Design and construction of a uniaxial cell stretcher

2000 ◽  
Vol 279 (6) ◽  
pp. H3124-H3130 ◽  
Author(s):  
Michael J. Yost ◽  
David Simpson ◽  
Kimberly Wrona ◽  
Stephen Ridley ◽  
Harry J. Ploehn ◽  
...  
Keyword(s):  
Mechanical Stimulation ◽  
High Performance ◽  
Cardiac Fibroblasts ◽  
Computer Control ◽  
Mechanical Stretch ◽  
Chemical Degradation ◽  
Neonatal Rat ◽  
Element Analysis ◽  
Accuracy And Repeatability

In vitro mechanical cell stimulators are used for the study of the effect of mechanical stimulation on anchorage-dependent cells. We developed a new mechanical cell stimulator, which uses stepper motor technology and computer control to achieve a high degree of accuracy and repeatability. This device also uses high-performance plastic components that have been shown to be noncytotoxic, dimensionally stable, and resistant to chemical degradation from common culture laboratory chemicals. We show that treatment with glow discharge for 25 s at 20 mA is sufficient to modify the surface of the rubber to allow proper adhesion for polymerization of aligned collagen. We show through finite element analysis that the middle area of the membrane, away from the clamped ends, is predictable, homogeneous, and has negligible shear strain. To test the efficacy of the mechanical stretch, we examined the effect of mechanical stimulation on the production of β1-integrin by neonatal rat cardiac fibroblasts. Mechanical stimulation was tested in the range of 0–12% stretch and 0–10-cycles/min stretch frequency. The fibroblasts respond with an increase in β1-integrin at 3% stretch and a decrease at 6 and 12% stretch. Stretch frequency was found to not significantly effect the concentration of β1-integrin. These studies yield a new and improved mechanical cell stimulator and demonstrate that mechanical stimulation has an effect on the expression of β1-integrin.

Norepinephrine and ANG II stimulate secretion of TGF-beta by neonatal rat cardiac fibroblasts in vitro

1995 ◽  
Vol 268 (4) ◽  
pp. C910-C917 ◽  
Author(s):  
S. A. Fisher ◽  
M. Absher
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Cardiac Fibroblasts ◽  
Neonatal Rat ◽  
Heart Cells ◽  
Ang Ii ◽  
Tgf Beta ◽  
Hypertrophic Growth ◽  
Beta 2

Transforming growth factor-beta (TGF-beta) is a ubiquitous growth-regulating protein that is capable of influencing the growth and function of heart cells in vitro. To better understand the role TGF-beta might play as a paracrine mediator of cardiac hypertrophy, the expression, secretion, and growth effects of TGF-beta were examined. Neonatal cardiac fibroblasts in vitro secreted latent TGF-beta 1 and TGF-beta 2 as high as 15 ng/10(6) cells. Angiotensin II (ANG II) and norepinephrine (NE) each augmented up to threefold the expression and secretion of latent TGF-beta 1 and TGF-beta 2 and also induced a shift in isoform predominance from beta 1 to beta 2. Each agent individually produced hypertrophic growth of neonatal cardiocytes and hyperplastic growth of cardiac fibroblasts. Paradoxically, the combination of NE and ANG II at intermediate and high concentrations resulted in less TGF-beta secretion (compared with either agent alone) and in hypertrophic growth of fibroblasts. These results suggest that the growth-promoting effects of ANG II and NE may in part be mediated via a paracrine stimulation of TGF-beta secretion.

