Embryonic Mouse Heart Research Articles

Front Cover: Speckle variance optical coherence tomography of blood flow in the beating mouse embryonic heart (J. Biophotonics 5/2017)

Front Cover: Speckle variance optical coherence tomography of blood flow in the beating mouse embryonic heart (J. Biophotonics 5/2017)

Speckle variance optical coherence tomography of blood flow in the beating mouse embryonic heart.

Efficient separation of blood and cardiac wall in the beating embryonic heart is essential and critical for experiment-based computational modelling and analysis of early-stage cardiac biomechanics. Although speckle variance optical coherence tomography (SV-OCT) relying on calculation of intensity variance over consecutively acquired frames is a powerful approach for segmentation of fluid flow from static tissue, application of this method in the beating embryonic heart remains challenging because moving structures generate SV signal indistinguishable from the blood. Here, we demonstrate a modified four-dimensional SV-OCT approach that effectively separates the blood flow from the dynamic heart wall in the beating mouse embryonic heart. The method takes advantage of the periodic motion of the cardiac wall and is based on calculation of the SV signal over the frames corresponding to the same phase of the heartbeat cycle. Through comparison with Doppler OCT imaging, we validate this speckle-based approach and show advantages in its insensitiveness to the flow direction and velocity as well as reduced influence from the heart wall movement. This approach has a potential in variety of applications relying on visualization and segmentation of blood flow in periodically moving structures, such as mechanical simulation studies and finite element modelling. Picture: Four-dimensional speckle variance OCT imaging shows the blood flow inside the beating heart of an E8.5 mouse embryo.

Assessment of Pep, Fndc5, C2c12, Pgc1α Pattern of Genes Expression during Differentiation of Human Embryonic Stem Cells into Heart Cells

FNDC5 gene with another protein called Xuemei Proxy (PEP) is a 209 amino acid protein coding. These genes mainly in heart tissue, skeletal muscle and brain expressed. This study aimed to clarify the pattern of expression of this gene in mouse embryonic cells Heart cells taken. The mouse embryonic stem cells as a model for cardiac differentiation induced by ascorbic acid used and the pattern of expression of PEP at certain stages of differentiation were analyzed by Real-Time PCR technique. The results show a dramatic increase in PEP gene expression in the adult cardiomyocytes. PEP increased expression of genes may have a role in later stages cardiogenesis is possible that further studies are needed to identify it.

Bioinformatics characteristics of lncRNA-uc.167 and its temporal and spatial expression pattern for mouse embryonic development

Objective To explore the basic biological characteristics of lncRNA-uc.167, and its spatial distribution, temporal expression pattern during the mouse embryonic development. Methods The UCSC genome browser of ENCODE was used to analyze preliminary bioinformatics of lncRNAs.Real-time (RT)-PCR was applied to detect the expression of uc.167 and neighboring genes in the embryonic mouse heart in different stages (P7.5, P11.5, P14.5, P18.5). Dimethyl sulphoxide was used to induce P19 cell differentiation into the cardiomyocytes.RT-PCR was applied to detect the expression changes in uc.167 and neighboring genes on differential day 0, 4, 6, 8 and 10. Results Full-length of human uc.167 was 201 bp, and human uc.167 was located in the genome 5q14.3 (chr5: 88179623-88179824, GRCh37/hg19). uc.167 mainly expressed in the ventricular muscle tissue.The expression of uc.167 was gradually decreased in the mouse embryonic heart development process(P7.5: 1.000±0.100, P11.5: 0.714±0.107, P14.5: 0.393±0.043, P18.5: 0.125±0.013), while the expression of its neighboring Mef2c gene was gradually increased(P7.5: 1.081±0.118, P11.5: 2.340±0.351, P14.5: 3.958±0.542, P18.5: 9.361±0.722), which showed an opposite trend.The expression of uc.167 during P19 cell differentiation into cardiomyocytes showed a an increase at first and then a decrease pattern, and the highest level expression of uc.167 was on differential day 4(d0: 1.071±0.117, d4: 4.714±0.501, d6: 3.572±0.414, d8: 2.550±0.314, d10: 0.786±0.085). The expression of neighboring gene Mef2c was in an opposite trend(d0: 1.012±0.041, d4: 0.353±0.037, d6: 2.470±0.329, d8: 6.706±0.682, d10: 7.765±0.705). Conclusions It is suggested that uc.167 may take part in the process of embryonic heart development and may play a role through negatively regulating its neighboring gene Mef2c. Key words: Bioinformatics analysis; Cardiac development; LncRNA

