Cardiac Tissue Formation Research Articles

Expression of Osteopontin and Gremlin 1 Proteins in Cardiomyocytes in Ischemic Heart Failure.

A relevant role of osteopontin (OPN) and gremlin 1 (Grem1) in regulating cardiac tissue remodeling and formation of heart failure (HF) are documented, with the changes of OPN and Grem1 levels in blood plasma due to acute ischemia, ischemic heart disease-induced advanced HF or dilatative cardiomyopathy being the primary focus in most of these studies. However, knowledge on the early OPN and Grem1 proteins expression changes within cardiomyocytes during remodeling due to chronic ischemia remains insufficient. The aim of this study was to determine the OPN and Grem1 proteins expression changes in human cardiomyocytes at different stages of ischemic HF. A semi-quantitative immunohistochemical analysis was performed in 105 myocardial tissue samples obtained from the left cardiac ventricles. Increased OPN immunostaining intensity was already detected in the stage A HF group, compared to the control group (p < 0.001), and continued to increase in the stage B HF (p < 0.001), achieving the peak of immunostaining in the stages C/D HF group (p < 0.001). Similar data of Grem1 immunostaining intensity changes in cardiomyocytes were documented. Significantly positive correlations were detected between OPN, Grem1 expression in cardiomyocytes and their diameter as well as the length, in addition to positive correlation between OPN and Grem1 expression changes within cardiomyocytes. These novel findings suggest that OPN and Grem1 contribute significantly to reorganization of cellular geometry from the earliest stage of cardiomyocyte remodeling, providing new insights into the ischemic HF pathogenesis.

Abstract P1119: Deep Learning-based Cardiac Microphysiological Systems For Studying Reentry Arrhythmia

Arrhythmias can be caused by reentry and focal activities, leading to a disruption of the normal sinus rhythm. Understanding these mechanisms and their contributions to arrhythmias in individual patients is crucial for developing effective treatments. To achieve this, in vitro human models based on induced pluripotent stem cell-derived cardiomyocytes have been developed to replicate arrhythmogenic activities and identify pathophysiological mechanisms specific to patients. However, the current methods for analyzing electrophysiology (EP) videos are limited and subjective, making the interpretation of clinical EP videos challenging. To address these limitations, we have developed a deep cardiac EP system called cEP-Net. The system comprises a cardiac microphysiological system designed for reproducing various forms of reentry and focal activities in vitro and a deep learning-based EP video classifier with a spatial and temporal streams architecture. The deep EP classifier analyzes cardiac EP videos, identifies arrhythmogenic activities, and categorizes videos into four categories based on their characteristics: (1) localized activities, (2) normal linear propagation, (3) focal activity, and (4) reentrant activity. We pre-trained cEP-Net using 111,110 annotated in vitro EP videos produced by a cardiac microphysiological system and evaluated its performance for in vitro and in vivo clinical settings. Our pre-trained cEP-Net demonstrated high Kappa (91.0%) and concordance rate (94.7%) for all categories. cEP-Net visualized in vitro EP videos in UMAP domains and successfully clustered them into four categories. Additionally, cEP-Net successfully captured the development and evolution of reentry during early in vitro cardiac tissue formation and the initiation of focal activities in the HCN4 (a pacemaker protein)-expressing tissues. We also used cEP-Net to quantify the arrhythmogenic risks of cardiac agents and found that cEP-Net could capture Flecainide-induced vulnerability to reentry. Lastly, we applied cEP-Net to analyze electroanatomical mapping videos in vivo. The cEP-Net demonstrated its capability to identify phase singularities in hearts with atrial fibrillation (AF) and capture the EP patterns of persistent AF hearts.

Flexible 3D printed microwires and 3D microelectrodes for heart-on-a-chip engineering

We developed a heart-on-a-chip platform that integrates highly flexible, vertical, 3D micropillar electrodes for electrophysiological recording and elastic microwires for the tissue’s contractile force assessment. The high aspect ratio microelectrodes were 3D-printed into the device using a conductive polymer, poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS). A pair of flexible, quantum dots/thermoplastic elastomer nanocomposite microwires were 3D printed to anchor the tissue and enable continuous contractile force assessment. The 3D microelectrodes and flexible microwires enabled unobstructed human iPSC-based cardiac tissue formation and contraction, suspended above the device surface, under both spontaneous beating and upon pacing with a separate set of integrated carbon electrodes. Recording of extracellular field potentials using the PEDOT:PSS micropillars was demonstrated with and without epinephrine as a model drug, non-invasively, along with in situ monitoring of tissue contractile properties and calcium transients. Uniquely, the platform provides integrated profiling of electrical and contractile tissue properties, which is critical for proper evaluation of complex, mechanically and electrically active tissues, such as the heart muscle under both physiological and pathological conditions.

