Virtual blebbistatin: A robust and rapid software approach to motion artifact removal in optical mapping of cardiomyocytes.

Fluorescent reporters of cardiac electrophysiology provide valuable information on heart cell and tissue function. However, motion artifacts caused by cardiac muscle contraction interfere with accurate measurement of fluorescence signals. Although drugs such as blebbistatin can be applied to stop cardiac tissue from contracting by uncoupling calcium-contraction, their usage prevents the study of excitation-contraction coupling and, as we show, impacts cellular structure. We therefore developed a robust method to remove motion computationally from images of contracting cardiac muscle and to map fluorescent reporters of cardiac electrophysiological activity onto images of undeformed tissue. When validated on cardiomyocytes derived from human induced pluripotent stem cells (iPSCs), in both monolayers and engineered tissues, the method enabled efficient and robust reduction of motion artifact. As with pharmacologic approaches using blebbistatin for motion removal, our algorithm improved the accuracy of optical mapping, as demonstrated by spatial maps of calcium transient decay. However, unlike pharmacologic motion removal, our computational approach allowed direct analysis of calcium-contraction coupling. Results revealed calcium-contraction coupling to be more uniform across cells within engineered tissues than across cells in monolayer culture. The algorithm shows promise as a robust and accurate tool for optical mapping studies of excitation-contraction coupling in heart tissue.

A review on qualifications and cost effectiveness of induced pluripotent stem cells (IPSCs)-induced cardiomyocytes in drug screening tests.

Human induced pluripotent stem cells (hIPSCs) have initiated a higher degree of successes in disease modelling, preclinical evaluation of drug therapy and pharmaco-toxicological testing. Since the discovery of iPSCs in 2006, many advanced techniques have been introduced to differentiate iPSCs to cardiomyocytes, which have been progressively improved. The disease models from iPSC-induced cardiomyocytes (iPSC-CM) have been successfully helping to study a variety of cardiac diseases such as long QT syndrome, drug-induced long QT, different cardiomyopathies related to mutations in mitochondria or desmosomal proteins and other rare genetic diseases. IPSC-CMs have also been used to screen the role of chemicals in cardiovascular drug discovery and individualisation of drug dosages. In this review, the quality of current procedures for characterisation and maturation of iPSC-CM lines will be discussed. Also, we will focus on time efficiency and cost of standard differentiation methods after reprogramming.

Fluorescence Correlation Spectroscopy and Photon Counting Histograms in Finite, Bounded Domains.

Analysis of fluctuations arising as fluorescent particles pass through a focused laser beam has enabled quantitative characterization of a broad range of molecular kinetic processes. Two key mathematical frameworks that have enabled these quantifications are fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis. Although these frameworks are effective and accurate when the focused laser beam is well approximated by an infinite Gaussian beam with a waist that is small compared to the size of the region over which the fluorescent particles can diffuse, they cannot be applied to situations in which this region is bounded at the nanoscale. We therefore derived general forms of the FCS and PCH frameworks for bounded systems. The finite-domain form of FCS differs from the classical form in its boundary and initial conditions and requires development of a new Fourier space solution for fitting data. Our finite-domain FCS predicts simulated data accurately and reduces to a previous model for the special case when the system is much larger than the Gaussian beam and can be considered to be infinite. We also derived the PCH form for the bounded systems. Our approach enables estimation of the concentration of diffusing fluorophores within a finite domain for the first time, to our knowledge. The method opens the possibility of quantification of kinetics in several systems for which this has never been possible.

The Balance between Actomyosin Contractility and Microtubule Polymerization Regulates Hierarchical Protrusions That Govern Efficient Fibroblast-Collagen Interactions.

