Resting-state functional connectivity of emotion regulation networks in euthymic and non-euthymic bipolar disorder patients.

BACKGROUND: Previous functional magnetic resonance imaging studies in bipolar disorder (BD) have evidenced changes in functional connectivity (FC) in brain areas associated with emotion processing, but how these changes vary with mood state and specific clinical symptoms is not fully understood. METHODS: We investigated resting-state FC between a priori regions of interest (ROIs) from the default-mode network and key structures for emotion processing and regulation in 27 BD patients and 27 matched healthy controls. We further compared connectivity patterns in subgroups of 15 euthymic and 12 non-euthymic patients and tested for correlations of the connectivity strength with measures of mood, anxiety, and rumination tendency. No correction for multiple comparisons was applied given the small population sample and pre-defined target ROIs. RESULTS: Overall, regardless of mood state, BD patients exhibited increased FC of the left amygdala with left sgACC and PCC, relative to controls. In addition, non-euthymic BD patients showed distinctive decrease in FC between right amygdala and sgACC, whereas euthymic patients showed lower FC between PCC and sgACC. Euthymic patients also displayed increased FC between sgACC and right VLPFC. The sgACC-PCC and sgACC-left amygdala connections were modulated by rumination tendency in non-euthymic patients, whereas the sgACC-VLPFC connection was modulated by both the current mood and tendency to ruminate. CONCLUSION: Our results suggest that sgACC-amygdala coupling is critically affected during mood episodes, and that FC of sgACC play a pivotal role in mood normalization through its interactions with the VLPFC and PCC. However, these preliminary findings require replication with larger samples of patients.

Differentiation markers of mouse C2C12 and rat L6 myogenic cell lines and the effect of the differentiation medium.

The differentiation grade of cells in culture is dependent on the composition of the culture medium. Two commonly used myogenic cell lines, mouse C2C12 and rat L6, usually differentiate at a low concentration of horse serum. In this study we compared the effect of horse serum with a medium containing a low percentage of Ultroser G and rat brain extract. The maturation grade was evaluated on the basis of various biochemical, (immuno)histochemical and cell-physiological parameters. Substitution of horse serum by Ultroser G and rat brain extract during the differentiation phase resulted in a higher maturation grade of the myotubes of both cell lines, on the basis of creatine kinase activity and the diameter of the myotubes. In addition, the C2C12 myotubes display cross-striation, contain a higher percentage of creatine kinase muscle-specific isoenzyme MM, show a ninefold increase in acetylcholine receptor (AChR) clusters, form a continuous basement membrane, and have a lower resting cytosolic Ca2+ concentration. L6 myotubes show a fivefold increase in AChR clusters and a twofold increase in the expression of the mRNA of the epsilon-subunit of AChR.C2C12 cells show spontaneous contraction and response of cytosolic Ca2+ to various stimulants in contrast to L6 cells which do not. These studies established that the Ultroser G/brain extract medium leads to a higher differentiation grade of both cell lines, but parameters appropriate for use as differentiation markers appear to differ between both cell lines.

Cytoarchitectural and metabolic adaptations in muscles with mitochondrial and cytosolic creatine kinase deficiencies.

We have blocked creatine kinase (CK) mediated phosphocreatine (PCr) <==> ATP transphosphorylation in mitochondria and cytosol of skeletal muscle by knocking out the genes for the mitochondrial (ScCKmit) and the cytosolic (M-CK) CK isoforms in mice. Animals which carry single or double mutations, if kept and tested under standard laboratory conditions, have surprisingly mild changes in muscle physiology. Strenuous ex vivo conditions were necessary to reveal that MM-CK absence in single and double mutants leads to a partial loss of tetanic force output. Single ScCKmit deficiency has no noticeable effects but in combination the mutations cause slowing of the relaxation rate. Importantly, our studies revealed that there is metabolic and cytoarchitectural adaptation to CK defects in energy metabolism. The effects involve mutation type-dependent alterations in the levels of AMP, IMP, glycogen and phosphomonoesters, changes in activity of metabolic enzymes like AMP-deaminase, alterations in mitochondrial volume and contractile protein (MHC isoform) profiles, and a hyperproliferation of the terminal cysternae of the SR (in tubular aggregates). This suggests that there is a compensatory resiliency of loss-of-function and redirection of flux distributions in the metabolic network for cellular energy in our mutants.

