Cardiac Tissue Structure Research Articles

Mechanical memory based biofabrication of hierarchical elastic cardiac tissue

Mimicking the multilayered, anisotropic, elastic structure of cardiac tissues for controlled guidiance of 3D cellular orientation is essential in designing bionic scaffolds for cardiac tissue biofabrication. Here, a hierarchically organized, anisotropic, wavy and conductive polycaprolactone/Au scaffold was created in a facile fashion based on mechanical memory during fabrication. The bionic 3D scaffold shows good biocompatibility, excellent biomimetic mechanical properties that guide myoblast alignment, support the hyperelastic behavior observed in native cardiac muscle tissue, and promote myotube maturation, which holds potential for cardiac muscle engineering and the establishment of anin vitroculture platform for drug screening.

Hypoxia and re-oxygenation effects on human cardiomyocytes cultured on polycaprolactone and polyurethane nanofibrous mats

Heart diseases are caused mainly by chronic oxygen insufficiency (hypoxia), leading to damage and apoptosis of cardiomyocytes. Research into the regeneration of a damaged human heart is limited due to the lack of cellular models that mimic damaged cardiac tissue. Based on the literature, nanofibrous mats affect the cardiomyocyte morphology and stimulate the growth and differentiation of cells cultured on them; therefore, nanofibrous materials can support the production of in vitro models that faithfully mimic the 3D structure of human cardiac tissue. Nanofibrous mats were used as scaffolds for adult primary human cardiomyocytes (HCM) and immature human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). This work focuses on understanding the effects of hypoxia and re-oxygenation on human cardiac cells cultured on polymer nanofibrous mats made of poly(ε-caprolactone) (PCL) and polyurethane (PU). The expression of selected genes and proteins in cardiomyocytes during hypoxia and re-oxygenation were evaluated. In addition, the type of cell death was analyzed. To the best of our knowledge, there are no studies on the effects of hypoxia on cardiomyocyte cells cultured on nanofibrous mats. The present study aimed to use nanofiber mats as scaffolds that structurally could mimic cardiac extracellular matrix. Understanding the impact of 3D structural properties in vitro cardiac models on different human cardiomyocytes is crucial for advancing cardiac tissue engineering and regenerative medicine. Observing how 3D scaffolds affect cardiomyocyte function under hypoxic conditions is necessary to understand the functioning of the entire human heart.

Maturation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) on polycaprolactone and polyurethane nanofibrous mats

Investigating the potential of human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) in in vitro heart models is essential to develop cardiac regenerative medicine. iPSC-CMs are immature with a fetal-like phenotype relative to cardiomyocytes in vivo. Literature indicates methods for enhancing the structural maturity of iPSC-CMs. Among these strategies, nanofibrous scaffolds offer more accurate mimicry of the functioning of cardiac tissue structures in the human body. However, further research is needed on the use of nanofibrous mats to understand their effects on iPSC-CMs. Our research aimed to evaluate the suitability of poly(ε-caprolactone) (PCL) and polyurethane (PU) nanofibrous mats with different elasticities as materials for the maturation of iPSC-CMs. Analysis of cell morphology and orientation and the expression levels of selected genes and proteins were performed to determine the effect of the type of nanofibrous mats on the maturation of iPSC-CMs after long-term (10-day) culture. Understanding the impact of 3D structural properties in in vitro cardiac models on induced pluripotent stem cell-derived cardiomyocyte maturation is crucial for advancing cardiac tissue engineering and regenerative medicine because it can help optimize conditions for obtaining more mature and functional human cardiomyocytes.

GelMA micropattern enhances cardiomyocyte organization, maturation, and contraction via contact guidance.

