scholarly journals Multiscale Characterization of Engineered Cardiac Tissue Architecture

2016 ◽  
Vol 138 (11) ◽  
Author(s):  
Nancy K. Drew ◽  
Nicholas E. Johnsen ◽  
Jason Q. Core ◽  
Anna Grosberg
Keyword(s):  
Self Assembly ◽  
Cardiac Tissue ◽  
Multiple Scales ◽  
Ventricular Myocytes ◽  
Orientational Order ◽  
Cardiac Cells ◽  
Neonatal Rat ◽  
Length Scales ◽  
Cardiac Tissues ◽  
The Relationship

In a properly contracting cardiac muscle, many different subcellular structures are organized into an intricate architecture. While it has been observed that this organization is altered in pathological conditions, the relationship between length-scales and architecture has not been properly explored. In this work, we utilize a variety of architecture metrics to quantify organization and consistency of single structures over multiple scales, from subcellular to tissue scale as well as correlation of organization of multiple structures. Specifically, as the best way to characterize cardiac tissues, we chose the orientational and co-orientational order parameters (COOPs). Similarly, neonatal rat ventricular myocytes were selected for their consistent architectural behavior. The engineered cells and tissues were stained for four architectural structures: actin, tubulin, sarcomeric z-lines, and nuclei. We applied the orientational metrics to cardiac cells of various shapes, isotropic cardiac tissues, and anisotropic globally aligned tissues. With these novel tools, we discovered: (1) the relationship between cellular shape and consistency of self-assembly; (2) the length-scales at which unguided tissues self-organize; and (3) the correlation or lack thereof between organization of actin fibrils, sarcomeric z-lines, tubulin fibrils, and nuclei. All of these together elucidate some of the current mysteries in the relationship between force production and architecture, while raising more questions about the effect of guidance cues on self-assembly function. These types of metrics are the future of quantitative tissue engineering in cardiovascular biomechanics.

scholarly journals Quantification of Cardiomyocyte Alignment from Three-Dimensional (3D) Confocal Microscopy of Engineered Tissue

2017 ◽  
Vol 23 (4) ◽  
pp. 826-842 ◽  
Author(s):  
William J. Kowalski ◽  
Fangping Yuan ◽  
Takeichiro Nakane ◽  
Hidetoshi Masumoto ◽  
Marc Dwenger ◽  
...  
Keyword(s):  
Multiple Scales ◽  
Orientational Order ◽  
Three Dimensional ◽  
Biological Tissues ◽  
Cardiac Cells ◽  
Cell Alignment ◽  
Cardiac Tissues ◽  
Automated Method ◽  
Synthetic Images ◽  
Induced Pluripotent

AbstractBiological tissues have complex, three-dimensional (3D) organizations of cells and matrix factors that provide the architecture necessary to meet morphogenic and functional demands. Disordered cell alignment is associated with congenital heart disease, cardiomyopathy, and neurodegenerative diseases and repairing or replacing these tissues using engineered constructs may improve regenerative capacity. However, optimizing cell alignment within engineered tissues requires quantitative 3D data on cell orientations and both efficient and validated processing algorithms. We developed an automated method to measure local 3D orientations based on structure tensor analysis and incorporated an adaptive subregion size to account for multiple scales. Our method calculates the statistical concentration parameter,κ, to quantify alignment, as well as the traditional orientational order parameter. We validated our method using synthetic images and accurately measured principal axis and concentration. We then applied our method to confocal stacks of cleared, whole-mount engineered cardiac tissues generated from human-induced pluripotent stem cells or embryonic chick cardiac cells and quantified cardiomyocyte alignment. We found significant differences in alignment based on cellular composition and tissue geometry. These results from our synthetic images and confocal data demonstrate the efficiency and accuracy of our method to measure alignment in 3D tissues.

