Developing Rat Heart Research Articles

The effects of maternal hypoxia on the heart development in fetal and neonatal rats

Fetal hypoxia leads to progressive cardiac remodeling in the rat offspring. Our objective is to determine the effects of hypoxia on heart remodeling in fetus and neonates. Pregnant rats were treated with normoxia or hypoxia (10.5% O2) from day 15 to 21 of gestation. Hearts were isolated from 21‐day fetuses (E21) and 7‐day‐old pups (PD7). The fibrillar collagen was examined by scanning electron microscopy (SEM). The expression of matrix metalloproteinases (MMP) 1, 2, 9 & 13, tissue inhibitors of metalloproteinases (TIMP) 1, 2, 3 and collagen 1 & 3 in the heart was measured by Western blots. Maternal hypoxia caused a significant decrease in the body weight of the pups at both E21 and PD7. The heart weight was decreased at PD7, but not in E21. SEM indicated less amount of fibrillar collagen at E21 but greater amount of collagen at PD7 in the heart of hypoxic pups, as compared with those of control animals. Myocardium wall thickness was decreased by hypoxia in both E21 and PD7. Hypoxia also reduced collagen‐1 and MMP‐1 in fetuses, while increased collagen‐1 and MMP‐1 in neonates. MMP‐13 was enhanced in fetuses but reduced in neonates, while TIMP‐3 level was increased in both developmental stages. There was no change in MMP‐2 & 9, TIMP‐1 & 2 and collagen‐3. Maternal hypoxia results in differential remodeling of fibrillar collagen in fetal and neonatal hearts, which may be mediated by the altered expression patterns of MMPs and TIMPs.

Relationship of hyperhomocysteinemia in pregnant rats and congenital heart defects in the newborn rats

To investigate the relationship between homocysteine (HCY) and congenital heart defects, and to observe the toxic effect of different doses of HCY on embryonic heart development in mammalian. A total of 30 SD pregnant rats were randomly divided into 3 groups: a high dose group [200 mg/(kg.d)], a low dose group [(100 mg/(kg.d)] and a control group (equal volume of physiologic saline, n=10 in each group). The HCY or vehicle was given intraperitoneally from 7 to 20 days after uterineincision delivery. The contents of HCY in serum were analyzed by high performance liquid chromatogram electrochem before the pregnancy and 20 days after the pregnancy. The structure changes of the newborn rats heart were observed by stereoscope. The ultrastructure changes of cadiomyocyte were observed through transmission electron microscope. Comparing with the control, serum HCY in rats 20 days after pregnancy was significantly increased in the high or low dose group [(30.47 ± 1.12), (20.90 ± 1.08)vs(10.98 ± 0.77)μmol/L, P<0.01)], indicating that the hyperhomocysteinemia animal model was successfully established. The incidence rate of congenital heart defects in neonatal was significantly increased in the high or low dose group(14.13%, 9.57% vs 0.76%, P<0.01). The number of apoptotic cells were significantly increased in the high dose group. Hyperhomocysteinemia may exert toxic effect on embryonic heart development in pregnancy rats, which led to congenital heart defects in the newborn rats. Hyperhomocysteinemia induced cardiomyocyte apoptosis may, at least partially, contribute to the heart defects.

Aldosterone Modulates Cell Proliferation and Apoptosis in the Neonatal Rat Heart

In the present study, we investigated whether and how the mineralocorticoid receptor antagonist spironolactone affects cardiac growth and development through apoptosis and cell proliferation in the neonatal rat heart. Newborn rat pups were treated with spironolactone (200 mg/kg/d) for 7 days. The cell proliferation was studied by PCNA immunostaining. The treatment with spironolactone decreased proliferating myocytes by 32% (P<0.05), and reduced myocytes apoptosis by 29% (P<0.05). Immunoblot and immunohistochemistry for the expression of p38, p53, clusterin, TGF-β2, and extracellular signal-regulated kinase were performed. In the spironolactone group, p38, p53, clusterin, and TGF-β2 protein expression was significantly decreased (P<0.05). These results indicate that aldosterone inhibition in the developing rat heart induces cardiac growth impairment by decreasing proliferation and apoptosis of myocytes.

