182 results found
Vasavan T, Deepak S, Jayawardane IA, et al., 2021, Fetal cardiac dysfunction in intrahepatic cholestasis of pregnancy is associated with elevated serum bile acid concentrations, JOURNAL OF HEPATOLOGY, Vol: 74, Pages: 1087-1096, ISSN: 0168-8278
Kondrashov A, Mohd Yusof NAN, Hasan A, et al., 2021, CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of β2AR in isogenic hPSC-derived cardiomyocytes, Molecular Therapy - Methods and Clinical Development, Vol: 20, Pages: 39-53, ISSN: 2329-0501
During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (β2AR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, in vitro experiments with primary cells, and transgenic overexpression models. However, heterogeneous genetic background and non-physiological transgene expression levels confound interpretation, leading to conflicting mechanistic conclusions. To overcome these limitations, we used CRISPR/Cas9 gene editing technology in human pluripotent stem cells (hPSCs) to create a unique suite of four isogenic homozygous variants at amino acid positions 16(G/R) and 27(G/Q), which reside in the N terminus of the β2AR. By producing cardiomyocytes from these hPSC lines, we determined that at a functional level β2AR signaling dominated over β1AR . Examining changes in beat rates and responses to isoprenaline, Gi coupling, cyclic AMP (cAMP) production, downregulation, and desensitization indicated that responses were often heightened for the GE variant, implying differential dominance of both polymorphic location and amino acid substitution. This finding was corroborated, since GE showed hypersensitivity to doxorubicin-induced cardiotoxicity relative to GQ and RQ variants. Thus, understanding the effect of β2AR polymorphisms on cardiac response to anticancer therapy may provide a route for personalized medicine and facilitate immediate clinical impact.
Medvedev R, Sanchez Alonso J, Mansfield C, et al., 2021, Local hyperactivation of L-type Ca2+ channels increases spontaneous Ca2+ release activity and cellular hypertrophy in right ventricular myocytes from heart failure rats, Scientific Reports, Vol: 11, ISSN: 2045-2322
Right ventricle (RV) dysfunction is an independent predictor of patient survival in heart failure (HF). However, the mechanisms of RV progression towards failing are not well understood. We studied cellular mechanisms of RV remodelling in a rat model of left ventricle myocardial infarction (MI)-caused HF. RV myocytes from HF rats show significant cellular hypertrophy accompanied with a disruption of transverse-axial tubular network and surface flattening. Functionally these cells exhibit higher contractility with lower Ca2+ transients. The structural changes in HF RV myocytes correlate with more frequent spontaneous Ca2+ release activity than in control RV myocytes. This is accompanied by hyperactivated L-type Ca2+ channels (LTCCs) located specifically in the T-tubules of HF RV myocytes. The increased open probability of tubular LTCCs and Ca2+ sparks activation is linked to protein kinase A-mediated channel phosphorylation that occurs locally in T-tubules. Thus, our approach revealed that alterations in RV myocytes in heart failure are specifically localized in microdomains. Our findings may indicate the development of compensatory, though potentially arrhythmogenic, RV remodelling in the setting of LV failure. These data will foster better understanding of mechanisms of heart failure and it could promote an optimized treatment of patients.
