Treatment with the essential amino acid L-tryptophan reduces masticatory impairments in experimental cerebral palsy.

Purpose Children with cerebral palsy (CP) often exhibit difficulties in feeding resulting from deficits in chewing. This study investigates the therapeutic potential of L-tryptophan (TRI) to reduce deficits in chewing in rats subjected to an experimental model of CP.Methods A total of 80 Wistar albino rats were used. Pups were randomly assigned to 4 experimental groups: Control Saline, Control TRI, CP Saline, and CP TRI groups. The experimental model of CP was based on the combination of perinatal anoxia associated with postnatal sensorimotor restriction of the hind limbs. TRI was administered subcutaneously during the lactation period. Anatomical and behavioral parameters were evaluated during maturation, including body weight gain, food intake, chewing movements, relative weight and the distribution of the types of masseter muscle fibers.Results The induction of CP limited body weight gain, decreased food intake and led to impairment in the morphological and functional parameters of chewing. Moreover, for a comparable amount of food ingested, CP TRI animals grew the most. In addition, supplementation with TRI improved the number of chewing movements, and increased the weight and proportion of type IIB fibers of the masseter in rats subjected to CP.Conclusion These results demonstrate that experimental CP impaired the development of mastication and that TRI supplementation increased masticatory maturation in animals subjected to CP.

Phosphatidylinositol 3-kinase inhibition restores Ca2+ release defects and prolongs survival in myotubularin-deficient mice.

Mutations in the gene encoding the phosphoinositide 3-phosphatase myotubularin (MTM1) are responsible for a pediatric disease of skeletal muscle named myotubular myopathy (XLMTM). Muscle fibers from MTM1-deficient mice present defects in excitation-contraction (EC) coupling likely responsible for the disease-associated fatal muscle weakness. However, the mechanism leading to EC coupling failure remains unclear. During normal skeletal muscle EC coupling, transverse (t) tubule depolarization triggers sarcoplasmic reticulum (SR) Ca2+ release through ryanodine receptor channels gated by conformational coupling with the t-tubule voltage-sensing dihydropyridine receptors. We report that MTM1 deficiency is associated with a 60% depression of global SR Ca2+ release over the full range of voltage sensitivity of EC coupling. SR Ca2+ release in the diseased fibers is also slower than in normal fibers, or delayed following voltage activation, consistent with the contribution of Ca2+-gated ryanodine receptors to EC coupling. In addition, we found that SR Ca2+ release is spatially heterogeneous within myotubularin-deficient muscle fibers, with focally defective areas recapitulating the global alterations. Importantly, we found that pharmacological inhibition of phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity rescues the Ca2+ release defects in isolated muscle fibers and increases the lifespan and mobility of XLMTM mice, providing proof of concept for the use of PtdIns 3-kinase inhibitors in myotubular myopathy and suggesting that unbalanced PtdIns 3-kinase activity plays a critical role in the pathological process.

Impaired excitation-contraction coupling in muscle fibres from the dynamin2R465W mouse model of centronuclear myopathy.

KEY POINTS: Dynamin 2 is a ubiquitously expressed protein involved in membrane trafficking processes. Mutations in the gene encoding dynamin 2 are responsible for a congenital myopathy associated with centrally located nuclei in the muscle fibres. Using muscle fibres from a mouse model of the most common mutation responsible for this disease in humans, we tested whether altered Ca2+ signalling and excitation-contraction coupling contribute to muscle weakness. The plasma membrane network that carries the electrical excitation is moderately perturbed in the diseased muscle fibres. The excitation-activated Ca2+ input fluxes across both the plasma membrane and the membrane of the sarcoplasmic reticulum are defective in the diseased fibres, which probably contributes to muscle weakness in patients. ABSTRACT: Mutations in the gene encoding dynamin 2 (DNM2) are responsible for autosomal dominant centronuclear myopathy (AD-CNM). We studied the functional properties of Ca2+ signalling and excitation-contraction (EC) coupling in muscle fibres isolated from a knock-in (KI) mouse model of the disease, using confocal imaging and the voltage clamp technique. The transverse-tubule network organization appeared to be unaltered in the diseased fibres, although its density was reduced by ∼10% compared to that in control fibres. The density of Ca2+ current through CaV1.1 channels and the rate of voltage-activated sarcoplasmic reticulum Ca2+ release were reduced by ∼60% and 30%, respectively, in KI vs. control fibres. In addition, Ca2+ release in the KI fibres reached its peak value 10-50 ms later than in control ones. Activation of Ca2+ transients along the longitudinal axis of the fibres was more heterogeneous in the KI than in the control fibres, with the difference being exacerbated at intermediate membrane voltages. KI fibres exhibited spontaneous Ca2+ release events that were almost absent from control fibres. Overall, the results of the present study demonstrate that Ca2+ signalling and EC coupling exhibit a number of dysfunctions likely contributing to muscle weakness in DNM2-related AD-CNM.

