Are transgenic mice the 'alkahest' to understanding myocardial hypertrophy and failure?

Murine transgenesis using cardioselective promoters has become increasingly common in studies of cardiac hypertrophy and heart failure, with expression mediated by pronuclear microinjection being the commonest format. Without wishing to decry their usefulness, in our view, such studies are not necessarily as unambiguous as sometimes portrayed and clarity is not always their consequence. We describe broadly the types of approach undertaken in the heart and point out some of the drawbacks. We provide three arbitrarily-chosen examples where, in spite of a number of often-independent studies, no consensus has yet been achieved. These include glycogen synthase kinase 3, the extracellular signal-regulated kinase pathway and the ryanodine receptor 2. We believe that the transgenic approach should not be viewed in an empyreal light and, depending on the questions asked, we suggest that other experimental systems provide equal (or even more) valuable outcomes.

Is optical imaging spectroscopy a viable measurement technique for the investigation of the negative BOLD phenomenon? A concurrent optical imaging spectroscopy and fMRI study at high field (7T)

Traditionally functional magnetic resonance imaging (fMRI) has been used to map activity in the human brain by measuring increases in the Blood Oxygenation Level Dependent (BOLD) signal. Often accompanying positive BOLD fMRI signal changes are sustained negative signal changes. Previous studies investigating the neurovascular coupling mechanisms of the negative BOLD phenomenon have used concurrent 2D-optical imaging spectroscopy (2D-OIS) and electrophysiology (Boorman et al., 2010). These experiments suggested that the negative BOLD signal in response to whisker stimulation was a result of an increase in deoxy-haemoglobin and reduced multi-unit activity in the deep cortical layers. However, Boorman et al. (2010) did not measure the BOLD and haemodynamic response concurrently and so could not quantitatively compare either the spatial maps or the 2D-OIS and fMRI time series directly. Furthermore their study utilised a homogeneous tissue model in which is predominantly sensitive to haemodynamic changes in more superficial layers. Here we test whether the 2D-OIS technique is appropriate for studies of negative BOLD. We used concurrent fMRI with 2D-OIS techniques for the investigation of the haemodynamics underlying the negative BOLD at 7 Tesla. We investigated whether optical methods could be used to accurately map and measure the negative BOLD phenomenon by using 2D-OIS haemodynamic data to derive predictions from a biophysical model of BOLD signal changes. We showed that despite the deep cortical origin of the negative BOLD response, if an appropriate heterogeneous tissue model is used in the spectroscopic analysis then 2D-OIS can be used to investigate the negative BOLD phenomenon.

Modulation of stretch-induced myocyte remodeling and gene expression by nitric oxide: a novel role for lipoma preferred partner in myofibrillogenesis

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signalling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors which convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain, 1Hz for 48 hours to mimic in vivo mechanical stress. Cells were also treated with and without L-NAME, a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres which was rescued by LNAME (P<0.05, n≥5 cultures). We hypothesized that the mechanism was through NOinduced alteration of myocyte gene expression. L-NAME up-regulated the mechanosensing proteins Muscle LIM protein (MLP (by 100%, p<0.05, n=4 cultures)) and lipoma preferred partner, a novel cardiac protein (LPP (by 80%, p<0.05, n=4 cultures)). L-NAME also significantly altered the subcellular localisation of LPP and MLP in a manner that favoured growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between iNOS and nNOS isoforms regulate gene expression. LPP knockdown by siRNA led to formation of α-actinin aggregates and Z-bodies showing that myofibrillogenesis was impaired. There was an up-regulation of E3 ubiquitin ligase (MUL1) by 75% (P<0.05, n=5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure. Keywords: Mechanotransduction, heart failure, stretch, heart, hypertrophy

Habitual energy expenditure modifies the association between NOS3 gene polymorphisms and blood pressue

