Annette Draeger

Association between statin-associated myopathy and skeletal muscle damage

Background: Many patients taking statins often complain of muscle pain and weakness. The extent to which muscle pain reflects muscle injury is unknown. Methods: We obtained biopsy samples from the vastus lateralis muscle of 83 patients. Of the 44 patients with clinically diagnosed statin-associated myopathy, 29 were currently taking a statin, and 15 had discontinued statin therapy before the biopsy (minimal duration of discontinuation 3 weeks). We also included 19 patients who were taking statins and had no myopathy, and 20 patients who had never taken statins and had no myopathy. We classified the muscles as injured if 2% or more of the muscle fibres in a biopsy sample showed damage. Using reverse transcriptase polymerase chain reaction, we evaluated the expression levels of candidate genes potentially related to myocyte injury. Results: Muscle injury was observed in 25 (of 44) patients with myopathy and in 1 patient without myopathy. Only 1 patient with structural injury had a circulating level of creatine phosphokinase that was elevated more than 1950 U/L (10× the upper limit of normal). Expression of ryanodine receptor 3 was significantly upregulated in patients with biopsy evidence of structural damage (1.7, standard error of the mean 0.3). Interpretation: Persistent myopathy in patients taking statins reflects structural muscle damage. A lack of elevated levels of circulating creatine phosphokinase does not rule out structural muscle injury. Upregulation of the expression of ryanodine receptor 3 is suggestive of an intracellular calcium leak.

Statin therapy induces ultrastructural damage in skeletal muscle in patients without myalgia

Muscle pain and weakness are frequent complaints in patients receiving 3‐hydroxymethylglutaryl coenzymeA (HMG CoA) reductase inhibitors (statins). Many patients with myalgia have creatine kinase levels that are either normal or only marginally elevated, and no obvious structural defects have been reported in patients with myalgia only. To investigate further the mechanism that mediates statin‐induced skeletal muscle damage, skeletal muscle biopsies from statin‐treated and non‐statin‐treated patients were examined using both electron microscopy and biochemical approaches. The present paper reports clear evidence of skeletal muscle damage in statin‐treated patients, despite their being asymptomatic. Though the degree of overall damage is slight, it has a characteristic pattern that includes breakdown of the T‐tubular system and subsarcolemmal rupture. These characteristic structural abnormalities observed in the statin‐treated patients were reproduced by extraction of cholesterol from skeletal muscle fibres in vitro. These findings support the hypothesis that statin‐induced cholesterol lowering per se contributes to myocyte damage and suggest further that it is the specific lipid/protein organization of the skeletal muscle cell itself that renders it particularly vulnerable. Copyright

The annexins: spatial and temporal coordination of signaling events during cellular stress

Annexins are a family of structurally related, Ca2+-sensitive proteins that bind to negatively charged phospholipids and establish specific interactions with other lipids and lipid microdomains. They are present in all eukaryotic cells and share a common folding motif, the “annexin core”, which incorporates Ca2+- and membrane-binding sites. Annexins participate in a variety of intracellular processes, ranging from the regulation of membrane dynamics to cell migration, proliferation, and apoptosis. Here we focus on the role of annexins in cellular signaling during stress. A chronic stress response triggers the activation of different intracellular pathways, resulting in profound changes in Ca2+ and pH homeostasis and the production of lipid second messengers. We review the latest data on how these changes are sensed by the annexins, which have the ability to simultaneously interact with specific lipid and protein moieties at the plasma membrane, contributing to stress adaptation via regulation of various signaling pathways.

The NK1 Receptor Localizes to the Plasma Membrane Microdomains, and Its Activation Is Dependent on Lipid Raft Integrity