Intravenous immunoglobulin preparation increases myoplasmic calcium concentration by activating the dihydropyridine-ryanodine receptor complex.

A human intravenous immunoglobulin preparation (IVIg) released Ca2+ from the sarcoplasmic reticulum of cultured human skeletal muscle cells in a dose-dependent manner. Blocking the dihydropyridine-ryanodine receptor complex abrogated the IVIg-mediated Ca2+ response, whereas inhibition of the voltage-operated Na+-channels or acetylcholine receptors did not. This effect of IVIg was not mediated by its main component, the IgG molecules, and differed between preparations from different manufacturers. Heating destroyed the activity. Data shows that an unidentified serum protein present in IVIg can influence human muscle cells by an effect on the dihydropyridine receptor. This phenomenon may be important in interpreting the (side) effects of IVIg in neuromuscular diseases.

Myotonic dystrophy protein kinase is involved in the modulation of the Ca2+ homeostasis in skeletal muscle cells.

Myotonic dystrophy (DM), the most prevalent muscular disorder in adults, is caused by (CTG)n-repeat expansion in a gene encoding a protein kinase (DM protein kinase; DMPK) and involves changes in cytoarchitecture and ion homeostasis. To obtain clues to the normal biological role of DMPK in cellular ion homeostasis, we have compared the resting [Ca2+]i, the amplitude and shape of depolarization-induced Ca2+ transients, and the content of ATP-driven ion pumps in cultured skeletal muscle cells of wild-type and DMPK[-/-] knockout mice. In vitro-differentiated DMPK[-/-] myotubes exhibit a higher resting [Ca2+]i than do wild-type myotubes because of an altered open probability of voltage-dependent l-type Ca2+ and Na+ channels. The mutant myotubes exhibit smaller and slower Ca2+ responses upon triggering by acetylcholine or high external K+. In addition, we observed that these Ca2+ transients partially result from an influx of extracellular Ca2+ through the l-type Ca2+ channel. Neither the content nor the activity of Na+/K+ ATPase and sarcoplasmic reticulum Ca2+-ATPase are affected by DMPK absence. In conclusion, our data suggest that DMPK is involved in modulating the initial events of excitation-contraction coupling in skeletal muscle.

Altered Ca2+ responses in muscles with combined mitochondrial and cytosolic creatine kinase deficiencies.

We have blocked creatine kinase (CK)-mediated phosphocreatine (PCr) -->/<-- ATP transphosphorylation in skeletal muscle by combining targeted mutations in the genes encoding mitochondrial and cytosolic CK in mice. Contrary to expectation, the PCr level was only marginally affected, but the compound was rendered metabolically inert. Mutant muscles in vivo showed significantly impaired tetanic force output, increased relaxation times, altered mitochondrial volume and location, and conspicuous tubular aggregates of sarcoplasmic reticulum membranes, as seen in myopathies with electrolyte disturbances. In depolarized myotubes cultured in vitro, CK absence influenced both the release and sequestration of Ca2+. Our data point to a direct link between the CK-PCr system and Ca2+-flux regulation during the excitation and relaxation phases of muscle contraction.

Excitation-contraction coupling of cultured human skeletal muscle cells and the relation between basal cytosolic Ca2+ and excitability.