BACKGROUND: The endothelial nitric-oxide synthase (NOS3) gene encodes the enzyme (eNOS) that synthesizes the molecule nitric oxide, which facilitates endothelium-dependent vasodilation in response to physical activity. Thus, energy expenditure may modify the association between the genetic variation at NOS3 and blood pressure. METHODS: To test this hypothesis, we genotyped 11 NOS3 polymorphisms, capturing all common variations, in 726 men and women from the Medical Research Council (MRC) Ely Study (age (mean +/- s.d.): 55 +/- 10 years, body mass index: 26.4 +/- 4.1 kg/m(2)). Habitual/non-resting energy expenditure (NREE) was assessed via individually calibrated heart rate monitoring over 4 days. RESULTS: The intronic variant, IVS25+15 [G-->A], was significantly associated with blood pressure; GG homozygotes had significantly lower levels of diastolic blood pressure (DBP) (-2.8 mm Hg; P = 0.016) and systolic blood pressure (SBP) (-1.9 mm Hg; P = 0.018) than A-allele carriers. The interaction between NREE and IVS25+15 was also significant for both DBP (P = 0.006) and SBP (P = 0.026), in such a way that the effect of the GG-genotype on blood pressure was stronger in individuals with higher NREE (DBP: -4.9 mm Hg, P = 0.02. SBP: -3.8 mm Hg, P= 0.03 for the third tertile). Similar results were observed when the outcome was dichotomously defined as hypertension. CONCLUSIONS: In summary, the NOS3 IVS25+15 is directly associated with blood pressure and hypertension in white Europeans. However, the associations are most evident in the individuals with the highest NREE. These results need further replication and have to be ideally tested in a trial before being informative for targeted disease prevention. Eventually, the selection of individuals for lifestyle intervention programs could be guided by knowledge of genotype.

The effects of dietary nitrate on blood pressure and endothelial function: a review of human intervention studies.

Evidence has accumulated in recent years that suggests that nitrate from the diet, particularly vegetables, is capable of producing bioactive NO in the vasculature, following bioconversion to nitrite by oral bacteria. The aim of the present review was to consider the current body of evidence for potential beneficial effects of dietary nitrate on blood pressure and endothelial function, with emphasis on evidence from acute and chronic human intervention studies. The studies to date suggest that dietary nitrate acutely lowers blood pressure in healthy humans. An inverse relationship was seen between dose of nitrate consumed and corresponding systolic blood pressure reduction, with doses of nitrate as low as 3 mmol of nitrate reducing systolic blood pressure by 3 mmHg. Moreover, the current studies provide some promising evidence on the beneficial effects of dietary nitrate on endothelial function. In vitro studies suggest a number of potential mechanisms by which dietary nitrate and its sequential reduction to NO may reduce blood pressure and improve endothelial function, such as: acting as a substrate for endothelial NO synthase; increasing vasodilation; inhibiting mitochondrial reactive oxygen species production and platelet aggregation. In conclusion, the evidence for beneficial effects of dietary nitrate on blood pressure and endothelial function is promising. Further long-term randomised controlled human intervention studies assessing the potential effects of dietary nitrate on blood pressure and endothelial function are needed, particularly in individuals with hypertension and at risk of CVD.

NK1 receptor stimulation causes contraction and inositol phosphate increase in medium-size human isolated bronchi

Although contraction of human isolated bronchi is mediated mainly by tachykinin NK2 receptors, NK1 receptors, via prostanoid release, contract small-size (approximately 1 mm in diameter) bronchi. Here, we have investigated the presence and biological responses of NK1 receptors in medium-size (2-5 mm in diameter) human isolated bronchi. Specific staining was seen in bronchial sections with an antibody directed against the human NK1 receptor. The selective NK1 receptor agonist, [Sar(9), Met(O2)(11)]SP, contracted about 60% of human isolated bronchial rings. This effect was reduced by two different NK1 receptor antagonists, CP-99,994 and SR 140333. Contraction induced by [Sar(9), Met(O2)(11)]SP was independent of acetylcholine and histamine release and epithelium removal, and was not affected by nitric oxide synthase and cyclooxygenase (COX) inhibition. [Sar(9), Met(O2)(11)]SP increased inositol phosphate (IP) levels, and SR 140333 blocked this increase, in segments of medium- and small-size (approximately 1 mm in diameter) human bronchi. COX inhibition blocked the IP increase induced by [Sar(9), Met(O2)(11)]SP in small-size, but not in medium-size, bronchi. NK1 receptors mediated bronchoconstriction in a large proportion of medium-size human bronchi. Unlike small-size bronchi this effect is independent of prostanoid release, and the results are suggestive of a direct activation of smooth muscle receptors and IP release.