Involvement of Cyclophilin D and Calcium in Isoflurane-induced Preconditioning.

BACKGROUND: The mitochondrial permeability transition pore (PTP) has been established as an important mediator of ischemia-reperfusion-induced cell death. The matrix protein cyclophilin D (CypD) is the best known regulator of PTP opening. Therefore, the authors hypothesized that isoflurane, by inhibiting the respiratory chain complex I, another regulator of PTP, might reinforce the myocardial protection afforded by CypD inhibition. METHODS: Adult mouse or isolated cardiomyocytes from wild-type or CypD knockout (CypD-KO) mice were subjected to ischemia or hypoxia followed by reperfusion or reoxygenation. Infarct size was assessed in vivo. Mitochondrial membrane potential and PTP opening were assessed using tetramethylrhodamine methyl ester perchlorate and calcein-cobalt fluorescence, respectively. Fluo-4 AM and rhod-2 AM staining allowed the measurement, by confocal microscopy, of Ca transient and Ca transfer from sarcoplasmic reticulum (SR) to mitochondria after caffeine stimulation. RESULTS: Both inhibition of CypD and isoflurane significantly reduced infarct size (-50 and -37%, respectively) and delayed PTP opening (+63% each). Their combination had no additive effect (n = 6/group). CypD-KO mice displayed endogenous protection against ischemia-reperfusion. Isoflurane depolarized the mitochondrial membrane (-28%, n = 5), decreased oxidative phosphorylation (-59%, n = 5), and blunted the caffeine-induced Ca transfer from SR to mitochondria (-22%, n = 7) in the cardiomyocytes of wild-type mice. Importantly, this transfer was spontaneously decreased in the cardiomyocytes of CypD-KO mice (-25%, n = 4 to 5). CONCLUSIONS: The results suggest that the partial inhibitory effect of isoflurane on respiratory complex I is insufficient to afford a synergy to CypD-induced protection. Isoflurane attenuates the Ca transfer from SR to mitochondria, which is also the prominent role of CypD, and finally prevents PTP opening.

Synergistic protective effect of cyclosporin A and rotenone against hypoxia-reoxygenation in cardiomyocytes.

Reperfusion of the heart after an ischemic event leads to the opening of a nonspecific pore in the inner mitochondrial membrane, the mitochondrial permeability transition pore (mPTP). Inhibition of mPTP opening is an effective strategy to prevent cardiomyocyte death. The matrix protein cyclophilin-D (CypD) is the best-known regulator of mPTP opening. In this study we confirmed that preconditioning and postconditioning with CypD inhibitor cyclosporin-A (CsA) reduced cell death after hypoxia-reoxygenation (H/R) in wild-type (WT) cardiomyocytes and HL-1 mouse cardiac cell line as measured by nuclear staining with propidium iodide. The complex I inhibitor rotenone (Rot), alone, had no effect on HL-1 and WT cardiomyocyte death after H/R, but enhanced the native protection of CypD-knocked-out (CypD KO) cardiomyocytes. Reduction of cell death was associated with a delay of mPTP opening challenged by H/R and observed by the calcein loading CoCl(2)-quenching technique. Simultaneous inhibition of complex I and CypD increased in a synergistic manner the calcium retention capacity in permeabilized cardiomyocytes and cardiac mitochondria. These results demonstrated that protection by complex I inhibition was CypD dependent.

