Maternal high fat diet induces early cardiac hypertrophy and alters cardiac metabolism in Sprague Dawley rat offspring.

BACKGROUND AND AIM: Maternal high fat diets (mHFD) have been associated with an increased offspring cardiovascular risk. Recently we found that the class IIa HDAC-MEF2 pathway regulates gene programs controlling fatty acid oxidation in striated muscle. This same pathway controls hypertrophic responses in the heart. We hypothesized that mHFD is associated with activation of signal controlling class II a HDAC activity and activation of genes involved in fatty acid oxidation and cardiac hypertrophy in offspring. METHODS AND RESULTS: Female Sprague Dawley rats were fed either normal fat diet (12%) or high fat diet (43%) three weeks prior to mating, remaining on diets until study completion. Hearts of postnatal day 1 (PN1) and PN10 pups were collected. Bioenergetics and respiration analyses were performed in neonatal ventricular cardiomyocytes (NVCM). In offspring exposed to mHFD, body weight was increased at PN10 accompanied by increased body fat percentage and blood glucose. Heart weight and heart weight to body weight ratio were increased at PN1 and PN10, and were associated with elevated signalling through the AMPK-class IIa HDAC-MEF2 axis. The expression of the MEF2-regulated hypertrophic markers ANP and BNP were increased as were expression of genes involved in fatty acid oxidation. However this was only accompanied by an increased protein expression of fatty acid oxidation enzymes at PN10. NVCM isolated from these pups exhibited increased glycolysis and an impaired substrate flexibility. CONCLUSION: Combined, these results suggest that mHFD induces signalling and transcriptional events indicative of reprogrammed cardiac metabolism and of cardiac hypertrophy in Sprague Dawley rat offspring.

Safety and efficacy of multipolar pulmonary vein ablation catheter vs. irrigated radiofrequency ablation for paroxysmal atrial fibrillation: a randomized multicentre trial.

AIMS: The current challenge in atrial fibrillation (AF) treatment is to develop effective, efficient, and safe ablation strategies. This randomized controlled trial assesses the medium-term efficacy of duty-cycled radiofrequency ablation via the circular pulmonary vein ablation catheter (PVAC) vs. conventional electro-anatomically guided wide-area circumferential ablation (WACA). METHODS AND RESULTS: One hundred and eighty-eight patients (mean age 62 ± 12 years, 116 M : 72 F) with paroxysmal AF were prospectively randomized to PVAC or WACA strategies and sequentially followed for 12 months. The primary endpoint was freedom from symptomatic or documented >30 s AF off medications for 7 days at 12 months post-procedure. One hundred and eighty-three patients completed 12 m follow-up. Ninety-four patients underwent PVAC PV isolation with 372 of 376 pulmonary veins (PVs) successfully isolated and all PVs isolated in 92 WACA patients. Three WACA and no PVAC patients developed tamponade. Fifty-six percent of WACA and 60% of PVAC patients were free of AF at 12 months post-procedure (P = ns) with a significant attrition rate from 77 to 78%, respectively, at 6 months. The mean procedure (140 ± 43 vs. 167 ± 42 min, P<0.0001), fluoroscopy (35 ± 16 vs. 42 ± 20 min, P<0.05) times were significantly shorter for PVAC than for WACA. Two patients developed strokes within 72 h of the procedure in the PVAC group, one possibly related directly to PVAC ablation in a high-risk patient and none in the WACA group (P = ns). Two of the 47 patients in the PVAC group who underwent repeat ablation had sub-clinical mild PV stenoses of 25-50% and 1 WACA patient developed delayed severe PV stenosis requiring venoplasty. CONCLUSION: The pulmonary vein ablation catheter is equivalent in efficacy to WACA with reduced procedural and fluoroscopy times. However, there is a risk of thrombo-embolic and pulmonary stenosis complications which needs to be addressed and prospectively monitored. CLINICALTRIALSGOV IDENTIFIER: NCT00678340.

Channel structures of gramicidin: characterization of succinyl derivatives.

Succinyl derivatives of gramicidin were tested for their ability to form channels in planar artificial lipid bilayers. Both N-succinyldeformylgramicidin methyl ester and charged O-succinylgramicidin formed channels, but the channels had markedly different sizes and lifetimes. This implies that gramicidin forms channels by end-to-end association. However, the doubly charged N,O-bissuccinyldeformylgramicidin was inactive, which suggests that only end-to-end association of gramicidin may result in channel formation.

Single channel properties of D-Leu2-gramicidin A. Side chain modulation of channel lifetime.

