Changes in extracellular matrix in failing human non-ischemic and ischemic hearts with mechanical unloading.

Ischemic and non-ischemic cardiomyopathies have distinct etiologies and underlying disease mechanisms, which require in-depth investigation for improved therapeutic interventions. The goal of this study was to use clinically obtained myocardium from healthy and heart failure patients, and characterize the changes in extracellular matrix (ECM) in ischemic and non-ischemic failing hearts, with and without mechanical unloading. Using tissue engineering methodologies, we also investigated how diseased human ECM, in the absence of systemic factors, can influence cardiomyocyte function. Heart tissues from heart failure patients with ischemic and non-ischemic cardiomyopathy were compared to explore differential disease phenotypes and reverse remodeling potential of left ventricular assisted device (LVAD) support at transcriptomic, proteomic and structural levels. The collected data demonstrated that the differential ECM compositions recapitulated the disease microenvironment and induced cardiomyocytes to undergo disease-like functional alterations. In addition, our study also revealed molecular profiles of non-ischemic and ischemic heart failure patients and explored the underlying mechanisms of etiology-specific impact on clinical outcome of LVAD support and tendency towards reverse remodeling.

Behavior of CMPCs in unidirectional constrained and stress-free 3D hydrogels.

Cardiomyocyte progenitor cells (CMPCs) are a candidate cell source for cardiac regenerative therapy. However, like other stem cells, after transplantation in the heart, cell retention and differentiation capacity of the CMPCs are low. Combining cells with biomaterials might overcome this problem. By serving as a (temporal) environment, the biomaterial can retain the cells and provide signals that enhance survival, proliferation and differentiation of the cells. To gain more insight into the effect that the encapsulation of CMPCs in a biomaterial has on their behavior, we cultured CMPCs in unidirectional constrained and stress-free collagen/Matrigel hydrogels. CMPCs cultured in 3D hydrogels stay viable and keep their cardiomyogenic profile independent of the application of strain. Moreover, the increased expression of Nkx2.5, myocardin and cTnT in 3D hydrogels compared to 2D cultures, suggests enhanced cardiomyogenic differentiation capacity of cells in 3D. Furthermore, increased expression of collagen I, collagen III, elastin and fibronectin and of the matrix remodeling enzymes MMP-1, MMP-2, MMP-9, and TIMP-1 and TIMP-2 in the 3D hydrogels is indicative of an enhanced matrix remodeling capacity of CMPCs in a 3D environment, independent of the application of strain. Interestingly, the additional application of static strain to the 3D hydrogels, as imposed by hydrogel constrainment, stabilized CMPC viability and proliferation, resulted in enhanced cardiac marker protein expression and appeared crucial for cellular organization and morphology. More specifically, CMPCs cultured in 3D collagen/Matrigel constrained hydrogels became readily mechanosensitive, had a rod-shaped morphology, and responded to the applied strain by orienting in the direction of the constraint. Overall, our data demonstrate the applicability of CMPCs in a 3D environment since encapsulation of CMPCs may stabilize survival and proliferation, can enhance the differentiation and remodeling capacity of the cells, and could induce cellular re-organization, which all may contribute to an improved efficiency of cardiac stem cell therapy.

The epicardium as modulator of the cardiac autonomic response during early development.

The cardiac autonomic nervous system (cANS) modulates heart rate, contraction force and conduction velocity. The embryonic chicken heart already responds to epinephrine prior to establishment of the cANS. The aim of this study was to define the regions of the heart that might participate in modulating the early autonomic response to epinephrine. Immunofluorescence analysis reveals expression of neural markers tubulin beta-3 chain and neural cell adhesion molecule in the epicardium during early development. In addition, expression of the ß2 adrenergic receptor, the receptor for epinephrine, was found in the epicardium. Ex-ovo micro-electrode recordings in hearts with inhibition of epicardial outgrowth showed a significantly reduced response of the heart rate to epinephrine compared to control hearts. This study suggests a role for the epicardium as autonomic modulator during early cardiac development.