Ciliary beating recovery in deficient human airway epithelial cells after lentivirus ex vivo gene therapy.

Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia. Our goal was to restore normal ciliary beating in DNAI1-deficient human airway epithelial cells. A lentiviral vector based on Simian Immunodeficiency Virus pseudotyped with Vesicular Stomatitis Virus Glycoprotein was used to transduce cultured human airway epithelial cells with a cDNA of DNAI1 driven by the Elongation Factor 1 promoter. Transcription and translation of the transduced gene were tested by RT-PCR and western blot, respectively. Human airway epithelial cells that were DNAI1-deficient due to compound heterozygous mutations, and consequently had immotile cilia and no outer dynein arm, were transduced by the lentivirus. Cilia beating was recorded and electron microscopy of the cilia was performed. Transcription and translation of the transduced DNAI1 gene were detected in human cells treated with the lentivirus. In addition, immotile cilia recovered a normal beat and outer dynein arms reappeared. We demonstrated that it is possible to obtain a normalization of ciliary beat frequency of deficient human airway epithelial cells by using a lentivirus to transduce cells with the therapeutic gene. This preliminary step constitutes a conceptual proof that is indispensable in the perspective of Primary Ciliary Dyskinesia's in vivo gene therapy. This is the first time that recovery of cilia beating is demonstrated in this disease.

Detection of Ca2+ transients near ryanodine receptors by targeting fluorescent Ca2+ sensors to the triad.

In intact muscle fibers, functional properties of ryanodine receptor (RYR)-mediated sarcoplasmic reticulum (SR) Ca2+ release triggered by activation of the voltage sensor CaV1.1 have so far essentially been addressed with diffusible Ca2+-sensitive dyes. Here, we used a domain (T306) of the protein triadin to target the Ca2+-sensitive probe GCaMP6f to the junctional SR membrane, in the immediate vicinity of RYR channels, within the triad region. Fluorescence of untargeted GCaMP6f was distributed throughout the muscle fibers and experienced large Ca2+-dependent changes, with obvious kinetic delays, upon application of voltage-clamp depolarizing pulses. Conversely, T306-GCaMP6f localized to the triad and generated Ca2+-dependent fluorescence transients of lower amplitude and faster kinetics for low and intermediate levels of Ca2+ release than those of untargeted GCaMP6f. By contrast, model simulation of the spatial gradients of Ca2+ following Ca2+ release predicted limited kinetic differences under the assumptions that the two probes were present at the same concentration and suffered from identical kinetic limitations. At the spatial level, T306-GCaMP6f transients within distinct regions of a same fiber yielded a uniform time course, even at low levels of Ca2+ release activation. Similar observations were made using GCaMP6f fused to the γ1 auxiliary subunit of CaV1.1. Despite the probe's limitations, our results point out the remarkable synchronicity of voltage-dependent Ca2+ release activation and termination among individual triads and highlight the potential of the approach to visualize activation or closure of single groups of RYR channels. We anticipate targeting of improved Ca2+ sensors to the triad will provide illuminating insights into physiological normal RYR function and its dysfunction under stress or pathological conditions.

Effects of maternal protein restriction on central and peripheral renin-angiotensin systems in male rat offspring.

