Krzysztof Zabłocki

Increased susceptibility to ATP via alteration of P2X receptor function in dystrophic mdx mouse muscle cells

Pathological cellular hallmarks of Duchenne muscular dystrophy (DMD) include, among others, abnormal calcium homeostasis. Changes in the expression of specific receptors for extracellular ATP in dystrophic muscle have been recently documented: here, we demonstrate that at the earliest, myoblast stage of developing dystrophic muscle a purinergic dystrophic phenotype arises. In myoblasts of a dystrophin‐negative muscle cell line established from the mdx mouse model of DMD but not in normal myoblasts, exposure to extracellular ATP triggered a strong increase in cytoplasmic Ca2+ concentrations. Influx of extracellular Ca2+ was stimulated by ATP and BzATP and inhibited by zinc, Coomassie Brilliant Blue‐G, and KN‐62, demonstrating activation of P2X7 receptors. Significant expression of P2X4 and P2X7 proteins was immunodetected in dystrophic myoblasts. Therefore, full‐length dystrophin appears, surprisingly, to play an important role in myoblasts in controlling responses to ATP. Our results suggest that altered function of P2X receptors may be an important contributor to pathogenic Ca2+ entry in dystrophic mouse muscle and may have implications for the pathogenesis of muscular dystrophies. Treatments aiming at inhibition of specific ATP receptors could be of a potential therapeutic benefit.‐Yeung, D., Zabłocki, K., Lien, C.‐F., Jiang, T., Arkle, S., Brutkowski, W., Brown, J., Lochmuller, H., Simon, J., Barnard, E. A., Górecki, D. C. Increased susceptibility to ATP via alteration of P2X receptor function in dystrophic mdx mouse muscle cells. FASEB J. 20, 610–620 (2006)

Mutation in dystrophin-encoding gene affects energy metabolism in mouse myoblasts

Duchenne Muscular Dystrophy is characterized by severe defects in differentiated muscle fibers, including abnormal calcium homeostasis and impaired cellular energy metabolism. Here we demonstrate that myoblasts derived from dystrophic (mdx) mouse exhibit reduced oxygen consumption, increased mitochondrial membrane potential, enhanced reactive oxygen species formation, stimulated glycolysis but unaffected total cellular ATP content. Moreover, reduced amounts of specific subunits of the mitochondrial respiratory complexes and ATP-synthase as well as disorganized mitochondrial network were observed. Both the dystrophic and control myoblasts used were derived from a common inbred mouse strain and the only difference between them is a point mutation in the dystrophin-encoding gene, thus these data indicate that this mutation results in multiple phenotypic alterations demonstrating as early as in undifferentiated myoblasts. This finding sheds new light on the molecular mechanisms of Duchenne Muscular Dystrophy pathogenesis.

Mitochondrial mechanisms of endothelial dysfunction

Endothelial cells play an important physiological role in vascular homeostasis. They are also the first barrier that separates blood from deeper layers of blood vessels and extravascular tissues. Thus, they are exposed to various physiological blood components as well as challenged by pathological stimuli, which may exert harmful effects on the vascular system by stimulation of excessive generation of reactive oxygen species (ROS). The major sources of ROS are NADPH oxidase and mitochondrial respiratory chain complexes. Modulation of mitochondrial energy metabolism in endothelial cells is thought to be a promising target for therapy in various cardiovascular diseases. Uncoupling protein 2 (UCP2) is a regulator of mitochondrial ROS generation and can antagonise oxidative stress-induced endothelial dysfunction. Several studies have revealed the important role of UCP2 in hyperglycaemia-induced modifications of mitochondrial function in endothelial cells. Additionally, potassium fluxes through the inner mitochondrial membrane, which are involved in ROS synthesis, affect the mitochondrial volume and change both the mitochondrial membrane potential and the transport of calcium into the mitochondria. In this review, we concentrate on the mitochondrial role in the cytoprotection phenomena of endothelial cells.

