scholarly journals Calcium mediated functional interplay between myocardial cells upon laser-induced single-cell injury: an in vitro study of cardiac cell death signaling mechanisms

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Krishna Chander Sridhar ◽  
Nils Hersch ◽  
Georg Dreissen ◽  
Rudolf Merkel ◽  
Bernd Hoffmann
Keyword(s):  
Cell Death ◽  
Single Cell ◽  
Cell Injury ◽  
Specific Effect ◽  
Myocardial Cell ◽  
Myocardial Tissue ◽  
Cell Clusters ◽  
Signaling Events ◽  
Intracellular Ca

Abstract Background The electromechanical function of myocardial tissue depends on the intercellular communication between cardiomyocytes (CMs) as well as their crosstalk with other cell types. Cell injury, and subsequent death trigger inflammation as in myocardial infarction (MI) resulting in myocardial remodeling. Although mechanisms underlying myocardial cell death have been studied so far, the signaling events following single cell death and spontaneous response of connected cells in the myocardial tissue is still barely understood. Methods Here, we investigated the effect of laser-induced single cell death on Calcium (Ca2+) concentrations and transport in myocardial cell clusters in vitro. Spatial and temporal changes in intracellular Ca2+ concentrations [Ca2+]i were studied using a fluorescent calcium indicator, Fluo-4AM. Spontaneous signaling events following cell death were studied in rat embryonic cardiomyocytes and non-myocytes using separate cell culture systems. Results Cell death triggered spontaneous increase in intracellular Ca2+ levels ([Ca2+]i) of surrounding cells. The spread of the observed propagating Ca2+ signal was slow and sustained in myocytes while it was rapid and transient in fibroblasts (Fbs). Further, sustained high Ca2+ levels temporarily impaired the contractility in CMs. The cell-type specific effect of ablation was confirmed using separate cultures of CMs and Fbs. Comparing Ca2+ propagation speed in myocytes and fibroblasts, we argue for a diffusion-driven Ca2+ propagation in myocytes, but not in fibroblasts. Radial and sequential Ca2+ diffusion across the CMs through cell–cell contacts and presence of Cx43-based intercellular junctions indicated a gap junction flow of Ca2+. Conclusions These findings illustrate the spontaneous Ca2+-mediated functional interplay in myocardial cell clusters upon mechanical injury and, further, the difference in Ca2+ signaling in cardiomyocytes and fibroblasts.

scholarly journals Single Cell Transcriptome Analysis Reveals Disease-Defining T Cell Subsets in the Tumor Microenvironment of Classic Hodgkin Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 547-547
Author(s):  
Tomohiro Aoki ◽  
Lauren C. Chong ◽  
Katsuyoshi Takata ◽  
Katy Milne ◽  
Monirath Hav ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Research Funding ◽  
Cell Clusters ◽  
Mhc Ii ◽  
Spatial Profiling ◽  
Classic Hodgkin Lymphoma ◽  
Cellular Components

