Publications
205 results found
Kane C, Dias P, Gorelik J, et al., 2015, Human Cardiac Fibroblasts Increase SR-Dependency of Induced Pluripotent Stem Cell-Derived Cardiomyocyte Calcium Handling by Increasing SR Calcium Uptake and SERCA2a Expression via Direct Physical Contact, Scientific Sessions and Resuscitation Science Symposium of the American-Heart-Association (AHA), Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Sanchez-Alonso JL, Poulet CE, Schobesberger S, et al., 2015, L-type Calcium Channels Localization and Function are Affected by Microdomain Remodelling in Human Failing Cardiomyocytes, Scientific Sessions and Resuscitation Science Symposium of the American-Heart-Association (AHA), Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Glukhov AV, Balycheva M, Sanchez-Alonso J, et al., 2015, Loss of adrenergic and adenosine regulation of extradyadic L-type calcium channels in rat atrial myocytes in heart failure, Scientific Sessions and Resuscitation Science Symposium of the American-Heart-Association (AHA), Publisher: American Heart Association, ISSN: 0009-7322
Gorelik J, 2015, Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes, Circulation, ISSN: 0009-7322
Leo-Macias A, Agullo-Pascual E, Sanchez-Alonso JL, et al., 2015, Nanoscale Visualization of Functional Adhesion/Excitability Nodes at the Intercalated Disc., 69th Annual Meeting of the Society-of-General-Physiologists - Macromolecular Local Signaling Complexes, Publisher: ROCKEFELLER UNIV PRESS, Pages: 9A-10A, ISSN: 0022-1295
Kane C, Dias P, Gorelik J, et al., 2015, Human cardiac fibroblasts increase SR-dependency of induced pluripotent stem cell-derived cardiomyocyte calcium handling by manipulating SR uptake and SERCA2a expression via direct physical contact, Congress of the European-Society-of-Cardiology (ESC), Publisher: OXFORD UNIV PRESS, Pages: 776-776, ISSN: 0195-668X
Wright PT, Gorelik J, Schobesberger S, 2015, Studying GPCR/cAMP pharmacologyfrom the perspective of cellularstructure, Frontiers in Pharmacology, Vol: 6, ISSN: 1663-9812
Signal transduction via G-protein coupled receptors (GPCRs) relies upon the productionof cAMP and other signaling cascades. A given receptor and agonist pair, producemultiple effects upon cellular physiology which can be opposite in different cell types.The production of variable cellular effects via the signaling of the same GPCR in differentcell types is a result of signal organization in space and time (compartmentation).This organization is usually based upon the physical and chemical properties of themembranes in which the GPCRs reside and the repertoire of downstream effectors andco-factors that are available at that location. In this review we explore mechanisms ofGPCR signal compartmentation and broadly review the state-of-the-art methodologieswhich can be utilized to study them. We provide a clear rationale for a “localized”approach to the study of the pharmacology and physiology of GPCRs and particularlythe secondary messenger cAMP.
