Human Fetal Heart Research Articles

Interaction between 52 kDa SSA/Ro and deubiquitinating enzyme UnpEL: a clue to function

The detection of isolated heart block in utero strongly predicts the presence of maternal autoantibodies reactive with 52 kDa SSA/Ro. The mechanisms that underlie this observation may be elucidated by defining the function of the target antigen. The initial approach was to identify proteins interactive with 52Ro using transcriptional activity in the yeast 2-hybrid system. A cDNA library was constructed using RNA isolated from human fetal hearts (12–23 weeks) and cloned into the HybriZAP vector encoding the activation domain of GAL4(AD) as target. Approximately 7×10 6 cDNAs were cotransformed with the bait into YRG-2. Plasmids from five interactive colonies were sequenced and three identified as the specific human deubiquitinating enzyme, UnpEL. UnpEL did not interact with bait plasmid encoding 52β, an alternative leucine zipper-minus form of 52 kDa SSA/Ro which is maximally expressed in fetal life. The mammalian 2-hybrid assay confirmed the interaction between full-length 52Ro and UnpEL. Further support for a biologic interaction was the marked redistribution in cellular localization of UnpEL following cotransfection of the two proteins into cultured human cardiocytes, human renal carcinoma cells (293 cells), and monkey kidney fibroblasts (COS-1). In conclusion, the interaction of full-length 52Ro and UnpEL implies that the former may also be involved in the ubiquitin pathway, an observation of particular interest since 52Ro contains a RING finger domain, a motif common to several recently reported proteins involved in modulating ubiquitination. The absence of an interaction with 52β raises the consideration that regulation of protein ubiquitination might differ in fetal life.

Connexin expression in the developing mouse heart and human foetal heart

Connexin expression in the developing mouse heart and human foetal heart

Alternative promoter usage and splicing of ZNF74 multifinger gene produce protein isoforms with a different repressor activity and nuclear partitioning.

We have previously shown that ZNF74, a candidate gene for DiGeorge syndrome, encodes a developmentally expressed zinc finger gene of the Kruppel-associated box (KRAB) multifinger subfamily. Using RACE, RT-PCR, and primer extension on human fetal brain and heart mRNAs, we here demonstrate the existence of six mRNA variants resulting from alternative promoter usage and splicing. These transcripts encode four protein isoforms differing at their N terminus by the composition of their KRAB motif. One isoform, ZNF74-I, which corresponds to the originally cloned cDNA, was found to be encoded by two additional mRNA variants. This isoform, which contains a KRAB motif lacking the N terminus of the KRAB A box, was devoid of transcriptional activity. In contrast, ZNF74-II, a newly identified form of the protein that is encoded by a single transcript and contains an intact KRAB domain with full A and B boxes, showed strong repressor activity. Deconvolution immunofluorescence microscopy using transfected human neuroblastoma cells and nonimmortalized HS68 fibroblasts revealed a distinct subcellular distribution for ZNF74-I and ZNF74-II. In contrast to ZNF74-I, which largely colocalizes with SC-35 in nuclear speckles enriched in splicing factors, the transcriptionally active ZNF74-II had a more diffuse nuclear distribution that is more characteristic of transcriptional regulators. Taken with the previously described RNA-binding activity of ZNF74-I and direct interaction with a hyperphosphorylated form of the RNA polymerase II participating in pre-mRNA processing, our results suggest that the two ZNF74 isoforms exert different or complementary roles in RNA maturation and in transcriptional regulation.