Human Induced Pluripotent Stem Cell Encapsulation Geometry Impacts Three-Dimensional Developing Human Engineered Cardiac Tissue Functionality.

Cardiac tissue engineering has been working to alleviate the immense burden of cardiovascular disease for several decades. To improve cardiac tissue homogeneity and cardiomyocyte (CM) maturation, in this study, we investigated altering initial encapsulation geometry in a three-dimensional (3D) direct cardiac differentiation platform. Traditional engineered cardiac tissue production utilizes predifferentiated CMs to produce 3D cardiac tissue and often involves various cell selection and exogenous stimulation methods to promote CM maturation. Starting tissue formation directly with human induced pluripotent stem cells (hiPSCs), rather than predifferentiated CMs, simplifies the engineered cardiac tissue formation process, making it more applicable for widespread implementation and scale-up. In this study, hiPSCs were encapsulated in poly (ethylene glycol)-fibrinogen in three tissue geometries (disc-shaped microislands, squares, and rectangles) and subjected to established cardiac differentiation protocols. Resulting 3D engineered cardiac tissues (3D-ECTs) from each geometry displayed similar CM populations (∼65%) and gene expression over time. Notably, rectangular tissues displayed less tissue heterogeneity and suggested more advanced features of maturing CMs, including myofibrillar alignment and Z-line formation. In addition, rectangular tissue showed significantly higher anisotropic contractile properties compared to square and microisland tissues (MI 0.28 ± 0.03, SQ 0.35 ± 0.05, RT 0.79 ± 0.04). This study demonstrates a straightforward method for simplifying and improving 3D-ECT production without the use of exogenous mechanical or electrical pacing and has the potential to be utilized in bioprinting and drug testing applications. Impact statement Current methods for improving cardiac maturation postdifferentiation remain tedious and complex. In this study, we examined the impact of initial encapsulation geometry on improvement of three-dimensional engineered cardiac tissue (3D-ECT) production and postdifferentiation maturation for three tissue geometries, including disc-shaped microislands, squares, and rectangles. Notably, rectangular 3D-ECTs displayed less tissue heterogeneity and more advanced features of maturing cardiomyocytes, including myofibrillar alignment, Z-line formation, and anisotropic contractile properties, compared to microisland and square tissues. This study demonstrates an initial human induced pluripotent stem cell-encapsulated rectangular tissue geometry can improve cardiac maturation, rather than implementing cell selection or tedious postdifferentiation manipulation, including exogenous mechanical and/or electrical pacing.

Directional dependence on concomitant pressure and volume increases during left ventricular filling

Myocardial infarction continues to be a leading cause of mortality and morbidity globally. A major challenge post-myocardial infarction is scar tissue growth, which eventually can lead to heart failure. Cardiovascular regenerative strategies to minimize scar tissue growth and promote cardiac tissue formation are currently being actively pursued via the development of cardiac patches. However, the patch must have viscoelastic properties that mimic healthy cardiac tissues to facilitate proper cardiac patch-to-cell communications. To this end, we investigated the tissue microstructure and the stress relaxation properties of the porcine left ventricle (LV) along its long and short axes using a nanoindentation technique. We found significantly higher collagen density along the long axis than the short axis (p < 0.05). We then identified a much more rapid stress relaxation along the porcine LV's short axis compared to its long axis during the diastolic filling timeframe. Therefore, these findings show that concomitant LV pressure and volume increases from blood filling during diastole are directional dependent, with its short axis responsible for increase in LV volume and the long axis responsible for increase in LV pressure. These directional-dependent stress relaxation properties are essential in the design of structurally, bio-mimetic cardiac patches to support cardiac function and regeneration.

