A superfusion system to study border zones in confluent cultures of neonatal rat heart cells

1998 ◽  
Vol 274 (6) ◽  
pp. H2001-H2008 ◽  
Author(s):  
Christopher J. Hyatt ◽  
John J. Lemasters ◽  
Barbara J. Muller-Borer ◽  
Timothy A. Johnson ◽  
Wayne E. Cascio
Keyword(s):  
Cardiac Tissue ◽  
Fluorescent Microscopy ◽  
Cell Monolayer ◽  
Neonatal Rat ◽  
Border Zone ◽  
Heart Cells ◽  
Fluid Viscosity ◽  
Ion Regulation ◽  
Border Zones ◽  
Superfusion System

We present a new experimental method to study intracellular ion regulation in cultured cardiomyocytes at a border zone separating two different and distinct environments. Our system uses a dual-flow superfusion chamber to produce two different but adjacent environments over a monolayer of cardiomyocytes. Fluorescent microscopy of fluorescein showed that the transition between the two environments was nearly linear and was 220–320 μm wide depending on fluid viscosity and velocity. We superfused cultured monolayers on one side with a solution at pH 6.5 and on the other side with a solution at pH 7.4. We observed a sharply demarcated difference in intracellular pH (pHi) between the two halves of the cell monolayer as measured with the fluorescent pHi indicator carboxy-seminaphthorhodafluor-1. The demarcation of pHi corresponded well with the demarcation of the border measured with fluorescein. We conclude that our superfusion system will facilitate the study of intercellular communication and interactions across boundaries of cardiac tissue where different ionic or metabolic conditions are present, for example, between ischemic and nonischemic myocardium.

Blood pressure and cardiac tissue responses to prostacyclin (PGI2) in various species

1982 ◽  
Vol 60 (2) ◽  
pp. 134-139 ◽  
Author(s):  
G. A. Collins ◽  
B. A. MacLeod ◽  
M. J. A. Walker
Keyword(s):  
Blood Pressure ◽  
Heart Rate ◽  
Cardiac Tissue ◽  
Isolated Heart ◽  
Heart Tissue ◽  
Neonatal Rat ◽  
Heart Cells ◽  
Camp Content ◽  
Rat Atria

The effect of prostacyclin (PGI2) on blood pressure and heart rate (in vivo) and on isolated heart tissue has been investigated in different species. Isolated cardiac tissue had limited resposes to PGI2 tested at 10−13 to 10−5 M. Cultured neonatal rat heart cells did not respond to PGI2, neither did intact rat hearts or rabbit cardiac tissue. Guinea pig and rat atria showed limited dose-dependent responses to PGI2 at concentrations greater than 10−7 M. In rat atria, 10−5 M PGI2 produced a limited elevation of tissue cAMP content. When given by intravenous injection or infusion, PGI2 produced hypotension in anaesthetized primates (three species), rat, rabbit, pig, and dog. As a vasodepressor in all species, PGI2 (on a weight basis) was more active than prostaglandins of the B or E type and, in most species tested, it was approximately five times more active than PGE2. Heart responses in intact animals were often paradoxical in that decreases in heart rate often accompanied blood pressure falls.

scholarly journals Biomimetic approach to cardiac tissue engineering

2007 ◽  
Vol 362 (1484) ◽  
pp. 1357-1368 ◽  
Author(s):  
M Radisic ◽  
H Park ◽  
S Gerecht ◽  
C Cannizzaro ◽  
R Langer ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Culture Medium ◽  
Cardiac Tissue ◽  
Cardiac Tissue Engineering ◽  
Neonatal Rat ◽  
Ultrastructural Organization ◽  
Heart Cells ◽  
Cell Alignment ◽  
Human Stem Cells ◽  
Biomimetic Approach

Here, we review an approach to tissue engineering of functional myocardium that is biomimetic in nature, as it involves the use of culture systems designed to recapitulate some aspects of the actual in vivo environment. To mimic the capillary network, subpopulations of neonatal rat heart cells were cultured on a highly porous elastomer scaffold with a parallel array of channels perfused with culture medium. To mimic oxygen supply by haemoglobin, the culture medium was supplemented with a perfluorocarbon (PFC) emulsion. Constructs cultivated in the presence of PFC contained higher amounts of DNA and cardiac markers and had significantly better contractile properties than control constructs cultured without PFC. To induce synchronous contractions of cultured constructs, electrical signals mimicking those in native heart were applied. Over only 8 days of cultivation, electrical stimulation induced cell alignment and coupling, markedly increased the amplitude of synchronous construct contractions and resulted in a remarkable level of ultrastructural organization. The biomimetic approach is discussed in the overall context of cardiac tissue engineering, and the possibility to engineer functional human cardiac grafts based on human stem cells.