Medvedev R, Sanchez-Alonso JL, Alvarez-Laviada A, et al., 2021, Nanoscale Study of Calcium Handling Remodeling in Right Ventricular Cardiomyocytes Following Pulmonary Hypertension, HYPERTENSION, Vol: 77, Pages: 605-616, ISSN: 0194-911X
Gross P, Johnson J, Romero CM, et al., 2021, Interaction of the Joining Region in Junctophilin-2 With the L-Type Ca2+ Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca2+ Dynamics, CIRCULATION RESEARCH, Vol: 128, Pages: 92-114, ISSN: 0009-7330
Poulet C, Sanchez-Alonso J, Swiatlowska P, et al., 2021, Junctophilin-2 tethers T-tubules and recruits functional L-type calcium channels to lipid rafts in adult cardiomyocytes, Cardiovascular Research, Vol: 117, Pages: 149-161, ISSN: 0008-6363
Aim: In cardiomyocytes, transverse tubules (T-tubules) associate with the sarcoplasmic reticulum (SR), forming junctional membrane complexes (JMCs) where L-type calcium channels (LTCCs) are juxtaposed to Ryanodine receptors (RyR). Junctophilin-2 (JPH2) supports the assembly of JMCs by tethering T-tubules to the SR membrane. T-tubule remodeling in cardiac diseases is associated with down-regulation of JPH2 expression suggesting that JPH2 plays a crucial role in T-tubule stability. Furthermore, increasing evidence indicate that JPH2 might additionally act as a modulator of calcium signaling by directly regulating RyR and LTCCs. This study aimed at determining whether JPH2 overexpression restores normal T-tubule structure and LTCC function in cultured cardiomyocytes.Methods and results: Rat ventricular myocytes kept in culture for 4 days showed extensive T-tubule remodeling with impaired JPH2 localization and relocation of the scaffolding protein Caveolin3 (Cav3) from the T-tubules to the outer membrane. Overexpression of JPH2 restored T-tubule structure and Cav3 relocation. Depletion of membrane cholesterol by chronic treatment with Methyl-β-cyclodextrin (MβCD) countered the stabilizing effect of JPH2 overexpression on T-tubules and Cav3. Super-resolution scanning patch-clamp showed that JPH2 overexpression greatly increased the number of functional LTCCs at the plasma membrane. Treatment with MβCD reduced LTCC open probability and activity. Proximity ligation assays showed that MβCD did not affect JPH2 interaction with RyR and the pore-forming LTCC subunit Cav1.2, but strongly impaired JPH2 association with Cav3 and the accessory LTCC subunit Cavβ2. Conclusions: JPH2 promotes T-tubule structural stability and recruits functional LTCCs to the membrane, most likely by directly binding to the channel. Cholesterol is involved in the binding of JPH2 to T-tubules as well as in the modulation of LTCC activity. We propose a model where cholesterol an
Hasan A, Mohammadi N, Nawaz A, et al., 2020, Age-dependent maturation of iPSC-CMs leads to the enhanced compartmentation of beta(2)AR-cAMP signalling, Cells, Vol: 9, ISSN: 2073-4409
The ability to differentiate induced-pluripotent stem cells into cardiomyocytes (iPSC-CMs) has opened up novel avenues for potential cardiac therapies. However, iPSC-CMs exhibit a range of somewhat immature functional properties. This study explored the development of the beta-adrenergic receptor (βAR) pathway, which is crucial in regulating contraction and signifying the health and maturity of myocytes. We explored the compartmentation of β2AR-signalling and phosphodiesterases (PDEs) in caveolae, as functional nanodomains supporting the mature phenotype. Förster Resonance Energy Transfer (FRET) microscopy was used to study the cyclic adenosine monophosphate (cAMP) levels in iPSC-CMs at day 30, 60, and 90 following βAR subtype-specific stimulation. Subsequently, the PDE2, PDE3, and PDE4 activity was investigated using specific inhibitors. Cells were treated with methyl-β-cyclodextrin (MβCD) to remove cholesterol as a method of decompartmentalising β2AR. As iPSC-CMs mature with a prolonged culture time, the caveolae density is increased, leading to a reduction in the overall cytoplasmic cAMP signal stimulated through β2AR (but not β1AR). Pan-phosphodiesterase inhibition or caveolae depletion leads to an increase in the β2AR-stimulated cytoplasmic cAMP. Moreover, with time in culture, the increase in the βAR-dependent cytoplasmic cAMP becomes more sensitive to cholesterol removal. The regulation of the β2AR response by PDE2 and 4 is similarly increased with the time in culture. We conclude that both the β2AR and PDEs are restricted to the caveolae nanodomains, and thereby exhibit a tighter spatial restriction over the cAMP signal in late-stage compared to early iPSC-CMs.