Age-related changes in parvalbumin in the heart of female rats

Changes of parvalbumin protein levels and immunolocalisation during the postnatal development of the female rat heart were investigated in order to determine if they were correlated with age-related changes in cardiac function. Hearts from newborn, 3-month-old (young), 6-month-old (young adult) and 12-month-old (adult) female Wistar rats were processed for immunohistochemical localization of parvalbumin and for Western blotting assay. Parvalbumin was detected by both methods in all age groups from newborn to 12-month-old rats. In the newborn rat heart, parvalbumin immunoreactivity did not fully fill the sarcoplasm of the cardiac myocytes and the amount of parvalbumin was low compared to the adult levels. In contrast, in 3-12-month-old rats, strong parvalbumin immunoreactivity was detected throughout the sarcoplasm of all cardiac myocytes and the amount of parvalbumin increased with increasing age (from newborn to adult). Our study indicates that an increase of parvalbumin levels in the female rat heart with increasing age may be associated with maintenance of proper relaxation of the cardiac myocytes needed to cope with the increasing workload of the heart during postnatal growth.

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.

The area composita of adhering junctions connecting heart muscle cells of vertebrates – IV: Coalescence and amalgamation of desmosomal and adhaerens junction components – Late processes in mammalian heart development

In the adult mammalian heart, the cardiomyocytes and thus their terminally anchored myofibrillar bundles are connected by large arrays of closely spaced or even fused adhering junctions (AJs), termed “intercalated disks” (IDs). In recent years, the ID complex has attracted special attention as it has become clear that several human hereditary cardiomyopathies are caused by mutations of genes encoding ID marker proteins, in particular some that are also known as constituents of epithelial desmosomes. Previously, we have shown that in the mature myocardial ID the compositional differences between desmosome-like and adhaerens junctions are, by and large, lost and a composite hybrid structure, the area composita, is formed. We now report results from immunofluorescence and (immuno-)electron microscopic studies of heart formation during mouse embryogenesis and postnatal growth and show that the formation of the IDs with extended area composita structures is a late, primarily postnatal process. While up to birth small distinct desmosomes and AJs are resolved as predominant ID structures, areae compositae of increasing sizes and merged marker protein patterns occupy most of the IDs in the mature heart. Differences in the patterns of ID formation and amalgamation of the two ensembles of junction proteins in time and space are also demonstrated. Together with corresponding observations during rat and human heart development our results indicate that ID topogenesis and area composita formation are also late developmental processes in other mammals. We discuss the importance of the ID and the areae compositae in cardiac functions and, consequently, in cardiomyopathies and possible myocardial regeneration processes.

High‐pressure freezing and freeze substitution of rat myocardium for immunogold labeling of connexin 43

The value of high-pressure freezing (HPF) and freeze substitution (FS) for immunoelectron microscopy (immuno-EM) of the heart was investigated in bioptic specimens taken from isolated hearts of 0-, 5-, and 14-day-old rats at baseline and at 15, 30, 45, and 60 min after induction of ischemia. The target antigen chosen here was the gap junction protein connexin 43 (Cx43). After HPF and FS, immunogold labeling was applied for detection of Cx43. Gold particles associated with gap junction areas, free plasma membrane, and annular gap junctions (AGJs) were counted and distributions compared by contingency table analysis. HPF and FS resulted in excellent preservation of antigenicity for Cx43. The mostly good preservation of the ultrastructure was limited by mechanical damage at the border and by ice crystal formation in the center of the tissue blocks. In normal myocardium of newborns, gold particles associated with free plasma membrane were frequently observed, with AGJs only seldom. In older rats, the opposite relation was found. During ischemia, no distribution changes occurred in newborn or 14-day-old rats. In 5-day-old rats, however, ischemia induced a shift of Cx43 from gap junction plaques to AGJs. In conclusion, HPF and FS are an ideal alternative to chemical fixation for immuno-EM as the excellent preservation of antigenicity is combined with a well-preserved ultrastructure. The results indicate that the process of degradation of gap junctions via AGJs gradually increases during postnatal rat heart development, a process that may be accelerated by ischemia in an early developmental state.

