Proliferation of Cardiomyocytes and Interstitial Cells In the Cardiac Muscle of the Mouse During Pre- and Postnatal Development

Proliferation of cardiomyocytes and interstitial cells in the cardiac ventricle of the mouse during pre- and postnatal development was studied. Furthermore, the number of cardiomyocyte and interstitial cell nuclei per unit area was determined on histological sections. The labelling index of cardiomyocytes decreases from 23% on day 14 of gestation to about zero at 3 weeks after birth. the number of cardiomyocyte nuclei per unit area increases up to day 16 of gestation and then continuously declines. This coincides with the concept that the increase in size of the heart during early fetal life is mainly due to hyperplasia, while during late fetal life and after birth it is mainly, and during adult life exclusively, due to hypertrophy of cardiomyocytes. Proliferation of interstitial cells continues up to 5 days after birth and then decreases. the ratio of cardiomyocytes to interstitial cells decreases by a factor of about 10 between day 14 of gestation and 3 weeks after birth.     

Alcohol exposure during late gestation adversely affects myocardial development with implications for postnatal cardiac function

Prenatal exposure to high levels of ethanol is associated with cardiac malformations, but the effects of lower levels of exposure on the heart are unclear. Our aim was to investigate the effects of daily exposure to ethanol during late gestation, when cardiomyocytes are undergoing maturation, on the developing myocardium. Pregnant ewes were infused with either ethanol (0.75 g/kg) or saline for 1 h each day from gestational days 95 to 133 (term ～145 days); tissues were collected at 134 days. In sheep, cardiomyocytes mature during late gestation as in humans. Within the left ventricle (LV), cardiomyocyte number was determined using unbiased stereology and cardiomyocyte size and nuclearity determined using confocal microscopy. Collagen deposition was quantified using image analysis. Genes relating to cardiomyocyte proliferation and apoptosis were examined using quantitative real-time PCR. Fetal plasma ethanol concentration reached 0.11 g/dL after EtOH infusions. Ethanol exposure induced significant increases in relative heart weight, relative LV wall volume, and cardiomyocyte cross-sectional area. Ethanol exposure advanced LV maturation in that the proportion of binucleated cardiomyocytes increased by 12%, and the number of mononucleated cardiomyocytes was decreased by a similar amount. Apoptotic gene expression increased in the ethanol-exposed hearts, although there were no significant differences between groups in total cardiomyocyte number or interstitial collagen. Daily exposure to a moderate dose of ethanol in late gestation accelerates the maturation of cardiomyocytes and increases cardiomyocyte and LV tissue volume in the fetal heart. These effects on cardiomyocyte growth may program for long-term cardiac vulnerability.     

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.     

The fate of fetal Leydig cells during the development of the fetal and postnatal rat testis

The ultrastructure and developmental fate of the fetal generation of Leydig cells of the rat testis was studied from the 17th day of fetal life up to 100 days after birth. The number of fetal Leydig cells per testis was determined by light microscopic morphometric analysis of semithin plastic sections. In fetal testes (days 17-22 postconception), Leydig cells exhibited a characteristic ultrastructure, containing smooth endoplasmic reticulum, many lipid inclusions and glycogen. Testes of 17-day-old fetuses contained about 25 x 10(3) fetal Leydig cells, rapidly increasing to 90 x 10(3) per testis in 21-day-old fetuses. After birth, fetal Leydig cells per testis remained relatively constant up to 2 weeks (80-90 x 10(3) per testis) and were identified by light and electron microscopy which showed their numerous lipid inclusions, their tendency for clustering and their association with interstitial tissue fibroblasts which partly encapsulated the fetal Leydig cells. From 21-100 days after birth, fetal Leydig cell numbers were quite variable with a mean of 45-60 x 10(3) per testis. These results are the first to show that the fetal generation of Leydig cells persist in the adult testis and do not undergo early postnatal degeneration or dedifferentiation into other interstitial cells. The simultaneous occurrence of the fetal Leydig cells and the adult population of Leydig cells indicates that these cells are distinct cell generations which are developmentally unrelated.     