P2X7 purinoceptor alterations in dystrophic mdx mouse muscles: Relationship to pathology and potential target for treatment.

Duchenne muscular dystrophy (DMD) is a lethal inherited muscle disorder. Pathological characteristics of DMD skeletal muscles include, among others, abnormal Ca2+ homeostasis and cell signalling. Here, in the mdx mouse model of DMD, we demonstrate significant P2X7 receptor abnormalities in isolated primary muscle cells and cell lines and in dystrophic muscles in vivo. P2X7 mRNA expression in dystrophic muscles was significantly up‐regulated but without alterations of specific splice variant patterns. P2X7 protein was also up‐regulated and this was associated with altered function of P2X7 receptors producing increased responsiveness of cytoplasmic Ca2+ and extracellular signal‐regulated kinase (ERK) phosphorylation to purinergic stimulation and altered sensitivity to NAD. Ca2+ influx and ERK signalling were stimulated by ATP and BzATP, inhibited by specific P2X7 antagonists and insensitive to ivermectin, confirming P2X7 receptor involvement. Despite the presence of pannexin‐1, prolonged P2X7 activation did not trigger cell permeabilization to propidium iodide or Lucifer yellow. In dystrophic mice, in vivo treatment with the P2X7 antagonist Coomassie Brilliant Blue reduced the number of degeneration–regeneration cycles in mdx skeletal muscles. Altered P2X7 expression and function is thus an important feature in dystrophic mdx muscle and treatments aiming to inhibit P2X7 receptor might slow the progression of this disease.

Calcium Signals Are Affected by Ciprofloxacin as a Consequence of Reduction of Mitochondrial DNA Content in Jurkat Cells

ABSTRACT The effects of ciprofloxacin on mitochondrial DNA (mtDNA) content, oxygen consumption, mitochondrial membrane potential, cellular ATP formation, and capacitative Ca2+ entry into Jurkat cells were investigated. In cells incubated for several days with 25 μg/ml ciprofloxacin, a 60% reduction of mtDNA content, inhibition of the respiratory chain, and a significant decrease in mitochondrial membrane potential were observed. These changes led to a decrease in the calcium buffering capacity of mitochondria which, in turn, resulted in a gradual inhibition of the capacitative Ca2+ entry. On days 4, 7, and 11 of incubation with ciprofloxacin, the initial rates of Ca2+ entry were reduced by 33%, 50%, and 50%, respectively. Ciprofloxacin caused a transient decrease in the cellular capability for ATP formation. In cells incubated for 15 min with glucose, pyruvate, and glutamine as exogenous fuel, ciprofloxacin reduced ATP content by 16% and 35% on days 4 and 7, respectively, of incubation with the drug. However, on day 11 of incubation with ciprofloxacin, a recovery of cellular ATP formation was observed. In conclusion, long-term exposure of Jurkat cells to ciprofloxacin at a concentration of 25 μg/ml seriously affects cellular energy metabolism and calcium homeostasis.

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein Is PAM involved in the capacitative calcium entry

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

Role of Annexin A6 Isoforms in Catecholamine Secretion by PC12 Cells: Distinct Influence on Calcium Response

Noradrenaline and adrenaline are secreted by adrenal medulla chromaffin cells via exocytosis. Exocytosis of catecholamines occurs after cell stimulation with various endogenous activators such as nicotine or after depolarization of the plasma membrane and is regulated by calcium ions. Cytosolic [Ca2+] increases in response to cell excitation and triggers a signal‐initiated secretion. Annexins are known to participate in the regulation of membrane dynamics and are also considered to be involved in vesicular trafficking. Some experimental evidence suggests that annexins may participate in Ca2+‐regulated catecholamine secretion. In this report the effect of annexin A6 (AnxA6) isoforms 1 and 2 on catecholamine secretion has been described. Overexpression of AnxA6 isoforms and AnxA6 knock‐down in PC12 cells were accompanied by almost complete inhibition or a 20% enhancement of dopamine secretion, respectively. AnxA6‐1 and AnxA6‐2 overexpression reduced Δ[Ca2+]c upon depolarization by 32% and 58%, respectively, while AnxA6 knock‐down increased Δ[Ca2+]c by 44%. The mechanism of AnxA6 action on Ca2+ signalling is not well understood. Experimental evidence suggests that two AnxA6 isoforms interact with different targets engaged in regulation of calcium homeostasis in PC12 cells. J. Cell. Biochem. 111: 168–178, 2010.

