In the first part of this study, the kinetics of decay of TNP-nucleotide superfluorescence was investigated with a view to understanding the role of nucleotides and Lys492 in later steps in the catalytic cycle of the skeletal muscle Ca²⁺ATPase. It has been found previously, and verified here, that tethering TNP-8N₃-AMP to the Ca²⁺ATPase via Lys492 retarded the Ca²⁺ initiated decay of Pᵢ-induced superfluorescence 10-fold compared with untethered nucleotide. The rapidity of the decay upon addition of EDTA suggested that the E₂ ↔ E₁ → E₁Ca₂ steps were being monitored rather than dephosphorylation per se. Tethered diand triphospho species did not accelerate the decay. While monophasic kinetics was observed with untethered TNP-AMP and TNP-8N₃-AMP, complex kinetics were observed with the di- and triphospho TNP-nucleotides. This was shown to be due to the utilization of TNP-ADP and -ATP, and the azido derivatives, as coupled substrates of the Ca²⁺ATPase in the forward direction of catalysis in the presence of Ca²⁺. The hydrolysis rates of TNP-ADP, TNP-ATP, TNP-8N₃ -ADP, and TNP-8N₃ -ATP were 10, 5, 15 and 10 nomoles/min/mg of protein, respectively, at room temperature and pH 5.5. Ca²⁺ transport was supported by all four nucleotides. This is the first time that a diphosphonucleotide has been shown to support Ca²⁺ transport. A new nonhydrolysable triphospho TNPnucleotide, TNP-AMP-PCP was synthesized and shown to interact with the Ca²⁺ATPase in a similar way, in terms of superfluorescence, as the other TNP-nucleotides. It did not show the complex kinetics on inhibition of the Pcinduced superfluorescence by Ca²⁺, but neither did it accelerate the kinetics. It was concluded that TNP-nucleotides do not accelerate the E₂ ↔ E₁ transition under these conditions, possibly because of the presence of glycerol in the medium. In the second part of the study, it was shown that addition of small amounts of chelators EGTA, EDTA, BAPTA, DTPA, HEDTA and NTA to a Ca²⁺ transport assay in which the free Ca²⁺ concentration is monitored by Fluo-3 causes the Ca²⁺ATPase to pump to apparently lower levels as seen in the [Ca²⁺] lim fluorescence. Addition of chelator retards pump function in the sense that it takes longer for 50 nmols Ca²⁺ to be accumulated. Increased thermodynamic efficiency of the pump and contaminating heavy metal ions were considered as possible mechanisms. To some extend Zn²⁺ and Cd²⁺, but not Fe²⁺ and Cu²⁺, appeared to reverse the partial inhibition. While interpretation of the results is difficult, it is suggested that heavy metal ions interact with luminal loops of the Ca²⁺ATPase and enhance Ca²⁺ release under conditions of high luminal Ca²⁺ concentrations.

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No / Catecholamines that accompany acute physiological stress are also involved in mediating the development of hypertrophy and failure. However, the cellular mechanisms involved in catecholamine-induced cardiac hypertrophy, particularly Ca2+ handling, are largely unknown. We therefore investigated the effects of cardiac hypertrophy, produced by isoprenaline, on INa/Ca and sarcoplasmic reticulum (SR) function in isolated myocytes. Methods: INa/Ca was studied in myocytes from Wistar rats, using descending (+80 to ¿110 mV) voltage ramps under steady state conditions. Myocytes were also loaded with fura-2 and either field stimulated or voltage clamped to assess [Ca2+]i and SR Ca2+ content. Results: Ca2+-dependent, steady state INa/Ca density was increased in hypertrophied myocytes (P<0.05). Ca2+ release from the SR was also increased, whereas resting [Ca2+]i and the rate of decline of [Ca2+]i to control levels were unchanged. SR Ca2+ content, estimated by using 10.0 mmol/l caffeine, was also significantly increased in hypertrophied myocytes, but only when myocytes were held and stimulated from their normal resting potential (¿80 mV) but not from ¿40 mV. However, the rate of decline of caffeine-induced Ca2+ transients or INa/Ca was not significantly different between control and hypertrophied myocytes. Ca2+-dependence of INa/Ca, examined by comparing the slope of the descending phase of the hysteresis plots of INa/Ca vs. [Ca2+]i, was also similar in the two groups of cells. Conclusion: Data show that SR Ca2+ release and SR Ca2+ content were increased in hypertrophied myocytes, despite an increase in the steady state INa/Ca density. The observation that increased SR function occurred only when myocytes were stimulated from ¿80 mV suggests that Na+ influx may play a role in altering Ca2+ homeostasis in hypertrophied cardiac muscle, possibly through increased reverse Na+/Ca2+ exchange, particularly at low stimulation frequencies.

