scholarly journals Tissue engineering of functional cardiac muscle: molecular, structural, and electrophysiological studies

2001 ◽  
Vol 280 (1) ◽  
pp. H168-H178 ◽  
Author(s):  
M. Papadaki ◽  
N. Bursac ◽  
R. Langer ◽  
J. Merok ◽  
G. Vunjak-Novakovic ◽  
...  
Keyword(s):  
Cardiac Muscle ◽  
Cardiac Myocytes ◽  
Conduction Velocity ◽  
Capture Rate ◽  
Signal Amplitude ◽  
Model System ◽  
Neonatal Rat ◽  
Excitation Threshold ◽  
Electrophysiological Properties

The primary aim of this study was to relate molecular and structural properties of in vitro reconstructed cardiac muscle with its electrophysiological function using an in vitro model system based on neonatal rat cardiac myocytes, three-dimensional polymeric scaffolds, and bioreactors. After 1 wk of cultivation, we found that engineered cardiac muscle contained a 120- to 160-μm-thick peripheral region with cardiac myocytes that were electrically connected through gap junctions and sustained macroscopically continuous impulse propagation over a distance of 5 mm. Molecular, structural, and electrophysiological properties were found to be interrelated and depended on specific model system parameters such as the tissue culture substrate, bioreactor, and culture medium. Native tissue and the best experimental group (engineered cardiac muscle cultivated using laminin-coated scaffolds, rotating bioreactors, and low-serum medium) were comparable with respect to the conduction velocity of propagated electrical impulses and spatial distribution of connexin43. Furthermore, the structural and electrophysiological properties of the engineered cardiac muscle, such as cellularity, conduction velocity, maximum signal amplitude, capture rate, and excitation threshold, were significantly improved compared with our previous studies.

Cardiac muscle tissue engineering: toward an in vitro model for electrophysiological studies

1999 ◽  
Vol 277 (2) ◽  
pp. H433-H444 ◽  
Author(s):  
N. Bursac ◽  
M. Papadaki ◽  
R. J. Cohen ◽  
F. J. Schoen ◽  
S. R. Eisenberg ◽  
...  
Keyword(s):  
Cardiac Muscle ◽  
Cardiac Myocytes ◽  
In Vitro Model ◽  
Neonatal Rat ◽  
Electrophysiological Properties ◽  
Impulse Propagation ◽  
Ventricular Cells ◽  
Electrophysiological Studies ◽  
Vitro Model

The objective of this study was to establish a three-dimensional (3-D) in vitro model system of cardiac muscle for electrophysiological studies. Primary neonatal rat ventricular cells containing lower or higher fractions of cardiac myocytes were cultured on polymeric scaffolds in bioreactors to form regular or enriched cardiac muscle constructs, respectively. After 1 wk, all constructs contained a peripheral tissue-like region (50–70 μm thick) in which differentiated cardiac myocytes were organized in multiple layers in a 3-D configuration. Indexes of cell size (protein/DNA) and metabolic activity (tetrazolium conversion/DNA) were similar for constructs and neonatal rat ventricles. Electrophysiological studies conducted using a linear array of extracellular electrodes showed that the peripheral region of constructs exhibited relatively homogeneous electrical properties and sustained macroscopically continuous impulse propagation on a centimeter-size scale. Electrophysiological properties of enriched constructs were superior to those of regular constructs but inferior to those of native ventricles. These results demonstrate that 3-D cardiac muscle constructs can be engineered with cardiac-specific structural and electrophysiological properties and used for in vitro impulse propagation studies.

scholarly journals Cardiomyocyte functional screening: interrogating comparative electrophysiology of high-throughput model cell systems

2019 ◽  
Vol 317 (6) ◽  
pp. C1256-C1267 ◽  
Author(s):  
Simon P. Wells ◽  
Helen M. Waddell ◽  
Choon Boon Sim ◽  
Shiang Y. Lim ◽  
Gabriel B. Bernasochi ◽  
...  
Keyword(s):  
Conduction Velocity ◽  
High Throughput ◽  
Microelectrode Array ◽  
Ventricular Myocytes ◽  
Field Potential ◽  
Induced Pluripotent Stem Cell ◽  
Neonatal Rat ◽  
Functional Screening ◽  
Electrophysiological Properties