The channel forming properties of synthetic gramicidin A and DLeu2-gramicidin A were compared in black lipid membranes. The most probable single channel conductance was identical for both derivatives but in each case a distribution of smaller channel sizes was observed. However, the lifetime of the channel formed by DLeu2-gramicidin A was considerably shorter than for gramicidin A. The DLeu2 substitution is considered to interfere with the head to head hydrogen bonding which forms the conducting dimer, thus destabilizing the dimeric structure of the channel and reducing the lifetime. This represents the first demonstration of side-chain modulation of channel lifetime.

Spectral analysis of mouse EEG after the administration of N,N-dimethyltryptamine.

Mice were implanted with permanent cortical electrodes. The EEG was recorded during the sleep-wake cycle and after the administration of N,N-dimethyltryptamine (DMT). Based upon spectral analysis the EEG was classified into three categories: awake, slow-wave sleep (SWS), and rapid eye movement sleep (REM). The dominant frequencies were located below 2 Hz in the case of SWS and between 6 and 9 Hz for REM. When DMT was administered intraperitoneally at 20 or 40 mg/kg, a dose-dependent hypersynchrony at 2.5-4.5 Hz was always observed lasting for up to 60 min. Hypersynchronous activity in the same range was occasionally observed during awake. In this range DMT induced a different but unique frequency for each animal. This individual frequency was closely reproduced by repeated administrations of DMT to the same animal.

The behavioral effects of L-methionine and related compounds in rats and mice.

Several groups of investigators have reported that the administration of L-methionine, with or without a monoamine oxidase inhibitor, induced an acute florid psychotic reaction in 40 percent of schizophrenics tested. The mode of action of L-methionine in brain is unknown, but may be via one or more of three mechanisms: the excess methionine (i) may lead to the production by transmethylation of some psychotomimetic methylated derivative of dopamine or serotonin, or (ii) could result in an increase in the levels of a metabolite of methionine (e.g., homocysteine, cystathionine, or cysteine), or (iii) may effect the cellular uptake of other amino acids. In order to test the first two hypotheses, L-methionine, betaine (another metyl group donor), L-methionine plus L-serine, L-cysteine, L-serine, and saline (as a control) were studied on the sleep-wake cycles of random-bred Swiss mice and on the avoidance behavior or rats. L-methionine plus L-histidine, L-methionine plus nicotinamide, L-histidine, and nicotinamide were also tested in the mice. Daily injections of 250 mg/kg of these compounds were administered for at least 21 consecutive days. Schedule performance in the rat and the sleep-wake cycles of the mice were monitored during this period and compared to controls. L-Methionine induced behavioral and sleep cycle disturbances which were removed by the simultaneous administration of L-serine but not by the addition of L-histidine or nicotinamide. These data suggest that the disruption may be due to an increase in the levels of one of the metabolities of methionine, homocysteine, rather than to an increase in the number of available methyl groups.

Multiple effects of alpha-toxins on the nicotinic acetylcholine receptor.

Very low concentrations (5 nM) of alpha-toxin from the venom of Naja naja atra produced a characteristic fade in muscle compound action potential and tetanus induced by repetitive nerve stimulation which was identical to the effects of curare. High concentrations of alpha-toxin and all concentrations of alpha-bungarotoxin reduced the response but produced very little fade in comparison to curare. These results suggest that alpha-toxins have more than one effect at the neuromuscular junction.

Overlap myasthenic syndrome: combined myasthenia gravis and Eaton-Lambert syndrome.

A patient with a known history of pernicious anemia had the combined features of autoimmune myasthenia gravis (MG) and the Eaton-Lambert syndrome (ELS). Initially, this patient had all the features typical of MG, and after thymectomy developed all the typical features of ELS. In view of the coexistence of two autoimmune neuromuscular transmission disorders in one patient, we termed this disorder "overlap myasthenic syndrome."

Juvenile myasthenia gravis.

We studied 32 children with myasthenia gravis over a period of 12 years. The mean age at onset was 7.7 years. Presentation was ocular in 63% of patients. Another major disease in addition to myasthenia occurred in 44% of patients; a seizure disorder was the most commonly associated disease. Serum IgG antibody to nicotinic acetylcholine receptor was present in 53% of patients and did not correlate with severity of disease or treatment. Medical management was effective in 63%; thymectomy was effective in only 28%. We conclude that myasthenia gravis appears commonly before age 10 and is associated with the risk of some disease other than hyperthyroidism. Serum IgG nicotinic acetylcholine receptor antibody is present less frequently than in normal adults, and vigorous medical management should be attempted before thymectomy.