Cardiac tissue engineering has emerged as a promising approach for restoring the functionality of damaged cardiac tissues following myocardial infarction. To effectively replicate the native anisotropic structure of cardiac tissues in vitro, this study focused on the fabrication of micropatterned gelatin methacryloyl hydrogels with varying geometric parameters. These substrates were evaluated for their ability to guide induced pluripotent stem cell-derived cardiomyocytes (CMs). The findings demonstrate that the mechanical properties of this hydrogel closely resemble those of native cardiac tissues, and it exhibits high fidelity in micropattern fabrication. Micropatterned hydrogel substrates lead to enhanced organization, maturation, and contraction of CMs. A microgroove with 20-μm-width and 20-μm-spacing was identified as the optimal configuration for maximizing the contact guidance effect, supported by analyses of nuclear orientation and F-actin organization. Furthermore, this specific micropattern design was found to promote CMs' maturation, as evidenced by increased expression of connexin 43 and vinculin, along with extended sarcomere length. It also enhanced CMs' contraction, resulting in larger contractile amplitudes and greater contractile motion anisotropy. In conclusion, these results underscore the significant benefits of optimizing micropatterned gelatin methacryloyl for improving CMs' organization, maturation, and contraction. This valuable insight paves the way for the development of highly organized and functionally mature cardiac tissues in vitro.

Suxiao Jiuxin Pill alleviates myocardial ischemia/reperfusion-induced autophagy via miR-193a-3p/ALKBH5 pathway

BackgroundMyocardial ischemia/reperfusion injury (MIRI) poses a formidable challenge to cardiac reperfusion therapy due to the absence of effective clinical interventions. Methylation of N6-methyladenosine (m6A), which is the most common post-transcriptional modifications occurring within mammalian mRNA, is believed to be involved in MIRI by modulating autophagy. MicroRNAs (miRNAs) play a crucial role in regulating gene expression at the post-transcriptional level and have been implicated in the regulation of m6A methylation. Suxiao Jiuxin Pill (SJP) is extensively used in China for the clinical treatment of angina pectoris and confers benefits to patients with acute coronary syndrome who have received percutaneous coronary intervention. However, the precise mechanisms underlying SJP intervention in MIRI remain unclear. PurposeThis study aimed to demonstrate, both in vivo and in vitro, that SJP could alleviate autophagy in MIRI by regulating miR-193a-3p to target and upregulate the demethylase ALKBH5. MethodsAn in vitro hypoxia/reoxygenation model was established using H9c2 cells, while an in vivo MIRI model was established using Wistar rats. A lentivirus harboring the precursor sequence of miR-193a-3p was employed for its overexpression. Adeno-associated viruses were used to silence both miR-193a-3p and ALKBH5 expressions. Cardiac function, infarct size, and tissue structure in rats were assessed using echocardiography, triphenyl tetrazolium chloride (TTC) staining, and HE staining, respectively. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was employed to detect the levels of apoptosis in rat cardiac tissue. m6A methylation levels were assessed using colorimetry. GFP-RFP-LC3B was used to monitor autophagic flux and transmission electron microscopy was used to evaluate the development of autophagosomes. Western Blot and qRT-PCR were respectively employed to assess the levels of autophagy-related proteins and miR-193a-3p. ResultsSJP alleviated autophagy, preserved cardiac function, and minimized myocardial damage in the hearts of MIRI rats. SJP attenuated autophagy in H/R H9C2 cells. Elevated levels of miR-193a-3p were observed in the cardiac tissues of MIRI rats and H/R H9C2 cells, whereas SJP downregulated miR-193a-3p levels in these models. ALKBH5, a target gene of miR-193, is negatively regulated by miR-193a-3p. Upon overexpression of miR-193a-3p or silencing of ALKBH5, m6A methylation decreased, and the autophagy-attenuating effects of SJP and its components, senkyunolide A and l-borneol, were lost in H/R H9C2 cells, whereas in MIRI rats, these effects were not abolished but merely weakened. Further investigation indicated that the METTL3 inhibitor STM2475, combined with the silencing of miR-193a-3p, similarly attenuated autophagy in the hearts of MIRI rats. This suggests that a reduction in m6A methylation is involved in autophagy alleviation. ConclusionWe demonstrated that SJP mitigates autophagy in MIRI by downregulating miR-193a-3p, enhancing ALKBH5 expression, and reducing m6A methylation, a mechanism potentially attributed to its constituents, senkyunolide A and l-borneol.