Tbx20 Is an Essential Regulator of Embryonic Heart Growth in Zebrafish.

The molecular mechanisms that regulate cardiomyocyte proliferation during embryonic heart growth are not completely deciphered yet. In a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified the recessive embryonic-lethal zebrafish mutant line weiches herz (whz). Homozygous mutant whz embryos display impaired heart growth due to diminished embryonic cardiomyocyte proliferation resulting in cardiac hypoplasia and weak cardiac contraction. By positional cloning, we found in whz mutant zebrafish a missense mutation within the T-box 20 (Tbx20) transcription factor gene leading to destabilization of Tbx20 protein. Morpholino-mediated knock-down of Tbx20 in wild-type zebrafish embryos phenocopies whz, indicating that the whz phenotype is due to loss of Tbx20 function, thereby leading to significantly reduced cardiomyocyte numbers by impaired proliferation of heart muscle cells. Ectopic overexpression of wild-type Tbx20 in whz mutant embryos restored cardiomyocyte proliferation and heart growth. Interestingly, ectopic overexpression of Tbx20 in wild-type zebrafish embryos resulted, similar to the situation in the embryonic mouse heart, in significantly reduced proliferation rates of ventricular cardiomyocytes, suggesting that Tbx20 activity needs to be tightly fine-tuned to guarantee regular cardiomyocyte proliferation and embryonic heart growth in vivo.

CITED2 Cooperates with ISL1 and Promotes Cardiac Differentiation of Mouse Embryonic Stem Cells.

SummaryThe transcriptional regulator CITED2 is essential for heart development. Here, we investigated the role of CITED2 in the specification of cardiac cell fate from mouse embryonic stem cells (ESC). The overexpression of CITED2 in undifferentiated ESC was sufficient to promote cardiac cell emergence upon differentiation. Conversely, the depletion of Cited2 at the onset of differentiation resulted in a decline of ESC ability to generate cardiac cells. Moreover, loss of Cited2 expression impairs the expression of early mesoderm markers and cardiogenic transcription factors (Isl1, Gata4, Tbx5). The cardiogenic defects in Cited2-depleted cells were rescued by treatment with recombinant CITED2 protein. We showed that Cited2 expression is enriched in cardiac progenitors either derived from ESC or mouse embryonic hearts. Finally, we demonstrated that CITED2 and ISL1 proteins interact physically and cooperate to promote ESC differentiation toward cardiomyocytes. Collectively, our results show that Cited2 plays a pivotal role in cardiac commitment of ESC.

Back Cover: Four‐dimensional live imaging of hemodynamics in mammalian embryonic heart with Doppler optical coherence tomography (J. Biophotonics 8/2016)

Detailed volumetric measurement of hemodynamics in early developing mammalian heart can provide great insights for improved understanding of normal cardiogenesis and management of congenital cardiac disease. In this study, Doppler optical coherence tomography is performed in live mouse embryo culture to obtain the first four-dimensional high-resolution reconstruction and quantitative analysis of hemodynamic features in the mouse embryonic heart. This provides a powerful approach to investigate biomechanical regulation of early mammalian cardiogenesis. Further details can be found in the article by Shang Wang et al. on pp. 837–847.

Apigenin reduce lipoteichoic acid-induced inflammatory response in rat cardiomyoblast cells.