INTRODUCTION: Classic Hodgkin lymphoma (cHL) is uniquely characterized by an extensively dominant microenvironment composed primarily of different types of non-cancerous immune cells with a rare population (~1%) of tumor cells. Detailed characterization of these cellular components and their spatial relationship is crucial to understand crosstalk and therapeutic targeting in the cellular ecosystem of the tumor microenvironment (TME). METHODS: In this study, we performed high dimensional and spatial profiling of immune cells in the TME of cHL. Single cell RNA sequencing (scRNA-seq) was performed with the 10x Genomics platform on cell suspensions collected from lymph nodes of 22 cHL patients, including 12 of nodular sclerosis subtype, 9 of mixed cellularity subtype and 1 of lymphocyte-rich subtype, with 5 reactive lymph nodes (RLNs) serving as normal controls. Illumina sequencing (HiSeq 2500) was performed to yield single-cell expression profiles for 127,786 cells. We also performed multicolor IHC and imaging mass cytometry (IMC) on TMA slides from the same patients. RESULTS: Unsupervised clustering using PhenoGraph identified 22 cell clusters including 12 T cell clusters, 7 B cell clusters and 1 macrophage cluster. While most immune cell populations were common between cHL and RLN, we observed an enrichment of cells from cHL in all 3 regulatory T cell (Treg) clusters. The most cHL-enriched cluster was characterized by high expression of LAG3, in addition to common Treg markers such as IL2RA (CD25) and TNFRSF18 (GITR), but lacked expression of FOXP3, consistent with a type 1 regulatory (Tr1) T cell population. LAG3+ T cells in cHL had high expression of immune-suppressive cytokines IL-10 and TGF-b . In vitro exposure of T cells to cHL cell line supernatant induced significantly higher levels of LAG3 in naïve T cells compared to co-culture with other lymphoma cell line supernatant or medium only. CD4+ LAG3+ T cells isolated by FACS also suppressed the proliferation of responder CD4+ T cells when co-cultured in vitro. Additionally, Luminex analysis revealed that cHL cell lines secrete substantial amounts of cytokines and chemokines that can promote Tr1 cell differentiation (e.g. IL-6). Our scRNA-seq analysis revealed that LAG3 expression was significantly higher in cHL cases with loss of major histocompatibility class II (MHC-II) expression on HRS cells as compared to MHC-II positive cases (P = 0.019), but was not correlated with EBV status or histological subtype. Strikingly, LAG3 was identified as the most up-regulated gene in cells from MHC-II negative cases compared to MHC-II positive cases. Topological analysis using multicolor IHC and IMC revealed that in MHC-II negative cases, HRS cells were surrounded by LAG3+ T cells. In these cases, the density of LAG3+ T cells in HRS cell-rich regions was significantly increased, and the average distance between an HRS cell and its closest LAG3+ T cell neighbor was significantly shorter. These associations were confirmed in an independent cohort of 166 cHL patients. Finally, we observed a trend towards an inferior disease-specific survival (DSS; P = 0.072) and overall survival (OS; P = 0.12) in cases with an increased number of LAG3+ T cells. A high proportion of LAG3+ T cells (> 20%) was identified as an independent prognostic factor for DSS by multivariate Cox regression. CONCLUSIONS: Our results reveal a diverse TME composition with inflammatory and immunosuppressive cellular components that are linked to MHC class II expression status on HRS cells (Figure). Unprecedented transcriptional and spatial profiling at the single cell level has established the pathogenic importance of HRS cell-induced CD4+ LAG3+ T cells as a mediator of immunosuppression in cHL, with potential implications for novel therapeutic approaches. Figure Disclosures Savage: Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding; BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria. Scott:Roche/Genentech: Research Funding; Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

scholarly journals Comparative transcriptional analysis of the satellite glial cell injury response

2021 ◽  
Author(s):  
Sara Elgaard Jager ◽  
Lone Tjener Pallesen ◽  
Lin Lin ◽  
Francesca Izzi ◽  
Alana Miranda Pinheiro ◽  
...  
Keyword(s):  
Sciatic Nerve ◽  
Single Cell ◽  
Nerve Injury ◽  
Cell Injury ◽  
Cholesterol Biosynthesis ◽  
Transcriptional Analysis ◽  
Sciatic Nerve Crush ◽  
Injury Response ◽  
Satellite Glial Cells

Satellite glial cells (SGCs) tightly surround and support primary sensory neurons in the peripheral nervous system and are increasingly recognized for their involvement in the development of neuropathic pain following nerve injury. The SGCs are difficult to investigate due to their flattened shape and tight physical connection to neurons in vivo and their rapid changes in phenotype and protein expression when cultured in vitro. Consequently, several aspects of SGC function under normal conditions as well as after a nerve injury remain to be explored. The recent advance in single cell RNAseq technologies has enabled a new approach to investigate SGCs. Here we publish a dataset from mice subjected to sciatic nerve injury as well as a dataset from dorsal root ganglia cells after 3 days in culture. We use a meta-analysis approach to compare the injury response with that in other published datasets and conclude that SGCs share a common signature following sciatic nerve crush and sciatic ligation, involving transcriptional regulation of cholesterol biosynthesis. We also observed a considerable transcriptional change when culturing SGCs, suggesting that some differentiate into a specialised in vitro state, while others start resembling Schwann cell-like precursors. The datasets are available via the Broad Institute Single Cell Portal.