Balycheva M, Faggian G, Glukhov AV, et al., 2015, Microdomain-specific localization of functional ion channels in cardiomyocytes: an emerging concept of local regulation and remodelling., Biophys Rev, Vol: 7, Pages: 43-62, ISSN: 1867-2450
Cardiac excitation involves the generation of action potential by individual cells and the subsequent conduction of the action potential from cell to cell through intercellular gap junctions. Excitation of the cellular membrane results in opening of the voltage-gated L-type calcium ion (Ca2+) channels, thereby allowing a small amount of Ca2+ to enter the cell, which in turn triggers the release of a much greater amount of Ca2+ from the sarcoplasmic reticulum, the intracellular Ca2+ store, and gives rise to the systolic Ca2+ transient and contraction. These processes are highly regulated by the autonomic nervous system, which ensures the acute and reliable contractile function of the heart and the short-term modulation of this function upon changes in heart rate or workload. It has recently become evident that discrete clusters of different ion channels and regulatory receptors are present in the sarcolemma, where they form an interacting network and work together as a part of a macro-molecular signalling complex which in turn allows the specificity, reliability and accuracy of the autonomic modulation of the excitation-contraction processes by a variety of neurohormonal pathways. Disruption in subcellular targeting of ion channels and associated signalling proteins may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure and certain arrhythmias. Recent methodological advances have made it possible to routinely image the topography of live cardiomyocytes, allowing the study of clustering functional ion channels and receptors as well as their coupling within a specific microdomain. In this review we highlight the emerging understanding of the functionality of distinct subcellular microdomains in cardiac myocytes (e.g. T-tubules, lipid rafts/caveolae, costameres and intercalated discs) and their functional role in the accumulation and regulation of different subcellular populations of sodium, Ca2+ and potassium ion channels and th
Padala M, Zamani MH, Sarathchandra P, et al., 2014, Filamin-A Mutations Causing Mitral Valve Prolapse Alters Integrin Expression and Causes Breakdown of Mechanotransduction, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Balycheva M, Glukhov A, Schobesberger S, et al., 2014, Increased Open Probability of L-type Calcium Channels Localized in T-tubules in Patients With Chronic Atrial Fibrillation: Role of Channel Subunits, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Miragoli M, Schultz F, Pijnappels D, et al., 2014, Connexin43 Expression Modulation Affects Myofibroblast-Cardiomyocyte Coupling: Implications for Infarct Borderzone Expansion, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Glukhov AV, Balycheva M, Bhogal N, et al., 2014, Caveolae Specific Location of L-type Calcium Channels and Their Role in Atrial Calcium Cycling, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Miragoli M, Schultz F, Vite A, et al., 2014, Connexin43 expression modulate myofibroblasts mechanical coupling: implication for the infarct borderzone expansion, CARDIOVASCULAR RESEARCH, Vol: 103, ISSN: 0008-6363
Kane C, Dias P, Helen N, et al., 2014, Direct contact between human cardiac fibroblasts and human induced pluripotent stem cell-derived cardiomyocytes counteracts changes in calcium cycling induced by soluble mediators, CARDIOVASCULAR RESEARCH, Vol: 103, ISSN: 0008-6363
Miragoli M, Yacoub MH, El-Hamamsy I, et al., 2014, Side-specific mechanical properties of valve endothelial cells, AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY, Vol: 307, Pages: H15-H24, ISSN: 0363-6135
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- Citations: 15
Hong T, Yang H, Zhang S-S, et al., 2014, Cardiac BIN1 folds T-tubule membrane, controlling ion flux and limiting arrhythmia, Nature Medicine, Vol: 20, Pages: 624-632, ISSN: 1078-8956
Cardiomyocyte T tubules are important for regulating ion flux. Bridging integrator 1 (BIN1) is a T-tubule protein associated with calcium channel trafficking that is downregulated in failing hearts. Here we find that cardiac T tubules normally contain dense protective inner membrane folds that are formed by a cardiac isoform of BIN1. In mice with cardiac Bin1 deletion, T-tubule folding is decreased, which does not change overall cardiomyocyte morphology but leads to free diffusion of local extracellular calcium and potassium ions, prolonging action-potential duration and increasing susceptibility to ventricular arrhythmias. We also found that T-tubule inner folds are rescued by expression of the BIN1 isoform BIN1+13+17, which promotes N-WASP–dependent actin polymerization to stabilize the T-tubule membrane at cardiac Z discs. BIN1+13+17 recruits actin to fold the T-tubule membrane, creating a 'fuzzy space' that protectively restricts ion flux. When the amount of the BIN1+13+17 isoform is decreased, as occurs in acquired cardiomyopathy, T-tubule morphology is altered, and arrhythmia can result.
