Proteoliposome as the model for the study of membrane-bound enzymes and transport proteins

Published data regarding the interaction of long-chain acyl CoA derivatives with the protein and phospholipid constituents of biological membranes is reviewed and discussed in relationship to the premise that such interactions may lead to membrane damage during pathological situations. The topics considered include: the detergent properties of long-chain CoA, the interaction with membrane-associated enzymes, biological membranes, or model membrane systems, and the binding to a soluble protein that may facilitate intracellular transport. The effects of long-chain acyl CoA on heart mitochondria and the relevance of such studies to myocardial ischemia also is emphasized.     

Cell envelope architecture in the Chloroflexi: a shifting frontline in a phylogenetic turf war

It is important that attempts to understand bacterial phylogeny take into account fundamental bacterial characteristics such as cell envelope composition and organization. Several prominent phylogenetic studies have assumed that the cell envelopes of members of the phylum Chloroflexi are ‘Gram‐negative’ (diderm, i.e. defined by both an inner plasma membrane and an outer membrane) and some of these studies have placed the branch leading to the extant Chloroflexi near the root of the bacterial phylogenetic tree. This Correspondence summarizes the compelling evidence that the Chloroflexi are in fact monoderm, i.e. have only a single cellular membrane. The phylogenetic implications of this conclusion are discussed. The data reviewed also shed interesting light on the distribution of protein secretion systems in diderm bacteria.     

Sequence relationships between integral inner membrane proteins of binding protein-dependent transport systems: evolution by recurrent gene duplications.

Periplasmic binding protein-dependent transport systems are composed of a periplasmic substrate-binding protein, a set of 2 (sometimes 1) very hydrophobic integral membrane proteins, and 1 (sometimes 2) hydrophilic peripheral membrane protein that binds and hydrolyzes ATP. These systems are members of the superfamily of ABC transporters. We performed a molecular phylogenetic analysis of the sequences of 70 hydrophobic membrane proteins of these transport systems in order to investigate their evolutionary history. Proteins were grouped into 8 clusters. Within each cluster, protein sequences displayed significant similarities, suggesting that they derive from a common ancestor. Most clusters contained proteins from systems transporting analogous substrates such as monosaccharides, oligopeptides, or hydrophobic amino acids, but this was not a general rule. Proteins from diverse bacteria are found within each cluster, suggesting that the ancestors of current clusters were present before the divergence of bacterial groups. The phylogenetic trees computed for hydrophobic membrane proteins of these permeases are similar to those described for the periplasmic substrate-binding proteins. This result suggests that the genetic regions encoding binding protein-dependent permeases evolved as whole units. Based on the results of the classification of the proteins and on the reconstructed phylogenetic trees, we propose an evolutionary scheme for periplasmic permeases. According to this model, it is probable that these transport systems derive from an ancestral system having only 1 hydrophobic membrane protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiac fibroblasts and the mechano-electric feedback mechanism in healthy and diseased hearts

Cardiac arrhythmia is a serious clinical condition, which is frequently associated with abnormalities of mechanical loading and changes in wall tension of the heart. Recent novel findings suggest that fibroblasts may function as mechano-electric transducers in healthy and diseased hearts. Cardiac fibroblasts are electrically non-excitable cells that respond to spontaneous contractions of the myocardium with rhythmical changes of their resting membrane potential. This phenomenon is referred to as mechanically induced potential (MIP) and has been implicated in the mechano-electric feedback mechanism of the heart. Mechano-electric feedback is thought to adjust the frequency of spontaneous myocardial contractions to changes in wall tension, which may result from variable filling pressure. Electrophysiological recordings of single atrial fibroblasts indicate that mechanical compression of the cells may activate a non-selective cation conductance leading to depolarisation of the membrane potential. Reduced amplitudes of MIPs due to pharmacological disruption of F-actin and tubulin suggest a role for the cytoskeleton in the mechano-electric signal transduction process. Enhanced sensitivity of the membrane potential of the fibroblasts to mechanical stretch after myocardial infarction correlates with depression of heart rates. It is assumed that altered electrical function of cardiac fibroblasts may contribute to the increased risk of post-infarct arrhythmia.     