Cardiac Cytochrome-c Oxidase Deficiency Occurs During Late Postnatal Development in Progeny of Copper-Deficient Rats

Although cytochrome-c oxidase (CCO) is a copper-dependent enzyme, the effect of maternal copper deficiency on the expression of CCO activity during postnatal development of the neonatal rat heart has not been investigated extensively. Here, we show that CCO activity in heart mitochondria isolated from neonates of copper-deficient dams did not exhibit significant reductions until postnatal days (PND) 15 and 21. In addition, immunoblot analysis indicated that the CCO subunit (Cox-1) was reduced on postnatal Days 10 and 21, and that Cox-4 was reduced on PND 21 in heart mitochondria of the neonates from copper-deficient dams. These findings indicate that the impairment of CCO activity in neonatal heart by maternal copper deficiency occurs late in the postnatal heart development. Furthermore, the concurrent reductions in Cox-1 and Cox-4 suggest that the impaired CCO activity reflects a CCO deficiency in heart mitochondria. CCO activity and Cox-1 in heart mitochondria were not fully restored by 6 weeks of postweaning copper repletion in the pups of copper-deficient dams. This indicates that prolonged maternal intake of moderately low dietary copper produces CCO deficiency in cardiac mitochondria of neonates during late postnatal heart development, after terminal differentiation of cardiomyocytes occurs. The resistance of CCO deficiency to repair by dietary copper supplementation may be related to the relatively slow turnover of the affected mitochondria in the terminally differentiated heart.

Balance between hypertrophic and hypoxic stimulus in caspase-3 activation during rat heart development

During heart development, cell hyperplasia and hypertrophy are the main mechanisms by which cardiac mass grows. Both these processes along with programmed cell death lead to complete growth and function. In addition, since the establishment of cardiac function depends on the relationship between oxygen supply and demand, we investigated some of the molecular mechanisms at the basis of rat myocardial cell response to hypoxic stress at different times of neonatal life. In particular, the role played by hypertrophic and survival factors like NF-kB and IAP-1 (Inhibiting Apoptosis Protein) and by death factors ASK-1 (Apoptosis Signal Regulating Kinase), JNK/SAPK (Jun-N-Terminal-Kinase/Stress-Activated Protein Kinase) pathways in regulating caspase-3 expression and activity has been evaluated by immunohistochemical and Western blotting analyses, respectively. Level of phosphorylation of IkBalpha and IAP-1 expression were substantial in 8-day-old hypoxic hearts, suggesting the persistence of NF-kB driven hypertrophic signal along with a rescue attempt against hypoxic stress. In contrast, ASK-1 mediated JNK/SAPK activation, regulating Bcl(2) levels, allows Bax homodimerization and caspase-3 activation in the same experimental conditions. Thus, a regulation carried out by NF-kB and JNK/SAPK pathways on caspase-3 activation at day 8 of neonatal life can be suggested as the main factor for the heart 'adaptive' response to hypoxia.

Expression and localization of caveolins during postnatal development in rat heart: implication of thyroid hormone