The bile acid taurocholate impairs rat cardiomyocyte function: a proposed mechanism for intra-uterine fetal death in obstetric cholestasis

Obstetric cholestasis is a liver disease of pregnancy that can be complicated by sudden, hitherto unexplained, intra-uterine fetal death. Because intra-uterine death occurs suddenly, and because fetal heart rate abnormalities have been reported in obstetric cholestasis, we hypothesized that intra-uterine death is caused by impaired fetal cardiomyocyte function, resulting in fetal cardiac arrest. Obstetric cholestasis is associated with raised levels of maternal and fetal serum bile acids, and we propose that these may alter cardiomyocyte function. It was not possible to investigate the effects of bile acids on the intact human fetal heart at a cellular level. Therefore we used the closest available model of fetal myocardium at term: a primary culture of neonatal rat cardiomyocytes in which cells beat synchronously and develop pacemaker activity. The effect of the primary bile acid taurocholate (0.3 mM and 3 mM) on cultures of single cardiomyocytes, each with its own independent rate of contraction, was a reversible decrease in the rate of contraction and in the proportion of beating cells (P < 0.001). Addition of taurocholate to a network of synchronously beating cells caused a similar decrease in the rate of contraction. Furthermore, the integrity of the network was destroyed, and cells ceased to beat synchronously. Taurocholate also resulted in altered calcium dynamics and loss of synchronous beating. These data suggest that raised levels of the bile acid taurocholate in the fetal serum in obstetric cholestasis may result in the development of a fetal dysrhythmia and in sudden intra-uterine death.

Doppler echocardiographic studies of diastolic cardiac function in the human fetal heart.

Fetal echocardiography has been used for the noninvasive evaluation of human fetal cardiac anatomy, function, and hemodynamics. The purpose of the present study was to use Doppler echocardiographic methods to measure diastolic flow velocity patterns across the tricuspid and mitral valves in human fetuses during gestation. Fifty normal fetuses, 35 fetuses with intrauterine growth retardation (IUGR), and 30 fetuses of diabetic mothers (DM) were studied. Peak flow velocities during early diastole (peak E wave) and peak flow velocities during atrial contraction (peak A wave) were measured, and the peak E/A ratio was calculated. The peak E/A ratio of the left ventricle in fetuses increased gradually with increasing gestational age (r = 0.57, p < 0.05), and the peak E/A ratio of the right ventricle in fetuses increased linearly with increasing gestational age (r = 0.48, p < 0.05). In early gestational age, the peak E/A ratios of both the left and right ventricle in fetuses with IUGR and of DM were not significantly different from those in the controls. However, in late gestational age, the peak E/A ratios of both the left and right ventricle in fetuses with IUGR and of DM were significantly smaller than those in the controls. This gradual decrease in the peak E/A ratio during gestation may represent the maturational or developmental alteration of diastolic cardiac properties in utero. Fetuses with IUGR and of DM may have abnormal diastolic cardiac function in later gestation.

Neonatal and adult human heart tissues from normal subjects and patients with ischemic, dilated or hypertrophic cardiomyopathy express insulin-like growth factor binding protein-3 (IGFBP-3).

In the human fetus IGFBP-3 mRNA expression is most abundant in the skin, muscle and heart but circulating IGFBP-3 levels show age-related variations. In human heart tissues from controls and patients with either ischemic, dilated or hypertrophic cardiomyopathy (no.: 20, age-range from fetuses to elderly subjects) we determined the expression of cardiac IGFBP-3 mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) and the protein by Western blotting. The same parameters were also determined in human livers. We detected IGFBP-3 mRNA in neonatal and adult as well as in fetal human heart tissues in both ventricles. Western blotting revealed the presence of IGFBP-3 in all the examined cardiac tissues. IGFBP-3 appeared to be more abundant in the heart than in the liver and in the failing hearts from patients with ischemic heart disease than in those with hypertrophic cardiomyopathy. Thus both normal and pathological human heart tissues express IGFBP-3 across lifespan and IGFBP-3 could play IGF-dependent and/or -independent actions at the myocardial level.

Three-dimensional cartography of the pattern of the myofibres in the second trimester fetal human heart.

