Fluctuations in the concentration of extracellular ATP synchronized with intracellular Ca(2+) oscillatory rhythm in cultured cardiac myocytes

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The intracellular concentration of free Ca²⁺ also changes rhythmically in association with the rhythmic contraction of myocytes (Ca²⁺ oscillation). Both the contraction and Ca²⁺ oscillatory rhythms are synchronized among myocytes, and intercellular communication via gap junctions has been considered primarily responsible for the synchronization. However, a recent study has demonstrated that intercellular communication via extracellular ATP-purinoceptor signaling is also involved in the intercellular synchronization of intracellular Ca²⁺ oscillation. In this study, we aim to elucidate whether the concentration of extracellular ATP changes cyclically and contributes to the intercellular synchronization of Ca²⁺ oscillation among myocytes. In almost all the cultured cardiac myocytes at four days in vitro (4 DIV), intracellular Ca²⁺ oscillations were synchronized with each other. The simultaneous measurement of the concentration of extracellular ATP and intracellular Ca²⁺ revealed the extracellular concentration of ATP actually oscillated concurrently with the intracellular Ca²⁺ oscillation. In addition, power spectrum and cross-correlation analyses suggested that the treatment of cultured cardiac myocytes with suramin, a blocker of P2 purinoceptors, resulted in the asynchronization of Ca²⁺ oscillatory rhythms among cardiac myocytes. Treatment with suramin also resulted in a significant decrease in the amplitudes of the cyclic changes in both intracellular Ca²⁺ and extracellular ATP. Taken together, the present study demonstrated the possibility that the concentration of extracellular ATP changes cyclically in association with intracellular Ca²⁺, contributing to the intercellular synchronization of Ca²⁺ oscillation among cultured cardiac myocytes.     

Maintenance and characterization of spontaneous contraction rhythm in cultured cardiac myocytes fused with cardiac fibroblasts

Cardiomyocytes (CMs) fuse with various cells including endothelial cells, cardiac fibroblasts (CFs). In addition, recent studies have shown that stem cells fuse spontaneously with cells remaining in the damaged tissues, and restore tissue functions after myocardial infarction. In this study, we investigated whether cultured cardiomyocytes fused with proliferative cardiac fibroblasts maintained the phenotype of functional myocytes by analyzing the spontaneous contraction rhythm after fusion with CFs lacking a beating capability. CMs and CFs cultured for 4 days in vitro were used in this study. The fusion of cultured CMs and CFs was achieved with polyethylene glycol (PEG) and hemagglutinating virus of Japan (HVJ). Analyses of CMs fused with CFs by using either PEG or HVJ to imitate spontaneous fusion in vivo demonstrated that CMs and CFs actually fused together and fused cells expressed lineage marker proteins of both CMs and CFs. In addition, fused cells reentered the G2-M phase of the cell cycle. Furthermore, fused cells retained the spontaneous contraction activity. The present study demonstrated that CMs fused with proliferative CFs showed the phenotype of both CMs and CFs and spontaneous rhythmic contraction.     

Rhythmic contraction and intracellular Ca2 + oscillatory rhythm in spontaneously beating cultured cardiac myocytes

Cultured cardiac myocytes from neonatal rats show spontaneous and rhythmic contractions. The intracellular concentration of free Ca^2 + also changes rhythmically, associated with the rhythmic contraction of myocytes (Ca^2 + oscillation). This study aims to elucidate whether spontaneous rhythmic contraction affects the dynamics of intracellular Ca^2 + oscillation in cultured cardiac myocytes. In cultures at four days in vitro (4 DIV), spontaneous Ca^2 + oscillation was synchronized among myocytes. Treatment of cultures with an uncoupler of E - C coupling resulted in a cessation of the spontaneous contraction of cardiac myocytes, but did not abolish the Ca^2 + oscillation. The intercellular synchronization of intracellular Ca^2 + oscillation persisted, and both the intervals and the fluctuation of the oscillation tended to increase after the termination of rhythmic contraction. The present study demonstrated that mechanical factors associated with rhythmic contraction did not affect the intercellular synchronization of intracellular Ca^2 + oscillation, but possibly contributed to the stability of the oscillatory rhythm.     