No description available.

Several studies have shown that the loss in tension during fatigue can be virtually reversed by exposure of the muscle to agents which evoke Ca²⁺ release from the SR. The purpose of this study was to determine whether the SR Ca²⁺ release mechanism of fatigued muscle is less sensitive to caffeine than that of rested muscle. Following a fatigue bout of electrically evoked tetanic pulses, the functioning of the SR of chemically skinned muscle fibers was determined by the sensitivity of the SR to increasing concentrations of caffeine. Measurements of tension and rate of tension development were made at the maximal Ca²⁺ activated contracture(pCa4.5), the maximal caffeine(25mM) activated contracture and at the caffeine threshold for contraction. All tension and rate values were normalized per cross sectional area and expressed as percents of the maximal calcium activated values. Results of the maximal Ca²⁺ and caffeine data suggest that the both control and fatigue fibers are similar in maximal tension and Ca²⁺ loading characteristics. While no differences were found between rested or fatigued maximal Ca²⁺ or caffeine contractures, significant difference was found at the caffeine threshold (p<.05) with the fatigued muscle tending to contract at a higher caffeine concentration. This suggests that fatigued muscle is less sensitive to the caffeine stimulus for Ca²⁺ release from the SR. / Master of Science

We have synthesized a new class of photoaffinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP, -ADP and -AMP (TNP- 8N₃ATP, -ADP and -AMP), and their radiolabeled derivatives, and characterized their interaction with the sarcoplasmic reticulum Ca²⁺-ATPase. The TNP-8N₃-nucleotides were synthesized from ATP in three steps involving bromination in the 8-position of the adenine ring followed by displacement with an azido group and then trinitrophenylation of the resulting 8N₃-nucleotide with TNBS. Inclusion of the oxidizing agent, DTNB, in the final reaction was found to be necessary to prevent reduction of the azido group by the released sulfite anion and also elevated the yield of trinitrophenylation to about 80%. Purity was determined spectrophotometrically, as well as by anion exchange TLC and reversed phase HPLC. In the dark, the compounds were found to display most of the features of the parent TNP-nucleotides and interacted with the Ca²⁺-ATPase in a similar way. When activated by illumination, the probes were specifically incorporated into SR vesicles with high efficiency at alkaline pH. The site of labeling was identified as being on the A₁ tryptic fragment.

Properties of the nucleotide binding site of the Ca²⁺-ATPase of skeletal muscle sarcoplasmic reticulum have been investigated. The study centred around interaction of the high affinity ATP analog, 2'-3'-0-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, (TNP-ATP), with the Ca²⁺-ATPase. Defined fractions of the sarcoplasmic reticulum (SR), corresponding to the terminal cisternae (TC) and light SR (LSR), were isolated. The TC were shown to have distinctive morphological characteristics that differ from the LSR. The TC vesicles contained electron dense intravesicular material representative of Ca²⁺ binding proteins, and visible membranous "feet" structures, which are reported to interconnect with the transverse tubule. Functional characterisation of the isolated fractions provided evidence for the predominant localisation of Ca²⁺ release channels in TC, and concentration of Ca²⁺-ATPase molecules in LSR. These conclusions were based on the following observations: (a) decreased Ca²⁺ transport of TC versus LSR; ruthenium red, a Ca²⁺ channel blocker, enhanced Ca²⁺ transport and pumping efficiency in TC, (b) higher Ca²⁺-ATPase activity for LSR in the presence and absence of ionophore, (c) rapid Ca²⁺ efflux from TC which is inhibited by ruthenium red. Of special interest was the characterisation of the TC and LSR with respect to turnover-dependent TNP-ATP fluorescence. Fluorescence observed for TC was approximately 65% of that for LSR. This phenomenon may be attributable to either the decreased Ca²⁺ ATPase content of the TC vesicles or open Ca²⁺ release channels. Hence the TNP-ATP fluorescence characteristics appear to reflect the morphological and functional subspecialisation of the defined SR fractions.