High-throughput rhythmic regulation of cardiomyocytes by integrated electrical stimulation and video-based automated analysis biosensing platform

In cardiac tissue engineering, electric stimulation is an efficient approach to improve the formation of cardiac tissue from individual cardiomyocyte. The regulation conditions of electric stimulation should be screened in an efficient way. However, the lack of high-throughput and large-scale assessment platforms limited the effectively screen the regulation conditions. Here, we develop a high-throughput integrated electrical stimulation system to rhythmically regulate the cardiomyocytes in situ. The state of regulated cardiomyocytes is characterized by a video-based automated biosensing system to analyze the beating of cardiomyocytes. Electrical stimulation conditions are optimized to regulate the cardiomyocyte state in vitro to replace the complex bioactive molecules and materials. By the video analysis, the accurate beating rate and regularity of cardiomyocyte can be determined. The results show that electrical stimulation frequency is a significant factor to regulate the cardiomyocyte beating. The electrical stimulation with a frequency of 3 Hz can effectively regulate the primary rat cardiomyocytes with normal rhythm. This high-throughput electrical stimulation and a video-based automated biosensing system will be a promising and powerful tool to effectively optimize the regulation conditions of cardiomyocyte in vitro, and possess broad application prospects in cardiac tissue engineering and pharmaceutical industry.

Direct 3D printing of hiPSC-cardiomyocytes in collagen-based bioinks

Abstract Background Cardiac tissue engineering is an effective strategy to generate tissues for drug testing and disease modelling as well as for cardiac repair. Tissues produced by casting show good functionality and advanced maturation, but do not replicate the native tissue architecture and hierarchy. Additive manufacturing technologies, such as 3D bioprinting, enable the generation of hierarchically structured tissues with complex geometries. This technology has been used previously to generate models of the heart. However, these approaches either showed limited tissue functionality or required a two-step procedure using a structural and a cell-laden bioink. Purpose Here, we aimed to develop a collagen-based bioink, which enables direct 3D-bioprinting of hiPSC-derived cardiomyocytes and supports the formation of functional cardiac tissue. Methods To generate cardiac tissues, a commercial pneumatic extrusion bioprinter with custom modifications to enable passive cooling of the bioink was used. Gelatin/gum arabic microparticles were obtained through complex coacervation, compacted by centrifugation and utilized as support bath. Cardiomyocytes were differentiated in 2D monolayer and expanded by CHIR99021-treatment and regular passaging. Cells were encapsulated in a rat collagen-I based bioink and printed into support bath prior to gelation. After bioink gelation at 37°C, support bath was removed, and constructs cultivated free-floating for up to 30 days. Results We printed ring-shaped cardiac tissues measuring 5 x 5 x 1 mm, which remained stable over the course of cultivation. First contractions were observed after three days, which increased in magnitude and synchronized across the tissue with prolonged culture. HiPSC-cardiomyocytes displayed striated sarcomeres and were responsive to pharmacological stimulation. In addition, using two distinct bioinks, multi-layered constructs were generated. Conclusion 3D-bioprinting is a promising tool to generate engineered cardiac tissues with complex geometries and improved functionality through designed hierarchy. Our collagen-based bioink and associated printing strategy enables the fabrication of Collagen-based contractile cardiac tissues in a direct manner. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Contractions of printed cardiac tissue

Investigation of the formation of cardiac tissue on substrates of varying degrees of anisotropy and rigidity

In the last decade,invitroexperiments have shown that mechanical properties of the bases could markedly influence the efficacy of differentiation of the induced pluripotent and embryonic stem cells and their development into the mature phenotype. By changing of mechanical, elastic and structural characteristics of the base, it is possible to increase the percentage of stem cells that differentiate to cardiomyocytes. The study was aimed at evaluation of the effects induced by changing physical characteristics of the base on the formation of phenotypic characteristics of cardiac cells. This included the comparison of structural properties of the cultured layer of heart tissue obtained by changing of elasticity and structure of polymeric bases. The results showed significant differences in calcium activity and structural characteristics of cardiomyocytes depending on the base properties, as well as significant variation in the excitation conduction. As long as conduction abnormalities in the heart tissues can often lead to occurrence of life-threatening cardiac arrhythmias, the results can be used to determine patient groups at increased risk of death from heart failure.

Epicardial Contribution to the Developing and Injured Heart: Exploring the Cellular Composition of the Epicardium.