The aim of the present study was to describe the fibre architecture of the fetal heart at mid gestation, and to clarify some persistent controversies concerning the architecture of the myofibres in the right ventricular wall and the muscular ventricular septum. We used quantitative polarized light microscopy to obtain information about the orientation of myocardial cells in the ventricular mass. These cells, joined into a network by anastomoses, have at any point in the ventricular mass a principal direction--the fibre direction. We have quantitated this information in the form of maps of the azimuth and elevation angles, in 18 midgestation fetal hearts. Our findings show that the fibre architecture of the heart can be conceptualised as myocardial fibres running like geodesics on a nested set of warped "pretzels". This model is an extension to the whole ventricular mass of Krehl's Triebwerk, and Streeter's model which was restricted to the left ventricle. A "pretzel" itself can be considered as two doughnuts joined side-by-side, with the tunnel at the center of each doughnut corresponding to the ventricular cavity. Over and above the excellence of the fit between the data and the geodesic representation, three strong arguments support this model. First, it is the only existing model that explains the observed rolling over of fibres around the atrioventricular valvar orifices. Second, it explains the trajectory of the fibres from the epicardium to the endocardium at the basal parts of both ventricles and at the apical part of the left ventricle. Third, the predicted topological singularities of the model are systematically observed in each of the 18 hearts studied.

Role of the Adenomatous Polyposis Coli Gene Product in Human Cardiac Development and Disease

Expressed sequence tag (EST) and digital Northern analyses of human fetal, adult, and hypertrophic heart cDNA libraries revealed ESTs with high homology to adenomatosis polyposis coli (APC) and its associated protein, beta-catenin, as well as their differential expression. Thus, we hypothesize that the APC/beta-catenin pathway may play a role in cardiac development and disease. Reverse transcriptase-polymerase chain reaction analysis exhibited a higher APC expression in adult compared with fetal and hypertrophic heart but no significant difference in beta-catenin mRNA level. However, beta-catenin protein level was higher in fetal and hypertrophic heart compared with adult heart, suggesting the post-translational regulation of beta-catenin by APC in the cardiovascular system. In vitro antisense inhibition of APC resulted a higher beta-catenin protein expression leading to an incomplete myotube formation, suggesting APC/beta-catenin pathway involvement in myotube development. Western blot analysis further reveals three novel isoforms, APC-F, APC-A, and APC-D, ubiquitously expressed in fetal, adult, and hypertrophic heart, respectively. Isoform switching during development and disease pathogenesis suggests functionally distinct roles for each isoform. These data (i) demonstrate the usefulness of genome-based expression analysis for rapid discovery of differentially expressed genes, (ii) implicate the APC/beta-catenin pathway in the cardiovascular development, and (iii) demonstrate APC isoform switching during cardiac development and disease.

The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the Down critical region

We applied cDNA selection methods to a genomic clone (YAC 761B5) from chromosome 21 located in the so-called ‘Down critical region’ in 21q22.3. Starting from human fetal heart and brain mRNAs we obtained and sequenced several cDNA clones. One of these clones (Down region aspartic protease (DRAP), named also BACE2 according to the gene nomenclature) revealed a striking nucleotide and amino acid sequence identity with several motifs present in members of the aspartic protease family. In particular the amino acid sequences comprising the two catalytic sites found in all mammalian aspartic proteases are perfectly conserved. Interestingly, the predicted protein shows a typical membrane spanning region; this is at variance with most other known aspartic proteases, which are soluble molecules. We present preliminary evidence, on the basis of in vitro translation studies and cell transfection, that this gene encodes a glycosylated protein which localizes mainly intracellularly but to some extent also to the plasma membrane. Furthermore DRAP/BACE2 shares a high homology with a newly described β-secretase enzyme (BACE-1) which is a transmembrane aspartic protease. The implications of this finding for Down syndrome are discussed.

Endothelin-1 regulates cytosolic and nuclear Ca2+ in human endocardial endothelium.