Caveolins modulate signaling pathways involved in cardiac development. Caveolin-1 exists in two isoforms: the beta-isoform derivates from an alternative translational start site that creates a protein truncated by 31 amino acids, mainly expressed in endothelial cells, whereas caveolin-3 is present in muscle cells. Our aim was to define caveolin distribution and expression during cardiac postnatal development using immunofluorescence and Western blotting. Caveolin-3 sarcolemmal labeling appeared as dotted lines from days 1 to 5 and as continuous lines after 14 days of age. Caveolin-3 expression, low at birth, increased (4-fold) to reach a maximum (P < 0.05) by day 5 and then decreased to stabilize in adults. Total caveolin-1 and its alpha-isoform were codistributed at birth in endothelial and smooth muscle cells; afterward, only the caveolin-1alpha labeling became limited to endothelium. Quantitative analysis indicated a similar temporal pattern of both total caveolin-1 and caveolin-1alpha expression, suggesting that caveolin-1alpha and -1beta are coregulated; the caveolin-1alpha levels increased fourfold by day 5 to reach a maximum by day 14 (P < 0.05). Tyrosine-14-caveolin-1 phosphorylation, low at birth, increased suddenly around day 14 (8-fold vs. day 1) and returning afterward to basal level. Because the T3/T4 level is maximal by day 14, caveolin-1 expression/phosphorylation profiles were analyzed in hypothyroid heart. The levels of caveolin-1alpha and consequently tyrosine-14-caveolin-1 phosphorylation, but not that of caveolin-3, decreased (50%) in hypothyroid 14-day-old rats. Our data demonstrate that, during postnatal cardiac growth, 1) caveolins are distinctly regulated, and 2) thyroid hormones are involved in caveolin-1alpha expression.

The Effect of Vascular Endothelial Growth Factor on in vitro Embryonic Heart Development in Rats

In vitro effects of vascular endothelial growth factor (VEGF) on heart development and total embryonic growth were investigated in 84 rat embryos (obtained from nine pregnant females) at 9.5 days of gestation that were cultured in whole rat serum (WRS), in <30 kDa + >50 kDa serum fractions [retenate (R)], and in R + VEGF. After 24-h culture, the embryos from each group were harvested and divided into two groups. One group was analysed morphologically and biochemically to obtain embryo protein content, the second group was serially sectioned and examined by light microscopy. Morphological score, embryo protein content, somite number and crown-rump length of embryos indicated that embryos cultured in R had significant embryonic retardation, whereas the addition of VEGF to R increased embryonic growth and development. The morphological scores for WRS, R and R + VEGF were 57.7 +/- 0.87, 46.6 +/- 1.90 and 52.1 +/- 0.97, somite numbers were 26.5 +/- 0.47, 20.1 +/- 0.63 and 24.4 +/- 0.46, crown-rump lengths were 3 +/- 0.07, 2.4 +/- 0.06 and 2.7 +/- 0.06 mm, and embryo protein contents were 160.5 +/- 7.41, 98.2 +/- 4.81 and 141.1 +/- 10.96 mug per embryo, respectively. The results of histological examination of heart development were similar. The hearts of embryos grown in R were unseptated and tubular. The atrioventricular endocardial cushions were incompletely developed. The addition of VEGF to R improved heart development. There were no gross morphological differences in the cardiac development between embryos grown in WRS and R + VEGF. In both groups, development of the muscular interventricular septum had begun. Development of the atrioventricular cushions was also similar in both groups and had caused narrowing of the atrioventricular canals, but the atrial septation was not observed.

Neuroendocrine properties of intrinsic cardiac adrenergic cells in fetal rat heart.

Intrinsic cardiac adrenergic (ICA) cells in developing rat heart constitute a novel adrenergic signaling system involved in cardiac regulation. Regulatory mechanisms of ICA cells remain to be defined. Immunohistochemical study of fetal rat hearts demonstrated ICA cells with catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). The mRNA of TH and PNMP was also detected in fetal rat hearts before sympathetic innervation. Immunoreactivity of norepinephrine transporter (NET) was localized to ICA cells in rat heart tissue and primary cell culture. For the functional study, the activity of intracellular Ca2+ concentration ([Ca2+]i) transients was quantified by a ratio fluorescent spectrometer in cultured ICA cells and myocytes. ICA cells generated spontaneous [Ca2+]i transients that were eliminated by tetrodotoxin or Ca(2+)-free solutions and showed greatly reduced amplitude with the addition of L-type Ca2+ channel blocker nifedipine. [3H]norepinephrine studies demonstrate release and uptake of norepinephrine. Functional interaction between catecholamines produced by the ICA cells and cocultured myocytes was evident by the effect of the beta-adrenergic blocker atenolol eliciting a dose-dependent reduction in the amplitude and frequency of [Ca2+]i transients of beating myocytes. Hypoxia inhibited [Ca2+]i transient activity of ICA cells, which subsequently produced a reoxygenation-mediated rebound augmentation of [Ca2+]i transients. We conclude that ICA cells are capable of catecholamine synthesis, release, and uptake. They generate spontaneous [Ca2+]i transient activity that can be regulated by oxygen tension. ICA cells may provide an alternative adrenergic supply to maintain cardiac contractile and pacemaker function at rest and during stress in the absence of sympathetic innervation.

