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Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.

During heart formation, the heart grows and undergoes dramatic morphogenesis to achieve efficient embryonic function. Both in fish and amniotes, much of the growth occurring after initial heart tube formation arises from second heart field (SHF)-derived progenitor cell addition to the arterial pole, allowing chamber formation. In zebrafish, this process has been extensively studied during embryonic life, but it is unclear how larval cardiac growth occurs beyond 3 days post-fertilisation (dpf). By quantifying zebrafish myocardial growth using live imaging of GFP-labelled myocardium we show that the heart grows extensively between 3 and 5 dpf. Using methods to assess cell division, cellular development timing assay and Kaede photoconversion, we demonstrate that proliferation, CM addition, and hypertrophy contribute to ventricle growth. Mechanistically, we show that reduction in Mef2c activity (mef2ca +/ - ;mef2cb -/- ), downstream or in parallel with Nkx2.5 and upstream of Ltbp3, prevents some CM addition and differentiation, resulting in a significantly smaller ventricle by 3 dpf. After 3 dpf, however, CM addition in mef2ca +/ - ;mef2cb -/- mutants recovers to a normal pace, and the heart size gap between mutants and their siblings diminishes into adulthood. Thus, as in mice, there is an early time window when SHF contribution to the myocardium is particularly sensitive to loss of Mef2c activity., Competing Interests: Declaration of competing interest All authors have no competing financial interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Ca 2+ signaling is critical for heart development; however, the precise roles and regulatory pathways of Ca 2+ transport proteins in cardiogenesis remain largely unknown. Sodium-calcium exchanger 1 (Ncx1) is responsible for Ca 2+ efflux in cardiomyocytes. It is involved in cardiogenesis, while the mechanism is unclear. Here, using the forward genetic screening in zebrafish, we identified a novel mutation at a highly-conserved leucine residue in ncx1 gene (mutant LDD353 /ncx1h L154P ) that led to smaller hearts with reduced heart rate and weak contraction. Mechanistically, the number of ventricular but not atrial cardiomyocytes was reduced in ncx1h L154P zebrafish. These defects were mimicked by knockdown or knockout of ncx1h. Moreover, ncx1h L154P had cytosolic and mitochondrial Ca 2+ overloading and Ca 2+ transient suppression in cardiomyocytes. Furthermore, ncx1h L154P and ncx1h morphants downregulated cardiac transcription factors hand2 and gata4 in the cardiac regions, while overexpression of hand2 and gata4 partially rescued cardiac defects including the number of ventricular myocytes. These findings demonstrate an essential role of the novel 154th leucine residue in the maintenance of Ncx1 function in zebrafish, and reveal previous unrecognized critical roles of the 154th leucine residue and Ncx1 in the formation of ventricular cardiomyocytes by at least partially regulating the expression levels of gata4 and hand2.

While major coronary artery development and pathologies affecting them have been extensively studied, understanding the development and organization of the coronary microvasculature beyond the earliest developmental stages requires new tools. Without techniques to image the coronary microvasculature over the whole heart, it is likely we are underestimating the microvasculature's impact on normal development and diseases. We present a new imaging and analysis toolset to visualize the coronary microvasculature in intact embryonic hearts and quantify vessel organization. The fluorescent dyes DiI and DAPI were used to stain the coronary vasculature and cardiomyocyte nuclei in quail embryo hearts during rapid growth and morphogenesis of the left ventricular wall. Vessel and cardiomyocytes orientation were automatically extracted and quantified, and vessel density was calculated. The coronary microvasculature was found to follow the known helical organization of cardiomyocytes in the ventricular wall. Vessel density in the left ventricle did not change during and after compaction. This quantitative and automated approach will enable future cohort studies to understand the microvasculature's role in diseases such as hypertrophic cardiomyopathy where misalignment of cardiomyocytes has been observed in utero.

Mapping of the holistic cell behaviours sculpting the four-chambered mammalian heart has been a goal or previous studies, but so far only success in transparent invertebrates and lower vertebrates with two-chambered hearts has been achieved. Using a live-imaging system comprising a customized vertical light-sheet microscope equipped with a mouse embryo culture module, a heartbeat-gated imaging strategy and a digital image processing framework, we realized volumetric imaging of developing mouse hearts at single-cell resolution and with uninterrupted cell lineages for up to 1.5 d. Four-dimensional landscapes of Nppa + cardiomyocyte cell behaviours revealed a blueprint for ventricle chamber formation by which biased outward migration of the outermost cardiomyocytes is coupled with cell intercalation and horizontal division. The inner-muscle architecture of trabeculae was developed through dual mechanisms: early fate segregation and transmural cell arrangement involving both oriented cell division and directional migration. Thus, live-imaging reconstruction of uninterrupted cell lineages affords a transformative means for deciphering mammalian organogenesis.

If viewed as a movie, heart morphogenesis appears to unfold in a continuous and seamless manner. At the mechanistic level, however, a series of discreet and separable processes sequentially underlie heart development. This is evident in examining the expansion of the ventricular wall, which accounts for most of the contractile force of each heartbeat. Ventricular wall expansion is driven by cardiomyocyte proliferation coupled with a morphogenetic program that causes wall thickening rather than lengthening. Although most studies of these processes have focused on heart-intrinsic processes, it is increasingly clear that extracardiac events influence or even direct heart morphogenesis. In this review, we specifically consider mechanisms responsible for coordinating cardiomyocyte proliferation and ventricular wall expansion in mammalian development, relying primarily on studies from mouse development where a wealth of molecular and genetic data have been accumulated.

The roles of cellular orientation during trabecular and ventricular wall morphogenesis are unknown, and so are the underlying mechanisms that regulate cellular orientation. Myocardial-specific Numb and Numblike double-knockout (MDKO) hearts display a variety of defects, including in cellular orientation, patterns of mitotic spindle orientation, trabeculation, and ventricular compaction. Furthermore, Numb - and Numblike -null cardiomyocytes exhibit cellular behaviors distinct from those of control cells during trabecular morphogenesis based on single-cell lineage tracing. We investigated how Numb regulates cellular orientation and behaviors and determined that N-cadherin levels and membrane localization are reduced in MDKO hearts. To determine how Numb regulates N-cadherin membrane localization, we generated an mCherry:Numb knockin line and found that Numb localized to diverse endocytic organelles but mainly to the recycling endosome. Consistent with this localization, cardiomyocytes in MDKO did not display defects in N-cadherin internalization but rather in postendocytic recycling to the plasma membrane. Furthermore, N-cadherin overexpression via a mosaic model partially rescued the defects in cellular orientation and trabeculation of MDKO hearts. Our study unravels a phenomenon that cardiomyocytes display spatiotemporal cellular orientation during ventricular wall morphogenesis, and its disruption leads to abnormal trabecular and ventricular wall morphogenesis. Furthermore, we established a mechanism by which Numb modulates cellular orientation and consequently trabecular and ventricular wall morphogenesis by regulating N-cadherin recycling to the plasma membrane., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Multiple syndromes share congenital heart and craniofacial muscle defects, indicating there is an intimate relationship between the adjacent cardiac and pharyngeal muscle (PM) progenitor fields. However, mechanisms that direct antagonistic lineage decisions of the cardiac and PM progenitors within the anterior mesoderm of vertebrates are not understood. Here, we identify that retinoic acid (RA) signaling directly promotes the expression of the transcription factor Nr2f1a within the anterior lateral plate mesoderm. Using zebrafish nr2f1a and nr2f2 mutants, we find that Nr2f1a and Nr2f2 have redundant requirements restricting ventricular cardiomyocyte (CM) number and promoting development of the posterior PMs. Cre-mediated genetic lineage tracing in nr2f1a; nr2f2 double mutants reveals that tcf21+ progenitor cells, which can give rise to ventricular CMs and PM, more frequently become ventricular CMs potentially at the expense of posterior PMs in nr2f1a; nr2f2 mutants. Our studies reveal insights into the molecular etiology that may underlie developmental syndromes that share heart, neck and facial defects as well as the phenotypic variability of congenital heart defects associated with NR2F mutations in humans., Competing Interests: The authors have declared that no competing interests exist.