Endocardial endothelium is far less studied than its systemic and pulmonary counterparts and its role in cardiac function is not well known. In the study reported here, we succeeded in isolating and culturing endocardial endothelial cells (EECs) from 21-week-old human fetal heart and verified whether endothelin-1 (ET-1) receptors are present on these cells and whether their activation regulates cytosolic ([Ca]c) and nuclear calcium ([Ca]n). Using fluo-3 Ca2+ measurement and three-dimension confocal microscopy techniques, superfusion of fetal human EECs from right and left ventricles with increasing concentrations of ET-1 induced a dose-dependent sustained increase of free cytosolic and nuclear Ca2+ levels with an EC50 near 10(-10) M and 10(-11) M, respectively. The sustained increase of cytosolic and nuclear Ca2+ by ET-1 in both EEC preparations was completely blocked by the calcium chelator ethylene glycol-bis (beta-aminoethylether)-N,N,N',N'-tetra-acetic acid (EGTA) but was insensitive to the L-type Ca2+ channel blocker, nifedipine. The effect of ET-1 was prevented by the ET(A)-receptor antagonist BQ 123. However the ET(B)-receptor antagonist BQ 788 had no effect. Our results suggest that ET-1 receptors are present in human EECs and that their stimulation induces sustained Ca2+ influx through ET(A)-receptor stimulation of a nifedipine-insensitive Ca2+ channel, probably the R-type Ca2+ channel. The presence of Ca2+-dependent responses to ET-1 supports a possible modulatory role of the endocardial endothelium in myocardial functions.

HCPTPA, a Protein Tyrosine Phosphatase That Regulates Vascular Endothelial Growth Factor Receptor-mediated Signal Transduction and Biological Activity

Angiogenesis is a tightly controlled process in which signaling by the receptors for vascular endothelial growth factor (VEGF) plays a key role. In order to define signaling pathways downstream of VEGF receptors (VEGFR), the kinase domain of VEGFR2 (Flk-1) was used as a bait to screen a human fetal heart library in the yeast two-hybrid system. One of the signaling molecules identified in this effort was HCPTPA, a low molecular weight, cytoplasmic protein tyrosine phosphatase. Although HCPTPA possesses no identifiable phosphotyrosine binding domains (i.e. SH2 or phosphotyrosine binding domains), it bound specifically to active, autophosphorylated VEGFR2 but not to a mutated, kinase-inactive VEGFR2. Recombinant VEGFR2 and endogenous VEGFR2 were substrates for recombinant HCPTPA, and HCPTPA was co-expressed with VEGFR2 in endothelial cell lines, suggesting that HCPTPA may be a negative regulator of VEGFR2 signal transduction. To pursue this possibility, an adenovirus directing the expression of HCPTPA was constructed. When used to infect cultured endothelial cells, this adenovirus directed high level expression of HCPTPA that resulted in impairment of VEGF-mediated VEGFR2 autophosphorylation and mitogen-activated protein kinase activation. Adenovirus-mediated overexpression of HCPTPA also inhibited VEGF-induced cellular responses (endothelial cell migration and proliferation) and inhibited angiogenesis in the rat aortic ring assay. Taken together, these findings indicate that HCPTPA may be an important regulator of VEGF-mediated signaling and biological activity. Potential interactions with other signaling pathways and possible therapeutic implications are discussed.

Endothelial growth factor receptors in human fetal heart.

