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Marc N. Hirt Nils A. S?rensen Lena M. Bartholdt Jasper Boeddinghaus Sebastian Schaaf Alexandra Eder Ingra Vollert Andrea St?hr Thomas Schulze Anika Witten Monika Stoll Arne Hansen Thomas Eschenhagen 《Basic research in cardiology》2012,107(6):1-16
Increased afterload results in ‘pathological’ cardiac hypertrophy, the most important risk factor for the development of heart failure. Current in vitro models fall short in deciphering the mechanisms of hypertrophy induced by afterload enhancement. The aim of this study was to develop an experimental model that allows investigating the impact of afterload enhancement (AE) on work-performing heart muscles in vitro. Fibrin-based engineered heart tissue (EHT) was cast between two hollow elastic silicone posts in a 24-well cell culture format. After 2?weeks, the posts were reinforced with metal braces, which markedly increased afterload of the spontaneously beating EHTs. Serum-free, triiodothyronine-, and hydrocortisone-supplemented medium conditions were established to prevent undefined serum effects. Control EHTs were handled identically without reinforcement. Endothelin-1 (ET-1)- or phenylephrine (PE)-stimulated EHTs served as positive control for hypertrophy. Cardiomyocytes in EHTs enlarged by 28.4?% under AE and to a similar extent by ET-1- or PE-stimulation (40.6 or 23.6?%), as determined by dystrophin staining. Cardiomyocyte hypertrophy was accompanied by activation of the fetal gene program, increased glucose consumption, and increased mRNA levels and extracellular deposition of collagen-1. Importantly, afterload-enhanced EHTs exhibited reduced contractile force and impaired diastolic relaxation directly after release of the metal braces. These deleterious effects of afterload enhancement were preventable by endothelin-A, but not endothelin-B receptor blockade. Sustained afterload enhancement of EHTs alone is sufficient to induce pathological cardiac remodeling with reduced contractile function and increased glucose consumption. The model will be useful to investigate novel therapeutic approaches in a simple and fast manner. 相似文献
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Michael C. Puljung Hannah A. DeBerg William N. Zagotta Stefan Stoll 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(27):9816-9821
Binding of 3′,5′-cyclic adenosine monophosphate (cAMP) to hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels regulates their gating. cAMP binds to a conserved intracellular cyclic nucleotide-binding domain (CNBD) in the channel, increasing the rate and extent of activation of the channel and shifting activation to less hyperpolarized voltages. The structural mechanism underlying this regulation, however, is unknown. We used double electron–electron resonance (DEER) spectroscopy to directly map the conformational ensembles of the CNBD in the absence and presence of cAMP. Site-directed, double-cysteine mutants in a soluble CNBD fragment were spin-labeled, and interspin label distance distributions were determined using DEER. We found motions of up to 10 Å induced by the binding of cAMP. In addition, the distributions were narrower in the presence of cAMP. Continuous-wave electron paramagnetic resonance studies revealed changes in mobility associated with cAMP binding, indicating less conformational heterogeneity in the cAMP-bound state. From the measured DEER distributions, we constructed a coarse-grained elastic-network structural model of the cAMP-induced conformational transition. We find that binding of cAMP triggers a reorientation of several helices within the CNBD, including the C-helix closest to the cAMP-binding site. These results provide a basis for understanding how the binding of cAMP is coupled to channel opening in HCN and related channels.Ion channels are allosteric membrane proteins that open selective pores in response to various physiological stimuli, including binding of ligands and changes in transmembrane voltage (1). They are important for diverse physiological functions ranging from neurotransmission to muscle contraction. One such channel, the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel, underlies the current (termed Ih, If, or Iq) produced in response to hyperpolarization of cardiac pacemaker cells and neurons (2). In the heart, HCN channels are responsible for pace-making activity and may have a role in the autonomic regulation of the heart rate (3–5). In the brain, HCN channels are involved in repetitive firing of neurons and dendritic integration (6–8). Despite the important physiological roles of HCN channels, the structure of the channels and molecular mechanism of their function are not completely understood.HCN channels are part of the voltage-gated K+ channel superfamily (9). Like other members of this family, they are tetramers, with each subunit having a voltage-sensor domain of four transmembrane helices (S1–S4) and a pore-lining domain consisting of two transmembrane helices separated by a reentrant loop (S5-P-S6; Fig. 1A). However, HCN channels contain two key specializations that make them unique among the voltage-gated ion channels: (i) They are activated by membrane hyperpolarization instead of depolarization, and (ii) they are regulated by the direct binding of cyclic nucleotides, like the ubiquitous second messenger cAMP, to a cytoplasmic domain in the carboxyl-terminal region of the channel. The direct binding of the agonist cAMP to HCN channels increases the rate and extent of activation and shifts the voltage dependence of activation to more depolarizing voltages.Open in a separate windowFig. 