Cell cycle regulation in mouse heart during embryonic and postnatal stages

The regulation of cardiomyocyte proliferation is important for heart development and function. Proliferation levels of mouse cardiomyocytes are high during early embryogenesis and start to decrease at midgestation. Many cardiomyocytes undergo mitosis without cytokinesis, resulting in binucleated cardiomyocytes during early postnatal stages, following which the cell cycle arrests irreversibly. It remains unknown how the proliferation pattern is regulated, and how the irreversible cell cycle arrest occurs. To clarify the mechanisms, fundamental information about cell cycle regulators in cardiomyocytes and cell cycle patterns during embryonic and postnatal stages is necessary. Here, we show that the expression, complex formation, and activity of main cyclins and cyclin‐dependent kinases (CDKs) changed in a synchronous manner during embryonic and postnatal stages. These levels decreased from midgestation to birth, and then showed one wave in which the peak was around postnatal day 5. Detailed analysis of the complexes suggested that CDK activities were inhibited before the protein levels decreased. Analysis of DNA content distribution patterns in mono‐ and binucleated cardiomyocytes after birth revealed changes in cell cycle distribution patterns and the transition from mono‐ to binucleated cells. These analyses indicated that the wave of cell cycle regulator expression or activities during postnatal stages mainly produced binucleated cells from mononucleated cells. The data obtained should provide a basis for the analysis of cell cycle regulation in cardiomyocytes during embryonic and postnatal stages.     

Cyto- and synaptogenesis in the arcuate nucleus of the rat hypothalamus during fetal and early postnatal life

Summary Morphogenesis of the arcuate nucleus of the rat from the 15th fetal day to the 6th postnatal day was investigated light and electron microscopically. The arcuate neurons exhibit a gradual development after the 15th fetal day. All cytoplasmic constituents are present in these nerve cells already during the last days of gestation. Nevertheless, they are not fully differentiated at birth. The first synapse-like structures (presynapses) were observed in 17 day-old, the first synapses in 18 day-old fetuses. During the early postnatal period the number of presynapses decreases, but at the same time there is a gradual increase in the number of the relatively mature synapses. This process starts already during the last days of prenatal life. Although all structural elements of the arcuate nucleus of the adult rat appear to be present at birth, the extent of the neuropil area and the number of the presynapses indicate that the arcuate nucleus is still in a fairly undeveloped stage during the first postnatal days.     

Newborn Hypoxia/Anoxia Inhibits Cardiomyocyte Proliferation and Decreases Cardiomyocyte Endowment in the Developing Heart: Role of Endothelin-1

In the developing heart, cardiomyocytes undergo terminal differentiation during a critical window around birth. Hypoxia is a major stress to preterm infants, yet its effect on the development and maturation of the heart remains unknown. We tested the hypothesis in a rat model that newborn anoxia accelerates cardiomyocyte terminal differentiation and results in reduced cardiomyocyte endowment in the developing heart via an endothelin-1-dependent mechanism. Newborn rats were exposed to anoxia twice daily from postnatal day 1 to 3, and hearts were isolated and studied at postnatal day 4 (P4), 7 (P7), and 14 (P14). Anoxia significantly increased HIF-1α protein expression and pre-proET-1 mRNA abundance in P4 neonatal hearts. Cardiomyocyte proliferation was significantly decreased by anoxia in P4 and P7, resulting in a significant reduction of cardiomyocyte number per heart weight in the P14 neonates. Furthermore, the expression of cyclin D2 was significantly decreased due to anoxia, while p27 expression was increased. Anoxia has no significant effect on cardiomyocyte binucleation or myocyte size. Consistently, prenatal hypoxia significantly decreased cardiomyocyte proliferation but had no effect on binucleation in the fetal heart. Newborn administration of PD156707, an ET_A-receptor antagonist, significantly increased cardiomyocyte proliferation at P4 and cell size at P7, resulting in an increase in the heart to body weight ratio in P7 neonates. In addition, PD156707 abrogated the anoxia-mediated effects. The results suggest that hypoxia and anoxia via activation of endothelin-1 at the critical window of heart development inhibits cardiomyocyte proliferation and decreases myocyte endowment in the developing heart, which may negatively impact cardiac function later in life.     