Matrix production and remodeling capacity of cardiomyocyte progenitor cells during in vitro differentiation.

Cell-based therapy has emerged as a treatment modality for myocardial repair. Especially cardiac resident stem cells are considered a potential cell source since they are able to differentiate into cardiomyocytes and have improved heart function after injury in a preclinical model for myocardial infarction. To avoid or repair myocardial damage it is important not only to replace the lost cardiomyocytes, but also to remodel and replace the scar tissue by "healthy" extracellular matrix (ECM). Interestingly, the role of cardiac stem cells in this facet of cardiac repair is largely unknown. Therefore, we investigated the expression and production of ECM proteins, matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in human cardiomyocyte progenitor cells (CMPCs) undergoing differentiation towards the cardiomyogenic lineage. Our data suggest that CMPCs have the capacity to synthesize and modulate their own matrix environment, especially during differentiation towards the cardiomyogenic lineage. While undifferentiated CMPCs expressed collagen I, III, IV and fibronectin, but no elastin, during the process of differentiation the expression of collagen I, III, IV and fibronectin increased and interestingly also elastin expression was induced. Furthermore, undifferentiated CMPCs express MMP-1 -2 and -9 and upon differentiation the expression of MMP-1 decreased, while the expression of MMP-2 and MMP-9, although the latter only in the early stage of differentiation, increased. Additionally, the expression of TIMP-1, -2 and -4 was induced during differentiation. This study provides new insights into the matrix production and remodeling capacity of human CMPCs, with potential beneficial effects for the treatment of cardiac injury.

Dysregulation of the PDGFRA gene causes inflow tract anomalies including TAPVR: integrating evidence from human genetics and model organisms.

Total anomalous pulmonary venous return (TAPVR) is a congenital heart defect inherited via complex genetic and/or environmental factors. We report detailed mapping in extended TAPVR kindreds and mutation analysis in TAPVR patients that implicate the PDGFRA gene in the development of TAPVR. Gene expression studies in mouse and chick embryos for both the Pdgfra receptor and its ligand Pdgf-a show temporal and spatial patterns consistent with a role in pulmonary vein (PV) development. We used an in ovo function blocking assay in chick and a conditional knockout approach in mouse to knock down Pdgfra expression in the developing venous pole during the period of PV formation. We observed that loss of PDGFRA function in both organisms causes TAPVR with low penetrance (approximately 7%) reminiscent of that observed in our human TAPVR kindreds. Intermediate inflow tract anomalies occurred in a higher percentage of embryos (approximately 30%), suggesting that TAPVR occurs at one end of a spectrum of defects. We show that the anomalous pulmonary venous connection seen in chick and mouse is highly similar to TAPVR discovered in an abnormal early stage embryo from the Kyoto human embryo collection. Whereas the embryology of the normal venous pole and PV is becoming understood, little is known about the embryogenesis or molecular pathogenesis of TAPVR. These models of TAPVR provide important insight into the pathogenesis of PV defects. Taken together, these data from human genetics and animal models support a role for PDGF-signaling in normal PV development, and in the pathogenesis of TAPVR.

Ultrastructural Characteristics of Myocardial Reperfusion Injury and Effect of Selective Intracoronary Hypothermia: An Observational Study in Isolated Beating Porcine Hearts.