Recovering intestinal redox homeostasis to resolve systemic inflammation for preventing remote myocardial injury by oral fullerenes.

The unbalanced immune state is the dominant feature of myocardial injury. However, the complicated pathology of cardiovascular diseases and the unique structure of cardiac tissue lead to challenges for effective immunoregulation therapy. Here, we exploited oral fullerene nanoscavenger (OFNS) to maintain intestinal redox homeostasis to resolve systemic inflammation for effectively preventing distal myocardial injury through bidirectional communication along the heart-gut immune axis. Observably, OFNS regulated redox microenvironment to repair cellular injury and reduce inflammation in vitro. Subsequently, OFNS prevented myocardial injury by regulating intestinal redox homeostasis and recovering epithelium barrier integrity in vivo. Based on the profiles of transcriptomics and proteomics, we demonstrated that OFNS balanced intestinal and systemic immune homeostasis for remote cardioprotection. Of note, we applied this principle to intervene myocardial infarction in mice and mini-pigs. These findings highlight that locally addressing intestinal redox to inhibit systemic inflammation could be a potent strategy for resolving remote tissue injury.

Biomimetic Cardiac Tissue Models for In Vitro Arrhythmia Studies.

Cardiac arrhythmias are a major cause of cardiovascular mortality worldwide. Many arrhythmias are caused by reentry, a phenomenon where excitation waves circulate in the heart. Optical mapping techniques have revealed the role of reentry in arrhythmia initiation and fibrillation transition, but the underlying biophysical mechanisms are still difficult to investigate in intact hearts. Tissue engineering models of cardiac tissue can mimic the structure and function of native cardiac tissue and enable interactive observation of reentry formation and wave propagation. This review will present various approaches to constructing cardiac tissue models for reentry studies, using the authors' work as examples. The review will highlight the evolution of tissue engineering designs based on different substrates, cell types, and structural parameters. A new approach using polymer materials and cellular reprogramming to create biomimetic cardiac tissues will be introduced. The review will also show how computational modeling of cardiac tissue can complement experimental data and how such models can be applied in the biomimetics of cardiac tissue.

A simple approach for image-based modelling of the heart that enables robust simulation of highly heterogeneous electrical excitation

Remodelling of cardiac tissue structure, including intercellular electrical coupling, is a major determinant of the complex and heterogeneous excitation patterns associated with cardiac arrhythmias. Evaluation of the precise mechanisms by which local tissue structure determines global arrhythmic excitation patterns is a major challenge that may be critically important for the development of effective treatment strategies. Computational modelling is a key tool in the study of cardiac arrhythmias, yet the established approaches for organ-scale modelling are unsuitable to capture the impact of local conduction heterogeneities; a novel approach is required to provide this multi-scale mechanistic insight. We present a fundamentally simple yet powerful approach to simulate electrical excitation in highly heterogeneous whole-heart models that exploits the underlying discreteness of the myocardium. Preliminary simulations demonstrate that this approach can capture lower conduction velocities and reproduce wave breakdown and the development of re-entry in a range of conditions.

Optimization of 3D printing and in vitro characterization of alginate/gelatin lattice and angular scaffolds for potential cardiac tissue engineering.