The epicardium is an essential cell population during cardiac development. It contributes different cell types to the developing heart through epithelial-to-mesenchymal transition (EMT) and it secretes paracrine factors that support cardiac tissue formation. In the adult heart the epicardium is a quiescent layer of cells which can be reactivated upon ischemic injury, initiating an embryonic-like response in the epicardium that contributes to post-injury repair processes. Therefore, the epicardial layer is considered an interesting target population to stimulate endogenous repair mechanisms. To date it is still not clear whether there are distinct cell populations in the epicardium that contribute to specific lineages or aid in cardiac repair, or that the epicardium functions as a whole. To address this putative heterogeneity, novel techniques such as single cell RNA sequencing (scRNA seq) are being applied. In this review, we summarize the role of the epicardium during development and after injury and provide an overview of the most recent insights into the cellular composition and diversity of the epicardium.

The role of bFGF in preventing the shrinkage of cardiac progenitor cell-engineered conduction tissue by downregulating α-SMA expression

AimsEngineered conduction tissues (ECTs) fabricated from cardiac progenitor cells (CPCs) and collagen sponges were precisely targeted for the treatment of atrioventricular conduction block in our previous studies. However, obvious shrinkage and deformation of ECTs was observed during in vitro culture. According to the literature, it can be speculated that basic fibroblast growth factor (bFGF) may downregulate alpha-smooth muscle actin (α-SMA) produced by CPCs to prevent the shrinkage of CPC-engineered conduction tissues. Main methodsIn this study, culture media with or without bFGF were used for both cell culture and 3D tissue construction. The expression of α-SMA and the size change of engineered tissue were analyzed to evaluate the feasibility of adding bFGF to regulate α-SMA expression and shrinkage of constructs. In addition, cardiac-specific examinations were performed to evaluate the effect of bFGF on cardiac tissue formation. Key findingsSupplementation with bFGF efficiently relieved shrinkage of engineered tissue by downregulating the expression of α-SMA at both the cellular and 3D tissue levels. Moreover, bFGF had a positive influence on cardiac tissue formation in terms of cell viability, tissue organization and electrical conduction velocity. SignificanceThis study provides a guide for both shape control and quality improvement of CPC-engineered cardiac tissues.

Engineered human cardiac microtissues: The state-of-the-(he)art.

Due to the integration of recent advances in stem cell biology, materials science, and engineering, the field of cardiac tissue engineering has been rapidly progressing toward developing more accurate functional 3D cardiac microtissues from human cell sources. These engineered tissues enable screening of cardiotoxic drugs, disease modeling (eg, by using cells from specific genetic backgrounds or modifying environmental conditions) and can serve as novel drug development platforms. This concise review presents the most recent advances and improvements in cardiac tissue formation, including cardiomyocyte maturation and disease modeling.

Dual Function of iPSC-Derived Pericyte-Like Cells in Vascularization and Fibrosis-Related Cardiac Tissue Remodeling In Vitro.

Myocardial interstitial fibrosis (MIF) is characterized by excessive extracellular matrix (ECM) deposition, increased myocardial stiffness, functional weakening, and compensatory cardiomyocyte (CM) hypertrophy. Fibroblasts (Fbs) are considered the principal source of ECM, but the contribution of perivascular cells, including pericytes (PCs), has gained attention, since MIF develops primarily around small vessels. The pathogenesis of MIF is difficult to study in humans because of the pleiotropy of mutually influencing pathomechanisms, unpredictable side effects, and the lack of available patient samples. Human pluripotent stem cells (hPSCs) offer the unique opportunity for the de novo formation of bioartificial cardiac tissue (BCT) using a variety of different cardiovascular cell types to model aspects of MIF pathogenesis in vitro. Here, we have optimized a protocol for the derivation of hPSC-derived PC-like cells (iPSC-PCs) and present a BCT in vitro model of MIF that shows their central influence on interstitial collagen deposition and myocardial tissue stiffening. This model was used to study the interplay of different cell types—i.e., hPSC-derived CMs, endothelial cells (ECs), and iPSC-PCs or primary Fbs, respectively. While iPSC-PCs improved the sarcomere structure and supported vascularization in a PC-like fashion, the functional and histological parameters of BCTs revealed EC- and PC-mediated effects on fibrosis-related cardiac tissue remodeling.

Natural Biomaterials for Cardiac Tissue Engineering: A Highly Biocompatible Solution.

