scholarly journals Optogenetic current in myofibroblasts acutely alters electrophysiology and conduction of co-cultured cardiomyocytes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Geran M. Kostecki ◽  
Yu Shi ◽  
Christopher S. Chen ◽  
Daniel H. Reich ◽  
Emilia Entcheva ◽  
...  
Keyword(s):  
Electrical Conductance ◽  
Tissue Level ◽  
Neonatal Rat ◽  
Light Levels ◽  
In Silico Modeling ◽  
Life Threatening ◽  
Experimental Findings ◽  
Rat Study ◽  
Cardiac Myofibroblasts

AbstractInteractions between cardiac myofibroblasts and myocytes may slow conduction and generate spontaneous beating in fibrosis, increasing the chance of life-threatening arrhythmia. While co-culture studies have shown that myofibroblasts can affect cardiomyocyte electrophysiology in vitro, the extent of myofibroblast-myocyte electrical conductance in a syncytium is unknown. In this neonatal rat study, cardiac myofibroblasts were transduced with Channelrhodopsin-2, which allowed acute and selective increase of myofibroblast current, and plated on top of cardiomyocytes. Optical mapping revealed significantly decreased conduction velocity (− 27 ± 6%, p < 10–3), upstroke rate (− 13 ± 4%, p = 0.002), and action potential duration (− 14 ± 7%, p = 0.004) in co-cultures when 0.017 mW/mm2 light was applied, as well as focal spontaneous beating in 6/7 samples and a decreased cycle length (− 36 ± 18%, p = 0.002) at 0.057 mW/mm2 light. In silico modeling of the experiments reproduced the experimental findings and suggested the light levels used in experiments produced excess current similar in magnitude to endogenous myofibroblast current. Fitting the model to experimental data predicted a tissue-level electrical conductance across the 3-D interface between myofibroblasts and cardiomyocytes of ~ 5 nS/cardiomyocyte, and showed how increased myofibroblast-myocyte conductance, increased myofibroblast/myocyte capacitance ratio, and increased myofibroblast current, which occur in fibrosis, can work in tandem to produce pro-arrhythmic increases in conduction and spontaneous beating.

scholarly journals Optogenetic currents in myofibroblasts acutely alter electrophysiology and conduction of co-cultured cardiomyocytes

2020 ◽  
Author(s):  
Geran Kostecki ◽  
Yu Shi ◽  
Christopher Chen ◽  
Daniel H. Reich ◽  
Emilia Entcheva ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Cardiac Injury ◽  
Neonatal Rat ◽  
Culture Studies ◽  
Electrophysiological Properties ◽  
Inward Currents ◽  
Life Threatening ◽  
Cultured Cardiomyocytes ◽  
Cardiac Myofibroblasts

AbstractInteractions between cardiac myofibroblasts and myocytes may slow conduction after cardiac injury, increasing the chance of life-threatening arrhythmia. While co-culture studies have shown that myofibroblasts can affect cardiomyocyte electrophysiology in vitro, the mechanism(s) remain debatable. In this study, primary neonatal rat cardiac myofibroblasts were transduced with the light-activated ion channel Channelrhodopsin-2, which allowed acute and selective modulation of myofibroblast currents in co-cultures with cardiomyocytes. Optical mapping revealed that myofibroblast-specific optogenetically induced inward currents decreased conduction velocity in the co-cultures by 27±6% (baseline = 17.7±5.3 cm/s), and shortened the cardiac action potential duration by 14±7% (baseline = 161±11 ms) when 0.017 mW/mm2 light was applied. When light irradiance was increased to 0.057 mW/mm2, the myofibroblast currents led to spontaneous beating in 6/7 co-cultures. Experiments showed that optogenetic perturbation did not lead to changes in myofibroblast strain and force generation, suggesting purely electrical effects in this model. In silico modeling of optogenetically modified myofibroblast-cardiomyocyte co-cultures largely reproduced these results and enabled a comprehensive study of relevant parameters. These results clearly demonstrate that myofibroblasts are sufficiently electrically connected to cardiomyocytes to effectively alter macroscopic electrophysiological properties in this model of cardiac tissue.

scholarly journals Proarrhythmic Electrical Remodeling by Noncardiomyocytes at Interfaces With Cardiomyocytes Under Oxidative Stress