Infective endocarditis is caused by Streptococcus sanguinis present in dental plaque, which can induce inflammatory responses in the endocardium. The present study depicts research on the properties of apigenin in embryonic mouse heart cells (H9c2) treated with lipoteichoic acid (LTA) obtained from S.sanguinis. Interleukin-1β and cyclooxygenase (COX)-2 expression were detected by reverse transcriptase polymerase chain reaction. In addition, western blot assays and immuno-fluorescence staining were used to assess translocation of nuclear factor kappa beta (NF-κB), degradation of IκB, as well as activity of the mitogen activated protein kinases: extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). Effect of apigenin on cell viability was equally assessed in other experimental series. Our results showed that apigenin blocked activation of ERK, JNK, and p38 in cardiomyocytes treated with LTA in a dose-dependent fashion. Moreover, apigenin showed no cytotoxic effects; it blocked NF-κB translocation and IκB degradation. Our findings suggested that apigenin possessed potential value in the treatment of infectious endocarditis.

Comparison of Different Tissue Clearing Methods and 3D Imaging Techniques for Visualization of GFP‐expressing Mouse Embryos and Embryonic Hearts

AimsTo find a suitable tissue clearing protocol preserving GFP fluorescence for whole mount imaging of embryonic and adult hearts of transgenic mice.MethodsWe tested various published organic solvent‐based or water‐based clearing protocols claiming to preserve GFP fluorescence in central nervous system: tetrahydrofurane dehydration and dibenzyl ether protocol (DBE), SCALE, CLARITY, and CUBIC to evaluate their use for embryonic heart tissue. Tissue transparency and preservation of GFP fluorescence was evaluated in hearts of Cx40:GFP knock in mice at stages: ED10.5 – ED18.5, and adults. We also tested several imaging techniques and evaluated their use for embryonic hearts visualization. We compared an Upright Single Photon Confocal Microscope (Olympus) with 4×, 10×, and 25× objectives after mounting the specimens into cavity slides or custom imaging chambers with an Optical Projection Tomography (OPT, custom made) and with a Single Plane Ilumination Microscopy (SPIM) (constructed according to the OpenSPIM project).ResultsDespite careful control of all critical parameters, DBE clearing protocol did not preserve GFP fluorescence; in addition, it caused considerable tissue shrinking and deformation. The SCALE clearing resulted in good tissue transparency up to ED12.5; at later stages and in the adults the useful depth of imaging was limited by tissue light scattering. The CLARITY method considerably improved tissue transparency at later stages, but also decreased GFP fluorescence intensity. The CUBIC method resulted in very well cleared specimens even at the adult stages, and GFP fluorescence was also preserved. In addition, it decolorized the blood and myocardium by removing iron from the tissues. High‐resolution images were thus obtained even from deep tissue layers with a confocal microscope with a Scale View immersion 25× objective. Simultaneously, intact specimens were imaged with OPT and SPIM that have lower resolution, but are more suitable for observing large samples.ConclusionsFrom the tested methods, the CUBIC protocol turned out to be the best for whole mount GFP cardiac specimens. SCALE technique is also suitable for younger embryos. Confocal microscopy is needed for detailed view, while OPT and SPIM enable to observe larger samples. These protocols will be used for three dimensional reconstructions of normal and abnormal specimens to visualize the developing cardiac conduction system and coronary vasculature.Support or Funding InformationSupported by 13‐12412S from the Czech Science Foundation, Ministry of Education PRVOUK P35/LF1/5, institutional support RVO:67985823, and Charles University UNCE.