Effects of free radicals on the electrophysiological function of cardiac membranes.

Abnormal electrical activity in heart cells can result in irregular heart rhythms or arrhythmias. Any form of pathological or toxicological damage to the sarcolemmal membrane presents the risk of precipitating arrhythmias and compromise of the heart's function as a pump. An array of cardiovascular conditions from coronary artery disease and myocardial infarction to cardiomyopathies and hypertrophy, can induce arrhythmias. Many of these conditions recently have been linked to increases in free radical production. Early studies suggesting a role for free radicals in the abnormal function of ischemic and reperfused hearts use anti-free radical interventions to reduce arrhythmias. More recent works have taken advantage of different free radical-generating systems to show a reproducible sequence of changes in the cellular action potential; these data suggest changes in the transmembrane movement of ions through membrane channels. Biochemical evidence supports a possible involvement of ion exchange mechanisms in the cardiac sarcolemma. All the evidence indicate that free radical injury may have profound effects on the electrical function of myocardial cells.     

Lasalocid immediately and completely prevents the myocardial damage caused by coronary ischemia reperfusion in rat heart

Lasalocid, a specific mobile membrane ionophore for calcium, dopamine and norepinephrine was assayed in its capacity to reduce or maintain unaltered the cardiovascular function in conditions of imminent myocardial injury. In experiments of coronary blockade and reperfusion carried out in rat heart, it was found that when administered from 5 to 30 minutes prior to the induction of coronary blockade, at a concentration of 2 mg/kg of body weight, the ionophore immediately, simultaneously, and completely interrupts the blood pressure decay, cardiac frequency increase, electrical ventricular tachycardia and fibrillation, as well as the fall of mitochondrial oxidative phosphorylation and decay of mitochondrial oxygen uptake provoked by the induced myocardial injury. It appears that the molecular mode of action of the lasalocid is associated with its unique ability to transport both calcium and the catecholamines, dopamine and norepinephrine, across mitochondrial and bimolecular lipid membranes, as well as through synaptic cell membrane terminals from rat heart, myocardial fibers of the heart and heart chromaffin membrane vesicles. It is suggested that for the potential medical use of lasalocid to detain incoming ischemic myocardial damage, there exists a need to develop a personal electronic device able to simultaneously monitor, detect, and inform on the very early and simultaneous signs of cardiac alterations of electrical, mechano-chemical, metabolic and hydraulic nature, all which precede heart failure and to administer the lasalocid.

     

Myocardial Depolarizing Response to Glutamate in the Myogenic Heart of the Branchiopod Crustacean Triops longicaudatus

Fine structure of the heart and the effects on the heartbeat of some transmitter candidates in crustacean cardioregulatory system were examined in the myogenic heart of the branchiopod crustacean Triops longicaudatus. Electron microscopy revealed that, in each myocardial cell, myofibrils are confined in the part facing the epicardium and intercalated disks are present between the myofibrillar regions of adjacent myocardial cells. No neural elements were found in the heart, suggesting lack of extrinsic cardioregulatory nerves from the central nervous system. Gamma aminobutyric acid and acetylcholine produced no detect-able changes in the myogenic activity of the heart at concentrations up to 10(-3) M, respectively. Glutamate induced a depolarizing membrane response in the cardiac muscle with a threshold concentration of approximately 1x10(-5) M. The amplitude of the depolarizing response was concetration-dependent and saturated at approximately 1x10(-4) M. The myogenic activity of the heart increased in frequency with glutamate of less than approximately 3x10(-5) M. With higher dose of glutamate, action potential adaptation occurred in the cardiac muscle and the heart exhibited a systolic arrest.     