Background: Engineering cardiac tissue that mimics the hierarchical structure of cardiac tissue remains challenging, raising the need for developing novel methods capable of creating structures with high complexity. Three-dimensional (3D)-printing techniques are among promising methods for engineering complex tissue constructs with high precision. By means of 3D printing, this study aims to develop cardiac constructs with a novel angular structure mimicking cardiac architecture from alginate (Alg) and gelatin (Gel) composite. The 3D-printing conditions were optimized and the structures were characterized in vitro, with human umbilical vein endothelial cells (HUVECs) and cardiomyocytes (H9c2 cells), for potential cardiac tissue engineering. Methods: We synthesized the composites of Alg and Gel with varying concentrations and examined their cytotoxicity with both H9c2 cells and HUVECs, as well as their printability for creating 3D structures of varying fibre orientations (angular design). The 3D-printed structures were characterized in terms of morphology by both scanning electron microscopy (SEM) and synchrotron radiation propagation-based imaging computed tomography (SR-PBI-CT), and elastic modulus, swelling percentage, and mass loss percentage as well. The cell viability studies were conducted via measuring the metabolic activity of the live cells with MTT assay and visualizing the cells with live/dead assay kit. Results: Among the examined composite groups of Alg and Gel, two combinations with ratios of 2 to 1 and 3 to 1 (termed as Alg2Gel1 and Alg3Gel1) showed the highest cell survival; they accordingly were used to fabricate two different structures: a novel angular and a conventional lattice structure. Scaffolds made of Alg3Gel1 showed higher elastic modulus, lower swelling percentage, less mass loss, and higher cell survival compared to that of Alg2Gel1. Although the viability of H9c2 cells and HUVECs on all scaffolds composed of Alg3Gel1 was above 99%, the group of the constructs with the angular design maintained significantly more viable cells compared to other investigated groups. Conclusion: The group of angular 3D-ptinted constructs has illustrated promising properties for cardiac tissue engineering by providing high cell viability for both endothelial and cardiac cells, high mechanical strength as well as appropriate swelling, and degradation properties during 21days of incubation. Statement of Significance: 3D-printing is an emerging method to create complex constructs with high precision in a large scale. In this study, we have demonstrated that 3D-printing can be used to create compatible constructs from the composite of Alg and Gel with endothelial cells and cardiac cells. Also, we have demonstrated that these constructs are able to enhance the viability of cardiac and endothelial cells via creating a 3D structure mimicking the alignment and orientation of the fibers in the native heart.

Placental Inflammation Leads to Abnormal Embryonic Heart Development.

Placental heart development and embryonic heart development occur in parallel, and these organs have been proposed to exert reciprocal regulation during gestation. Poor placentation has been associated with congenital heart disease, an important cause of infant mortality. However, the mechanisms by which altered placental development can lead to congenital heart disease remain unresolved. In this study, we use an in vivo neutrophil-driven placental inflammation model through antibody depletion of maternal circulating neutrophils at key stages during time-mated murine pregnancy: embryonic days 4.5 and 7.5. Pregnant mice were culled at embryonic day 14.5 to assess placental and embryonic heart development. A combination of flow cytometry, histology, and bulk RNA sequencing was used to assess placental immune cell composition and tissue architecture. We also used flow cytometry and single-cell sequencing to assess embryonic cardiac immune cells at embryonic day 14.5 and histology and gene analyses to investigate embryonic heart structure and development. In some cases, offspring were culled at postnatal days 5 and 28 to assess any postnatal cardiac changes in immune cells, structure, and cardiac function, as measured by echocardiography. In the present study, we show that neutrophil-driven placental inflammation leads to inadequate placental development and loss of barrier function. Consequently, placental inflammatory monocytes of maternal origin become capable of migration to the embryonic heart and alter the normal composition of resident cardiac macrophages and cardiac tissue structure. This cardiac impairment continues into postnatal life, hindering normal tissue architecture and function. Last, we show that tempering placental inflammation can prevent this fetal cardiac defect and is sufficient to promote normal cardiac function in postnatal life. Taken together, these observations provide a mechanistic paradigm whereby neutrophil-driven inflammation in pregnancy can preclude normal embryonic heart development as a direct consequence of poor placental development, which has major implications on cardiac function into adult life.

Effect of epithelial-mesenchymal transition on cardiac fibrosis induced by oil mist particulate matter