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 Nicotine Causes Mitochondrial Dynamics Imbalance and Apoptosis Through ROS Mediated Mitophagy Impairment in Cardiomyocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Ting-ting Meng ◽  
Wei Wang ◽  
Fan-liang Meng ◽  
Shu-ya Wang ◽  
Hui-hui Wu ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Ventricular Myocytes ◽  
Mitochondrial Dynamics ◽  
Cathepsin L ◽  
Mitochondrial Fission ◽  
Treated Group ◽  
Superoxide Production ◽  
Neonatal Rat ◽  
Nicotine Treatment ◽  
The Relationship

Nicotine contained in traditional cigarettes, hookahs, and e-cigarettes is an important risk factor for cardiovascular disease. Our previous study showed that macroautophagic flux impairment occurred under nicotine stimulation. However, whether nicotine influences mitochondrial dynamics in neonatal rat ventricular myocytes (NRVMs) is unclear. The purpose of this study was to explore the effects and potential mechanism of nicotine on mitophagy, mitochondrial dynamics, apoptosis, and the relationship between these processes in NRVMs. Our results showed that nicotine exposure increased mitochondria-derived superoxide production, decreased mitochondrial membrane potential, and impaired PINK1/Parkin-mediated mitophagic flux in NRVMs. Interestingly, nicotine significantly promoted dynamin-related protein 1 (Drp1)-mediated mitochondrial fission and suppressed mitofusin (MFN)-mediated fusion, which was also observed in the bafilomycin A1-treated group. These results suggest that mitophagic flux impairment may contribute to Drp-1-mediated mitochondrial fission. Finally, nicotine caused excessive mitochondrial fission and contributed to apoptosis, which could be alleviated by mdivi-1, an inhibitor of Drp1. In addition to CTSB, as we previously reported, the enzyme activity of cathepsin L (CTSL) was also decreased in lysosomes after stimulation with nicotine, which may be the main cause of the hindered mitophagic flux induced by nicotine in NRVMs. Pretreatment with Torin 1, which is an inhibitor of mTOR, activated CTSL and ameliorated nicotine-induced mTOR activation and mitophagy impairment, decreased mitochondria-derived superoxide production, and blunted mitochondrial fission and apoptosis. Pretreatment with the ROS scavenger N-acetyl-cysteine (NAC) or inhibitors of p38 and JNK, which could also alleviate mitophagy impairment, exhibited similar effects as Torin1 on mitochondria. Taken together, our study demonstrated that nicotine treatment may lead to an increase in Drp1-mediated mitochondrial fission by blocking mitophagic flux by weakening the enzyme activity of CTSL and activating the ROS/p38/JNK signaling pathway. Excessive mitochondrial fission induced by nicotine ultimately leads to apoptosis. Torin1 restored the decreased CTSL enzyme activity by removing excessive ROS and alleviated the effects of nicotine on mitophagic flux, mitochondrial dynamics, and apoptosis. These results may provide new evidence on the relationship between mitophagic flux and mitochondrial dynamics and new perspectives on nicotine’s effects on mitochondrial dynamics in cardiomyocytes.

scholarly journals Channeled ECM-Based Nanofibrous Hydrogel for Engineering Vascularized Cardiac Tissues

Nanomaterials ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 689
Author(s):  
Smadar Arvatz ◽  
Lior Wertheim ◽  
Sharon Fleischer ◽  
Assaf Shapira ◽  
Tal Dvir
Keyword(s):  
Mathematical Model ◽  
Tissue Engineering ◽  
Mass Transfer ◽  
Cell Viability ◽  
Cardiac Tissue ◽  
Cardiac Cells ◽  
Cardiac Tissues ◽  
The Core ◽  
Engineered Tissue ◽  
Cardiac Patch

Hydrogels are widely used materials for cardiac tissue engineering. However, once the cells are encapsulated within hydrogels, mass transfer to the core of the engineered tissue is limited, and cell viability is compromised. Here, we report on the development of a channeled ECM-based nanofibrous hydrogel for engineering vascularized cardiac tissues. An omentum hydrogel was mixed with cardiac cells, patterned to create channels and closed, and then seeded with endothelial cells to form open cellular lumens. A mathematical model was used to evaluate the necessity of the channels for maintaining cell viability and the true potential of the vascularized hydrogel to form a viable cardiac patch was studied.