Fibroblasts undergo a critical transformation from an initially inactive state to a morphologically different and contractile state after several hours of being embedded within a physiologically relevant three-dimensional (3D) fibrous collagen-based extracellular matrix (ECM). However, little is known about the critical mechanisms by which fibroblasts adapt themselves and their microenvironment in the earliest stage of cell-matrix interaction. Here, we identified the mechanisms by which fibroblasts interact with their 3D collagen fibrous matrices in the early stages of cell-matrix interaction and showed that fibroblasts use energetically efficient hierarchical micro/nano-scaled protrusions in these stages as the primary means for the transformation and adaptation. We found that actomyosin contractility in these protrusions in the early stages of cell-matrix interaction restricts the growth of microtubules by applying compressive forces on them. Our results show that actomyosin contractility and microtubules work in concert in the early stages of cell-matrix interaction to adapt fibroblasts and their microenvironment to one another. These early stage interactions result in responses to disruption of the microtubule network and/or actomyosin contractility that are opposite to well-known responses to late-stage disruption and reveal insight into the ways that cells adapt themselves and their ECM recursively.

A model for positive feedback control of the transformation of fibroblasts to myofibroblasts.

The phenotypic conversion of normal fibroblasts to myofibroblasts is central to normal wound healing and to pathological fibrosis that can occur in the heart and many other tissues. The transformation occurs in two stages. The first stage is driven mainly by mechanical changes such as increased stiffness of the heart due to hypertension and cellular contractility. The second stage requires both increasing stiffness and biochemical factors such as the growth factor, TGFß. As more and more cells convert from weakly contractile fibroblasts to strongly contractile myofibroblasts, the stiffness of the ventricular muscle increases. We propose a simple model for the establishment of non-equilibrium steady states with different compositions of fibroblasts and myofibroblasts. Under some conditions a positive feedback loop resulting from the increasing stiffness caused by increasing numbers of myofibroblasts can produce a bifurcation between steady states with low and high myofibroblast content. We illustrate the large mechanical differences between normal fibroblasts and myofibroblasts with measurements in engineered tissue constructs.

Energy dissipation in quasi-linear viscoelastic tissues, cells, and extracellular matrix.

Characterizing how a tissue's constituents give rise to its viscoelasticity is important for uncovering how hidden timescales underlie multiscale biomechanics. These constituents are viscoelastic in nature, and their mechanics must typically be assessed from the uniaxial behavior of a tissue. Confounding the challenge is that tissue viscoelasticity is typically associated with nonlinear elastic responses. Here, we experimentally assessed how fibroblasts and extracellular matrix (ECM) within engineered tissue constructs give rise to the nonlinear viscoelastic responses of a tissue. We applied a constant strain rate, "triangular-wave" loading and interpreted responses using the Fung quasi-linear viscoelastic (QLV) material model. Although the Fung QLV model has several well-known weaknesses, it was well suited to the behaviors of the tissue constructs, cells, and ECM tested. Cells showed relatively high damping over certain loading frequency ranges. Analysis revealed that, even in cases where the Fung QLV model provided an excellent fit to data, the the time constant derived from the model was not in general a material parameter. Results have implications for design of protocols for the mechanical characterization of biological materials, and for the mechanobiology of cells within viscoelastic tissues.

Investigation of Nanoscopic Phase Separations in Lipid Membranes Using Inverse FCS.

Measurement of the sizes of nanoscopic particles is a difficult challenge, especially in two-dimensional systems such as cell membranes. We have extended inverse fluorescence correlation spectroscopy (iFCS) to endow it with unique advantages for measuring particle size from the nano- to the microscale. We have augmented iFCS with an analysis of moments of fluorescence fluctuations and used it to measure stages of phase separation in model lipid bilayer membranes. We observed two different pathways for the growth of phase domains. In one, nanoscopic gel domains appeared first and then gradually grew to micrometer size. In the other, the domains reached micrometer size quickly, and their number gradually increased. These measurements demonstrate the value of iFCS measurements through their ability, to our knowledge, to provide new information about the mechanism of lipid phase separation and potentially about the physical basis of naturally occurring nanodomains such as lipid rafts.

Discrete quasi-linear viscoelastic damping analysis of connective tissues, and the biomechanics of stretching.