Mid-gestation mouse ventricles (E11.5) contain a larger number of Nkx2.5 + cardiac progenitor cells (CPCs). The proliferation rates are consistently higher in CPCs compared to myocyte population of developing ventricles. Recent studies suggested that CPCs are an ideal donor cell type for replacing damaged tissue in diseased hearts. Thus, the ability to isolate and expand CPCs from embryos or stem cell cultures could be useful for cell fate studies and regenerative therapies. Since embryonic CPCs possess fewer mitochondria compared to cardiomyocytes, we reasoned that CPCs can be fractionated using a fluorescent mitochondrial membrane potential dye (TMRM) and these cells may retain cardiomyogenic potential even in the absence of cardiomyocytes (CMs). FACS sorting of TMRM stained embryonic ventricular cells indicated that over 99% of cells in TMRM high fraction stained positive for sarcomeric myosin (MF20) and all of them expressed Nkx2.5. Although majority of cells present in TMRM low fraction expressed Nkx2.5, very few cells (~1%) stained positive for MF20. Further culturing of TMRM low cells over a period of 48 h showed a progressive increase in MF20 positive cells. Additional analyses revealed that MF20 negative cells in TMRM low fraction do not express markers for endothelial cells (vWF, CD31) or smooth muscle cells (SM myosin). Treatment of TMRM low cells with known cardiogenic factors DMSO and dynorphin B significantly increased the percentage of MF20 + cells compared to untreated cultures. Collectively, these studies suggest that embryonic CPCs can be separated as a TMRM low fraction and their differentiation potential can be enhanced by exogenous addition of known cardiomyogenic factors., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Trabecular morphogenesis is a key morphologic event during cardiogenesis and contributes to the formation of a competent ventricular wall. Lack of trabeculation results in embryonic lethality. The trabecular morphogenesis is a multistep process that includes, but is not limited to, trabecular initiation, proliferation/growth, specification, and compaction. Although a number of signaling molecules have been implicated in regulating trabeculation, the cellular processes underlying mammalian trabecular formation are not fully understood. Recent works show that the myocardium displays polarity, and oriented cell division (OCD) and directional migration of the cardiomyocytes in the monolayer myocardium are required for trabecular initiation and formation. Furthermore, perpendicular OCD is an extrinsic asymmetric cell division that contributes to trabecular specification, and is a mechanism that causes the trabecular cardiomyocytes to be distinct from the cardiomyocytes in compact zone. Once the coronary vasculature system starts to function in the embryonic heart, the trabeculae will coalesce with the compact zone to thicken the heart wall, and abnormal compaction will lead to left ventricular non-compaction (LVNC) and heart failure. There are many reviews about compaction and LVNC. In this review, we will focus on the roles of myocardial polarity and OCD in trabecular initiation, formation, and specification.

Trabeculation is crucial for cardiac muscle growth in vertebrates. This process requires the Erbb2/4 ligand Neuregulin (Nrg), secreted by the endocardium, as well as blood flow/cardiac contractility. Here, we address two fundamental, yet unresolved, questions about cardiac trabeculation: why does it initially occur in the ventricle and not the atrium, and how is it modulated by blood flow/contractility. Using loss-of-function approaches, we first show that zebrafish Nrg2a is required for trabeculation, and using a protein-trap line, find that it is expressed in both cardiac chambers albeit with different spatiotemporal patterns. Through gain-of-function experiments, we show that atrial cardiomyocytes can also respond to Nrg2a signalling, suggesting that the cardiac jelly, which remains prominent in the atrium, represents a barrier to Erbb2/4 activation. Furthermore, we find that blood flow/contractility is required for Nrg2a expression, and that while non-contractile hearts fail to trabeculate, non-contractile cardiomyocytes are also competent to respond to Nrg2a/Erbb2 signalling.

The developmental program of the heart requires accurate regulation to ensure continuous circulation and simultaneous cardiac morphogenesis, because any functional abnormalities may progress to congenital heart malformation. Notably, energy metabolism in fetal ventricular cells is regulated in a manner that differs from adult ventricular cells: fetal cardiomyocytes generally have immature mitochondria and fetal ventricular cells show greater dependence on glycolytic ATP production. However, although various characteristics of energy metabolism in fetal ventricular cells have been reported, to our knowledge, a quantitative description of the contributions of these factors to fetal ventricular cell functions has not yet been established. Here, we constructed a mathematical model to integrate various characteristics of fetal ventricular cells and predicted the contribution of each characteristic to the maintenance of intracellular ATP concentration and sarcomere contraction under anoxic conditions. Our simulation results demonstrated that higher glycogen content, higher hexokinase activity, and lower creatine concentration helped prolong the time for which ventricular cell contraction was maintained under anoxic conditions. The integrated model also enabled us to quantitatively assess the contributions of factors related to energy metabolism in ventricular cells. Because fetal cardiomyocytes exhibit similar energy metabolic profiles to stem cell-derived cardiomyocytes and those in the failing heart, an improved understanding of these fetal ventricular cells will contribute to a better comprehension of the processes in stem cell-derived cardiomyocytes or under pathological conditions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The mouse fetal and adult hearts express two adenine nucleotide translocator (ANT) isoform genes. The predominant isoform is the heart-muscle-brain ANT-isoform gene 1 (Ant1) while the other is the systemic Ant2 gene. Genetic inactivation of the Ant1 gene does not impair fetal development but results in hypertrophic cardiomyopathy in postnatal mice. Using a knockin X-linked Ant2 allele in which exons 3 and 4 are flanked by loxP sites combined in males with a protamine 1 promoter driven Cre recombinase we created females heterozygous for a null Ant2 allele. Crossing the heterozygous females with the Ant2(fl), PrmCre(+) males resulted in male and female ANT2-null embryos. These fetuses proved to be embryonic lethal by day E14.5 in association with cardiac developmental failure, immature cardiomyocytes having swollen mitochondria, cardiomyocyte hyperproliferation, and cardiac failure due to hypertrabeculation/noncompaction. ANTs have two main functions, mitochondrial-cytosol ATP/ADP exchange and modulation of the mitochondrial permeability transition pore (mtPTP). Previous studies imply that ANT2 biases the mtPTP toward closed while ANT1 biases the mtPTP toward open. It has been reported that immature cardiomyocytes have a constitutively opened mtPTP, the closure of which signals the maturation of cardiomyocytes. Therefore, we hypothesize that the developmental toxicity of the Ant2 null mutation may be the result of biasing the cardiomyocyte mtPTP to remain open thus impairing cardiomyocyte maturation and resulting in cardiomyocyte hyperproliferation and failure of trabecular maturation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Many organs are composed of complex tissue walls that are structurally organized to optimize organ function. In particular, the ventricular myocardial wall of the heart comprises an outer compact layer that concentrically encircles the ridge-like inner trabecular layer. Although disruption in the morphogenesis of this myocardial wall can lead to various forms of congenital heart disease and non-compaction cardiomyopathies, it remains unclear how embryonic cardiomyocytes assemble to form ventricular wall layers of appropriate spatial dimensions and myocardial mass. Here we use advanced genetic and imaging tools in zebrafish to reveal an interplay between myocardial Notch and Erbb2 signalling that directs the spatial allocation of myocardial cells to their proper morphological positions in the ventricular wall. Although previous studies have shown that endocardial Notch signalling non-cell-autonomously promotes myocardial trabeculation through Erbb2 and bone morphogenetic protein (BMP) signalling, we discover that distinct ventricular cardiomyocyte clusters exhibit myocardial Notch activity that cell-autonomously inhibits Erbb2 signalling and prevents cardiomyocyte sprouting and trabeculation. Myocardial-specific Notch inactivation leads to ventricles of reduced size and increased wall thickness because of excessive trabeculae, whereas widespread myocardial Notch activity results in ventricles of increased size with a single-cell-thick wall but no trabeculae. Notably, this myocardial Notch signalling is activated non-cell-autonomously by neighbouring Erbb2-activated cardiomyocytes that sprout and form nascent trabeculae. Thus, these findings support an interactive cellular feedback process that guides the assembly of cardiomyocytes to morphologically create the ventricular myocardial wall and more broadly provide insight into the cellular dynamics of how diverse cell lineages organize to create form.