A possible role for atrial fibroblasts in postinfarction bradycardia

Atrial fibroblasts are considered to modulate the contractile activity of the heart in response to mechanical stretch. In this study we examined whether atrial fibroblasts are possibly involved in bradyarrhythmia, which is a severe complication after myocardial infarction. For this purpose, transmembrane electrical potentials were recorded in cardiac fibroblasts near the sinoatrial node from sham-operated rats and from rats with myocardial infarction. Twenty days after infarction due to coronary artery ligation, the right atrial tissue weights and the sensitivity of the fibroblast membrane potential to mechanical stretch correlated positively with the infarct size. Cardiac growth was enhanced, but the stretch sensitivity and the resting membrane potential of the atrial fibroblasts declined between 8 and 30 days after infarction. The frequency of spontaneous atrial contractions was significantly reduced 8 days after myocardial infarction and recovered in parallel with the membrane potential of the fibroblasts. These findings suggest that changes in the susceptibility of atrial fibroblasts to mechanical stretch may contribute to bradyarrhythmia during postinfarct remodeling of the heart.     

Organ arrest, protection and preservation: natural hibernation to cardiac surgery

Cardiac surgery continues to be limited by an inability to achieve complete myocardial protection from ischemia-reperfusion injury. This paper considers the following questions: (1) what lessons can be learned from mammalian hibernators to improve current methods of human myocardial arrest, protection and preservation? and (2) can the human heart be pharmacologically manipulated during acute global ischemia to act more like the heart of a hibernating mammal? After reviewing the major entropy-slowing strategies of hibernation, a major player identified in the armortarium is maintenance of the membrane potential. The resting membrane potential of the hibernator's heart appears to be maintained close to its pre-torpid state of around -85 mV. In open-heart surgery, 99% of all surgical heart arrest solutions (cardioplegia) employ high potassium (>16 mM) which depolarises the membrane voltage from -85 to around -50 mV. However, depolarising potassium cardioplegia has been increasingly linked to myocyte and microvascular damage leading to functional loss during reperfusion. Our recent work has been borrowed from hibernation biology and is focused on a very different arrest strategy which 'clamps' the membrane near its resting potential and depresses O2 consumption from baseline by about 90%. The new 'polarising' cardioplegia incorporates adenosine and lidocaine (AL) as the arresting combination, not high potassium. Studies in the isolated rat heart show that AL cardioplegia delivered at 37 degrees C can arrest the heart for up to 4 h with 70-80% recovery of the cardiac output, 85-100% recovery of heart rate, systolic pressure and rate-pressure product and 70-80% of baseline coronary flows. Only 14% of hearts arrested with crystalloid St. Thomas' solution No. 2 cardioplegia survived after 4 h. In conclusion, maintenance of the myocardial membrane potential near or close to its resting state appears to be an important feature of the hibernator's heart that may find great utility in surgical arrest and cellular preservation strategies. Identifying and safely turning 'off' and 'on' the entropy-slowing genes to down-regulate the hibernator's heart and applying this to human organs and tissues remains a major challenge for future genomics and proteomics.     

Activity of ectoenzymes of the heart breaking up ATP in the period of myocardial reperfusion after ischemia

Nowadays the activity of heart ectoenzymes, breaking up ATP, are determined in the undamaged tissue to eliminate the effect to the ATP of intracellular enzymes. Our aim is to study the activity of ecto-adenosine triphosphatase in the heart damaged by ischemia. It was established that the activity of ectoenzymes, breaking up ATP, may be measured in the tissue damaged by ischemia only in case when the cellular membrane becomes impermeable for the intracellular enzymes. The activity of adenosine triphosphatase is significantly reduced during the myocardial reperfusion after ischemia and appeared as one of the criteria by which we can assume the degree of the heart reperfusion damage.     

External transport of beta-adrenergic binding sites in ischemic myocardium

The properties of beta-adrenergic receptors were studied in normal and in flow restricted regions of the dog heart. Purified cardiac membrane preparations and papillary muscle preparations were isolated from control and ischemic areas and tested a) following chronic beta-receptor blockade with metipranolol or exaprolol, and b) after acute regional myocardial ischemia. A significant reduction in the sensitivity of the heart muscle preparations from compromised heart for isoprenaline resulting in a reduced affinity of beta-adrenergic receptors to exaprolol was observed. Quantitative ligand binding data showed higher numbers of (3H) dihydroalprenolol/(3H) DHA/binding sites in the membrane fraction obtained from compromised compared to control myocardium. The ratio of intra- to extracellular beta-adrenergic receptors decreased from 1.35 to 0.55 in the membrane fractions obtained from the compromised hearts. Pretreatment of experimental animals with metipranolol or propranolol attenuated the observed increase in the total number of beta-adrenergic receptor sites in myocardial membrane fractions from ischemic hearts. These data suggest preferential distribution of beta-adrenergic binding sites from intracellular to membrane fractions in flow restricted regions of the dog heart after coronary occlusion.     