Ferraro E, Pozhidaeva L, Pitcher DS, et al., 2020, Prolonged ursodeoxycholic acid administration reduces acute ischaemia-induced arrhythmias in adult rat hearts, Scientific Reports, Vol: 10, Pages: 1-13, ISSN: 2045-2322
Acute myocardial ischaemiaand reperfusion (I-R) are major causes of ventricular arrhythmias in patients with a history of coronary artery disease. Ursodeoxycholic acid (UDCA)has previously been shown to be antiarrhythmic in fetal hearts.This study was performed to investigate if UDCA protects against ischaemia-induced and reperfusion-induced arrhythmias in the adult myocardium,43andcomparesthe effect of acute (perfusion only) versus prolonged (2 weeks pre-treatment plus perfusion) UDCA administration. Langendorff-perfused adult Sprague-Dawley rat hearts were subjected to acute regional ischaemia by ligation of the left anterior descending artery(10 minutes), followed by reperfusion (2 minutes), and arrhythmia incidence quantified. Prolonged UDCA administration reduced the incidence of acute ischaemia-induced arrhythmias (p=0.028),with a eduction in number of ventricular ectopic beats during the ischaemic phase compared with acute treatment(10±3 vs 58±15, p=0.036).No antiarrhythmic effect was observed in the acute UDCA administration group. Neither acute nor prolonged UDCA treatment altered the incidence of reperfusion arrhythmias.The antiarrhythmic effect of UDCA maybe partially mediated by an increase in cardiac wavelength, due tothe attenuation of conduction velocity slowing (p=0.03), and the preservation of Connexin43 phosphorylation during acute ischaemia(p=0.0027). The potential antiarrhythmic effects of prolonged UDCA administration merit further investigation.
Sintou A, Mansfield C, Iacob A-O, et al., 2020, Mediastinal lymphadenopathy, class-switched auto-antibodies and myocardial immune-complexes during heart failure in rodents and humans, Frontiers in Cell and Developmental Biology, Vol: 8, Pages: 1-12, ISSN: 2296-634X
Mediastinal lymphadenopathy and auto-antibodies are clinical phenomena during ischemicheart failure pointing to an autoimmune response against the heart. T and B cell have beenconvincingly demonstrated to be activated after myocardial infarction, a prerequisite for thegeneration of mature auto-antibodies. Yet, little is known about the immunoglobulin isotyperepertoire thus pathological potential of anti-heart auto-antibodies during heart failure.We obtained human myocardial tissue from ischemic heart failure patients and inducedexperimental MI in rats. We found that anti-heart autoimmunity persists during heart failure.Rat mediastinal lymph nodes are enlarged and contain active secondary follicles with matureisotype-switched IgG2a B cells. Mature IgG2a auto-antibodies specific for cardiac antigens arepresent in rat heart failure serum, and IgG and complement C3 deposits are evident in heartfailure tissue of both rats and human patients.Previously established myocardial inflammation, and the herein provided proof of B cellmaturation in lymph nodes and myocardial deposition of mature auto-antibodies, provide allthe hallmark signs of an established autoimmune response in chronic heart failure.
Swiatlowska P, Sanchez-Alonso JL, Mansfield C, et al., 2020, Short-term angiotensin II treatment regulates cardiac nanomechanics via microtubule modifications., Nanoscale, Vol: 12, Pages: 16315-16329, ISSN: 2040-3364
Mechanical properties of single myocytes contribute to the whole heart performance, but the measurement of mechanics in living cells at high resolution with minimal force interaction remains challenging. Angiotensin II (AngII) is a peptide hormone that regulates a number of physiological functions, including heart performance. It has also been shown to contribute to cell mechanics by inducing cell stiffening. Using non-contact high-resolution Scanning Ion Conductance Microscopy (SICM), we determine simultaneously cell topography and membrane transverse Young's modulus (YM) by a constant pressure application through a nanopipette. While applying pressure, the vertical position is recorded and a deformation map is generated from which YM can be calculated and corrected for the uneven geometry. High resolution of this method also allows studying specific membrane subdomains, such as Z-grooves and crests. We found that short-term AngII treatment reduces the transversal YM in isolated adult rat cardiomyocytes acting via an AT1 receptor. Blocking either a TGF-β1 receptor or Rho kinase abolishes this effect. Analysis of the cytoskeleton showed that AngII depletes microtubules by decreasing long-lived detyrosinated and acetylated microtubule populations. Interestingly, in the failing cardiomyocytes, which are stiffer than controls, the short-term AngII treatment also reduces the YM, thus normalizing the mechanical state of cells. This suggests that the short-term softening effect of AngII on cardiac cells is opposite to the well-characterized long-term hypertrophic effect. In conclusion, we generate a precise nanoscale indication map of location-specific transverse cortical YM within the cell and this can substantially advance our understanding of cellular mechanics in a physiological environment, for example in isolated cardiac myocytes.