The spatial targeting of receptors to discrete domains within the plasma membrane allows their preferential coupling to specific effectors, which is essential for rapid and accurate discrimination of signals. Efficiency of signaling is further increased by protein and lipid segregation within the plasma membrane. We have previously demonstrated the importance of raft-mediated signaling in the regulation of smooth and skeletal muscle cell contraction. Since G protein-coupled receptors (GPCRs) are key components in the regulation of smooth muscle contraction-relaxation cycles, it is important to determine whether GPCR signaling is mediated by lipid rafts and raft-associated molecules. Neurokinin 1 receptor (NK1R) is expressed in central and peripheral nervous system as well as in endothelial and smooth muscle cells and involved in mediation of pain, inflammation, exocrine secretion, and smooth muscle contraction. The NK1 receptor was transiently expressed in HEK293 and HepG2 cell lines and its localization in membrane microdomains investigated using biochemical methods and immunofluorescent labeling. We show that the NK1 receptor, similar to the earlier described β2-adrenergic receptor and G proteins, localizes to lipid rafts and caveolae. Protein kinase C (PKC) is one of the downstream effectors of the NK1 activation. Its active form translocates from the cytoplasm to the plasma membrane. Upon stimulation of the NK1 receptor with Substance P, the activated PKC relocated to lipid rafts. Using cholesterol extraction and replenishment assays we show that activation of NK1 receptor is dependent on the microarchitecture of the plasma membrane: NK1R-mediated signaling was abolished after cholesterol depletion of the receptor-expressing cells with methyl-β-cyclodextrin. Our results demonstrate that reorganization of the plasma membrane has an effect on the activation of the raft-associated NK1R and the down-stream events such as recruitment of protein kinases.

Modulating signaling events in smooth muscle: cleavage of annexin 2 abolishes its binding to lipid rafts

Cell membrane compartmentalization, which is believed to involve association of cholesterol‐and glycosphingolipid‐enriched membrane rafts, represents an important means of transmitting information across the plasma membrane. We have previously shown that raft association is mediated by the Ca2+dependent binding of annexin 2 to the plasma membrane. In the present study, we demonstrate that the association of annexins 1 and 2 with the smooth muscle cell membrane can be terminated by their proteolytic cleavage. This proteolysis is thought to be triggered by calpain and occurs at non‐raft regions of the plasma membrane. It is critically dependent on the intracellular concentration of free Ca2+ and requires an intact contractile apparatus. Annexins 1 and 2 interact with different membrane microcompartments–the former with non‐raft, glycerolipid regions, the latter preferentially with membrane rafts. We demonstrate that PKC and RhoA, major signaling molecules that regulate smooth muscle contraction, are spatially segregated and interact with distinct membrane microcompartments. Proteolysis abolishes annexin binding to the plasma membrane and might result in rearrangement of membrane constituents followed by the interruption of segregation‐dependent signaling events.—Babiychuk, E. B., Monastyrskaya, K., Burkhard, F. C., Wray, S., Draeger, A. Modulating signaling events in smooth muscle: cleavage of annexin 2 abolishes its binding to lipid rafts. FASEB J. 16, 1177–1184 (2002)

MMP‐19: cellular localization of a novel metalloproteinase within normal breast tissue and mammary gland tumours

Matrix metalloproteinases (MMPs) are instrumental in promoting and facilitating the spread of malignant diseases and in the de novo formation of blood vessels. This study has mapped the immunoreactivity of a novel, angiogenesis‐related metalloproteinase – MMP‐19 – in normal breast tissue and in benign and malignant breast lesions and compared this pattern of expression with that of MMP‐2. In the normal resting mammary gland, MMP‐19 was strongly expressed in the myoepithelial layer of the ductal system; the alveolar and ductal epithelia displayed considerable, but lobule‐specific, variations in labelling intensity. MMP‐19 was also present within the smooth muscle and endothelial layers of large and medium‐sized blood vessels, as well as within capillary walls. In benign lesions, all tumour cells and their surrounding vasculature were uniformly and strongly immunoreactive for MMP‐19. Progression towards an invasive phenotype and neoplastic dedifferentiation led to the disappearance of MMP‐19 from tumour cells and blood vessels and a concomitant rise in the levels of MMP‐2. In vitro experiments conducted with isolated smooth muscle cells cultivated on a solid substratum, or within the interstices of a collagen matrix, indicated that the expression of MMP‐19 is influenced by the architecture of the surrounding extracellular matrix. Copyright

MicroRNAs May Mediate the Down-Regulation of Neurokinin-1 Receptor in Chronic Bladder Pain Syndrome