Engineered Membranes Improve Cardiac Function in Ischemic Rat Hearts

2010 ◽  
Author(s):  
Monica Sandri ◽  
Anna Tampieri ◽  
Joung H. Levialdi Ghiron ◽  
Gianluigi Condorelli
Keyword(s):  
Cardiac Function ◽  
Cardiac Tissue ◽  
Diglycidyl Ether ◽  
Beating Heart ◽  
Cardiac Cells ◽  
Neonatal Rat ◽  
Heart Cell ◽  
Collagen Membrane ◽  
Heart Cells ◽  
Type I

In the relatively young field of cardiac tissue engineering, different biomaterials, methods and techniques have been tested for cardiac repair. In this study we examined the validity of a series of new preformed membrane scaffolds, based on collagen type I, for the transplantation of cardiac cells. One type of membrane, cross-linked with 1,4-butanediol diglycidyl ether (BDDGE) and fibronectin-enriched, gave rise to spontaneously beating heart cell constructs 5–9 days after seeding with neonatal rat cardiac cells. This membrane was then grafted, with and without beating cardiac cells, onto the infarcted area of rat models of heart failure. Seriate echocardiography, performed on rats before transplantation and at 4 and 8 weeks after transplantation, showed that rats that received collagen membranes with beating cells showed an improvement in cardiac function after 8 weeks. These results suggest that this new type of collagen membrane can be used as vector for the transplantation of beating heart cells to the injured myocardium, hence representing an important potential tool for cardiac tissue repair technologies.

Confocal imaging of calcium in intact ventricular muscle provides a new view of excitation-contraction coupling

1996 ◽  
Vol 54 ◽  
pp. 738-739
Author(s):  
W.G. Wier
Keyword(s):  
Local Control ◽  
Cardiac Tissue ◽  
Cell Function ◽  
Isolated Heart ◽  
Cardiac Cells ◽  
Confocal Imaging ◽  
Ion Concentration ◽  
Heart Cell ◽  
Free Calcium ◽  
Heart Cells

A fundamentally new understanding of cardiac excitation-contraction (E-C) coupling is being developed from recent experimental work using confocal microscopy of single isolated heart cells. In particular, the transient change in intracellular free calcium ion concentration ([Ca2+]i transient) that activates muscle contraction is now viewed as resulting from the spatial and temporal summation of small (∼ 8 μm3), subcellular, stereotyped ‘local [Ca2+]i-transients' or, as they have been called, ‘calcium sparks'. This new understanding may be called ‘local control of E-C coupling'. The relevance to normal heart cell function of ‘local control, theory and the recent confocal data on spontaneous Ca2+ ‘sparks', and on electrically evoked local [Ca2+]i-transients has been unknown however, because the previous studies were all conducted on slack, internally perfused, single, enzymatically dissociated cardiac cells, at room temperature, usually with Cs+ replacing K+, and often in the presence of Ca2-channel blockers. The present work was undertaken to establish whether or not the concepts derived from these studies are in fact relevant to normal cardiac tissue under physiological conditions, by attempting to record local [Ca2+]i-transients, sparks (and Ca2+ waves) in intact, multi-cellular cardiac tissue.

Abstract P279: YAP, a Transcriptional Cofactor of the Hippo Pathway, Regulates Cardiomyocyte Growth, Survival, and Proliferation

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Yanfei Yang ◽  
Noritsugu Nakano ◽  
Junichi Sadoshima
Keyword(s):  
Cardiac Hypertrophy ◽  
Hippo Pathway ◽  
Neonatal Rat ◽  
Border Zone ◽  
Luciferase Reporter ◽  
Tunel Staining ◽  
H2o2 Treatment ◽  
Rat Hearts ◽  
The Hippo Pathway