Cultured human skeletal muscle cells are frequently used as a model to study muscle pathology, in which Ca2+ homeostasis might be affected. However, their excitation-contraction (E-C) coupling has been poorly investigated. In order to elucidate E-C coupling of cultured muscle cells, we activated the acetylcholine receptors, voltage-dependent Na+ channels, dihydropyridine receptors or ryanodine receptors both in the presence and absence of external Ca2+, as well as after specific inhibition, and measured the effects on the cytosolic Ca2+ concentration ([Ca2+]i) using Fura-2. Furthermore, we examined the excitability of these cells during iterative high (125 mM) K+ stimulation with various repolarisation intervals. The resting [Ca2+]i in muscle cells of controls is about 130 nM. Acetylcholine, veratridine, high K+ and caffeine elicit dose-dependent Ca2+ transients, which are independent of extracellular Ca2+ and can be inhibited by alpha-bungarotoxin, tetrodotoxin, nifedipine or ryanodine. During repetitive K+ stimulation, the excitability of the muscle cells depends on the repolarisation interval between successive stimulations. Upon shortening the repolarisation time the Ca2+ transients become smaller and slower. Thereby, the basal [Ca2+]i rises, the Ca2+ response amplitude declines and both the half-increase and half-decay time increase. However, if the basal [Ca2+]i equals the resting [Ca2+]i the initial Ca2+ response can be recovered. The intracellular pH of 7.23, measured by BCECF, is unaffected by repeated K+ stimulation, whatever the repolarisation interval was. In conclusion, cultured human skeletal muscle cells possess a 'skeletal muscle type' of E-C coupling and their excitability at iterative stimulation is set by their basal [Ca2+]i.

Effects of HMG-CoA reductase inhibitors on growth and differentiation of cultured rat skeletal muscle cells.

HMG-CoA reductase inhibitors have been associated with skeletal muscle myopathy, ranging from asymptomatic elevations of serum creatine kinase (CK) activity to rhabdomyolysis. In this study, we assessed the effects of addition of different concentrations of simvastatin and pravastatin on growth and differentiation of cultured primary rat skeletal muscle cells. Protein concentrations, CK activity and percentage CK-MM, which is a parameter for maturation, were determined. Effects were generally stronger if inhibitors were added to both growth and differentiation medium rather than only to differentiation medium. Addition of 25 microM pravastatin caused only a decrease of CK activity. Addition of 1-5 microM simvastatin resulted in a decrease of protein concentration, CK activity and percentage CK-MM, whereas 25 microM simvastatin resulted in cell death. Addition of mevalonic acid or cholesterol could not prevent the effects of 1 microM simvastatin. In addition, 1 microM simvastatin did not influence the cholesterol and phospholipid content of the cells. Superfusion of cultured cells with simvastatin concentrations of 10 microM and higher caused a transient increase of the cytoplasmic calcium concentration followed by an apparent second rise and cell puncture. The results indicate that HMG-CoA reductase inhibitors may affect skeletal muscle cell regeneration in vivo by a direct toxic effect on growth and differentiation.

Ion transport in human skeletal muscle cells: disturbances in myotonic dystrophy and Brody's disease.

After excitation of skeletal muscle, the disturbed ion homeostasis is restored by Na+, K+ ATPase of the sarcolemma and Ca2+ ATPase of the sarcoplasmic reticulum (SR). Contrary to Na+, K+ ATPase, the concentration and isoenzyme distribution of SR Ca2+ ATPase in human skeletal muscle depend on fibre type and age. In cultured human muscle cells the concentration and activity of Na+, K+ ATPase and SR Ca2+ ATPase increase with maturation. In skeletal muscle and cultured muscle cells of patients suffering from myotonic dystrophy (MyD), the activity and the concentration of both Na+, K+ ATPase and SR Ca2+ ATPase are decreased by about 40%. In addition, we measured in cultured MyD muscle cells at rest an increased cytosolic Ca2+ concentration ([Ca2+]i) caused by active voltage-operated Ca2+ channels, which are inactive in resting control cells. However, the restoration of a stimulus-induced Ca2+ transient is unaffected. A differentiation-related disturbance of membranes or a modulation defect of membrane proteins may play a role in MyD. In skeletal muscle and cultured muscle cells of patients suffering from Brody's disease, which is characterized by impaired muscle relaxation, the SR Ca2+ ATPase activity is reduced by about 50%, but the concentrations of total SR Ca2+ ATPase and the predominant SERCA1 isoform are normal. Diseased muscle cells show a delayed restoration of [Ca2+]i after stimulation, which might be explained by structural modifications of SERCA1. Reduction of the Ca2+ release by drugs balances the excitation-relaxation cycle of the pathological cells.