Abstract 65: Desmin Preamyloid Oligomers in Experimental Heart Failure and Cultured Cardiac Myocytes

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Peter Rainer ◽  
Dong I Lee ◽  
Matteo Sorge ◽  
Carlo Guarnieri ◽  
Charles G Glabe ◽  
...  
Keyword(s):  
Heart Failure ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Molecular Mechanisms ◽  
Ventricular Myocytes ◽  
Preliminary Evidence ◽  
Cardiac Cells ◽  
Neonatal Rat ◽  
Genetic Mutations

Background: Heart Failure (HF) is one of the main causes of morbidity and mortality in westernized countries but the molecular mechanisms underlying its development are still unclear. A paradigm-shifting view focuses on the accumulation of preamyloid oligomers (PAOs), similar to those observed in Alzheimer disease, as a potential mechanism of cardiac toxicity. We reported that differential desmin phosphorylation at serines (S) 27 and 31 could drive the formation of PAOs in the heart, in the absence of genetic mutations. We sought to establish the identity of the molecular seed triggering the nucleation of cardiac PAOs in an experimental model of HF and in cultured cardiac cells. Methods: Mice were subjected to transverse aortic constriction (TAC) for 4 weeks (FS% = 29.3±2.6, P=0.0001). Alternatively, neonatal rat ventricular myocytes were transduced with lentiviral vectors carrying alanine (A) or phospho-mimetic aspartate (D) desmin double mutants at S27 and S31, fused with GFP. Protein homogenates were subjected to western blot analysis with fluorescent co-staining using the A11 anti-PAOs and anti-desmin antibodies. Transduced cells were also subjected to live imaging to assess phenotype. Results: Co-western blot analysis of both TAC mice and phospho-mimetic mutant cells revealed the colocalization of PAOs with desmin modified (potentially cleaved) forms. Preamyloid oligomers and a desmin fragment were both increased in TAC mice vs. controls (2.8-fold, P=0.023 and 1.8-fold, P=0.038, respectively). The DD mutant, mimicking the doubly phosphorylated desmin that we hypothesized is the physiological form, showed a healthier phenotype in terms of number of spontaneously contracting cells (P=0.041) and incorporation of GFP-desmin at the Z-discs (P=0.0027), whereas the mono-phosphomimetic mutant (AD) resulted in the increase of desmin positive aggregates (P=0.0014). Conclusions: This preliminary evidence suggests that desmin modified forms represent the seed triggering the formation of cardiac PAOs, in the absence of genetic mutations. The accumulation of desmin PAOs could therefore represent an overarching mechanism underlying the deterioration of cardiac function in HF.

scholarly journals Mitochondrial function in engineered cardiac tissues is regulated by extracellular matrix elasticity and tissue alignment

2017 ◽  
Vol 313 (4) ◽  
pp. H757-H767 ◽  
Author(s):  
Davi M. Lyra-Leite ◽  
Allen M. Andres ◽  
Andrew P. Petersen ◽  
Nethika R. Ariyasinghe ◽  
Nathan Cho ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Oxygen Consumption ◽  
Cardiac Myocytes ◽  
Mitochondrial Function ◽  
Stress Responses ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Sprague Dawley ◽  
Cardiac Tissues ◽  
Matrix Elasticity

Mitochondria in cardiac myocytes are critical for generating ATP to meet the high metabolic demands associated with sarcomere shortening. Distinct remodeling of mitochondrial structure and function occur in cardiac myocytes in both developmental and pathological settings. However, the factors that underlie these changes are poorly understood. Because remodeling of tissue architecture and extracellular matrix (ECM) elasticity are also hallmarks of ventricular development and disease, we hypothesize that these environmental factors regulate mitochondrial function in cardiac myocytes. To test this, we developed a new procedure to transfer tunable polydimethylsiloxane disks microcontact-printed with fibronectin into cell culture microplates. We cultured Sprague-Dawley neonatal rat ventricular myocytes within the wells, which consistently formed tissues following the printed fibronectin, and measured oxygen consumption rate using a Seahorse extracellular flux analyzer. Our data indicate that parameters associated with baseline metabolism are predominantly regulated by ECM elasticity, whereas the ability of tissues to adapt to metabolic stress is regulated by both ECM elasticity and tissue alignment. Furthermore, bioenergetic health index, which reflects both the positive and negative aspects of oxygen consumption, was highest in aligned tissues on the most rigid substrate, suggesting that overall mitochondrial function is regulated by both ECM elasticity and tissue alignment. Our results demonstrate that mitochondrial function is regulated by both ECM elasticity and myofibril architecture in cardiac myocytes. This provides novel insight into how extracellular cues impact mitochondrial function in the context of cardiac development and disease. NEW & NOTEWORTHY A new methodology has been developed to measure O2 consumption rates in engineered cardiac tissues with independent control over tissue alignment and matrix elasticity. This led to the findings that matrix elasticity regulates basal mitochondrial function, whereas both matrix elasticity and tissue alignment regulate mitochondrial stress responses.