Endothelial receptor tyrosine kinases include 3 members of the vascular endothelial growth factor receptor (VEGFR) family and 2 members of the angiopoietin receptor (Tie) family. In addition, the VEGF(165) isoform binds to neuropilin-1 (NP-1), a receptor for collapsins/semaphorins. The importance of these receptors for vasculogenesis and angiogenesis has been shown in gene-targeted mice, but so far, little is known about their exact expression patterns in the human vasculature. Frozen sections of human fetal heart were stained immunohistochemically with receptor-specific monoclonal (VEGFR, Tie) or polyclonal (NP-1) antibodies. The following patterns were observed: The endocardium was positive for VEGFR-1, VEGFR-2, NP-1, Tie-1, and Tie-2 but negative for VEGFR-3. The coronary vessels were positive for Tie-1, Tie-2, VEGFR-1, and NP-1 and negative for VEGFR-2 and VEGFR-3. Myocardial capillaries and epicardial blood vessels stained for VEGFR-1, VEGFR-2, NP-1, and Tie-1; myocardial capillaries and epicardial veins weakly for Tie-2; and epicardial lymphatic vessels for VEGFR-2 and VEGFR-3, weakly for Tie-1 and Tie-2, but not for VEGFR-1 or NP-1. The results demonstrate differential expression of the endothelial growth factor receptors in distinct types of vessels in the human heart. This information is useful for the understanding of their roles in physiological and pathological processes and for their diagnostic and therapeutic application in cardiovascular medicine.

Comparison between right and left ventricular myocardia during the human fetal period. Stereological evaluation.

To develop a stereological comparison between right (RV) and left ventricle (LV) myocardium during the third human gestational trimester. Five human fetal hearts of the third trimester provided representative samples of 5 RV myocardium and 4 LV myocardium. The material was fixed in 10% buffered formaldehyde, and processed through routine methods. Fifteen microscopic fields were randomly chosen and counted in each ventricular myocardium using an "M-42" test system. The following stereological parameters were assessed: Vv (%), Lv (micron 2), Sv (micron 2/micron 3), Vp (micron 3), Nv (1/mm3) and total N. No significant difference between the stereological parameters of the myocardial structures assessed was evidenced, when comparing RV and LV. Right and left human ventricular myocardium are very similar during the fetal period at least in regard to their structural aspects.

Unitary current analysis of L-type Ca2+ channels in human fetal ventricular myocytes.

L-type calcium channels were studied in cell-attached patches from ventricular cell membranes of human fetal heart. Experiments were performed in the presence of 70 mM Ba2+ as the charge carrier at 22 degrees C to 24 degrees C. Unitary current sweeps were evoked by 300-msec depolarizing pulses to 0 mV from a holding potential of -50 mV at 0.5 Hz. Recorded currents were blocked by nisoldipine (1 microM) and stimulated by (-)Bay K 8644 (1 microM). During control, channel activity was seen in 13.9%+/-4.2% of the total 200 sweeps. Ensemble average current amplitude was 0.03+/-0.01 pA (n = 6) and average conductance was 20.4+/-0.2 pS (n = 5). Analysis of single channel kinetics showed open time and closed time histograms were best fit by one and two exponentials, respectively. Mean open time was tau(o) = 0.99+/-0.05 msec (n = 6). Mean closed time fast (tau(cf)) and slow (tau(cs)) component values were tau(cf) = 0.85+/-0.09 msec and tau(cs) = 8.0+/-0.94 msec (n = 6), respectively. With intrapipette (-)Bay K 8644 (1 microM), mean open time was best fit by two exponentials, tau(of) = 0.9+/-0.2 msec (n = 10) and tau(os) = 13.4+/-2.6 msec (n = 10); mean close time values were tau(cf) = 0.6+/-0.1 msec (n = 10) and tau(cs) = 9.8+/-1.9 msec (n = 10), respectively. With (-)Bay K 8644, channel activity was 66.5%+/-7.4%, the ensemble average current was 0.52+/-0.04 pA (n = 10) and the conductance 20.7+/-0.5 pS (n = 5). (1) the data establishes the characteristics of L-type Ca channels of human fetal hearts and their modulation by dihydropyridines; (2) the open time kinetics differ from those of avian embryonic and rat fetal hearts; and (3) the findings provide new and relevant information for understanding the physiologic behavior of unitary Ca2+ channels in the developing human heart and the baseline comparison for diseases that implicate Ca2+ channels in their etiology, such as autoimmune-associated congenital heart block.