Mechanical stimulation of organogenic cardiomyocyte growth in vitro

1996 ◽  
Vol 270 (5) ◽  
pp. C1284-C1292 ◽  
Author(s):  
H. H. Vandenburgh ◽  
R. Solerssi ◽  
J. Shansky ◽  
J. W. Adams ◽  
S. A. Henderson
Keyword(s):  
Mechanical Load ◽  
Mechanical Stretch ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Heart Cells ◽  
Mechanical Stimuli ◽  
Rat Cardiomyocytes ◽  
Morphological Alterations

Adherent cultures of neonatal rat cardiomyocytes were subjected to progressive, unidirectional lengthening for 2-4 days in serum-containing medium. This mechanical stretch (25% increase in initial length each day) simulates the eccentric mechanical load placed on in vivo heart cells by increases in postnatal blood pressure and volume. The in vitro mechanical stimuli initiated a number of morphological alterations in the confluent cardiomyocyte population which were similar to those occurring during in vivo heart growth. These include cardiomyocyte organization into parallel arrays of rod-shaped cells, increased cardiomyocyte binucleation, and cardiomyocyte hypertrophy by longitudinal cell growth. Stretch stimulated DNA synthesis in the noncardiomyocyte population but not in the cardiomyocytes. Myosin heavy chain (MHC) content increased 62% over 4 days of stretch and included increased accumulation of both fetal beta-MHC and adult alpha-MHC isoforms. This new model of stretch-induced cardiomyocyte hypertrophy may assist in examining some of the complex mechanogenic growth processes that occur in the rapidly enlarging neonatal heart.

scholarly journals Synthesis and Biological Evaluation of Novel Thiazolyl-Coumarin Derivatives as Potent Histone Deacetylase Inhibitors with Antifibrotic Activity

Molecules ◽  
2019 ◽  
Vol 24 (4) ◽  
pp. 739 ◽  
Author(s):  
Viviana Pardo-Jiménez ◽  
Patricio Navarrete-Encina ◽  
Guillermo Díaz-Araya
Keyword(s):  
Histone Deacetylases ◽  
Histone Deacetylase Inhibitors ◽  
Cardiac Fibrosis ◽  
Cardiac Fibroblasts ◽  
Hdac Inhibitors ◽  
Biological Evaluation ◽  
Neonatal Rat ◽  
Zinc Binding ◽  
Coumarin Derivatives

New histone deacetylases (HDAC) inhibitors with low toxicity to non-cancerous cells, are a prevalent issue at present because these enzymes are actively involved in fibrotic diseases. We designed and synthesized a novel series of thiazolyl-coumarins, substituted at position 6 (R = H, Br, OCH3), linked to classic zinc binding groups, such as hydroxamic and carboxylic acid moieties and alternative zinc binding groups such as disulfide and catechol. Their in vitro inhibitory activities against HDACs were evaluated. Disulfide and hydroxamic acid derivatives were the most potent ones. Assays with neonatal rat cardiac fibroblasts demonstrated low cytotoxic effects for all compounds. Regarding the parameters associated to cardiac fibrosis development, the compounds showed antiproliferative effects, and triggered a strong decrease on the expression levels of both α-SMA and procollagen I. In conclusion, the new thiazolyl-coumarin derivatives inhibit HDAC activity and decrease profibrotic effects on cardiac fibroblasts.

Abstract 220: miR-486 Mediates the Benefits of Exercise in Attenuating Cardiac Fibrosis

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Dongchao Lv ◽  
Yihua Bei ◽  
Qiulian Zhou ◽  
Qi Sun ◽  
Tianzhao Xu ◽  
...  
Keyword(s):  
Cardiac Fibrosis ◽  
Cardiac Fibroblasts ◽  
Mrna Level ◽  
Neonatal Rat ◽  
Cardiac Growth ◽  
Non Coding Rnas ◽  
Exercise Induced ◽  
Proliferation Assays

