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Physiol. Genomics 31: 53-62, 2007. First published June 5, 2007; doi:10.1152/physiolgenomics.00026.2007 Free Article
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Received 6 February 2007; accepted in final form 23 May 2007.
Physiological Genomics 31:53-62 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Fgl2 deficiency causes neonatal death and cardiac dysfunction during embryonic and postnatal development in mice

Junwu Mu7, Dawei Qu7, Agata Bartczak5, M. James Phillips5, Justin Manuel5, Wei He5, Cheryl Koscik5, Michael Mendicino5, Li Zhang5, David A. Clark5, David R. Grant5, Peter H. Backx2,3,4,6, Gary A. Levy2,5 and S. Lee Adamson1,3,4,7

1 Departments of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada
2 Department of Medicine, University of Toronto, Toronto, Ontario, Canada
3 Department of Physiology, University of Toronto, Toronto, Ontario, Canada
4 Heart and Stroke/Richard Lewar Centre, University of Toronto, Toronto, Ontario, Canada
5 Multi-Organ Transplant Program, Toronto, Ontario, Canada
6 Cardiology, University Health Network, Toronto, Ontario, Canada
7 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
We hypothesized that cardiac dysfunction was responsible for the high perinatal lethality that we previously reported in fibrinogen-like protein 2 (Fgl2) knockout (KO) mice. We therefore used ultrasound biomicroscopy to assess left ventricular (LV) cardiac structure and function during development in Fgl2 KO and wild-type (WT) mice. The only deaths observed between embryonic day (E)8.5 (onset of heart beating) and postnatal day (P)28 (weaning) were within 3 days after birth, when 33% of Fgl2 KO pups died. Histopathology and Doppler assessments suggested that death was due to acute congestive cardiac failure without evidence of valvular or other obvious cardiac structural abnormalities. Heart rates in Fgl2 KO embryos were significantly reduced at E8.5 and E17.5, and irregular heart rhythms were significantly more common in Fgl2 KO (21/26) than WT (2/21) embryos at E13.5. Indexes of systolic and/or diastolic cardiac function were also abnormal in KO mice at E13.5 and E17.5, in postnatal mice studied at P1, and in KO mice surviving to P28. M-mode analysis showed no difference in LV diastolic chamber dimension, although posterior wall thickness was thinner at P7 and P28 in Fgl2 KO mice. We conclude that Fgl2 deficiency is not associated with obvious structural cardiac defects but is associated with a high incidence of neonatal death as well as contractile dysfunction and rhythm abnormalities during embryonic and postnatal development in mice.

mutant; knockout; embryo; newborn; ultrasound biomicroscopy; mouse; echocardiography; fibrinogen-like protein 2; fibroleukin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
FIBRINOGEN-LIKE PROTEIN 2 (Fgl2; also known as fibroleukin and pT49 gene product musfblp) is a member of the fibrinogen-related protein superfamily that includes fibrinogen, angiopoietin, ficolin, and tenascin (40, 45, 49), proteins that are involved in coagulation, cell adhesion, transendothelial migration, cell proliferation, and regulation of transcription factors (27, 44). Fgl2 is expressed and differentially regulated in many cell types including endothelial cells (9, 25, 26), T cells (29), and macrophages (26, 27) and is constitutively expressed in many organs including the liver, spleen, lungs, kidney, ovary, myometrium, and brain (6, 41) and at lower levels in the heart (9, 41). The physiological role of constitutively expressed Fgl2 in these tissues is currently unknown.

Work in our laboratory (23, 30) has shown that Fgl2 has serine protease activity and functions as an immune coagulant directly cleaving prothrombin to thrombin. As such, Fgl2 has been shown to have important immune-related functions including promotion of hepatic fibrin deposition and thrombosis in both experimental and human viral hepatitis (30) and promotion of immune-mediated thrombosis in xenograft and allograft rejection (31, 33). We recently (30) generated Fgl2 knockout (KO) mice by homologous recombination and showed that fibrin deposition and liver necrosis were markedly reduced and survival markedly increased in Fgl2 KO mice infected with murine hepatitis virus (MHV)-3 coronavirus. More recently, we and others have shown that whereas membrane-associated Fgl2 expressed primarily by reticuloendothelial cells has prothrombinase activity, Fgl2 secreted by T cells has immunomodulatory activity (2, 3, 29).