Cardiac developmental disorders represent the most common of human birth defects, and anomalies in cardiomyocyte proliferation drive many of these disorders. This review highlights the molecular mechanisms of prenatal cardiac growth. Trabeculation represents the initial ventricular growth phase and is necessary for embryonic survival. Later in development, the bulk of the ventricular wall derives from the compaction process, yet the arrest of this process can still be compatible with life. Cardiomyocyte proliferation and growth form the basis of both trabeculation and compaction, and mouse models indicate that cardiomyocyte interactions with the surrounding environment are critical for these proliferative processes. The human genetics of left ventricular noncompaction cardiomyopathy suggest that cardiomyocyte cell-autonomous mechanisms contribute to the compaction process. Understanding the determinants of prenatal or early postnatal cardiomyocyte proliferation and growth provides critical information that identifies risk factors for cardiovascular disease, including heart failure and its associated complications of arrhythmias and thromboembolic events.

One major obstacle to the application of stem cell-derived cardiomyocytes (CMs) for disease modeling and clinical therapies is the inability to identify the developmental stage of these cells without the need for genetic manipulation or utilization of exogenous markers. In this study, we demonstrate that Raman microspectroscopy can non-invasively identify embryonic stem cell (ESC)-derived chamber-specific CMs and monitor cell maturation. Using this marker-free approach, Raman peaks were identified for atrial and ventricular CMs, ESCs were successfully discriminated from their cardiac derivatives, a distinct phenotypic spectrum for ESC-derived CMs was confirmed, and unique spectral differences between fetal versus adult CMs were detected. The real-time identification and characterization of CMs, their progenitors, and subpopulations by Raman microspectroscopy strongly correlated to the phenotypical features of these cells. Due to its high molecular resolution, Raman microspectroscopy offers distinct analytical characterization for differentiating cardiovascular cell populations., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The mechanisms that govern specification of various cell types that constitute vertebrate heart are not fully understood. Whilst most studies of heart development have utilised the mouse embryo, we have used an alternative model, embryos of the frog Xenopus laevis, which permits direct experimental manipulation of a non-essential heart. We show that in this model pluripotent animal cap explants injected with cardiogenic factor GATA4 mRNA express pan-myocardial as well as ventricular and proepicardial markers. We found that cardiac cell fate diversification, as assessed by ventricular and proepicardial markers, critically depends on tissue integrity, as it is disrupted by dissociation but can be fully restored by inhibition of the BMP pathway and partially by Dkk-1. Ventricular and proepicardial cell fates can also be restored in reaggregated GATA4-expressing cells upon transplantation into a host embryo. The competence of the host embryo to induce ventricular and proepicardial markers gradually decreases with the age of the transplant and is lost by the onset of myocardial differentiation at the late tailbud stage (st. 28). The influence of the host on the transplant was not limited to diversification of cardiac cell fates, but also included induction of growth and rhythmic beating, resulting in generation of a secondary heart-like structure. Our results additionally show that efficient generation of secondary heart requires normal axial patterning of the host embryo. Furthermore, secondary hearts can be induced in a wide range of locations within the host, arguing that the host embryo provides a permissive environment for development of cardiac patterning, growth and physiological maturation. Our results have implications for a major goal of cardiac regenerative medicine, differentiation of ventricular myocardium., (Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Rationale: Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers., Methods and Results: In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis., Conclusion: Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.

The neonatal heart is a very useful tool for the study of biochemical pathways and properties of cardiomyocytes and as it has the potential to regenerate for a brief period of time from birth; it is also useful to study cardiac regeneration. However, as the heart matures, this proficiency for regeneration is reduced. This regenerative potential may be influenced by the microenvironment of the heart in the early stages of postnatal development and therefore, cell cultures derived at this stage may contain functional cardiomyocytes and progenitor cells. The aim of this study was to identify key steps in the isolation and culture of such early stage-neonatal mouse hearts to allow maximum migration of cardiomyocytes from the explant and their maintenance as functional, long term cultures. Explant cultures of mouse ventricles preserved 3-dimensional structure and generated migrating layers of cardiomyocytes that expressed alpha sarcomeric actin which could be further sub-cultured by enzymatic dissociation. Western blotting demonstrated expression of c-KIT, GATA4, alpha sarcomeric actin and connexin43 proteins after 20 days of explant culture. ACTA1, GATA4, and CX43 continued to express in five weeks old explant cultures while the c-KIT protein was expressed up to two passages during sub-culture. Real time PCR and SQRT PCR also demonstrated gene expression of cardiomyocyte markers in long term cultures. Migrating cells from the explants assembled into contracting spheroids after subculture and expressed the c-KIT protein. Progenitor markers CD44, CD90, and extracellular proteins, periostin and vimentin demonstrated the preservation of cellular heterogeneity in such cultures. Supplementation with Hydrocortisone maintained a cardioprotective environment and reduced the non-myocyte population. This is an optimized and efficient method for the generation of neonatal heart cultures that is not labor intensive and does not require supplementation with cytokines., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The vertebrate heart muscle (myocardium) develops from the first heart field (FHF) and expands by adding second heart field (SHF) cells. While both lineages exist already in teleosts, the primordial contributions of FHF and SHF to heart structure and function remain incompletely understood. Here we delineate the functional contribution of the FHF and SHF to the zebrafish heart using the cis-regulatory elements of the draculin (drl) gene. The drl reporters initially delineate the lateral plate mesoderm, including heart progenitors. Subsequent myocardial drl reporter expression restricts to FHF descendants. We harnessed this unique feature to uncover that loss of tbx5a and pitx2 affect relative FHF versus SHF contributions to the heart. High-resolution physiology reveals distinctive electrical properties of each heart field territory that define a functional boundary within the single zebrafish ventricle. Our data establish that the transcriptional program driving cardiac septation regulates physiologic ventricle partitioning, which successively provides mechanical advantages of sequential contraction.