scholarly journals Human Erbb2-induced Erk Activity Robustly Stimulates Cycling and Functional Remodeling of Rat and Human Cardiomyocytes

2020 ◽  
Author(s):  
Nicholas Strash ◽  
Sophia DeLuca ◽  
Geovanni Janer Carattini ◽  
Soon Chul Heo ◽  
Ryne Gorsuch ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Induced Pluripotent Stem Cell ◽  
Neonatal Rat ◽  
Mutant Form ◽  
Cardiac Tissues ◽  
Human Cardiomyocytes ◽  
Heart Repair ◽  
Induced Pluripotent ◽  
Species Specific

Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte (CM) proliferation have been identified as potential candidates for functional heart repair following myocardial infarction. However, it is unclear whether the effects of these mitogens are species-specific and how they directly compare in the same cardiac setting. Here, we examined how CM-specific lentiviral expression of various candidate mitogens affects human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) in vitro. In 2D-cultured CMs from both species, and in highly mature 3D-engineered cardiac tissues generated from NRVMs, a constitutively-active mutant form of the human gene Erbb2 (cahErbb2) was the most potent tested mitogen. Persistent expression of cahErbb2 induced CM proliferation, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of Erk signaling. These results suggest transient activation of Erbb2/Erk axis in cardiomyocytes as a potential strategy for regenerative heart repair.

Abstract 262: Depolarization Stimulates Neonatal Cardiomyocyte Proliferation In Vitro

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Corin Williams ◽  
Michael Levin ◽  
Lauren D Black
Keyword(s):  
Cardiac Tissue ◽  
Heart Defects ◽  
Cell Types ◽  
Cardiac Cells ◽  
Neonatal Rat ◽  
Resting Membrane Potential ◽  
Cardiomyocyte Proliferation ◽  
Potassium Gluconate ◽  
Hypertrophic Growth

Cardiac tissue engineering is a promising approach for treating children with congenital heart defects. However, as cardiomyocytes (CMs) undergo a rapid transition from hyperplastic to hypertrophic growth after birth, a major challenge to the development of engineered cardiac tissue is the limited proliferation of CMs. Mature CMs and other terminally differentiated cell types tend to have a highly negative resting membrane potential (Vmem) while stem cells and less mature cells tend to have Vmem closer to zero. Vmem has been shown to play an important role in cell differentiation and proliferation. We hypothesized that depolarization of cardiac cells would stimulate CM proliferation in vitro . To test our hypothesis, we isolated neonatal rat cardiac cells and cultured them for 24 hr under standard conditions. Cells were then subjected to depolarization treatment for 72 hr using potassium gluconate or ouabain at various concentrations. Samples were fixed and stained for cardiac α-actin (Fig 1A, red) and phospho-histone H3 (Fig 1A, green) to assess CM mitosis. We found that potassium gluconate had no significant effect while ouabain significantly increased CM mitosis, suggesting Vmem regulation via Na/K-ATPase. CM-specific proliferation was significantly higher with 10nM (p= 0.015) and 100nM (p=0.008) ouabain treatment compared to controls (n=3) (Fig 1B). Cell density was significantly higher with 100μM ouabain versus controls (2656 ± 50 vs. 2026 ± 117 cells/mm 2 ), indicating increased cardiac cell proliferation (Fig 1C). Our findings suggest that depolarization promotes CM proliferation and may be a novel approach to encourage growth of engineered cardiac tissue in vitro .

