Echocardiographic Assessment of Embryonic and Fetal Mouse Heart Development: A Focus on Haemodynamics and Morphology

Background. Heart development is a complex process, and abnormal development may result in congenital heart disease (CHD). Currently, studies on animal models mainly focus on cardiac morphology and the availability of hemodynamic data, especially of the right heart half, is limited. Here we aimed to assess the morphological and hemodynamic parameters of normal developing mouse embryos/fetuses by using a high-frequency ultrasound system. Methods. A timed breeding program was initiated with a WT mouse line (Swiss/129Sv background). All recordings were performed transabdominally, in isoflurane sedated pregnant mice, in hearts of sequential developmental stages: 12.5, 14.5, and 17.5 days after conception (n=105). Results. Along development the heart rate increased significantly from 125 ± 9.5 to 219 ± 8.3 beats per minute. Reliable flow measurements could be performed across the developing mitral and tricuspid valves and outflow tract. M-mode measurements could be obtained of all cardiac compartments. An overall increase of cardiac systolic and diastolic function with embryonic/fetal development was observed. Conclusion. High-frequency echocardiography is a promising and useful imaging modality for structural and hemodynamic analysis of embryonic/fetal mouse hearts

Differential Temporal and Spatial Progerin Expression during Closure of the Ductus Arteriosus in Neonates

Closure of the ductus arteriosus (DA) at birth is essential for the transition from fetal to postnatal life. Before birth the DA bypasses the uninflated lungs by shunting blood from the pulmonary trunk into the systemic circulation. The molecular mechanism underlying DA closure and degeneration has not been fully elucidated, but is associated with apoptosis and cytolytic necrosis in the inner media and intima. We detected features of histology during DA degeneration that are comparable to Hutchinson Gilford Progeria syndrome and ageing. Immunohistochemistry on human fetal and neonatal DA, and aorta showed that lamin A/C was expressed in all layers of the vessel wall. As a novel finding we report that progerin, a splicing variant of lamin A/C was expressed almost selectively in the normal closing neonatal DA, from which we hypothesized that progerin is involved in DA closure. Progerin was detected in 16.2%±7.2 cells of the DA. Progerin-expressing cells were predominantly located in intima and inner media where cytolytic necrosis accompanied by apoptosis will develop. Concomitantly we found loss of α-smooth muscle actin as well as reduced lamin A/C expression compared to the fetal and non-closing DA. In cells of the adjacent aorta, that remains patent, progerin expression was only sporadically detected in 2.5%±1.5 of the cells. Data were substantiated by the detection of mRNA of progerin in the neonatal DA but not in the aorta, by PCR and sequencing analysis. The fetal DA and the non-closing persistent DA did not present with progerin expressing cells. Our analysis revealed that the spatiotemporal expression of lamin A/C and progerin in the neonatal DA was mutually exclusive. We suggest that activation of LMNA alternative splicing is involved in vascular remodeling in the circulatory system during normal neonatal DA closure

Deficient Myocardial Organization and Pathological Fibrosis in Fetal Aortic Stenosis—Association of Prenatal Ultrasound with Postmortem Histology

In fetal aortic stenosis (AS), it remains challenging to predict left ventricular development over the course of pregnancy. Myocardial organization, differentiation and fibrosis could be potential biomarkers relevant for biventricular outcome. We present four cases of fetal AS with varying degrees of severity and associate myocardial deformation on fetal ultrasound with postmortem histopathological characteristics. During routine fetal echocardiography, speckle tracking recordings of the cardiac four-chamber view were performed to assess myocardial strain as parameter for myocardial deformation. After pregnancy termination, postmortem cardiac specimens were examined using immunohistochemical labeling (IHC) of key markers for myocardial organization, differentiation and fibrosis and compared to normal fetal hearts. Two cases with critical AS presented extremely decreased left ventricular (LV) strain on fetal ultrasound. IHC showed overt endocardial fibro-elastosis, which correlated with pathological fibrosis patterns in the myocardium and extremely disturbed cardiomyocyte organization. The LV in severe AS showed mildly reduced myocardial strain and less severe disorganization of the cardiomyocytes. In conclusion, the degree of reduction in myocardial deformation corresponded with high extent to the amount of pathological fibrosis patterns and cardiomyocyte disorganization. Myocardial deformation on fetal ultrasound seems to hold promise as a potential biomarker for left ventricular structural damage in AS

Spatial and temporal expression of progerin is associated with development of CN in the DA.

<p>A. Overview of the wall of the DA of an 18-week-old fetus depicting α-smooth muscle actin (αSM actin) staining of the vascular smooth muscle cells. Details of intima (i) and outer media (om) show the absence of progerin and the overall expression of lamin A/C in the cells. B. Overview of the DA of a 10-day-old neonate with onset of degeneration of the inner media (im). In this layer there is loss of α-SM actin staining. Details of intima and inner media show progerin expressing nuclei (arrowheads) and reduction of lamin A/C expressing cells compared to the fetal DA in A. C. Overview of the DA of a 10-day-old neonate with marked cytolytic necrosis (CN) in the inner media with loss of cells. Smooth muscle cells are still detectable in the intima and outer media. Detail of the intima shows some progerin expressing nuclei (arrowheads) and absence of these nuclei in the outer media. Lamin A/C expression is found in the inner and outer media. D. Overview of a non-closing persistent DA (PDA) of a 2-year-old infant. In all layers there is abundant α-SM actin expression. Details of the intima and outer media show absence of progerin and presence of lamin A/C expressing cells. Scale bars: overviews A–D 200 µm, details 50 µm.</p