Bladder pain syndrome (BPS) is a clinical syndrome of pelvic pain and urinary urgency-frequency in the absence of a specific cause. Investigating the expression levels of genes involved in the regulation of epithelial permeability, bladder contractility, and inflammation, we show that neurokinin (NK)1 and NK2 tachykinin receptors were significantly down-regulated in BPS patients. Tight junction proteins zona occludens-1, junctional adherins molecule -1, and occludin were similarly down-regulated, implicating increased urothelial permeability, whereas bradykinin B(1) receptor, cannabinoid receptor CB1 and muscarinic receptors M3-M5 were up-regulated. Using cell-based models, we show that prolonged exposure of NK1R to substance P caused a decrease of NK1R mRNA levels and a concomitant increase of regulatory micro(mi)RNAs miR-449b and miR-500. In the biopsies of BPS patients, the same miRNAs were significantly increased, suggesting that BPS promotes an attenuation of NK1R synthesis via activation of specific miRNAs. We confirm this hypothesis by identifying 31 differentially expressed miRNAs in BPS patients and demonstrate a direct correlation between miR-449b, miR-500, miR-328, and miR-320 and a down-regulation of NK1R mRNA and/or protein levels. Our findings further the knowledge of the molecular mechanisms of BPS, and have relevance for other clinical conditions involving the NK1 receptor.

Tailored Protection against Plasmalemmal Injury by Annexins with Different Ca2+ Sensitivities

The annexins, a family of Ca2+- and lipid-binding proteins, are involved in a range of intracellular processes. Recent findings have implicated annexin A1 in the resealing of plasmalemmal injuries. Here, we demonstrate that another member of the annexin protein family, annexin A6, is also involved in the repair of plasmalemmal lesions induced by a bacterial pore-forming toxin, streptolysin O. An injury-induced elevation in the intracellular concentration of Ca2+ ([Ca2+]i) triggers plasmalemmal repair. The highly Ca2+-sensitive annexin A6 responds faster than annexin A1 to [Ca2+]i elevation. Correspondingly, a limited plasmalemmal injury can be promptly countered by annexin A6 even without the participation of annexin A1. However, its high Ca2+ sensitivity makes annexin A6 highly amenable to an unproductive binding to the uninjured plasmalemma; during an extensive injury accompanied by a massive elevation in [Ca2+]i, its active pool is severely depleted. In contrast, annexin A1 with a much lower Ca2+ sensitivity is ineffective at the early stages of injury; however, it remains available for the repair even at high [Ca2+]i. Our findings highlight the role of the annexins in the process of plasmalemmal repair; a number of annexins with different Ca2+-sensitivities provide a cell with the means to react promptly to a limited injury in its early stages and, at the same time, to withstand a sustained injury accompanied by the continuous formation of plasmalemmal lesions.

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24–33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance.

Plasma Membrane-associated Annexin A6 Reduces Ca2+ Entry by Stabilizing the Cortical Actin Cytoskeleton

The annexins are a family of Ca2+- and phospholipid-binding proteins, which interact with membranes upon increase of [Ca2+]i or during cytoplasmic acidification. The transient nature of the membrane binding of annexins complicates the study of their influence on intracellular processes. To address the function of annexins at the plasma membrane (PM), we fused fluorescent protein-tagged annexins A6, A1, and A2 with H- and K-Ras membrane anchors. Stable PM localization of membrane-anchored annexin A6 significantly decreased the store-operated Ca2+ entry (SOCE), but did not influence the rates of Ca2+ extrusion. This attenuation was specific for annexin A6 because PM-anchored annexins A1 and A2 did not alter SOCE. Membrane association of annexin A6 was necessary for a measurable decrease of SOCE, because cytoplasmic annexin A6 had no effect on Ca2+ entry as long as [Ca2+]i was below the threshold of annexin A6-membrane translocation. However, when [Ca2+]i reached the levels necessary for the Ca2+-dependent PM association of ectopically expressed wild-type annexin A6, SOCE was also inhibited. Conversely, knockdown of the endogenous annexin A6 in HEK293 cells resulted in an elevated Ca2+ entry. Constitutive PM localization of annexin A6 caused a rearrangement and accumulation of F-actin at the PM, indicating a stabilized cortical cytoskeleton. Consistent with these findings, disruption of the actin cytoskeleton using latrunculin A abolished the inhibitory effect of PM-anchored annexin A6 on SOCE. In agreement with the inhibitory effect of annexin A6 on SOCE, constitutive PM localization of annexin A6 inhibited cell proliferation. Taken together, our results implicate annexin A6 in the actin-dependent regulation of Ca2+ entry, with consequences for the rates of cell proliferation.