Cardiac arrhythmias of both atrial and ventricular origin are an important feature of cardiovascular disease. Novel antiarrhythmic therapies are required to overcome current drug limitations related to effectiveness and pro-arrhythmia risk in some contexts. Cardiomyocyte culture models provide a high-throughput platform for screening antiarrhythmic compounds, but comparative information about electrophysiological properties of commonly used types of cardiomyocyte preparations is lacking. Standardization of cultured cardiomyocyte microelectrode array (MEA) experimentation is required for its application as a high-throughput platform for antiarrhythmic drug development. The aim of this study was to directly compare the electrophysiological properties and responses to isoproterenol of three commonly used cardiac cultures. Neonatal rat ventricular myocytes (NRVMs), immortalized atrial HL-1 cells, and custom-generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured on microelectrode arrays for 48–120 h. Extracellular field potentials were recorded, and conduction velocity was mapped in the presence/absence of the β-adrenoceptor agonist isoproterenol (1 µM). Field potential amplitude and conduction velocity were greatest in NRVMs and did not differ in cardiomyocytes isolated from male/female hearts. Both NRVMs and hiPSC-CMs exhibited longer field potential durations with rate dependence and were responsive to isoproterenol. In contrast, HL-1 cells exhibited slower conduction and shorter field potential durations and did not respond to 1 µM isoproterenol. This is the first study to compare the intrinsic electrophysiologic properties of cultured cardiomyocyte preparations commonly used for in vitro electrophysiology assessment. These findings offer important comparative data to inform methodological approaches in the use of MEA and other techniques relating to cardiomyocyte functional screening investigations of particular relevance to arrhythmogenesis.

Medium perfusion enables engineering of compact and contractile cardiac tissue

2004 ◽  
Vol 286 (2) ◽  
pp. H507-H516 ◽  
Author(s):  
Milica Radisic ◽  
Liming Yang ◽  
Jan Boublik ◽  
Richard J. Cohen ◽  
Robert Langer ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Troponin I ◽  
Capture Rate ◽  
Initial Density ◽  
Neonatal Rat ◽  
Excitation Threshold ◽  
Interstitial Flow ◽  
Rat Cardiomyocytes ◽  
Gap Junctional

We hypothesized that functional constructs with physiological cell densities can be engineered in vitro by mimicking convective-diffusive oxygen transport normally present in vivo. To test this hypothesis, we designed an in vitro culture system that maintains efficient oxygen supply to the cells at all times during cell seeding and construct cultivation and characterized in detail construct metabolism, structure, and function. Neonatal rat cardiomyocytes suspended in Matrigel were cultured on collagen sponges at a high initial density (1.35 × 108 cells/cm3) for 7 days with interstitial flow of medium; constructs cultured in orbitally mixed dishes, neonatal rat ventricles, and freshly isolated cardiomyocytes served as controls. Constructs were assessed at timed intervals with respect to cell number, distribution, viability, metabolic activity, cell cycle, presence of contractile proteins (sarcomeric α-actin, troponin I, and tropomyosin), and contractile function in response to electrical stimulation [excitation threshold (ET), maximum capture rate (MCR), response to a gap junctional blocker]. Interstitial flow of culture medium through the central 5-mm-diameter × 1.5-mm-thick region resulted in a physiological density of viable and differentiated, aerobically metabolizing cells, whereas dish culture resulted in constructs with only a 100- to 200-μm-thick surface layer containing viable and differentiated but anaerobically metabolizing cells around an acellular interior. Perfusion resulted in significantly higher numbers of live cells, higher cell viability, and significantly more cells in the S phase compared with dish-grown constructs. In response to electrical stimulation, perfused constructs contracted synchronously, had lower ETs, and recovered their baseline function levels of ET and MCR after treatment with a gap junctional blocker; dish-grown constructs exhibited arrhythmic contractile patterns and failed to recover their baseline MCR levels.

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.

Chronic cardiotrophin-1 stimulation impairs contractile function in reconstituted heart tissue

2005 ◽  
Vol 288 (6) ◽  
pp. E1214-E1221 ◽  
Author(s):  
Oliver Zolk ◽  
Sven Engmann ◽  
Felix Münzel ◽  
Rasti Krajcik
Keyword(s):  
Cardiac Myocytes ◽  
Structural Changes ◽  
Contractile Function ◽  
Heart Tissue ◽  
Neonatal Rat ◽  
In Vitro Test ◽  
Human Heart Failure ◽  
Inotropic Effects ◽  
Neonatal Rat Cardiac Myocytes