Gorelik J, Sanchez-Alonso J, Loucks A, et al., 2020, Nanoscale regulation of L-type calcium channels differentiates between ischemic and dilated cardiomyopathies., EBioMedicine, Vol: 57, Pages: 1-14, ISSN: 2352-3964
BackgroundSubcellular localization and function of L-type calcium channels (LTCCs) play an important role in regulating contraction of cardiomyocytes. Understanding how this is affected by the disruption of transverse tubules during heart failure could lead to new insights into the disease.MethodsCardiomyocytes were isolated from healthy donor hearts, as well as from patients with cardiomyopathies and with left ventricular assist devices. Scanning ion conductance and confocal microscopy was used to study membrane structures in the cells. Super-resolution scanning patch-clamp was used to examine LTCC function in different microdomains. Computational modeling predicted the impact of these changes to arrhythmogenesis at the whole-heart level.FindingsWe showed that loss of structural organization in failing myocytes leads to re-distribution of functional LTCCs from the T-tubules to the sarcolemma. In ischemic cardiomyopathy, the increased LTCC open probability in the T-tubules depends on the phosphorylation by protein kinase A, whereas in dilated cardiomyopathy, the increased LTCC opening probability in the sarcolemma results from enhanced phosphorylation by calcium-calmodulin kinase II. LVAD implantation corrected LTCCs pathophysiological activity, although it did not improve their distribution. Using computational modeling in a 3D anatomically-realistic human ventricular model, we showed how LTCC location and activity can trigger heart rhythm disorders of different severity.InterpretationOur findings demonstrate that LTCC redistribution and function differentiate between disease aetiologies. The subcellular changes observed in specific microdomains could be the consequence of the action of distinct protein kinases.FundingThis work was supported by NIH grant (ROI-HL 126802 to NT-JG) and British Heart Foundation (grant RG/17/13/33173 to JG, project grant PG/16/17/32069 to RAC). Funders had no role in study design, data collection, data analysis, interpretation, writing
Lang D, Calaghan SC, Gorelik J, et al., 2020, Editorial: cardiomyocyte microdomains: an emerging concept of local regulation and remodeling, Frontiers in Physiology, Vol: 11, Pages: 1-3, ISSN: 1664-042X
Rifai SE, Sintou A, Mansfield C, et al., 2020, Humoral factors in serum of rats with chronic heart failure induce cardiomyocyte hypertrophy and reduce viability., Clinical medicine (London, England), Vol: 20, Pages: s107-s107, ISSN: 1470-2118
Schobesberger S, Wright PT, Poulet C, et al., 2020, beta(3)-Adrenoceptor redistribution impairs NO/cGMP/PDE2 signalling in failing cardiomyocytes, eLife, Vol: 9, Pages: 1-15, ISSN: 2050-084X
Cardiomyocyte β3-adrenoceptors (β3-ARs) coupled to soluble guanylyl cyclase (sGC)-dependent production of the second messenger 3’,5’-cyclic guanosine monophosphate (cGMP) have been shown to protect from heart failure. However, the exact localization of these receptors to fine membrane structures and subcellular compartmentation of β3-AR/cGMP signals underpinning this protection in health and disease remain elusive. Here, we used a Förster Resonance Energy Transfer (FRET)-based cGMP biosensor combined with scanning ion conductance microscopy (SICM) to show that functional β3-ARs are mostly confined to the T-tubules of healthy rat cardiomyocytes. Heart failure, induced via myocardial infarction, causes a decrease of the cGMP levels generated by these receptors and a change of subcellular cGMP compartmentation. Furthermore, attenuated cGMP signals led to impaired phosphodiesterase two dependent negative cGMP-to-cAMP cross-talk. In conclusion, topographic and functional reorganization of the β3-AR/cGMP signalosome happens in heart failure and should be considered when designing new therapies acting via this receptor.
Tikhomirov R, Donnell BR-O, Catapano F, et al., 2020, Exosomes: From potential culprits to new therapeutic promise in the setting of cardiac fibrosis, Cells, Vol: 9, ISSN: 2073-4409
Fibrosis is a significant global health problem associated with many inflammatory and degenerative diseases affecting multiple organs, individually or simultaneously. Fibrosis develops when extracellular matrix (ECM) remodeling becomes excessive or uncontrolled and is associated with nearly all forms of heart disease. Cardiac fibroblasts and myofibroblasts are the main effectors of ECM deposition and scar formation. The heart is a complex multicellular organ, where the various resident cell types communicate between themselves and with cells of the blood and immune systems. Exosomes, which are small extracellular vesicles, (EVs), contribute to cell-to-cell communication and their pathophysiological relevance and therapeutic potential is emerging. Here, we will critically review the role of endogenous exosomes as possible fibrosis mediators and discuss the possibility of using stem cell-derived and/or engineered exosomes as anti-fibrotic agents.