Previous studies of Fgl2 KO mice found embryonic and/or early neonatal lethality that affected males and females equally (5, 30) and that could not be explained by infectious agents. Given that Fgl2 is expressed in the heart (9, 31, 41), we hypothesized that cardiac deficiency of Fgl2 might be responsible for the embryonic and neonatal lethality observed in Fgl2-deficient mice. Thus the purpose of the present study was to determine the role of Fgl2 in the heart by systematically evaluating cardiac structure and function during embryonic and early postnatal development in Fgl2 KO mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Animals.
All studies were approved by the University of Toronto, University Health Network, or Mount Sinai Hospital Animal Care Committees and were performed in accordance with the guidelines of the Canadian Council on Animal Care.

The generation and characterization of mice with a targeted disruption of the Fgl2 gene have been described previously (30). Fgl2 heterozygous mice were backcrossed to C57BL/6 mice for 10 successive generations. Fgl2 heterozygous mice were then crossed to obtain male and female Fgl2 KO mice (Fgl2 –/–) or Fgl2 wild-type (WT) (Fgl2 +/+) mice that were identified by PCR genotyping. KO males were bred with KO females, and WT males were bred with WT females at 8 and 12 wk of age, and the offspring were studied in utero and during postnatal development to weaning. Day 0.5 of pregnancy was defined as the day a vaginal plug was found after overnight mating.

Neonatal O2 saturation, hematology, and histopathology.
O2 saturation in awake Fgl2 KO (n = 13) and WT (n = 11) mice at postnatal day (P)1 was measured by pulse oximetry (model N-100c, Nellcor, Pleasanton, CA; www.nellcor.com). A transmittance probe was placed across the chest, and the signal was continuously monitored (PowerLab; www.adinstruments.com) until stable readings free of movement artifacts were obtained. Trunk blood (20 µl) was then obtained by decapitation. The sample was analyzed in a hematology analyzer (Coulter AcTdiff, Miami, Florida) to obtain red blood cell, white blood cell, and platelet counts and to measure hematocrit and hemoglobin concentration.

Fgl2 KO neonates were collected after spontaneous death (n = 10), and same-age WT controls (n = 10) were killed at predetermined times. For this examination, serial 4-µm sections at 100-µm intervals in a sagittal plane through the midline were made through the entire animal after fixation (20 sections/animal) so that an in situ histological examination of the entire animal could be made. The chest organs including heart, lungs, aorta, superior and inferior vena cava, great vessels entering and leaving the heart, major veins, and all other structures were examined histologically. All sections were stained with hematoxylin and eosin and examined by light microscopy (12). Masson trichrome was used to evaluate cardiac fibrosis. In a second series, embryos at embryonic day (E)17.5 and neonates at 1 day after birth were killed and fixed in formalin for 10 days without dissection. In three animals from each group, serial step sections in a horizontal plane were made in a rostral to caudal direction to visualize and evaluate the overall structure of the heart and great vessels entering and leaving the heart. The remainder of the animals were placed on their sides and subserially sagittally sectioned. Sections were stained with hematoxylin and eosin (33).

Immunohistochemistry.
Immunohistochemistry for FGL2 was performed on 6-µm cryosections of hearts snap frozen in liquid nitrogen in optimum cutting temperature (OCT) compound (Sadura Finetek, Torrance, CA). Hearts were collected at E13.5 and P1 and from 6-wk-old animals. Immunohistochemistry was performed with a rabbit anti-FGL2 antibody (1/10,000 dilution) or a rabbit IgG antibody (negative control) and a peroxidase-labeled goat anti-rabbit antibody as previously described (33).

The targeting construct used to generate Fgl2 KO mice contained a ß-galactosidase reporter gene under the transcriptional control of the native Fgl2 promoter. This allows the use of ß-galactosidase to localize Fgl2 transcriptional activation in vivo in Fgl2-deficient mice. ß-Galactosidase staining was performed on 6-µm cryostat sections of tissues snap frozen in liquid nitrogen in OCT compound (Sakura Finetek). A ß-galactosidase staining kit (Invitrogen; www.invitrogen.com) was used, with technical modifications required for use of the protocol with tissue sections. WT littermates were used as negative controls.