In acute myocardial infarction (AMI), myocardial reperfusion injury may undo part of the recovery after revascularization of the occluded coronary artery. Selective intracoronary hypothermia is a novel method aimed at reducing myocardial reperfusion injury, but its presumed protective effects in AMI still await further elucidation. This proof-of-concept study assesses the potential protective effects of selective intracoronary hypothermia in an ex-vivo, isolated beating heart model of AMI. In four isolated Langendorff perfused beating pig hearts, an anterior wall myocardial infarction was created by inflating a balloon in the mid segment of the left anterior descending (LAD) artery. After one hour, two hearts were treated with selective intracoronary hypothermia followed by normal reperfusion (cooled hearts). In the other two hearts, the balloon was deflated after one hour, allowing normal reperfusion (control hearts). Biopsies for histologic and electron microscopic evaluation were taken from the myocardium at risk at different time points: before occlusion (t = BO); 5 minutes before reperfusion (t = BR); and 10 minutes after reperfusion (t = AR). Electron microscopic analysis was performed to evaluate the condition of the mitochondria. Histological analyses included evaluation of sarcomeric collapse and intramyocardial hematoma. Electron microscopic analysis revealed intact mitochondria in the hypothermia treated hearts compared to the control hearts where mitochondria were more frequently damaged. No differences in the prespecified histological parameters were observed between cooled and control hearts at t = AR. In the isolated beating porcine heart model of AMI, reperfusion was associated with additional myocardial injury beyond ischemic injury. Selective intracoronary hypothermia preserved mitochondrial integrity compared to nontreated controls.

Epithelial-to-mesenchymal transformation alters electrical conductivity of human epicardial cells.

The myocardium of the developing heart tube is covered by epicardium. These epicardial cells undergo a process of epithelial-to-mesenchymal transformation (EMT) and develop into epicardium-derived cells (EPDCs). The ingrowing EPDCs differentiate into several celltypes of which the cardiac fibroblasts form the main group. Disturbance of EMT of the epicardium leads to serious hypoplasia of the myocardium, abnormal coronary artery differentiation and Purkinje fibre paucity. Interestingly, the electrophysiological properties of epicardial cells and whether EMT influences electrical conductivity of epicardial cells is not yet known. We studied the electrophysiological aspects of epicardial cells before and after EMT in a dedicated in vitro model, using micro-electrode arrays to investigate electrical conduction across epicardial cells. Therefore, human adult epicardial cells were placed between two neonatal rat cardiomyocyte populations. Before EMT the epicardial cells have a cobblestone (epithelium-like) phenotype that was confirmed by staining for the cell-adhesion molecule ß-catenin. After spontaneous EMT in vitro the EPDCs acquired a spindle-shaped morphology confirmed by vimentin staining. When comparing both types we observed that the electrical conduction is influenced by EMT, resulting in significantly reduced conductivity of spindle-shaped EPDCs, associated with a conduction block. Furthermore, the expression of both gap junction (connexins 40, Cx43 and Cx45) and ion channel proteins (SCN5a, CACNA1C and Kir2.1) was down-regulated after EMT. This study shows for the first time the conduction differences between epicardial cells before and after EMT. These differences may be of relevance for the role of EPDCs in cardiac development, and in EMT-related cardiac dysfunction.

In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGFß-signaling and WT1.

Adult epicardial cells are required for endogenous cardiac repair. After myocardial injury, they are reactivated, undergo epithelial-to-mesenchymal transformation (EMT) and migrate into the injured myocardium where they generate various cell types, including coronary smooth muscle cells and cardiac interstitial fibroblasts, which contribute to cardiac repair. To understand what drives epicardial EMT, we used an in vitro model for human adult epicardial cells. These cells have an epithelium-like morphology and markedly express the cell surface marker vascular cell adhesion marker (VCAM-1). In culture, epicardial cells spontaneously undergo EMT after which the spindle-shaped cells now express endoglin. Both epicardial cells before and after EMT express the epicardial marker, Wilms tumor 1 (WT1). Adding transforming growth factor beta (TGFß) induces loss of epithelial character and initiates the onset of mesenchymal differentiation in human adult epicardial cells. In this study, we show that TGFß-induced EMT is dependent on type-1 TGFß receptor activity and can be inhibited by soluble VCAM-1. We also show that epicardial-specific knockdown of Wilms tumor-1 (WT1) induces the process of EMT in human adult epicardial cells, through transcriptional regulation of platelet-derived growth factor receptor alpha (Pdgfrα), Snai1 and VCAM-1. These data provide new insights into the process of EMT in human adult epicardial cells, which might provide opportunities to develop new strategies for endogenous cell-based cardiac repair.

Cardiac malformations in Pdgfralpha mutant embryos are associated with increased expression of WT1 and Nkx2.5 in the second heart field.