The time- and frequency-dependent properties of connective tissue define their physiological function, but are notoriously difficult to characterize. Well-established tools such as linear viscoelasticity and the Fung quasi-linear viscoelastic (QLV) model impose forms on responses that can mask true tissue behavior. Here, we applied a more general discrete quasi-linear viscoelastic (DQLV) model to identify the static and dynamic time- and frequency-dependent behavior of rabbit medial collateral ligaments. Unlike the Fung QLV approach, the DQLV approach revealed that energy dissipation is elevated at a loading period of ∼10s. The fitting algorithm was applied to the entire loading history on each specimen, enabling accurate estimation of the material's viscoelastic relaxation spectrum from data gathered from transient rather than only steady states. The application of the DQLV method to cyclically loading regimens has broad applicability for the characterization of biological tissues, and the results suggest a mechanistic basis for the stretching regimens most favored by athletic trainers.

Fibroblasts Slow Conduction Velocity in a Reconstituted Tissue Model of Fibrotic Cardiomyopathy.

Myocardial function deteriorates over the course of fibrotic cardiomyopathy, due to electrophysiological and mechanical effects of myofibroblasts that are not completely understood. Although a range of experimental model systems and associated theoretical treatments exist at the levels of isolated cardiomyocytes and planar co-cultures of myofibroblasts and cardiomyocytes, interactions between these cell types at the tissue level are less clear. We studied these interactions through an engineered heart tissue (EHT) model of fibrotic myocardium and a mathematical model of the effects of cellular composition on EHT impulse conduction velocity. The EHT model allowed for modulation of cardiomyocyte and myofibroblast volume fractions, and observation of cell behavior in a three-dimensional environment that is more similar to native heart tissue than is planar cell culture. The cardiomyocyte and myofibroblast volume fractions determined the retardation of impulse conduction (spread of the action potential) in EHTs as measured by changes of the fluorescence of the Ca2+ probe, Fluo-2. Interpretation through our model showed retardation far in excess of predictions by homogenization theory, with conduction ceasing far below the fibroblast volume fraction associated with steric percolation. Results point to an important multiscale structural role of myofibroblasts in attenuating impulse conduction in fibrotic cardiomyopathy.

Remodeling by fibroblasts alters the rate-dependent mechanical properties of collagen.

UNLABELLED: The ways that fibroblasts remodel their environment are central to wound healing, development of musculoskeletal tissues, and progression of pathologies such as fibrosis. However, the changes that fibroblasts make to the material around them and the mechanical consequences of these changes have proven difficult to quantify, especially in realistic, viscoelastic three-dimensional culture environments, leaving a critical need for quantitative data. Here, we observed the mechanisms and quantified the mechanical effects of fibroblast remodeling in engineered tissue constructs (ETCs) comprised of reconstituted rat tail (type I) collagen and human fibroblast cells. To study the effects of remodeling on tissue mechanics, stress-relaxation tests were performed on ETCs cultured for 24, 48, and 72h. ETCs were treated with deoxycholate and tested again to assess the ECM response. Viscoelastic relaxation spectra were obtained using the generalized Maxwell model. Cells exhibited viscoelastic damping at two finite time constants over which the ECM showed little damping, approximately 0.2s and 10-30s. Different finite time constants in the range of 1-7000s were attributed to ECM relaxation. Cells remodeled the ECM to produce a relaxation time constant on the order of 7000s, and to merge relaxation finite time constants in the 0.5-2s range into a single time content in the 1s range. Results shed light on hierarchical deformation mechanisms in tissues, and on pathologies related to collagen relaxation such as diastolic dysfunction. STATEMENT OF SIGNIFICANCE: As fibroblasts proliferate within and remodel a tissue, they change the tissue mechanically. Quantifying these changes is critical for understanding wound healing and the development of pathologies such as cardiac fibrosis. Here, we characterize for the first time the spectrum of viscoelastic (rate-dependent) changes arising from the remodeling of reconstituted collagen by fibroblasts. The method also provides estimates of the viscoelastic spectra of fibroblasts within a three-dimensional culture environment. Results are of particular interest because of the ways that fibroblasts alter the mechanical response of collagen at loading frequencies associated with cardiac contraction in humans.