Presence and bronchomotor activity of protease-activated receptor-2 in guinea pig airways

The protease activated receptor-2 (PAR-2) belongs to a family of G-protein-coupled receptors that are activated by proteolysis. Trypsin cleaves PAR-2, exposing an N-terminal tethered ligand (SLIGRL) that activates the receptor. Messenger RNA (mRNA) for PAR-2 was found in guinea pig airway tissue by reverse transcription-polymerase chain reaction, and PAR-2 was found by immunohistochemistry in airway epithelial and smooth-muscle cells. In anesthetized guinea pigs, trypsin and SLIGRL-NH(2) (given intratracheally or intravenously) caused a bronchoconstriction that was inhibited by the combination of tachykinin-NK(1) and -NK(2) receptor antagonists and was potentiated by inhibition of nitric oxide synthase (NOS). Trypsin and SLIGRL-NH(2) relaxed isolated trachea and main bronchi, and contracted intrapulmonary bronchi. Relaxation of main bronchi was abolished or reversed to contraction by removal of epithelium, administration of indomethacin, and NOS inhibition. PAR-1, PAR-3, and PAR-4 were not involved in the bronchomotor action of either trypsin or SLIGRL-NH(2), because ligands of these receptors were inactive either in vitro or in vivo, and because thrombin (a PAR-1 and PAR-3 agonist) did not show cross-desensitization with PAR-2 agonists in vivo. Thus, we have localized PAR-2 to the guinea-pig airways, and have shown that activation of PAR-2 causes multiple motor effects in these airways, including in vivo bronchoconstriction, which is in part mediated by a neural mechanism.

Differential effect of beetroot bread on postprandial DBP according to Glu298Asp polymorphism in the eNOS gene: a pilot study.

Our objective was to investigate whether the presence of Glu298Asp polymorphism in the endothelial NO synthase (eNOS) gene differentially affects the postprandial blood pressure response to dietary nitrate-rich beetroot bread. A randomised, single-blind, controlled, crossover acute pilot study was performed in 14 healthy men (mean age: 34±9 years) who were retrospectively genotyped for Glu298Asp polymorphism (7GG; T carriers 7). Volunteers were randomised to receive 200 g beetroot-enriched bread (1.1 mmol nitrate) or control bread (no beetroot; 0.01 mmol nitrate) on two separate occasions 10 days apart. Baseline and incremental area under the curve of blood pressure and NOx (nitrate/nitrite) were measured for a 6-h postprandial period. A treatment × genotype interaction was observed for diastolic blood pressure (P<0.02), which was significantly lower in T carriers (P<0.01) after consumption of beetroot bread compared with control bread. No significant differences were observed in the GG group. The beneficial diastolic blood pressure reduction was observed only in the T carriers of the Glu298Asp polymorphism in the eNOS gene after consumption of nitrate-rich beetroot bread. These data require confirmation in a larger population group.

Regulation of KCa2.3 andendothelium-dependent hyperpolarization (EDH) in the rat middle cerebral artery: the role of lipoxygenase metabolites and isoprostanes

Background and Purpose. In rat middle cerebral arteries, endothelium-dependent hyperpolarization (EDH) is mediated by activation of calcium-activated potassium(KCa) channels specifically KCa2.3 and KCa3.1. Lipoxygenase (LOX) products function as endothelium-derived hyperpolarizing factors (EDHFs) in rabbit arteries by stimulating KCa2.3. We investigated if LOX products contribute to EDH in rat cerebral arteries. Methods. Arachidonic acid (AA) metabolites produced in middle cerebral arteries were measured using HPLC and LC/MS. Vascular tension and membrane potential responses to SLIGRL were simultaneously recorded using wire myography and intracellular microelectrodes. Results. SLIGRL, an agonist at PAR2 receptors, caused EDH that was inhibited by a combination of KCa2.3 and KCa3.1 blockade. Non-selective LOX-inhibition reduced EDH, whereas inhibition of 12-LOX had no effect. Soluble epoxide hydrolase (sEH) inhibition enhanced the KCa2.3 component of EDH. Following NO synthase (NOS) inhibition, the KCa2.3 component of EDH was absent. Using HPLC, middle cerebral arteries metabolized 14C-AA to 15- and 12-LOX products under control conditions. With NOS inhibition, there was little change in LOX metabolites, but increased F-type isoprostanes. 8-iso-PGF2α inhibited the KCa2.3 component of EDH. Conclusions. LOX metabolites mediate EDH in rat middle cerebral arteries. Inhibition of sEH increases the KCa2.3 component of EDH. Following NOS inhibition,loss of KCa2.3 function is independent of changes in LOX production or sEH inhibition but due to increased isoprostane production and subsequent stimulation of TP receptors. These findings have important implications in diseases associated with loss of NO signaling such as stroke; where inhibition of sEH and/or isoprostane formation may of benefit.