The human NADH: ubiquinone oxidoreductase NDUFS5 (15 kDa) subunit: cDNA cloning, chromosomal localization, tissue distribution and the absence of mutations in isolated complex I-deficient patients.

We have cloned the cDNA of the NDUFS5 subunit (15 kDa) of the human mitochondrial respiratory chain complex NADH: ubiquinone oxidoreductase (complex I). The open reading frame consists of 321 base-pairs, coding for 106 amino acids, with a calculated molecular mass of 12.5 kDa. There is an 81.0% identity with the bovine equivalent on cDNA level and 74.5% identity on amino acid basis. PCR analysis of rodent-human somatic cell hybrids revealed that the human NDUFS5 gene maps to chromosome 1. The NDUFS5 mRNA is expressed ubiquitously in human tissues, with a relative higher expression in human heart, skeletal muscle, liver, kidney and fetal heart. A mutation detection study of twenty isolated enzymatic complex I-deficient patients revealed no mutations, nor polymorphisms.

Fibre Orientation in Human Fetal Heart and Ventricular Mechanics : A Small Perturbation

The study of the topological organisation of myocardial cells is a basic requirement for understanding the mechanical design of the normal and pathological heart. Anatomical observations show that cardiac muscle tissue has a highly specialized architecture. We have made new quantitative measurements of fibre orientation through the heart wall by means of polarized light analysis on some thick sections of human fetal heart embedded in a resin and polymerized. A small perturbation method to find an equilibrium solution in a cylindrical left ventricular (LV) geometry with fibres running on toroidal shells of revolution is used to investigate the mechanical behaviour of three human fetal hearts (FH) of 14, 20 and 33 weeks of gestational age.|The results of fibre strains and stresses presented for end-systolic state show significant differences when compared to results of the cylindrical geometry with regular helicoidal fibres running on cylindrical surfaces. Moreover, the toroidal shells of revolution explain shear stresses and strains in the transverse plane which also exist in the adult heart.

Expression of extracellular matrix components fibronectin and laminin in the human fetal heart.

It has been well documented that the extracellular matrix components fibronectin and laminin promote or regulate morphogenesis of the myocardial cells in mammalian heart. However, their chronological change of expression (or localization) in the human heart remains elusive. In this study, fibronectin and laminin in the left ventricle of forty-two human fetuses aged from 8 to 26 weeks gestation and left ventricular tissues obtained from a 2-week old infant and two adults were investigated by Western blot analyses and indirect immunofluorescence technique with monoclonal antibodies. In the fetal heart, fibronectins were present along the endocardium, epicardium, and linings of larger blood vessels. In 14-16 weeks gestation, fibronectin immunofluorescence became stronger but not evenly dispersed in the interstitium. After 24 weeks gestation, they were strongly positive only in the relatively larger blood vessels, as well as those in the infant and adult cardiac tissues. Laminins were strongly positive along the endocardium and basement membrane of the myocardial cells and fibroblasts during fetal life. After birth, laminins formed fine fibrillar network along the basement membrane in association with the transverse tubules of myocardial cell; these morphological characteristics remained in the adult cardiac tissues. These results indicate that fibronectin expression is relatively constant during fetal life but decreases after birth; in contrast, laminin expression is not age-dependent and constant throughout the life.

Fatty acid binding proteins from developing human fetal brain: Characterization and binding properties