Myocardial cell relationships during morphogenesis in normal and cardiac lethal mutant axolotls, Ambystoma mexicanum

Sarcomere formation has been shown to be deficient in the myocardium of axolotl embryos homozygous for the recessive cardiac lethal gene c. We examined the developing hearts of normal and cardiac mutant embryos from tailbud stage 33 to posthatching stage 43 by scanning electron microscopy in order to determine whether that deficiency has any effect on heart morphogenesis. Specifically, we investigated the relationships of myocardial cells during the formation of the heart tube (stage 33), the initiation of dextral looping (stages 34-36), and the subsequent flexure of the elongating heart (stages 38-43). In addition, we compared the morphogenetic events in the axolotl to the published accounts of comparable stages in the chick embryo. In the axolotl (stage 33), changes in cell shape and orientation accompany the closure of the myocardial trough to form the tubular heart. The ventral mesocardium persists longer in the axolotl embryo than in the chick and appears to contribute to the asymmetry of dextral looping (stages 34-36) in two ways. First, as a persisting structure it places constraints on the simple elongation of the heart tube and the ability of the heart to bend. Second, after it is resorbed, the ventral myocardial cells that contributed to it are identifiable by their orientation, which is orthogonal to adjacent cells: a potential source of shearing effects. Cardiac lethal mutant embryos behave identically during these events, indicating that functional sarcomeres are not necessary to these processes. The absence of dynamic apical myocardial membrane changes, characteristic of the chick embryo (Hamburger and Hamilton stages 9-11), suggests that sudden hydration of the cardiac jelly is less likely to be a major factor in axolotl cardiac morphogenesis. Subsequent flexure (stages 38-43) of the axolotl heart is the same in normal and cardiac lethal mutant embryos as the myocardial tube lengthens within the confines of a pericardial cavity of fixed length. However, the cardiac mutant begins to exhibit abnormalities at this time. The lack of trabeculation (normally beginning at stage 37) in the mutant ventricle is evident at the same time as an increase in myocardial surface area, manifest in extra bends of the heart tube at stage 39. Nonbeating mutant hearts (stage 41) have an abnormally large diameter in the atrioventricular region, possibly the result of the accumulation of ascites fluid. In addition, mutant myocardial cells have a larger apical surface area compared to normals.     

Enzyme histochemical studies on the Purkinje fibres of the arrioventricular system of the bovine and porcine hearts

Synopsis In this communication the results of applying various histochemical semipermeable membrane techniques to the localization of several enzymes in bovine and procine heart are presented. The Purkinje fibres of the atrioventricular conducting system of the bovine heart differ from the myocardium proper in containing a greater activity of the glycolytic and gluconeogenetic enzymes—lactate dehydrogenase, glyceraldehyde-phosphate dehydrogenase, hexokinase, glucosephosphate isomerase and phosphoglucomutase, and less activity of the aerobic enzymes-NADH: nitroBT oxidoreductase and isocitrate dehydrogenase (NADP⁺). The metabolic reactions obtained with Purkinje fibres of the porcine heart are less pronounced. These histochemical findings are in accordance with the impression that Purkinje fibres, compared with the common myocardial fibres, have a higher rate of anaerobic metabolism and a lower rate of aerobic metabolism.The activity of the NADPH regenerating enzymes, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating), and the activity of acid hydrolases such as non-specific esterase and acid phosphatase is higher in the Purkinje fibres of both the bovine and porcine heart.     

