scholarly journals Functional Maturation of Human iPSC-based Cardiac Microphysiological Systems with Tunable Electroconductive Decellularized Extracellular Matrices

2019 ◽  
Author(s):  
Jonathan H. Tsui ◽  
Andrea Leonard ◽  
Nathan D. Camp ◽  
Joseph T. Long ◽  
Zeid Y. Nawas ◽  
...  
Keyword(s):  
Action Potential ◽  
Contractile Function ◽  
Protein Profile ◽  
Induced Pluripotent Stem Cell ◽  
Specific Protein ◽  
Calcium Handling ◽  
Cardiac Tissues ◽  
Hydrogel Scaffolds

AbstractHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer tremendous potential for use in engineering human tissues for regenerative therapy and drug screening. However, differentiated cardiomyocytes are phenotypically immature, reducing assay reliability when translating in vitro results to clinical studies and precluding hiPSC-derived cardiac tissues from therapeutic use in vivo. To address this, we have developed hybrid hydrogels comprised of decellularized porcine myocardial extracellular matrix (dECM) and reduced graphene oxide (rGO) to provide a more instructive microenvironment for proper cellular and tissue development. A tissue-specific protein profile was preserved post-decellularization, and through the modulation of rGO content and degree of reduction, the mechanical and electrical properties of the hydrogels could be tuned. Engineered heart tissues (EHTs) generated using dECM-rGO hydrogel scaffolds and hiPSC-derived cardiomyocytes exhibited significantly increased twitch forces at 14 days of culture and had increased the expression of genes that regulate contractile function. Similar improvements in various aspects of electrophysiological function, such as calcium-handling, action potential duration, and conduction velocity, were also induced by the hybrid biomaterial. We also demonstrate that dECM-rGO hydrogels can be used as a bioink to print cardiac tissues in a high-throughput manner, and these tissues were utilized to assess the proarrhythmic potential of cisapride. Action potential prolongation and beat interval irregularities was observed in dECM-rGO tissues at clinical doses of cisapride, indicating that the enhanced maturation of these tissues corresponded well with a capability to produce physiologically relevant drug responses.

scholarly journals Effects of dietary omega–3 fatty acids on ventricular function in dogs with healed myocardial infarctions: in vivo and in vitro studies

2010 ◽  
Vol 298 (4) ◽  
pp. H1219-H1228 ◽  
Author(s):  
George E. Billman ◽  
Yoshinori Nishijima ◽  
Andriy E. Belevych ◽  
Dmitry Terentyev ◽  
Ying Xu ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Fatty Acids ◽  
Ventricular Function ◽  
Contractile Function ◽  
Left Ventricular ◽  
Calcium Currents ◽  
Calcium Handling ◽  
Omega 3

Since omega–3 polyunsaturated fatty acids (n-3 PUFAs) can alter ventricular myocyte calcium handling, these fatty acids could adversely affect cardiac contractile function, particularly following myocardial infarction. Therefore, 4 wk after myocardial infarction, dogs were randomly assigned to either placebo (corn oil, 1 g/day, n = 16) or n-3 PUFAs supplement [docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) ethyl esters; 1, 2, or 4 g/day; n = 7, 8, and 12, respectively] groups. In vivo, ventricular function was evaluated by echocardiography before and after 3 mo of treatment. At the end of the 3-mo period, hearts were removed and in vitro function was evaluated using right ventricular trabeculae and isolated left ventricular myocytes. The treatment elicited significant ( P < 0.0001) dose-dependent increases (16.4-fold increase with 4 g/day) in left ventricular tissue and red blood cell n-3 PUFA levels (EPA + DHA, placebo, 0.42 ± 0.04; 1 g/day, 3.02 ± 0.23; 2 g/day, 3.63 ± 0.17; and 4 g/day, 6.97 ± 0.33%). Regardless of the dose, n-3 PUFA treatment did not alter ventricular function in the intact animal (e.g., 4 g/day, fractional shortening: pre, 42.9 ± 1.6 vs. post, 40.1 ± 1.7%; placebo: pre, 39.2 ± 1.3 vs. post, 38.4 ± 1.6%). The developed force per cross-sectional area, changes in length- and frequency-dependent behavior in contractile force, and the inotropic response to β-adrenoceptor activation were also similar for trabeculae obtained from placebo- or n-3 PUFA-treated dogs. Finally, calcium currents and calcium transients were the same in myocytes from n-3 PUFA- and placebo-treated dogs. Thus dietary n-3 PUFAs did not adversely alter either in vitro or in vivo ventricular contractile function in dogs with healed infarctions.