Goetz KR, Sprenger JU, Perera RK, et al., 2014, Transgenic mice for real-time visualization of cGMP in intact adult cardiomyocytes, Circulation Research, Vol: 114, Pages: 1235-1245, ISSN: 0009-7330
Rationale:3′,5′-Cyclic guanosine monophosphate (cGMP) is an important second messenger that regulates cardiac contractility and protects the heart from hypertrophy. However, because of the lack of real-time imaging techniques, specific subcellular mechanisms and spatiotemporal dynamics of cGMP in adult cardiomyocytes are not well understood.Objective:Our aim was to generate and characterize a novel cGMP sensor model to measure cGMP with nanomolar sensitivity in adult cardiomyocytes.Methods and Results:We generated transgenic mice with cardiomyocyte-specific expression of the highly sensitive cytosolic Förster resonance energy transfer–based cGMP biosensor red cGES-DE5 and performed the first Förster resonance energy transfer measurements of cGMP in intact adult mouse ventricular myocytes. We found very low (≈10 nmol/L) basal cytosolic cGMP levels, which can be markedly increased by natriuretic peptides (C-type natriuretic peptide >> atrial natriuretic peptide) and, to a much smaller extent, by the direct stimulation of soluble guanylyl cyclase. Constitutive activity of this cyclase contributes to basal cGMP production, which is balanced by the activity of clinically established phosphodiesterase (PDE) families. The PDE3 inhibitor, cilostamide, showed especially strong cGMP responses. In a mild model of cardiac hypertrophy after transverse aortic constriction, PDE3 effects were not affected, whereas the contribution of PDE5 was increased. In addition, after natriuretic peptide stimulation, PDE3 was also involved in cGMP/cAMP crosstalk.Conclusions:The new sensor model allows visualization of real-time cGMP dynamics and pharmacology in intact adult cardiomyocytes. Förster resonance energy transfer imaging suggests the importance of well-established and potentially novel PDE-dependent mechanisms that regulate cGMP under physiological and pathophysiological conditions.
Novak P, Shevchuk A, Ruenraroengsak P, et al., 2014, Imaging single nanoparticle interactions with human lung cells using fast ion conductance microscopy, Nano Letters: a journal dedicated to nanoscience and nanotechnology, Vol: 14, Pages: 1202-1207, ISSN: 1530-6984
Experimental data on dynamic interactions between individual nanoparticles and membrane processes at nanoscale, essential for biomedical applications of nanoparticles, remain scarce due to limitations of imaging techniques. We were able to follow single 200 nm carboxyl-modified particles interacting with identified membrane structures at the rate of 15 s/frame using a scanning ion conductance microscope modified for simultaneous high-speed topographical and fluorescence imaging. The imaging approach demonstrated here opens a new window into the complexity of nanoparticle–cell interactions.
Wright PT, Nikolaev VO, O'Hara T, et al., 2014, Caveolin-3 regulates compartmentation of cardiomyocyte beta2-adrenergic receptor-mediated cAMP signaling, JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, Vol: 67, Pages: 38-48, ISSN: 0022-2828
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- Citations: 82
Glukhov AV, Balycheva M, Schobesberger S, et al., 2013, Distinct Distribution of Functional Calcium Channels Revealed by Super-resolution Scanning Patch-clamp in Adult Rat Atrial Cardiomyocytes, Scientific Sessions and Resuscitation Science Symposium of the American-Heart-Association, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Schobesberger S, Tokar S, Bhargava A, et al., 2013, Alteration in b2-ARs Dependent cAMP Signalling Linked to Post Infarction Remodelling of T-tubules in Rat Cardiomyocytes, Scientific Sessions and Resuscitation Science Symposium of the American-Heart-Association, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
O'Hara T, Wright PT, Nikolaev VO, et al., 2013, Caveolin-3 Restores Local cAMP Signaling Without Restoring T-Tubules in Response to ß2 Adrenergic Receptor Stimulation in Heart Failure, Scientific Sessions and Resuscitation Science Symposium of the American-Heart-Association, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Novak P, Gorelik J, Vivekananda U, et al., 2013, Nanoscale-targeted patch-clamp recordings of functional presynaptic ion channels, Neuron, Vol: 79, Pages: 1067-1077, ISSN: 0896-6273
Direct electrical access to presynaptic ion channels has hitherto been limited to large specialized terminals such as the calyx of Held or hippocampal mossy fiber bouton. The electrophysiology and ion-channel complement of far more abundant small synaptic terminals (≤1 μm) remain poorly understood. Here we report a method based on superresolution scanning ion conductance imaging of small synapses in culture at approximately 100–150 nm 3D resolution, which allows presynaptic patch-clamp recordings in all four configurations (cell-attached, inside-out, outside-out, and whole-cell). Using this technique, we report presynaptic recordings of K+, Na+, Cl−, and Ca2+ channels. This semiautomated approach allows direct investigation of the distribution and properties of presynaptic ion channels at small central synapses.