Objective: To investigate the effects of oil-mist particulate matter (OMPM) on cardiac tissue structure fibrosis in rats and the role of epithelial-mesenchymal transition (EMT). Methods: Six-week-old Wistar rats (half male and half female) were randomly divided into 3 groups: control group (without OMPM exposure), low-dose exposure group (50 mg/m3) and high-dose exposure group (100 mg/m3), 18 rats in each group, with 6.5 hours per day of dynamic inhalation exposure. After 42 days of continuous exposure, cardiac tissues were collected for morphological observation; Western blot was used to detect fibrosis markers collagen I and collagen III levels, epithelial marker E-cadherin levels, interstitial markers N-cadherin, fibronectin, vimentin, alpha-smooth muscle actin (α-SMA) levels, and EMT transcription factor Twist protein levels; Real-time polymerase chain reaction (RT-qPCR) was used to detect collagen I and collagen III mRNA levels. Results: After OMPM exposure, myocardial cell edema and collagen fiber deposition were increased gradually with increasing exposure dose. Western blot results showed that compared with the control group, the expression levels of collagen I, collagen III, N-Cadherin, fibronectin, vimentin, α-SMA, and Twist protein were increased significantly in the low-dose exposure group and the high-dose exposure group (P＜0.01), and protein expression levels were higher in the high-dose exposure group than those in the low-dose exposure group (P＜0.01). In contrast, E-Cadherin protein expression levels were decreased significantly, and lower in the high-dose exposure group (P＜0.01). RT-qPCR results showed that compared with the control group, collagen I and collagen III mRNA levels were increased significantly in the low-dose exposure group and the high-dose exposure group (P＜0.01), and were increased with increasing exposure dose. (P＜0.01). Conclusion: OMPM may induce cardiac fibrosis in rats by promoting EMT process.

Repercussions of pathway inhibition response of Panaxginseng fractions ameliorate cardiac dysfunction in hypertensive model rats

This study examined the preventive and therapeutic benefits of various Panax ginseng fractions against NG-nitro-L-arginine methyl ester (L-NAME)-induced hypertension in rats. Rats injected with L-NAME plus vehicle exhibited persistently elevated SBP, serum concentrations of the cardiac injury biomarkers creatine kinase (CK), CK-MB, and troponin, total cholesterol, triglyceride (TG), low-density lipoprotein (LDL), and interleukin (IL)-6, but lower high-density lipoprotein (HDL) compared to the vehicle control group. Furthermore, L-NAME disrupted the normal histological structure and function of rat cardiac tissue. All ginseng fractions and losartan restored SBP to a normal level, reversed the changes in CK, CK-MB, troponin, total cholesterol, TG, HDL, and LDH, and preserved normal myocardial histology, with the WGF demonstrating the greatest cardioprotective and antihypertensive effects. Compounds within multiple ginseng fractions, but especially the aqueous fraction, possess potent and effective antihypertensive, antilipidemic, anti-inflammatory, and cardioprotective properties.

Implication of P2Y12 receptor in uc.48+-mediated abnormal sympathoexcitatory reflex via superior cervical ganglia in myocardial ischemic rats

Purinergic 2Y12 (P2Y12) receptor antagonists are used as platelet aggregation inhibitors. Long non-coding RNAs (lncRNAs) play an important role in neuropathological events. Satellite glial cells (SGCs) in the superior cervical ganglia (SCGs) encircle the somata of neurons. This study explored if the upregulated P2Y12 receptor in SCGs was relevant to lncRNA uc.48+ during myocardial ischemia (MI). The results showed that upregulation of P2Y12 receptor was accompanied by increased expression of uc.48+ in the SCGs of MI rats which displayed abnormal changes in cervical sympathetic nerve activity, blood pressure, heart rate, electrocardiograms and cardiac tissue structure. The P2Y12 antagonist clopidogrel improved abnormal alterations in cardiac function and tissue structure in MI rats. Short hairpin RNA (shRNA) against uc.48+ significantly inhibited P2Y12 receptor upregulation and its co-expression with glial fibrillary acidic protein (GFAP) in SCGs, and ameliorated the cardiac dysfunction in MI rats. By contrast, overexpression of uc.48+ increased the expression of P2Y12 in SCGs and enhanced cervical sympathetic nerve activity in control rats. Direct interaction between uc.48+ and the P2Y12 receptor was predicted using the bioinformatic tool CatRAPID and confirmed by RNA immunoprecipitation. Moreover, overexpression of the P2Y12 receptor reversed the protective effect of uc.48+ shRNA on cardiac dysfunction in MI rats. Uc.48 shRNA treatment also inhibited the enhanced rise of intracellular free Ca2+ level ([Ca2+]i) evoked by the P2Y12 agonist 2-methylthio-adenosine-5′-diphosphate (2-MeSADP) in SGCs of SCGs after oxygen-glucose deprivation (OGD) treatment. These data demonstrated that uc.48+ shRNA could counteract the P2Y12 upregulation and improve P2Y12-implicated cardiac dysfunction due to MI.