Influence of a mitochondrial genetic defect on capacitative calcium entry and mitochondrial organization in the osteosarcoma cells

Effects of T8993G mutation in mitochondrial DNA (mtDNA), associated with neurogenical muscle weakness, ataxia and retinitis pigmentosa (NARP), on the cytoskeleton, mitochondrial network and calcium homeostasis in human osteosarcoma cells were investigated. In 98% NARP and ρ 0 (lacking mtDNA) cells, the organization of the mitochondrial network and actin cytoskeleton was disturbed. Capacitative calcium entry (CCE) was practically independent of mitochondrial energy status in osteosarcoma cell lines. The significantly slower Ca2+ influx rates observed in 98% NARP and ρ 0, in comparison to parental cells, indicates that proper actin cytoskeletal organization is important for CCE in these cells.

Hyperglycaemia modifies energy metabolism and reactive oxygen species formation in endothelial cells in vitro

There is significant evidence for an involvement of reactive oxygen species (ROS) in the pathogenesis of diabetic vascular complications through many metabolic and structural derangements. However, despite the advanced knowledge on the crucial role of ROS in cardiovascular damage, their intracellular source in endothelial cells exposed to high concentrations of glucose has not been precisely defined. Moreover, the molecular mechanism of action of elevated glucose on mitochondria has not been fully elucidated. The main aim of this study was to describe changes in the mitochondrial metabolism of human umbilical vein endothelial cells (HUVECs) treated with high glucose concentrations and to indicate the actual source of ROS in these cells. HUVECs exposed to 30 mM glucose exhibited an increased content of vascular adhesive molecule-1 (VCAM-1) and an excessive ROS production. Faster oxygen consumption and increased abundance of selected respiratory complexes coexist with slightly declined mitochondrial membrane potential and substantially elevated amount of uncoupling protein-2 (UCP2). Inhibition of NADPH oxidase (NOX) and modification of mitochondrial ROS generation with a mitochondrial uncoupler or respiratory chain inhibitors allowed concluding that the major source of ROS in HUVECs exposed to hyperglycaemic conditions is NOX. The mitochondrial respiratory chain seems not to participate in this phenomenon.

TNFα affects energy metabolism and stimulates biogenesis of mitochondria in EA.hy926 endothelial cells

Mitochondrial response of EA.hy926 endothelial cells to tumour necrosis factor alpha (TNFα) was investigated. It was confirmed that TNFα stimulates reactive oxygen species (ROS) generation and increases intercellular adhesion molecule-1 (ICAM-1) level. These changes were paralleled by elevated oxygen consumption, slightly raised total mitochondrial mass and increased manganese superoxide dismutase (Mn-SOD) and uncoupling protein 2 (UCP2) content. They also correlated with a rise of mitochondrial transcription factor 1 (TFAM), nuclear respiratory factor-1 (NRF-1) and peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, which are involved in regulation of mitochondrial biogenesis and an elevated level of selected respiratory chain proteins. Thus, the apparent stimulatory effect of TNFα on mitochondrial metabolism probably reflects an increased amount of mitochondria rather than activation of biochemical processes per se, although the latter cannot be excluded definitely. These observations are similar to those described for cardiac muscle cells challenged with bacterial lipopolysaccharide (LPS), in which mitochondrial biogenesis was postulated. Stimulation of mitochondrial biogenesis could be a mechanism activated to prevent TNFα-induced cell death.