An enzyme-linked immunoabsorbent assay for measuring antibodies against muscle acetylcholine receptor.

An enzyme-linked immunoabsorbent assay has been developed for measuring anti-acetylcholine receptor antibodies from sera of patients with myasthenia gravis or tissue culture supernatants from hybridomas. Acetylcholine receptor from a detergent extract of muscle tissue was bound indirectly to microtiter plates via a monoclonal anti-receptor antibody already coupled to the polyvinyl plates. Myasthenic sera or antibodies in tissue culture media were then tested for binding to the acetylcholine receptor attached to the monoclonal antibody. Anti-receptor antibodies were detected in the serum of 80% of myasthenic patients when assayed by this method and the levels of antibody corresponded fairly well with antibody titers determined by an immunoprecipitation assay. Occasional myasthenic patients had serum antibodies which reacted specifically with the monoclonal antibody attached to the microtiter plate. The assay described here was far less time-consuming than immunoprecipitation assays, required only small quantities of receptor, and did not require the use of radioisotopes such as 125I-alpha-bungarotoxin.

Stereospecific actions of 2,5-dimethoxy-4-methylamphetamine (DOM) on colonic temperature in the rat at various ambient temperatures.

The R(-) and S(+)-isomers of 2,5-dimethoxy-4-methylamphetamine (DOM) produce a dose-dependent hypothermia in rats kept in the cold (6 degrees C). 2 This hypothermia was linearly dependent upon ambient temperature and the R(-)-isomer was considerably more potent than the S(+)-isomer. 3 A statistically significant tachyphylaxis was observed when R(-)-DOM was administered on two successive days. The response seven days after the second injection was similar to that on the first day of injection. 4 The hypothermia induced by R(-) and S(+)-DOM was antagonized by methysergide but not by p-chlorophenylalanine (PCPA) or pimozide. Methysergide, PCPA or pimozide alone did not elicit hypothermia at the doses used. The results indicate that R(-) and S(+)-DOM act at post-synaptic 5-hydroxytryptamine receptors.

Lamprey brain contains globular and asymmetric forms of acetylcholinesterase.

To obtain more information about the evolution of acetylcholinesterase in the vertebrates, we studied the cholinesterase activity from the brain of the lamprey Petromyzon marinus. We found that the enzyme is true acetylcholinesterase and that 98% of it is present in the G(4) globular form. Only 1% of the enzyme was found distributed among the asymmetric forms A(4), A(8) and A(12); an additional 1% of the activity could not be extracted from the brain. The identity of the asymmetric forms was confirmed by collagenase digestion. These data demonstrate that asymmetric acetylcholinesterase is present in the CNS of organisms representing all classes of vertebrates. However, our results are inconsistent with an evolutionary trend that has been observed for vertebrate brain acetylcholinesterase.

Batrachotoxinin-A N-methylanthranilate, a new fluorescent ligand for voltage-sensitive sodium channels.

Batrachotoxin and its derivatives have become important tools for the study of membrane excitability by virtue of their effects on voltage-sensitive sodium channels. Recent studies have shown that the 2,4-dimethylpyrrole carboxylate ester normally occurring in the 20-alpha position of batrachotoxin may be replaced by a benzoate moiety without loss of activity. We have now extended this series of active batrachotoxin derivatives and report here the synthesis of batrachotoxinin-A 20-alpha-N-methylanthranilate. The new fluorescent compound is highly toxic (LD50 approx. 15 micrograms/kg, i.p. mouse) and binds to the same receptor site of voltage-sensitive sodium channels as batrachotoxin with an equilibrium dissociation constant Kd = 180 nM (in the presence of scorpion toxin). These fluorescence and binding properties have been exploited to achieve successful visualization of sodium channels at mammalian nodes of Ranvier. Batrachotoxinin-A 20-alpha-N-methylanthranilate should prove to be a useful compound for studies of sodium channel distribution and for investigations of the microenvironment of the batrachotoxin binding site.

Reversal of organophosphate-induced muscle block by neomycin.

The organophosphate, diisopropyl fluorophosphate, and the aminoglycoside antibiotic, neomycin, both independently block neuromuscular transmission. At the neuromuscular junction, neomycin reduces the presynaptic release of acetylcholine, whereas diisopropyl fluorophosphate irreversibly inhibits acetylcholinesterase, thereby increasing the acetylcholine concentration. In the rat diaphragm preparation, the block in neuromuscular transmission caused by diisopropyl fluorophosphate could be reversed by adding neomycin.