AIMS: We investigated the involvement of the renin angiotensin system (RAS) on the cardiorespiratory control in rats from dams fed with a low-protein diet. MAIN METHODS: Male offspring were obtained from dams fed a normoprotein diet (NP, 17% casein) and low-protein diet (LP, 8% casein) during pregnancy and lactation. Direct measurements of arterial pressure (AP), heart rate (HR) and respiratory frequency (RF) were recorded in awake 90-day-old at resting and after losartan potassium through either intracerebroventricular (ICV) microinjections or intravenous (IV) administration. Cardiovascular variability was evaluated by spectral analysis. Peripheral chemoreflex sensitivity was assessed through the potassium cyanide (KCN; 40 µg/0.1 ml/rat, IV). Gene expression was evaluated by qPCR, and MAPK (Mitogen Activated Protein Kinase) expression was evaluated by western blot. KEY FINDINGS: The LP offspring had higher mean AP (MAP) and RF than NP offspring. In the spectral analysis, the LP rats also showed higher low frequency of systolic AP (NP: 2.7 ± 0.3 vs. LP: 5.0 ± 1.0 mmHg). After ICV losartan, MAP and RF in LP rats remained higher than those in NP rats, but without changes in HR. The peripheral chemoreflex was similar between the groups. LP group had lower gene expression of Rac1 (Ras-related C3 botulinum toxin substrate 1) (NP: 1.13 ± 0.06 vs. LP: 0.88 ± 0.08). Peripherally, LP rats had larger delta of MAP after IV losartan (NP: -9.8 ± 2 vs. LP: -23 ± 6 mmHg), without changes in HR and RF. SIGNIFICANCE: In rats, the RAS participates peripherally, but not centrally, in the maintenance of arterial hypertension in male offspring induced by maternal protein restriction.

Mitochondrial dynamics in heart cells: very low amplitude high frequency fluctuations in adult cardiomyocytes and flow motion in non beating Hl-1 cells.

The arrangement and movement of mitochondria were quantitatively studied in adult rat cardiomyocytes and in cultured continuously dividing non beating (NB) HL-1 cells with differentiated cardiac phenotype. Mitochondria were stained with MitoTracker Green and studied by fluorescent confocal microscopy. High speed scanning (one image every 400 ms) revealed very rapid fluctuation of positions of fluorescence centers of mitochondria in adult cardiomyocytes. These fluctuations followed the pattern of random walk movement within the limits of the internal space of mitochondria, probably due to transitions between condensed and orthodox configurational states of matrix and inner membrane. Mitochondrial fusion or fission was seen only in NB HL-1 cells but not in adult cardiomyocytes. In NB HL-1 cells, mitochondria were arranged as a dense tubular network, in permanent fusion, fission and high velocity displacements of approximately 90 nm/s. The differences observed in mitochondrial dynamics are related to specific structural organization and mitochondria-cytoskeleton interactions in these cells.

Twice-a-day training improves mitochondrial efficiency, but not mitochondrial biogenesis, compared with once-daily training.

Exercise training performed with lowered muscle glycogen stores can amplify adaptations related to oxidative metabolism, but it is not known if this is affected by the "train-low" strategy used (i.e., once-daily versus twice-a-day training). Fifteen healthy men performed 3 wk of an endurance exercise (100-min) followed by a high-intensity interval exercise 2 (twice-a-day group, n = 8) or 14 h (once-daily group, n = 7) later; therefore, the second training session always started with low muscle glycogen in both groups. Mitochondrial efficiency (state 4 respiration) was improved only for the twice-a-day group (group × training interaction, P < 0.05). However, muscle citrate synthase activity, mitochondria, and lipid area in intermyofibrillar and subsarcolemmal regions, and PGC1α, PPARα, and electron transport chain relative protein abundance were not altered with training in either group (P > 0.05). Markers of aerobic fitness (e.g., peak oxygen uptake) were increased, and plasma lactate, O2 cost, and rating of perceived exertion during a 100-min exercise task were reduced in both groups, although the reduction in rating of perceived exertion was larger in the twice-a-day group (group × time × training interaction, P < 0.05). These findings suggest similar training adaptations with both training low approaches; however, improvements in mitochondrial efficiency and perceived effort seem to be more pronounced with twice-a-day training.NEW & NOTEWORTHY We assessed, for the first time, the differences between two "train-low" strategies (once-daily and twice-a-day) in terms of training-induced molecular, functional, and morphological adaptations. We found that both strategies had similar molecular and morphological adaptations; however, only the twice-a-day strategy increased mitochondrial efficiency and had a superior reduction in the rating of perceived exertion during a constant-load exercise compared with once-daily training. Our findings provide novel insights into skeletal muscle adaptations using the "train-low" strategy.