CONTEXTE: Au cours de l'infarctus du myocarde, l'homéostasie du calcium entre leréticulum sarcoplasmique (SR), les mitochondries et le cytosol est altérée chez lescardiomyocytes (CM) et conduit à la mort cellulaire. Les canaux de fuite de calcium sontconsidérés comme des régulateurs clés de l'homéostasie calcique réticulaire. Le translocon(TLC), un composant majeur de la machine de la traduction protéique, est un important canalde fuite calcique réticulaire.METHODES: Par des moyens optiques, nous avons d'abord évalué l'organisation spatiale etla fonction du TLC dans le SR de souris adultes CM. Dans un second temps, nous avonsinterrogé si et comment la modulation pharmacologique TLC pourrait réduire les lésionsd'ischémie/reperfusion (I/R) cardiaque dans un modèle d'infarctus du myocarde de souris.RÉSULTATS: Nos données montrent que le TLC est spécifiquement localisée dans le SRlongitudinale des CM chez la souris adulte. Nous démontrons que la puromycine (activateurpharmacologique du TLC) induit une réduction partielle des réserves de calcium dans le SRlongitudinale, alors que nous n'observons aucune altération des réserves de calcium dépendantdu récepteur ryanodine dans le SR jonctionnelle. Le préconditionnement de la souris par lapuromycine, soumis à un infarctus du myocarde, diminue significativement la zone d'infarctusde près de 30,9±6,3%. Ceci est corrélé à une diminution de l'activation des protéines proapoptotiquesmitochondriales et à une augmentation d'un mécanisme de pro-survie:l'autophagie. Nous avons également démontré que le préconditionnement de la puromycinediminue la vitesse d'augmentation du calcium dans le cytosol du CM adulte pendant la duréede l'ischémie en corrélation avec la diminution de l'activation des calpains calciques.CONCLUSIONS: Dans cette étude, nous avons caractérisé le TLC comme un canal de fuitespécifiquement situé dans le compartiment longitudinale du SR dans les CM de souris adultes.Nous avons constaté que l'activation pharmacologique de la TLC avant l'infarctus dumyocarde exerce un effet de préconditionnement sur le myocarde sans altérer les réserves de calcium dépendant de la ryanodine. Dans l'ensemble, ces résultats mettent l'accent sur les connaissances actuelles sur la dualité entre le SR jonctionnelle et le SR longitudinale et ouvrent de nouvelles perspectives thérapeutiques / BACKGROUND: During myocardial infarction, alteration of calcium homeostasis between sarcoplasmic reticulum (SR), mitochondria and cytosol occurs in cardiomyocytes (CM) and leads to cell death. Calcium leak channels are thought to be key regulators of the reticular calcium homeostasis. Translocon (TLC), a major component of the translation machinery, is a major reticular calcium leak channel.METHODS: By the mean of photonics, we first assessed the spatial organization and the function of TLC in the SR of adult mouse CM. In a second time, we questioned if and how the pharmacological TLC modulation could reduce ischemia/reperfusion (I/R)-mediated heart injury in a model of mouse myocardial infarction.RESULTS: Our data show that TLC is specifically located in the longitudinal SR in adult mouse CM. We demonstrate that puromycin induces a partial reduction of calcium stores in the longitudinal SR, while we observe no alteration in the ryanodine receptor-dependent calcium stores in the junctional SR. Puromycin preconditioning of mouse subjected to myocardial infarction significantly decreases the infarct area by near 30.9±6.3%. This is correlated with a decrease in the activation of mitochondrial pro-apoptotic proteins and an increase of a pro-survival mechanism: autophagy. We further demonstrated that puromycin preconditioning decreases the rate of calcium increase in the cytosol of adult CM during the ischemia duration in correlation with the decreased activation of calcium-dependent calpains.CONCLUSIONS: In this study, we characterized TLC as a leak channel specifically located in the longitudinal SR compartment of adult mouse CM. We found that the pharmacological activation of TLC before myocardial infarction exerts a preconditioning effect on myocardium without altering the ryanodine-dependent calcium stores. Altogether, these findings emphasize the present knowledge on the duality between junctional and longitudinal SR in CM and open up new therapeutic perspectives