Platelet-derived growth factor receptor alpha (Pdgfralpha) identifies cardiac progenitor cells in the posterior part of the second heart field. We aim to elucidate the role of Pdgfralpha in this region. Hearts of Pdgfralpha-deficient mouse embryos (E9.5-E14.5) showed cardiac malformations consisting of atrial and sinus venosus myocardium hypoplasia, including venous valves and sinoatrial node. In vivo staining for Nkx2.5 showed increased myocardial expression in Pdgfralpha mutants, confirmed by Western blot analysis. Due to hypoplasia of the primary atrial septum, mesenchymal cap, and dorsal mesenchymal protrusion, the atrioventricular septal complex failed to fuse. Impaired epicardial development and severe blebbing coincided with diminished migration of epicardium-derived cells and myocardial thinning, which could be linked to increased WT1 and altered alpha4-integrin expression. Our data provide novel insight for a possible role for Pdgfralpha in transduction pathways that lead to repression of Nkx2.5 and WT1 during development of posterior heart field-derived cardiac structures.

Epicardium-derived cells enhance proliferation, cellular maturation and alignment of cardiomyocytes.

During heart development, cells from the proepicardial organ spread over the naked heart tube to form the epicardium. From here, epicardium-derived cells (EPDCs) migrate into the myocardium. EPDCs proved to be indispensable for the formation of the ventricular compact zone and myocardial maturation, by largely unknown mechanisms. In this study we investigated in vitro how EPDCs affect cardiomyocyte proliferation, cellular alignment and contraction, as well as the expression and cellular distribution of proteins involved in myocardial maturation. Embryonic quail EPDCs induced proliferation of neonatal mouse cardiomyocytes. This required cell-cell interactions, as proliferation was not observed in transwell cocultures. Western blot analysis showed elevated levels of electrical and mechanical junctions (connexin43, N-cadherin), sarcomeric proteins (Troponin-I, alpha-actinin), extracellular matrix (collagen I and periostin) in cocultures of EPDCs and cardiomyocytes. Immunohistochemistry indicated more membrane-bound expression of Cx43, N-cadherin, the mechanotransduction molecule focal adhesion kinase, and higher expression of the sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a). Newly developed software for analysis of directionality in immunofluorescent stainings showed a quantitatively determined enhanced cellular alignment of cardiomyocytes. This was functionally related to increased contraction. The in vitro effects of EPDCs on cardiomyocytes were confirmed in three reciprocal in vivo models for EPDC-depletion (chicken and mice) in which downregulation of myocardial N-cadherin, Cx43, and FAK were observed. In conclusion, direct interaction of EPDCs with cardiomyocytes induced proliferation, correct mechanical and electrical coupling of cardiomyocytes, ECM-deposition and concurrent establishment of cellular array. These findings implicate that EPDCs are ideal candidates as adjuvant cells for cardiomyocyte integration during cardiac (stem) cell therapy.

Electrical activation of sinus venosus myocardium and expression patterns of RhoA and Isl-1 in the chick embryo.

UNLABELLED: Electrical Activity and RhoA in the Embryo. INTRODUCTION: Myocardium at the venous pole (sinus venosus) of the heart has gained clinical interest as arrhythmias can be initiated from this area. During development, sinus venosus myocardium is incorporated to the primary heart tube and expresses different markers than primary myocardium. We aimed to elucidate the development of sinus venosus myocardium, including the sinoatrial node (SAN), by studying expression patterns of RhoA in relation to other markers, and by studying electrical activation patterns of the developing sinus venosus myocardium. METHODS AND RESULTS: Expression of RhoA, myocardial markers cTnI and Nkx2.5, transcription factors Isl-1 and Tbx18, and cation channel HCN4 were examined in sequential stages in chick embryos. Electrical activation patterns were studied using microelectrodes and optical mapping. Embryonic sinus venosus myocardium is cTnI and HCN4 positive, Nkx2.5 negative, complemented by distinct patterns of Isl-1 and Tbx18. During development, initial myocardium-wide expression of RhoA becomes restricted to right-sided sinus venosus myocardium, comprising the SAN. Electrophysiological measurements revealed initial capacity of both atria to show electrical activity that in time shifts to a right-sided dominance, coinciding with persistence of RhoA, Tbx18, and HCN4 and absence of Nkx2.5 expression in the definitive SAN. CONCLUSION: Results show an initially bilateral electrical potential of sinus venosus myocardium evolving into a right-sided activation pattern during development, and suggest a role for RhoA in conduction system development. We hypothesize an initial sinus venosus-wide capacity to generate pacemaker signals, becoming confined to the definitive SAN. Lack of differentiation toward a chamber phenotype would explain ectopic pacemaker foci.