Copper toxicity in cultured human skeletal muscle cells: the involvement of Na+/K(+)-ATPase and the Na+/Ca(2+)-exchanger.

Copper (Cu2+) intoxication has been shown to induce pathological changes in various tissues. The mechanism underlying Cu2+ toxicity is still unclear. It has been suggested that the Na+/K(+)-ATPase and/or a change of the membrane permeability may be involved. In this study we examined the effects of Cu2+ on the Na+ and Ca2+ homeostasis of cultured human skeletal muscle cells using the ion-selective fluorescent probes Na(+)-binding benzofuran isophatalate (SBFI) and Fura-2, respectively. In addition, we measured the effect of Cu2+ on the Na+/K(+)-ATPase activity. Cu2+ and ouabain increase the cytoplasmic free Na+ concentration ([Na+]i). Subsequent addition of Cu2+ after ouabain does not affect the rate of [Na+]i increase. Cu2+ inhibits the Na+/K(+)-ATPase activity with an IC50 of 51 microM. The cytoplasmic free Ca2+ concentration ([Ca2+]i) remains unaffected for more than 10 min after the administration of Cu2+. Thereafter, [Ca2+]i increases as a result of the Na+/Ca(2+)-exchanger operating in the reversed mode. The effects of Cu2+ on the Na+ homeostasis are reversed by the reducing and chelating agent dithiothreitol and the heavy metal chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN). In conclusion, SBFI is a good tool to examine Na+ homeostasis in cultured human skeletal muscle cells. Under the experimental conditions used, Cu2+ does not modify the general membrane permeability, but inhibits the Na+/K(+)-pump leading to an increase of [Na+]i. As a consequence the operation mode of the Na+/Ca(2+)-exchanger reverses and [Ca2+]i rises.

Ca2+ homeostasis in Brody's disease. A study in skeletal muscle and cultured muscle cells and the effects of dantrolene an verapamil.

Brody's disease, i.e., sarcoplasmic reticulum (SR) Ca(2+)-dependent Mg(2+)-ATPase (Ca(2+)-ATPase) deficiency, is a rare inherited disorder of skeletal muscle function. Pseudo-myotonia is the most important clinical feature. SR Ca(2+)-ATPase and Ca2+ homeostasis are examined in m. quadriceps and/or cultured muscle cells of controls and 10 patients suffering from Brody's disease. In both m. quadriceps and cultured muscle cells of patients, the SR Ca(2+)-ATPase activity is decreased by approximately 50%. However, the concentration of SR Ca(2+)-ATPase and SERCA1 are normal. SERCA1 accounts for 83 and 100% of total SR Ca(2+)-ATPase in m. quadriceps and cultured muscle cells, respectively. This implies a reduction of the molecular activity of SERCA1 in Brody's disease. The cytosolic Ca2+ concentration ([Ca2+]i) at rest and the increase of [Ca2+]i after addition of acetylcholine are the same in cultured muscle cells of controls and patients. The half-life of the maximal response, however, is raised three times in the pathological muscle cells. Addition of dantrolene or verapamil after the maximal response accelerates the restoration of the [Ca2+]i in these muscle cells. The differences in Ca2+ handling disappear by administration of dantrolene or verapamil concomitantly with acetylcholine. The reduced Ca2+ re-uptake from the cytosol presumably due to structural modification(s) of SERCA1 may explain the pseudo-myotonia in Brody's disease. Single cell measurements suggest a beneficial effect of dantrolene or verapamil in treating patients suffering from Brody's disease.

Deficiency of Na+/K(+)-ATPase and sarcoplasmic reticulum Ca(2+)-ATPase in skeletal muscle and cultured muscle cells of myotonic dystrophy patients.