Abstract 146: Biphasic Effect Of Cyclic Amp Axis On Cardiomyocyte Survival

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Morihiko Aoyama ◽  
Yasuko K Bando ◽  
Haruya Kawase ◽  
Akio Monji ◽  
Toyoaki Murohara
Keyword(s):  
Cell Death ◽  
Cyclic Amp ◽  
Pressure Overload ◽  
Myocardial Cell ◽  
Threshold Concentration ◽  
High Concentration ◽  
Neonatal Cardiomyocytes ◽  
Myocardial Apoptosis ◽  
Vitro Analysis

Background: Persistent cardiac hypertrophy in response to pathological stimuli is results in maladaptive myocardial remodeling and cell death. Clinical evidence revealed that one of the most significantly beneficial medications for targeting heart failure with pathologic myocardial hypertrophy is beta1-adrenergic receptor (β1R) blockers. The molecular pathway of β1R is mediated by the second messenger cyclic AMP (cAMP). However, there are some debate regarding the role of cAMP in myocardial survival. We hypothesized whether there may be threshold concentration of cAMP in cell susceptibility to cardiomyocyte cell death. Methods: Male 14-week-old C57BL6 mice were subjected to the surgery of thoracic aortic constriction (TAC) to induce pressure overload. Changes in apoptosis were evaluated in each heart section and in vitro culture of neonatal cardiomyocytes using TUNEL. To elucidate the concentration-dependent distinct effect of cAMP on myocardial cell death, we tested the different concentration of cell-permeable cAMP (8-br-cAMP) at low (60 μM) and high concentration (6 mM), and receptor-mediated cAMP-stimulators (Ex4; exendin-4, ISO; isoproterenol). Results: In vitro analysis revealed that the high-cAMP and ISO exhibited marked increase in TUNEL-positivity (15.46%±3.09% for high-cAMP versus 6.71%±0.33% for ISO), which was reversed by Rp-cAMP (1.80%±0.17% and 2.05%±0.25%, respectively). Unexpectedly, the 8-p-Methoxyphenylthon-2-O-methyl-cAMP (pMe-cAMP, 50 μM), the specific activator of another cAMP-sensitive target Epac, reversed the high-cAMP-induced cell death even at a less extent compared to that observed by PKA-inhibitor Rp-cAMP (3.73%±0.70%). Serum depletion induced 3.22±0.24% of TUNEL-positive cell count of NRVM, which was reversed by pMe-cAMP, (50 μM) and Ex4 (1.74±0.18%, n=6, P<0.01), which was insensitive to PKA inhibition by Rp-cAMP (100 μM). TAC increased myocardial apoptosis. TAC-CON heart exhibited 1.66-fold decrease in cardiac cAMP concentration compared to sham-CON. Ex4 ameliorated the TAC-induced cardiac dysfunction and apoptosis by increase in cAMP. Conclusions: The cAMP-related cell death was mediated by PKA activation, which were reversed by Epac activation.

Abstract 346: Lipophilic Statins Attenuate Human Atrial Fibroblast Viability In Vitro

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Kiranjit K Sran ◽  
Yun Li ◽  
Saeid Ghavami ◽  
Melanie Ngo ◽  
Rakesh C Arora ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Heart Surgery ◽  
Cardiac Fibrosis ◽  
Therapeutic Option ◽  
Open Heart Surgery ◽  
Myocardial Cell ◽  
Dependent Manner ◽  
Vitro Effect