Knockdown of cyclin-dependent kinase inhibitors induces cardiomyocyte re-entry in the cell cycle

Proliferation of mammalian cardiomyocytes stops rapidly after birth and injured hearts do not regenerate adequately. High cyclin-dependent kinase inhibitor (CKI) levels have been observed in cardiomyocytes, but their role in maintaining cardiomyocytes in a post-mitotic state is still unknown. In this report, it was investigated whether CKI knockdown by RNA interference induced cardiomyocyte proliferation. We found that triple transfection with p21(Waf1), p27(Kip1), and p57(Kip2) siRNAs induced both neonatal and adult cardiomyocyte to enter S phase and increased the nuclei/cardiomyocyte ratio; furthermore, a subpopulation of cardiomyocytes progressed beyond karyokynesis, as assessed by the detection of mid-body structures and by straight cardiomyocyte counting. Intriguingly, cardiomyocyte proliferation occurred in the absence of overt DNA damage and aberrant mitotic figures. Finally, CKI knockdown and DNA synthesis reactivation correlated with a dramatic change in adult cardiomyocyte morphology that may be a prerequisite for cell division. In conclusion, CKI expression plays an active role in maintaining cardiomyocyte withdrawal from the cell cycle.     

Regulation of the cardiomyocyte population in the developing heart

During fetal life the myocardium expands through replication of cardiomyocytes. In sheep, cardiomyocytes begin the process of becoming terminally differentiated at about 100 gestation days out of 145 days term. In this final step of development, cardiomyocytes become binucleated and stop dividing. The number of cells at birth is important in determining the number of cardiomyocytes for life. Therefore, the regulation of cardiomyocyte growth in the womb is critical to long term disease outcome. Growth factors that stimulate proliferation of fetal cardiomyocytes include angiotensin II, cortisol and insulin-like growth factor-1. Increased ventricular wall stress leads to short term increases in proliferation but longer-term loss of cardiomyocyte generative capacity. Two normally circulating hormones have been identified that suppress proliferation: atrial natriuretic peptide (ANP) and tri-iodo-l-thyronine (T₃). Atrial natriuretic peptide signals through the NPRA receptor that serves as a guanylate cyclase and signals through cGMP. ANP powerfully suppresses mitotic activity in cardiomyocytes in the presence of angiotensin II in culture. Addition of a cGMP analog has the same effect as ANP. ANP suppresses both the extracellular receptor kinases and the phosphoinositol-3 kinase pathways. T₃ also suppresses increased mitotic activity of stimulated cardiomyocytes but does so by increasing the cell cycle suppressant, p21, and decreasing the cell cycle activator, cyclin D1.     

Temporal Characteristics of the Differentiation of Embryonic Erythroid Cells in Fetal Peripheral Blood of the Syrian Hamster

The morphological changes in erythroid cells and their nuclei in the circulation of fetuses of the Syrian hamster were investigated by use of an image-processing system. The analysis included monitoring of nuclear condensation, nuclear periphralization (access of the nucleus to the cell membrane), enucleation, density of cells, and changes in cell size from day 9 of gestation to day 5 after birth. The yolk-sac-derived erythroid cells made rapid progress in nuclear condensation on day 11, while this process proceeded at a much lower rate after day 12 of gestation. The peripheralization of nuclei started on day 10 and reached a maximum on day 11. The frequency of enucleated cells was below 2% on day 11, while it increased to 30% on day 12. Extruded nuclei, most of which were accompanied by a small quantity of cytoplasm, appeared in the circulation on day 12. The most frequently observed diameter of enucleated erythrocytes, which was 10–10.5 μm on day 12, fell gradually to 8–9 μm on day 14. By contrast, the shift from fetal liver erythrocytes to adult erythrocytes occurred in a discontinuous manner. Adult-type erythrocytes were detected after birth with diameters of 5.5–6 μm. Our data allows us to present the schedule of morphological changes during embryonic erythropoiesis and show that the developmental behavior of "primitive" yolk-sac-derived erythroid cells is more closely correlated with that of the "definitive" fetal liver cells than has been considered to be the case to date.     