Cardiac fibroblast growth factor‐16 increases efflux drug (ABC) transporter production and resistance to doxorubicin‐induced cardiomyocyte death

Doxorubicin (DOX) is a cytotoxic anthracycline antibiotic that is widely used in cancer treatment. However, its cardiotoxic side effects can be fatal, and have long been an issue for cancer patients both in the short and longer term. Strategies are needed to protect the heart. Fibroblast growth factor (FGF) 16 plays an important role in embryonic mouse heart development. After birth, FGF‐16 is predominantly expressed in the myocardium, and is linked to both anti‐fibrotic and anti‐hypertrophic activities. Supplementation with FGF‐16 increases resistance to DOX‐induced loss of ventricular function in an isolated mouse heart model. However, there is no report on the effects of DOX on endogenous FGF‐16 levels or the mechanism(s) for cardioprotection by FGF‐16. ATP‐binding cassette (ABC) transporters are a family of ATP‐dependent membrane proteins that play an important role in drug distribution and excretion. ABC transporters work as pumps effluxing drugs out of the cell immediately after drug entry thereby reducing cellular drug concentration. Drug concentration positively determines the ABC transporter levels. Previously we showed that FGF‐16 can compete with FGF‐2 binding to the same FGF receptors (FGFRs). Unlike FGF‐16, FGF‐2 is not cardiac‐specific but is cardioprotective and known to induce p‐glycoprotein (MDRs) and multidrug resistance‐associated protein (MRPs) expression in neonatal rat cardiomyocytes. Our hypothesis is that FGF‐16 protects cardiomyocytes from DOX‐induced cell death by increasing the levels of ABC drug transporters. Our results show that 1 μM DOX induces a time‐dependent decrease in endogenous FGF‐16 RNA levels with a rapid 80% reduction by 2 hours in neonatal rat cardiomyocytes. Knockdown of endogenous FGF‐16 levels using siRNA results in a significant increase in apoptotic (Annexin‐V positive) and necrotic (LDH release) cells; this is consistent with endogenous FGF‐16 serving as a cardiac maintenance/survival factor. Overexpression of FGF‐16 using adenoviral delivery significantly and specifically increases efflux drug transporter Mdr1a levels in a dose‐dependent manner. Significant decrease in calcein fluorescence intensity in a functional analysis using calcein AM as a substrate for the efflux drug transporters was consistent with the increased levels of drug transporters RNA post FGF‐16 overexpression. In addition, FGF‐16 adenovirus overexpression in cardiomyocytes treated with 1μM DOX significantly decreased the intracellular DOX concentration as measured by DOX autofluorescence. This was associated with a significant reduction in the apoptotic and necrotic cell population, consistent with a decrease in DOX cytotoxicity. Furthermore, inhibition of FGFR signaling using 20 μM SU5402 blocked the increased efflux effect of the ABC transporters. In conclusion, FGF‐16 increases resistance to DOX‐induced injury by increasing efflux drug transporters via binding to the FGFRs. Understanding the effect of FGF‐16 on drug transporter production and function may offer a further target for intervention in the context of DOX and cardiotoxicity.Support or Funding InformationFunded by the Canadian Institutes of Health Research and Research Manitoba.

Four-dimensional live imaging of hemodynamics in mammalian embryonic heart with Doppler optical coherence tomography.

Hemodynamic analysis of the mouse embryonic heart is essential for understanding the functional aspects of early cardiogenesis and advancing the research in congenital heart defects. However, high-resolution imaging of cardiac hemodynamics in mammalian models remains challenging, primarily due to the dynamic nature and deep location of the embryonic heart. Here we report four-dimensional micro-scale imaging of blood flow in the early mouse embryonic heart, enabling time-resolved measurement and analysis of flow velocity throughout the heart tube. Our method uses Doppler optical coherence tomography in live mouse embryo culture, and employs a post-processing synchronization approach to reconstruct three-dimensional data over time at a 100 Hz volume rate. Experiments were performed on live mouse embryos at embryonic day 9.0. Our results show blood flow dynamics inside the beating heart, with the capability for quantitative flow velocity assessment in the primitive atrium, atrioventricular and bulboventricular regions, and bulbus cordis. Combined cardiodynamic and hemodynamic analysis indicates this functional imaging method can be utilized to further investigate the mechanical relationship between blood flow dynamics and cardiac wall movement, bringing new possibilities to study biomechanics in early mammalian cardiogenesis. Four-dimensional live hemodynamic imaging of the mouse embryonic heart at embryonic day 9.0 using Doppler optical coherence tomography, showing directional blood flows in the sinus venosus, primitive atrium, atrioventricular region and vitelline vein.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration.