Activated Drp1 regulates p62-mediated autophagic flux and aggravates inflammation in cerebral ischemia-reperfusion via the ROS-RIP1/RIP3-exosome axis

BackgroundCerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury remains elusive.MethodsThe 150 male C57 mice underwent middle cerebral artery occlusion (MCAO) for 1 h and reperfusion for 24 h, Among them, 50 MCAO mice were further treated with Mitochondrial division inhibitor 1 (Mdivi-1) and 50 MCAO mice were further treated with N-acetylcysteine (NAC). SH-SY5Y cells were cultured in a low-glucose culture medium for 4 h under hypoxic conditions and then transferred to normal conditions for 12 h. Then, cerebral blood flow, mitochondrial structure, mitochondrial DNA (mtDNA) copy number, intracellular and mitochondrial reactive oxygen species (ROS), autophagic flux, aggresome and exosome expression profiles, cardiac tissue structure, mitochondrial length and cristae density, mtDNA and ROS content, as well as the expression of Drp1-Ser616/Drp1, RIP1/RIP3, LC3 II/LC3 I, TNF-α, IL-1β, etc., were detected under normal or Drp1 interference conditions.ResultsThe mtDNA content, ROS levels, and Drp1-Ser616/Drp1 were elevated by 2.2, 1.7 and 2.7 times after CIRI (P < 0.05). However, the high cytoplasmic LC3 II/I ratio and increased aggregation of p62 could be reversed by 44% and 88% by Drp1 short hairpin RNA (shRNA) (P < 0.05). The low fluorescence intensity of autophagic flux and the increased phosphorylation of RIP3 induced by CIRI could be attenuated by ROS scavenger, NAC (P < 0.05). RIP1/RIP3 inhibitor Necrostatin-1 (Nec-1) restored 75% to a low LC3 II/LC3 I ratio and enhanced 2 times to a high RFP-LC3 after Drp1 activation (P < 0.05). In addition, although CIRI-induced ROS production caused no considerable accumulation of autophagosomes (P > 0.05), it increased the packaging and extracellular secretion of exosomes containing p62 by 4 – 5 times, which could be decreased by Mdivi-1, Drp1 shRNA, and Nec-1 (P < 0.05). Furthermore, TNF-α and IL-1β increased in CIRI-derived exosomes could increase RIP3 phosphorylation in normal or oxygen–glucose deprivation/reoxygenation (OGD/R) conditions (P < 0.05).ConclusionsCIRI activated Drp1 and accelerated the p62-mediated formation of autophagosomes while inhibiting the transition of autophagosomes to autolysosomes via the RIP1/RIP3 pathway activation. Undegraded autophagosomes were secreted extracellularly in the form of exosomes, leading to inflammatory cascades that further damaged mitochondria, resulting in excessive ROS generation and the blockage of autophagosome degradation, triggering a vicious cycle.

Silk-Based Matrices and c-Kit-Positive Cardiac Progenitor Cells for a Cellularized Silk Fibroin Scaffold: Study of an in vivo Model