2021 ◽  
Vol 11 ◽  
Author(s):  
Yali Zhao ◽  
Shankar Iyer ◽  
Maryam Tavanaei ◽  
Nicole T. Nguyen ◽  
Andrew Lin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
High Speed ◽  
Cardiac Fibrosis ◽  
Neonatal Rat ◽  
Electrical Remodeling ◽  
Life Threatening ◽  
Electrical Impulses ◽  
Regenerative Therapies ◽  
Safe By Design

Life-threatening ventricular arrhythmias, typically arising from interfaces between fibrosis and surviving cardiomyocytes, are feared sequelae of structurally remodeled hearts under oxidative stress. Incomplete understanding of the proarrhythmic electrical remodeling by fibrosis limits the development of novel antiarrhythmic strategies. To define the mechanistic determinants of the proarrhythmia in electrical crosstalk between cardiomyocytes and noncardiomyocytes, we developed a novel in vitro model of interface between neonatal rat ventricular cardiomyocytes (NRVMs) and controls [NRVMs or connexin43 (Cx43)-deficient HeLa cells] vs. Cx43+ noncardiomyocytes [aged rat ventricular myofibroblasts (ARVFs) or HeLaCx43 cells]. We performed high-speed voltage-sensitive optical imaging at baseline and following acute H2O2 exposure. In NRVM-NRVM and NRVM-HeLa controls, no arrhythmias occurred under either experimental condition. In the NRVM-ARVF and NRVM-HeLaCx43 groups, Cx43+ noncardiomyocytes enabled passive decremental propagation of electrical impulses and impaired NRVM activation and repolarization, thereby slowing conduction and prolonging action potential duration. Following H2O2 exposure, arrhythmia triggers, automaticity, and non-reentrant and reentrant arrhythmias emerged. This study reveals that myofibroblasts (which generate cardiac fibrosis) and other noncardiomyocytes can induce not only structural remodeling but also electrical remodeling and that electrical remodeling by noncardiomyocytes can be particularly arrhythmogenic in the presence of an oxidative burst. Synergistic electrical remodeling between H2O2 and noncardiomyocytes may account for the clinical arrhythmogenicity of myofibroblasts at fibrotic interfaces with cardiomyocytes in ischemic/non-ischemic cardiomyopathies. Understanding the enhanced arrhythmogenicity of synergistic electrical remodeling by H2O2 and noncardiomyocytes may guide novel safe-by-design antiarrhythmic strategies for next-generation iatrogenic interfaces between surviving native cardiomyocytes and exogenous stem cells or engineered tissues in cardiac regenerative therapies.

Cytochemical Evidence for Presynatic β-Adrenergic Regulation of Secretion in the Developing Rat Submandibular Gland

1977 ◽  
Vol 35 ◽  
pp. 430-431
Author(s):  
L.S. Cutler
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Submandibular Gland ◽  
Neonatal Rat ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Parotid Glands ◽  
Adrenergic Agonist ◽  
Rat Submandibular Gland

Many studies previously have shown that the B-adrenergic agonist isoproterenol and the a-adrenergic agonist norepinephrine will stimulate secretion by the adult rat submandibular (SMG) and parotid glands. Recent data from several laboratories indicates that adrenergic agonists bind to specific receptors on the secretory cell surface and stimulate membrane associated adenylate cyclase activity which generates cyclic AMP. The production of cyclic AMP apparently initiates a cascade of events which culminates in exocytosis. During recent studies in our laboratory it was observed that the adenylate cyclase activity in plasma membrane fractions derived from the prenatal and early neonatal rat submandibular gland was retractile to stimulation by isoproterenol but was stimulated by norepinephrine. In addition, in vitro secretion studies indicated that these prenatal and neonatal glands would not secrete peroxidase in response to isoproterenol but would secrete in response to norepinephrine. In contrast to these in vitro observations, it has been shown that the injection of isoproterenol into the living newborn rat results in secretion of peroxidase by the SMG (1).