Abstract 156: Hydrogel Mattress, an in vitro Platform to Enhance Maturation and Evaluate Contractile Function of Individual hiPSC-CMs

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Tromondae K Feaster ◽  
Charles H Williams ◽  
Adrian G Cadar ◽  
Young W Chun ◽  
Lili Wang ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Disease Modeling ◽  
Contractile Function ◽  
Traction Force ◽  
Induced Pluripotent Stem Cell ◽  
Contractile Properties ◽  
Calcium Handling ◽  
Cell Shortening ◽  
Induced Pluripotent

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have great potential as tools for human heart disease modeling and drug discovery. However, their contractile properties have not been routinely evaluated; as current methods are not accessible for most laboratories. We sought to develop a more efficient method to evaluate hiPSC-CM mechanical properties, at the single cell level. Individual hiPSC-CMs were cultured on a hydrogel based platform, termed the “hydrogel mattress,” and their cellular contractile properties evaluated using video-based edge detection. We found that hiPSC-CMs maintained on the mattress reproducibly exhibited robust cell shortening, in dramatic contrast to hiPSC-CMs maintained in a standard manner. We further found that contraction and peak cell shortening amplitude of hiPSC-CMs on mattress was comparable to that of freshly isolated adult ventricular mouse CM. Importantly, hiPSC-CMs maintained on the mattress exhibited several characteristics of a native CM, in terms of myocyte elongation, calcium handling and pharmacological response. Finally, using this platform, we could calculate the traction force generated by individual CMs. In summary, the Hydrogel mattress platform is a simple and reliable in vitro platform that not only enables the quantification of contractile performance of isolated hiPSC-CMs, but also enhances CM maturation. This flexible platform can be extended to in vitro disease modeling, drug discovery and cardiotoxicity testing.

Abstract 542: Chronic Doxorubicin Cardiotoxicity Assessed in Engineered Cardiac Tissues Generated in Biowire™ II Platform

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Roozbeh Aschar-sobbi ◽  
Julia E Napolitano ◽  
Danielle R Bogdanowicz ◽  
Michael P Graziano
Keyword(s):  
Action Potential ◽  
Action Potential Duration ◽  
Cardiac Fibroblasts ◽  
Left Ventricular ◽  
Left Ventricular Ejection ◽  
Resting Membrane Potential ◽  
Ventricular Ejection Fraction ◽  
Cardiac Tissues

The anthracycline doxorubicin is an effective anti-tumor agent widely used in both adults and children. One major adverse effect of doxorubicin therapy is dose-dependent cardiotoxicity, ranging from asymptomatic reduction in left ventricular ejection fraction to more serious, potentially fatal symptoms including arrythmias and congestive heart failure. The exact mechanism of doxorubicin-induced cardiotoxicity remains unknown. Recently, human induced pluripotent stem cells (hiPSC) have emerged as a potential tool to model cardiac toxicity, but their fetal-like phenotype raises concerns about the translatability of in vitro data to in vivo cardiotoxicity. To overcome this limitation, Biowire™ II platform was used to generate 3D engineered cardiac tissues (ECTs) from hiPSC-derived cardiomyocytes and human cardiac fibroblasts. Using long-term electrical stimulation, ECTs with a phenotype approaching that of adult human myocardium were obtained. The ECTs were then exposed to 1 μM doxorubicin for 8 days followed by 7 days of washout. Measurements of contractile force amplitude at 1 Hz stimulation showed a transient increase in force within 24 hours of doxorubicin exposure followed by decrease in force after 2 days. Intracellular recordings of action potential (AP) showed a decrease in maximum upstroke velocity (dV/dt), AP amplitude (APA), and resting membrane potential (RMP) after 8 days of doxorubicin treatment. In addition, action potential duration (APD) at 30% (APD30) repolarization was increased in doxorubicin-treated ECTs, whereas APD50 and APD90 were decreased. Following 7 days of washout, no difference in force or AP parameters was found between doxorubicin and vehicle-treated ECTs with the exception of APD50 and APD90 which remained abbreviated. A global untargeted analysis of the conditioned media from doxorubicin-treated ECTs identified 204 analytes and revealed an upregulation of redox homeostasis, differential fatty acid metabolism, altered glycolysis and TCA cycle metabolites, and decreased nucleoside metabolism compared to vehicle-treated ECTs. These results show that doxorubicin not only increases oxidative stress, but also irreversibly affects action potential duration which may predispose to cardiac arrhythmias.