Intercellular calcium signalling in cultured renal epithelia: a theoretical study of synchronization mode and pacemaker activity

We investigate a two-dimensional lattice model representation of intercellular Ca²⁺ signalling in a population of epithelial cells coupled by gap junctions. The model is based on and compared with Ca²⁺ imaging data from globally bradykinin-stimulated MDCK-I (Madin-Darby canine kidney)-I cell layers. We study large-scale synchronization of relevance to our laboratory experiments. The system is found to express a wealth of dynamics, including quasiperiodic, chaotic and multiply-periodic behaviour for intermediate couplings. We take a particular interest in understanding the role of pacemaker cells in the synchronization process. It has been hypothesized that a few highly hormone-sensitive cells control the collective frequency of oscillation, which is close to the natural frequencies (without coupling) of these cells. The model behaviour is consistent with the conjectures of the pacemaker cell hypothesis near the critical coupling where the cells lock onto a single frequency. However, the simulations predict that the frequency in globally connected systems decreases with increasing coupling. It is found that a pacemaker is not defined by its natural frequency alone, but that other intrinsic or local factors must be considered. Inclusion of partly sensitized cells that do not oscillate autonomously in the cell layer increases the coupling necessary for global synchronization. For not excessively high coupling, these cells oscillate irregularly and with distinctive lower frequencies. In summary, the present study shows that the frequency of synchronized oscillations is not dictated by one or few fast-responding cells. The collective frequency is the result of a two-way communication between the phase-advanced pacemaker and its environment.     

Involvement of oxygen free radicals in the respiratory uncoupling induced by free calcium and ADP-magnesium in isolated cardiac mitochondria: Comparing reoxygenation in cultured cardiomyocytes

Recently, we have observed that the simultaneous application of free calcium (fCa) and ADP-magnesium (Mg) reduced the ADP:O ratio in isolated cardiac mitochondria. The uncoupling was prevented by cyclosporin A, an inhibitor of the permeability transition pore. The purpose of this study was to know if the generation of oxygen free radicals (OFR) is involved in this phenomenon and if it occurs during reoxygenation (Reox) of cultured cardiomyocytes. Cardiac mitochondria were harvested from male Wistar rats. Respiration was assessed in two media with different fCa concentrations (0 or 0.6 M) with palmitoylcarnitine and ADP-Mg as respiration substrates. The production of Krebs cycle intermediates (KCI) was determined. Without fCa in the medium, the mitochondria displayed a large production of citrate + isocitrate + -ketoglutarate. fCa drastically reduced these KCI and promoted the accumulation of succinate. To know if OFR are involved in the respiratory uncoupling, the effect of 4OH-TEMPO (250 M), a hydrosoluble scavenger of OFR, was tested. 4OH-TEMPO completely abolished the fCa- and ADP-Mg-induced uncoupling. Conversely, vitamin E contributed to further decreasing the ADP:O ratio. Since no hydrosoluble electron acceptor was added in our experiment, the oxygen free radical-induced oxidized vitamin E was confined near the mitochondrial membranes, which should reduce the ADP:O ratio by opening the permeability transition pore. The generation of OFR could result from the matrix accumulation of succinate. Taken together, these results indicate that mitochondrial Ca uptake induces a slight increase in membrane permeability. Thereafter, Mg enters the matrix and, in combination with Ca, stimulates the isocitrate and/or -ketoglutarate dehydrogenases. Matrix succinate favors oxygen free radical generation that further increases membrane permeability and allows respiratory uncoupling through proton leakage. To determine whether the phenomenon takes place during Reox, cultured cardiomyocytes were subjected to hypoxia and Reox. ¹⁴C-palmitate was added during Reox to determine the KCI profile. Succinate had not increased during Reox. In conclusion, calcium- and ADP-Mg-induced respiratory uncoupling is due to oxygen free radical generation through excess matrix accumulation of succinate. The phenomenon does not occur during reoxygenation because of a total restoration of mitochondrial magnesium and/or ADP concentration.     

