/- 2 bursts/min P=0.0001). MSNA in Chagas` patients with normal ejection fraction and normal controls was not different. During exercise, MSNA was similar in all 3 heart failure groups. And, was lower in the Chagas` patients with normal ejection fraction than in patients with Chagas` disease and heart failure (28 +/- 1 vs. 63 +/- 5 bursts/min, respectively). Conclusion: MSNA is not elevated in patients with Chagas` disease with normal ejection fraction. These findings support the concept of remodeling and neurohumoral activation as a common pathway following significant cardiac injury. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Regulation and crystallization of phosphorylated and dephosphorylated forms of truncated dimeric phenylalanine hydroxylase

Phenylalanine hydroxylase is regulated in a complex manner, including activation by phosphorylation. It is normally found as an equilibrium of dimeric and tetrameric species, with the tetramer thought to be the active form. We converted the protein to the dimeric form by deleting the C-terminal 24 residues and show that the truncated protein remains active and regulated by phosphorylation. This indicates that changes in the tetrameric quaternary structure of phenylalanine hydroxylase are not required for enzyme activation. Truncation also facilitates crystallization of both phosphorylated and dephosphorylated forms of the enzyme.

Increased muscle sympathetic nerve activity predicts mortality in heart failure patients

Background: Previous studies have associated neurohumoral excitation, as estimated by plasma norepinephrine levels, with increased mortality in heart failure. However, the prognostic value of neurovascular interplay in heart failure (HF) is unknown. We tested the hypothesis that the muscle sympathetic nerve activity (MSNA) and forearm blood flow would predict mortality in chronic heart failure patients. Methods: One hundred and twenty two heart failure patients, NYHA II-IV, age 50 +/- 1 ys, LVEF 33 +/- 1%, and LVDD 7.1 +/- 0.2 mm, were followed up for one year. MSNA was directly measured from the peroneal nerve by microneurography. Forearm blood flow was obtained by venous occlusion plethysmography. The variables were analyzed by using univariate, stepwise multivariate Cox proportional hazards analysis, and Kaplan-Meier analysis. Results: After one year, 34 pts died from cardiac death. The univariate analysis showed that MSNA, forearm blood flow, LVDD, LVEF, and heart rate were significant predictors of mortality. The multivariate analysis showed that only MSNA (P = 0.001) and forearm blood flow (P = 0.003) were significant independent predictors of mortality. On the basis of median levels of MSNA, survival rate was significantly lower in pts with >49 bursts/min. Similarly, survival rate was significantly lower in pts with forearm blood flow <1.87 ml/min/100 ml (P = 0.002). Conclusion: MSNA and forearm blood flow predict mortality rate in patients with heart failure. It remains unknown whether therapies that specifically target these abnormalities will improve survival in heart failure. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Glu298Asp eNOS gene polymorphism causes attenuation in nonexercising muscle vasodilatation

Dias RG, Alves MJ, Pereira AC, Rondon MU, dos Santos MR, Krieger JE, Krieger MH, Negrao CE. Glu298Asp eNOS gene polymorphism causes attenuation in nonexercising muscle vasodilatation. Physiol Genomics 37: 99-107, 2009. First published January 21, 2009; doi:10.1152/physiolgenomics.90368.2008.-The influence of Glu298Asp endothelial nitric oxide synthase (eNOS) polymorphism in exercise-induced reflex muscle vasodilatation is unknown. We hypothesized that nonexercising forearm blood flow (FBF) responses during handgrip isometric exercise would be attenuated in individuals carrying the Asp298 allele. In addition, these responses would be mediated by reduced eNOS function and NO-mediated vasodilatation or sympathetic vasoconstriction. From 287 volunteers previously genotyped, we selected 33 healthy individuals to represent three genotypes: Glu/Glu [n = 15, age 43 +/- 3 yr, body mass index (BMI) 22.9 +/- 0.3 kg/m(2)], Glu/Asp (n = 9, age 41 +/- 3 yr, BMI 23.7 +/- 1.0 kg/m(2)), and Asp/Asp (n = 9, age 40 +/- 4 yr, BMI 23.5 +/- 0.9 kg/m(2)). Heart rate (HR), mean blood pressure (MBP), and FBF (plethysmography) were recorded for 3 min at baseline and 3 min during isometric handgrip exercise. Baseline HR, MBP, FBF, and forearm vascular conductance (FVC) were similar among genotypes. FVC responses to exercise were significantly lower in Asp/Asp when compared with Glu/Asp and Glu/Glu (Delta = 0.07 +/- 0.14 vs. 0.64 +/- 0.20 and 0.57 +/- 0.09 units, respectively; P = 0.002). Further studies showed that intra-arterial infusion of N(G)-monomethyl-L-arginine (L-NMMA) did not change FVC responses to exercise in Asp/Asp, but significantly reduced FVC in Glu/Glu (Delta = 0.79 +/- 0.14 vs. 0.14 +/- 0.09 units). Thus the differences between Glu/Glu and Asp/Asp were no longer observed (P = 0.62). L-NMMA + phentolamine increased similarly FVC responses to exercise in Glu/Glu and Asp/Asp (P = 0.43). MBP and muscle sympathetic nerve activity increased significant and similarly throughout experimental protocols in Glu/Glu and Asp/Asp. Individuals who are homozygous for the Asp298 allele of the eNOS enzyme have attenuated nonexercising muscle vasodilatation in response to exercise. This genotype difference is due to reduced eNOS function and NO-mediated vasodilatation, but not sympathetic vasoconstriction.