MicroRNAs (miRNAs, miRs), a novel group of small non-coding RNAs, play important roles in cardiac fibrosis. Exercise-induced physiological cardiac growth is associated with hypertrophy and proliferation of cardiomyocytes. In addition, exercise has been shown to inhibit cardiac fibrosis. However, relative little is known about whether exercise could attenuating cardiac fibrosis via targeting miRNA. miR-486 is a muscle enriched miRNAs, however, its role in heart is relative unclear. The current study aimed to investigate the role of miR-486 in exercise-induced cardiac growth in a 3-week swimming training murine model as well as in the function of cardiac fibroblasts and production of extracellular matrix (ECM) using neonatal rat cardiac fibroblasts in primary culture. Our data showed that exercised mice displayed increased about three-fold expression of miR-486 in hearts as measured by microarray analysis and qRT-PCRs. EdU proliferation assays demonstrated that miR-486 mimics decreased (5.90%±0.57% vs 4.02%±0.27% in nc-mimics vs miR-486-mimics, respectively), while miR-486 inhibitor increased the proliferation of cardiac fibroblasts in vitro (5.87%±0.16% vs 9.60%±0.58% in nc-inhibitor vs miR-486-inhibitor, respectively). Although downregulation of miR-486 had no regulatory effect on α-sma and collagen-1 gene expression in cardiac fibroblasts, overexpression of miR-486 significantly reduced the mRNA level of α-sma (1.01±0.08 vs 0.28±0.04 in nc-mimics vs miR-486-mimics, respectively) and collagen-1(1.02±0.12 vs 0.58±0.09 in nc-mimics vs miR-486-mimics, respectively), indicative of attenuated activation of fibroblasts and reduced production of ECM. These data reveal that miR-486 is essentially involved in the proliferation and activation of cardiac fibroblasts, and might be a key regulator mediating the benefit of exercise in preventing cardiac fibrosis.

M-CAT element mediates mechanical stretch-activated transcription of B-type natriuretic peptide via ERK activation

2011 ◽  
Vol 89 (8) ◽  
pp. 539-550 ◽  
Author(s):  
Elina Koivisto ◽  
Laura Karkkola ◽  
Theresa Majalahti ◽  
Jani Aro ◽  
Heikki Tokola ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Mechanical Stretch ◽  
Neonatal Rat ◽  
Luciferase Reporter ◽  
Transcriptional Enhancer ◽  
Luciferase Reporter Construct ◽  
Rat Cardiomyocytes ◽  
Erk Activation ◽  
Enhancer Factor

The muscle-CAT (M-CAT) promoter element is found on promoters of most muscle-specific cardiac genes, but its role in cardiac pathology is poorly understood. Here we studied whether the M-CAT element is involved in hypertrophic process activated by mechanical stretch, and identified the intracellular pathways mediating the response. When an in vitro stretch model of cultured neonatal rat cardiomyocytes and luciferase reporter construct driven by rat B-type natriuretic peptide (BNP) promoter were used, mutation of M-CAT element inhibited not only the basal reporter activity (88%), but also the stretch-activated BNP transcription (58%, p < 0.001). Stretch-induced BNP promoter activation was associated with an increase in transcriptional enhancer factor-1 (TEF-1) binding activity after 24 h mechanical stretch (p < 0.05). Inhibition of mitogen-activated protein kinases ERK, JNK, or p38 attenuated stretch-induced BNP activation. Interestingly, as opposed to p38 and JNK, inhibition of ERK had no additional effect on transcriptional activity of BNP promoter harboring the M-CAT mutation, suggesting a pivotal role for ERK in regulating stretch-induced BNP transcription via M-CAT binding site. Finally, immunoprecipitation studies showed that mechanical stretch induced myocyte enhancer factor-2 (MEF-2) binding to TEF-1. These data suggest a central role for M-CAT element in regulation of mechanical stretch-induced hypertrophic response via ERK activation.

scholarly journals (Pro)renin receptor involves in myocardial fibrosis and oxidative stress in diabetic cardiomyopathy via the PRR–YAP pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shiran Yu ◽  
Xuefei Dong ◽  
Min Yang ◽  
Qingtao Yu ◽  
Jie Xiong ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Myocardial Fibrosis ◽  
Cardiac Fibroblasts ◽  
Animal Experiments ◽  
Specific Inhibitor ◽  
Neonatal Rat ◽  
Rnai Silencing ◽  
The Impact