Judina A, Gorelik J, Wright PT, 2020, Studying signal compartmentation in adult cardiomyocytes., Biochemical Society Transactions, Vol: 48, Pages: 61-70, ISSN: 0300-5127
Multiple intra-cellular signalling pathways rely on calcium and 3'-5' cyclic adenosine monophosphate (cAMP) to act as secondary messengers. This is especially true in cardiomyocytes which act as the force-producing units of the cardiac muscle and are required to react rapidly to environmental stimuli. The specificity of functional responses within cardiomyocytes and other cell types is produced by the organellar compartmentation of both calcium and cAMP. In this review, we assess the role of molecular localisation and relative contribution of active and passive processes in producing compartmentation. Active processes comprise the creation and destruction of signals, whereas passive processes comprise the release or sequestration of signals. Cardiomyocytes display a highly articulated membrane structure which displays significant cell-to-cell variability. Special attention is paid to the way in which cell membrane caveolae and the transverse-axial tubule system allow molecular localisation. We explore the effects of cell maturation, pathology and regional differences in the organisation of these processes. The subject of signal compartmentation has had a significant amount of attention within the cardiovascular field and has undergone a revolution over the past two decades. Advances in the area have been driven by molecular imaging using fluorescent dyes and genetically encoded constructs based upon fluorescent proteins. We also explore the use of scanning probe microscopy in the area. These techniques allow the analysis of molecular compartmentation within specific organellar compartments which gives researchers an entirely new perspective.
Francis AJ, Firth JM, Islam N, et al., 2020, The Effect of Oestrogen Withdrawal on Ca2+ Regulation and the Influence of GPER1, 64th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 403A-404A, ISSN: 0006-3495
Yang H-Q, Perez-Hernandez M, Sanchez-Alonso JL, et al., 2020, Ankyrin-G Mediates Targeting of both Na+ and K-ATP Channels to the Cardiac Intercalated Disc, 64th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 270A-270A, ISSN: 0006-3495
Swiatlowska P, Sanchez-Alonso JL, Wright PT, et al., 2020, Microtubules regulate cardiomyocyte transversal Young's modulus., Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 2764-2766, ISSN: 0027-8424
The field of cardiomyocyte mechanobiology is gaining significant attention, due to accumulating evidence concerning the significant role of cellular mechanical effects on the integrated function of the heart. To date, the protein titin has been demonstrated as a major contributor to the cardiomyocytes Young's modulus (YM). The microtubular network represents another potential regulator of cardiac mechanics. However, the contribution of microtubules (MTs) to the membrane YM is still understudied and has not been interrogated in the context of myocardial infarction (MI) or mechanical loading and unloading. Using nanoscale mechanoscanning ion conductance microscopy, we demonstrate that MTs contribute to cardiomyocyte transverse YM in healthy and pathological states with different mechanical loading. Specifically, we show that posttranslational modifications of MTs have differing effects on cardiomyocyte YM: Acetylation provides flexibility, whereas detyrosination imparts rigidity. Further studies demonstrate that there is no correlation between the total protein amount of acetylated and detyrosinated MT. Yet, in the polymerized-only populations, an increased level of acetylation results in a decline of detyrosinated MTs in an MI model.
Yang H-Q, Perez-Hernandez M, Sanchez-Alonso J, et al., 2020, Ankyrin-G mediates targeting of both Na+ and K-ATP channels to the rat cardiac intercalated disc, eLife, Vol: 9, ISSN: 2050-084X
We investigated targeting mechanisms of Na+ and KATP channels to the intercalated disk (ICD) of cardiomyocytes. Patch clamp and surface biotinylation data show reciprocal downregulation of each other’s surface density. Mutagenesis of the Kir6.2 ankyrin binding site disrupts this functional coupling. Duplex patch clamping and Angle SICM recordings show that INa and IKATP functionally co-localize at the rat ICD, but not at the lateral membrane. Quantitative STORM imaging show that Na+ and KATP channels are localized close to each other and to AnkG, but not to AnkB, at the ICD. Peptides corresponding to Nav1.5 and Kir6.2 ankyrin binding sites dysregulate targeting of both Na+ and KATP channels to the ICD, but not to lateral membranes. Finally, a clinically relevant gene variant that disrupts KATP channel trafficking also regulates Na+ channel surface expression. The functional coupling between these two channels need to be considered when assessing clinical variants and therapeutics.