Platelet-derived growth factor is involved in the differentiation of second heart field-derived cardiac structures in chicken embryos.

For the establishment of a fully functional septated heart, addition of myocardium from second heart field-derived structures is important. Platelet-derived growth factors (PDGFs) are known for their role in cardiovascular development. In this study, we aim to elucidate this role of PDGF-A, PDGF-C, and their receptor PDGFR-alpha. We analyzed the expression patterns of PDGF-A, -C, and their receptor PDGFR-alpha during avian heart development. A spatiotemporal pattern of ligands was seen with colocalization of the PDGFR-alpha. This was found in second heart field-derived myocardium as well as the proepicardial organ (PEO) and epicardium. Mechanical inhibition of epicardial outgrowth as well as chemical disturbance of PDGFR-alpha support a functional role of the ligands and the receptor in cardiac development.

In vivo and in vitro Approaches Reveal Novel Insight Into the Ability of Epicardium-Derived Cells to Create Their Own Extracellular Environment.

Human epicardium-derived cells (hEPDCs) transplanted in the NOD-SCID mouse heart after myocardial infarction (MI) are known to improve cardiac function, most likely orchestrated by paracrine mechanisms that limit adverse remodeling. It is not yet known, however, if hEPDCs contribute to preservation of cardiac function via the secretion of matrix proteins and/or matrix proteases to reduce scar formation. This study describes the ability of hEPDCs to produce human collagen type I after transplantation into the infarct border zone, thereby creating their own extracellular environment. As the in vivo environment is too complex to investigate the mechanisms involved, we use an in vitro set-up, mimicking biophysical and biochemical cues from the myocardial tissue to unravel hEPDC-induced matrix remodeling. The in vivo contribution of hEPDCs to the cardiac extracellular matrix (ECM) was assessed in a historical dataset of the NOD-SCID murine model of experimentally induced MI and cell transplantation. Analysis showed that within 48 h after transplantation, hEPDCs produce human collagen type I. The build-up of the human collagen microenvironment was reversed within 6 weeks. To understand the hEPDCs response to the pathologic cardiac microenvironment, we studied the influence of cyclic straining and/or transforming growth beta (TGFß) signaling in vitro. We revealed that 48 h of cyclic straining induced collagen type I production via the TGFß/ALK5 signaling pathway. The in vitro approach enables further unraveling of the hEPDCs ability to secrete matrix proteins and matrix proteases and the potential to create and remodel the cardiac matrix in response to injury.

Origin, fate, and function of epicardium-derived cells (EPDCs) in normal and abnormal cardiac development.

