Heart Of Day Research Articles

Direct Inhibitory Effect of Hypoxia on Cardiomyocyte Proliferation in Fetal Rat Hearts

Maternal hypoxia inhibits cell proliferation in the heart of fetal and neonatal rats. This study determined the direct effect of hypoxia on cardiomyocyte proliferation in fetal rat hearts. Isolated hearts of day 17 fetal rats and rat embryonic ventricular myocyte H9c2 cells were treated ex vivo with 21% or 1% O2 for 48 or 24 h. Bromodeoxyuridine (BrdU) and Ki‐67 were examined by immunohistochemistry (IHC) and immunofluorescence (IF), respectively. The expression of tissue inhibitors of metalloproteinases (TIMP) −3 & −4, cell division marker cyclin D2 and cell division inhibitor p27 was measured by Western blots. IHC staining indicated decreased numbers of BrdU positive nuclei in hypoxic‐treated fetal hearts. Analysis of double IF revealed a predominant distribution of Ki‐67 in cardiomyocytes in fetal hearts, and hypoxia caused a reduction of Ki‐67 colocalization with cardiomytocytes. In accordance, decreased Ki‐67 positive nuclei were demonstrated in H9c2 cells under the hypoxic condition. Cyclin D2 was decreased in hypoxic fetal hearts and H9c2 cells. In contrast, hypoxia enhanced the expression of p27, TIMP‐3 and TIMP‐4 in fetal hearts and H9c2 cells. The results suggest that hypoxia has direct inhibitory effect on cardiomyocyte proliferation in the developing heart, which may be mediated by the upregulation of TIMP‐3 and/or TIMP‐4. (Supported in part by NIH grants HL82779 and HL83966).

Transient Expression of Phospholipase D1 during Heart Development in Rats

The expression of phospholipase D (PLD) isozymes was examined in the hearts of rats at different stages of development. Immunoprecipitation and Western blot analysis revealed weak PLD1 expression in the hearts of day 17 embryos. The level of PLD1 protein increased transiently 0 and 3 days postpartum, and declined gradually beginning 7 days after birth. Immunohistochemistry revealed weak PLD1 immunostaining in some cells at embryonic day 17. In contrast, some vascular endothelial cells and cardiomyocytes were immunostained typically at days 0, 3, and 7 after birth. After postnatal day 21, weak PLD1 expression was immunodetected in some vascular endothelial cells and cardiomyocytes. This suggests that the PLD1 protein in the heart is strongly associated with the early postnatal development of the heart in rats.

Change in the cells that express connective tissue growth factor in acute Coxsackievirus-induced myocardial fibrosis in mouse

Cardiac fibrosis and inflammation are major pathologic conditions that result from viral myocarditis. Connective tissue growth factor (CTGF) stimulates fibroblast proliferation and induces production of extracellular matrix molecules. We studied the correlation between CTGF and cardiac fibrosis in an acute Coxsackievirus B3 (CVB3) myocarditis animal model. Eight-week-old BALB/c mice were infected intraperitoneally with 10 4 plaque forming units (PFU) of CVB3. Myocardial inflammation peaked on day 7 and decreased markedly by day 14 post-infection (pi); cardiac fibrosis was noted from day 7 and peaked on day 14. By contrast, CTGF was weakly expressed by the interstitial cells in uninfected control hearts and also in the hearts of day 3 pi. CTGF expression measured by real-time PCR was elevated on day 3 and peaked on day 7 pi. TGF-β expression peaked at day 7 pi. The cell type of CTGF expression changed from interstitial cells to myocytes after virus infection. On day 7, CTGF was strongly expressed by myocytes and inflammatory cells surrounding calcified necrotic areas. In addition, cardiac myocytes expressed CTGF on day 14. Our results, based on an acute CVB3 model of myocarditis, provide evidence that CTGF may mediate the development of fibrosis after viral myocarditis, and that the cells expressed CTGF changes during the course of viral myocarditis.