In Vitro Studies on the Mechanism of the Antifibrino-lytic Action of E-Aminocaproic Acid (EACA)

1968 ◽  
Vol 19 (03/04) ◽  
pp. 584-592 ◽  
Author(s):  
Hanna Lukasiewicz ◽  
S Niewiarowski
Keyword(s):  
Aminocaproic Acid ◽  
Test Tube ◽  
In Vitro Studies ◽  
Lytic Action ◽  
Human Plasminogen ◽  
Experimental Findings ◽  
Fibrin Clots

Summary and Conclusion1. It has been found that EACA does not inhibit activation of human plasminogen into plasmin by SK and UK in a concentration of 5 × 10–2 M. The activation of bovine plasminogen by SK and UK is inhibited by this concentration of EACA but not by a lower one.2. EACA in concentrations of 1,5 × 10–1 – 10–4 M does not inhibit casein proteolysis by plasmin. The proteolysis of fibrinogen and fibrin measured by the release of TCA soluble tyrosine is inhibited by EACA in concentrations of 1,5 × 10–1 – 10–2 M.3. The lysis of non-stabilized clots by plasmin measured in a test tube was inhibited by an EACA concentration of 5 × 10–3 – 5 × 10–4 M. The lysis of stabilized clots by plasmin was inhibited by an EACA concentration of 10–5 M.4. On the basis of experimental findings and data given in literature the authors postulate that the mechanism of the antifibrinolytic effects of EACA consists mainly in a modification of plasmin action on fibrin. These effects are dependent on the structure of the fibrin clots.

The in vitro neonatal rat spinal cord preparation: a new insight into mammalian locomotor mechanisms

2004 ◽  
Vol 190 (5) ◽  
pp. 343-357 ◽  
Author(s):  
F. Clarac ◽  
E. Pearlstein ◽  
J. F. Pflieger ◽  
L. Vinay
Keyword(s):  
Spinal Cord ◽  
Neonatal Rat ◽  
Rat Spinal Cord ◽  
Insight Into

scholarly journals Impairing flow-mediated endothelial remodeling reduces extravasation of tumor cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Gautier Follain ◽  
Naël Osmani ◽  
Valentin Gensbittel ◽  
Nandini Asokan ◽  
Annabel Larnicol ◽  
...  
Keyword(s):  
Blood Flow ◽  
Tumor Cells ◽  
Molecular Mechanisms ◽  
Metastatic Progression ◽  
Metastatic Dissemination ◽  
Secondary Tumors ◽  
Flow Profiles ◽  
Endothelial Response ◽  
Life Threatening

AbstractTumor progression and metastatic dissemination are driven by cell-intrinsic and biomechanical cues that favor the growth of life-threatening secondary tumors. We recently identified pro-metastatic vascular regions with blood flow profiles that are permissive for the arrest of circulating tumor cells. We have further established that such flow profiles also control endothelial remodeling, which favors extravasation of arrested CTCs. Yet, how shear forces control endothelial remodeling is unknown. In the present work, we aimed at dissecting the cellular and molecular mechanisms driving blood flow-dependent endothelial remodeling. Transcriptomic analysis of endothelial cells revealed that blood flow enhanced VEGFR signaling, among others. Using a combination of in vitro microfluidics and intravital imaging in zebrafish embryos, we now demonstrate that the early flow-driven endothelial response can be prevented upon specific inhibition of VEGFR tyrosine kinase and subsequent signaling. Inhibitory targeting of VEGFRs reduced endothelial remodeling and subsequent metastatic extravasation. These results confirm the importance of VEGFR-dependent endothelial remodeling as a driving force of CTC extravasation and metastatic dissemination. Furthermore, the present work suggests that therapies targeting endothelial remodeling might be a relevant clinical strategy in order to impede metastatic progression.

scholarly journals 2′FL and LNnT Exert Antipathogenic Effects against C. difficile ATCC 9689 In Vitro, Coinciding with Increased Levels of Bifidobacteriaceae and/or Secondary Bile Acids

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 927
Author(s):  
Louise Kristine Vigsnaes ◽  
Jonas Ghyselinck ◽  
Pieter Van den Van den Abbeele ◽  
Bruce McConnell ◽  
Frédéric Moens ◽  
...  
Keyword(s):  
Antimicrobial Effect ◽  
Secondary Bile Acid ◽  
Clostridioides Difficile ◽  
Life Threatening ◽  
Intermediate Time ◽  
Indigenous Microbiota ◽  
Hospital Acquired ◽  
Gut Microbial Community ◽  
Secondary Bile Acids