Temporally controlled mechanisms that define the unique features of ventricular and atrial cardiomyocyte identities are essential for the construction of a coordinated, morphologically intact heart. We have previously demonstrated an important role for nkx genes in maintaining ventricular identity, however, the specific timing of nkx2.5 function in distinct cardiomyocyte populations has yet to be elucidated. Here, we show that heat-shock induction of a novel transgenic line, Tg(hsp70l:nkx2.5-EGFP), during the initial stages of cardiomyocyte differentiation leads to rescue of chamber shape and identity in nkx2.5(-/-) embryos as chambers emerge. Intriguingly, our findings link an early role of this essential cardiac transcription factor with a later function. Moreover, these data reveal that nkx2.5 is also required in the second heart field as the heart tube forms, reflecting the temporal delay in differentiation of this population. Thus, our results support a model in which nkx genes induce downstream targets that are necessary to maintain chamber-specific identity in both early- and late-differentiating cardiomyocytes at discrete stages in cardiac morphogenesis. Furthermore, we show that overexpression of nkx2.5 during the first and second heart field development not only rescues the mutant phenotype, but also is sufficient for proper function of the adult heart. Taken together, these results shed new light on the stage-dependent mechanisms that sculpt chamber-specific cardiomyocytes and, therefore, have the potential to improve in vitro generation of ventricular cells to treat myocardial infarction and congenital heart disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aims: The strategies that control formation of the ventricular wall during heart development are not well understood. In previous studies, we documented IGF2 as a major mitogenic signal that controls ventricular cardiomyocyte proliferation and chamber wall expansion. Our objective in this study was to define the tissue source of IGF2 in heart development and the upstream pathways that control its expression., Methods and Results: Using a number of mouse genetic tools, we confirm that the critical source of IGF2 is the epicardium. We find that epicardial Igf2 expression is controlled in a biphasic manner, first induced by erythropoietin and then regulated by oxygen and glucose with onset of placental function. Both processes are independently controlled by retinoic acid signalling., Conclusions: Our results demonstrate that ventricular wall cardiomyocyte proliferation is subdivided into distinct regulatory phases. Each involves instructive cues that originate outside the heart and thereby act on the epicardium in an endocrine manner, a mode of regulation that is mostly unknown in embryogenesis., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Rationale: Islet-1 positive (Islet-1(+)) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1(+) cells retain proliferative activity and may therefore play a role in regenerating specialized regions in the heart., Methods and Results: DNA synthesis was analyzed by the incorporation of tritiated thymidine ((3)H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of (3)H-thymidine for 5 days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1(+) cells. Whereas Islet(-) non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed., Conclusions: Islet-1(+) cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Protein Phosphatase 2A (PP2A) function is controlled by regulatory subunits that modulate the activity of the catalytic subunit and direct the PP2A complex to specific intracellular locations. To study PP2A's role in signal transduction pathways that control growth and differentiation in vivo, a transgenic mouse lacking the B56γ regulatory subunit of PP2A was made., Results: Lack of PP2A activity specific to the PP2A-B56γ holoenzyme, resulted in the formation of an incomplete ventricular septum and a decrease in the number of ventricular cardiomyocytes. During cardiac development, B56γ is expressed in the nucleus of α-actinin-positive cardiomyocytes that contain Z-bands. The pattern of B56γ expression correlated with the cardiomyocyte apoptosis we observed in B56γ-deficient mice during mid to late gestation. In addition to the cardiac phenotypes, mice lacking B56γ have a decrease in locomotive coordination and gripping strength, indicating that B56γ has a role in controlling PP2A activity required for efficient neuromuscular function., Conclusions: PP2A-B56γ activity is required for efficient cardiomyocyte maturation and survival. The PP2A B56γ regulatory subunit controls PP2A substrate specificity in vivo in a manner that cannot be fully compensated for by other B56 subunits., (© 2014 Wiley Periodicals, Inc.)

Background: In cardiac muscle, the intercalated disk (ID) at the longitudinal cell-edges of cardiomyocytes provides as a macromolecular infrastructure that integrates mechanical and electrical coupling within the heart. Pathophysiological disturbance in composition of this complex is well known to trigger cardiac arrhythmias and pump failure. The mechanisms underlying assembly of this important cellular domain in human heart is currently unknown., Methods: We collected 18 specimens from individuals that died from non-cardiovascular causes. Age of the specimens ranged from a gestational age of 15 weeks through 11 years postnatal. Immunohistochemical labeling was performed against proteins comprising desmosomes, adherens junctions, the cardiac sodium channel and gap junctions to visualize spatiotemporal alterations in subcellular location of the proteins., Results: Changes in spatiotemporal localization of the adherens junction proteins (N-cadherin and ZO-1) and desmosomal proteins (plakoglobin, desmoplakin and plakophilin-2) were identical in all subsequent ages studied. After an initial period of diffuse and lateral labelling, all proteins were fully localized in the ID at approximately 1 year after birth. Nav1.5 that composes the cardiac sodium channel and the gap junction protein Cx43 follow a similar pattern but their arrival in the ID is detected at (much) later stages (two years for Nav1.5 and seven years for Cx43, respectively)., Conclusion: Our data on developmental maturation of the ID in human heart indicate that generation of the mechanical junctions at the ID precedes that of the electrical junctions with a significant difference in time. In addition arrival of the electrical junctions (Nav1.5 and Cx43) is not uniform since sodium channels localize much earlier than gap junction channels.

Background: The ventricular myocardium is the most prominent layer of the heart, and the most important for mediating cardiac physiology. Although the ventricular myocardium is critical for heart function, the cellular hierarchy responsible for ventricle-specific myocardium development remains unresolved., Results: To determine the pattern and time course of ventricular myocardium development, we investigated IRX4 protein expression, which has not been previously reported. We identified IRX4+ cells in the cardiac crescent, and these cells were positive for markers of the first or second heart fields. From the onset of chamber formation, IRX4+ cells were restricted to the ventricular myocardium. This expression pattern persisted into adulthood. Of interest, we observed that IRX4 exhibits developmentally regulated dynamic intracellular localization. Throughout prenatal cardiogenesis, and up to postnatal day 4, IRX4 was detected in the cytoplasm of ventricular myocytes. However, between postnatal days 5–6, IRX4 translocated to the nucleus of ventricular myocytes., Conclusions: Given the ventricle-specific expression of Irx4 in later stages of heart development, we hypothesize that IRX4+ cells in the cardiac crescent represent the earliest cell population in the cellular hierarchy underlying ventricular myocardium development.

Stem cell-derived cardiomyocytes represent unique tools for cell- and tissue-based regenerative therapies, drug discovery and safety, and studies of fundamental heart-failure mechanisms. However, the degree to which stem cell-derived cardiomyocytes compare to mature cardiomyocytes is often debated. We reasoned that physiological metrics of engineered cardiac tissues offer a means of comparison. We built laminar myocardium engineered from cardiomyocytes that were differentiated from mouse embryonic stem cell-derived cardiac progenitors or harvested directly from neonatal mouse ventricles, and compared their anatomy and physiology in vitro. Tissues assembled from progenitor-derived myocytes and neonate myocytes demonstrated similar cytoskeletal architectures but different gap junction organization and electromechanical properties. Progenitor-derived myocardium had significantly less contractile stress and slower longitudinal conduction velocity than neonate-derived myocardium, indicating that the developmental state of the cardiomyocytes affects the electromechanical function of the resultant engineered tissue. These data suggest a need to establish performance metrics for future stem cell applications.