Brezovjakova H, Tomlinson C, Mohd Naim N, et al., 2019, Junction Mapper is a novel computer vision tool to decipher cell-cell contact phenotypes., eLife, Vol: 8, Pages: 1-48, ISSN: 2050-084X
Stable cell-cell contacts underpin tissue architecture and organization. Quantification of junctions of mammalian epithelia requires laborious manual measurements that are a major roadblock for mechanistic studies. We designed Junction Mapper as an open access, semi-automated software that defines the status of adhesiveness via the simultaneous measurement of pre-defined parameters at cell-cell contacts. It identifies contacting interfaces and corners with minimal user input and quantifies length, area and intensity of junction markers. Its ability to measure fragmented junctions is unique. Importantly, junctions that considerably deviate from the contiguous staining and straight contact phenotype seen in epithelia are also successfully quantified (i.e. cardiomyocytes or endothelia). Distinct phenotypes of junction disruption can be clearly differentiated among various oncogenes, depletion of actin regulators or stimulation with other agents. Junction Mapper is thus a powerful, unbiased and highly applicable software for profiling cell-cell adhesion phenotypes and facilitate studies on junction dynamics in health and disease.
Ramuz M, Hasan A, Gruscheski L, et al., 2019, A software tool for high-throughput real-time measurement of intensity-based ratio-metric FRET, Cells, Vol: 8, ISSN: 2073-4409
Förster resonance energy transfer (FRET) is increasingly used for non-invasive measurement of fluorescently tagged molecules in live cells. In this study, we have developed a freely available software tool MultiFRET, which, together with the use of a motorised microscope stage, allows multiple single cells to be studied in one experiment. MultiFRET is a Java plugin for Micro-Manager software, which provides real-time calculations of ratio-metric signals during acquisition and can simultaneously record from multiple cells in the same experiment. It can also make other custom-determined live calculations that can be easily exported to Excel at the end of the experiment. It is flexible and can work with multiple spectral acquisition channels. We validated this software by comparing the output of MultiFRET to that of a previously established and well-documented method for live ratio-metric FRET experiments and found no significant difference between the data produced with the use of the new MultiFRET and other methods. In this validation, we used several cAMP FRET sensors and cell models: i) isolated adult cardiomyocytes from transgenic mice expressing the cytosolic epac1-camps and targeted pmEpac1 and Epac1-PLN sensors, ii) isolated neonatal mouse cardiomyocytes transfected with the AKAP79-CUTie sensor, and iii) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) transfected with the Epac-SH74 sensor. The MultiFRET plugin is an open source freely available package that can be used in a wide area of live cell imaging when live ratio-metric calculations are required.
Reilly-O'Donnell B, Patel L, Ferraro E, et al., 2019, Ursodeoxycholic acid and INT-777 reduce markers of fibrosis in heart fibroblasts, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 3039-3039, ISSN: 0007-1188
Schultz F, Swiatlowska P, Alvarez-Laviada A, et al., 2019, Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43., FASEB Journal, Pages: 1-16, ISSN: 0892-6638
Healthy cardiomyocytes are electrically coupled at the intercalated discs by gap junctions. In infarcted hearts, adverse gap-junctional remodeling occurs in the border zone, where cardiomyocytes are chemically and electrically influenced by myofibroblasts. The physical movement of these contacts remains unquantified. Using scanning ion conductance microscopy, we show that intercellular contacts between cardiomyocytes and myofibroblasts are highly dynamic, mainly owing to the edge dynamics (lamellipodia) of the myofibroblasts. Decreasing the amount of functional connexin-43 (Cx43) at the membrane through Cx43 silencing, suppression of Cx43 trafficking, or hypoxia-induced Cx43 internalization attenuates heterocellular contact dynamism. However, we found decreased dynamism and stabilized membrane contacts when cellular coupling was strengthened using 4-phenylbutyrate (4PB). Fluorescent-dye transfer between cells showed that the extent of functional coupling between the 2 cell types correlated with contact dynamism. Intercellular calcein transfer from myofibroblasts to cardiomyocytes is reduced after myofibroblast-specific Cx43 down-regulation. Conversely, 4PB-treated myofibroblasts increased their functional coupling to cardiomyocytes. Consistent with lamellipodia-mediated contacts, latrunculin-B decreases dynamism, lowers physical communication between heterocellular pairs, and reduces Cx43 intensity in contact regions. Our data show that heterocellular cardiomyocyte-myofibroblast contacts exhibit high dynamism. Therefore, Cx43 is a potential target for prevention of aberrant cardiomyocyte coupling and myofibroblast proliferation in the infarct border zone.-Schultz, F., Swiatlowska, P., Alvarez-Laviada, A., Sanchez-Alonso, J. L., Song, Q., de Vries, A. A. F., Pijnappels, D. A., Ongstad, E., Braga, V. M. M., Entcheva, E., Gourdie, R. G., Miragoli, M., Gorelik, J. Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43.