During heart development, cells of the primary and secondary heart field give rise to the myocardial component of the heart. The neural crest and epicardium provide the heart with a considerable amount of nonmyocardial cells that are indispensable for correct heart development. During the past 2 decades, the importance of epicardium-derived cells (EPDCs) in heart formation became increasingly clear. The epicardium is embryologically formed by the outgrowth of proepicardial cells over the naked heart tube. Following epithelial-mesenchymal transformation, EPDCs form the subepicardial mesenchyme and subsequently migrate into the myocardium, and differentiate into smooth muscle cells and fibroblasts. They contribute to the media of the coronary arteries, to the atrioventricular valves, and the fibrous heart skeleton. Furthermore, they are important for the myocardial architecture of the ventricular walls and for the induction of Purkinje fiber formation. Whereas the exact signaling cascades in EPDC migration and function still need to be elucidated, recent research has revealed several factors that are involved in EPDC migration and specialization, and in the cross-talk between EPDCs and other cells during heart development. Among these factors are the Ets transcription factors Ets-1 and Ets-2. New data obtained with lentiviral antisense constructs targeting Ets-1 and Ets-2 specifically in the epicardium indicate that both factors are independently involved in the migratory behavior of EPDCs. Ets-2 seems to be especially important for the migration of EPDCs into the myocardial wall, and to subendocardial positions in the atrioventricular cushions and the trabeculae. With respect to the clinical importance of correct EPDC development, the relation with coronary arteriogenesis has been noted well before. In this review, we also propose a role for EPDCs in cardiac looping, and emphasize their contribution to the development of the valves and myocardial architecture. Lastly, we focus on the congenital heart anomalies that might be caused primarily by an epicardial developmental defect.

Cardiac Progenitor Cells and the Interplay with Their Microenvironment.

The microenvironment plays a crucial role in the behavior of stem and progenitor cells. In the heart, cardiac progenitor cells (CPCs) reside in specific niches, characterized by key components that are altered in response to a myocardial infarction. To date, there is a lack of knowledge on these niches and on the CPC interplay with the niche components. Insight into these complex interactions and into the influence of microenvironmental factors on CPCs can be used to promote the regenerative potential of these cells. In this review, we discuss cardiac resident progenitor cells and their regenerative potential and provide an overview of the interactions of CPCs with the key elements of their niche. We focus on the interaction between CPCs and supporting cells, extracellular matrix, mechanical stimuli, and soluble factors. Finally, we describe novel approaches to modulate the CPC niche that can represent the next step in recreating an optimal CPC microenvironment and thereby improve their regeneration capacity.

Glycosylated Cell Surface Markers for the Isolation of Human Cardiac Progenitors.

The aim of stem cell therapy after cardiac injury is to replace damaged cardiac tissue. Human cardiac progenitor cells (CPCs) represent an interesting cell population for clinical strategies to treat cardiac disease and human CPC-specific antibodies would aid in the clinical implementation of cardiac progenitor-based cell therapy. However, the field of CPC biology suffers from the lack of human CPC-specific markers. Therefore, we raised a panel of monoclonal antibodies (mAb) against CPCs. Of this panel of antibodies, we show that mAb C1096 recognizes a progenitor-like population in the fetal and adult human heart and partially colocalize with reported CPC populations in vitro. Furthermore, mAb C1096 can be used to isolate a multipotent progenitor population from human heart tissue. Interestingly, the two lead candidates, mAb C1096 and mAb C19, recognize glycosylated residues on PECAM1 (platelet and endothelial cell adhesion molecule 1) and GRP78, respectively, and de-N-glycosylation significantly abolishes their binding. Thereby, this report describes new clinically applicable antibodies against human CPCs, and for the first time demonstrates the importance of glycosylated residues as CPCs specific markers.

Mechanically Robust Electrospun Hydrogel Scaffolds Crosslinked via Supramolecular Interactions.

One of the major challenges in the processing of hydrogels based on poly(ethylene glycol) (PEG) is to create mechanically robust electrospun hydrogel scaffolds without chemical crosslinking postprocessing. In this study, this is achieved by the introduction of physical crosslinks in the form of supramolecular hydrogen bonding ureido-pyrimidinone (UPy) moieties, resulting in chain-extended UPy-PEG polymers (CE-UPy-PEG) that can be electrospun from organic solvent. The resultant fibrous meshes are swollen in contact with water and form mechanically stable, elastic hydrogels, while the fibrous morphology remains intact. Mixing up to 30 wt% gelatin with these CE-UPy-PEG polymers introduce bioactivity into these scaffolds, without affecting the mechanical properties. Manipulating the electrospinning parameters results in meshes with either small or large fiber diameters, i.e., 0.63 ± 0.36 and 2.14 ± 0.63 µm, respectively. In that order, these meshes provide support for renal epithelial monolayer formation or a niche for the culture of cardiac progenitor cells.