Gorelik J, Wright PT, Lyon AR, et al., 2013, Spatial control of the βAR system in heart failure: the transverse tubule and beyond, CARDIOVASCULAR RESEARCH, Vol: 98, Pages: 216-224, ISSN: 0008-6363
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- Citations: 36
Miragoli M, Novak P, Ruenraroengsak P, et al., 2013, Functional interaction between charged nanoparticles and cardiac tissue: a new paradigm for cardiac arrhythmia?, NANOMEDICINE, Vol: 8, Pages: 725-737, ISSN: 1743-5889
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- Citations: 40
Ibrahim M, Navaratnarajah M, Siedlecka U, et al., 2013, Mechanical unloading reverses transverse tubule remodelling and normalises local calcium-induced calcium release in a rodent model of heart failure, Spring Meeting for Clinician Scientists in Training, Publisher: ELSEVIER SCIENCE INC, Pages: 54-54, ISSN: 0140-6736
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- Citations: 1
Bhargava A, Lin X, Novak P, et al., 2013, Super-Resolution Scanning Patch-Clamp Reveals Clustering of Functional Ion Channels in the Adult Ventricular Myocyte, Circ Res, ISSN: 1524-4571
Rationale: Compartmentation of ion channels on the cardiomyocyte surface is important for electrical propagation and electromechanical coupling. The specialized T-tubule and costameric structures facilitate spatial coupling of various ion channels and receptors. Existing methods like immunofluorescence and patch-clamp techniques are limited in their ability to localize functional ion channels. As such, a correlation between channel protein location and channel function remains incomplete. Objective: To validate a method that permits to routinely image the topography of a live cardiomyocyte, and then study clustering of functional ion channels from a specific microdomain. Methods and Results: We used scanning ion conductance microscopy and conventional cell-attached patch-clamp with a software modification that allows controlled increase of pipette tip diameter. The sharp nanopipette used for topography scan was modified into a larger patch pipette which can be positioned with nanoscale precision to a specific site of interest (crest, groove or T-tubules of cardiomyocytes), and sealed to the membrane for cell-attached recording of ion channels. Using this method, we significantly increased the probability of detecting activity of L-type calcium channels in the T-tubules of ventricular cardiomyocytes. We also demonstrated that active sodium channels do not distribute homogenously on the sarcolemma but rather, they segregate into clusters of various densities -most crowded in the crest region- that are surrounded by areas virtually free of functional sodium channels. Conclusions: Our new method substantially increases the throughput of recording location-specific functional ion channels on the cardiomyocyte sarcolemma, thus allowing characterization of ion channels in relation to the microdomain in which they reside.