Clostridioides difficile (formerly Clostridium difficile) infection (CDI) is one of the most common hospital-acquired infections, which is often triggered by a dysbiosed indigenous gut microbiota (e.g., upon antibiotic therapy). Symptoms can be as severe as life-threatening colitis. The current study assessed the antipathogenic potential of human milk oligosaccharides (HMOs), i.e., 2′-O-fucosyllactose (2′FL), lacto-N-neotetraose (LNnT), and a combination thereof (MIX), against C. difficile ATCC 9689 using in vitro gut models that allowed the evaluation of both direct and, upon microbiota modulation, indirect effects. During a first 48 h fecal batch study, dysbiosis and CDI were induced by dilution of the fecal inoculum. For each of the three donors tested, C. difficile levels strongly decreased (with >4 log CFU/mL) upon treatment with 2′FL, LNnT and MIX versus untreated blanks, coinciding with increased acetate/Bifidobacteriaceae levels. Interindividual differences among donors at an intermediate time point suggested that the antimicrobial effect was microbiota-mediated rather than being a direct effect of the HMOs. During a subsequent 11 week study with the PathogutTM model (specific application of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®)), dysbiosis and CDI were induced by clindamycin (CLI) treatment. Vancomycin (VNC) treatment cured CDI, but the further dysbiosis of the indigenous microbiota likely contributed to CDI recurrence. Upon co-supplementation with VNC, both 2′FL and MIX boosted microbial activity (acetate and to lesser extent propionate/butyrate). Moreover, 2′FL avoided CDI recurrence, potentially because of increased secondary bile acid production. Overall, while not elucidating the exact antipathogenic mechanisms-of-action, the current study highlights the potential of HMOs to combat CDI recurrence, help the gut microbial community recover after antibiotic treatment, and hence counteract the adverse effects of antibiotic therapies.

scholarly journals Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin N. Nelson ◽  
Savannah G. Beakley ◽  
Sierra Posey ◽  
Brittney Conn ◽  
Emma Maritz ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Dose Dependent ◽  
Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

scholarly journals Conditioned medium-preconditioned EPCs enhanced the ability in oligovascular repair in cerebral ischemia neonatal rats

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ning Zhou ◽  
Lei Wang ◽  
Ping Fu ◽  
Zihao Cui ◽  
Yuhang Ge ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Conditioned Medium ◽  
Cognitive Outcome ◽  
Adult Brain ◽  
Neonatal Rat ◽  
Vascular Density ◽  
Cxcr4 Axis

Abstract Background Oligovascular niche mediates interactions between cerebral endothelial cells and oligodendrocyte precursor cells (OPCs). Disruption of OPC-endothelium trophic coupling may aggravate the progress of cerebral white matter injury (WMI) because endothelial cells could not provide sufficient support under diseased conditions. Endothelial progenitor cells (EPCs) have been reported to ameliorate WMI in the adult brain by boosting oligovascular remodeling. It is necessary to clarify the role of the conditioned medium from hypoxic endothelial cells preconditioned EPCs (EC-pEPCs) in WMI since EPCs usually were recruited and play important roles under blood-brain barrier disruption. Here, we investigated the effects of EC-pEPCs on oligovascular remodeling in a neonatal rat model of WMI. Methods In vitro, OPC apoptosis induced by the conditioned medium from oxygen-glucose deprivation-injured brain microvascular endothelial cells (OGD-EC-CM) was analyzed by TUNEL and FACS. The effects of EPCs on EC damage and the expression of cytomokine C-X-C motif ligand 12 (CXCL12) were examined by western blot and FACS. The effect of the CM from EC-pEPCs against OPC apoptosis was also verified by western blot and silencing RNA. In vivo, P3 rat pups were subjected to right common carotid artery ligation and hypoxia and treated with EPCs or EC-pEPCs at P7, and then angiogenesis and myelination together with cognitive outcome were evaluated at the 6th week. Results In vitro, EPCs enhanced endothelial function and decreased OPC apoptosis. Meanwhile, it was confirmed that OGD-EC-CM induced an increase of CXCL12 in EPCs, and CXCL12-CXCR4 axis is a key signaling since CXCR4 knockdown alleviated the anti-apoptosis effect of EPCs on OPCs. In vivo, the number of EPCs and CXCL12 protein level markedly increased in the WMI rats. Compared to the EPCs, EC-pEPCs significantly decreased OPC apoptosis, increased vascular density and myelination in the corpus callosum, and improved learning and memory deficits in the neonatal rat WMI model. Conclusions EC-pEPCs more effectively promote oligovascular remodeling and myelination via CXCL12-CXCR4 axis in the neonatal rat WMI model.

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