Echocardiography.
Echocardiography was performed between 1:00 and 5:00 PM with an Ultrasound Biomicroscope (UBM) and a 30- or 40-MHz transducer (Vevo 660, VisualSonics, Toronto, ON, Canada). Mice were anesthetized with isoflurane in oxygen by face mask, using the minimum concentration required to suppress spontaneous body movements (~1.5% isoflurane).

Recordings were obtained from three to five embryos in each pregnant mouse studied at E8.5, E13.5, and E17.5 (where E18.5 is full term). Three pregnant Fgl2 KO and two pregnant WT mice were studied serially from E8.5. Two additional pregnant mice in each group were studied serially at E13.5 and E17.5. Body temperature was monitored via a rectal thermometer and maintained at 36–38°C with a heating pad and lamp, and heart rate was monitored via transcutaneous electrodes (Indus Instruments; Houston, TX). All hair was removed from the abdomen by shaving followed by a chemical hair remover (Nair, Carter-Horner; Mississauga, ON, Canada). Prewarmed gel was used as an ultrasound coupling medium. At E8.5, noninvasive B-mode imaging was used to look for implantation sites containing nonviable and viable embryos (Fig. 1A). The pulsed-Doppler sample volume was placed in the heart region of viable embryos to quantify embryonic heart rates (Fig. 1B). At E13.5, nonviable embryos were sought and the heart rate of viable embryos was determined in the same way. In addition, the pulsed-Doppler sample volume was placed within the left ventricle (LV) to record the atrioventricular inflow and aortic outflow blood velocity waveforms when embryos were in a suitable orientation for obtaining a four-chamber view showing both ventricles and both atria (e.g., as in Fig. 2C). At E17.5, nonviable embryos were counted and LV intraventricular Doppler waveforms were obtained as above. In addition, an M-mode recording was obtained from the LV when embryos were in a suitable orientation for obtaining a long-axis view of the LV and right ventricle (Fig. 2, A and B). Doppler and M-mode recordings were saved for subsequent analysis. Each ultrasound exam took 30–45 min per pregnant mouse (from the onset of anesthesia to arousal). After recordings were obtained at E17.5, the pregnant mice were killed by cervical dislocation, the embryos were collected to measure body weight and heart weight, and the heart was immersion fixed for histology.


Figure 1
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  		Fig. 1. Ultrasound images and Doppler waveforms from embryos at day 8.5 (E8.5) or 13.5 (E13.5) of gestation. A: embryos (E) visible within implantation sites in the uterus with noninvasive B-mode ultrasound imaging at E8.5. The presence of detectable cardiac motion was used to identify viable embryos. B: the pulsed Doppler sample volume (between horizontal lines) was placed in the heart region of E8.5 embryos, and the Doppler blood velocity was recorded (shown in C) to quantify heart rate. C: Doppler blood velocity recordings from two Fgl2 knockout (KO) embryos at E13.5 showing examples of irregular cardiac rhythms (top: intraventricular waveform; bottom: aortic waveform).

 

Figure 2
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  		Fig. 2. Ultrasound images (A, D, G, and H), M-mode recordings (B, E), and intraventricular Doppler waveforms (C, F) from the left ventricle (LV) of embryos at E17.5 (A–C) and mice at postnatal day 28 (P28; D–H). In A and D, the M-mode cursor (dashed line) was positioned across the LV in the ultrasound image to obtain the M-mode recordings shown in B and E. Smallest division in scale bars in B and E is 100 µm. C and F show intraventricular inflow and outflow waveforms recorded simultaneously from the LV. G: short-axis view of the aorta and longitudinal view of the pulmonary trunk showing the pulsed Doppler sample volume (between horizontal lines) in the pulmonary trunk. H: longitudinal view of the ascending aorta showing the pulsed Doppler sample volume in the proximal aorta and the dashed line used to measure the angle of insonation for angle correction of velocity data. I: Doppler waveforms obtained from the pulmonary artery (top) and aorta (bottom) from a mouse at 28 days after birth. A, peak velocity during atrial contraction; Ao, aorta; AW, anterior wall; Dd, diastolic dimension; Ds, systolic dimension; E, peak velocity during early filling; ET, ejection time; ICT, isovolumetric contraction time; IRT, isovolumetric relaxation time; IVS, interventricular septum; LA, left atrium; LV, left ventricle; PA, pulmonary artery; PW, posterior wall; RV, right ventricle.