Lab MJ, Bhargava A, Wright PT, et al., 2013, The scanning ion conductance microscope for cellular physiology, Am J Physiol Heart Circ Physiol, Vol: 304, Pages: H1-H11, ISSN: 1522-1539
The quest for nonoptical imaging methods that can surmount light diffraction limits resulted in the development of scanning probe microscopes. However, most of the existing methods are not quite suitable for studying biological samples. The scanning ion conductance microscope (SICM) bridges the gap between the resolution capabilities of atomic force microscope and scanning electron microscope and functional capabilities of conventional light microscope. A nanopipette mounted on a three-axis piezo-actuator, scans a sample of interest and ion current is measured between the pipette tip and the sample. The feedback control system always keeps a certain distance between the sample and the pipette so the pipette never touches the sample. At the same time pipette movement is recorded and this generates a three-dimensional topographical image of the sample surface. SICM represents an alternative to conventional high-resolution microscopy, especially in imaging topography of live biological samples. In addition, the nanopipette probe provides a host of added modalities, for example using the same pipette and feedback control for efficient approach and seal with the cell membrane for ion channel recording. SICM can be combined in one instrument with optical and fluorescent methods and allows drawing structure-function correlations. It can also be used for precise mechanical force measurements as well as vehicle to apply pressure with precision. This can be done on living cells and tissues for prolonged periods of time without them loosing viability. The SICM is a multifunctional instrument, and it is maturing rapidly and will open even more possibilities in the near future.
Ibrahim M, Siedlecka U, Buyandelger B, et al., 2013, A critical role for Telethonin in regulating t-tubule structure and function in the mammalian heart, Hum Mol Genet, Vol: 22, Pages: 372-383, ISSN: 1460-2083
The transverse (t)-tubule system plays an essential role in healthy and diseased heart muscle, particularly in Ca(2+)-induced Ca(2+) release (CICR), and its structural disruption is an early event in heart failure. Both mechanical overload and unloading alter t-tubule structure, but the mechanisms mediating the normally tight regulation of the t-tubules in response to load variation are poorly understood. Telethonin (Tcap) is a stretch-sensitive Z-disc protein that binds to proteins in the t-tubule membrane. To assess its role in regulating t-tubule structure and function, we used Tcap knockout (KO) mice and investigated cardiomyocyte t-tubule and cell structure and CICR over time and following mechanical overload. In cardiomyocytes from 3-month-old KO (3mKO), there were isolated t-tubule defects and Ca(2+) transient dysynchrony without whole heart and cellular dysfunction. Ca(2+) spark frequency more than doubled in 3mKO. At 8 months of age (8mKO), cardiomyocytes showed progressive loss of t-tubules and remodelling of the cell surface, with prolonged and dysynchronous Ca(2+) transients. Ca(2+) spark frequency was elevated and the L-type Ca(2+) channel was depressed at 8 months only. After mechanical overload obtained by aortic banding constriction, the Ca(2+) transient was prolonged in both wild type and KO. Mechanical overload increased the Ca(2+) spark frequency in KO alone, where there was also significantly more t-tubule loss, with a greater deterioration in t-tubule regularity. In conjunction, Tcap KO showed severe loss of cell surface ultrastructure. These data suggest that Tcap is a critical, load-sensitive regulator of t-tubule structure and function.
Bhargava A, Gorelik J, 2013, Recording single-channel currents using "smart patch-clamp" technique., Methods Mol Biol, Vol: 998, Pages: 189-197
Microdomains that form on the plasma membrane of cells are essential for signalling compartmentation within cells. The localization of ion channels in these surface microdomains is important in defining what signalling cascades will be generated. For example, in cardiomyocytes, similar to other excitable cells, action potential propagation depends essentially on the properties of ion channels that are functionally and spatially coupled. In this chapter we describe a novel advanced patch-clamp technique, "Smart patch-clamp," which enables the study of functional ion channels in the cell surface microdomains in a wide variety of biological cells and tissues. Smart patch-clamp combines conventional patch-clamp and Scanning Ion Conductance Microscopy (SICM). SICM uses a glass micropipette as scanning probe and generates a high-resolution topography image of the cell surface. Next, same micropipette is used as a patch-clamp pipette to record ion channel signals from specific spots on the cell surface as determined by SICM. In this chapter we focus on recording single channel L-type calcium channel currents from T-tubules of adult rat cardiomyocytes.
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