Abstract(Pro)renin receptor (PRR) and Yes-associated protein (YAP) play an important role in cardiovascular diseases. However, the role of PRR–YAP pathway in the pathogenesis of DCM is also not clear. We hypothesized that PRR–YAP pathway may promote pathological injuries in DCM by triggering redox. Wistar rats and neonatal rat cardiac fibroblasts were respectively used in vivo and in vitro studies. In order to observe the effects of PRR mediated YAP pathway on the pathogenesis of DCM, animal experiments were divided into 3 parts, including the evaluation the effects of PRR overexpression, PRR RNAi silencing and YAP RNAi silencing. Recombinant-adenoviruses-carried-PRR-gene (Ad-PRR), Ad-PRR-shRNA and lentivirus-carried-YAP-shRNA were constructed and the effects of PRR mediated YAP on the pathogenesis of DCM were evaluated. YAP specific inhibitor Verteporfin was also administrated in cardiac fibroblasts to explore the impact of PRR–YAP pathway on oxidative stress and myocardial fibrosis. The results displayed that PRR overexpression could enhance YAP expression but PRR RNAi silencing down-regulated its expression. Moreover, PRR overexpression could exacerbate oxidative stress and myocardial fibrosis in DCM, and these pathological changes could be rescued by YAP blockade. We concluded that PRR–YAP pathway plays a key role in the pathogenesis of DCM.

Response of Neonatal Rat Cardiomyocytes to Repetitive Mechanical Stimulation In Vitro

1995 ◽  
Vol 752 (1 Cardiac Growt) ◽  
pp. 19-29 ◽  
Author(s):  
HERMAN H. VANDENBURGH ◽  
ROSA SOLERSSI ◽  
JANET SHANSKY ◽  
JOHN W. ADAMS ◽  
SCOTT A. HENDERSON ◽  
...  
Keyword(s):  
Mechanical Stimulation ◽  
Neonatal Rat ◽  
Rat Cardiomyocytes ◽  
Neonatal Rat Cardiomyocytes

Enhancement and Co-Localization of Erk and Collagens in Human Myocardium with Congestive Heart Failure

2001 ◽  
Vol 7 (S2) ◽  
pp. 648-649
Author(s):  
Y. Dong ◽  
R. Lin ◽  
C Wei
Keyword(s):  
Intracellular Signaling ◽  
Cardiac Fibroblasts ◽  
Biological Effects ◽  
Pressure Overload ◽  
Mechanical Stretch ◽  
Collagen Type I ◽  
Type I ◽  
Mechanical Stimuli ◽  
Adult Rat

Mechanical stimuli frequently trigger signals leading to increase gene expression, protein synthesis, or mitogenesis. Cardiac fibroblasts respond to changes in ventricular hemodynamic loading by increasing matrix production in models of pressure-overload hypertrophy and myocardial infarction. Recent studies have identified some of the intracellular signaling pathways that mediate the biological effects observed upon mechanical stimulation in vitro. These include mitogenactivated protein kinases (MAPKs) and protein kinase C (PKC) family. MAPKs include extracellular signal-regulated kinase (ERK), C-Jun NH2-terminal kinase (JNK), and p38 MAPK. ERK pathway is frequently stimulated in response to mitogen.Previous studies demonstrated that mechanical stretch stimulated MAPK activities in normal adult rat cardiac fibroblasts. On the other hand, mechanical stretch also increases transcription and production of the matrix proteins collagen (type I and HI) and fibronectin in cardiac fibroblasts. MAPKs are activated by mechanical stretch during cardiac myocytes hypertrophy through both PKCdependent and independent pathways.

scholarly journals NOTCH1 is critical for fibroblast-mediated induction of cardiomyocyte specialization into ventricular conduction system-like cells in vitro

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Agatha Ribeiro da Silva ◽  
Elida A. Neri ◽  
Lauro Thiago Turaça ◽  
Rafael Dariolli ◽  
Miriam H. Fonseca-Alaniz ◽  
...  
Keyword(s):  
Cardiac Fibroblasts ◽  
3D Culture ◽  
Fold Increase ◽  
Calcium Transient ◽  
Conduction System ◽  
Cardiac Cells ◽  
Neonatal Rat ◽  
Connexin 40 ◽  
Ventricular Conduction