Diffusion MRI Tractography of the Developing Human Fetal Heart

Objective

Human myocardium has a complex and anisotropic 3D fiber pattern. It remains unknown, however, when in fetal life this anisotropic pattern develops and whether the human heart is structurally fully mature at birth. We aimed here to use diffusion tensor MRI (DTI) tractography to characterize the evolution of fiber architecture in the developing human fetal heart.

Methods

Human fetal hearts (n = 5) between 10–19 weeks of gestation were studied. The heart from a 6-day old neonate and an adult human heart served as controls. The degree of myocardial anisotropy was measured by calculating the fractional anisotropy (FA) index. In addition, fiber tracts were created by numerically integrating the primary eigenvector field in the heart into coherent streamlines.

Results

At 10–14 weeks the fetal hearts were highly isotropic and few tracts could be resolved. Between 14–19 weeks the anisotropy seen in the adult heart began to develop. Coherent fiber tracts were well resolved by 19 weeks. The 19-week myocardium, however, remained weakly anisotropic with a low FA and no discernable sheet structure.

Conclusions

The human fetal heart remains highly isotropic until 14–19 weeks, at which time cardiomyocytes self-align into coherent tracts. This process lags 2–3 months behind the onset of cardiac contraction, which may be a prerequisite for cardiomyocyte maturation and alignment. No evidence of a connective tissue scaffold guiding this process could be identified by DTI. Maturation of the heart’s sheet structure occurs late in gestation and evolves further after birth.     

Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover

Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously ¹⁴C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that ¹⁴C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans.     

Protein synthesis in cardiac cells and the ultrastructural dynamics of cardiomyocytes of hibernating animals during the hibernation cycle

A significant increase in protein synthesis correlating with ultrastructural dynamics of cardiomyocyte organelle convertions has been demonstrated in cardiomyocytes of ground squirrel during arousal from hibernation. In hibernating ground squirrels, the ultrastructure of protein-synthesizing organelles and of the cardiomyocyte nucleus points out to the readiness of cells to active synthesis of proteins. In the perinuclear area of cardiomyocytes abundant ribosomes, elements of endoplasmic reticulum and Golgi complex, mitochondria and high-energy substrates--glycogen and lipid inclusions--are seen. The cardiomyocyte nuclei are large, with highly convoluted borders and abundant pores, their nucleolar structure is granular, the chromatin is mainly diffuse. The potency of cardiomyocyte protein-synthesizing system of hibernating ground squirrels is realized every time at periodical arousals during hibernation. The role of cyclic changes of protein synthesis rate in adaptation of cells of hibernating mammals to functioning at various temperatures is discussed.     

Cytochemical and biochemical studies on the differentiation of microperoxisomes in the small intestine of the fetal mouse

The localization of catalase activity during the morphogenesis of duodenum and ileum has been studied in Swiss ICR mouse embryos from the 16th day of fetal life until birth. Catalase activity was also measured by a spectrophotometric method. Few diaminobenzidine-positive microperoxisomes are present at 15 days of gestation in undifferentiated cells of the stratified epithelium lining the lumen of duodenum and ileum. The number of microperoxisomes increases considerably in the duodenal enterocytes at 17 days; the highest concentration of microperoxisomes is attained at 18 days, after which time their number becomes stable until 4 weeks after birth. Biochemically, catalase activity is barely detected at 15 days in the first half of the small intestine, but afterwards it increases steadily up to 1 day after birth. In the ileum, the increase in microperoxisome number is far less important than in the duodenal enterocytes and reaches a maximum at 19 days of gestation, that is, immediately at birth. The level of catalase activity in the second half of the small intestine is also much lower than that measured in the first half. These results are discussed in relation to the biogenesis of microperoxisimes in the small intestine before birth.     