Mst1 and Lats2, components of the mammalian Hippo pathway, stimulate apoptosis and inhibit hypertrophy of cardiomyocytes (CMs), thereby mediating reperfusion injury and heart failure. YAP, a transcription factor co-factor, is negatively regulated by the Hippo pathway, and controls cell survival, proliferation and tissue regeneration. The role of YAP in regulating growth and death of CMs is poorly understood. YAP overexpression in CMs induced cardiac hypertrophy, as indicated by increases in cell size (+1.2 fold, p<0.01), protein content (+1.1 fold, p<0.01) and ANF (luciferase reporter activity +1.7 fold, mRNA +2.2 fold, and staining +2.7 fold, p<0.01). Lats2 phosphorylates YAP at Serine 127, which induces cytoplasmic translocation of YAP, whereas YAP(S127A) is localized constitutively in the nucleus. Expression of YAP(S127A) enhanced hypertrophy in cultured CMs compared to that of wild type YAP (+1.87 fold ANF staining, p<0.05), suggesting that the Mst1/Hippo pathway negatively regulates cardiac hypertrophy through YAP. YAP inhibited cell death induced by H2O2 treatment, as evaluated with TUNEL staining (-65%, p<0.05) and CellTiter Blue assays (+34.9%, p<0.01), indicating that YAP plays an essential role in mediating CM survival. Interestingly, YAP also significantly increased Ki67 positive cells in cultured CMs compared to LacZ (+2.65 fold, p<0.05). We used a mouse model of chronic myocardial infarction (MI) to evaluate the function of YAP in the heart in vivo. Although YAP is diffusely localized both in the nucleus and cytosol in CMs in control hearts, CMs in the border zone of MI exhibited nuclear localization of YAP whereas YAP was excluded from the nucleus in CMs in the remodeling area four days after MI (+6.52 fold and +1.28 fold). Some of the YAP positive CMs in the border zone exhibited positive co-staining with Ki67, suggesting that YAP potentially induces CM proliferation. A significant increase in nuclear YAP and Ki67 positive CMs (+2.95 fold, p<0.01 and +2.18 fold, p<0.05) was also observed in neonatal rat hearts whose apex was surgically resected three days before euthanasia. These results suggest that YAP plays an important role in mediating not only hypertrophy and survival, but also proliferation of CMs in response to myocardial injury.

scholarly journals Hexokinase–mitochondrial interactions regulate glucose metabolism differentially in adult and neonatal cardiac myocytes

2013 ◽  
Vol 142 (4) ◽  
pp. 425-436 ◽  
Author(s):  
Guillaume Calmettes ◽  
Scott A. John ◽  
James N. Weiss ◽  
Bernard Ribalet
Keyword(s):  
Cardiac Tissue ◽  
Ventricular Myocytes ◽  
Glycogen Synthesis ◽  
Neonatal Rat ◽  
Metabolic Profiles ◽  
Rat Ventricular Myocytes ◽  
Glycolytic Activity ◽  
Extracellular Glucose ◽  
Functional Consequences ◽  
Neonatal Rat Ventricular Myocytes

In mammalian tumor cell lines, localization of hexokinase (HK) isoforms to the cytoplasm or mitochondria has been shown to control their anabolic (glycogen synthesis) and catabolic (glycolysis) activities. In this study, we examined whether HK isoform differences could explain the markedly different metabolic profiles between normal adult and neonatal cardiac tissue. We used a set of novel genetically encoded optical imaging tools to track, in real-time in isolated adult (ARVM) and neonatal (NRVM) rat ventricular myocytes, the subcellular distributions of HKI and HKII, and the functional consequences on glucose utilization. We show that HKII, the predominant isoform in ARVM, dynamically translocates from mitochondria and cytoplasm in response to removal of extracellular glucose or addition of iodoacetate (IAA). In contrast, HKI, the predominant isoform in NRVM, is only bound to mitochondria and is not displaced by the above interventions. In ARVM, overexpression of HKI, but not HKII, increased glycolytic activity. In neonatal rat ventricular myocytes (NVRM), knockdown of HKI, but not HKII, decreased glycolytic activity. In conclusion, differential interactions of HKI and HKII with mitochondria underlie the different metabolic profiles of ARVM and NRVM, accounting for the markedly increased glycolytic activity of NRVM.

scholarly journals “Buoyant Cartographies”: Strata-Mapping the Detroit River Border

2019 ◽  
Vol 1 ◽  
pp. 1-1
Author(s):  
Taien Ng-Chan
Keyword(s):  
Digital Media ◽  
Cultural Landscape ◽  
Border Crossing ◽  
Border Zone ◽  
City Image ◽  
Detroit River ◽  
Border Zones ◽  
Nature Park ◽  
A Site ◽  
International Border

