MicroRNA-1 and -133 increase arrhythmogenesis in heart failure by dissociating phosphatase activity from RyR2 complex

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.     

Neurochemistry of GABAergic System in Cerebral Cortex Chronically Exposed to Bromide In Vivo

Neurochemical correlates of GABAergic synaptic transmission [binding, uptake, metabolism, and tissue content of gamma-aminobutyric acid (GABA)] were investigated in the cortex of rats that had been given 27 mM bromide in drinking water for periods of time ranging from 1 day to 1 month. No effect of bromide on any of the parameters was found and it is concluded that chronic administration of bromide has no profound effect on GABAergic inhibitory system in the rat cortex.     

Elevated Levels of Asymmetric Dimethylarginine (ADMA) in the Pericardial Fluid of Cardiac Patients Correlate with Cardiac Hypertrophy

Background

Pericardial fluid (PF) contains several biologically active substances, which may provide information regarding the cardiac conditions. Nitric oxide (NO) has been implicated in cardiac remodeling. We hypothesized that L-arginine (L-Arg) precursor of NO-synthase (NOS) and asymmetric dimethylarginine (ADMA), an inhibitor of NOS, are present in PF of cardiac patients and their altered levels may contribute to altered cardiac morphology.

Methods

L-Arg and ADMA concentrations in plasma and PF, and echocardiographic parameters of patients undergoing coronary artery bypass graft (CABG, n = 28) or valve replacement (VR, n = 25) were determined.

Results

We have found LV hypertrophy in 35.7% of CABG, and 80% of VR patients. In all groups, plasma and PF L-Arg levels were higher than that of ADMA. Plasma L-Arg level was higher in CABG than VR (75.7±4.6 μmol/L vs. 58.1±4.9 μmol/L, p = 0.011), whereas PF ADMA level was higher in VR than CABG (0.9±0.0 μmol/L vs. 0.7±0.0 μmol/L, p = 0.009). L-Arg/ADMA ratio was lower in the VR than CABG (VR_plasma: 76.1±6.6 vs. CABG_plasma: 125.4±10.7, p = 0.004; VR_PF: 81.7±4.8 vs. CABG_PF: 110.4±7.2, p = 0.009). There was a positive correlation between plasma L-Arg and ADMA in CABG (r = 0.539, p = 0.015); and plasma and PF L-Arg in CABG (r = 0.357, p = 0.031); and plasma and PF ADMA in VR (r = 0.529, p = 0.003); and PF L-Arg and ADMA in both CABG and VR (CABG: r = 0.468, p = 0.006; VR: r = 0.371, p = 0.034). The following echocardiographic parameters were higher in VR compared to CABG: interventricular septum (14.7±0.5 mm vs. 11.9±0.4 mm, p = 0.000); posterior wall thickness (12.6±0.3 mm vs. 11.5±0.2 mm, p = 0.000); left ventricular (LV) mass (318.6±23.5 g vs. 234.6±12.3 g, p = 0.007); right ventricular (RV) (33.9±0.9 cm² vs. 29.7±0.7 cm², p = 0.004); right atrial (18.6±1.0 cm² vs. 15.4±0.6 cm², p = 0.020); left atrial (19.8±1.0 cm² vs. 16.9±0.6 cm², p = 0.033) areas. There was a positive correlation between plasma ADMA and RV area (r = 0.453, p = 0.011); PF ADMA and end-diastolic (r = 0.434, p = 0.015) and systolic diameter of LV (r = 0.487, p = 0.007); and negative correlation between PF ADMA and LV ejection fraction (r = -0.445, p = 0.013) in VR.

Conclusion

We suggest that elevated levels of ADMA in the PF of patients indicate upregulated RAS and reduced bioavailability of NO, which can contribute to the development of cardiac hypertrophy and remodeling.     

Complexity reduction of polymorphic sequences (CRoPS): a novel approach for large-scale polymorphism discovery in complex genomes

Application of single nucleotide polymorphisms (SNPs) is revolutionizing human bio-medical research. However, discovery of polymorphisms in low polymorphic species is still a challenging and costly endeavor, despite widespread availability of Sanger sequencing technology. We present CRoPS as a novel approach for polymorphism discovery by combining the power of reproducible genome complexity reduction of AFLP with Genome Sequencer (GS) 20/GS FLX next-generation sequencing technology. With CRoPS, hundreds-of-thousands of sequence reads derived from complexity-reduced genome sequences of two or more samples are processed and mined for SNPs using a fully-automated bioinformatics pipeline. We show that over 75% of putative maize SNPs discovered using CRoPS are successfully converted to SNPWave assays, confirming them to be true SNPs derived from unique (single-copy) genome sequences. By using CRoPS, polymorphism discovery will become affordable in organisms with high levels of repetitive DNA in the genome and/or low levels of polymorphism in the (breeding) germplasm without the need for prior sequence information.     