 
In a second series, echocardiography was performed serially on Fgl2 KO (n = 24–30 at each age) and WT (n = 15–20 at each age) offspring on P1, P3, P7, P14, and P28 (weaning), where P0 is the day of birth (Table 1). Pups were tattooed at P1 for subsequent identification with published methods (21). Body temperature was monitored via a rectal thermometer and maintained at 36–38°C with a heating pad and lamp on and after P14, and heart rate was monitored via transcutaneous electrodes (Indus Instruments; Houston, TX) on P28. Heat and anesthetic settings were the same for the younger age groups but heart rate and body temperature were not monitored.


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  		Table 1. Body weight and echocardiographic variables measured from the left ventricle of Fgl2 wild-type and knockout mice

 
In postnatal mice, the short-axis view of the heart was used to image the aorta in cross section and the pulmonary trunk in longitudinal section (Fig. 2G). The pulsed-Doppler sample volume was placed in the pulmonary trunk, and the Doppler waveform was recorded (Fig. 2, G and I). The transducer was then moved in the apical direction to obtain the short-axis view of the right and left ventricles and an LV M-mode recording was obtained (Fig, 2, D and E). A four-chamber view showing both ventricles and both atria was obtained from the apex of the heart, and a LV intraventricular Doppler waveform was recorded (Fig. 2, C and F). Finally, the ascending aorta was imaged with an upper right parasternal approach, the transducer was adjusted to minimize the Doppler intercept angle, the pulsed-Doppler sample volume was placed in the proximal aorta, and waveforms were recorded (Fig. 2, H and I). Doppler and M-mode recordings were saved for subsequent analysis. The ultrasound exam, from the onset of anesthesia to arousal, took ~10–15 min. The mouse was then returned to its cage.

M-mode recordings were analyzed to obtain LV end-diastolic and end-systolic inner chamber dimensions (LVDd and LVDs, respectively). Fractional shortening (FS) was calculated with the formula %FS = 100 x (LVDd – LVDs)/LVDd. LV end-diastolic posterior wall thickness (LVWT) was measured in postnatal mice. In fetuses, LV end-diastolic wall thickness of the LV free wall was measured (LVWT).

Cardiac time intervals and peak ventricular filling velocities were measured from the intraventricular Doppler waveforms in late embryonic and postnatal mice (Fig. 2, C and F). For LV intraventricular waveforms, we measured the isovolumetric contraction time (ICT; end of ventricular filling to onset of ventricular ejection), ejection time (ET; onset to end of ventricular ejection), and isovolumetric relaxation time (IRT; end of ventricular ejection to onset of ventricular filling) (Fig. 2, C and F) for three successive cardiac cycles, and results were averaged. The Tei index was then calculated with the formula Tei = (ICT + IRT)/ET to provide an index of global heart function (48). Peak ventricular filling velocities during early filling (E wave) and during filling due to atrial contraction (A wave) were measured and the peak E-to peak-A ratio (E/A) was determined (Fig. 2, C and F). Fusion of the E and A waves was uncommon and did not result in exclusion of any subjects from the study. E/A and IRT are indexes of diastolic function (43), and FS and ICT are indexes of systolic cardiac function. Doppler peak systolic velocities in the proximal aorta and pulmonary artery were also obtained.

Statistical analysis.
Variables measured at multiple age points were compared with a two-way analysis of variance (ANOVA) with group and age as factors. If statistical significance was shown by ANOVA, then a Student-Newman-Keuls test was used as a post hoc test for multiple comparisons. Student's two-tailed t-tests were used to test for significance between groups for variables measured at single age points, and Fisher's exact test was used to test differences in proportions. Differences with a P value of <0.05 were considered statistically significant. All data are expressed as means ± SE, where n is the number of embryos or postnatal mice. All histological sections were examined independently by four experienced pathologists, three of whom were cardiac pathologists. There was full agreement with the stated findings.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Neonatal lethality.
All implantation sites observed by ultrasound B-mode imaging at E8.5 (n = 16 in 3 Fgl2 KO and n = 10 in 2 WT pregnancies) contained viable embryos based on the presence of a beating heart. Similarly, no dead embryos were observed at E13.5 or E17.5 in either group by ultrasound or in E17.5 animals at necropsy. The body weights of KO embryos at E17.5 and of KO pups at P1 did not differ significantly from WT (Table 1). However, within 3 days after birth, 19 of 57 (33%) Fgl2 KO pups from 10 litters of Fgl2 KO mothers were found dead or moribund compared with 1 of 24 (4%) from three litters in the WT group (P < 0.005). The 38 surviving Fgl2 KO neonates were from five litters, and all survived into adulthood.