Carotid body removal normalizes arterial blood pressure and respiratory frequency in offspring of protein-restricted mothers.

The aim of this study is to evaluate the short-term and long-term effects elicited by carotid body removal (CBR) on ventilatory function and the development of hypertension in the offspring of malnourished rats. Wistar rats were fed a normo-protein (NP, 17% casein) or low-protein (LP, 8% casein) diet during pregnancy and lactation. At 29 days of age, the animals were submitted to CBR or a sham surgery, according to the following groups: NP-cbr, LP-cbr, NP-sham, or LP-sham. In the short-term, at 30 days of age, the respiratory frequency (RF) and immunoreactivity for Fos on the retrotrapezoid nucleus (RTN; brainstem site containing CO2 sensitive neurons) after exposure to CO2 were evaluated. In the long term, at 90 days of age, arterial pressure (AP), heart rate (HR), and cardiovascular variability were evaluated. In the short term, an increase in the baseline RF (~6%), response to CO2 (~8%), and Fos in the RTN (~27%) occurred in the LP-sham group compared with the NP-sham group. Interestingly, the CBR in the LP group normalized the RF in response to CO2 as well as RTN cell activation. In the long term, CBR reduced the mean AP by ~20 mmHg in malnourished rats. The normalization of the arterial pressure was associated with a decrease in the low-frequency (LF) oscillatory component of AP (~58%) and in the sympathetic tonus to the cardiovascular system (~29%). In conclusion, carotid body inputs in malnourished offspring may be responsible for the following: (i) enhanced respiratory frequency and CO2 chemosensitivity in early life and (ii) the production of autonomic imbalance and the development of hypertension.

Differential effects of maternal high-fat/high-caloric or isocaloric diet on offspring's skeletal muscle phenotype.

Aim This study sought to investigate the effects of two different maternal high-fat diets, during gestation and lactation, on the morphology of the skeletal muscle of the adult offspring rats. METHODS: Female Wistar rats were fed Control (C) or High-fat/high-caloric (HH) or High-fat/isocaloric (HI) diet during gestation and lactation. The somatic growth of the offspring was measured throughout lactation. Glucose and insulin tolerance tests were performed at PND61 and PND65, respectively. At PND70, soleus and extensor digitorum longus (EDL) muscles were removed for myofibrillar ATPase staining analysis. KEY FINDINGS: HH pups were heavier and longer at weaning but presented same body weight at PND70. No difference among groups in glucose or insulin tolerance tests was observed. In the soleus muscle, HH offspring showed increased proportion and size of type 1 fibres and reduced proportion and number with increased size of type 2A fibres. In EDL muscle, there was no difference in proportion and number of fibres. HH and HI animals presented reduced type 1 and 2A fibres size while HH animals presented increased type 2B fibres size, in EDL muscle. SIGNIFICANCE: Maternal HH diet promoted a more oxidative profile in soleus muscle. Though, maternal high-fat/isocaloric diet influenced only fibres size. Glycolytic muscle is more resistant to maternal diet influence. These results emphasize the importance of maternal diet during the critical period of development on muscle morphology of the offspring.

Different kinetics of the regulation of respiration in permeabilized cardiomyocytes and in HL-1 cardiac cells. Importance of cell structure/organization for respiration regulation.