Separating heart and brain: on the reduction of physiological noise from multichannel functional near-infrared spectroscopy (fNIRS) signals

Objective. Functional near-infrared spectroscopy (fNIRS) is an emerging technique for the in vivo assessment of functional activity of the cerebral cortex as well as in the field of brain–computer interface (BCI) research. A common challenge for the utilization of fNIRS in these areas is a stable and reliable investigation of the spatio-temporal hemodynamic patterns. However, the recorded patterns may be influenced and superimposed by signals generated from physiological processes, resulting in an inaccurate estimation of the cortical activity. Up to now only a few studies have investigated these influences, and still less has been attempted to remove/reduce these influences. The present study aims to gain insights into the reduction of physiological rhythms in hemodynamic signals (oxygenated hemoglobin (oxy-Hb), deoxygenated hemoglobin (deoxy-Hb)). Approach. We introduce the use of three different signal processing approaches (spatial filtering, a common average reference (CAR) method; independent component analysis (ICA); and transfer function (TF) models) to reduce the influence of respiratory and blood pressure (BP) rhythms on the hemodynamic responses. Main results. All approaches produce large reductions in BP and respiration influences on the oxy-Hb signals and, therefore, improve the contrast-to-noise ratio (CNR). In contrast, for deoxy-Hb signals CAR and ICA did not improve the CNR. However, for the TF approach, a CNR-improvement in deoxy-Hb can also be found. Significance. The present study investigates the application of different signal processing approaches to reduce the influences of physiological rhythms on the hemodynamic responses. In addition to the identification of the best signal processing method, we also show the importance of noise reduction in fNIRS data.

Transcriptome analysis for discovering candidate genes involve in embryogenesis in coconut (Cocos nucifera L.) through 454 pyrosequencing

Coconut, Cocos nucifera L. is a major plantation crop, which ensures income for millions of people in the tropical region. Detailed molecular studies on zygotic embryo development would provide valuable clues for the identification of molecular markers to improve somatic embryogenesis. Since there is no ongoing genome project for this species, coconut expressed sequence tags (EST) would be an interesting technique to identify important coconut embryo specific genes as well as other functional genes in different biochemical pathways. The goal of this study was to analyse the ESTs by examining the transcriptome data of the different embryo tissue types together with one somatic tissue. Here, four cDNA libraries from immature embryo, mature embryo, microspore derived embryo and mature leaves were constructed. cDNA was sequenced by the Roche-454 GS-FLX system and assembled into 32621 putative unigenes and 155017 singletons. Of these unigenes, 18651 had significant sequence similarities to non-redundant protein database, from which 16153 were assigned to one or more gene ontology categories. Homologue genes, which are responsible for embryo development such as chitinase, beta-1,3-glucanase, ATP synthase CF0 subunit, thaumatin-like protein and metallothionein-like protein were identified among the embryo EST collection. Of the unigenes, 6694 were mapped into 139 KEGG pathways including carbohydrate metabolism, energy metabolism, lipid metabolism, amino acid metabolism and nucleotide metabolism. This collection of 454-derived EST data generated from different tissue types provides a significant resource for genome wide studies and gene discovery of coconut, a non-model species.

Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A.

Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 microM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 microM), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the alpha-adrenoceptor, but, at high concentrations (100 microM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the beta-adrenoceptor. Phenylephrine (100 microM) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both alpha(1)- and beta-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the beta-adrenoceptor. Inhibitor studies and partial purification of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, efficient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and 8-(4-chlorophenylthio)-cAMP.