Cardiovascular diseases (CVD) constitute a major fraction of the current major global diseases and lead to about 30% of the deaths, i.e., 17.9 million deaths per year. CVD include coronary artery disease (CAD), myocardial infarction (MI), arrhythmias, heart failure, heart valve diseases, congenital heart disease, and cardiomyopathy. Cardiac Tissue Engineering (CTE) aims to address these conditions, the overall goal being the efficient regeneration of diseased cardiac tissue using an ideal combination of biomaterials and cells. Various cells have thus far been utilized in pre-clinical studies for CTE. These include adult stem cell populations (mesenchymal stem cells) and pluripotent stem cells (including autologous human induced pluripotent stem cells or allogenic human embryonic stem cells) with the latter undergoing differentiation to form functional cardiac cells. The ideal biomaterial for cardiac tissue engineering needs to have suitable material properties with the ability to support efficient attachment, growth, and differentiation of the cardiac cells, leading to the formation of functional cardiac tissue. In this review, we have focused on the use of biomaterials of natural origin for CTE. Natural biomaterials are generally known to be highly biocompatible and in addition are sustainable in nature. We have focused on those that have been widely explored in CTE and describe the original work and the current state of art. These include fibrinogen (in the context of Engineered Heart Tissue, EHT), collagen, alginate, silk, and Polyhydroxyalkanoates (PHAs). Amongst these, fibrinogen, collagen, alginate, and silk are isolated from natural sources whereas PHAs are produced via bacterial fermentation. Overall, these biomaterials have proven to be highly promising, displaying robust biocompatibility and, when combined with cells, an ability to enhance post-MI cardiac function in pre-clinical models. As such, CTE has great potential for future clinical solutions and hence can lead to a considerable reduction in mortality rates due to CVD.

Hydrogel scaffolds with elasticity-mimicking embryonic substrates promote cardiac cellular network formation.

Hydrogels are a class of biomaterials used for a wide range of biomedical applications, including as a three-dimensional (3D) scaffold for cell culture that mimics the extracellular matrix (ECM) of native tissues. To understand the role of the ECM in the modulation of cardiac cell function, alginate was used to fabricate crosslinked gels with stiffness values that resembled embryonic (2.66 ± 0.84kPa), physiologic (8.98 ± 1.29kPa) and fibrotic (18.27 ± 3.17kPa) cardiac tissues. The average pore diameter and hydrogel swelling were seen to decrease with increasing substrate stiffness. Cardiomyocytes cultured within soft embryonic gels demonstrated enhanced cell spreading, elongation, and network formation, while a progressive increase in gel stiffness diminished these behaviors. Cell viability decreased with increasing hydrogel stiffness. Furthermore, cells in fibrotic gels showed enhanced protein expression of the characteristic cardiac stress biomarker, Troponin-I, while reduced protein expression of the cardiac gap junction protein, Connexin-43, in comparison to cells within embryonic gels. The results from this study demonstrate the role that 3D substrate stiffness has on cardiac tissue formation and its implications in the development of complex matrix remodeling-based conditions, such as myocardial fibrosis.

Mechanical stimulation of cell microenvironment for cardiac muscle tissue regeneration: a 3D in-silico model

The processes in which cardiac cells are reorganized for tissue regeneration is still unclear. It is a complicated process that is orchestrated by many factors such as mechanical, chemical, thermal, and/or electrical cues. Studying and optimizing these conditions in-vitro is complicated and time costly. In such cases, in-silico numerical simulations can offer a reliable solution to predict and optimize the considered conditions for the cell culture process. For this aim, a 3D novel and enhanced numerical model has been developed to study the effect of the mechanical properties of the extracellular matrix (ECM) as well as the applied external forces in the process of the cell differentiation and proliferation for cardiac muscle tissue regeneration. The model has into account the essential cellular processes such as migration, cell–cell interaction, cell–ECM interaction, differentiation, proliferation and/or apoptosis. It has employed to study the initial stages of cardiac muscle tissue formation within a wide range of ECM stiffness (8–50 kPa). The results show that, after cell culture within a free surface ECM, cells tend to form elongated aggregations in the ECM center. The formation rate, as well as the aggregation morphology, have been found to be a function of the ECM stiffness and the applied external force. Besides, it has been found that the optimum ECM stiffness for cardiovascular tissue regeneration is in the range of 29–39 kPa, combined with the application of a mechanical stimulus equivalent to deformations of 20–25%.

A Percutaneous Catheter for In Vivo Hyperspectral Imaging of Cardiac Tissue: Challenges, Solutions and Future Directions.