Cardiovascular diseases (CVD) leading to heart failure are associated with myocardial cell loss and cardiac fibrosis. Hydroxymethylglutaryl-Coenzyme-A Reductase (HMGR) inhibitors ("statins") are widely used to limit cardiovascular events in patients with hypercholesterolemia and CVD by altering their lipid profile. HMGR inhibition reduces cholesterol precursor L-mevalonate production, whose depletion induces autophagy, apoptosis, and endoplasmic reticulum stress in various cell types. However it is unclear if this is a class effect or a phenomenon specific to various compounds. We examined the in vitro effect of HMGR inhibition on human atrial fibroblast (hATF) viability with particular reference to hydrophilic vs lipophilic compounds. Hypothesis- Lipophilic statins induce cell death in primary hATF via mevalonate depletion; whereas hydrophilic statins do not have any effect on hATF viability. IRB approval was obtained for collection of hATF from consenting patients undergoing open heart surgery. Cells were treated with atorvastatin, simvastatin or pravastatin (0.1, 1.0 or 10 λM) for 24, 48, 72 or 96 hours. Expression of proteins involved in the regulation of apoptosis and autophagy was assessed using immunoblotting. Cell viability was assessed using MTT assay. Treatment of hATF with 0.1 - 10 λM atorvastatin or simvastatin (lipophilic statins) resulted in progressively reduced cell viability in time and dose-dependent manner. Viability could be rescued by coincubation with mevalonate. Expression of key apoptotic cascade proteins -Bcl2, Bax and cleaved Caspase3 showed a clear induction of apoptosis. Also, there was an increase in Atg5-12 expression at 24h indicating induction of early autophagic response. Pravastatin (hydrophilic statin) did not affect cell viability or autophagy and apoptosis. We conclude that statin-induced cell death is mediated by mevalonate depletion, which activates intrinsic apoptotic pathways in hATF. Lipophilic statins impair the viability of hATFs in vitro, whereas hydrophilic statins have no effect on cell growth and cell viability of hATFs. This may represent an additional pleiotropic effect of statins, and may represent a novel therapeutic option for the prevention and treatment of cardiac fibrosis.

scholarly journals Role of Meconium and Hypoxia in Meconium Aspiration-Induced Lung Injury in Neonatal Rabbits

2010 ◽  
Vol 2010 ◽  
pp. 1-6 ◽  
Author(s):  
Alex Zagariya ◽  
Monica Sierzputovska ◽  
Shan Navale ◽  
Dharmapuri Vidyasagar
Keyword(s):  
Cell Death ◽  
Lung Injury ◽  
Cell Injury ◽  
Apoptotic Cell ◽  
A549 Cells ◽  
Cell Loss ◽  
Lung Lavage ◽  
Cytokine Expression ◽  
Meconium Aspiration

Background. We previously showed that meconium cuases lung cell death by apoptosis and inflammatory cytokine expression. Whether this is due to meconium exposure itself, or meconium related hypoxia remains unclear.Objectives. To elucidate the effects of meconium, saline, milk, hypoxia and hyperoxia induced lung injury.Design/Methods. We studied 5 groups of rabbit pups: (I) normal saline; (II) Milk; (III) 10% solution of meconium; (IV) only to 15 minutes of hypoxia (10% O2), and (V) 5 minutes of hypoxia (95% O2). After exposure lung lavage cells were used for apoptotic cell count and cytokine expression.In vitroresponse of human A 549 epithelial cells to meconium-and milk exposure was also studied.Results. There was no difference in cell death between saline and milk groups. However, meconium caused a significant cell loss compared to saline and milk—Inflammatory cytokines increased significantly in meconium group compared to saline or milk group. Although hypoxic and hyperoxic lungs showed increased inflammatory reaction compared to saline-treated lungs, this injury was not significant compared to meconium group. Studies with A549 cells also showed similar results.Conclusions. We conclude that lung cell injury in meconium aspiration is maily from meconium itself.

scholarly journals Evaluation of the Cytopathicity (Fusion/Hemifusion) of Patient-Derived HIV-1 Envelope Glycoproteins Comparing Two Effector Cell Lines