Oxytocin in cardiac ontogeny.

Previous studies demonstrated the presence of oxytocin (OT) and oxytocin receptors (OTRs) in the heart. The present work provides results supporting a potential role of OT in cardiomyogenesis. Here, we show a maximal OT and OTR protein level in the developing rat heart at day 21 of gestation and postnatal days 1-4, when cardiac myocytes are at a stage of intense hyperplasia. Between postnatal days 1 and 66, OT decreased linearly in all heart chambers (4.1- to 6.6-fold). Correspondingly, immunocytochemistry demonstrated that OTRs, which were eminent in postnatal cardiomyocytes, declined with age to low levels in adults. Interestingly, in coronary vasculature, OTRs developed in endothelial cells at postnatal days 12 and 22 and achieved a plateau in adult rats. These findings suggest that OT can be involved in developmental formation of the coronary vessels. In vivo, the OT/OTR system in the fetal heart was sensitive to the actions of retinoic acid (RA), recognized as a major cardiac morphogen. RA treatment produced a significant increase (2- to 3-fold) both in the OT concentration and in the OT mRNA levels. Ex vivo, an OT antagonist inhibited RA-mediated cardiomyocyte differentiation of P19 embryonic stem cells. The decline of cardiac OT expression from infancy to adulthood of the rat and changes in cell types expressing OTR indicate a dynamic regulation of the OT system in the heart rather than constitutive expression. The results support the hypothesis that RA induces cardiomyogenesis by activation of the cardiac OT system.

Expression of the IKr components KCNH2 (rERG) and KCNE2 (rMiRP1) during late rat heart development.

To understand molecular mechanisms that regulate formation and maintenance of cardiac IKr (rapidly activating component of the delayed rectifier K+ current), we have investigated the spatiotemporal expression pattern of two rat potassium voltage-gated channels, namely subfamily H (eag-related), member2 (KCNH2) (alias name: rERG) and Isk-related family, member2 (KCNE2) (alias name: rMiRP1) during late embryonic development by means of the in situ hybridization technique. KCNE2 is transcribed predominantly in atrial und ventricular myocardium at stages E14.5-E18.5dpc and only a minor signal emerged in the tongue at E16.5dpc. In contrast, KCNH2 transcripts appeared in a less confined pattern with intense signals in atrial and ventricular myocardium, somites, spinal cord, bowel system, central nervous system and thymus at stages E14.5-E18.5dpc. Non-cardiac expression even exceeds the intensity of the cardiac signal, indicating that KCNH2 contributes to K+ currents in non-cardiac tissue as well. Transcription of the rat b-subunit KCNE2 is present in all regions of the fetal myocardium and co-distributes perfectly with transcription of the pore forming a-subunit KCNH2. It seems likely that KCNH2 and KCNE2 are linked to form cardiac IKr channels, associated to cardiogenesis and cardiomyocyte excitability.

The slow skeletal muscle troponin T gene is expressed in developing and diseased human heart.