A possible calcium binding site in D1 protein: A fluorescence and FTIR study of the interaction between lanthanides and a synthetic peptide

A peptide ranging from residue 229 to 240 of the D₁ protein of Photosystem (PS) II was synthesized and lanthanides were used as candidates of calcium. Fluorescence and FTIR spectroscopy were used to test the conformational adaptation after lanthanide additions. Fluorescence spectroscopy showed that the synthetic peptide provides lanthanide binding site, and that glutamic acids are involved in lanthanide binding. Resolution enhancement techniques were combined with band curve-fitting procedures to quantitate the FTIR spectral information from the amide 1 bands. The relative areas of these component bands indicate that lanthanide induced a substantial decrease in the amount of unordered structure and turns, while a corresponding increase in the amount of -helix and open loop was also observed. This indicates that a relatively compact structure of the synthetic peptide is formed if lanthanides are applied. The results may reflect on the physiological and biochemical function of calcium in PS II, including preventing D₁ from trypsin digestion.Abbreviations DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea - FTIR Fourier transform infrared - FSD Fourier self-deconvolution - PS Photosystem - Q_B Secondary plastoquinone electron acceptor of PS II     

No association between the vitamin D pathway gene polymorphisms and bone biomarkers response to calcium and low dose calcitriol supplementation in postmenopausal Chinese women: a one-year prospective study

Abstract

Introduction: The aim of the study was to explore the association between the vitamin D pathway gene variations and the bone biomarkers response to calcium and low dose calcitriol supplementation in postmenopausal Chinese women.

Methods: A total of 110 healthy postmenopausal Chinese women (61.51?±?6.93?years) were enrolled. The participants were supplemented with calcium (600?mg/d) and calcitriol (0.25?μg/d), for 1?year. Four biomarkers, serum levels of beta C-terminal cross-linked telopeptides of type I collagen (β-CTX), amino-terminal propeptide of type I collagen (P1NP), parathyroid hormone (PTH) and 25-hydroxyvitamin D [25(OH)D] were measured at baseline and 12-month follow-up. Multivariate regression models were established to explore the statistical association between the change rate of the four biomarkers and 15?key genes within the vitamin D metabolic pathway.

Results: This exclusion process left 98 participants for analysis. Serum levels of P1NP, β-CTX and PTH were significantly decreased at the 12-month follow-up (all p?<?0.05). Serum 25(OH)D level had no significant change (p?>?0.05). No association was found between the vitamin D pathway gene polymorphisms and bone biomarkers response to calcium and low dose calcitriol supplementation.

Conclusions: Genetic background of postmenopausal Chinese women might not influence supplemental response of the biomarkers to calcium and low dose calcitriol.     

Construction and analysis of a BAC library in the grass<Emphasis Type="Italic"> Brachypodium sylvaticum</Emphasis>: its use as a tool to bridge the gap between rice and wheat in elucidating gene content

A BAC library of 30,228 clones with an average insert size of 102 kb was constructed in the grass Brachypodium sylvaticum. Brachypodium has a simple genome, similar in size and repetitive DNA content to that of rice, and is more closely related than rice both to the major temperate cereals wheat and barley, and to the forage grasses. The library represents 6.6 genome equivalents, implying a 99.9% probability of recovering any specific sequence. The library was arrayed onto two high-density colony filters, which were screened with heterologous DNA probes from rice chromosome nine and from syntenous regions of wheat, barley, maize and oat. The construction of Brachypodium BAC contigs revealed that synteny between rice, wheat and Brachypodium was largely maintained over several regions of rice chromosome nine. This suggests that Brachypodium will be a useful tool in the elucidation of gene content in agronomically important cereal crops, complementing rice as a grass genome model.