The aim of this study was to investigate the mechanism of cellular regulation of mitochondrial respiration in permeabilized cardiac cells with clearly different structural organization: (i) in isolated rat cardiomyocytes with very regular mitochondrial arrangement, (ii) in HL-1 cells from mouse heart, and (iii) in non-beating (NB HL-1 cells) without sarcomeres with irregular and dynamic filamentous mitochondrial network. We found striking differences in the kinetics of respiration regulation by exogenous ADP between these cells: the apparent Km for exogenous ADP was by more than order of magnitude (14 times) lower in the permeabilized non-beating NB HL-1 cells without sarcomeres (25+/-4 microM) and seven times lower in normally cultured HL-1 cells (47+/-15 microM) than in permeabilized primary cardiomyocytes (360+/-51 microM). In the latter cells, treatment with trypsin resulted in dramatic changes in intracellular structure that were associated with 3-fold decrease in apparent Km for ADP in regulation of respiration. In contrast to permeabilized cardiomyocytes, in NB HL-1 cells creatine kinase activity was low and the endogenous ADP fluxes from MgATPases recorded spectrophotometrically by the coupled enzyme assay were not reduced after activation of mitochondrial oxidative phosphorylation by the addition of mitochondrial substrates, showing the absence of ADP channelling in the NB HL-1 cells. While in the permeabilized cardiomyocytes creatine strongly activated mitochondrial respiration even in the presence of powerful competing pyruvate kinase-phosphoenolpyruvate system, in the NB HL-1 cells the stimulatory effect of creatine was not significant. The results of this study show that in normal adult cardiomyocytes and HL-1 cells intracellular local restrictions of diffusion of adenine nucleotides and metabolic feedback regulation of respiration via phosphotransfer networks are different, most probably related to differences in structural organization of these cells.

Non-beating HL-1 cells for confocal microscopy: application to mitochondrial functions during cardiac preconditioning.

HL-1, the first cell line with a cardiac phenotype for biological experiments, displays spontaneous electrophysiological and mechanical regular activity, and cyclic calcium movements. We isolated a derived line, devoid of transient movements, for confocal microscopy experiments. These cells do express cardiac proteins: connexin 43, the cardiac isoform of dihydropyridine receptors, desmin, and developmental myosin but have no sarcomeric arrangement. They still possess the electrophysiological characteristics and ionic currents of cardiac cells, among them the cardiac potassium current IKr. We also found diazoxide and glibenclamide sensitive potassium channels with properties similar to IK(ATP) in adult cardiac myocytes. The pacemaker current I(f) was not observed, in agreement with the cells showing excitability but lacking in pacemaker activity. The absence of movement is an advantage for studies which include changes of media in order to follow morphological changes under continuous perfusion. We observed however a basal spontaneous movement of mitochondria and we developed a method to quantify its amplitude using confocal microscopy. No mitochondrial depolarization could be detected when the membrane potential was measured by using very low light photomultiplier and confocal fluorescence imaging under the K(ATP) channel opener diazoxide. Thus cardiac pharmacological preconditioning by K(ATP) channel openers might involve other routes than mitochondrial K channels targeting.

Confocal laser scanning microscopy using dialkylcarbocyanine dyes for cell tracing in hard and soft biomaterials.

The aim of this work was to study, in vitro, cell colonization of two biomaterials currently used for bone and cartilage repair, this step being important to understand the function of engineered tissues. Current methods that use histological approaches are not always suited to tissue-engineering analysis. We, therefore, set up a protocol to assess cell distribution, utilizing noninvasive confocal microscopy and fluorescent labels with a far red emission wavelength to optimize scaffold transparency and minimize light scattering. Hard (ceramic substitute) and soft (collagen sponge) biomaterials were seeded respectively, on one side of the scaffold, with human fibroblasts and bovine chondrocytes labelled with carbocyanine dyes (DiD and DiR). The mean penetration depth for DiR labelled fibroblasts and chondrocytes in the two scaffolds, around 270 m, was greater than for DiD (136-218 microm) labelled cells. These depths were independent of cell origin but were influenced by the nature of the scaffolds. Collagen sponge is transparent in contrast to ceramic substitutes where measurements could only be made in opened macropores. Besides the limits of the equipment, the limits of the supports were diffusion for collagen sponges and transmission for ceramic substitutes. Confocal microscopy techniques could thus be used to address the question of cell colonization of porous biomaterials in a noninvasive manner.

Structure-function relationships in the regulation of energy transfer between mitochondria and ATPases in cardiac cells.