Mimicking Cardiac Fibrosis in a Dish: Fibroblast Density Rather than Collagen Density Weakens Cardiomyocyte Function.

Cardiac fibrosis is one of the most devastating effects of cardiac disease. Current in vitro models of cardiac fibrosis do not sufficiently mimic the complex in vivo environment of the cardiomyocyte. We determined the local composition and mechanical properties of the myocardium in established mouse models of genetic and acquired fibrosis and tested the effect of myocardial composition on cardiomyocyte contractility in vitro by systematically manipulating the number of fibroblasts and collagen concentration in a platform of engineered cardiac microtissues. The in vitro results showed that while increasing collagen content had little effect on microtissue contraction, increasing fibroblast density caused a significant reduction in contraction force. In addition, the beating frequency dropped significantly in tissues consisting of 50% cardiac fibroblasts or higher. Despite apparent dissimilarities between native and in vitro fibrosis, the latter allows for the independent analysis of local determinants of fibrosis, which is not possible in vivo.

Epicardium-derived cells are important for correct development of the Purkinje fibers in the avian heart.

During embryonic development, the proepicardial organ (PEO) grows out over the heart surface to form the epicardium. Following epithelial-mesenchymal transformation, epicardium-derived cells (EPDCs) migrate into the heart and contribute to the developing coronary arteries, to the valves, and to the myocardium. The peripheral Purkinje fiber network develops from differentiating cardiomyocytes in the ventricular myocardium. Intrigued by the close spatial relationship between the final destinations of migrating EPDCs and Purkinje fiber differentiation in the avian heart, that is, surrounding the coronary arteries and at subendocardial sites, we investigated whether inhibition of epicardial outgrowth would disturb cardiomyocyte differentiation into Purkinje fibers. To this end, epicardial development was inhibited mechanically with a membrane, or genetically, by suppressing epicardial epithelial-to-mesenchymal transformation with antisense retroviral vectors affecting Ets transcription factor levels (n=4, HH39-41). In both epicardial inhibition models, we evaluated Purkinje fiber development by EAP-300 immunohistochemistry and found that restraints on EPDC development resulted in morphologically aberrant differentiation of Purkinje fibers. Purkinje fiber hypoplasia was observed both periarterially and at subendocardial positions. Furthermore, the cells were morphologically abnormal and not aligned in orderly Purkinje fibers. We conclude that EPDCs are instrumental in Purkinje fiber differentiation, and we hypothesize that they cooperate directly with endothelial and endocardial cells in the development of the peripheral conduction system.

Cardiomyocyte progenitor cell mechanoresponse unrevealed: strain avoidance and mechanosome development.

For emerging cardiac regeneration strategies, it is essential to know if and how cardiac stem cells sense and respond to the mechanical stimuli provided by their environment in the beating heart. Here, we study the response to cyclic strain of undifferentiated and predifferentiated human cardiomyocyte progenitor cells (CMPCs), as well as the formation and activation of the cellular structures involved in mechanosensing, that we termed 'mechanosome'. Once verified that the applied uniaxial cyclic strain (10%, 0.5 Hz) did not alter the cardiac lineage commitment and differentiation state of CMPCs, the cellular mechanoresponse to the applied strain was quantified by cellular orientation. While undifferentiated cells maintained their original (random) orientation, upon early cardiomyogenic differentiation (predifferentiated) CMPCs exhibited a distinct strain avoidance response after 48 h of cyclic straining. Interestingly, the mechanosome development and the activation of the mechanotransduction pathways also occurred with early cardiac differentiation of the CMPCs, regardless of the substrate or the applied cyclic strain. These results indicate that the mechanoresponse of CMPCs depends on the presence of a developed mechanosome, which only develops during early cardiomyogenic differentiation Our findings provide the first understanding of mechanotransduction in human CMPCs and as such can contribute to the improvement of cardiac regeneration strategies.