The production of a cellularized silk fibroin scaffold is very difficult because it is actually impossible to differentiate cells into a well-organized cardiac tissue. Without vascularization, not only do cell masses fail to grow, but they may also exhibit an area of necrosis, indicating a lack of oxygen and nutrients. In the present study, we used the so-called tyrosine protein kinase kit (c-Kit)-positive cardiac progenitor cells (CPCs) to generate cardiac cellularized silk fibroin scaffolds, multipotent cells isolated from the adult heart to date that can show some degree of differentiation toward the cardiac phenotype. To test their ability to differentiate into the cardiac phenotype in vivo as well, CPC and collagen organoid-like masses were implanted into nude mice and their behavior observed. Since the 3-dimensional structure of cardiac tissue can be preserved by scaffolds, we prepared in parallel different silk fibroin scaffolds with 3 different geometries and tested their behavior in 3 different models of immunosuppressed animals. Unfortunately, CPC cellularized silk fibroin scaffolds cannot be used in vivo. CPCs implanted alone or in collagen type I gel were destroyed by CD3+ lymphocyte aggregates, whereas the porous and partially oriented scaffolds elicited a consistent foreign body response characterized by giant cells. Only the electrospun meshes were resistant to the foreign body reaction. In conclusion, c-Kit-positive CPCs, although expressing a good level of cardiac differentiation markers in vitro with or without fibroin meshes, are not suitable for an in vivo model of cardiac organoids because they are degraded by a T-cell-mediated immune response. Even scaffolds which may preserve the survival of these cells in vivo also induced a host response. However, among the tested scaffolds, the electrospun meshes (F-scaffold) induced a lower response compared to all the other tested structures.

Scaffold-free 3D printing for fabrication of biomimetic branched multinucleated cardiac tissue construct: A promising ex vivo model for in situ detection of drug-induced sodium ion channel responses

3D bioprinted cardiac constructs have the potential to develop into functional native tissue. Although bioengineered cardiac constructs can be a promising tool for evaluating cardiac responses, the intrinsic limitation in reproducing the well-defined structural and functional properties of native cardiac tissue within the generated substitute restricts their applications. Herein, for the first time, we have introduced a sophisticated scaffold-free de novo three-dimensional (3D) printing model (SFP) to regenerate biomimetic cardiac tissue with a multinucleated branched cellular structure similar to native tissue–like phenotypes. Furthermore, the formation of tight intracellular junctions expressing connexin 43 certifies the maturation of the fabricated structure. In addition, in situ measurement of the bioengineered cardiac construct's physiologic sodium ion channel responses to known cardiac drugs was successfully performed. Hence, the proposed methodology recapitulates a significant milestone in developing biomimetic cardiac tissue structure and functionality in vitro and could be applied for regenerative therapies, disease modeling, and drug screening.

3D virtual histopathology of cardiac tissue from Covid-19 patients based on phase-contrast X-ray tomography.

For the first time, we have used phase-contrast X-ray tomography to characterize the three-dimensional (3d) structure of cardiac tissue from patients who succumbed to Covid-19. By extending conventional histopathological examination by a third dimension, the delicate pathological changes of the vascular system of severe Covid-19 progressions can be analyzed, fully quantified and compared to other types of viral myocarditis and controls. To this end, cardiac samples with a cross-section of 3.5mm were scanned at a laboratory setup as well as at a parallel beam setup at a synchrotron radiation facility the synchrotron in a parallel beam configuration. The vascular network was segmented by a deep learning architecture suitable for 3d datasets (V-net), trained by sparse manual annotations. Pathological alterations of vessels, concerning the variation of diameters and the amount of small holes, were observed, indicative of elevated occurrence of intussusceptive angiogenesis, also confirmed by high-resolution cone beam X-ray tomography and scanning electron microscopy. Furthermore, we implemented a fully automated analysis of the tissue structure in the form of shape measures based on the structure tensor. The corresponding distributions show that the histopathology of Covid-19 differs from both influenza and typical coxsackie virus myocarditis.

Abnormal Expression of Connexin43 in Cardiac Injury Induced by S-Band and X-Band Microwave Exposure in Rats