The present study discusses the role of structural organization of cardiac cells in determining the mechanisms of regulation of oxidative phosphorylation and interaction between mitochondria and ATPases. In permeabilized adult cardiomyocytes, the apparent K(m) (Michaelis-Menten constant) for ADP in the regulation of respiration is far higher than in mitochondria isolated from the myocardium. Respiration of mitochondria in permeabilized cardiomyocytes is effectively activated by endogenous ADP produced by ATPases from exogenous ATP, and the activation of respiration is associated with a decrease in the apparent K(m) for ATP in the regulation of ATPase activity compared with this parameter in the absence of oxidative phosphorylation. It has also been shown that a large fraction of the endogenous ADP stimulating respiration remains inaccessible for the exogenous ADP trapping system, consisting of pyruvate kinase and phosphoenolpyruvate, unless the mitochondrial structures are modified by controlled proteolysis. These data point to the endogenous cycling of adenine nucleotides between mitochondria and ATPases. Accordingly, the current hypothesis is that in cardiac cells, mitochondria and ATPases are compartmentalized into functional complexes (ie, intracellular energetic units [ICEUs]), which appear to represent a basic pattern of organization of energy metabolism in these cells. Within the ICEUs, the mitochondria and ATPases interact via different routes: creatine kinase-mediated phosphoryltransfer; adenylate kinase-mediated phosphoryltransfer; and direct ATP and ADP channelling. The function of ICEUs changes not only after selective proteolysis, but also during contraction of cardiomyocytes caused by an increase in cytosolic Ca(2+) concentration up to micromolar levels. In these conditions, the apparent K(m) for exogenous ADP and ATP in the regulation of respiration markedly decreases, and more ADP becomes available for the exogenous pyruvate kinase-phosphoenolpyruvate system, which indicates altered barrier functions of the ICEUs. Thus, structural changes transmitted from the contractile apparatus to mitochondria clearly participate in the regulation of mitochondrial function due to alterations in localized restriction of the diffusion of adenine nucleotides. The importance of strict structural organization in cardiac cells emerged drastically from experiments in which the regulation of mitochondrial respiration was assessed in a novel cardiac cell line, that is, beating and nonbeating HL-1 cells. In these cells, the mitochondrial arrangement is irregular and dynamic, whereas the sarcomeric structures are either absent (in nonbeating HL-1 cells) or only rarely present (in beating HL-1 cells). In parallel, the apparent K(m) for exogenous ADP in the regulation of respiration was much lower than that in permeabilized primary cardiomyocytes, and trypsin treatment exerted no impact on the low K(m) value for ADP, in contrast to adult cardiomyocytes where it caused a marked decrease in this parameter. The HL-1 cells were also characterized by the absence of direct exchange of adenine nucleotides. The results further support the concept that the ICEUs in adult cardiomyocytes are products of complex structural organization developed to create the most optimal conditions for effective energy transfer and feedback between mitochondria and ATPases.

High-resolution tomographic diffractive microscopy of biological samples.

The authors have developed a tomographic diffractive microscope that combines microholography with illumination from an angular synthetic aperture. It images specimens relative to their complex index of refraction distribution (index and absorption) and permits imaging of unlabelled specimens, with high lateral resolution. The authors now study its use for biological applications, and imaged several preparations with fluorescence confocal microscopy and tomographic diffractive microscopy. The results highlight some interesting features of this instrument, which should attract the interest of biologists for this new technique.

Iron nanoparticles increase 7-ketocholesterol-induced cell death, inflammation, and oxidation on murine cardiac HL1-NB cells.