scholarly journals Expression of magA in Legionella pneumophila Philadelphia-1 Is Developmentally Regulated and a Marker of Formation of Mature Intracellular Forms

2004 ◽  
Vol 186 (10) ◽  
pp. 3038-3045 ◽  
Author(s):  
Margot F. Hiltz ◽  
Gary R. Sisson ◽  
Ann Karen C. Brassinga ◽  
Elizabeth Garduno ◽  
Rafael A. Garduno ◽  
...  
Keyword(s):  
Hela Cells ◽  
Legionella Pneumophila ◽  
Protein Profile ◽  
Methionine Sulfoxide ◽  
Growth Cycle ◽  
Specific Protein ◽  
Methionine Sulfoxide Reductase ◽  
Developmental Cycle

ABSTRACT Legionella pneumophila displays a biphasic developmental cycle in which replicating forms (RFs) differentiate postexponentially into highly infectious, cyst-like mature intracellular forms (MIFs). Using comparative protein profile analyses (MIFs versus RFs), we identified a 20-kDa protein, previously annotated as “Mip-like” protein, that was enriched in MIFs. However, this 20-kDa protein shared no similarity with Mip, a well-characterized peptidyl-prolyl isomerase of L. pneumophila, and for clarity we renamed it MagA (for “MIF-associated gene”). We monitored MagA levels across the growth cycle (in vitro and in vivo) by immunoblotting and established that MagA levels increased postexponentially in vitro (∼3-fold) and nearly 10-fold during MIF morphogenesis in HeLa cells. DNA sequence analysis of the magA locus revealed an upstream divergently transcribed gene, msrA, encoding a peptide methionine sulfoxide reductase and a shared promoter region containing direct and indirect repeat sequences as well as −10 hexamers often associated with stationary-phase regulation. While MagA has no known function, it contains a conserved CXXC motif commonly found in members of the thioredoxin reductase family and in AhpD reductases that are associated with alkylhydroperoxide reductase (AhpC), suggesting a possible role in protection from oxidative stress. MIFs from L. pneumophila strain Lp02 containing a magA deletion exhibited differences in Giménez staining, as well as an apparent increase in cytopathology to HeLa cells, but otherwise were unaltered in virulence traits. As demonstrated by this study, MagA appears to be a MIF-specific protein expressed late in intracellular growth that may serve as a useful marker of development.

Lethal ischemia due to intracoronary endothelin in pigs

1989 ◽  
Vol 257 (1) ◽  
pp. H339-H343 ◽  
Author(s):  
D. Ezra ◽  
R. E. Goldstein ◽  
J. F. Czaja ◽  
G. Z. Feuerstein
Keyword(s):  
Diastolic Pressure ◽  
Contractile Function ◽  
Left Ventricular ◽  
Systemic Hemodynamic ◽  
Progressive Decline ◽  
Cardiac Tissues ◽  
Persistent Hypotension ◽  
Net Release

Endothelin is a recently discovered endothelium-derived peptide with potent coronary constrictor properties in vitro. To evaluate endothelin's cardiac actions in vivo, we measured coronary flow and regional myocardial shortening when intracoronary porcine endothelin was given to anesthetized open-chested pigs. Bolus adminstration into the left anterior descending (LAD) coronary artery of six pigs caused dose-related rapidly reversing depression of LAD flow and local shortening. Marked reductions in flow [-71 +/- 8 (SE) %] and shortening (-83 +/- 2%) after 30 pmol/kg demonstrated endothelin's potency in cardiac tissues. Systemic hemodynamic values were unaltered except for transient rises in left ventricular end-diastolic pressure. Endothelin-induced decrement in LAD flow was accompanied by electrocardiographic signs of myocardial ischemia and net release of local myocardial lactate. Intracoronary infusion of endothelin, 15 pmol.kg-1.min-1, caused progressive decline in LAD flow and local shortening followed by severe persistent hypotension and terminal ventricular fibrillation in four of five pigs. Unlike intracoronary delivery of other potent coronary constrictors, intracoronary administration of endothelin did not lead to rapid escape from the peptide's deleterious influence. Coronary exposure to endothelin under pathophysiological circumstances could result in uniquely persistent decrements in myocardial perfusion and contractile function.