<p><strong>Abstract.</strong> In the early days of September 2018, a group of artists and researchers converged on the Detroit River (an international border between Detroit, Michigan, USA and Windsor, Ontario, Canada) to investigate the “Buoyant Cartographies” that this particular site demanded. As one of the parties involved in this participatory event (along with Lead Investigators <i>In/Terminus Creative Research Group</i> and <i>Float School</i>), my artist-research collective Hamilton Perambulatory Unit (HPU) undertook an experimental mapping project that investigated the different “strata” of the place and the development of a “city-image.” The HPU Strata-Walk is an experimental and performative mapping methodology that focuses on how spatial meaning is created through a “stratigraphic” sensing of a site. The Detroit-Windsor border makes an especially compelling site for a Strata-Walk, in light of the conflicts over borders and walls in the current political environment, which presents an urgent need towards understanding and envisioning alternate possibilities for border zones. As a material site and geo-political space, the Detroit River border particularly benefits from intermedial investigations into spatial meanings and their construction. Notably, the role of folklore and local narratives on the internet and social media (and the erasure of Indigenous knowledge) figures large in developing one's knowledge of place. Experimental cartographies can thus help to develop alternate ways of seeing such sites.</p><p>This presentation is an attempt to trace this particular event of “discovery,” an account of how a place becomes known and how intermedial practices influence the manifestation of space and experience. Inherent in this research is the overarching question of how one begins to decolonize public narratives of place, how gaps and erasures in knowledge can be located in order to demarcate a way forward for further study and action. With these concerns in mind, I conduct a preliminary analysis of the border site through the activities of the HPU and our specific “strata-walking” framework, which focuses on different approaches of mapping-as-process, from phenomenological, ethnographic and cultural landscape reading methodologies to networked, social and digital media research. This performative mapping can shape individual experiences of the border through the revealing of complex networks, flows, and narratives, and point to fissures where alternative imaginings might be possible. I will first begin with a brief introduction to the HPU’s methodologies, before situating them in a survey of relevant literatures around experimental and critical processes of mapping. Then, using photographic and textual documentation, I delve into some very preliminary results of the investigation, focusing on touristic experiences of border crossing as well as a look at the specific “imageability” of Peche Island in the Detroit River, a place of rumour and mystery, now a nature park maintained by the city of Windsor. The overall goal will be to demonstrate the necessity of experimental cartographies in the creation of alternate experiences and more reflexive narratives about the border zone, with the Detroit-Windsor border as a case study.</p>

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 The role of dye affinity in optical measurements of Cai2+ transients in cardiac muscle

2014 ◽  
Vol 307 (1) ◽  
pp. H73-H79 ◽  
Author(s):  
Wei Kong ◽  
Vladimir G. Fast
Keyword(s):  
Optical Fibers ◽  
Cardiac Tissue ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Optical Measurements ◽  
Neonatal Rat ◽  
High Affinity ◽  
Cycle Lengths ◽  
Rat Myocytes

Previous experiments in cultures of neonatal rat myocytes demonstrated that the shape of Cai2+ transients measured using high-affinity Ca2+-sensitive dyes may be misrepresented. The purpose of this study was to examine the role of dye affinity in Cai2+ measurements in intact adult cardiac tissue by comparing optical recordings obtained with high- and low-affinity dyes. Experiments were carried out in porcine left ventricular (LV) wedge preparations stained locally by intramural injection via microcapillaries (diameter = 150 μm) with a low-affinity Ca2+-sensitive dye Fluo-4FF or Fluo-2LA (nominal Kd, ∼7–10 μmol/l), high-affinity dye Rhod-2 ( Kd = 0.57 μmol/l), and Fluo-4 or Fluo-2MA ( Kd, ∼0.4 μmol/l); in addition, tissue was stained with transmembrane potential ( Vm)-sensitive dye RH-237. Optical recordings of Vm and Cai2+ were made using optical fibers (diameter = 325 μm) glued with the microcapillaries. The durations of Cai2+ transients measured at 50% level of recovery (CaD50) using high-affinity Fluo-4/Fluo-2MA dyes were up to ∼81% longer than those measured with low-affinity Fluo-4FF/Fluo-2LA at long pacing cycle lengths (CL). In Fluo-4/Fluo-2MA measurements at long CLs, Cai2+ transients often (∼50% of cases) exhibited slow upstroke rise and extended plateau. In Rhod-2 measurements, CaD50 was moderately longer (up to ∼35%) than in Fluo-4FF recordings, but Cai2+ transient shapes were similar. In all series of measurements, mean action potential duration values were not significantly different ( P > 0.05). The delays between Vm and Cai2+ upstrokes were comparable for low- and high-affinity dyes ( P > 0.05). In conclusion, measurements of Cai2+ transient in ventricular myocardium are strongly affected by the affinity of Ca2+ dyes. The high-affinity dyes may overestimate the duration and alter the shape of Cai2+ transients.

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