Molecular Cloning and Characterization of a Novel ATP P2X Receptor Subtype from Embryonic Chick Skeletal Muscle

We have cloned a new P2X ligand-gated ion channel receptor from embryonic chick skeletal muscle, which is tentatively named as chick P2X(8) (cP2X(8)) receptor. The cloned cDNA encodes a protein with 402 amino acids. Electrophysiological study of the recombinant cP2X(8) receptor expressed in Xenopus oocytes showed that 10 microm ATP induced a fast inward current followed by rapid and long lasting desensitization in medium containing 1.8 mm Ca(2+). In medium with 0. 3 mm Ca(2+) ATP induced a bi-phasic response as follows: a slower inward current succeeded the initial fast one. 2-Methylthio-ATP, alpha,beta-methylene-ATP, and adenosine 5'-O-(thio)triphosphate were potent agonists, whereas ADP was a very weak agonist. ATP-induced currents were blocked by 100 microm suramin and pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid. Northern blot analysis and reverse transcription-polymerase chain reaction showed that cP2X(8) RNA transcripts were mainly expressed in skeletal muscle, brain, and heart of Day 10 chick embryos. A moderate level of expression was also detected in gizzard and retina. Whole mount in situ hybridization showed that cP2X(8) RNA transcripts were expressed mainly in neurotube, notochord, and stomach in Day 3 embryos. In Day 4 and Day 6 embryos, the cP2X(8) RNA transcripts were highly expressed in the myotome and premuscle mass. The physiological role of this receptor in the establishment of the skeletal muscle innervation will be studied.

Expression of the Mf1 gene in developing mouse hearts: implication in the development of human congenital heart defects.

The transcription factor FKHL7 gene has recently been associated with the anterior segment dysgenesis disorder of the eye known as Axenfeld-Rieger anomaly (ARA). A growing body of evidence indicates that mutations in FKHL7 cause not only defects in the anterior segment of the eye but defects in the heart valves and septa as well. In order to evaluate its contribution to normal heart septation and valve formation, expression of the mouse homologue Mf1 in embryonic hearts was analyzed by in situ hybridization. A weak but significant level of Mf1 expression could be detected in the endocardium of mouse embryos as early as day 8.5 post-conception (p.c.). Mf1 expression was undetectable in the hearts of day 9.5 p.c. embryos, but by day 10.5-11 p.c., Mf1 transcripts could be found again in the endocardium of both the atrium and ventricle and a relatively strong signal was observed in the dorsal portion of the septum primum, in what appeared to be the spinal vestibule. At day 13 p.c. when aortic and pulmonary trunks are separated, relatively more Mf1 transcripts were detected in the leaflets of aortic, pulmonary, and venous valves, the ventral portion of the septum primum, as well as in the single layer of cells on the edges of the atrioventricular cushion tissues. Surprisingly, there was no signal detected in the developing interventricular septum. At day 15 p.c., overall Mf1 signals were greatly decreased. However, significant levels of expression could still be observed in the atrial septum, the tricuspid valve, the mitral valve, and in the venous valve but not in the interventricular septum. The temporal and spatial expression patterns of the Mf1 gene in developing mouse hearts suggest that Mf1 may play a critical role in the formation of valves and septa with the exception of the interventricular septum. This is further supported by our studies showing that mutations in the FKHL7 gene were associated with defects in the anterior segment of the eye as well as atrial septal defects or mitral valve defects. Dev Dyn 1999;216:16-27.

Expression of the Mf1 gene in developing mouse hearts: Implication in the development of human congenital heart defects

The transcription factor FKHL7 gene has recently been associated with the anterior segment dysgenesis disorder of the eye known as Axenfeld-Rieger anomaly (ARA). A growing body of evidence indicates that mutations in FKHL7 cause not only defects in the anterior segment of the eye but defects in the heart valves and septa as well. In order to evaluate its contribution to normal heart septation and valve formation, expression of the mouse homologue Mf1 in embryonic hearts was analyzed by in situ hybridization. A weak but significant level of Mf1 expression could be detected in the endocardium of mouse embryos as early as day 8.5 post-conception (p.c.). Mf1 expression was undetectable in the hearts of day 9.5 p.c. embryos, but by day 10.5–11 p.c., Mf1 transcripts could be found again in the endocardium of both the atrium and ventricle and a relatively strong signal was observed in the dorsal portion of the septum primum, in what appeared to be the spinal vestibule. At day 13 p.c. when aortic and pulmonary trunks are separated, relatively more Mf1 transcripts were detected in the leaflets of aortic, pulmonary, and venous valves, the ventral portion of the septum primum, as well as in the single layer of cells on the edges of the atrioventricular cushion tissues. Surprisingly, there was no signal detected in the developing interventricular septum. At day 15 p.c., overall Mf1 signals were greatly decreased. However, significant levels of expression could still be observed in the atrial septum, the tricuspid valve, the mitral valve, and in the venous valve but not in the interventricular septum. The temporal and spatial expression patterns of the Mf1 gene in developing mouse hearts suggest that Mf1 may play a critical role in the formation of valves and septa with the exception of the interventricular septum. This is further supported by our studies showing that mutations in the FKHL7 gene were associated with defects in the anterior segment of the eye as well as atrial septal defects or mitral valve defects. Dev Dyn 1999;216:16–27. © 1999 Wiley-Liss, Inc.