Human (h) embryonic stem cells (ESC) represent an unlimited source of cardiomyocytes (CMs); however, these differentiated cells are immature. Thus far, gene profiling studies have been performed with non-purified or non-chamber specific CMs. Here we took a combinatorial approach of using systems biology to guide functional discoveries of novel biological properties of purified hESC-derived ventricular (V) CMs. We profiled the transcriptomes of hESCs, hESC-, fetal (hF) and adult (hA) VCMs, and showed that hESC-VCMs displayed a unique transcriptomic signature. Not only did a detailed comparison between hESC-VCMs and hF-VCMs confirm known expression changes in metabolic and contractile genes, it further revealed novel differences in genes associated with reactive oxygen species (ROS) metabolism, migration and cell cycle, as well as potassium and calcium ion transport. Following these guides, we functionally confirmed that hESC-VCMs expressed IKATP with immature properties, and were accordingly vulnerable to hypoxia/reoxygenation-induced apoptosis. For mechanistic insights, our coexpression and promoter analyses uncovered a novel transcriptional hierarchy involving select transcription factors (GATA4, HAND1, NKX2.5, PPARGC1A and TCF8), and genes involved in contraction, calcium homeostasis and metabolism. These data highlight novel expression and functional differences between hESC-VCMs and their fetal counterparts, and offer insights into the underlying cell developmental state. These findings may lead to mechanism-based methods for in vitro driven maturation.

Background: Substance P is a sensory nerve neuropeptide located near coronary vessels in the heart. Therefore, substance P may be one of the first mediators released in the heart in response to hypertension, and can contribute to adverse myocardial remodeling via interactions with the neurokinin-1 receptor. We asked: 1) whether substance P promoted cardiac hypertrophy, including the expression of fetal genes known to be re-expressed during pathological hypertrophy; and 2) the extent to which substance P regulated collagen production and fibrosis., Methods and Results: Spontaneously hypertensive rats (SHR) were treated with the neurokinin-1 receptor antagonist L732138 (5mg/kg/d) from 8 to 24 weeks of age. Age-matched WKY served as controls. The gene encoding substance P, TAC1, was up-regulated as blood pressure increased in SHR. Fetal gene expression by cardiomyocytes was increased in SHR and was prevented by L732138. Cardiac fibrosis also occurred in the SHR and was prevented by L732138. Endothelin-1 was up-regulated in the SHR and this was prevented by L732138. In isolated cardiac fibroblasts, substance P transiently up-regulated several genes related to cell-cell adhesion, cell-matrix adhesion, and extracellular matrix regulation, however, no changes in fibroblast function were observed., Conclusions: Substance P activation of the neurokinin-1 receptor induced expression of fetal genes related to pathological hypertrophy in the hypertensive heart. Additionally, activation of the neurokinin-1 receptor was critical to the development of cardiac fibrosis. Since no functional changes were induced in isolated cardiac fibroblasts by substance P, we conclude that substance P mediates fibrosis via up-regulation of endothelin-1., (© 2013.)

In mammals, cardiac development proceeds from the formation of the linear heart tube, through complex looping and septation, all the while increasing in mass to provide the oxygen delivery demands of embryonic growth. The developing heart must orchestrate regional differences in cardiomyocyte proliferation to control cardiac morphogenesis. During ventricular wall formation, the compact myocardium proliferates more vigorously than the trabecular myocardium, but the mechanisms controlling such regional differences among cardiomyocyte populations are not understood. Control of definitive cardiomyocyte proliferation is of great importance for application to regenerative cell-based therapies. We have used murine and human pluripotent stem cell systems to demonstrate that, during in vitro cellular differentiation, early ventricular cardiac myocytes display a robust proliferative response to β-catenin-mediated signaling and conversely accelerate differentiation in response to inhibition of this pathway. Using gain- and loss-of-function murine genetic models, we show that β-catenin controls ventricular myocyte proliferation during development and the perinatal period. We further demonstrate that the differential activation of the Wnt/β-catenin signaling pathway accounts for the observed differences in the proliferation rates of the compact versus the trabecular myocardium during normal cardiac development. Collectively, these results provide a mechanistic explanation for the differences in localized proliferation rates of cardiac myocytes and point to a practical method for the generation of the large numbers of stem cell-derived cardiac myocytes necessary for clinical applications.

Epigenetic regulation is implicated in embryonic development and the control of gene expression in a cell-specific manner. However, little is known about the role of histone methylation changes on human cardiac differentiation and maturation. Using human embryonic stem cells (hESCs) and their derived ventricular (V) cardiomyocytes (CMs) as a model, we examined trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) on promoters of genes associated with cardiac electrophysiology, contraction, and Ca(2+) handling. To avoid ambiguities due to heterogeneous chamber-specific types, hESC-derived ventricular cardiomyocytes (VCMs) were selected by dual zeocin-GFP expression under the transcriptional control of the MLC2v promoter and confirmed electrophysiologically by its signature action potential phenotype. High levels of H3K4me3 are present on pluripotency genes in hESCs with an absence of H3K27me3. Human ESC-VCMS, relative to hESCs, were characterized by a profound loss of H3K27me3 and an enrichment of H3K4me3 marks on cardiac-specific genes, including MYH6, MYH7, MYL2, cTNT, and ANF. Gene transcripts encoding key voltage-gated ion channels and Ca(2+)-handling proteins in hESC-VCMs were significantly increased, which could be attributed to a distinct pattern of differential H3K4me3 and H3K27me3 profiles. Treatment of hESC-VCMs with the histone deacetylase inhibitor valproic acid increased H3K4me3 on gene promoters, induced hypertrophic growth (as gauged by cell volume and capacitance), and augmented cardiac gene expression, but it did not affect electrophysiological properties of these cells. Hence, cardiac differentiation of hESCs involves a dynamic shift in histone methylation, which differentially affects VCM gene expression and function. We conclude that the epigenetic state of hESC-VCMs is dynamic and primed to promote growth and developmental maturation, but that proper environmental stimuli with chromatin remodeling will be required to synergistically trigger global CM maturation to a more adult-like phenotype.

Most of the mammalian heart is formed from mesodermal progenitors in the first and second heart fields (FHF and SHF), whereby the FHF gives rise to the left ventricle and parts of the atria and the SHF to the right ventricle, outflow tract and parts of the atria. Whereas SHF progenitors have been characterized in detail, using specific molecular markers, comprehensive studies on the FHF have been hampered by the lack of exclusive markers. Here, we present Hcn4 (hyperpolarization-activated cyclic nucleotide-gated channel 4) as an FHF marker. Lineage-traced Hcn4+/FHF cells delineate FHF-derived structures in the heart and primarily contribute to cardiomyogenic cell lineages, thereby identifying an early cardiomyogenic progenitor pool. As a surface marker, HCN4 also allowed the isolation of cardiomyogenic Hcn4+/FHF progenitors from human embryonic stem cells. We conclude that a primary purpose of the FHF is to generate cardiac muscle and support the contractile activity of the primitive heart tube, whereas SHF-derived progenitors contribute to heart cell lineage diversification.