Abstract Cardiac fibroblasts are present throughout the myocardium and are enriched in the microenvironment surrounding the ventricular conduction system (VCS). Several forms of arrhythmias are linked to VCS abnormalities, but it is still unclear whether VCS malformations are cardiomyocyte autonomous or could be linked to crosstalk between different cell types. We reasoned that fibroblasts influence cardiomyocyte specialization in VCS cells. We developed 2D and 3D culture models of neonatal rat cardiac cells to assess the influence of cardiac fibroblasts on cardiomyocytes. Cardiomyocytes adjacent to cardiac fibroblasts showed a two-fold increase in expression of VCS markers (NAV1.5 and CONTACTIN 2) and calcium transient duration, displaying a Purkinje-like profile. Fibroblast-conditioned media (fCM) was sufficient to activate VCS-related genes (Irx3, Scn5a, Connexin 40) and to induce action potential prolongation, a hallmark of Purkinge phenotype. fCM-mediated response seemed to be spatially-dependent as cardiomyocyte organoids treated with fCM had increased expression of connexin 40 and NAV1.5 primarily on its outer surface. Finally, NOTCH1 activation in both cardiomyocytes and fibroblasts was required for connexin 40 up-regulation (a proxy of VCS phenotype). Altogether, we provide evidence that cardiac fibroblasts influence cardiomyocyte specialization into VCS-like cells via NOTCH1 signaling in vitro.

Abstract 298: Flavin Containing Monooxygenase 2 in Cardiac Fibrosis

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Xinyang Hu ◽  
Cheng Ni
Keyword(s):  
Cardiac Fibrosis ◽  
Nuclear Translocation ◽  
Heart Function ◽  
Cardiac Fibroblasts ◽  
Neonatal Rat ◽  
Critical Determinant ◽  
Ubiquitin Protein Ligase ◽  
Over Expression ◽  
First Time

Cardiac fibrosis is associated with clinical outcome in patients with heart failing. There is no information concerning whether flavin containing monooxygenase 2 (FMO2) is involved in the fibrotic response. Here, we demonstrate that FMO2 is consistently down-regulated after myocardial infarction (MI) in rats, non-human primates and patients with chronic MI. Furthermore, FMO2 knockdown or knockout results in significantly increased cardiac fibrosis accompanied with impaired heart function in rats. Compensation of non-myocyte FMO2 using the strategy of miR133a/1a TS aided lentivirus inhibits the cardiac fibrosis and restores the deteriorated cardiac function following MI in rats. In vitro studies showed FMO2 expression in neonatal rat cardiac fibroblasts (NRCFs) is dramatically decreased after treatment of TGF-β. FMO2 deficiency in CFs isolated from FMO2-/- rats induces augmented collagen deposition, fibroblast-myofibroblast transdifferentiation and increased phosphorylated SMAD2/3. However, overexpression of FMO2 in NRCFs or CFs from MI patients suppresses this exact process. Mechanistically, we demonstrate using mass spectrometry that FMO2 combines with cytochrome p450 superfamily 2J3 (CYP2J3) at the ubiquitination site of the latter one after TGF-β stimulation, and then blocks the CYP2J3 ubiquitination. The accumulated CYP2J3 expression induces the up-regulation and nuclear translocation of SMURF2, the E3 ubiquitin-protein ligase specifically degrades phosphorylated SMAD2/3, through a negative feedback as another substrate of SMURF2, in addition, FMO2 can competitively exploit CYP2J3 from SMURF2, thus in turn promotes nuclear translocation of SMURF2 to degrade phosphorylated SMAD2/3. Furthermore, in non-human primate MI model, delivery of non-myocyte FMO2 over-expression lentivirus significantly decreases the cardiac fibrosis and improves heart function. In summary, our study demonstrates for the first time that FMO2 is a critical determinant of cardiac fibrosis by interfering TGF-β/SMAD2/3 and provides a novel potential target for treating cardiac fibrosis.

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