NFAT1 transcription factor is central in the regulation of tissue microenvironment for tumor metastasis

Members of the nuclear factor of activated T cell (NFAT) family of transcription factors were originally described in T lymphocytes but later shown to be expressed in several immune and non-immune cell types. NFAT proteins can modulate cellular transformation intrinsically, and NFAT-deficient (NFAT1-/-) mice are indeed more susceptible to transformation than wild-type counterparts. However, the contribution of an NFAT1-/- microenvironment to tumor progression has not been studied. We have addressed this question by inoculating NFAT1-/- mice with B16F10 melanoma cells intravenously, an established model of tumor homing and growth. Surprisingly, NFAT1-/- animals sustained less tumor growth in the lungs after melanoma inoculation than wild-type counterparts. Even though melanoma cells equally colonize NFAT1-/- and wild-type lungs, tumors do not progress in the absence of NFAT1 expression. A massive mononuclear perivascular infiltrate and reduced expression of TGF-beta in the absence of NFAT1 suggested a role for tumor-infiltrating immune cells and the cytokine milieu. However, these processes are independent of an IL-4-induced regulatory tumor microenvironment, since lack of this cytokine does not alter the phenotype in NFAT1-/- animals. Bone marrow chimera experiments meant to differentiate the contributions of stromal and infiltrating cells to tumor progression demonstrated that NFAT1-induced susceptibility to pulmonary tumor growth depends on NFAT1-expressing parenchyma rather than on bone marrow-derived cells. These results suggest an important role for NFAT1 in radio-resistant tumor-associated parenchyma, which is independent of the anti-tumor immune response and Th1 versus Th2 cytokine milieu established by the cancer cells, but able to promote site-specific tumor growth.

The cell biology of atherosclerosis - new developments

In the development of atherosclerotic lesions, three basic processes occur: 1) invasion of the artery wall by leucocytes, particularly monocytes and T-lymphocytes; 2) smooth muscle phenotypic modulation, proliferation, and synthesis of extracellular matrix; and 3) intracellular (macrophage and smooth muscle) lipoprotein uptake and lipid accumulation. Invasion of the vessel wall by leucocytes is mediated through the expression of adhesion molecules on both leucocytes and the endothelium making them 'sticky'. The adhesion molecules are induced by high serum cholesterol levels or complement fragments. Leucocytes which have adhered to the endothelium are chemo-attracted into the vessel wall by cytokines produced by early arriving leucocytes or by low density lipoprotein which has passively passed into the wall, in the process being trapped and oxidised. The oxidised low density lipoprotein is taken up by scavenger receptors (which are not subject to down-regulation) on both macrophages and smooth muscle cells. The overaccumulation of lipid is toxic to the cells and they die contributing to the central necrotic core. The macrophages and T-lymphocytes produce substances which induce smooth muscle cells of the artery wall to change from a 'contractile' (high volume fraction of myofilaments [V(v)myo]) to a 'synthetic' (low V(v)myo) phenotype. In this altered state they respond to growth factors released from macrophages, platelets, regenerating endothelial cells and smooth muscle cells; produce large amounts of matrix; express lipoprotein scavenger receptors; express adhesion molecules for leucocytes; and express HLA-DR following exposure to the T-lymphocyte product, IFN-delta, suggesting that they can become involved in a generalised immune reaction.

Physical Training Leads to Remodeling of Diaphragm Muscle in Asthma Model

Matrix metalloproteinases (MMPs) are crucial to the development and maintenance of healthy tissue and are mainly involved in extracellular matrix (ECM) remodeling of skeletal muscle. This study evaluated the effects of chronic allergic airway inflammation (CAAI), induced by ovalbumin, and aerobic training in the MMPs activity in mouse diaphragm muscle. Thirty mice were divided into 6 groups: 1) control; 2) ovalbumin; 3) treadmill trained at 50% of maximum speed; 4) ovalbumin and trained at 50%; 5) trained at 75%; 6) ovalbumin and trained at 75%. CAAI did not after MMPs activities in diaphragm muscle. Nevertheless, both treadmill aerobic trainings, associated with CAAI increased the MMP-2 and -1 activities. Furthermore, MMP-9 was not detected in any group. Together, these findings suggest an ECM remodeling in diaphragm muscle of asthmatic mice submitted to physical training. This result may be useful for a better understanding of functional significance of changes in the MMPs activity in response to physical training in asthma.