Multiple studies have shown that spectral analysis of tissue autofluorescence can be used as a live indicator for various pathophysiological states of cardiac tissue, including ischemia, ablation-induced damage, or scar formation. Yet today there are no percutaneous devices that can detect autofluorescence signals from inside a beating heart. Our aim was to develop a prototype catheter to demonstrate the feasibility of doing so. Here we summarize technical solutions leading to the development of a percutaneous catheter capable of multispectral imaging of intracardiac surfaces. The process included several iterations of light sources, optical filtering, and image acquisition techniques. The developed system included a compliant balloon, 355 nm laser irradiance, a high-sensitivity CCD, bandpass filtering, and image acquisition synchronized with the cardiac cycle. It enabled us to capture autofluorescence images from multiple spectral bands within the visible range while illuminating the endocardial surface with ultraviolet light. Principal component analysis and other spectral unmixing post-processing algorithms were then used to reveal target tissue. Based on the success of our prototype system, we are confident that the development of ever more sensitive cameras, recent advances in tunable filters, fiber bundles, and other optical and computational components makes it possible to create percutaneous catheters capable of acquiring hyper or multispectral hypercubes, including those based on autofluorescence, in real-time. This opens the door for widespread use of this methodology for high-resolution intraoperative imaging of internal tissues and organs-including cardiovascular applications.

Reprogramming Cardiac Cells Into Cardiac Purkinje‐like Cells For The Treatment Of Heart Failure

Heart failure (HF) is a chronic condition where the heart loses its ability to efficiently pump blood to other organs in the body. Currently, there is no cure for HF, other than a heart transplant, which may not be available for most patients. Other treatments available for HF focus on reducing the symptoms, but do not properly address the loss of cardiac tissue and scar tissue formation commonly observed in this condition. We believe that a novel therapeutic approach for HF can be achieved through direct administration of cardiac electrical conducting cells to failing hearts. This is because cardiac conducting cells, such as Purkinje cells, can intrinsically propagate electrical signals in synchronicity with other cardiac cells, allowing the heart to contract and pump blood. Thus, direct injection of cardiac Purkinje cells into failing hearts may overcome mechanical issues from scar tissue, as well as restore some of the cardiac tissue that has been lost in HF. To create Purkinje‐like cells, a reporter gene containing a Connexin 30.2 promoter and mCherry fluorescent tag was first introduced into the cardiac cells. This was followed by 5 days treatment with specific small molecules. The reprogrammed cardiac Purkinje‐like cells are then being characterized by transcriptome, epigenetic state, and functional analyses through FACS sorting, immunostaining, qPCR analysis, RNA sequencing, ChIP‐seq and Patch clamp. Successfully reprogrammed cardiac cells are expected to exhibit Purkinje‐like properties such as genetic make‐up and functionality. Upon creating Purkinje‐like cells, the next step will be to test the effectiveness of the Purkinje‐like cells in restoring the electrical conduction, as well as improving cardiac function, in small animal models of HF.Support or Funding InformationRobert J. Kleberg, Jr. and Helen C. Kleberg FoundationNational Heart, Lung, and Blood Institute (NHLBI) of the NIH (R15HL141963)

Solvent-Free Fabrication of Carbon Nanotube/Silk Fibroin Electrospun Matrices for Enhancing Cardiomyocyte Functionalities.

Cardiac tissue engineering holds great potential in regenerating functional cardiac tissues for various applications. The major strategy is to design scaffolds recapitulating the native cardiac microenvironment to enhance cell and tissue functionalities. Among various biomaterial systems, nanofibrous matrices with aligned morphologies and enhanced conductivity incline to induce the formation of oriented engineered cardiac tissues with enhanced functionalities. The challenge is to functionalize the scaffolds with conductive additives without influencing their biocompatibility. In this study, we developed a fully aqueous process for the fabrication of conductive carbon nanotube/silk fibroin (CNT/silk) electrospun scaffolds. The carbon nanotubes are well dispersed within the nanofibers, providing the scaffolds with enhanced conductivity and excellent biocompatibility for the culture of neonatal rat cardiomyocytes with improved cell spreading and enhanced expression of cardiac-specific proteins. Moreover, the aligned CNT/silk fibroin composite scaffolds exhibit abilities to guide the oriented organization of cardiac tissues and the biomimicking distribution of sarcomeres and gap junctions. The findings demonstrate the great potential of the CNT/silk scaffolds prepared through this aqueous processing method in supporting the formation of cardiac tissues with enhanced functionalities.