2012 ◽  
Vol 17 (6) ◽  
pp. 727-737 ◽  
Author(s):  
Francesc Cunyat ◽  
Marta Curriu ◽  
Silvia Marfil ◽  
Elisabet García ◽  
Bonaventura Clotet ◽  
...  
Keyword(s):  
Cell Death ◽  
Single Cell ◽  
Cell Lines ◽  
Effector Cell ◽  
Cell Loss ◽  
Biological Properties ◽  
In Vitro Assays ◽  
Envelope Glycoproteins ◽  
Hiv 1

HIV-1 envelope glycoprotein (Env) is a major determinant of viral pathogenicity. The evaluation of the biological properties of patient-derived envelopes by comparing two effector cell lines (293T and HeLa) is reported. A standard cell-to-cell fusion assay was used to evaluate fusogenicity, whereas a coculture with CD4+ cells was used to evaluate absolute cell loss, single cell death, and hemifusion events. Fusion and absolute cell loss assays showed that Env-expressing 293T and HeLa cells had different fusion efficiencies; fusion was magnified in 293T cells despite a significantly lower cell-surface Env expression. Conversely, gp41-mediated single cell death and hemifusion induced in CD4+ cells by 293T-Env-positive cells were significantly lower than that induced by HeLa-Env-positive cells. These data showed that the effector cell line used in the in vitro assays is crucial, and a combination of assays is recommended to evaluate the biological properties of patient-derived envelope glycoproteins: preferentially, 293T-Env-positive cells for the evaluation of fusogenicity and HeLa-Env-positive cells for the evaluation of cell death parameters. The combination of assays described in our work could be a valuable tool for dual screenings of large collections of primary Envs or Env mutants and drugs acting on these Envs.

Intracellular pH regulation of normal and hypertrophic rat myocardium

1979 ◽  
Vol 47 (4) ◽  
pp. 651-656 ◽  
Author(s):  
N. C. Gonzalez ◽  
H. G. Wemken ◽  
N. Heisler
Keyword(s):  
Myocardial Hypertrophy ◽  
Ph Regulation ◽  
Myocardial Cell ◽  
Cellular Level ◽  
Myocardial Tissue ◽  
Intracellular Ph Regulation ◽  
Buffer Value ◽  
Bicarbonate Uptake ◽  
Tissue Homogenates

The myocardial cell pH (pHi) observed during breathing of 0, 7.5, or 10% CO2 in air for 3 h was studied in rats with myocardial hypertrophy due to aortic stenosis and in sham-operated rats. The change in pHi during hypercapnia was significantly smaller in the rats with myocardial hypertrophy, with the apparent nonbicarbonate buffer value (delta [HCO3-]i/delta pHi) being almost three times that of the sham-operated rats. In vitro CO2 equilibrium of myocardial tissue homogenates showed no difference in nonbicarbonate buffer value between homogenates obtained from normal rats and from rats with myocardial hypertrophy. Therefore, it appears that the increased ability of the myocardial cell to regulate its pH during hypertrophy is not due to an increase in the cellular level of nonbicarbonate buffers, but seems to be related to a larger bicarbonate uptake by the myocardial cell during hypercapnia.

Abstract 23: Disruption of TRAF2-TAK1-NF-κB Signaling Axis Triggers K-48 Linked Poly-Ubiquitylation of RIP1 and Necrotic Cell Death in Doxorubicin Cardiotoxicity

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Rimpy Dhingra ◽  
Victoria Margulets ◽  
Floribeth Aguilar ◽  
Lorrie A. Kirshenbaum
Keyword(s):  
Cell Death ◽  
Cardiac Myocytes ◽  
Cell Injury ◽  
Ventricular Myocytes ◽  
Necrotic Cell Death ◽  
Ros Production ◽  
Necrotic Cell ◽  
Interacting Protein