Ali T, Bednarska J, Vassilopoulos S, et al., 2019, Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin, The FASEB Journal, Vol: 33, Pages: 8504-8518, ISSN: 0892-6638
Dynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a wholeclass of dynamin-dependent endocytosis, including clathrin-mediated endocytosis bycaveoli. It has been suggestedthat mutations in theDNM2gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactlymutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used livecorrelative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study howdisease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly followingtheir dynamics of formation, maturation, and internalizationinskinfibroblastsfrompatients with centronuclearmyopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we havedeveloped, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, andsignificantly increases the lifetime of CCPs. Differently,p.R522H slows down the formation of CCPs without affectingtheir internalization. We also found that CNM mutations inDNM2affect the distribution of caveoli and reduce dorsalruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electronmicroscopy data,argue for the impairment of several forms of endocytosis inDNM2-linked CNM.—Ali,T.,Bednarska,J.,Vassilopoulos,S.,Tran,M.,Diakonov,I.A.,Ziyadeh-Isleem,A.,Guicheney,P.,Gorelik,J.,Korchev,Y.E.,Reilly,M.M.,Bitoun,M.,Shevchuk,A.CorrelativeSICM-FCMreveals changes in morphology and kinetics of endocytic pitsinduced by disease-associated mutations in dynamin.
Swiatlowska PP, Sanchez-Alonso J, Wright P, et al., 2019, Single cell mechanics in failing heart: role of microtubules and mitochondria re-arrangement, ESC, Publisher: WILEY, Pages: 316-316, ISSN: 1388-9842
Sintou A, Rifai SE, Mansfield C, et al., 2019, Persistent anti-heart autoimmunity causes cardiomyocyte damage in chronic heart failure, Publisher: bioRxiv
Although clinicians and researchers have long appreciated the detrimental effects of excessive acute inflammation after myocardial infarction (MI), less is known about the role of the adaptive immune system in MI complications including heart failure. Yet, abundant cardiac self-antigens released from necrotic cardiomyocytes in a highly inflammatory environment are likely to overwhelm peripheral mechanisms of immunological self-tolerance and adaptive auto-reactivity against the heart may cause ongoing tissue destruction and exacerbate progression to chronic heart failure (CHF). Here, we confirm that the adaptive immune system is indeed persistently active in CHF due to ischemic heart disease triggered by MI in rats. Heart draining mediastinal lymph nodes contain active secondary follicles with mature class-switched IgG2a positive cells, and mature anti-heart auto-antibodies binding to cardiac epitopes are still present in serum as late as 16 weeks after MI. When applied to healthy cardiomyocytes in vitro, humoral factors present in CHF serum promoted apoptosis, cytotoxicity and signs of hypertrophy. These findings directly implicate post-MI autoimmunity as an integral feature of CHF progression, constituting a roadblock to effective regeneration and a promising target for therapeutic intervention.