P2Y2 Nucleotide Receptor Is a Regulator of the Formation of Cardiac Adipose Tissue and Its Fat-Associated Lymphoid Clusters.

The formation of pericardial adipose tissue (PAT) and its regulatory function in cardiac inflammation are not well understood. We investigated the potential role of the ubiquitous ATP/UTP nucleotide receptor P2Y2 in the PAT by using P2Y2-null mice. We observed that P2Y2-null mice displayed a lower mass of PAT and a reduced density of its fat-associated lymphoid clusters (FALCs) and, more particularly, B cells. Loss of P2Y2 receptor in pericardial preadipocytes decreased their adipogenic differentiation and maturation abilities in vitro. Gene profiling identified P2Y2 target genes in PAT linked to immunomodulation. These data led to the identification of an increase of M2c anti-inflammatory macrophages correlated with increased apoptosis of B lymphocytes in P2Y2-null pericardial fat. In addition, follicular helper T cells, which contribute to B cell expansion in germinal centers, were dramatically decreased. The effect of P2Y2 loss was also investigated after ischemia-mediated expansion of FALCs in a model of myocardial infarct. Loss of P2Y2 led to reduced expansion of B and neutrophil populations in these clusters, whereas density of M2c anti-inflammatory macrophages was increased. Our study defines the P2Y2 nucleotide receptor as a regulator of the formation and inflammatory status of pericardial fat. The P2Y2 receptor could represent a therapeutic target in the regulation of PAT function before and during cardiac ischemia.

P6307Harnessing epicardial-derived cells for myocardial repair: the importance of epithelial-to-mesenchymal transition

Abstract Background The epicardium, the outer layer of the heart, is an indispensable source of cells and paracrine factors during embryonic heart formation. In the adult heart, the epicardium is quiescent unless there is injury. Cardiac damage results in partial recapitulation of developmental processes including epithelial-to-mesenchymal transition (EMT), expression of Wilms' Tumor-1 (WT1), proliferation, and migration of epicardial-derived cells (EPDCs). Aim Given their vital role during development, EPDCs represent an appealing source for endogenous cardiovascular repair. However, EPDC contribution to cardiac tissue formation in the adult is less efficient than during embryonic development. Our aim is to determine the requirements to optimize the adult epicardial response to injury. Methods Human foetal and adult EPDCs were isolated from cardiac specimens and cultured as epithelial-like cells in the presence of an Alk5-kinase inhibitor (A5ki). EMT was induced by adding 1 ng/mL TGFβ for 5 days. Immunofluorescent staining, qPCR, and cytokine arrays were performed. Cultured adult EPDCs pre- and post-EMT were transplanted into the myocardial wall of NOD-SCID mice after inducing myocardial infarction (MI), and cardiac function was measured by high-frequency ultrasound. Hearts were histologically analysed 3 days and 6 weeks post-MI. Results Both foetal and adult human EPDCs can be expanded in culture and undergo EMT after TGFβ stimulation leading to morphological changes accompanied by downregulation of WT1 and E-cadherin, and upregulation of mesenchymal genes. Importantly, upon removal of Alk5ki, foetal EPDCs display instant spontaneous EMT, suggesting the importance of this process for EPDCs' developmental potential. In vivo, animals receiving intramyocardial transplantation of post-EMT EPDCs displayed a higher ejection fraction 6 weeks after MI compared to pre-EMT EPDC receiving animals (26%±11 n=8 vs. 11%±5 n=9 respectively P&lt;0.05). This corresponded to a smaller infarct size in the post-EMT group (16,4%±4 of the left ventricle versus 26,9%±5 in pre-EMT, p&lt;0.05). This could not be explained by a difference in cell grafting, analysed at 3 days post-MI. After 6 weeks, we observed a small difference in human collagen deposition in the post-EMT group, however very low numbers of human cells were detected suggesting a predominantly short-acting paracrine effect. Analysis of cytokine production of cultured cells revealed a higher production of factors involved in angiogenesis and chemotaxis like VEGF and MCP-3 in post-EMT EPDCs in comparison to pre-EMT EPDCs. Effects on local angiogenesis and inflammation in vivo are being investigated Conclusion EPDCs require EMT to acquire the ability to contribute to cardiac repair, which appears to be predominantly through paracrine processes. Our research now focuses on enhancing EMT of endogenous epicardial cells. Acknowledgement/Funding AMS is funded by a Dekker fellowship from the Dutch Heart Foundation