Early embryonic rodent ventricular cells exhibit spontaneous action potential (AP), which disappears in later developmental stages. Here, we used 3 mathematical models-the Kyoto, Ten Tusscher-Panfilov, and Luo-Rudy models-to present an overview of the functional landscape of developmental changes in embryonic ventricular cells. We switched the relative current densities of 9 ionic components in the Kyoto model, and 160 of 512 representative combinations were predicted to result in regular spontaneous APs, in which the quantitative changes in Na(+) current (I Na) and funny current (I f) made large contributions to a wide range of basic cycle lengths. In all three models, the increase in inward rectifier current (I K1) before the disappearance of I f was predicted to result in abnormally high intracellular Ca(2+) concentrations. Thus, we demonstrated that the developmental changes in APs were well represented, as I Na increased before the disappearance of I f, followed by a 10-fold increase in I K1.

In recent years, the contribution of I(f), an important pacemaker current, and intracellular Ca(2+) release (ICR) from sarcoplasmic reticulum to pacemaking and arrhythmia has been intensively studied. However, their functional roles in embryonic heart remain uncertain. Using patch clamp, Ca(2+) imaging, and RT-PCR, we found that I(f) regulated the firing rate in early and late stage embryonic ventricular cells, as ivabradine (30 µM), a specific blocker of I(f), slowed down action potential (AP) frequency. This inhibitory effect was even stronger in late stage cells, though I(f) was down-regulated. In contrast to I(f), ICR was found to be indispensable for the occurrence of APs in ventricular cells of different stages, because abolishment of ICR with ryanodine and 2-aminoethoxydiphenyl borate (2-APB), specific blockers of ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs), completely abolished APs. In addition, we noticed that RyR- and IP3R-mediated ICR coexisted in early-stage ventricular cells and RyRs functionally dominated. While at late stage RyRs, but not IP3Rs, mediated ICR. In both early and late stage ventricular cells, Na-Ca exchanger current (I(Na/Ca)) mediated ICR-triggered depolarization of membrane potential and resulted in the initiation of APs. We also observed that different from I(f), which presented as the substantial component of the earlier diastolic depolarization current, application of ryanodine, and/or 2-APB slowed the late phase of diastolic depolarization. Thus, we conclude that in murine embryonic ventricular cells I(f) regulates firing rate, while RyRs and IP3Rs (early stage) or RyRs (late stage)-mediated ICR determines the occurrence of APs., (Copyright © 2013 Wiley Periodicals, Inc.)

Cardiomyocyte organization is a critical determinant of coordinated cardiac contractile function. Because of the acute opening of the pulmonary circulation, the relative workload of the left ventricle (LV) and right ventricle (RV) changes substantially immediately after birth. We hypothesized that three-dimensional cardiomyocyte architecture might be required to adapt rapidly to accommodate programmed perinatal changes of cardiac function. Isolated fixed hearts from pig fetuses or pigs at midgestation, preborn, postnatal day 1 (P1), postnatal day 5, postnatal day 14 (P14), and adulthood (n = 5 for each group) were acquired for diffusion-weighted magnetic resonance imaging. Cardiomyocyte architecture was visualized by three-dimensional fiber tracking and was quantitatively evaluated by the measured helix angle (α(h)). Upon the completion of MRI, hearts were sectioned and stained with hematoxylin/eosin (H&E) to evaluate cardiomyocyte alignment, with picrosirius red to evaluate collagen content, and with anti-Ki67 to evaluate postnatal cell proliferation. The helical architecture of cardiomyocyte was observed as early as the midgestational period. Postnatal changes of cardiomyocyte architecture were observed from P1 to P14, which primary occurred in the septum and RV free wall (RVFW). In the septum, the volume ratio of LV- vs. RV-associated cardiomyocytes rapidly changed from RV-LV balanced pattern at birth to LV dominant pattern by P14. In the RVFW, subendocardial α(h) decreased by ~30° from P1 to P14. These findings indicate that the helical architecture of cardiomyocyte is developed as early as the midgestation period. Substantial and rapid adaptive changes in cardiac microarchitecture suggested considerable developmental plasticity of cardiomyocyte form and function in the postnatal period in response to altered cardiac mechanical function.

Developmental changes in excitation-contraction mechanisms were examined in the ventricular myocardium from fetal, neonatal, and 1-, 2-, and 4-week-old mice. In isolated tissue, the negative inotropic effect of nifedipine decreased, while that of ryanodine increased with age. Action potential duration decreased with age, especially during the late fetal period. In ventricular cardiomyocytes, fluorescence imaging revealed that the sarcoplasmic reticulum increases progressively during pre- and postnatal development. t-Tubules were absent in the fetus and neonate, were observed only in the subsarcolemmal region at 1 week after birth, and were present throughout the cytoplasm at 2 and 4 weeks after birth. The amplitude of Ca(2+) transients, as well as its ryanodine-sensitive component, increased with age. In the neonate and 1-week-old mice, Ca(2+) at the cell center showed slower rise than the subsarcolemmal region, but in 2- and 4-week-old mice, Ca(2+) increased simultaneously across the entire width of the cell. These results suggest that in the mouse ventricular myocardium, the shortening of the action potential during the late fetal period and the development of t-tubule-sarcoplasmic reticulum coupling during the second postnatal week largely contribute to the developmental increase in the dependence of contraction on sarcoplasmic reticulum function.

Vertebrate cardiac progenitor cells are initially allocated in two distinct domains, the first and second heart fields. It has been demonstrated that first heart field cells give rise to the myocardial cells in the left ventricle and part of the atria, whereas second heart field cells move into the developing heart tube and contribute to the myocardium of the outflow tract and right ventricle and the majority of atria. In this study, we have examined the expression of the mouse Cripto gene and the lineage of Cripto-expressing cells, focusing on its relationship with cardiac myocyte differentiation. The mouse Cripto gene is initially expressed at late head fold (LHF) stages in the cardiac crescent region, known as the first heart field; later in the medial region of the early heart tube, and by embryonic day 8.5, it is localized to the outflow tract. Using a Cripto-LacZ allele, we found that Cripto- expressing progeny cells contribute to the myocardium of the entire outflow tract and right ventricle, as well as to a majority of cells within the left ventricle. In contrast, no Cripto- expressing progeny cells were found in the atria or atrio-ventricular canal. Therefore, Cripto is transiently expressed in early differentiating myocardial cells of the left ventricle, right ventricle and outflow tract between LHF stages and E8.5. Cripto expression is subsequently downregulated as cells undergo further differentiation.