Cardiac muscle development is characterised by the activation of contractile protein genes and subsequent modulation of expression resulting, ultimately, in the formation of a mature four-chambered organ. Myocardial gene expression is also altered in the adult in response to pathological stimuli and this is thought to contribute to the altered contractile characteristics of the diseased heart. We have examined the expression of the slow skeletal troponin T (TnT) gene in the human heart during development and in disease using whole mount in situ hybridisation and real-time quantitative (TaqMan) polymerase chain reaction (PCR). Slow skeletal TnT mRNA shows transitory and regional expression in the early foetal heart, which occurs at different times in atria and ventricles. In ventricular myocardium, expression is seen in the outer epicardial layer at a time when the coronary circulation is being established. Expression was detected at low levels in the adult human heart and was significantly increased in end-stage heart failure. Similarly, expression was readily detectable during early rat heart development and was up-regulated in pressure overload hypertrophy in adult. Together these data show for the first time that slow skeletal TnT mRNA is readily detectable during early human heart development. They further suggest that slow skeletal TnT may be responsive to myocardial stress and that elevated levels may contribute to myocardial dysfunction in adult disease. (Mol Cell Biochem 263: 91-97, 2004).

Expression of genes participating in regulation of fatty acid and glucose utilization and energy metabolism in developing rat hearts.

The heart is a unique organ that can use several fuels for energy production. During development, the heart undergoes changes in fuel supply, and it must be able to respond to these changes. We have examined changes in the expression of several genes that regulate fuel transport and metabolism in rat hearts during early development. At birth, there was increased expression of fatty acid transporters and enzymes of fatty acid metabolism that allow fatty acids to become the major source of energy for cardiac muscle during the first 2 wk of life. At the same time, expression of genes that control glucose transport and oxidation was downregulated. After 2 wk, expression of genes for glucose uptake and oxidation was increased, and expression of genes for fatty acid uptake and utilization was decreased. Expression of carnitine palmitoyltransferase I (CPT I) isoforms during development was different from published data obtained from rabbit hearts. CPT Ialpha and Ibeta isoforms were both highly expressed in hearts before birth, and both increased further at birth. Only after the second week did CPT Ialpha expression decrease appreciably below the level of CPT Ibeta expression. These results represent another example of different expression patterns of CPT I isoforms among various mammalian species. In rats, changes in gene expression followed nutrient availability during development and may render cardiac fatty acid oxidation less sensitive to factors that influence malonyl-CoA content (e.g., fluctuations in glucose concentration) and thereby favor fatty acid oxidation as an energy source for cardiomyocytes in early development.

Developmentally regulated switching of titin size alters myofibrillar stiffness in the perinatal heart.

Before birth, the compliance of the heart is limited predominantly by extracardiac constraint. Reduction of this constraint at birth requires that myocardial compliance be determined mainly by the heart's own constituents. Because titin is a principal contributor to ventricular passive tension (PT), we studied the expression and mechanics of cardiac-titin isoforms during perinatal rat heart development. Gel electrophoresis and immunoblotting revealed a single, 3.7-MDa, N2BA isoform present 6 days before birth and an additional, also previously unknown, N2BA isoform of 3.5 to 3.6 MDa expressed in the near-term fetus. These large isoforms rapidly disappear after birth and are replaced by a small N2B isoform (3.0 MDa) predominating in 1-week-old and adult rats. In addition, neonatal pig hearts showed large N2BA-titin isoforms distinct from those present in the adult porcine myocardium. By quantitative reverse transcriptase-polymerase chain reaction, developmentally expressed titin-mRNA species were detected in rat heart. Titin-based PT was much lower (approximately 15 times) in fetal than adult rat cardiomyocytes, and measured PT levels were readily predictable with a model of worm-like chain titin elasticity. Immunofluorescence microscopy showed the extensibility of the differentially spliced molecular spring regions of fetal/neonatal titin isoforms in isolated rat cardiomyofibrils. Whereas the titin-isoform shift by 700 kDa ensures high passive stiffness of the postnatal cardiac myofibrils, the expression of specific fetal/neonatal cardiac-titin isoforms may also have important functions for contractile properties, myofibril assembly or turnover, and myocardial signaling during perinatal heart development.