Neonatal O2 saturation, hematology, and histopathology.
Pulse oximetry and hematology performed on a separate series of pups on P1 revealed no significant differences in oxygen saturation, red blood cell counts, hemoglobin concentration or hematocrit, or platelet counts between Fgl2 KO and WT mice (Table 2), although white blood cell counts were significantly reduced in KO pups (18 ± 1 x 109/l) relative to WT controls (28 ± 2 x 109/l). The normal oxygen saturation levels suggest that pulmonary vasodilation had occurred normally and lend further support to the histopathology finding of no cardiac morphological abnormalities. The lack of elevation in red blood cell counts, hemoglobin concentration, and hematocrit suggests that KO fetuses had not been chronically hypoxic in utero (20).


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  		Table 2. Oxygen saturation and hematology results for Fg12 WT and KO mice at P1

 
Histopathological examination of sagittal sections of whole neonates revealed dilation and engorgement of all cardiac chambers and great vessels and lung and liver congestion in all 10 Fgl2 KO mice examined after death. In particular, the heart had a more globular shape in contrast to a normal triangular shape seen in the 10 WT age-matched controls (Fig. 3). In addition, there were focal, scattered hemorrhages in the skin, subcutaneous tissue, and muscles of the thorax and head and neck regions. There was no detectable cardiac fibrosis in Masson trichrome-stained sections.


Figure 3
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  		Fig. 3. Pathology findings in Fgl2 KO mice and wild-type (WT) control mice. A–C: hematoxylin and eosin-stained sagittal sections of chest regions of newborn WT pups (A) and newborn Fgl2 KO pups (B, C) that were found dead in the early postnatal period. In A, note the normal triangular shape of the WT heart in this plane of section that is through the apex of the heart. Shown are the great vessels entering and leaving the heart, the right atrium (RA) and right ventricle (RV), the chest wall structures, and the dome of the liver (bottom left below diaphragm). All structures are normal. B: representative section from a Fgl2 KO animal. This section was cut in almost the exact same plane as the control heart in A and is a good comparison. Note the more globular shape of the heart. Note in particular the apex of the heart, which is more rounded than in the control heart and note that the chambers of the heart, especially the right atrium, and the great vessels are distended and blood filled. C: same Fgl2 KO animal as in B, but the section was deeper in the block and it shows the extent of the right atrial distension and the distension of the inferior and superior vena cava (IVC, SVC). These congestive changes were present in all Fgl2 KO animals, but varied from mild to severe. On deeper cuts, enormous distension of the right atrium, superior vena cava, and inferior vena cava as well as engorgement of the pulmonary vasculature were seen. Similar findings were obtained in all Fgl2 KO animals that died (n = 10). D: Fgl2 WT control. Photomicrograph (tangential) of the heart showing a portion of the right atrium, the right ventricular wall, and the pleural (pl) and pericardial (per) spaces. Pleural and pericardial spaces are clearly empty. E: representative Fgl2 KO animal. Note the presence of uniform pink-staining material in the pleural and pericardial spaces. The pink material (arrows) is interpreted as protein-rich fluid in the pleural and pericardial spaces in Fgl2 KO embryos. It was found in 4 of the 6 animals in this group. A mild, moderate, or marked degree of pulmonary congestion was noted in all Fgl2 KO animals, and in 3 there was pink-staining fluid in pulmonary air spaces consistent with pulmonary edema. F: higher-magnification micrograph of E showing the fluid in the pleural space. Note as well the marked pulmonary congestion and edema in E and F. A–E: x40; F: x80 (n = 10 animals in each group).

 
Together, these findings are consistent with death due to acute congestive cardiac failure. To investigate further the possibility of heart failure, serial sections of the hearts and lungs from E17.5 WT and KO embryos were examined. The great vessels entering the heart, the aorta, the pulmonary artery, the mitral and tricuspid valves, the ductus arteriosis, and the atrial and ventricular septa and walls were all examined and found to be normal in both Fgl2 KO and Fgl2 WT animals. Visible cardiac structures were also normal in Fgl2 WT and Fgl2 KO animals. The lungs, pleura, and pericardium of Fgl2 KO animals were, however, found to be abnormal compared with Fgl2 WT mice. Specifically, a uniform pink-staining material was seen in the pleural and pericardial spaces in four of the six Fgl2 KO animals, but not in any of the Fgl2 WT animals. The pink material was consistent with protein-rich fluid as seen in pleural and pericardial effusions. In addition, varying degrees of pulmonary congestion and edema were invariably noted in Fgl2 KO animals (Fig. 3).

By immunohistochemistry, Fgl2 was localized to both the cytoplasm and membranes of cardiomyocytes in WT mice (Fig. 4A). Independent confirmation of the presence of Fgl2 staining was the presence of ß-galactosidase staining of cardiomyocytes but not endothelial cells in Fgl2 KO mice (Fig. 4B). Negative control sections from WT mice lacked ß-galactosidase staining (data not shown). Similar results were obtained in all age groups examined.


Figure 4
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  		Fig. 4. Fgl2 immunohistochemistry showing expression in cardiomyocytes. A: Fgl2 protein (brown) in WT cardiomyocytes was primarily localized in the cytoplasm (x40). B: ß-galactosidase staining (blue) in the nucleus and cytoplasm of cardiomyocytes from Fgl2 KO mice (x10).

 
Cardiac phenotype of Fgl2 KO mice.
In an attempt to determine the basis for the deaths in the Fgl2 KO mice at birth, we further assessed the mice with echocardiography. Heart rates in Fgl2 KO embryos were lower (P < 0.01) than in WT embryos at E8.5 [92 ± 4 (n = 12) vs. 115 ± 7 (n = 8) min–1] and at E17.5 but not at postnatal ages (Table 1). At E13.5, an irregular cardiac rhythm was more common (P < 0.001) in Fgl2 KO embryos (21 of 26) (Fig. 1C) than WT embryos (2 of 21). Irregularities included isolated premature beats, bigeminy, and trigeminy. A regular cardiac rhythm was observed in all mice of both groups at all other ages.

ICT were prolonged in both pre- and postnatal Fgl2 KO mice (Fig. 5E) in conjunction with reduced FS (Fig. 5B), establishing impaired systolic function in the Fgl2 KO mice. LV diastolic function was also impaired as indicated by significantly prolonged IRT in pre- and postnatal Fgl2 KO mice (Fig. 5D), although E/A during LV filling was not abnormal in Fgl2 KO mice (Fig. 3A). Nevertheless, a significantly elevated Tei index, indicating global myocardial dysfunction (48), was observed at E13.5 and E17.5 and at all postnatal ages examined (Fig. 5C). Interestingly, cardiac morphology appeared to be relatively unaffected, with the diastolic LV inner chamber dimension not differing between KO and WT mice, although LVWT was significantly thinner at P7 and P28 (Table 1) in Fgl2 KO hearts. There was no evidence of valvular regurgitation in cardiac Doppler waveforms, suggesting that valves were competent, and peak systolic blood velocities in the aorta and pulmonary artery were not elevated, suggesting that aortic and pulmonary valves were not stenotic (Table 1). Cardiac dysfunction was unlikely to be caused by abnormal plasma K+ because blood electrolytes were found to be normal in postnatal KO mice (unpublished observations).


Figure 5
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  		Fig. 5. Prenatal (E13.5, E17.5) and postnatal (1–28 days) growth and LV cardiac function in Fgl2 KO mice and WT controls. A: ratio of peak LV filling velocities during early filling (E) and atrial contraction (A). B: % LV fractional shortening (%FS). C: LV Tei index. D: LV isovolumetric relaxation time (IRT). E: LV isovolumetric contraction time (ICT). F: LV ejection time (ET). Values are means ± SE. *Significant difference between Fgl2 KO mice and same-age WT controls (P < 0.05).