Data_Sheet_2_Acute myocardial infarction induces remodeling of the murine superior cervical ganglia and the carotid body.pdf

A role for cardiac sympathetic hyperinnervation in arrhythmogenesis after myocardial infarction (MI) has increasingly been recognized. In humans and mice, the heart receives cervical as well as thoracic sympathetic contributions. In mice, superior cervical ganglia (SCG) have been shown to contribute significantly to myocardial sympathetic innervation of the left ventricular anterior wall. Of interest, the SCG is situated adjacent to the carotid body (CB), a small organ involved in oxygen and metabolic sensing. We investigated the remodeling of murine SCG and CB over time after MI. Murine SCG were isolated from control mice, as well as 24 h, 3 days, 7 days and 6 weeks after MI. SCG and CBs were stained for the autonomic nervous system markers β3-tubulin, tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), as well as for the neurotrophic factors brain derived neurotropic factor (BDNF), nerve growth factor (NGF) and their tyrosine receptor kinase (pan TRK). Results show that after MI a significant increase in neuron size occurs, especially in the region bordering the CB. Co-expression of TH and ChAT is observed in SCG neuronal cells, but not in the CB. After MI, a significant decrease in ChAT intensity occurs, which negatively correlated with the increased cell size. In addition, an increase of BDNF and NGF at protein and mRNA levels was observed in both the CB and SCG. This upregulation of neurotropic factors coincides with the upregulation of their receptor within the SCG. These findings were concomitant with an increase in GAP43 expression in the SCG, which is known to contribute to axonal outgrowth and elongation. In conclusion, neuronal remodeling toward an increased adrenergic phenotype occurs in the SCG, which is possibly mediated by the CB and might contribute to pathological hyperinnervation after MI.</p

Data_Sheet_1_Acute myocardial infarction induces remodeling of the murine superior cervical ganglia and the carotid body.docx

A role for cardiac sympathetic hyperinnervation in arrhythmogenesis after myocardial infarction (MI) has increasingly been recognized. In humans and mice, the heart receives cervical as well as thoracic sympathetic contributions. In mice, superior cervical ganglia (SCG) have been shown to contribute significantly to myocardial sympathetic innervation of the left ventricular anterior wall. Of interest, the SCG is situated adjacent to the carotid body (CB), a small organ involved in oxygen and metabolic sensing. We investigated the remodeling of murine SCG and CB over time after MI. Murine SCG were isolated from control mice, as well as 24 h, 3 days, 7 days and 6 weeks after MI. SCG and CBs were stained for the autonomic nervous system markers β3-tubulin, tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), as well as for the neurotrophic factors brain derived neurotropic factor (BDNF), nerve growth factor (NGF) and their tyrosine receptor kinase (pan TRK). Results show that after MI a significant increase in neuron size occurs, especially in the region bordering the CB. Co-expression of TH and ChAT is observed in SCG neuronal cells, but not in the CB. After MI, a significant decrease in ChAT intensity occurs, which negatively correlated with the increased cell size. In addition, an increase of BDNF and NGF at protein and mRNA levels was observed in both the CB and SCG. This upregulation of neurotropic factors coincides with the upregulation of their receptor within the SCG. These findings were concomitant with an increase in GAP43 expression in the SCG, which is known to contribute to axonal outgrowth and elongation. In conclusion, neuronal remodeling toward an increased adrenergic phenotype occurs in the SCG, which is possibly mediated by the CB and might contribute to pathological hyperinnervation after MI.</p

Spatial distribution of TUNEL staining in the DA.

<p>(a) A sister section of the neonatal DA from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023975#pone-0023975-g003" target="_blank">figure 3A</a> that was stained with TUNEL (green). Details of the staining are shown in magnifications of the boxed areas b–d. TUNEL staining is predominantly found in the inner media (c) and the bordering intima (b). TUNEL positive nuclei were not found in the outer media (d). Nuclei were counter stained with DAPI. Scale bars are: scale bar 100 µm (a); 10 µm (b,c,d).</p

Spatial changes of progerin expression in a neonatal DA.

<p><b>A.</b> (b) Confocal images of immunofluorescence show even nuclear distribution of lamin A/C (red) and (c) nuclear envelope localization of progerin (green) in the media of DA. (a) Nuclei are counterstained with DAPI. (d) Merging of the staining patterns. Scale bars: 5 µm. <b>B.</b> Microscopic image of immunofluorescence in DA and aorta (Ao) show (a,d) the tissue specific expression of lamin A/C and (b,e) progerin. (c,f) in the media. Nuclei expressing both proteins as shown in the merged images. Scale bars: 20 µm. <b>C.</b> (a) Box-plot shows the percentage of progerin positive cells in DA and aorta. Statistical analysis represents 1302 nuclei in DA and 302 nuclei in aortas from 5 individuals. P-value indicates significant difference in progerin expression between DA and aorta. (b) Histograms show the average ratio of progerin positive cells in intima and inner media <i>versus</i> the outer media. Statistical analysis represents 4 samples from each of the 5 individuals. P-value indicates that intima and inner media contain significantly more progerin expressing cells than the outer media.</p

RT-PCR reveals expression of progerin mRNA in DA.

<p><b>A.</b> RT-PCR analysis of <i>LMNA</i> and progerin mRNA in neonatal aorta and DA was performed with primers that amplify 270 and 185 bp of <i>LMNA</i>, and 120 bp of progerin. GUSB PCR product was used as a control. Aorta tissue was obtained from a 4-month patient and DA tissues from 6 neonates 8 days after birth. <b>B.</b> Direct sequencing of PCR fragments 270, 185 and 120 bp. Shown are the sequence histograms from position 2212 bp.</p