Induction of smooth muscle cell nitric oxide synthase by human leukaemia inhibitory factor: Effects in vitro and in vivo

We have previously shown that human leukaemia inhibitory factor (hLIF) inhibits perivascular cuff-induced neointimal formation in the rabbit carotid artery. Since nitric oxide (NO) is a known inhibitor of smooth muscle growth, NO synthase (NOS) activity in the presence of hLIF was examined in vivo and in vitro. In rabbit aortic smooth muscle cell (SMC) culture, significant NOS activity was observed at 50 pg/ml hLIF, with maximal activity at 5 ng/ml. In the presence of the NOS inhibitor L-NAME, hLIF-induced activation of NOS was greatly decreased, however it was still 63-fold higher than in control (p < 0.05). SMC-DNA synthesis was significantly reduced (-47%) following incubation with hLIF plus L-arginine, the substrate required for NO production (p < 0.05), with no effect observed in the absence of L-arginine. Silastic cuff placement over the right carotid artery of rabbits resulted in a neointima 19.3 +/- 5.4% of total wall cross-sectional area, which was increased in the presence of L-NAME (27.0 +/- 2.0%; p < 0.05) and reduced in the presence of L-arginine (11.3 +/- 2.0%; p < 0.05). The effect of L-arginine was ameliorated by co-administration of L-NAME (16.4 +/- 1.5%). However, administration of L-NAME with hLIF had no effect on the potent inhibition of neointimal formation by hLIF (3.2 +/- 2.5 vs. 2.1 +/- 5.4%, respectively). Similarly, with hLIF administration, NOS activity in the cuffed carotid increased to 269.0 +/- 14.0% of saline-treated controls and remained significantly higher with coadministration of L-NAME (188.5 +/- 14.7%). These results indicate that hLIF causes superinduction of NO by SMC, and that it is, either partially or wholly, through this mechanism that hLIF is a potent inhibitor of neointimal formation in vivo and of smooth muscle proliferation in vitro.

Down-regulation of the amyloid protein precursor of Alzheimer's disease by antisense oligonucleotides reduces neuronal adhesion to specific substrata

The hallmark of Alzheimer's disease is the cerebral deposition of amyloid which is derived from the amyloid precursor protein (APP). The function of APP is unknown but there is increasing evidence for the role of APP in cell-cell and/or cell-matrix interactions. Primary cultures of murine neurons were treated with antisense oligonucleotides to down-regulate APP. This paper presents evidence that APP mediates a substrate-specific interaction between neurons and extracellular matrix components collagen type I, laminin and heparan sulphate proteoglycan but not fibronectin or poly-L-lysine. It remains to be determined whether this effect is the direct result of APP-matrix interactions, or whether an intermediary pathway is involved. (C) 1997 Elsevier Science B.V.

Effect of saponin treatment on the sarcoplasmic reticulum of rat, cane toad and crustacean (yabby) skeletal muscle

1. Mechanically skinned fibres from skeletal muscles of the rat, toad and yabby were used to investigate the effect of saponin treatment on sarcoplasmic reticulum (SR) Ca2+ loading properties. The SR was loaded submaximally under control conditions before and after treatment with saponin and SR Ca2+ was released with caffeine. 2. Treatment with 10 mu g ml(-1) saponin greatly reduced the SR Ca2+ loading ability of skinned fibres from the extensor digitorum longus muscle of the rat with a rate constant of 0.24 min(-1). Saponin concentrations up to 150 mu g ml(-1) and increased exposure time up to 30 min did not further reduce the SR Ca2+ loading ability of the SR, which indicates that the inhibitory action of 10-150 mu g ml(-1) saponin is not dose dependent. The effect of saponin was also not dependent on the state of polarization of the transverse-tubular system. 3. Treatment with saponin at concentrations up to 100 mu g ml(-1) for 30 min did not affect the Ca2+ loading ability of SR in skinned skeletal muscle fibres from the twitch portion of the toad iliofibularis muscle but SR Ca2+ loading ability decreased markedly with a time constant of 0.22 min(-1) in the presence of 150 mu g ml(-1) saponin. 4. The saponin dependent increase in permeability could be reversed in both rat and toad fibres by short treatment with 6 mu M Ruthenium Red, a potent SR Ca2+ channel blocker, suggesting that saponin does affect the SR Ca2+ channel properties in mammalian and anuran skeletal muscle. 5. Treatment of skinned fibres of long sarcomere length (> 6 mu m) from the claw muscle of the yabby (a freshwater decapod crustacean) with 10 mu g ml(-1) saponin for 30 min abolished the ability of the SR to load Ca2+, indicating that saponin affects differently the SR from skeletal muscles of mammals, anurans and crustaceans. 6. is concluded that at relatively low concentrations, saponin causes inhibition of the skeletal SR Ca2+ loading ability in a species dependent manner, probably by increasing the Ca2+ loss through SR Ca2+ release channels.

Inactivation of a c-Myb/estrogen receptor fusion protein in transformed primary cells leads to granulocyte/macrophage differentiation and down regulation of c-kit but not c-myc or cdc2

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxyl-terminally truncated c-Myb protein (ERMYB), The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of beta-estradiol. Upon removal of beta-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and differentiate to cells exhibiting granulocyte or macrophage morphology, The expression of the c-myc, c-kit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of beta-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of beta-estradiol demonstrating that differentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of beta-estradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis, In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following differentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of beta-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.