An approach to quantifying 3D responses of cells to extreme strain.

The tissues of hollow organs can routinely stretch up to 2.5 times their length. Although significant pathology can arise if relatively large stretches are sustained, the responses of cells are not known at these levels of sustained strain. A key challenge is presenting cells with a realistic and well-defined three-dimensional (3D) culture environment that can sustain such strains. Here, we describe an in vitro system called microscale, magnetically-actuated synthetic tissues (micro-MASTs) to quantify these responses for cells within a 3D hydrogel matrix. Cellular strain-threshold and saturation behaviors were observed in hydrogel matrix, including strain-dependent proliferation, spreading, polarization, and differentiation, and matrix adhesion retained at strains sufficient for apoptosis. More broadly, the system shows promise for defining and controlling the effects of mechanical environment upon a broad range of cells.

Tissue constructs: platforms for basic research and drug discovery.

The functions, form and mechanical properties of cells are inextricably linked to their extracellular environment. Cells from solid tissues change fundamentally when, isolated from this environment, they are cultured on rigid two-dimensional substrata. These changes limit the significance of mechanical measurements on cells in two-dimensional culture and motivate the development of constructs with cells embedded in three-dimensional matrices that mimic the natural tissue. While measurements of cell mechanics are difficult in natural tissues, they have proven effective in engineered tissue constructs, especially constructs that emphasize specific cell types and their functions, e.g. engineered heart tissues. Tissue constructs developed as models of disease also have been useful as platforms for drug discovery. Underlying the use of tissue constructs as platforms for basic research and drug discovery is integration of multiscale biomaterials measurement and computational modelling to dissect the distinguishable mechanical responses separately of cells and extracellular matrix from measurements on tissue constructs and to quantify the effects of drug treatment on these responses. These methods and their application are the main subjects of this review.

A discrete spectral analysis for determining quasi-linear viscoelastic properties of biological materials.

The viscoelastic behaviour of a biological material is central to its functioning and is an indicator of its health. The Fung quasi-linear viscoelastic (QLV) model, a standard tool for characterizing biological materials, provides excellent fits to most stress-relaxation data by imposing a simple form upon a material's temporal relaxation spectrum. However, model identification is challenging because the Fung QLV model's 'box'-shaped relaxation spectrum, predominant in biomechanics applications, can provide an excellent fit even when it is not a reasonable representation of a material's relaxation spectrum. Here, we present a robust and simple discrete approach for identifying a material's temporal relaxation spectrum from stress-relaxation data in an unbiased way. Our 'discrete QLV' (DQLV) approach identifies ranges of time constants over which the Fung QLV model's typical box spectrum provides an accurate representation of a particular material's temporal relaxation spectrum, and is effective at providing a fit to this model. The DQLV spectrum also reveals when other forms or discrete time constants are more suitable than a box spectrum. After validating the approach against idealized and noisy data, we applied the methods to analyse medial collateral ligament stress-relaxation data and identify the strengths and weaknesses of an optimal Fung QLV fit.

Efficient and optimized identification of generalized Maxwell viscoelastic relaxation spectra.

Viscoelastic relaxation spectra are essential for predicting and interpreting the mechanical responses of materials and structures. For biological tissues, these spectra must usually be estimated from viscoelastic relaxation tests. Interpreting viscoelastic relaxation tests is challenging because the inverse problem is expensive computationally. We present here an efficient algorithm that enables rapid identification of viscoelastic relaxation spectra. The algorithm was tested against trial data to characterize its robustness and identify its limitations and strengths. The algorithm was then applied to identify the viscoelastic response of reconstituted collagen, revealing an extensive distribution of viscoelastic time constants.

Diffusion of human replication protein A along single-stranded DNA.

Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA-DNA interactions, we combined ensemble and single-molecule fluorescence approaches to examine human RPA (hRPA) diffusion along ssDNA and find that an hRPA heterotrimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D1~5000nt(2) s(-1) at 37°C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA.