Disclosing quantitative RT-PCR raw data during manuscript submission: a call for action.

Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.

Familial multiple discoid fibromas is linked to a locus on chromosome 5 including the FNIP1 gene.

Previously, we reported a series of families presenting with trichodiscomas, inherited in an autosomal dominant pattern. The phenotype was named familial multiple discoid fibromas (FMDF). The genetic cause of FMDF remained unknown so far. Trichodiscomas are skin lesions previously reported to be part of the same spectrum as the fibrofolliculoma observed in Birt-Hogg-Dubé syndrome (BHD), an inherited disease caused by pathogenic variants in the FLCN gene. Given the clinical and histological differences with BHD and the exclusion of linkage with the FLCN locus, the phenotype was concluded to be distinct from BHD. We performed extensive clinical evaluations and genetic testing in ten families with FMDF. We identified a FNIP1 frameshift variant in nine families and genealogical studies showed common ancestry for eight families. Using whole exome sequencing, we identified six additional rare variants in the haplotype surrounding FNIP1, including a missense variant in the PDGFRB gene that was found to be present in all tested patients with FMDF. Genome-wide linkage analysis showed that the locus on chromosome 5 including FNIP1 was the only region reaching the maximal possible LOD score. We concluded that FMDF is linked to a haplotype on chromosome 5. Additional evaluations in families with FMDF are required to unravel the exact genetic cause underlying the phenotype. When evaluating patients with multiple trichodisomas without a pathogenic variant in the FLCN gene, further genetic testing is warranted and can include analysis of the haplotype on chromosome 5.

Role of the Epicardium in the Development of the Atrioventricular Valves and Its Relevance to the Pathogenesis of Myxomatous Valve Disease.

This paper is dedicated to the memory of Dr. Adriana "Adri" Gittenberger-de Groot and in appreciation of her work in the field of developmental cardiovascular biology and the legacy that she has left behind. During her impressive career, Dr. Gittenberger-de Groot studied many aspects of heart development, including aspects of cardiac valve formation and disease and the role of the epicardium in the formation of the heart. In this contribution, we review some of the work on the role of epicardially-derived cells (EPDCs) in the development of the atrioventricular valves and their potential involvement in the pathogenesis of myxomatous valve disease (MVD). We provide an overview of critical events in the development of the atrioventricular junction, discuss the role of the epicardium in these events, and illustrate how interfering with molecular mechanisms that are involved in the epicardial-dependent formation of the atrioventricular junction leads to a number of abnormalities. These abnormalities include defects of the AV valves that resemble those observed in humans that suffer from MVD. The studies demonstrate the importance of the epicardium for the proper formation and maturation of the AV valves and show that the possibility of epicardial-associated developmental defects should be taken into consideration when determining the genetic origin and pathogenesis of MVD.

Identifying pathogenic variants in the Follistatin-like 1 gene (FSTL1) in patients with skeletal and atrioventricular valve disorders.

BACKGROUND: Follistatin-like 1 (Fstl1) is a glycoprotein expressed throughout embryonic development. Homozygous loss of Fstl1 in mice results in skeletal and respiratory defects, leading to neonatal death due to a collapse of the trachea. Furthermore, Fstl1 conditional deletion from the endocardial/endothelial lineage results in postnatal death due to heart failure and profound atrioventricular valve defects. Here, we investigated patients with phenotypes similar to the phenotypes observed in the transgenic mice, for variants in FSTL1. METHODS: In total, 69 genetically unresolved patients were selected with the following phenotypes: campomelic dysplasia (12), small patella syndrome (2), BILU (1), and congenital heart disease patients (54), of which 16 also had kyphoscoliosis, and 38 had valve abnormalities as their main diagnosis. Using qPCR, none of 69 patients showed copy number variations in FSTL1. The entire gene body, including microRNA-198 and three validated microRNA-binding sites, were analyzed using Sanger sequencing. RESULTS: No variants were found in the coding region. However, 8 intronic variants were identified that differed significantly in their minor allele frequency compared to controls. Variant rs2272515 was found to significantly correlate (p < 0.05) with kyphoscoliosis. CONCLUSION: We conclude that pathogenic variants in FSTL1 are unlikely to be responsible for skeletal or atrioventricular valve anomalies in humans.

Follistatin-like 1 in development and human diseases.

Follistatin-like 1 (FSTL1) is a secreted glycoprotein displaying expression changes during development and disease, among which cardiovascular disease, cancer, and arthritis. The cardioprotective role of FSTL1 has been intensively studied over the last years, though its mechanism of action remains elusive. FSTL1 is involved in multiple signaling pathways and biological processes, including vascularization and regulation of the immune response, a feature that complicates its study. Binding to the DIP2A, TLR4 and BMP receptors have been shown, but other molecular partners probably exist. During cancer progression and rheumatoid arthritis, controversial data have been reported with respect to the proliferative, apoptotic, migratory, and inflammatory effects of FSTL1. This controversy might reside in the extensive post-transcriptional regulation of FSTL1. The FSTL1 primary transcript also encodes for a microRNA (miR-198) in primates and multiple microRNA-binding sites are present in the 3'UTR. The switch between expression of the FSTL1 protein and miR-198 is an important regulator of tumour metastasis and wound healing. The glycosylation state of FSTL1 is a determinant of biological activity, in cardiomyocytes the glycosylated form promoting proliferation and the non-glycosylated working anti-apoptotic. Moreover, the glycosylation state shows differences between species and tissues which might underlie the differences observed in in vitro studies. Finally, regulation at the level of protein secretion has been described.

Single-site neural tube closure in human embryos revisited.

Since the multi-site closure theory was first proposed in 1991 as explanation for the preferential localizations of neural tube defects, the closure of the neural tube has been debated. Although the multi-site closure theory is much cited in clinical literature, single-site closure is most apparent in literature concerning embryology. Inspired by Victor Hamburgers (1900-2001) statement that "our real teacher has been and still is the embryo, who is, incidentally, the only teacher who is always right", we decided to critically review both theories of neural tube closure. To verify the theories of closure, we studied serial histological sections of 10 mouse embryos between 8.5 and 9.5 days of gestation and 18 human embryos of the Carnegie collection between Carnegie stage 9 (19-21 days) and 13 (28-32 days). Neural tube closure was histologically defined by the neuroepithelial remodeling of the two adjoining neural fold tips in the midline. We did not observe multiple fusion sites in neither mouse nor human embryos. A meta-analysis of case reports on neural tube defects showed that defects can occur at any level of the neural axis. Our data indicate that the human neural tube fuses at a single site and, therefore, we propose to reinstate the single-site closure theory for neural tube closure. We showed that neural tube defects are not restricted to a specific location, thereby refuting the reasoning underlying the multi-site closure theory. Clin. Anat. 30:988-999, 2017. © 2017 Wiley Periodicals, Inc.

Deletion of Fstl1 (Follistatin-Like 1) From the Endocardial/Endothelial Lineage Causes Mitral Valve Disease.

OBJECTIVE: Fstl1 (Follistatin-like 1) is a secreted protein that is expressed in the atrioventricular valves throughout embryonic development, postnatal maturation, and adulthood. In this study, we investigated the loss of Fstl1 in the endocardium/endothelium and their derived cells. APPROACH AND RESULTS: We conditionally ablated Fstl1 from the endocardial lineage using a transgenic Tie2-Cre mouse model. These mice showed a sustained Bmp and Tgfß signaling after birth. This resulted in ongoing proliferation and endocardial-to-mesenchymal transition and ultimately in deformed nonfunctional mitral valves and a hypertrophic dilated heart. Echocardiographic and electrocardiographic analyses revealed that loss of Fstl1 leads to mitral regurgitation and left ventricular diastolic dysfunction. Cardiac function gradually deteriorated resulting in heart failure with preserved ejection fraction and death of the mice between 2 and 4 weeks after birth. CONCLUSIONS: We report on a mouse model in which deletion of Fstl1 from the endocardial/endothelial lineage results in deformed mitral valves, which cause regurgitation, heart failure, and early cardiac death. The findings provide a potential molecular target for the clinical research into myxomatous mitral valve disease.

Resveratrol Inhibits Aortic Root Dilatation in the Fbn1C1039G/+ Marfan Mouse Model.

OBJECTIVE: Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the fibrillin-1 gene. Patients with MFS are at risk of aortic aneurysm formation and dissection. Usually, blood pressure-lowering drugs are used to reduce aortic events; however, this is not sufficient for most patients. In the aorta of smooth muscle cell-specific sirtuin-1-deficient mice, spontaneous aneurysm formation and senescence are observed. Resveratrol is known to enhance sirtuin-1 activity and to reduce senescence, which prompted us to investigate the effectiveness of resveratrol in inhibition of aortic dilatation in the Fbn1(C1039G/+) MFS mouse model. APPROACH AND RESULTS: Aortic senescence strongly correlates with aortic root dilatation rate in MFS mice. However, although resveratrol inhibits aortic dilatation, it only shows a trend toward reduced aortic senescence. Resveratrol enhances nuclear localization of sirtuin-1 in the vessel wall and, in contrast to losartan, does not affect leukocyte infiltration nor activation of SMAD2 and extracellular signal-regulated kinases 1/2 (ERK1/2). Interestingly, specific sirtuin-1 activation (SRT1720) or inhibition (sirtinol) in MFS mice does not affect aortic root dilatation rate, although senescence is changed. Resveratrol reduces aortic elastin breaks and decreases micro-RNA-29b expression coinciding with enhanced antiapoptotic Bcl-2 expression and decreased number of terminal apoptotic cells. In cultured smooth muscle cells, the resveratrol effect on micro-RNA-29b downregulation is endothelial cell and nuclear factor κB-dependent. CONCLUSIONS: Resveratrol inhibits aortic root dilatation in MFS mice by promoting elastin integrity and smooth muscle cell survival, involving downregulation of the aneurysm-related micro-RNA-29b in the aorta. On the basis of these data, resveratrol holds promise as a novel intervention strategy for patients with MFS.

Congenital coronary artery anomalies: a bridge from embryology to anatomy and pathophysiology--a position statement of the development, anatomy, and pathology ESC Working Group.

Congenital coronary artery anomalies are of major significance in clinical cardiology and cardiac surgery due to their association with myocardial ischaemia and sudden death. Such anomalies are detectable by imaging modalities and, according to various definitions, their prevalence ranges from 0.21 to 5.79%. This consensus document from the Development, Anatomy and Pathology Working Group of the European Society of Cardiology aims to provide: (i) a definition of normality that refers to essential anatomical and embryological features of coronary vessels, based on the integrated analysis of studies of normal and abnormal coronary embryogenesis and pathophysiology; (ii) an animal model-based systematic survey of the molecular and cellular mechanisms that regulate coronary blood vessel development; (iii) an organization of the wide spectrum of coronary artery anomalies, according to a comprehensive anatomical and embryological classification scheme; (iv) current knowledge of the pathophysiological mechanisms underlying symptoms and signs of coronary artery anomalies, with diagnostic and therapeutic implications. This document identifies the mosaic-like embryonic development of the coronary vascular system, as coronary cell types differentiate from multiple cell sources through an intricate network of molecular signals and haemodynamic cues, as the necessary framework for understanding the complex spectrum of coronary artery anomalies observed in human patients.

Cardiac myocyte-derived follistatin-like 1 prevents renal injury in a subtotal nephrectomy model.

Heart disease contributes to the progression of CKD. Heart tissue produces a number of secreted proteins, also known as cardiokines, which participate in intercellular and intertissue communication. We recently reported that follistatin-like 1 (Fstl1) functions as a cardiokine with cardioprotective properties. Here, we investigated the role of cardiac Fstl1 in renal injury after subtotal nephrectomy. Cardiac-specific Fstl1-deficient (cFstl1-KO) mice and wild-type mice were subjected to subtotal (5/6) nephrectomy. cFstl1-KO mice showed exacerbation of urinary albumin excretion, glomerular hypertrophy, and tubulointerstitial fibrosis after subtotal renal ablation compared with wild-type mice. cFstl1-KO mice also exhibited increased mRNA levels of proinflammatory cytokines, including TNF-α and IL-6, NADPH oxidase components, and fibrotic mediators, in the remnant kidney. Conversely, systemic administration of adenoviral vectors expressing Fstl1 (Ad-Fstl1) to wild-type mice with subtotal nephrectomy led to amelioration of albuminuria, glomerular hypertrophy, and tubulointerstitial fibrosis, accompanied by reduced expression of proinflammatory mediators, NADPH oxidase components, and fibrotic markers in the remnant kidney. In cultured human mesangial cells, treatment with recombinant FSTL1 attenuated TNF-α-stimulated expression of proinflammatory cytokines. Treatment of mesangial cells with FSTL1 augmented the phosphorylation of AMP-activated protein kinase (AMPK), and inhibition of AMPK activation abrogated the anti-inflammatory effects of FSTL1. These data suggest that Fstl1 functions in cardiorenal communication and that the lack of Fstl1 production by myocytes promotes glomerular and tubulointerstitial damage in the kidney.

Alk3 mediated Bmp signaling controls the contribution of epicardially derived cells to the tissues of the atrioventricular junction.

Recent studies using mouse models for cell fate tracing of epicardial derived cells (EPDCs) have demonstrated that at the atrioventricular (AV) junction EPDCs contribute to the mesenchyme of the AV sulcus, the annulus fibrosus, and the parietal leaflets of the AV valves. There is little insight, however, into the mechanisms that govern the contribution of EPDCs to these tissues. While it has been demonstrated that bone morphogenetic protein (Bmp) signaling is required for AV cushion formation, its role in regulating EPDC contribution to the AV junction remains unexplored. To determine the role of Bmp signaling in the contribution of EPDCs to the AV junction, the Bmp receptor activin-like kinase 3 (Alk3; or Bmpr1a) was conditionally deleted in the epicardium and EPDCs using the mWt1/IRES/GFP-Cre (Wt1(Cre)) mouse. Embryonic Wt1(Cre);Alk3(fl/fl) specimens showed a significantly smaller AV sulcus and a severely underdeveloped annulus fibrosus. Electrophysiological analysis of adult Wt1(Cre);Alk3(fl/fl) mice showed, unexpectedly, no ventricular pre-excitation. Cell fate tracing revealed a significant decrease in the number of EPDCs within the parietal leaflets of the AV valves. Postnatal Wt1(Cre);Alk3(fl/fl) specimens showed myxomatous changes in the leaflets of the mitral valve. Together these observations indicate that Alk3 mediated Bmp signaling is important in the cascade of events that regulate the contribution of EPDCs to the AV sulcus, annulus fibrosus, and the parietal leaflets of the AV valves. Furthermore, this study shows that EPDCs do not only play a critical role in early developmental events at the AV junction, but that they also are important in the normal maturation of the AV valves.

Muscle-derived follistatin-like 1 functions to reduce neointimal formation after vascular injury.

AIMS: It is well-established that exercise diminishes cardiovascular risk, but whether humoral factors secreted by muscle confer these benefits has not been conclusively shown. We have shown that the secreted protein follistatin-like 1 (Fstl1) has beneficial actions on cardiac and endothelial function. However, the role of muscle-derived Fstl1 in proliferative vascular disease remains largely unknown. Here, we investigated whether muscle-derived Fstl1 modulates vascular remodelling in response to injury. METHODS AND RESULTS: The targeted ablation of Fstl1 in muscle led to an increase in neointimal formation following wire-induced arterial injury compared with control mice. Conversely, muscle-specific Fstl1 transgenic (TG) mice displayed a decrease in the neointimal thickening following arterial injury. Muscle-specific Fstl1 ablation and overexpression increased and decreased, respectively, the frequency of BrdU-positive proliferating cells in injured vessels. In cultured human aortic smooth muscle cells (HASMCs), treatment with human FSTL1 protein decreased proliferation and migration induced by stimulation with PDGF-BB. Treatment with FSTL1 enhanced AMPK phosphorylation, and inhibition of AMPK abrogated the inhibitory actions of FSTL1 on HASMC responses to PDGF-BB. The injured arteries of Fstl1-TG mice exhibited an increase in AMPK phosphorylation, and administration of AMPK inhibitor reversed the anti-proliferative actions of Fstl1 on the vessel wall. CONCLUSION: Our findings indicate that muscle-derived Fstl1 attenuates neointimal formation in response to arterial injury by suppressing SMC proliferation through an AMPK-dependent mechanism. Thus, the release of protein factors from muscle, such as Fstl1, may partly explain why the maintenance of muscle function can have a therapeutic effect on the cardiovascular system.

Measurement and 3D-visualization of cell-cycle length using double labelling with two thymidine analogues applied in early heart development.

Organ development is a complex spatial process in which local differences in cell proliferation rate play a key role. Understanding this role requires the measurement of the length of the cell cycle at every position of the three-dimensional (3D) structure. This measurement can be accomplished by exposing the developing embryo to two different thymidine analogues for two different durations immediately followed by tissue fixation. This paper presents a method and a dedicated computer program to measure the resulting labelling indices and subsequently calculate and visualize local cell cycle lengths within the 3D morphological context of a developing organ. By applying this method to the developing heart, we show a large difference in cell cycle lengths between the early heart tube and the adjacent mesenchyme of the pericardial wall. Later in development, a local increase in cell size was found to be associated with a decrease in cell cycle length in the region where the chamber myocardium starts to develop. The combined application of halogenated-thymidine double exposure and image processing enables the automated study of local cell cycle parameters in single specimens in a full 3D context. It can be applied in a wide range of research fields ranging from embryonic development to tissue regeneration and cancer research.

Cardiac regeneration: different cells same goal.

Cardiovascular diseases are the leading cause of mortality, morbidity, hospitalization and impaired quality of life. In most, if not all, pathologic cardiac ischemia ensues triggering a succession of events leading to massive death of cardiomyocytes, fibroblast and extracellular matrix accumulation, cardiomyocyte hypertrophy which culminates in heart failure and eventually death. Though current pharmacological treatment is able to delay the succession of events and as a consequence the development of heart failure, the only currently available and effective treatment of end-stage heart failure is heart transplantation. However, donor heart availability and immunorejection upon transplantation seriously limit the applicability. Cardiac regeneration could provide a solution, making real a dream of both scientist and clinician in the previous century and ending an ongoing challenge for this century. In this review, we present a basic overview of the various cell types that have been used in both the clinical and research setting with respect to myocardial differentiation.

Comprehensive gene-expression survey identifies wif1 as a modulator of cardiomyocyte differentiation.

During chicken cardiac development the proepicardium (PE) forms the epicardium (Epi), which contributes to several non-myocardial lineages within the heart. In contrast to Epi-explant cultures, PE explants can differentiate into a cardiomyocyte phenotype. By temporal microarray expression profiles of PE-explant cultures and maturing Epi cells, we identified genes specifically associated with differentiation towards either of these lineages and genes that are associated with the Epi-lineage restriction. We found a central role for Wnt signaling in the determination of the different cell lineages. Immunofluorescent staining after recombinant-protein incubation in PE-explant cultures indicated that the early upregulated Wnt inhibitory factor-1 (Wif1), stimulates cardiomyocyte differentiation in a similar manner as Wnt stimulation. Concordingly, in the mouse pluripotent embryogenic carcinoma cell line p19cl6, early and late Wif1 exposure enhances and attenuates differentiation, respectively. In ovo exposure of the HH12 chicken embryonic heart to Wif1 increases the Tbx18-positive cardiac progenitor pool. These data indicate that Wif1 enhances cardiomyogenesis.

Wt1 and retinoic acid signaling in the subcoelomic mesenchyme control the development of the pleuropericardial membranes and the sinus horns.

RATIONALE: The cardiac venous pole is a common focus of congenital malformations and atrial arrhythmias, yet little is known about the cellular and molecular mechanisms that regulate its development. The systemic venous return myocardium (sinus node and sinus horns) forms only late in cardiogenesis from a pool of pericardial mesenchymal precursor cells. OBJECTIVE: To analyze the cellular and molecular mechanisms directing the formation of the fetal sinus horns. METHODS AND RESULTS: We analyzed embryos deficient for the Wt1 (Wilms tumor 1) gene and observed a failure to form myocardialized sinus horns. Instead, the cardinal veins become embedded laterally in the pleuropericardial membranes that remain tethered to the lateral body wall by the persisting subcoelomic mesenchyme, a finding that correlates with decreased apoptosis in this region. We show by expression analysis and lineage tracing studies that Wt1 is expressed in the subcoelomic mesenchyme surrounding the cardinal veins, but that this Wt1-positive mesenchyme does not contribute cells to the sinus horn myocardium. Expression of the Raldh2 (aldehyde dehydrogenase family 1, subfamily A2) gene was lost from this mesenchyme in Wt1(-/-) embryos. Phenotypic analysis of Raldh2 mutant mice rescued from early cardiac defects by retinoic acid food supply revealed defects of the venous pole and pericardium highly similar to those of Wt1(-/-) mice. CONCLUSIONS: Pericardium and sinus horn formation are coupled and depend on the expansion and correct temporal release of pleuropericardial membranes from the underlying subcoelomic mesenchyme. Wt1 and downstream Raldh2/retinoic acid signaling are crucial regulators of this process. Thus, our results provide novel insight into the genetic and cellular pathways regulating the posterior extension of the mammalian heart and the formation of its coelomic lining.

Absence of cardiotoxicity of the experimental cytotoxic drug cyclopentenyl cytosine (CPEC) in rats.

The experimental anticancer drug cyclopentenyl cytosine (CPEC) was associated with cardiotoxicity in a phase I study. The aim of the present study was twofold; first we investigated whether the observed effects could be reproduced in in-vitro and in-vivo rat models. Second, we intended to investigate the underlying mechanism of the possible cardiotoxicity of CPEC. Effects on frequency and contractility were studied on the isolated atria of 18 male Wistar rats. Atria were incubated with 0.1 mmol L(-1) (n = 6) or 1 mmol L(-1) (n = 6) CPEC for 1.5 h and compared with control atria (incubation with buffer solution, n = 6). The cardiac apoptosis-inducing potential was studied in-vivo on 66 rats by 99mTc-Annexin V scintigraphy, followed by postmortem determination of radioactivity in tissues, histological confirmation with the TUNEL assay (late-phase apoptosis), and immunohistochemical staining for cleaved caspase 3 and cytochrome C (early-phase apoptosis). Serum levels of the necrotic cardiomyopathy marker troponin T were also determined. No effect on heart frequency was found in the isolated atria after CPEC treatment. A trend towards a decrease of contraction force was observed. However, the differences were not statistically significant. 99mTc-Annexin V scintigraphy showed no increase in cardiac uptake ratio upon CPEC treatment in the in-vivo rat model, which was confirmed by determination of radioactivity in heart versus blood ratios. At each section a few individual isolated late apoptotic cells (< 5) could be identified by the TUNEL assay in the highest CPEC dose group (90 mg kg(-1)) but not in controls or in rats treated with 60 mg kg(-1) CPEC. Staining for the early apoptosis markers cleaved caspase 3 and cytochrome C did not reveal any significant differences between treated and control rats. Cardiac troponin T levels were not increased after CPEC treatment. CPEC does not affect heart frequency or contraction force in our cardiotoxicity models. Moreover, we did not find an indication of CPEC-induced apoptosis in heart tissue.

Lineage and morphogenetic analysis of the cardiac valves.

We used a genetic lineage-labeling system to establish the material contributions of the progeny of 3 specific cell types to the cardiac valves. Thus, we labeled irreversibly the myocardial (alphaMHC-Cre+), endocardial (Tie2-Cre+), and neural crest (Wnt1-Cre+) cells during development and assessed their eventual contribution to the definitive valvar complexes. The leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity, and the leaflets of the outflow tract valves were all found to be generated from mesenchyme derived from the endocardium, with no substantial contribution from cells of the myocardial and neural crest lineages. Analysis of chicken-quail chimeras revealed absence of any substantial contribution from proepicardially derived cells. Molecular and morphogenetic analysis revealed several new aspects of atrioventricular valvar formation. Marked similarities are seen during the formation of the mural leaflets of the mitral and tricuspid valves. These leaflets form by protrusion and growth of a sheet of atrioventricular myocardium into the ventricular lumen, with subsequent formation of valvar mesenchyme on its surface rather than by delamination of lateral cushions from the ventricular myocardial wall. The myocardial layer is subsequently removed by the process of apoptosis. In contrast, the aortic leaflet of the mitral valve, the septal leaflet of the tricuspid valve, and the atrioventricular fibrous continuity between these valves develop from the mesenchyme of the inferior and superior atrioventricular cushions. The tricuspid septal leaflet then delaminates from the muscular ventricular septum late in development.

Annexin V imaging of acute doxorubicin cardiotoxicity (apoptosis) in rats.

UNLABELLED: Anthracyclines are widely used in chemotherapy regimens for several malignancies, with cardiotoxicity being the major limiting factor in high-dose schedules. Recently, it was reported that doxorubicin induces apoptosis in cardiac muscle cells in vivo and, as such, is expected to be involved in the genesis of doxorubicin-induced cardiomyopathy. The aim of this study was to validate an animal model for in vivo monitoring of doxorubicin cardiotoxicity by means of scintigraphic detection of apoptosis. METHODS: Three groups of 5 male Wistar rats each were treated for 3, 4, and 5 times with a weekly intraperitoneal injection of doxorubicin at 2.5 mg/kg. At 24 h before and 24 h after the final treatment, (99m)Tc-annexin pinhole scintigraphy was performed. A control group of 5 rats was scanned without doxorubicin treatment. A cardiac uptake ratio was calculated from planar scintigraphy results with the following formula: (mediastinum - fat)/fat. After scintigraphy, the rats were sacrificed, and the heart was processed for histologic analysis. RESULTS: Incremental general signs of illness were observed with increasing total cumulative doxorubicin dose. Rats treated for 3, 4, and 5 wk with doxorubicin showed significantly higher uptake ratios of, respectively, 4.0 +/- 0.52 (mean +/- SEM), 4.8 +/- 0.46, and 5.2 +/- 0.17 after the final treatment; the ratio for controls was 1.84 +/- 0.05 (P < 0.05). Histologic analysis confirmed cardiac stress in treated groups, with an increasing left ventricular atrial natriuretic factor messenger RNA expression level with increasing cumulative doxorubicin dose. Late apoptosis was confirmed by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling in the rats treated for 5 wk. CONCLUSION: Acute doxorubicin-induced cardiomyopathy based on early apoptosis can be assessed and imaged with annexin V scintigraphy in rats. This finding makes it possible to use this animal model for repetitive noninvasive evaluation of cardioprotective regimens for anthracycline cardiotoxicity.

Recruitment of intra- and extracardiac cells into the myocardial lineage during mouse development.

The tubular heart differentiates from the bilateral cardiac fields in the splanchnic mesoderm. The expression of smooth muscle proteins has been shown to accompany the early phases of cardiac muscle formation. In this study we show that during elongation of the arterial pole of the mouse linear heart tube, alpha-smooth muscle actin (alpha-Sma) expression extends in the area that has been shown to become recruited into the myocardial lineage, but does not yet express myocardial markers. These data suggest that alpha-Sma identifies mesodermal cells that during subsequent development will be recruited into the myocardial lineage. Myocardium formation is not only observed at the arterial pole, but also at the venous pole and in the intracardiac mesenchyme. This results in the formation of the caval and pulmonary myocardium, the smooth-walled atrial myocardium, the myocardial atrioventricular septum, and the myocardial outlet septum. To determine whether recruitment into the myocardial lineage also takes place in these regions, the spatiotemporal pattern of expression of alpha-Sma and of the myocardial markers sarcoplasmatic reticulum calcium ATPase (Serca2a), alpha-myosin heavy chain (Mhc), and beta-Mhc were examined. We show that prior to the expression of myocardial markers, alpha-Sma is expressed in these regions, which suggests that these mesodermal cells become recruited into the cardiac lineage after formation of the linear heart tube.