Abstract
The respiratory epithelium is a significant target of inhaled, nano-sized particles, the biological reactivity of which will depend on its physicochemical properties. Surface-modified, 50 ...and 100 nm, polystyrene latex nanoparticles (NPs) were used as model particles to examine the effect of particle size and surface chemistry on transformed human alveolar epithelial type 1-like cells (TT1). Live images of TT1 exposed to amine-modified NPs taken by hopping probe ion conductance microscopy revealed severe damage and holes on cell membranes that were not observed with other types of NPs. This paralleled induction of cell detachment, cytotoxicity and apoptotic (caspase-3/7 and caspase-9) cell death, and increased release of CXCL8 (IL-8). In contrast, unmodified, carboxyl-modified 50 nm NPs and the 100 nm NPs did not cause membrane damage, and were less reactive. Thus, the susceptibility and membrane damage to respiratory epithelium following inhalation of NPs will depend on both surface chemistry (e.g., cationic) and nano-size.
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IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Current knowledge of the structural changes taking place during clathrin-mediated endocytosis is largely based on electron microscopy images of fixed preparations and x-ray crystallography data of ...purified proteins. In this paper, we describe a study of clathrin-coated pit dynamics in living cells using ion conductance microscopy to directly image the changes in pit shape, combined with simultaneous confocal microscopy to follow molecule-specific fluorescence. We find that 70% of pits closed with the formation of a protrusion that grew on one side of the pit, covered the entire pit, and then disappeared together with pit-associated clathrin-enhanced green fluorescent protein (EGFP) and actin-binding protein-EGFP (Abp1-EGFP) fluorescence. This was in contrast to conventionally closing pits that closed and cleaved from flat membrane sheets and lacked accompanying Abp1-EGFP fluorescence. Scission of both types of pits was found to be dynamin-2 dependent. This technique now enables direct spatial and temporal correlation between functional molecule-specific fluorescence and structural information to follow key biological processes at cell surfaces.
Abstract Human embryonic stem cell-derived cardiomyocytes (hESC-CM) are being developed for tissue repair and as a model system for cardiac physiology and pathophysiology. However, the signaling ...requirements of their growth have not yet been fully characterized. We showed that hESC-CM retain their capacity for increase in size in long-term culture. Exposing hESC-CM to hypertrophic stimuli such as equiaxial cyclic stretch, angiotensin II, and phenylephrine (PE) increased cell size and volume, percentage of hESC-CM with organized sarcomeres, levels of ANF, and cytoskeletal assembly. PE effects on cell size were separable from those on cell cycle. Changes in cell size by PE were completely inhibited by p38–MAPK, calcineurin/FKBP, and mTOR blockers. p38–MAPK and calcineurin were also implicated in basal cell growth. Inhibitors of ERK, JNK, and CaMK II partially reduced PE effects; PKG or GSK3β inhibitors had no effect. The role of p38–MAPK was confirmed by an additional pharmacological inhibitor and adenoviral infection of hESC-CM with a dominant-inhibitory form of p38–MAPK. Infection of hESC-CM with constitutively active upstream MAP2K3b resulted in an increased cell size, sarcomere and cytoskeletal assembly, elongation of the cells, and induction of ANF mRNA levels. siRNA knockdown of p38–MAPK inhibited PE-induced effects on cell size. These results reveal an important role for active protein kinase signaling in hESC-CM growth and hypertrophy, with potential implications for hESC-CM as a novel in vitro test system. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Intrahepatic cholestasis of pregnancy (ICP) is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that ...bile acid taurocholate (TC), which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM). Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart.
Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M(2) receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M(2) receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters.
We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M(2) receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cardiomyocytes from the apex but not the base of the heart increase their contractility in response to β2-adrenoceptor (β2AR) stimulation, which may underlie the development of Takotsubo ...cardiomyopathy. However, both cell types produce comparable cytosolic amounts of the second messenger cAMP. We investigated this discrepancy using nanoscale imaging techniques and found that, structurally, basal cardiomyocytes have more organized membranes (higher T-tubular and caveolar densities). Local membrane microdomain responses measured in isolated basal cardiomyocytes or in whole hearts revealed significantly smaller and more short-lived β2AR/cAMP signals. Inhibition of PDE4, caveolar disruption by removing cholesterol or genetic deletion of Cav3 eliminated differences in local cAMP production and equilibrated the contractile response to β2AR. We conclude that basal cells possess tighter control of cAMP because of a higher degree of signaling microdomain organization. This provides varying levels of nanostructural control for cAMP-mediated functional effects that orchestrate macroscopic, regional physiological differences within the heart.
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•Cardiomyocyte membrane organization varies in degree between regions of the heart•Differences in structural organization affect adrenergic signaling via β2AR•Reduced organization allows β2AR-cAMP to influence contractility in myocardial apex•Variability in cell structure may allow differential response of heart regions
Wright et al. present evidence that cardiomyocyte membrane organization (T-tubule regularity and caveolar number) varies between myocardial regions. The reduced membrane organization of cells from the myocardial apex allows β2AR-cAMP to influence PKA_RII domains. As a result, β2AR stimulation enhances apically but not basally derived cardiomyocyte contractility.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background:
The increased risk of post-menopausal women developing abnormalities of heart function emphasises the requirement to understand the effect of declining oestrogen levels on cardiac ...electrophysiology and structure, and investigate possible therapeutic targets, namely the G protein-coupled oestrogen receptor 1 (GPER).
Methods:
Female guinea pigs underwent sham or ovariectomy (OVx) surgeries. Cardiomyocytes were isolated 150-days post-operatively. Membrane structure was assessed using di-8-ANEPPs staining and scanning ion conductance microscopy. Imunnohistochemistry (IHC) determined the localisation of oestrogen receptors. The effect of GPER activation on excitation-contraction coupling mechanisms were assessed using electrophysiological and fluorescence techniques. Downstream signalling proteins were investigated by western blot.
Results:
IHC staining confirmed the presence of nuclear oestrogen receptors and GPER, the latter prominently localised to the peri-nuclear region and having a clear striated pattern elsewhere in the cells. Following OVx, GPER expression increased and its activation reduced Ca
2+
transient amplitude (by 40%) and sarcomere shortening (by 32%). In these cells, GPER activation reduced abnormal spontaneous Ca
2+
activity, shortened action potential duration and limited drug-induced early after-depolarisation formation.
Conclusion:
In an animal species with comparable steroidogenesis and cardiac physiology to humans, we show the expression and localisation of all three oestrogen receptors in cardiac myocytes. We found that following oestrogen withdrawal, GPER expression increased and its activation limited arrhythmogenic behaviours in this low oestrogen state, indicating a potential cardioprotective role of this receptor in post-menopausal women.
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
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