Forskolin counteracts the effects of the organophosphate soman at the neuromuscular junction.

When neuromuscular transmission had been blocked using an organophosphate, function could be restored by treatment of the muscle with forskolin which increases the rate of desensitization of the nicotinic acetylcholine receptor. This suggests that excess post-synaptic depolarization due to acetylcholinesterase inhibition might be reversed by procedures which promote receptor desensitization.

Agonist and inhibitory effects of pyridostigmine at the neuromuscular junction.

When all of the AChE at the endplate is irreversibly inhibited by phospholine iodide the ionophoretically induced ACh endplate currents are increased more than 10-fold in amplitude. The reversible AChE inhibitor pyridostigmine only increases the current to about half this value because its effects are obscured by receptor blocking. It was found that pyridostigmine can activate the receptor ion channels when released by ionophoresis at the endplate, thus suggesting that agonist-like desensitization could contribute to the blocking effects.

The effects of alcohols and diols at the nicotinic acetylcholine receptor of the neuromuscular junction.

A series of straight chain aliphatic alcohols from ethanol to octanol were tested at voltage-clamped frog endplates. In the presence of high concentrations of ethanol (greater than 1 M) the individual current responses to ionophoretic pulses of ethanol were reduced in amplitude and the dose-response curve for acetylcholine was shifted to the right. All the alcohols tested had this effect and their potency increased with the length of the carbon chain. The results were interpreted to indicate that as the molecular weight of the alcohol increased, its potency as a channel blocker also increased. The diol derivative of ethanol, which is ethylene glycol (ethanediol), was totally inactive up to 400 mM. However, 1,3-propanediol was a more potent blocker than propanol. After dose-response curves were carried out in high doses of ethanol and propanediol, the number of receptors was found to be permanently reduced. This effect could be due to irreversible denaturation of the receptor and therefore reversible denaturation could account for some of the reversible blocking effects caused by such drugs. An additional effect on the receptor was observed in that low concentrations of ethanol and propanol reduced the apparent dissociation constant for acetylcholine, thus increasing the amplitude of individual responses and shifting the dose-response curve to the left.

Curare can reverse the failure in muscle contraction caused by an AChE inhibitor.

Diisopropyl fluorophosphate, which is an organophosphate inhibitor of acetylcholinesterase, caused an irreversible block of neuromuscular transmission in the rat diaphragm preparation. This block could be reversed by the addition of a competitive antagonist of acetylcholine such as D-tubocurarine.

Agonist and blocking effects of choline at the neuromuscular junction.

The effects of choline chloride were studied at the voltage-clamped frog neuromuscular junction by measuring miniature endplate currents and equilibrium dose response curves for acetylcholine applied by microionophoresis. Choline reduced the amplitude and shortened the time constant of miniature endplate currents in a dose dependent manner. Dose response curves carried out in the presence of low doses of choline (200 microM) were shifted to the right and the apparent dissociation constant for ACh was increased without affecting the Hill coefficient or the maximum conductance at the endplate. Higher doses of choline shifted the curve even further to the right but reduced the Hill coefficient and maximum conductance. Choline ionophoretic dose response curves were carried out but the conductance response was only about 1% of the response to comparable concentrations of ACh. In the presence of ethanol, which reduces the agonist dissociation constant, choline responses were increased and the dose response curve analysis revealed that the efficacy of choline was about 17% in comparison to ACh. Similar effects were measured at rat endplates. Rat nerve-muscle preparations were used to investigate the effects of choline upon neuromuscular transmission.

Evidence that certain peripheral anti-acetylcholine receptor antibodies do not interact with brain BuTX binding sites.

The cross-reactivity of rat brain alpha-bungarotoxin (BuTX) binding sites with peripheral anti-acetylcholine receptor (nAChR) polyclonal and monoclonal antibodies was investigated in Triton extracts of several brain areas and purified whole brain putative receptors using radioimmunoassays and IgG/CNBr-Sepharose 4B immunoaffinity chromatography. The effects of the chronic intracerebral administration of antibodies on [125I]BuTX binding and food and water intake were also investigated. It was concluded that brain BuTX sites do not share antigenic determinants with peripheral nAChRs. Additionally, no evidence was found to support the suggestions that anti-nAChR antibodies affect food and water intake, or that chronic administration of antibodies alters [125I]BuTX binding in the hypothalamus of rats.