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Bioinformatic and Expression Analysis of Ventricular Septal Defect-associated Long Non-coding RNA TUC40-.

To explore the potential role of TUC40- in human and mouse embryonic heart development. Bioinformatics databases including NCBI,UCSC,and Uniprot and software including Clustal,DNAMAN,and MEGA 6 were used to collect information of TUC40- and uc.40-. The expression profile at key time points of heart development was investigated by strand-specific quantitative real time polymerase chain reaction. Uc.40- was conservative in sequence, genomic location, and transcription factor binding sites across human and mouse. Pbx1/TUC40- showed negative trend during embryonic mouse heart maturation. Various levels of conservation of uc.40- suggests similar functions of TUC40- in these two species. TUC40- may play its roles in human and mouse embryonic heart development by regulating Pbx1.

The Mitochondrial Permeability Transition Pore and ATP Synthase.

Mitochondrial ATP generation by oxidative phosphorylation combines the stepwise oxidation by the electron transport chain (ETC) of the reducing equivalents NADH and FADH2 with the generation of ATP by the ATP synthase. Recent studies show that the ATP synthase is not only essential for the generation of ATP but may also contribute to the formation of the mitochondrial permeability transition pore (PTP). We present a model, in which the PTP is located within the c-subunit ring in the Fo subunit of the ATP synthase. Opening of the PTP was long associated with uncoupling of the ETC and the initiation of programmed cell death. More recently, it was shown that PTP opening may serve a physiologic role: it can transiently open to regulate mitochondrial signaling in mature cells, and it is open in the embryonic mouse heart. This review will discuss how the ATP synthase paradoxically lies at the center of both ATP generation and cell death.

Mapping transcriptome profiles of in vitro iPSC-derived cardiac differentiation to in utero heart development.

The dataset includes microarray data (Affymetrix Mouse Genome 430 2.0 Array) from WT and Nos3−/− mouse embryonic heart ventricular tissues at 14.5 days post coitum (E14.5), induced pluripotent stem cells (iPSCs) derived from WT and Nos3−/− mouse tail tip fibroblasts, iPSC-differentiated cardiomyocytes at Day 11, and mouse embryonic stem cells (mESCs) and differentiated cardiomyocytes as positive controls for mouse iPSC differentiation. Both in utero (using embryonic heart tissues) and in vitro (using iPSCs and differentiated cells) microarray datasets were deposited to the NCBI Gene Expression Omnibus (GEO) database. The deposited data in GEO include raw microarray data, metadata for sample source information, experimental design, sample and data processing, and gene expression matrix. The data are available under GEO Access Number GSE69317 (GSE69315 for tissue sample microarray data, GSE69316 for iPSCs microarray data, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE69317).

Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

RationaleCentral 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 ResultsIn 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.ConclusionHere, 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.

Abstract 15317: Deletion of Low Molecular Weight Protein Tyrosine Phosphatase (Acp1) Protects Against Stress-induced Cardiomyopathy

