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A computer model of engineered cardiac monolayers.

ABSTRACT: Engineered monolayers created using microabrasion and micropatterning methods have provided a simplified in vitro system to study the effects of anisotropy and fiber direction on electrical propagation. Interpreting the behavior in these culture systems has often been performed using classical computer models with continuous properties. However, such models do not account for the effects of random cell shapes, cell orientations, and cleft spaces inherent in these monolayers on the resulting wavefront conduction. This work presents a novel methodology for modeling a monolayer of cardiac tissue in which the factors governing cell shape, cell-to-cell coupling, and degree of cleft space are not constant but rather are treated as spatially random with assigned distributions. This modeling approach makes it possible to simulate wavefront propagation in a manner analogous to performing experiments on engineered monolayer tissues. Simulated results are compared to previously published measured data from monolayers used to investigate the role of cellular architecture on conduction velocities and anisotropy ratios. We also present an estimate for obtaining the electrical properties from these networks and demonstrate how variations in the discrete cellular architecture affect the macroscopic conductivities. The simulations support the common assumption that under normal ranges of coupling strength, tissues with relatively uniform distributions of cell shapes and connectivity can be represented using continuous models with conductivities derived from random discrete cellular architecture using either global or local estimates. The results also reveal that in the presence of abrupt changes in cell orientation, local estimates of tissue properties predict smoother changes in conductivity that may not adequately predict the discrete nature of propagation at the transition sites.

SUBMITTER: Kim JM

PROVIDER: S-EPMC2862160 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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A computer model of engineered cardiac monolayers.

Kim Jong M JM Bursac Nenad N Henriquez Craig S CS

Biophysical journal 20100501 9

Engineered monolayers created using microabrasion and micropatterning methods have provided a simplified in vitro system to study the effects of anisotropy and fiber direction on electrical propagation. Interpreting the behavior in these culture systems has often been performed using classical computer models with continuous properties. However, such models do not account for the effects of random cell shapes, cell orientations, and cleft spaces inherent in these monolayers on the resulting wave ...[more]

PMID: 20441739

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Project description:Rationale and objectiveThe use of genetic engineering of unexcitable cells to enable expression of gap junctions and inward rectifier potassium channels has suggested that cell therapies aimed at establishing electrical coupling of unexcitable donor cells to host cardiomyocytes may be arrhythmogenic. Whether similar considerations apply when the donor cells are electrically excitable has not been investigated. Here we tested the hypothesis that adenoviral transfer of genes coding Kir2.1 (I(K1)), Na(V)1.5 (I(Na)) and connexin-43 (Cx43) proteins into neonatal rat ventricular myofibroblasts (NRVF) will convert them into fully excitable cells, rescue rapid conduction velocity (CV) and reduce the incidence of complex reentry arrhythmias in an in vitro model.Methods and resultsWe used adenoviral (Ad-) constructs encoding Kir2.1, Na(V)1.5 and Cx43 in NRVF. In single NRVF, Ad-Kir2.1 or Ad-Na(V)1.5 infection enabled us to regulate the densities of I(K1) and I(Na), respectively. At varying MOI ratios of 10/10, 5/10 and 5/20, NRVF co-infected with Ad-Kir2.1+ Na(V)1.5 were hyperpolarized and generated action potentials (APs) with upstroke velocities >100 V/s. However, when forming monolayers only the addition of Ad-Cx43 made the excitable NRVF capable of conducting electrical impulses (CV?=?20.71±0.79 cm/s). When genetically engineered excitable NRVF overexpressing Kir2.1, Na(V)1.5 and Cx43 were used to replace normal NRVF in heterocellular monolayers that included neonatal rat ventricular myocytes (NRVM), CV was significantly increased (27.59±0.76 cm/s vs. 21.18±0.65 cm/s, p<0.05), reaching values similar to those of pure myocytes monolayers (27.27±0.72 cm/s). Moreover, during reentry, propagation was faster and more organized, with a significantly lower number of wavebreaks in heterocellular monolayers formed by excitable compared with unexcitable NRVF.ConclusionViral transfer of genes coding Kir2.1, Na(V)1.5 and Cx43 to cardiac myofibroblasts endows them with the ability to generate and propagate APs. The results provide proof of concept that cell therapies with excitable donor cells increase safety and reduce arrhythmogenic potential.

Project description:BACKGROUND: The interest in introducing ecologically-clean, and efficient enzymes into modern industry has been growing steadily. However, difficulties associated with controlling their orientation, and maintaining their selectivity and reactivity is still a significant obstacle. We have developed precise immobilization of biomolecules, while retaining their native functionality, and report a new, fast, easy, and reliable procedure of protein immobilization, with the use of Adenylate kinase as a model system. METHODS: Self-assembled monolayers of hexane-1,6-dithiol were formed on gold surfaces. The monolayers were characterized by contact-angle measurements, Elman-reagent reaction, QCM, and XPS. A specifically designed, mutated Adenylate kinase, where cysteine was inserted at the 75 residue, and the cysteine at residue 77 was replaced by serine, was used for attachment to the SAM surface via spontaneously formed disulfide (S-S) bonds. QCM, and XPS were used for characterization of the immobilized protein layer. Curve fitting in XPS measurements used a Gaussian-Lorentzian function. RESULTS AND DISCUSSION: Water contact angle (65-70°), as well as all characterization techniques used, confirmed the formation of self-assembled monolayer with surface SH groups. X-ray photoelectron spectroscopy showed clearly the two types of sulfur atom, one attached to the gold (triolate) and the other (SH/S-S) at the ?-position for the hexane-1,6-dithiol SAMs. The formation of a protein monolayer was confirmed using XPS, and QCM, where the QCM-determined amount of protein on the surface was in agreement with a model that considered the surface area of a single protein molecule. Enzymatic activity tests of the immobilized protein confirmed that there is no change in enzymatic functionality, and reveal activity ~100 times that expected for the same amount of protein in solution. CONCLUSIONS: To the best of our knowledge, immobilization of a protein by the method presented here, with the resulting high enzymatic activity, has never been reported. There are many potential applications for selective localization of active proteins at patterned surfaces, for example, bioMEMS (MEMS--Micro-Electro-Mechanical Systems. Due to the success of the method, presented here, it was decided to continue a research project of a biosensor by transferring it to a high aspect ratio platform--nanotubes.

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A computer model of engineered cardiac monolayers.

Publications

A computer model of engineered cardiac monolayers.

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