Au cours du couplage excitation-contraction (CEC) cardiaque, une partie du calcium (Ca2+) libéré par le réticulum sarcoplasmique (RS) via les récepteurs de la ryanodine de type (RyR2) est captée par la mitochondrie. Une fois dans la matrice mitochondriale, le Ca2+ module l’activité de plusieurs enzymes du métabolisme oxydatif ainsi que la production d’ATP. Une altération de la libération de Ca2+ du RS et une dysfonction mitochondriale ont été décrites dans plusieurs pathologies cardiovasculaires associées à des troubles métaboliques comme la cardiomyopathie diabétique (CMD). Cependant, les mécanismes qui sous-tendent le remodelage du métabolisme et de l’homéostasie calcique au stade précoce de la CMD demeurent méconnus. Afin de déterminer dans quelle mesure la dynamique calcique et le couplage excitation métabolisme oxydatif sont altérés au stade précoce de la CMD, des souris C57Bl/6 ont été soumises à un régime enrichi en graisses et en sucres (HFS) pendant deux semaines. Au terme du régime, les souris HFS présentent un hyperinsulinisme, une euglycémie et une dyslipidémie. En plus de ce statut pré-diabétique, le flux mitral (E/A) évalué par échocardiographie, est significativement diminué chez les animaux HFS par rapport aux animaux Ctrl, suggérant une dysfonction diastolique précoce du ventricule gauche. A l’échelle du cardiomyocyte, une diminution du raccourcissement cellulaire sans modification de l’amplitude des transitoires calciques induits par stimulation électrique est responsable d’une diminution de l’efficacité du CEC en condition HFS. De plus, l’entrée dynamique de Ca2+ dans la mitochondrie, évaluée à l’aide de la technique de patch-clamp en configuration cellule entière, est également significativement diminuée. Bien que l’expression du canal anionique dépendant du voltage (VDAC) et de l’unipore calcique (MCU) demeure inchangée, l’expression de la protéine MICU1 augmente. Cette altération de l’entrée de Ca2+ dans la mitochondrie est responsable d’une diminution de l’activité de la pyruvate déshydrogénase via sa phosphorylation sur le résidu ser232, réduisant ainsi l’utilisation des glucides comme substrats énergétiques. Enfin, bien que les paramètres de base de l’ECG soient comparables entre les deux groupes, les souris HFS déclenchent spontanément des troubles du rythme et présentent une altération de la modulation de la fonction cardiaque par le système nerveux autonome.Ces travaux suggèrent que MICU1, en modulant l’homéostatisie calcique, le couplage excitation-métabolisme oxydatif et la flexibilité métabolique, pourrait jouer un rôle de senseur métabolique au sein des cardiomyocytes initiant ainsi la cardiomyopathie diabétique. / During cardiac excitation-contraction coupling (ECC) a fraction of the calcium (Ca2+) released by the sarcoplasmic reticulum (SR) through type 2 Ryanodine Receptor (RyR2) is taken up by mitochondria. Once in the matrix, Ca2+ modulates several enzymes of oxidative metabolism and ATP production. SR Ca2+ release alteration and mitochondrial dysfunction have been reported in several cardiovascular diseases associated with metabolic disorders such as diabetic cardiomyopathy (DCM). However, the mechanisms that govern the metabolic remodeling and Ca2+ handling at the early stage of DCM are currently unknown. To determine whether mitochondrial Ca2+ dynamics and excitation oxidative metabolism coupling are affected at the early stage of DCM, C57Bl/6 mice were fed a standard or high fat sucrose (HFS) diet for two weeks. At the end of the diet, HFS mice display hyperinsulinemia, euglycemia and dyslipidemia. In addition to this prediabetic state, the transmitral inflow (E/A), assessed by echocardiography, is significantly reduced in HFS mice compared to their control (Ctrl) littermates suggesting an early left ventricle diastolic dysfunction. At the single cardiomyocyte level, a decrease in peak cell shortening without any change regarding the amplitude of electrically evoked Ca2+ transients leads to reduced ECC efficiency under HFS conditions. Moreover, dynamic mitochondrial Ca2+ uptake measured using the whole cell patch-clamp technique is significantly decreased. While the expression of voltage-dependent anionic channel (VDAC) and mitochondrial uniporter (MCU) is unchanged, expression of mitochondrial Ca2+ uptake protein 1 (MICU1) increases. Impaired mitochondrial Ca2+ uptake leads to reduced pyruvate dehydrogenase activity through the phosphorylation of ser232, thus decreasing the use of carbohydrates as energetic substrate. Finally, although ECG basal parameters are comparable between the two groups, HFS mice trigger spontaneous arrhythmia and impaired autonomic modulation of cardiovascular function.This study suggests that MICU1 could act as a metabolic sensor by modulating mitochondrial Ca2+ handling, excitation–oxidative metabolic coupling and the metabolic flexibility paving the way to the DCM.

Heart failure represents an important cause of death in Western Countries. The pathophysiology of heart failure is mainly associated with abnormalities in intracellular calcium control. We previously showed that Egr-1 negatively regulates expression of sodium-calcium exchanger (NCX) in vivo and in vitro. Here we tested the hypothesis that Egr-1 regulates expression of calcium storage proteins in the sarco-endoplasmic reticulum (SER), calsequestrin (CSQ) and/or ER, calreticulin (CRT) directly or indirectly via Egr-1:NFAT (nuclear factor of activated T-cells) formation. Secondarily, we hypothesized that this will reduce calcium mobilization. We found that undifferentiated 1293F cells, overexpressing Egr-1, have reduced CSQ compared to control H9c2 cells. We demonstrated that Egr-1 negatively regulates CSQ but not CRT expression. The Egr-1 mediated decrease in CSQ is linked to decreased calcium availability. Repression is by a novel NAB-independent (NGFI-A binding protein) activity localized to a.a. region 1-307. We conclude that Egr-1-mediated reductions in calcium storage protein expression alter calcium availability for cardiac contraction/relaxation.