 
All of the 25 Fgl2 KO pups that underwent an ultrasound exam on P1 appeared normal based on gross behavior and appearance, but 3 died by P3. Ultrasonography revealed that Fgl2 KO pups that died by P3 (KOd) had the lowest LV FS on P1, suggesting that they had the worst LV systolic function (Fig. 6A). Their mean LV FS was 23 ± 0.8% versus 36 ± 0.4% in the Fgl2 KO group that remained alive (KOa) and 41 ± 1.5% in the WT controls. Similarly, these three were among the highest in the Fgl2 KO group for Tei index (0.90 ± 0.05 in KOd vs. 0.72 ± 0.02 in KOa and 0.57 ± 0.01 in WT) (Fig. 6B) and were among the highest for IRT and ICT (Fig. 6, C and D). Furthermore, the estimated cardiac output [(LVDd3 – LVDs3) x heart rate] of KO pups that later died was significantly lower (0.43 ± 0.09 ml/min) than that in KO pups that remained alive (0.97 ± 0.06 ml/min) despite a similar body weight [1.19 ± 0.01 (KOd) vs. 1.31 ± 0.02 (KOa) g (not significantly different)]. Thus, at P1, the cardiac function of pups that later died was among the most impaired in the Fgl2 KO group, adding further support for cardiac failure as the cause of early neonatal lethality in Fgl2 KO pups.


Figure 6
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  		Fig. 6. LV cardiac function 1 day after birth in WT pups ({circ}), Fgl2 KO pups alive (KOa; •), and Fgl2 KO pups that died or were moribund by day 2 (KOd; x). A: % FS of LV. B: LV Tei index. C: LV IRT. D: LV ICT.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
Consistent with prior reports (5, 30), we observed a high incidence of early postnatal lethality in Fgl2 KO neonates. Results were consistent with death due to congestive ventricular pump failure because prominent systolic dysfunction was observed before death and cardiac dilation and liver congestion were observed postmortem. The time course of pump failure appeared to be acute because, unlike chronic heart failure in adults, no compensatory cardiac hypertrophy or fibrosis was observed. It is conceivable that Fgl2 KO pups died within days of birth because their hearts were unable to meet the increased hemodynamic workload placed on the LV during the early postnatal period (14, 46). Obvious structures in the Fgl2 KO hearts were normal. However, abnormal cardiac rhythmicity was observed in embryos, and LV systolic and diastolic function was impaired throughout late embryonic and early postnatal development. Together these results suggest that Fgl2 is an important determinant of normal myocardial function during pre- and postnatal development but is not required for normal cardiac structure.

Heart rates of embryos at E8.5 and just before birth (E17.5) were slower in Fgl2 KO compared with WT controls, while no differences in heart rate were observed after birth. Interestingly, the heart rate of Fgl2 KO embryos nevertheless increased with prenatal cardiac development in parallel with WT controls, suggesting that bradycardia was superimposed on normal maturational events. Another interesting finding was the presence of irregular cardiac rhythms in Fgl2 KO embryos only at E13.5. At E13.5, the interventricular septum is nearly complete in the mouse, and marked changes in the cardiac conduction pathways (13) and myocardial activation patterns (37) of the heart are under way. Thus we speculate that Fgl2 plays a role in the normal development of the cardiac pacemaker and/or conduction systems and that its deletion delays, or otherwise transiently perturbs, the development of these systems.

The systolic and/or diastolic cardiac function of the Fgl2 KO mice appeared to be impaired at E13.5 and E17.5 and postnatally to weaning, as indicated by elevations in the Tei index [or myocardial performance index (MPI); Ref. 48]. The Tei index is inversely related to the maximum positive derivative of LV pressure with time (+dP/dtmax) and like +dP/dtmax is influenced by preload and afterload (1, 4). The Tei index is elevated by various cardiac pathologies including myocardial infarction in adult mice (from 0.4 to 0.7; Ref. 42) and congestive heart failure in adult humans (from 0.4 to 0.65; Ref. 36) and by a variety of etiologies in human fetuses (from 0.3 to as high as 0.7; Ref. 15). Thus the elevated Tei index from 0.47 in WT mice to 0.63 in KO mice suggests heart dysfunction in the Fgl2 KO mice, which is consistent with the observed lower FS in Fgl2 mutants (34%) compared with WT mice (37%). This degree of contractile dysfunction is similar to that observed in mice overexpressing a mutant form of phospholamban, although unlike our Fgl2 KO mice LV dilation develops progressively in adult phospholamban mutants (11). Interestingly, dilation also fails to occur in vivo in embryos dying of heart failure from other causes (22, 34), suggesting that the embryonic heart may respond differently to heart failure than the adult heart.