Since defective regulation of ion transport could initiate or contribute to the abnormal cellular function in myotonic dystrophy (MyD), Na+/K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase were examined in skeletal muscle and cultured skeletal muscle cells of controls and MyD patients. Na+/K(+)-ATPase was investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphate (3-O-MFPase). SR Ca(2+)-ATPase was analysed by e.l.i.s.a., Ca(2+)-dependent phosphorylation and its activities with ATP and 3-O-methylfluorescein phosphatase (3-O-MFP). In MyD muscle the K(+)-dependent 3-O-MFPase activity and the activity and concentration of SR Ca(2+)-ATPase were decreased by 40%. In cultured muscle cells from MyD patients the activities as well as the concentration of both Na+/K(+)-ATPase and SR Ca(2+)-ATPase were reduced by about 30-40%. The ouabain-binding constant and the molecular activities, i.e. catalytic-centre activities with ATP or 3-O-MFP, of Na+/K(+)-ATPase and SR Ca(2+)-ATPase were similar in muscle as well as in cultured cells from both controls and MyD patients. Thus the decreased activity of both ATPases in MyD muscle is caused by a reduction in the number of their molecules. To check whether the deficiency of ATP-dependent ion pumps is a general feature of the pathology of MyD, we examined erythrocytes from the same patients. In these cells the Ca2+ uptake rate and the Ca(2+)-ATPase activity were lower than in controls, but the Ca(2+)-ATPase concentration was normal. Thus the reduced Ca(2+)-ATPase activity is caused by a decrease in the molecular activity of the ion pump. The Na+/K(+)-ATPase activity is also lower in erythrocytes of MyD patients. It is concluded that the observed alterations in ion pumps may contribute to the pathological phenomena in the muscle and other tissues in patients with MyD.

Exertional rhabdomyolysis in a patient with calcium adenosine triphosphatase deficiency.

A patient with exertional rhabdomyolysis and continuously elevated serum creatine kinase (CK) was investigated. The known causes of recurrent attacks of rhabdomyolysis were ruled out by appropriate histochemical and biochemical investigations. During ischaemic exercise tests an abnormal K(+)-efflux from exercising muscles was observed. The patient was found to have a deficiency of muscular Ca(2+)-ATPase. Dantrolene sodium therapy gave relief of muscle symptoms and improved the exercise tolerance. Both the CK level and the K(+)-efflux in ischaemic forearm testing became normal on this therapy.

Adenosine triphosphatases during maturation of cultured human skeletal muscle cells and in adult human muscle.

Na+/K(+)-ATPase, Mg(2+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase are examined in cultured human skeletal muscle cells of different maturation grade and in human skeletal muscle. Na+/K(+)-ATPase is investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase). SR Ca(2+)-ATPase is examined by ELISA, Ca(2+)-dependent phosphorylation and its activities on ATP and 3-O-methylfluorescein phosphate. Na+/K(+)-ATPase and SR Ca(2+)-ATPase are localized by immunocytochemistry. The activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase show a good correlation with the other assayed parameters of these ion pumps. All ATPase parameters investigated increase with the maturation grade of the cultured muscle cells. The number of ouabain-binding sites and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-MFPase are significantly higher in cultured muscle cells than in muscle. The Mg(2+)-ATPase activity, the content of SR Ca(2+)-ATPase and the activities of SR Ca(2+)-ATPase and Ca(2+)-dependent 3-O-MFPase remain significantly lower in cultured cells than in muscle. The ouabain-binding constant and the molecular activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase are equal in muscle and cultured cells. During ageing of human muscle the activity as well as the concentration of SR Ca(2+)-ATPase decrease. Thus the changes of the activities of the ATPases are caused by variations of the number of their molecules. Na+/K(+)-ATPase is localized in the periphery of fast- and slow-twitch muscle fibers and at the sarcomeric I-band. SR Ca(2+)-ATPase is predominantly confined to the I-band, whereas fast-twitch fibers are much more immunoreactive than slow-twitch fibers. The presence of cross-striation for Na+/K(+)-ATPase and SR Ca(2+)-ATPase in highly matured cultured muscle cells indicate the development and subcellular organization of a transverse tubular system and SR, respectively, which resembles the in vivo situation.

Ultroser G and brain extract induce a continuous basement membrane with specific synaptic elements in aneurally cultured human skeletal muscle cells.