Trabecular myocardium accounts for the majority of the ventricles during early cardiogenesis, but compact myocardium is the primary component at later developmental stages. Elucidation of the genes regulating compact myocardium development is essential to increase our understanding of left ventricular noncompaction (LVNC), a cardiomyopathy characterized by increased ratios of trabecular to compact myocardium. 14-3-3ε is an adapter protein expressed in the lateral plate mesoderm, but its in vivo cardiac functions remain to be defined. Here we show that 14-3-3ε is expressed in the developing mouse heart as well as in cardiomyocytes. 14-3-3ε deletion did not appear to induce compensation by other 14-3-3 isoforms but led to ventricular noncompaction, with features similar to LVNC, resulting from a selective reduction in compact myocardium thickness. Abnormal compaction derived from a 50% decrease in cardiac proliferation as a result of a reduced number of cardiomyocytes in G(2)/M and the accumulation of cardiomyocytes in the G(0)/G(1) phase of the cell cycle. These defects originated from downregulation of cyclin E1 and upregulation of p27(Kip1), possibly through both transcriptional and posttranslational mechanisms. Our work shows that 14-3-3ε regulates cardiogenesis and growth of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via both cyclin E1 and p27(Kip1). These data are consistent with the long-held view that human LVNC may result from compaction arrest, and they implicate 14-3-3ε as a new candidate gene in congenital human cardiomyopathies.

As vertebrate embryos develop to adulthood, their organs undergo marked changes in size and tissue architecture. The heart acquires muscle mass and matures structurally to fulfil increasing circulatory needs, a process that is incompletely understood. Here we used multicolour clonal analysis to define the contributions of individual cardiomyocytes as the zebrafish heart undergoes morphogenesis from a primitive embryonic structure into its complex adult form. We find that the single-cardiomyocyte-thick wall of the juvenile ventricle forms by lateral expansion of several dozen cardiomyocytes into muscle patches of variable sizes and shapes. As juvenile zebrafish mature into adults, this structure becomes fully enveloped by a new lineage of cortical muscle. Adult cortical muscle originates from a small number of cardiomyocytes--an average of approximately eight per animal--that display clonal dominance reminiscent of stem cell populations. Cortical cardiomyocytes initially emerge from internal myofibres that in rare events breach the juvenile ventricular wall, and then expand over the surface. Our results illuminate the dynamic proliferative behaviours that generate adult cardiac structure, revealing clonal dominance as a key mechanism that shapes a vertebrate organ.

Cardiomyocyte hypertrophy is a complex cellular behavior involving coordination of cell size expansion and myofibril content increase. Here, we investigate the contribution of cardiomyocyte hypertrophy to cardiac chamber emergence, the process during which the primitive heart tube transforms into morphologically distinct chambers and increases its contractile strength. Focusing on the emergence of the zebrafish ventricle, we observed trends toward increased cell surface area and myofibril content. To examine the extent to which these trends reflect coordinated hypertrophy of individual ventricular cardiomyocytes, we developed a method for tracking cell surface area changes and myofibril dynamics in live embryos. Our data reveal a previously unappreciated heterogeneity of ventricular cardiomyocyte behavior during chamber emergence: although cardiomyocyte hypertrophy was prevalent, many cells did not increase their surface area or myofibril content during the observed timeframe. Despite the heterogeneity of cell behavior, we often found hypertrophic cells neighboring each other. Next, we examined the impact of blood flow on the regulation of cardiomyocyte behavior during this phase of development. When blood flow through the ventricle was reduced, cell surface area expansion and myofibril content increase were both dampened, and the behavior of neighboring cells did not seem coordinated. Together, our studies suggest a model in which hemodynamic forces have multiple influences on cardiac chamber emergence: promoting both cardiomyocyte enlargement and myofibril maturation, enhancing the extent of cardiomyocyte hypertrophy, and facilitating the coordination of neighboring cell behaviors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Prevailing data suggest that ATP-sensitive potassium channels (K(ATP)) contribute to a surprising resistance to hypoxia in mammalian embryos, thus we aimed to characterize the developmental changes of K(ATP) channels in murine fetal ventricular cardiomyocytes., Methods: Patch clamp was applied to investigate the functions of K(ATP). RT-PCR, Western blot were used to further characterize the molecular properties of K(ATP) channels., Results: Similar K(ATP) current density was detected in ventricular cardiomyocytes of late development stage (LDS) and early development stage (EDS). Molecular-biological study revealed the upregulation of Kir6.1/SUR2A in membrane and Kir6.2 remained constant during development. Kir6.1, Kir6.2, and SUR1 were detectable in the mitochondria without marked difference between EDS and LDS. Acute hypoxia-ischemia led to cessation of APs in 62.5% of tested EDS cells and no APs cessation was observed in LDS cells. SarcK(ATP) blocker glibenclamide rescued 47% of EDS cells but converted 42.8% of LDS cells to APs cessations under hypoxia-ischemic condition. MitoK(ATP) blocker 5-HD did not significantly influence the response to acute hypoxia-ischemia at either EDS or LDS. In summary, sarcK(ATP) played distinct functional roles under acute hypoxia-ischemic condition in EDS and LDS fetal ventricular cardiomyocytes, with developmental changes in sarcK(ATP) subunits. MitoK(ATP) were not significantly involved in the response of fetal cardiomyocytes to acute hypoxia-ischemia and no developmental changes of K(ATP) subunits were found in mitochondria., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Trabeculation is an integral component of cardiac ventricular morphogenesis and is dependent on the matrix metalloproteinase, ADAMTS1. A substrate of ADAMTS1 is the proteoglycan versican which is expressed in the developing ventricle and which has been implicated in trabeculation. Fibulin-1 is a versican and ADAMTS1-binding extracellular matrix protein required for ventricular morphogenesis. Here we investigated the involvement of fibulin-1 in ADAMTS1-mediated cleavage of versican in vitro, and the involvement of fibulin-1 in versican cleavage in ventricular morphogenesis., Results: We show that fibulin-1 is a cofactor for ADAMTS1-dependent in vitro cleavage of versican V1, yielding a 70-kDa amino-terminal fragment. Furthermore, fibulin-1-deficiency in mice was found to cause a significant reduction (>90%) in ventricular levels of the 70-kDa versican V1 cleavage product and a 2-fold increase in trabecular cardiomyocyte proliferation. Decreased versican V1 cleavage and augmented trabecular cardiomyocyte proliferation in fibulin-1 null hearts is accompanied by increased ventricular activation of ErbB2 and Erk1/2. By contrast, versican deficiency was found to lead to decreased cardiomyocyte proliferation and reduced ventricular trabeculation., Conclusion: We conclude that fibulin-1 regulates versican-dependent events in ventricular morphogenesis by promoting ADAMTS1 cleavage of versican leading to suppression of trabecular cardiomyocyte proliferation mediated by the ErbB2-Map kinase pathway., (Copyright © 2011 Wiley Periodicals, Inc.)

Normal heart formation requires reiterative phases of canonical Wnt/β-catenin (Wnt) signaling. Understanding the mechanisms by which Wnt signaling directs cardiomyocyte (CM) formation in vivo is critical to being able to precisely direct differentiated CMs from stem cells in vitro. Here, we investigate the roles of Wnt signaling in zebrafish CM formation using heat-shock inducible transgenes that increase and decrease Wnt signaling. We find that there are three phases during which CM formation is sensitive to modulation of Wnt signaling through the first 24 h of development. In addition to the previously recognized roles for Wnt signaling during mesoderm specification and in the pre-cardiac mesoderm, we find a previously unrecognized role during CM differentiation where Wnt signaling is necessary and sufficient to promote the differentiation of additional atrial cells. We also extend the previous studies of the roles of Wnt signaling during mesoderm specification and in pre-cardiac mesoderm. Importantly, in pre-cardiac mesoderm we define a new mechanism where Wnt signaling is sufficient to prevent CM differentiation, in contrast to a proposed role in inhibiting cardiac progenitor (CP) specification. The inability of the CPs to differentiate appears to lead to cell death through a p53/Caspase-3 independent mechanism. Together with a report for an even later role for Wnt signaling in restricting proliferation of differentiated ventricular CMs, our results indicate that during the first 3days of development in zebrafish there are four distinct phases during which CMs are sensitive to Wnt signaling., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs) are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the pan-retinoic acid receptor antagonist BMS-189453 (RAi) significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin+RAi-treated cultures with Noggin+RA-treated cultures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin+RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin+RAi-treated cultures, but not in the cardiomyocytes of Noggin+RA-treated cultures. Flow cytometry analysis and electrophysiological studies indicated that with 64.7 ± 0.88% (mean ±s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (APs). With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin+RA-treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca(2+) sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial- and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin and retinoid signals.