The basis for impaired systolic and diastolic heart function in Fgl2 KO mice is unclear. However, it is interesting that a similar degree of impairment was sustained throughout rapid maturational changes in pre- and postnatal systemic hemodynamics, and in the embryonic and postnatal myocardium. At a cellular level in the myocardium, there is electrical maturation (32, 47), myofibrillar alignment (12, 24), changes myofibrillar proteins (see, e.g., Ref. 28), and changes in excitation-contraction coupling (7, 50) during this period. At a hemodynamic level, birth reduces pericardial and right ventricular pressures, effectively increasing left ventricular preload, chamber dimensions, and stroke volume (10), and there are rapid increases in postnatal arterial pressure (14) that would tend to increase afterload. LV diastolic function (i.e., E/A) and heart rate show particularly pronounced developmental changes (53), and these were nevertheless exhibited by the Fgl2 KO mice. Thus the results are consistent with an effect of Fgl2 expressed in the myocardium on a fundamental intrinsic property of the myocardium, a property that is of sustained importance throughout perinatal development, although an indirect effect caused by changes in preload and/or afterload cannot be ruled out. Further work is required to uncover the specific mechanism(s) whereby Fgl2 deficiency impairs cardiac function.

Ultrasound biomicroscopy (8, 35) enables serial evaluation of heart function before embryonic heart failure (22, 34) as well as throughout perinatal development in mice (53). Assessing heart function noninvasively avoids the confounding effects of surgery and exposure of the embryo (16, 19) and the technical difficulty of making direct intraventricular +dP/dt measurements on the small, rapidly beating early postnatal heart. However, to maximally benefit from the 50- to 100-µm resolution of the ultrasound biomicroscope (54), it is necessary to use anesthesia to minimize body movements during ultrasound exams. Anesthesia in adult mice depresses cardiac function and alters systemic hemodynamics relative to restrained conscious mice (17, 18, 39, 52). How much of this difference is due to an augmentation in cardiac function in conscious mice caused by stress associated with restraint and how much to depression caused by anesthesia is unclear. In adult mice, the effects of isoflurane are modest (18) and cardiac function is more reproducible in repeat studies (38) than with injectable anesthetics. Less is known about the effects of isoflurane anesthesia in embryonic and early postnatal mice. Isoflurane anesthesia in P7 mouse pups caused a small decrement in heart rate and peak E and A blood velocities but no change in E/A in prior work (53), whereas isoflurane had no effect on heart rate and cardiac performance in chick embryos (51). Isoflurane anesthesia thus appears to have modest effects on cardiac function in embryonic and perinatal mice and was therefore used in the present study. Inhalation anesthesia had the added benefit of permitting a light level of anesthesia to be readily obtained regardless of animal size.

In conclusion, we found that Fgl2 has a novel and important role in normal cardiac function throughout embryonic and early postnatal development, and that its deficiency leads to significant early postnatal lethality apparently due to acute congestive cardiac failure. Despite marked abnormalities in systolic and diastolic cardiac function, obvious cardiac structures were unaffected. Thus our results show for the first time that Fgl2 is not critical for morphological cardiac development but is required for normal myocardial function during prenatal and postnatal development in mice.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
The authors gratefully acknowledge the support of the Canadian Institutes of Health Research, the Heart and Stroke Foundation, and the Richard Ivey Foundation.


   DISCLOSURES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
S. L. Adamson was a member of the Scientific Advisory Board of the VisualSonics Company but has no financial interest in the company.


   FOOTNOTES
 
Address for reprint requests and other correspondence: S. L. Adamson, Samuel Lunenfeld Research Inst., Mount Sinai Hosp., Rm. 138P. 600 University Ave., Toronto, ON, Canada M5G 1X5 (e-mail: adamson{at}mshri.on.ca).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

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