Maternal methamphetamine administration during pregnancy influences on fetal rat heart development

Methamphetamine (MAP) is one of the most abused drugs in Japan. The rate of MAP abuse by young women has recently reached more than 50 percent in adolescents. A major health concern is that these women will continue to use MAP during pregnancy. The purpose of this study was to investigate whether MAP administered to the mother during pregnancy would change the expression of α- and β- myosin heavy chain (MHC) mRNA in rat neonatal hearts, as detected by quantitative RT-PCR. In addition, morphological changes in the rat neonatal ventricles were examined. Pregnant rats were injected intraperitoneally with MAP (1 mg/kg/day) starting at day 0 of gestation and ending at day 21. There was a significant increase in α-MHC mRNA expression in the neonatal ventricular muscle in the experimental group compared with the control at postnatal day (P) 0 and 5. α-MHC mRNA expression in both groups was similar after P9. β-MHC mRNA expression was similar in both groups at P0. Postnatal β-MHC mRNA expression decreased rapidly, but significant alteration was not detected. Neonatal rats at P0 exhibited some cardiac changes, including hypertrophy, degeneration, and disarrangement of myofibers, but these lesions disappeared by P14. We conclude that chronic maternal administration of MAP changes the α- and β-MHC mRNA expression pattern in fetal and neonatal hearts, correlating with abnormal development, plasma level of hormones, and myocardial damage. At the same time, it is indicated that neonatal cardiomyocytes have reversibility.

Expression and distribution of the calcitonin receptor-like receptor in the developing rat heart.

During ontogenesis the 52 amino acid peptide adrenomedullin is first expressed in the heart and it is essential for normal cardiovascular development. Recent work suggests that most adrenomedullin effects are conveyed via the calcitonin receptor-like receptor (CRLR) in combination with appropriate receptor activity-modifying proteins (RAMPs). Here, we investigated the expression of these components during the development of the rat heart, focusing on the period of coronary vascular development. Using RT-PCR, transcripts for CRLR, RAMP1 and RAMP2 were detected at all stages from E 14 to adulthood. The distribution of CRLR was investigated by immunohistochemistry, and endothelial cells and their precursors identified with monoclonal antibodies against RECA-1 and flk-1. On E 14, intense CRLR immunoreactivity was observed in endothelial cells of the large vessels and the endocardial cushions at the AV-junction. Small CRLR immunoreactive cell clusters were located in the wall of the outflow tract and subepicardially in the ventricular wall. On E 16, tubes of CRLR immunoreactive cells formed a subepicardial plexus, from which they penetrated radially towards the trabecular network and entered at E 18. Smooth muscle cells of coronary arteries gained a moderate CRLR immunoreactivity at E 20 which persisted at this intensity up to P 8 and then decreased. At the same time, CRLR immunoreactivity of endothelial cells in coronary arteries vanished while those of coronary veins still exhibited intense CRLR immunoreactivity. These data suggest multiple functions of the adrenomedullin/CRLR signaling pathway in cardiac development, among which the most prominent appears to be the early outgrowth and proliferation of the immature endothelial cells of the coronary vasculature.

Correlations between protein kinase C ζ signaling and morphological modifications during rat heart development and aging

From birth to aging the heart undergoes functional changes reflecting biochemical and ultrastructural modifications which imply apoptosis. This is a physiological process resulting from genetic programs closely associated with development and aging. During development apoptosis eliminates redundant cells leading to heart remodeling, while during aging it eliminates damaged or exhausted cells. In the present paper we analyze some molecular mechanisms involved with heart morphological modifications, especially in the neonatal heart which displays different features in the subendocardial and myocardial area. The high number of subendocardial apoptotic cells and the inverted ratio of Bcl-2/Bax molecule expression in the two heart compartments led us to hypothesize a different metabolism in the myocardium as compared with subendocardium. Moreover, we propose that PKC ζ may mediate this different response by activating Nf-kB pathway and by maintaining the balance between hypertrophic growth and apoptosis involved with remodeling of neonatal heart. Further, we underline that in the aged heart, where this pathway is not activated, such balance is not maintained.