Immunocytochemical localization of the functional fraction of lipoprotein lipase in the perfused heart

The functional (heparin-releasable) fraction of myocardial lipoprotein lipase (LPL) has been located at the lumen surface of capillary endothelium by means of an indirect immunocytochemical perfusion method for electron microscopy. The primary step immunoreactant was an IgG fraction of goat antiserum directed against LPL from rat heart. The second step antibody, conjugated with horseradish peroxidase, was rabbit IgG directed against goat IgG. Peroxidase reaction product, when present, appeared at the surface an in invaginations of the lumenal plasma membrane of capillary endothelium and also on chylomicrons adherent to that membrane. The highest coverage by such product occurred when the highest heparin-releasable heart LPL activity was attained after fat-feeding of rats. Coverage was low when a low level of heparin-releasable heart LPL activity was induced by carbohydrate-feeding. Coverage was very low in the perfused hearts after heparin-release of functional LPL activity. The positive association between these immunocytochemical results and actual levels of functional LPL activities indicates that functional LPL in the isolated rat heart is at the lumen surface of capillary endothelium.     

Expression of extracellular matrix components fibronectin and laminin in the human fetal heart.

It has been well documented that the extracellular matrix components fibronectin and laminin promote or regulate morphogenesis of the myocardial cells in mammalian heart. However, their chronological change of expression (or localization) in the human heart remains elusive. In this study, fibronectin and laminin in the left ventricle of forty-two human fetuses aged from 8 to 26 weeks gestation and left ventricular tissues obtained from a 2-week old infant and two adults were investigated by Western blot analyses and indirect immunofluorescence technique with monoclonal antibodies. In the fetal heart, fibronectins were present along the endocardium, epicardium, and linings of larger blood vessels. In 14-16 weeks gestation, fibronectin immunofluorescence became stronger but not evenly dispersed in the interstitium. After 24 weeks gestation, they were strongly positive only in the relatively larger blood vessels, as well as those in the infant and adult cardiac tissues. Laminins were strongly positive along the endocardium and basement membrane of the myocardial cells and fibroblasts during fetal life. After birth, laminins formed fine fibrillar network along the basement membrane in association with the transverse tubules of myocardial cell; these morphological characteristics remained in the adult cardiac tissues. These results indicate that fibronectin expression is relatively constant during fetal life but decreases after birth; in contrast, laminin expression is not age-dependent and constant throughout the life.     

Cardiac pacemaker mechanisms in the ostracod crustacean Vargula hilgendorfii

The heart of the ostracod crustacean Vargula hilgendorfii has a single intrinsic neuron that morphologically appears to innervate the myocardium. We, therefore, examined the heart activity electrophysiologically to determine whether the heartbeat is neurogenic. Each heartbeat is associated with a myocardial action potential composed of a spike potential followed by a plateau potential. The frequency of the action potential is not stable but changes successively over a wide range. The action potential is not preceded by a pacemaker potential and has an inflection in its rising phase. The myocardial cells couple electrically and fire almost simultaneously. The frequency of the action potential was unchanged by injection of depolarizing or hyperpolarizing current into the myocardium. However, slow oscillatory potentials appeared during the depolarization and its frequency was higher with increasing current intensity. Application of 1-microM tetrodotoxin (TTX) depolarized the myocardial membrane and completely prevented the action potential. During this depolarization, slow oscillatory potentials often appeared spontaneously. These results suggest that, although the myocardium has a property of conditional oscillator, the heartbeat is driven by the single cell cardiac ganglion that has both pacemaker and motor functions.     

Mechanisms of attenuation of ischemic heart injury by means of a modified reperfusion

Mechanisms of attenuation of membrane injury and metabolic impairments in postischemic cardiomyocytes have been studied on a model of ischemic and reperfusion stress of rat heart using a modified early reperfusion. Optimization of the reperfusion infusate composition augmented recovery of cardiac pump and contractile function. This was accompanied by reduced release of lactate dehydrogenase activity and systems generating short-living reactive oxygen species into myocardial effluent and was associated with more efficient oxidative metabolism recovery and decreased losses of intracellular total creatine and amino acids pools. The results indicate perspectives of postischemic functional and metabolic myocardial injury correction by means of the controlled reperfusion.     