Wright PT, Sanchez-Alonso JL, Lucarelli C, et al., 2018, Partial mechanical unloading of the heart disrupts L-type calcium channel and beta-adrenoceptor signaling microdomains, Frontiers in Physiology, Vol: 9, ISSN: 1664-042X
Introduction: We investigated the effect of partial mechanical unloading (PMU) of the heart on the physiology of calcium and beta-adrenoceptor-cAMP (βAR-cAMP) microdomains. Previous studies have investigated PMU using a model of heterotopic-heart and lung transplantation (HTHAL). These studies have demonstrated that PMU disrupts the structure of cardiomyocytes and calcium handling. We sought to understand these processes by studying L-Type Calcium Channel (LTCC) activity and sub-type-specific βAR-cAMP signaling within cardiomyocyte membrane microdomains.Method: We utilized an 8-week model of HTHAL, whereby the hearts of syngeneic Lewis rats were transplanted into the abdomens of randomly assigned cage mates. A pronounced atrophy was observed in hearts after HTHAL. Cardiomyocytes were isolated via enzymatic perfusion. We utilized Förster Resonance Energy Transfer (FRET) based cAMP-biosensors and scanning ion conductance microscopy (SICM) based methodologies to study localization of LTCC and βAR-cAMP signaling.Results: β2AR-cAMP responses measured by FRET in the cardiomyocyte cytosol were reduced by PMU (loaded 28.51 ± 7.18% vs. unloaded 10.84 ± 3.27% N,n 4/10-13 mean ± SEM ∗p < 0.05). There was no effect of PMU on β2AR-cAMP signaling in RII_Protein Kinase A domains. β1AR-cAMP was unaffected by PMU in either microdomain. Consistent with this SICM/FRET analysis demonstrated that β2AR-cAMP was specifically reduced in t-tubules (TTs) after PMU (loaded TT 0.721 ± 0.106% vs. loaded crest 0.104 ± 0.062%, unloaded TT 0.112 ± 0.072% vs. unloaded crest 0.219 ± 0.084% N,n 5/6-9 mean ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001 vs. loaded TT). By comparison β1AR-cAMP responses in either TT or sarcolemmal crests were unaffected by the PMU. LTCC occurrence and open probability (Po) were reduced by PMU (loaded TT Po 0.073 ± 0.011% vs. load
Bastug-Özel Z, Wright PT, Kraft AE, et al., 2018, Heart failure leads to altered b2-adrenoceptor/cyclic adenosine monophosphate dynamics in the sarcolemmal phospholemman/Na,KATPase microdomain, Cardiovascular Research, Vol: 115, Pages: 546-555, ISSN: 1755-3245
Aims: Cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling by acting in microdomains associated with sarcolemmal ion channels. However, local real time cAMP dynamics in such microdomains has not been visualized before. We sought to directly monitor cAMP in a microdomain formed around sodium-potassium ATPase (NKA) in healthy and failing cardiomyocytes and to better understand alterations of cAMP compartmentation in heart failure. Methods and Results: A novel Förster resonance energy transfer (FRET)-based biosensor termed PLM-Epac1 was developed by fusing a highly sensitive cAMP sensor Epac1-camps to the C-terminus of phospholemman (PLM). Live cell imaging in PLM-Epac1 and Epac1-camps expressing adult rat ventricular myocytes revealed extensive regulation of NKA/PLM microdomain associated cAMP levels by β2-adrenoceptors (β2-ARs). Local cAMP pools stimulated by these receptors were tightly controlled by phosphodiesterase (PDE) type 3. In chronic heart failure following myocardial infarction, dramatic reduction of the microdomain-specific β2-AR/cAMP signals and β2-AR dependent PLM phosphorylation was accompanied by a pronounced loss of local PDE3 and an increase in PDE2 effects. Conclusions: NKA/PLM complex forms a distinct cAMP microdomain which is directly regulated by β2-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted β2-AR signaling to cAMP in the vicinity of PLM.
Veeraraghavan R, Hoeker GS, Alvarez-Laviada A, et al., 2018, The adhesion function of the sodium channel beta subunit (beta 1) contributes to cardiac action potential propagation, eLife, Vol: 7, ISSN: 2050-084X
Computational modeling indicates that cardiac conduction may involve ephaptic coupling – intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that β1(SCN1B) –mediated adhesion scaffolds trans-activating NaV1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential β1 localization at the perinexus, where it co-locates with NaV1.5. Smart patch clamp (SPC) indicated greater sodium current density (INa) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, βadp1, potently and selectively inhibited β1-mediated adhesion, in electric cell-substrate impedance sensing studies. βadp1 significantly widened perinexi in guinea pig ventricles, and selectively reduced perinexal INa, but not whole cell INa, in myocyte monolayers. In optical mapping studies, βadp1 precipitated arrhythmogenic conduction slowing. In summary, β1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies.
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