Although the effects of microwave exposure on the heart have gradually become the focus of domestic and foreign scholars, the biological effects caused by different doses and different frequency bands of exposure are still unclear. In this study, we will investigate the damaging effect of S-band and X-band microwave composite exposure on cardiac structure and function, as well as the pathophysiological significance of Cx43 in cardiac conduction dysfunction after exposure. We used S- and X-band radiation sources with the average power density of 5 and 10 mW/cm2 to expose Wistar rats to single or composite exposure. At the 6th hour, on the 7th, 14th, and 28th days after exposure, ECG was used to detect the electrical conduction of the heart, and the myocardial enzyme was measured by the automatic biochemical analyzer. We selected the observation time points and groups with severe damage to observe the changes of myocardial structure and ultrastructure with an optical microscope and TEM; and to detect the expression and distribution of Cx43 by western blotting and immunohistochemistry. After exposure, the heart rate increased, the P wave amplitude decreased, and the R wave amplitude increased; the content of the myocardial enzyme in serum increased; the structure and ultrastructure of cardiac tissue were damaged. The damage was dose-dependent and frequency-dependent. The expression of Cx43 in myocardial tissue decreased, and distribution was abnormal. Taken together, these findings suggested that the mechanism of abnormal electrical conduction in the heart of rats by S- and X-band microwave exposure might be related to the decreased expression and disordered distribution of Cx43 after microwave exposure.

Cardiac Cell Patterning on Customized Microelectrode Arrays for Electrophysiological Recordings.

Cardiomyocytes (CMs) and fibroblast cells are two essential elements for cardiac tissue structure and function. The interactions between them can alter cardiac electrophysiology and thus contribute to cardiac diseases, such as arrhythmogenesis. One possible explanation is that fibroblasts can directly affect cardiac electrophysiology through electrical coupling with CMs. Therefore, detecting the electrical activities in the CM-fibroblast network is vital for understanding the coupling dynamics among them. Current commercialized platforms for studying cardiac electrophysiology utilize planar microelectrode arrays (MEAs) to record the extracellular field potential (FP) in real-time, but the prearranged electrode configuration highly limits the measurement capabilities at specific locations. Here, we report a custom-designed MEA device with a novel micropatterning method to construct a controlled network of neonatal rat CMs (rCMs) and fibroblast connections for monitoring the electrical activity of rCM-fibroblast co-cultures in a spatially controlled fashion. For the micropatterning of the co-culture, surface topographical features and mobile blockers were used to control the initial attachment locations of a mixture of rCMs and fibroblasts, to form separate beating rCM-fibroblast clusters while leaving empty space for fibroblast growth to connect these clusters. Once the blockers are removed, the proliferating fibroblasts connect and couple the separate beating clusters. Using this method, electrical activity of both rCMs and human-induced-pluripotent-stem-cell-derived cardiomyocytes (iCMs) was examined. The coupling dynamics were studied through the extracellular FP and impedance profile recorded from the MEA device, indicating that the fibroblast bridge provided an RC-type coupling of physically separate rCM-containing clusters and enabled synchronization of these clusters.

Derivation of a new macroscopic bidomain model including three scales for the electrical activity of cardiac tissue

In the present paper, a new three-scale asymptotic homogenization method is proposed to study the electrical behavior of the cardiac tissue structure with multiple heterogeneities at two different levels. The first level is associated with the mesoscopic structure such that the cardiac tissue is composed of extracellular and intracellular domains. The second level is associated with the microscopic structure in such a way the intracellular medium can only be viewed as a periodical layout of unit cells (mitochondria). Then, we define two kinds of local cells that are obtained by upscaling methods. The homogenization method is based on a power series expansion which allows determining the macroscopic (homogenized) bidomain model from the microscopic bidomain problem at each structural level. First, we use the two-scale asymptotic expansion to homogenize the extracellular problem. Then, we apply a three-scale asymptotic expansion in the intracellular problem to obtain its homogenized equation at two levels. The first upscaling level of the intracellular structure yields the mesoscopic equation and the second step of the homogenization leads to obtain the intracellular homogenized equation. Both the mesoscopic and microscopic information are obtained by homogenization to capture local characteristics inside the cardiac tissue structure. Finally, we obtain the macroscopic bidomain model and the heart domain coincides with the intracellular medium and extracellular one, which are two inter-penetrating and superimposed continua connected at each point by the cardiac cellular membrane. The interest of the proposed method comes from the fact that it combines microscopic and mesoscopic characteristics to obtain a macroscopic description of the electrical behavior of the heart.