Two fatty acid binding proteins (FABPs) of identicalM r, 13 kDa, have been isolated from developing human fetal brain. A delipidated 105,000 g supernatant was incubated with [1 -14C]oleate and subjected to a Sephacryl S-200 column followed by gel filtration chromatography on a Sephadex G-75 column and ion-exchange chromatography using a DEAE-Sephacel column. Purity was checked by UV spectroscopy, SDS-PAGE, isoelectric focusing and immunological cross-reactivity. The two FABPs designated as DE-I (pI 5.4) and DE-II (pI 6.9) showed cross-reactivity with each other and no alteration at the antigenic site during intrauterine development. Anti-human fetal brain FABP does not cross-react with purified human fetal heart, gut, lung or liver FABPs. The molecular mass of DE-I and DE-II is lower than those of fetal lung and liver FABPs. Like liver FABP, these proteins bind organic anions, fatty acids and acyl CoAs but differ in their binding affinities. Both DE-I and DE-II have been found to exhibit higher affinity for oleate (K d = 0.23 μM) than palmitate (K d = 0.9μM) or palmitoyl-CoA (K d = 0.96 μM), with DE-I binding less fatty acids than DE-II. DE-II is more efficient in transferring fatty acid from phospholipid vesjcles than DE-I indicating that human fetal brain FABPs may play a significant role in fatty acid transport in developing fetal brain.

The nuclear-encoded human NADH:ubiquinone oxidoreductase NDUFA8 subunit: cDNA cloning, chromosomal localization, tissue distribution, and mutation detection in complex-I-deficient patients.

We report the cloning of the cDNA sequence of the nuclear-encoded NDUFA8 subunit of NADH: ubiquinone oxidoreductase, the first mitochondrial respiratory chain complex. The NDUFA8 open reading frame (ORF) includes 519 bp and encodes 172 amino acids (Mr=20.1 kDa). The human cDNA sequence shows 86.2% identity with the bovine sequence, whereas the human NDUFA8 amino acid sequence is 87.8% similar to its bovine PGIV protein counterpart. Both human and bovine NDUFA8 contain a conserved cysteine motif. Polymerase chain reaction analysis of rodent/human somatic cell hybrids maps the human NDUFA8 gene to chromosome 9. A multiple tissue blot has revealed the highest NDUFA8 mRNA expression in human heart, skeletal muscle, and fetal heart. Mutation analysis of the NDUFA8 fibroblast cDNA in 20 patients with an isolated enzymatic complex I deficiency in cultured skin fibroblasts has revealed two polymorphisms, one within the ORF and the other in the 3' untranslated region of the NDUFA8 cDNA sequence. The allelic frequency of both polymorphisms was similar in controls and complex-I-deficient patients.

Characterization of a Novel Gene Encoding Zinc Finger Domains Identified from Expressed Sequence Tags (ESTs) of a Human Heart cDNA Database

Seven types of zinc finger protein (ZFP) genes based on the combinations of cysteine and histidine residues were found in a human heart cDNA database. Here we report the isolation of 360 cDNA clones encoding putative ZFPs. Of these, 154 (42.8%) represent C2H2-type ZFPs, 101 (28.1%) represent C2C2-type, five (1.4%) represent C2HC-type, 71 (19.7%) represent C2HC4C(HD)-type, three (0.8%) represent C3H-type, eight (2.2%) represent C3HC4-type and 18 (0.5%) represent combination type (genes containing more than one type of zinc finger). Among these 360 ZFPs, a novel ZFP cDNA named HFHZ (human fetal heart ZFP) with sequence homology to a Kruppel-associated box (KRAB) was identified. Sequencing the full-length of this cDNA clone identified an open reading frame of 711 bp that encodes a 237 amino acid protein with a predicted molecular weight of 27.7 kDa. Sequence analysis indicated that HFHZ contained a truncated KRAB box at the N-terminus and two C2H2zinc fingers at the C-terminus. The transcript of HFHZ is highly expressed in fetal heart and moderately expressed in fetal brain but not expressed in fetal liver as revealed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis suggesting that HFHZ is not expressed ubiquitously. The 3.3-fold higher expression in the fetal heart than in the adult heart suggests that HFHZ mRNA is downregulated in the process of development. In addition, the relatively high expression (1.9-fold) of HFHZ observed in the hypertrophic as compared to the normal adult heart suggests that this fetal gene is reactivated in response to hypertrophic stimuli. Chromosomal localization byin situhybridization revealed that this gene is in 19q13.1, a region containing genes involved in both cell cycle and developmental regulation.