scholarly journals Engineering aligned human cardiac muscle using developmentally inspired fibronectin micropatterns

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ivan Batalov ◽  
Quentin Jallerat ◽  
Sean Kim ◽  
Jacqueline Bliley ◽  
Adam W. Feinberg
Keyword(s):  
Cardiac Fibroblasts ◽  
Induced Pluripotent Stem Cell ◽  
Confocal Imaging ◽  
Embryonic Chick ◽  
Cardiac Tissues ◽  
Density Dependent ◽  
Induced Pluripotent ◽  
Cell Cell

AbstractCardiac two-dimensional tissues were engineered using biomimetic micropatterns based on the fibronectin-rich extracellular matrix (ECM) of the embryonic heart. The goal of this developmentally-inspired, in vitro approach was to identify cell–cell and cell-ECM interactions in the microenvironment of the early 4-chambered vertebrate heart that drive cardiomyocyte organization and alignment. To test this, biomimetic micropatterns based on confocal imaging of fibronectin in embryonic chick myocardium were created and compared to control micropatterns designed with 2 or 20 µm wide fibronectin lines. Results show that embryonic chick cardiomyocytes have a unique density-dependent alignment on the biomimetic micropattern that is mediated in part by N-cadherin, suggesting that both cell–cell and cell-ECM interactions play an important role in the formation of aligned myocardium. Human induced pluripotent stem cell-derived cardiomyocytes also showed density-dependent alignment on the biomimetic micropattern but were overall less well organized. Interestingly, the addition of human adult cardiac fibroblasts and conditioning with T3 hormone were both shown to increase human cardiomyocyte alignment. In total, these results show that cardiomyocyte maturation state, cardiomyocyte-cardiomyocyte and cardiomyocyte-fibroblast interactions, and cardiomyocyte-ECM interactions can all play a role when engineering anisotropic cardiac tissues in vitro and provides insight as to how these factors may influence cardiogenesis in vivo.

scholarly journals Myofibrils in Cardiomyocytes Tend to Assemble Along the Maximal Principle Stress Directions

2017 ◽  
Vol 139 (12) ◽  
Author(s):  
Hongyan Yuan ◽  
Bahador Marzban ◽  
Kevin Kit Parker
Keyword(s):  
Rational Design ◽  
Spatial Organization ◽  
Cardiac Tissue ◽  
Contractile Function ◽  
Focal Adhesions ◽  
Cardiac Tissues ◽  
Mechanics Model ◽  
Stress Directions

The mechanisms underlying the spatial organization of self-assembled myofibrils in cardiac tissues remain incompletely understood. By modeling cells as elastic solids under active cytoskeletal contraction, we found a good correlation between the predicted maximal principal stress directions and the in vitro myofibril orientations in individual cardiomyocytes. This implies that actomyosin fibers tend to assemble along the maximal tensile stress (MTS) directions. By considering the dynamics of focal adhesion and myofibril formation in the model, we showed that different patterns of myofibril organizations in mature versus immature cardiomyocytes can be explained as the consequence of the different levels of force-dependent remodeling of focal adhesions. Further, we applied the mechanics model to cell pairs and showed that the myofibril organizations can be regulated by a combination of multiple factors including cell shape, cell–substrate adhesions, and cell–cell adhesions. This mechanics model can guide the rational design in cardiac tissue engineering where recapitulating in vivo myofibril organizations is crucial to the contractile function of the heart.

scholarly journals A Study of Gene Expression, Structure, and Contractility of iPSC-Derived Cardiac Myocytes from a Family with Heart Disease due to LMNA Mutation

2021 ◽  
Author(s):  
Mehrsa Mehrabi ◽  
Tessa A. Morris ◽  
Zixuan Cang ◽  
Cecilia H. H. Nguyen ◽  
Yutong Sha ◽  
...  
Keyword(s):  
Heart Disease ◽  
Disease Progression ◽  
Contractile Function ◽  
Induced Pluripotent Stem Cell ◽  
Stress Generation ◽  
Patient Specific ◽  
Single Experiment ◽  
Cardiac Tissues ◽  
Functional Changes