OBJECTIVE: To evaluate the cytotoxicity of iron nanoparticles on cardiac cells and to determine whether they can modulate the biological activity of 7-ketocholesterol (7KC) involved in the development of cardiovascular diseases. Nanoparticles of iron labeled with Texas Red are introduced in cultures of nonbeating mouse cardiac cells (HL1-NB) with or without 7-ketocholesterol 7KC, and their ability to induce cell death, pro-inflammatory and oxidative effects are analyzed simultaneously. STUDY DESIGN: Flow cytometry (FCM), confocal laser scanning microscopy (CLSM), and subsequent factor analysis image processing (FAMIS) are used to characterize the action of iron nanoparticles and to define their cytotoxicity which is evaluated by enhanced permeability to SYTOX Green, and release of lactate deshydrogenase (LDH). Pro-inflammatory effects are estimated by ELISA in order to quantify IL-8 and MCP-1 secretions. Pro-oxidative effects are measured with hydroethydine (HE). RESULTS: Iron Texas Red nanoparticles accumulate at the cytoplasmic membrane level. They induce a slight LDH release, and have no inflammatory or oxidative effects. However, they enhance the cytotoxic, pro-inflammatory and oxidative effects of 7KC. The accumulation dynamics of SYTOX Green in cells is measured by CLSM to characterize the toxicity of nanoparticles. The emission spectra of SYTOX Green and nanoparticles are differentiated, and corresponding factor images specify the possible capture and cellular localization of nanoparticles in cells. CONCLUSION: The designed protocol makes it possible to show how Iron Texas Red nanoparticles are captured by cardiomyocytes. Interestingly, whereas these fluorescent iron nanoparticles have no cytotoxic, pro-inflammatory or oxidative activities, they enhance the side effects of 7KC.

Analysis of the capture and influence of nanoparticles on the cytotoxic effects of 7-ketocholesterol on cardiac cells: investigation by flow cytometry and by dynamic and spectral imaging microscopy associated with factor analysis of medical image sequences.

OBJECTIVE: To evaluate the capture of nanoparticles (quantum dots [QDs], fluorospheres) by nonbeating mouse cardiac cells (HL1-NB) cultured without or with 7-ketocholesterol (7KC) found at an increased level in the plasma of atherosclerotic patients and to simultaneously analyze their cytotoxic, proinflammatory and oxidative properties. STUDY DESIGN: Flow cytometry (FCM), confocal laser scanning microscopy and subsequent factor analysis image processing were used to characterize the uptake of nanoparticles and to define their cytotoxicity, evaluated by enhanced permeability to SYTOX Green, release of lactate dehydrogenase (LDH) and morphologic nuclear changes determined with Hoechst 33342. Proinflammatory effects were estimated by enzyme linked immunoassay to quantify IL-8 and MCP-1 secretion. The overproduction of reactive oxygen species (ROS) was determined by FCM with hydroethidine. RESULTS: Whereas the nanoparticles had no cytotoxic or inflammatory effects, they could stimulate ROS production. QDs were not incorporated. When 7KC was used, LDH release was enhanced and QDs potentialized IL-8 secretion. The incorporation and exit dynamics of nanoparticles were visualized to differentiate the emission spectra of SYTOX Green and nanoparticles and to precisely determine the cellular localization of nanoparticles. CONCLUSION: The selected nanoparticles, which accumulate at the inner or outer cytoplasmic membrane level, can induce biologic activities and are able to interfere with those of chemically defined molecules such as 7KC.

Telomere uncapping during in vitro T-lymphocyte senescence.

Normal lymphocytes represent examples of somatic cells that are able to induce telomerase activity when stimulated. As previously reported, we showed that, during lymphocyte long-term culture and repeated stimulations, the appearance of senescent cells is associated with telomere shortening and a progressive drop in telomerase activity. We further showed that this shortening preferentially occured at long telomeres and was interrupted at each stimulation by a transitory increase in telomere length. In agreement with the fact that telomere uncapping triggers lymphocyte senescence, we observed an increase in gamma-H2AX and 53BP1 foci as well as in the percentage of cells exhibiting DNA damage foci in telomeres. Such a DNA damage response may be related to the continuous increase of p16(ink4a) upon cell stimulation and cell aging. Remarkably, at each stimulation, the expression of shelterin genes, such as hTRF1, hTANK1, hTIN2, hPOT1 and hRAP1, was decreased. We propose that telomere dysfunction during lymphocyte senescence caused by iterative stimulations does not only result from an excessive telomere shortening, but also from a decrease in shelterin content. These observations may be relevant for T-cell biology and aging.