There is scant information on the fate of cardiac progenitor cells (CPC) in the embryonic heart after chamber specification. Here we simultaneously tracked three lineage-specific markers (Nkx2.5, MLC2v, and ANF) and confirmed that CPCs with an Nkx2.5+MLC2v-ANF- phenotype are present in the embryonic (E) day 11.5 mouse ventricular myocardium. We demonstrated that these CPCs could give rise to working cardiomyocytes and conduction system cells. Using a two-photon imaging analysis, we found that the majority of CPCs are not capable of developing Ca2+ transients in response to beta-adrenergic receptor stimulation. In contrast, Nkx2.5+ cells expressing MLC2v but not ANF are capable of developing functional Ca2+ transients. We showed that Ca2+ transients could be invoked in Nkx2.5+MLC2v+ANF+ cells only upon inhibition of Gi, muscarinic receptors, or nitric oxide synthase (NOS) signaling pathways. Our data suggest that these inhibitory pathways may delay functional specification in a subset of developing ventricular cells., (Copyright 2009 Wiley-Liss, Inc.)

The mammalian heart is formed from distinct sets of first and second heart field (FHF and SHF, respectively) progenitors. Although multipotent progenitors have previously been shown to give rise to cardiomyocytes, smooth muscle, and endothelial cells, the mechanism governing the generation of large numbers of differentiated progeny remains poorly understood. We have employed a two-colored fluorescent reporter system to isolate FHF and SHF progenitors from developing mouse embryos and embryonic stem cells. Genome-wide profiling of coding and noncoding transcripts revealed distinct molecular signatures of these progenitor populations. We further identify a committed ventricular progenitor cell in the Islet 1 lineage that is capable of limited in vitro expansion, differentiation, and assembly into functional ventricular muscle tissue, representing a combination of tissue engineering and stem cell biology.

Rationale: T-box transcription factors play critical roles in the coordinated formation of the working chambers and the atrioventricular canal (AVC). Tbx2 patterns embryonic myocardial cells to form the AVC and suppresses their differentiation into chamber myocardium. Tbx20-deficient embryos, which fail to form chambers, ectopically express Tbx2 throughout the entire heart tube, providing a potential mechanism for the function of Tbx20 in chamber differentiation., Objective: To identify the mechanism of Tbx2 suppression by Tbx20 and to investigate the involvement of Tbx2 in Tbx20-mediated chamber formation., Methods and Results: We generated Tbx20 and Tbx2 single and double knockout embryos and observed that loss of Tbx2 did not rescue the Tbx20-deficient heart from failure to form chambers. However, Tbx20 is required to suppress Tbx2 in the developing chambers, a prerequisite to localize its strong differentiation-inhibiting activity to the AVC. We identified a bone morphogenetic protein (Bmp)/Smad-dependent Tbx2 enhancer conferring AVC-restricted expression and Tbx20-dependent chamber suppression of Tbx2 in vivo. Unexpectedly, we found in transfection and localization studies in vitro that both Tbx20 and mutant isoforms of Tbx20 unable to bind DNA attenuate Bmp/Smad-dependent activation of Tbx2 by binding Smad1 and Smad5 and sequestering them from Smad4., Conclusions: Our data suggest that Tbx20 directly interferes with Bmp/Smad signaling to suppress Tbx2 expression in the chambers, thereby confining Tbx2 expression to the prospective AVC region.

Over the past decade, much has been learned concerning the origin and development of the ventricles. However, most, if not all, of the new information has come from study of the mouse heart. Most of this information has yet to be assimilated by those who study ventricular function or diagnose congenitally malformed hearts. Nevertheless, the evidence available from recent studies, particularly if it can be shown relevant to human development, is remarkably pertinent to these topics. For example, knowledge of how each ventricle derives its inlet and outlet components, information available for human development (Lamers et al., Circulation 86:1194-1205, 1992), provides a firm foundation for understanding congenital cardiac malformations, particularly those dependent on a functionally univentricular circulation (Jacobs and Anderson, Cardiol Young 16(Suppl 1):3-8, 2006). Appreciation of ventricle development also is important with regard to understanding the basis of so-called ventricular noncompaction because this knowledge will elucidate whether the compact component of the ventricular walls is produced by consolidation of the initially extensive trabecular zone seen during early development or by defective formation and/or maturation of the compact myocardium (Anderson, Eur Heart J 29:10-11, 2008). Knowledge concerning the mechanism whereby ventricular myocytes are packed within the compact component of the ventricular walls then will help clarify the architectural arrangement of the aggregated myocytes, a topic of considerable recent interest. This review discusses all these topics.

Calcium homeostasis is critical for cardiac myocyte function and must be tightly regulated. The guiding hypothesis of this study is that a carboxyl-terminal cleavage product of the cardiac L-type calcium channel (Ca(V)1.2) autoregulates expression. First, we confirmed that the Ca(V)1.2 C terminus (CCt) is cleaved in murine cardiac myocytes from mature and developing ventricle. Overexpression of full-length CCt caused a 34+/-8% decrease of Ca(V)1.2 promoter activity, and truncated CCt caused an 80+/-3% decrease of Ca(V)1.2 promoter (n=12). The full-length CCt distributes into cytosol and nucleus. A deletion mutant of CCt has a greater relative affinity for the nucleus than full-length CCt, and this is consistent with increased repression of Ca(V)1.2 promoter activity by truncated CCt. Chromatin immunoprecipitation analysis revealed that CCt interacts with the Ca(V)1.2 promoter in adult ventricular cardiac myocytes at promoter modules containing Nkx2.5/Mef2, C/EBp, and a cis regulatory module. The next hypothesis tested was that CCt contributes to transcriptional signaling associated with cellular hypertrophy. We explored whether fetal cardiac myocyte Ca(V)1.2 was regulated by serum in vitro. We tested atrial natriuretic factor promoter activity as a positive control and measured the serum response of Ca(V)1.2 promoter, protein, and L-type current (I(Ca,L)) from fetal mouse ventricular myocytes. Serum increased atrial natriuretic factor promoter activity and cell size as expected. Serum withdrawal increased Ca(V)1.2 promoter activity, mRNA, and I(Ca,L). Moreover, serum withdrawal decreased the relative nuclear localization of CCt. A combination of promoter deletion mutant analyses, and the response of promoter mutants to serum withdrawal support the conclusion that CCt, a proteolytic fragment of Ca(V)1.2, autoregulates Ca(V)1.2 expression in cardiac myocytes. These data support the novel mechanism that a mobile segment of Ca(V)1.2 links Ca handling to nuclear signaling.