Background: The low molecular weight protein tyrosine phosphatase (LMPTP), encoded by the ACP1 gene, is a ubiquitously expressed phosphatase whose in vivo function remains poorly understood. Genetic studies revealed an association between ACP1 and a variety of human cardiovascular disorders. However, evidence of a direct involvement of LMPTP in cardiac diseases is still lacking. Methodology: We generated the first mouse model of Lmptp deficiency and studied the response of the mice under basal condition or to long-term pressure overload using comprehensive experimental approaches such as q-PCR, siRNA technology, fluorescent microscopy and immuneprecipitation technique. Results: Acp1 -/- mice develop normally, and aging mice do not show any obvious signs of pathology in major tissues under basal condition. However, Acp1 -/- mice are strikingly resistant to pressure overload hypertrophy and heart failure. Lmptp expression is high in the embryonic mouse heart, decreased in the post-natal stage, and increased in the adult mouse and human failing heart. Consistent with their protected phenotype, Acp1 -/- mice subjected to pressure overload hypertrophy have attenuated fibrosis and decreased expression of hypertrophic markers. Transcriptional profiling and analysis of molecular signaling shows that the resistance of Acp1 -/- mice to pathological cardiac stress correlates with marginal expression of fetal cardiac genes, increased insulin receptor beta phosphorylation and inactivation of CaMKIIδ pathway. Conclusion: Our data show that ablation of Lmptp inhibits pathological cardiac remodeling and suggest that inhibition of LMPTP may be of therapeutic relevance for the treatment of heart failure in human.

Cis-Regulatory control of Mesp1 expression by YY1 and SP1 during mouse embryogenesis.

Mesp1 is critical for early cardiomyocyte differentiation and heart development. We previously observed down-regulation of Mesp1 expression in YY1-ablated mouse embryonic hearts. However, how Mesp1 expression is mediated by YY1 is not well understood. We excised YY1 in the murine embryos using Sox2-cre and found that Mesp1 was down-regulated in the embryonic day (E) 7.5 mutant embryos. Also, YY1 activated the 6 kb Mesp1 regulatory element fused to a luciferase reporter. We identified two putative YY1 binding sites in the proximal promoter region of Mesp1 gene, and found that mutation of these sites significantly reduced YY1-induced activation of the Mesp1 promoter. We also uncovered one cognitive site for SP1, one of the earliest binding partners of YY1 identified. Mutation of this SP1 site repressed SP1-induced activation of the Mesp1 promoter. Moreover, YY1 and SP1 synergistically activated the Mesp1 promoter. Consistently, while Lacz expression driven by the wild-type 6 kb regulatory element of Mesp1 gene was robust in E7.5 mouse embryos, the mutation of these binding sites in the context of this 6 kb sequence substantially reduced the LacZ expression during embryogenesis. YY1 and SP1 independently and cooperatively govern the Mesp1 expression during embryogenesis.

Deletion of low molecular weight protein tyrosine phosphatase (Acp1) protects against stress-induced cardiomyopathy.

The low molecular weight protein tyrosine phosphatase (LMPTP), encoded by the ACP1 gene, is a ubiquitously expressed phosphatase whose in vivo function in the heart and in cardiac diseases remains unknown. To investigate the in vivo role of LMPTP in cardiac function, we generated mice with genetic inactivation of the Acp1 locus and studied their response to long‐term pressure overload. Acp1−/− mice develop normally and ageing mice do not show pathology in major tissues under basal conditions. However, Acp1−/− mice are strikingly resistant to pressure overload hypertrophy and heart failure. Lmptp expression is high in the embryonic mouse heart, decreased in the postnatal stage, and increased in the adult mouse failing heart. We also show that LMPTP expression increases in end‐stage heart failure in humans. Consistent with their protected phenotype, Acp1−/− mice subjected to pressure overload hypertrophy have attenuated fibrosis and decreased expression of fibrotic genes. Transcriptional profiling and analysis of molecular signalling show that the resistance of Acp1−/− mice to pathological cardiac stress correlates with marginal re‐expression of fetal cardiac genes, increased insulin receptor beta phosphorylation, as well as PKA and ephrin receptor expression, and inactivation of the CaMKIIδ pathway. Our data show that ablation of Lmptp inhibits pathological cardiac remodelling and suggest that inhibition of LMPTP may be of therapeutic relevance for the treatment of human heart failure. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes.

In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.