Cardiotrophin-1 (CT-1) is known to promote survival but also to induce an elongated morphology of isolated cardiac myocytes, leading to the hypothesis that CT-1, which is chronically augmented in human heart failure, might induce eccentric cardiac hypertrophy and contractile failure. To address this, we used heart tissues reconstituted from neonatal rat cardiac myocytes (engineered heart tissue, EHT) as multicellular in vitro test systems. CT-1 dose-dependently affected contractile function in EHTs. After treatment with 0.1 nM CT-1 (corresponds to plasma levels in humans) for 10 days, twitch tension significantly decreased to 0.30 ± 0.04 mN ( n = 15) vs. 0.45 ± 0.04 mN ( n = 16) in controls. Furthermore, positive inotropic effects of cumulative concentrations of Ca2+ and isoprenaline were significantly diminished. Maximum isoprenaline-induced increase in twitch tension amounted to 0.27 ± 0.04 mN ( n = 15) vs. 0.47 ± 0.06 mN ( n = 16) in controls ( P < 0.001). When EHTs were treated for only 5 days, qualitatively similar results were obtained but changes were less pronounced. Immunostaining of whole mount EHT preparations revealed that after CT-1 treatment, the number of nonmyocytes significantly increased by 98% (1 nM, 10 days), and myocytes did not form compact, longitudinally oriented muscle bundles. Interestingly, expression of the Ca2+-handling protein calsequestrin was markedly reduced (69 ± 7% of control) by treatment with CT-1 (0.1 nM, 10 days). In summary, long-term exposure to CT-1 induces contractile dysfunction in EHTs. Structural changes due to impaired differentiation and/or remodeling of heart tissue may play an important role.

scholarly journals Notch1 signaling stimulates proliferation of immature cardiomyocytes

2008 ◽  
Vol 183 (1) ◽  
pp. 117-128 ◽  
Author(s):  
Chiara Collesi ◽  
Lorena Zentilin ◽  
Gianfranco Sinagra ◽  
Mauro Giacca
Keyword(s):  
Notch Signaling ◽  
Cardiac Myocytes ◽  
Molecular Mechanisms ◽  
Notch Pathway ◽  
Constitutive Expression ◽  
Neonatal Rat ◽  
Proliferative Potential ◽  
Rat Cardiomyocytes

The identification of the molecular mechanisms controlling cardiomyocyte proliferation during the embryonic, fetal, and early neonatal life appears of paramount interest in regard to exploiting this information to promote cardiac regeneration. Here, we show that the proliferative potential of neonatal rat cardiomyocytes is powerfully stimulated by the sustained activation of the Notch pathway. We found that Notch1 is expressed in proliferating ventricular immature cardiac myocytes (ICMs) both in vitro and in vivo, and that the number of Notch1-positive cells in the heart declines with age. Notch1 expression in ICMs paralleled the expression of its Jagged1 ligand on non-myocyte supporting cells. The inhibition of Notch signaling in ICMs blocked their proliferation and induced apoptosis; in contrast, its activation by Jagged1 or by the constitutive expression of its activated form using an adeno-associated virus markedly stimulated proliferative signaling and promoted ICM expansion. Maintenance or reactivation of Notch signaling in cardiac myocytes might represent an interesting target for innovative regenerative therapy.

scholarly journals The Use of iPSC-Derived Cardiomyocytes and Optical Mapping for Erythromycin Arrhythmogenicity Testing

2019 ◽  
Author(s):  
A.D. Podgurskaya ◽  
V.A. Tsvelaya ◽  
M.M. Slotvitsky ◽  
E.V. Dementyeva ◽  
K.R. Valetdinova ◽  
...  
Keyword(s):  
Conduction Velocity ◽  
Potassium Current ◽  
Optical Mapping ◽  
Capture Rate ◽  
Torsade De Pointes ◽  
Induced Pluripotent Stem Cell ◽  
Experimental Models ◽  
Mapping Method ◽  
Neonatal Rat ◽  
Induced Pluripotent

AbstractErythromycin is an antibiotic that prolongs the QT-interval and causes Torsade de Pointes (TdP) by blocking the rapid delayed rectifying potassium current (IKr) without affecting either the slow delayed rectifying potassium current (IKs) or inward rectifying potassium current (IK1). Erythromycin exerts this effect in the range of 1.5 μM–100 μM. However, the mechanism of action underlying its cardiotoxic effect and its role in the induction of arrhythmias, especially in multicellular cardiac experimental models, remain unclear. In this study the re-entry formation, conduction velocity, and maximum capture rate were investigated in a monolayer of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes from a healthy donor and in a neonatal rat ventricular myocyte (NRVM) monolayer using the optical mapping method under erythromycin concentrations of 15, 30, and 45 μM. In the monolayer of human iPSC-derived cardiomyocytes, the conduction velocity (CV) varied up to 12±9% at concentrations of 15–45 μM as compared with that of the control, whereas the maximum capture rate (MCR) declined substantially up to 28±12% (p < 0.05). In contrast, the tests on the NRVM monolayer showed no significant effect on the MCR. The results of the arrhythmogenicity test provided evidence for a “window” of concentrations of the drug (15 to 30 μM) at which the probability of re-entry increased.

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