Basement membrane (BM) components were studied on human muscle and skeletal muscle cells cultured on different media by immunofluorescence and electron microscopy. Their topographical relation with acetylcholine receptors was investigated. Myotubes cultured on a combination of the serum substitute Ultroser G and brain extract show a continuous layer of heparan sulfate proteoglycans (HSPGs), laminin, and type IV collagen. In contrast, myotubes cultured on serum-containing media are associated with granular depositions of HSPG and laminin and only with wisps of type IV collagen. Omission of brain extract or substitution by chicken embryo extract results in an intermediate staining pattern. For all types of cultures, fibronectin is localized in and around mononuclear cells, but hardly associated with myotubes. A codistribution between clusters of acetylcholine receptors and HSPG and laminin and Vicia villosa B4 lectin-positive material exists only in Ultroser G/brain extract-based myotubes like in muscle in vivo. No clustering is observed in serum-based myotubes. Electron microscopy reveals that the former myotubes are surrounded by a continuous BM consisting of a lamina lucida, lamina densa, and lamina fibroreticularis. Proteoglycans are present on the external site of the lamina densa and associated in a regular fashion with collagen fibrils. In conclusion, BMs associated with myotubes cultured on Ultroser G/brain extract resemble in many ways the in vivo situation, including synaptic specializations. Cultured myotubes may serve as a model system for studies on the structure and function of human muscular (synaptic) BM under normal and pathological conditions.

Expression of different isoenzymes of adenylate deaminase in cultured human muscle cells. Relation to myoadenylate deaminase deficiency.

Adenylate deaminase activity was determined in cultured muscle cells of different maturation grades and muscle biopsies from normal subjects and four patients with a primary myoadenylate deaminase (MAD) deficiency. Adenylate deaminase activity was much lower in cultured human muscle cells than in normal muscle. The activity increased with maturation. The ratio of activities measured at 5 and 2 mM AMP decreased in the order: immature muscle cells greater than more mature muscle cells greater than muscle. Adenylate deaminase activity was detectable in muscle cell cultures of MAD-deficient patients. However, both at 2 and 5 mM AMP this activity was significantly lower than in cultured cells with the same high maturation grade obtained from control subjects, whereas the ratio between the activities at 5 and 2 mM AMP was higher. The observations indicate that transition from a fetal to an adult muscle isoenzyme of adenylate deaminase takes place in human cultured muscle cells during maturation. In cultures obtained from MAD-deficient patients this transition does not occur and only the fetal isoenzyme is present.

Effects of growth medium, electrical stimulation and paralysis on various enzyme activities in cultured rat muscle cells. Comparison with activities in rat muscles in vivo.

1. Replacement of fetal calf serum and chicken embryo extract by Ultroser G and rat brain extract during the proliferation phase resulted in a higher maturation grade of cultured rat muscle cells after 7 days of differentiation, on base of the percentage of the muscle specific isoenzyme of creatine kinase (CK-MM). 2. Furthermore, the activities of creatine kinase, citrate synthase, cytochrome c oxidase and hexokinase were significantly higher. 3. Compared to the enzyme activities in m. quadriceps of 10 day-old rat and m. quadriceps, m. soleus and m. extensor digitorum longus of young adult rats, the metabolic capacity of cultured myotubes most closely resembles that of the first muscle. 4. Paralysis with tetrodotoxin caused a slight decrease of the creatine kinase activity and the percentage of CK-MM of cultured myotubes and an increase of the activities of hexokinase, phosphorylase and AMP deaminase. 5. Electrical stimulation performed at different frequencies and time periods had no effect on the enzyme activities of cultured rat muscle cells. 6. Only the AMP deaminase activity was decreased after intense electrical stimulation.

In vitro contraction test for malignant hyperthermia in patients with unexplained recurrent rhabdomyolysis.

A few cases of non-anaesthetic-induced rhabdomyolysis in humans, predisposed to malignant hyperthermia (MH), have been described in literature. We studied a group of 6 consecutive patients with unexplained and recurrent attacks of rhabdomyolysis with the test used to determine susceptibility to MH, the in vitro contraction test (IVCT). The results of the IVCT showed 5 of these 6 patients to be MH susceptible. In cultured muscle cells from one of these patients a disturbed calcium homeostasis could be demonstrated. The relation between MH and recurrent rhabdomyolysis is discussed.