Morphometric and fine-structural studies of rat pancreatic acinar cells during early postnatal life

Summary Pancreatic acinar cells of rats obtained at 1,2, 3, 5, 7 and 14 days of age were examined using fine structural and morphometric techniques. From 5 days of age onwards, the acinar cells were analysed twice per day, at 20.00 h and 08.00 h.The present study demonstrates changes in the average volume of the cell, nucleus and cytoplasm, and volume densities of various cytoplasmic organelles during the first two weeks after birth. During early postnatal life, the volume density of rER increases, whereas that of zymogen granules decreases. From 5 days of age onwards, the volume densities of these two organelles differ significantly at 20.00 h and 08.00 h. During the first 2–3 days after birth, inclusion body-like structures appear in the cytoplasm of acinar cells; they contain aggregated zymogen granules and, sometimes, amorphous structures or cytoplasmic organelles. These structures also occur in interstitial cells and cells located in the intercalated region between acinar and ductal epithelial cells. Serum level of -amylase activity is high at birth, compared with other stages during the first three weeks. Degenerating acinar cells and cell debris can be seen in the acinar and ductal lumina during these stages, a feature suggesting holocrine secretion. Cellular polarity appears to be incomplete during the first two or three days after birth.     

Vitamin D-dependent calcium-binding protein. Changes during gestation, prenatal and postnatal development in rats

During the perinatal period, calcium metabolism is stressed. As intestinal Ca-binding protein is considered as a molecular expression of the hormonal effect of 1,25-dihydroxycholecalciferol (1,25(OH)2D3), Ca-binding protin measurements may document the vitamin D roles during this period. We describe the variations of Ca-binding protein concentrations in the rat during the last 5 days of gestation, in the maternal duodenum, placentas, fetal membranes and fetal intestines. We also report intestinal Ca-binding protein changes from birth until weaning. The evolution of the maternal intestinal Ca-binding protein, which increases on day 19.5 of gestation, is consistent with that of calcium intestinal absorption and may be explained by increased 1,25(OH)2D3 production. Placental Ca-binding protein rises from day 17.5 until the end of gestation, and may be related to the profile of calcium transfer from mother to fetuses. It is noteworthy that the placental Ca-binding protein is predominantly found in the fetal part of the organ where materno-fetal exchanges occur. The yolk sac synthesizes substantial amounts of Ca-binding protein. In the fetal membranes, Ca-binding protein plateaus from day 17.5 until day 20.5 and decreases on day 21.5. The Ca-binding protein presence in the fetal placenta and in the yolk sac may suggest that these tissues are also targets for vitamin D. In the fetus the intestinal Ca-binding protein s is detected as early as day 17.5 of gestation and increases markedly during the last day of gestation. From birth and during the first 3 weeks of postnatal life, the intestinal Ca-binding protein concentration does not change. It undergoes a sharp rise just at the time of weaning. We have also shown that the specific distribution of Ca-binding protein along the intestine is acquired during intrauterine life and does not change with sucking or weaning. The two main changes of intestinal Ca-binding protein, observed just before birth and at weaning, may reflect the intestinal maturation and/or variations in vitamin D metabolism.     