Repair of triple helix directed psoralen adducts in human cells.

Triple helix forming oligonucleotides can direct DNA damaging agents at specific sites in an intact double helix. In our study, triple helix formation was demonstrated in a SV40 based shuttle vector treated with psoralen linked to a 22-mer purine rich oligonucleotide. UVA irradiation caused a covalent linkage of the oligonucleotide through the psoralen to the mutational supF marker gene of the plasmid. After passage in the Jurkat human cell line the recovered vector was analysed in an indicator bacterial strain and mutants were collected. The presence of adducts in the target sequence did not reduce the yield of replicated progeny vector molecules, indicating repair of triple helix associated monoadducts and cross-links. Mutations were highly targeted to a six nucleotide long region of the target sequence. The number of target sequence mutants obtained after triple helix directed psoralen treatment was approximately 160 times higher than with free psoralen. A further investigation of the exact mechanism of the mutational process could make triple helix directed mutagenesis a more useful tool in gene therapy, antiviral therapy, and in studies on DNA repair and genome organisation.     

Functional consequences of stably expressing a mutant calsequestrin (CASQ2D307H) in the CASQ2 null background

The role of calsequestrin (CASQ2) in cardiac sarcoplasmic reticulum (SR) calcium (Ca(2+)) transport has gained significant attention since point mutations in CASQ2 were reported to cause ventricular arrhythmia. In the present study, we have critically evaluated the functional consequences of expressing the CASQ2(D307H) mutant protein in the CASQ2 null mouse. We recently reported that the mutant CASQ2(D307H) protein can be stably expressed in CASQ2 null hearts, and it targets appropriately to the junctional SR (Kalyanasundaram A, Bal NC, Franzini-Armstrong C, Knollmann BC, Periasamy M. J Biol Chem 285: 3076-3083, 2010). In this study, we found that introduction of CASQ2(D307H) protein in the CASQ2 null background partially restored triadin 1 levels, which were decreased in the CASQ2 null mice. Despite twofold expression (relative to wild-type CASQ2), the mutant protein failed to increase SR Ca(2+) load. We also found that the Ca(2+) transient decays slower in the CASQ2 null and CASQ2(D307H) cells. CASQ2(D307H) myocytes, when rhythmically paced and challenged with isoproterenol, exhibit spontaneous Ca(2+) waves similar to CASQ2 null myocytes; however, the stability of Ca(2+) cycling was increased in the CASQ2(D307H) myocytes. In the presence of isoproterenol, Ca(2+)-transient amplitude in CASQ2(D307H) myocytes was significantly decreased, possibly indicating an inherent defect in Ca(2+) buffering capacity and release from the mutant CASQ2 at high Ca(2+) concentrations. We also observed polymorphic ventricular tachycardia in the CASQ2(D307H) mice, although lesser than in the CASQ2 null mice. These data suggest that CASQ2(D307H) point mutation may affect Ca(2+) buffering capacity and Ca(2+) release. We propose that poor interaction between CASQ2(D307H) and triadin 1 could affect ryanodine receptor 2 stability, thereby increasing susceptibility to delayed afterdepolarizations and triggered arrhythmic activity.     

Effects of Moderate Aerobic Exercise Training on Hemorheological and Laboratory Parameters in Ischemic Heart Disease Patients

Background and Design

In this study we set out to determine the effects of long-term physical training on hemorheological, laboratory parameters, exercise tolerability, psychological factors in cardiac patients participating in an ambulatory rehabilitation program.

Methods

Before physical training, patients were examined by echocardiography, tested on treadmill by the Bruce protocol, and blood was drawn for laboratory tests. The enrolled 79 ischemic heart disease patients joined a 24-week cardiac rehabilitation training program. Blood was drawn to measure hematocrit (Hct), plasma and whole blood viscosity (PV, WBV), red blood cell (RBC) aggregation and deformability. Hemorheological, clinical chemistry and psychological measurements were repeated 12 and 24 weeks later, and a treadmill test was performed at the end of the program.