scholarly journals Rapid Prototyping of Patterned Multifunctional Nanostructures

2000 ◽  
Vol 625 ◽  
Author(s):  
Hongyou Fan ◽  
Gabriel P. Laópez ◽  
C. Jeffrey Brinker
Keyword(s):  
Rapid Prototyping ◽  
Self Assembly ◽  
Computer Aided Design ◽  
Multiple Scales ◽  
Sol Gel ◽  
Dip Coating ◽  
Length Scales ◽  
Form And Function ◽  
Micro Systems ◽  
And Function

AbstractThe ability to engineer ordered arrays of objects on multiple length scales has potential for applications such as microelectronics, sensors, wave guides, and photonic lattices with tunable band gaps. Since the invention of surfactant templated mesoporous sieves in 1992, great progress has been made in controlling different mesophases in the form of powders, particles, fibers, and films. To date, although there have been several reports of patterned mesostructures, materials prepared have been limited to metal oxides with no specific functionality. For many of the envisioned applications of hierarchical materials in micro-systems, sensors, waveguides, photonics, and electronics, it is necessary to define both form and function on several length scales. In addition, the patterning strategies utilized so far require hours or even days for completion. Such slow processes are inherently difficult to implement in commercial environments. We present a series of new methods of producing patterns within seconds. Combining sol-gel chemistry, Evaporation-Induced Self-Assembly (EISA), and rapid prototyping techniques like pen lithography, ink-jet printing, and dip-coating on micro-contact printed substrates, we form hierarchically organized silica structures that exhibit order and function on multiple scales: on the molecular scale, functional organic moieties are positioned on pore surfaces, on the mesoscale, mono-sized pores are organized into 1-, 2-, or 3-dimensional networks, providing size-selective accessibility from the gas or liquid phase, and on the macroscale, 2-dimensional arrays and fluidic or photonic systems may be defined. These rapid patterning techniques establish for the first time a link between computer-aided design and rapid processing of self-assembled nanostructures.

scholarly journals Rapid Prototyping of Patterned Multifunctional Nanostructures

2000 ◽  
Vol 624 ◽  
Author(s):  
Hongyou Fan ◽  
Gabriel P. López ◽  
C. Jeffrey Brinker
Keyword(s):  
Rapid Prototyping ◽  
Self Assembly ◽  
Computer Aided Design ◽  
Multiple Scales ◽  
Sol Gel ◽  
Dip Coating ◽  
Length Scales ◽  
Form And Function ◽  
Micro Systems ◽  
And Function

ABSTRACTThe ability to engineer ordered arrays of objects on multiple length scales has potential for applications such as microelectronics, sensors, wave guides, and photonic lattices with tunable band gaps. Since the invention of surfactant templated mesoporous sieves in 1992, great progress has been made in controlling different mesophases in the form of powders, particles, fibers, and films. To date, although there have been several reports of patterned mesostructures, materials prepared have been limited to metal oxides with no specific functionality. For many of the envisioned applications of hierarchical materials in micro-systems, sensors, waveguides, photonics, and electronics, it is necessary to define both form and function on several length scales. In addition, the patterning strategies utilized so far require hours or even days for completion. Such slowprocesses are inherently difficult to implement in commercial environments. We present a series of new methods of producing patterns within seconds. Combining sol-gel chemistry, Evaporation-Induced Self-Assembly (EISA), and rapid prototyping techniques like pen lithography, ink-jet printing, and dip-coating on micro-contact printed substrates, we form hierarchically organized silica structures that exhibit order and function on multiple scales: on the molecular scale, functional organic moieties are positioned on pore surfaces, on the mesoscale, mono-sized pores are organized into 1-, 2-, or 3-dimensional networks, providing size-selective accessibility from the gas or liquid phase, and on the macroscale, 2-dimensional arrays and fluidic or photonic systems may be defined. These rapid patterning techniques establish for the first time a link between computer-aided design and rapid processing of self-assembled nanostructures

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