1.Study of conformational changes in HCN2 using DEER. (A, Upper) Putative transmembrane topology of HCN2 channels highlighting the voltage sensor domain (S1–S4) and the pore domain (S5–S6). Only two subunits are shown. (A, Lower) Crystal structure [Protein Data Bank (PDB) ID code 3ETQ] of the cysteine-free cytoplasmic carboxyl-terminal domain of HCN2. One subunit of the tetramer is shown in color. (B) Schematic diagram showing the distance change between two cysteine-attached MTSL spin labels in a protein upon cAMP binding. In this example, the two positions are closer in the presence of cAMP. (C) Raw DEER time traces for HCN2cys-free V537C,A624C labeled with MTSL are shown in black in the absence or presence of cAMP, as indicated. The colored curves are distance-distribution fits to the data. The oscillation frequency is higher in the presence of cAMP, indicating that the two positions are closer together in the ligand-bound form.The crystal structure of the carboxyl-terminal region bound to cAMP has been solved for several HCN channels (10–14). The nearly identical structures consist of fourfold symmetrical tetramers predicted to connect directly to the S6 segments that form the ion-conducting pore (Fig. 1A). Each of the subunits contains two domains: the cyclic nucleotide-binding domain (CNBD) and the C-linker domain. The CNBD exhibits strong structural similarity to the CNBDs of other cyclic nucleotide-binding proteins, including cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac, and the Escherichia coli catabolite gene activator protein (CAP) (15–19). The CNBD consists of an eight-stranded antiparallel β-roll, followed by two α-helices (B-helix and C-helix). cAMP binds in the anticonformation between the β-roll and the C-helix. The C-linker is a unique domain found only in HCN channels and their close homologs, cyclic nucleotide-gated (CNG) channels, and KCNH family K+ channels (14, 20, 21). It is situated between the CNBD and membrane-spanning domains of the channel, and is the site of virtually all intersubunit interactions in the structure (Fig. 1A). The C-linker has been found to play a key role in coupling conformational changes in the CNBD to opening of the pore (9, 22, 23).The ligand-induced movement of the C-helix is widely thought to initiate the conformational changes that lead to opening of the channel pore, but the structural evidence in support of this hypothesis is equivocal (10, 24–29). The crystal structure of the HCN2 carboxyl-terminal region in the absence of ligand shows little difference from the cyclic nucleotide-bound structure (12). The only significant differences between the two structures are observed in the F′-helix of the C-linker and in the C-helix. The proximal half of the C-helix is in the same position in the cAMP-bound and unbound structures, whereas the distal half is missing from the apo structure, indicating that it is disordered or can access multiple conformations. In contrast, studies on the soluble carboxyl-terminal fragment using transition metal ion FRET (tmFRET) demonstrate a relatively large movement (∼5 Å) at the proximal end of the C-helix upon binding of cAMP (12). The tmFRET studies also indicate a smaller movement at the distal end of the C-helix and increased disorder in the C-helix in the absence of cyclic nucleotides (12, 26).In this study, we examined the cAMP-induced conformational transition in the CNBD of HCN2 using double electron–electron resonance (DEER) spectroscopy. DEER is a pulse electron paramagnetic resonance (EPR) method that can determine distances and resolve distance distributions between pairs of sites within proteins separated by about 15–80 Å (30–33). In a typical DEER experiment, two sites in a protein are mutated to cysteines and labeled with small magnetic spin labels (Fig. 1B). DEER measures the pair’s magnetic through-space coupling via excitation of one label and probing of the other with a series of short microwave pulses. This method yields an oscillating signal whose frequency falls off with the third power of the distance between the labels (Fig. 1C). Crucially, DEER measures full-distance distributions, rather than just an average distance, providing quantitative information on structural heterogeneity and variability that is not accessible from X-ray crystal structures or ensemble FRET experiments. Using DEER, we found that the binding of cAMP to the isolated C-linker/CNBD of HCN2 causes the C-helix to move substantially toward the β-roll and decreases the conformational heterogeneity of the protein. These observations are the first step in understanding the mechanisms of ligand gating of HCN channels and the activation of other CNBD-containing proteins. 相似文献
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Stoll Svenja Macha Kosmas Marsch Armin Gerner Stefan T. Siedler Gabriela Fröhlich Kilian Volbers Bastian Strasser Erwin F. Schwab Stefan Kallmünzer Bernd 《Journal of neurology》2020,267(7):2007-2012
Journal of Neurology - Oral Factor Xa inhibitors for the prevention of stroke in atrial fibrillation require dose adjustment based on certain clinical criteria, but the off-label use of the reduced... 相似文献