Results

After 12 weeks Hct, PV, WBV and RBC aggregation were significantly decreased, RBC deformability exhibited a significant increase (p<0.05). Laboratory parameters (triglyceride, uric acid, hsCRP and fibrinogen) were significantly decreased (p<0.05). After 24 weeks the significant results were still observed. By the end of the study, IL-6 and TNF-α levels displayed decreasing trends (p<0.06). There was a significant improvement in MET (p<0.001), and the BMI decrease was also significant (p<0.05). The vital exhaustion parameters measured on the fatigue impact scale indicated a significant improvement in two areas of the daily activities (p<0.05).

Conclusions

Regular physical training improved the exercise tolerability of patients with ischemic heart disease. Previous publications have demonstrated that decreases in Hct and PV may reduce cardiovascular risk, while a decrease in RBC aggregation and an increase in deformability improve the capillary flow. Positive changes in laboratory parameters and body weight may indicate better oxidative and inflammatory circumstances and an improved metabolic state. The psychological findings point to an improvement in the quality of life.     

Somatostatin stimulates clearance and hepatic uptake of colloidal carbon in the rat

Somatostatin exerts hormonal and neuroendocrine effects. Since it prevents several organ injuries and systemic intoxications, we tested the hypothesis that activation of Kupffer cells might be one mechanism of the beneficial actions of this peptide. The data presented here demonstrate that somatostatin given i.v. in rats dose- and time-dependently accelerated the clearance of colloidal carbon from the blood and enhanced the uptake of carbon in the liver. On a weight basis, somatostatin was more potent than the RES stimulant zymosan or RES blocker gadolinium chloride. Thus, somatostatin might modulate Kupffer cells and possibly other macrophages, and these effects may have a role in the physiologic or pharmacologic actions of this peptide.     

Butyrobetaine is equal to L-carnitine in elevating L-carnitine levels in rats

This work shows that butyrobetaine administered to rats in a single dose can be highly effective in elevating L-carnitine levels in all tissues. This ability of butyrobetaine was compared to that of L-carnitine. In an experiment with tracer dose of the compounds, 12 h following administration of [3H]butyrobetaine plasma and tissues contained radioactivity exclusively in L-carnitine and in similar amounts as in the other group of animals receiving L-[3H]carnitine. This was observed both after intraperitoneal and oral administration of the compounds. In the loading experiments 100 mumol [3H]butyrobetaine was administered orally to one group and 100 mumol L-[3H]carnitine to the other group of animals and 12 h later it was found that butyrobetaine caused the same increments in L-carnitine as L-carnitine administration. The increments in the organs of the butyrobetaine-treated group (in decreasing order) were as follows: kidney, 1227 nmol/g vs. 652 nmol/g; liver, 469 nmol/g vs. 258 nmol/g; muscle, 1043 nmol/g vs. 881 nmol/g; plasma, 79.4 nmol/ml vs. 39.3 nmol/ml. Butyrobetaine (100 mumol) caused similar increments when it was administered intraperitoneally. Based on these results butyrobetaine can be considered as a potential agent for L-carnitine supplementation therapy.     

Comparative study of the nesting behaviour of Tachysphex inconspicuus (Kirby) (Hymenoptera: Crabronidae) in two locations in southeast Brazil

The nesting behaviour of the solitary wasp Tachysphex inconspicuus (Kirby) was comparatively studied based on observations made in two environmentally distinct locations in southeast Brazil: upper portions of two sandy beaches and a dirt road in a forested area. Motor patterns related to nest construction were similar in all observed wasps, but some behavioural features, which were different among the observed wasps in the two environments, seem to be constrained by ecological factors. T. inconspicuus can be behaviourally differentiated from several other species of the genus by the following combination of features: they close the nest temporarily during its provision, do not let the excavated sand to form into a mound in front of the nest entrance, construct unicellular nests, and provision the nest with cockroaches. Moreover, females T. inscospicuus use their hind legs in a peculiar way to manipulate the prey, which allows them to associate the use of the mandibles in prey transport with the possibility of excavating the temporary plug and entering the nest without releasing the prey. Chorisoneura lopesi Albuquerque, C. excelsa Albuquerque & Silva, and unidentified species of Riatia (Blattodea: Blatellidae) were found as prey and Amobia floridensis (Towsend) (Diptera: Sarcophagidae) as parasitoid of T. insconspicuus.