大豆磷脂脂质体对再灌注心肌线粒体的影响

利用Ｌａｎｇｅｎｄｏｒｆｆ离体心脏灌流装置,研究在缺血－再灌注时补充大豆磷脂脂质体对心肌线粒体膜脂质特性和超微结构的影响。结果：在缺血－再灌注时补充大豆磷脂脂质体可提高线粒体膜磷脂含量,抑制胆固醇－磷脂摩尔比和膜脂质微粘度的增加,改善线粒体的超微结构。结果表明,补充大豆磷脂脂质体对再灌注心肌线粒体的脂质特性和超微结构的损伤性变化具有保护作用。     

Therapeutic potential of vitamin E against myocardial ischemic-reperfusion injury.

Myocardial ischemia is a disease process characterized by reduced coronary flow such that the supply of nutritive blood to heart muscle (myocardium) is insufficient for normal myocardial aerobic metabolism. Prompt reestablishment of coronary flow by invasive and noninvasive clinical procedures is the most direct and effective means of limiting myocardial damage in ischemic heart disease patients, although reperfusion carries with it an injury component which may reflect, at least to some degree, the toxic effects of partially reduced oxygen species and their participation in degenerative cellular processes such as membrane lipid peroxidation. Vitamin E, a lipophilic, chain-breaking antioxidant, is a prominent membrane constituent in heart muscle, where it modulates/regulates various aspects of heart muscle-cell metabolism and function. Vitamin E's beneficial effects against experimentally induced oxidative damage to the heart, along with inverse epidemiological correlations between plasma vitamin E level and either anginal pain or mortality due to ischemic heart disease, suggest that vitamin E might have protective and therapeutic roles against myocardial ischemic-reperfusion injury. Laboratory investigations aimed at addressing this possibility have demonstrated that vitamin E supplementation protects isolated hearts against ischemic-reperfusion injury, and relatively more inconsistent and limited data document cardioprotective effects of vitamin E in some animal models of myocardial ischemia-reperfusion, especially when administered prior to the ischemic period. Clinical attempts to establish whether vitamin E has therapeutic benefit in ischemic heart disease patients remain inconclusive, having relied upon a variety of nonuniformly controlled protocols and a single, rather subjective endpoint (anginal pain). Consequently, although laboratory data constitute a conceptual context for and indirect support of the idea that vitamin E could be a cardioprotectant against ischemic-reperfusion injury, compelling clinical evidence regarding vitamin E's therapeutic potential in the ischemic heart-disease patient is lacking. Elective coronary revascularization would appear to provide an attractive clinical setting for evaluating the therapeutic efficacy of vitamin E in the context of cardiac ischemia-reperfusion. Further biochemical work would still be required to define how vitamin E exerts any cardioprotective effect observed in these patients.     

Dopamine receptors in the guinea-pig heart. A binding study

The binding of dopaminergic agonists and antagonists to guinea-pig myocardial membrane preparations was studied using 3H-dopamine and 3H-spiperone as radioligand. 3H-Dopamine bound specifically to heart membranes while 3H-spiperone did not. A Scatchard analysis of 3H-dopamine binding showed a curvilinear plot indicating the presence of two dopamine receptor populations that we have termed high- (Kd = 1.2 nM, Bmx = 52.9 fmol/mg prot.) and low- (Kd = 11.8 nM, Bmx = 267.3 fmol/mg prot.) affinity binding sites, respectively. The characterisation of the high-affinity component of 3H-dopamine binding indicated that the binding is rapid, saturable, stereospecific, pH- and temperature-dependent, and displaced by dopaminergic agonists and antagonists known to act similarly in vivo. The finding that pretreatment with dibenamine (which has been described as an alpha-adrenoceptor irreversible blocker) did not affect the binding of dopamine to cardiac membrane preparations suggests that alpha-adrenoceptors and dopamine receptors have separate recognition sites in the heart. We conclude that 3H-dopamine binds to specific dopamine receptors in the heart of guinea-pigs.