AbstractGenetic mutations to the Lamin A/C gene (LMNA) can cause heart disease, but the mechanisms making cardiac tissues uniquely vulnerable to the mutations remain largely unknown. Further, patients with LMNA mutations have highly variable presentation of heart disease progression and type. In vitro patient-specific experiments could provide a powerful platform for studying this phenomenon, but the use of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) introduces heterogeneity in maturity and function thus complicating the interpretation of the results of any single experiment. We hypothesized that integrating single cell RNA sequencing (scRNA-seq) with analysis of the tissue architecture and contractile function would elucidate some of the probable mechanisms. To test this, we investigated five iPSC-CM lines, three controls and two patients with a (c.357-2A>G) mutation. The patient iPSC-CM tissues had significantly weaker stress generation potential than control iPSC-CM tissues demonstrating the viability of our in vitro approach. Through scRNA-seq, differentially expressed genes between control and patient lines were identified. Some of these genes, linked to quantitative structural and functional changes, were cardiac specific, explaining the targeted nature of the disease progression seen in patients. The results of this work demonstrate the utility of combining in vitro tools in exploring heart disease mechanics.

Abstract 123: Cardiac Beta-arrestin2 Promotes Contractility by Enhancing SERCA2a SUMOylation and Activity

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Anastasios Lymperopoulos ◽  
Malika Jafferjee ◽  
Thairy Reyes Valero ◽  
Christine Marrero ◽  
Katie A McCrink ◽  
...  
Keyword(s):  
G Protein ◽  
Contractile Function ◽  
Cardiac Contractility ◽  
Beta Agonist ◽  
Calcium Handling ◽  
Western World ◽  
Mouse Heart ◽  
Signaling Mechanism

Heart failure (HF) is the number one killer disease in the western world and new and innovative treatments are needed. Sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA)-2a is a crucial, for contractile function, calcium-handling protein expressed in the mammalian myocardium and its downregulation is one of the molecular hallmarks of chronic HF. Its activation is part of the signaling mechanism by which the β 1 -adrenergic receptors (ARs) increase cardiac contractility. Agonist-bound β 1 ARs however, like most G protein-coupled receptors (GPCRs), undergo functional desensitization/internalization due to the actions of βarrestin1 or -2. These two arrestins are universal GPCR adapter proteins, mediating G protein-independent signaling via multi-protein scaffolding, and, among the cellular processes they can regulate, is protein SUMO (small ubiquitin-like modifier)-ylation, which generally increases protein stability/levels. In the heart, βarrestin1 appears detrimental, whereas βarrestin2 beneficial, for structure and function post-myocardial infarction (MI). Post-MI βarrestin1 knockout mice also display elevated SERCA2a activity and better contractility than post-MI wild type mice. In addition, reduced cardiac SERCA2a SUMOylation is known to underlie its downregulation in HF, decreasing cardiac contractility. Thus, in the present study, we sought to investigate a potential involvement of cardiac β 1 AR-activated βarrestins in regulation of SERCA2a SUMOylation and activity. By studying individual βarrestin knockout heart extracts, we found that βarrestin2, but not βarrestin1, interacts with SERCA2a in the mouse heart in vivo, promoting the latter`s SUMOylation and activity. This interaction is direct, as indicated by pull-down and FRET experiments. Finally, via in vitro studies in the cardiomyocyte-like cell line H9c2, we found that this interaction is both β 1 AR-, and beta-agonist-specific, and leads to increased Ubc9-dependent SERCA2a SUMOylation, which, in turn, acutely enhances SERCA2a activity in H9c2 cells. These results suggest that βarrestin2, presumed to also decrease cardiac function by desensitizing βARs, may actually (directly) enhance cardiac contractility, thereby opposing βarrestin1 in that regard.

Specific Labeling of Protein Domains with Antibody Fragments

1981 ◽  
Vol 39 ◽  
pp. 416-417
Author(s):  
U. Aebi ◽  
L.E. Buhle ◽  
W.E. Fowler
Keyword(s):  
Image Processing ◽  
Specific Protein ◽  
Antibody Fragments ◽  
Supramolecular Organization ◽  
Two Dimensional ◽  
Ordered Structures ◽  
Virus Capsids ◽  
Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

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