Temporal and spatial regulation of a gene expressed during rat heart maturation • 123

Structural development and functional maturation in the rat heart occur over a short period of time between the 10th day of gestation and the third postnatal week. Researchers have been actively attempting to identify and characterize genes that are likely to be important in this process. We have used differential display for this purpose, comparing the mRNAs expressed in the outflow tract of the heart on day 12 of embryonic development to those in the early postnatal aorta of the rat. One of the differentially displayed products was subcloned into pBluescript for sequencing and probe generation. Total RNA was harvested from rat hearts at various developmental ages, and RNA from multiple tissues was harvested from adult rats; the RNA was electrophoresed through agarose gels and transferred to nylon membranes for northern analysis. By northern analysis, this fragment hybridizes to a single 2 kilobase transcript present abundantly in the heart, kidney, and brain of adult rat tissues, while it is barely detectable in liver and spleen, and undetectable in lung. A single transcript of identical size is detected at low levels in RNA from rat heart of day 14 to 20 of embryonic development, after which the abundance increases significantly to a maximum during the first postnatal week, reaching an intermediate level similar to the adult by the third postnatal week. A digoxigenin-tagged antisense riboprobe was synthesized and hybridized specifically to the atrioventricular canal and outflow tract regions of the heart of day 12 embryonic rats, as well as to the brain by in situ hybridization. Sequence analysis of the cloned fragment reveals significant homology to the 3′ untranslated region of an alpha integrin cloned from human and mouse sources. The transcript size of these known integrins are almost 4 kilobases, suggesting that the gene we are studying is likely to be novel. Its tissue-specific and developmentally regulated expression pattern, as well as its homology to a member of the integrin family of cell-surface receptors suggest that it may have a role in heart maturation. Cloning of this gene will facilitate determination of its function.

Maternal diabetes induces increased expression of extracellular matrix components in rat embryos.

The mechanisms responsible for the increased incidence of congenital malformations in offspring of diabetic mothers are poorly understood. Because the abnormal metabolic milieu of diabetes induces in the adult organism increased synthesis of basement membrane components, and these molecules play a prominent role in morphogenesis, we investigated whether maternal diabetes or high glucose levels disturb extracellular matrix synthesis in rat embryos. In gestational day 11 embryos, maternal diabetes induced a small but significant increase in laminin B1 (127 +/- 40% of control, mean +/- SD, P < 0.02) but not in fibronectin mRNA (101 +/- 26% of control). Day 12 embryos from diabetic mothers showed a larger increment in laminin B1 (179 +/- 91% of control, P < 0.02) and also an increase in fibronectin mRNA (172 +/- 73% of control, P < 0.02). A similar increase in the expression of fibronectin was observed in the kidneys and hearts of day 20 fetuses dissected from diabetic rats. High glucose levels mimicked in vitro the effects of maternal diabetes. Day 9 embryos cultured for 48 h in 50 mM D-glucose showed, akin to the day 11 embryos in vivo, an increase in laminin B1 mRNA (129 +/- 47% of control) and no changes in fibronectin mRNA (106 +/- 35% of control). The finding that maternal diabetes induces increased expression of extracellular matrix components in developing embryos establishes a link with the abnormalities occurring in the chronic complications of diabetes and proposes a new path of investigation for the mechanism of teratogenicity of the diabetic milieu.

Cell cycle analysis in the cardiac and neuroepithelial tissues of day 10 rat embryos and the effects of phosphoramide mustard, the major teratogenic metabolite of cyclophosphamide.

Using the autoradiographic technique of percent labeled mitoses (PLM), total cell cycle length (Tc) and length of the cell cycle phases G1, S, G2, and M have been estimated for the neuroepithelium and heart of day 10 rat embryos. The Tc for the neuroepithelium was estimated to be 9.45 hr, consisting of G1 of 1.24 hr, S of 6.15 hr, G2 of 1.34 hr, and M of 0.72 hr. The Tc for the heart was estimated to be 13.37 hr, consisting of G1 of 4.30 hr, S of 7.01 hr, G2 of 1.81 hr, and M of 0.25 hr. Comparison of cell cycle parameters in these two tissues indicates that the longer cell cycle in heart tissue is related primarily to an increase in G1. If embryos are exposed to the major teratogenic metabolite of cyclophosphamide, phosphoramide mustard, cell cycle analysis reveals that the S phase of the neuroepithelial cell cycle is lengthened and cells are either slowed or arrested in G2.