The anthracycline doxorubicin (Dox) is a highly effective anti-tumour agent, however, its use is limited by its severe cardiotoxic effects that manifests as heart failure. The decline in cardiac performance induced by doxorubicin remains poorly defined. A critical survival role for the canonical IKKβ -mTOR-NF-κB signaling pathway has been demonstrated in ventricular myocytes. In this report, we demonstrate that, Dox impairs IKKβ-mTOR- NF-κB signaling in ventricular myocytes accompanied by mitochondrial perturbations including mPTP, loss of mitochondrial membrane potential and ROS production. IKKβ- NF-κB signaling involves TRAF 2 mediated ligation of K63- ubiquitin chains to RIP1 (Receptor Interacting Protein 1) which serves as scaffold for recruitment of ubiquitylated Tak1 complexes and phosphorylation-dependent activation of IKKβ -NF-kB signaling. Interestingly, ventricular myocytes treated with dox demonstrated reduction in expression levels of TRAF2 and TAK1, in vivo and in vitro. This was accompanied by a decline in K63- and concomitant increase in K-48 linked polyubiquitination on RIP1, impaired NF-kB activation and necrotic cell death of cardiac myocytes. Interestingly, inhibiting the kinase activity of RIP1 with Necrostatin-1, (Nec1) suppressed necrotic cell injury induced by dox but not NF-kB activation. Concordant with these findings was a marked increase in necrotic cell death in cardiac myocytes defective for IKKB signaling or MEF cells deficient for p65 treated with dox. Notably, mitochondrial perturbations, including PT-pore opening , ROS production, calcium uptake, LDH, Tn(T) and HMGB-1 release and necrotic cell injury induced by dox were completely abrogated by restoring NF-kB signaling in cardiac myocytes or Nec-1. Herein, we provide novel evidence that K-48 linked poly ubiquitylation of RIP1 provides a functional switch that impairs NF-kB activation and signals necrosis in cells treated with dox. Interventions that modulate NF-kB activity may prove beneficial in mitigating the cardiotoxic effects of dox.

scholarly journals Exosomes from MiR-126-Overexpressing Adscs Are Therapeutic in Relieving Acute Myocardial Ischaemic Injury

2017 ◽  
Vol 44 (6) ◽  
pp. 2105-2116 ◽  
Author(s):  
Qiancheng Luo ◽  
Dongfeng Guo ◽  
Guorong Liu ◽  
Guo Chen ◽  
Min Hang ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cardiac Fibrosis ◽  
Cell Injury ◽  
Myocardial Cell ◽  
In Vivo Studies ◽  
Protective Mechanism ◽  
Ischaemic Injury ◽  
Myocardial Cell Injury

Background/Aims: Recent studies have indicated that exosomes play an important role in adipose-derived stem cell (ADSC) transplant-mediated ischaemic heart disease therapy. However, the treatment effect is not obvious. The aim of this study is to investigate whether ADSC-derived exosomes enriched with microRNA (miR)-126 have a more protective effect on acute myocardial infarction (AMI). Methods: Exosomes were characterized by transmission electron microscopy, and the exosome particles were further examined using nanoparticle tracking analyses. A rat model of myocardial infarction and in vitro model of hypoxia-induced H9c2 myocardial cell injury were established to study the protective mechanism of exosomes from miR-126-overexpressing ADSCs. Results: The in vitro results showed that exosomes derived from miR-126-overexpressing ADSCs decreased H9c2 myocardial cell injury by reducing inflammation factor expression during hypoxia induction. The miR-126-enriched exosomes also decreased the expression of fibrosis-related proteins of H9c2 cells under hypoxic conditions. Matrigel® and Transwell® assays showed that miR-126-enriched exosomes significantly promoted microvascular generation and migration, respectively. In vivo studies confirmed that exosomes derived from ADSCs significantly decreased the myocardial injury area of infarction, especially after miR-126-enriched exosome treatment. Cardiac fibrosis and inflammatory cytokine expression were also decreased after treatment with miR-126-enriched exosomes. However, blood vessel formation was promoted in the infarction region of AMI rats. Conclusions: The results suggested that the expression of miR-126-enhanced ADSC-derived exosomes prevented myocardial damage by protecting myocardial cells from apoptosis, inflammation, fibrosis, and increased angiogenesis.

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