Cell proliferation patterns during development of the equine placenta

Placentation involves considerable growth and reorganization of both maternal and fetal tissues. In this investigation, immunohistochemical localization of the proliferation marker Ki-67 antigen was used to monitor cell division during placentation in mares. Endometrial biopsies were obtained from eight mares between day 14 and day 26 of pregnancy and from eight anoestrous mares that had been treated with various combinations of progesterone and oestrogen. Samples of endometrium and fetal membranes were obtained from 19 mares carrying normal horse conceptuses between day 30 and day 250 of gestation and from three failing extraspecific donkey-in-horse pregnancies. Proliferation in the superficial strata of the endometrium was increased by day 18 of gestation and this effect could be mimicked by supplementing with oestradiol benzoate during the last 6 days of a prolonged period (18-36 days) of progesterone administration. Fetal chorionic girdle cells were proliferating vigorously at days 30-32 of gestation, but stopped dividing after they invaded the endometrium, while the trophoblast cells of the allantochorion showed an increase in mitotic activity after day 38. The luminal epithelium of the endometrium started to proliferate only after the primary villi of the true epitheliochorial placenta had been formed, and during days 58-70 this effect was seen only in the pregnant horn in which placentation was further advanced. During the second half of gestation, most of the mitotic activity was confined to the periphery of the microcotyledons which were still growing. In the donkey-in-horse pregnancies, proliferation rates of the maternal and fetal epithelial at day 70 of gestation were markedly reduced in areas of heavy endometrial lymphocyte infiltration and poor placentation. These results provide a basis for further studies on factors that influence invasive and non-invasive placentation.     

IGF-2R-mediated signaling results in hypertrophy of cultured cardiomyocytes from fetal sheep

Activation of the insulin-like growth factor-1 receptor (IGF-1R) is known to play a role in cardiomyocyte hypertrophy. While IGF-2R is understood to be a clearance receptor for IGF-2, there is also evidence that it may play a role in the induction of pathological cardiomyocyte hypertrophy. It is not known whether IGF-2R activates cardiomyocyte hypertrophy during growth of the fetal heart. Fetal sheep hearts (125 ± 0.4 days gestation) were dissected, and the cardiomyocytes isolated from the left and right ventricles for culturing. Cultured cardiomyocytes were treated with either LONG R(3)IGF-1, an IGF-1R agonist; picropodophyllin, an IGF-1R autophosphorylation inhibitor; U0126, an inhibitor of extracellular signal-regulated protein kinase (ERK); Leu(27)IGF-2, an IGF-2R agonist; G?6976, a protein kinase C inhibitor; KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII); or KN-92, an L-type calcium channel inhibitor and negative control for KN-93. The cross-sectional area of cultured cardiomyocytes was determined relative to control cardiomyocytes treated with serum-free culture medium. IGF-1R and IGF-2R activation each resulted in ERK signaling, but IGF-2R activation alone induced CaMKII signaling, resulting in hypertrophy of cardiomyocytes in the late gestation sheep fetus. These data suggest that changes in the intrauterine environment that result in increased cardiac IGF-2R may also lead to cardiomyocyte hypertrophy in the fetus and potentially an increased risk of cardiovascular disease in adult life.     

Changes in cardiac troponins with gestational age explain changes in cardiac muscle contractility in the sheep fetus

The development of the adult cardiac troponin complex in conjunction with changes in cardiac function and cardiomyocyte binucleation has not been systematically characterized during fetal life in a species where maturation of the cardiomyocytes occurs prenatally as it does in the human. The aim of this study was to correlate the expression of each of the major adult troponin isoforms (T, I, and C) during late gestation (term of 150 days) to changes in both Ca(2+) sensitivity and maximum Ca(2+)-activated force of the contractile apparatus and the maturation of cardiomyocytes. The percentage of mononucleated cardiomyocytes in the right ventricle decreased with gestational age to 46% by 137-142 days of gestation. The length of binucleated cardiomyocytes did not change with gestational age, but the length of binucleated cardiomyocytes relative to heart weight decreased with gestational age. There was no change in the expression of adult cardiac troponin T with increasing gestation. The contractile apparatus was significantly more sensitive to Ca(2+) at 90 days compared with either 132 or 139 days of gestation, consistent with an ～30% increase in the expression of adult cardiac troponin I between 90 and 110 days of gestation. Maximum Ca(2+)-activated force significantly increased from 90 days compared with 130 days consistent with an increase of ～40% in cardiac troponin C protein expression. These data show that increased adult cardiac troponin I and C protein expression across late gestation is consistent with reduced Ca(2+) sensitivity and increased maximum Ca(2+)-activated force. Furthermore, changes in cardiac troponin C, not I, protein expression track with the timing of cardiomyocyte binucleation.     

Normal lactational environment restores cardiomyocyte number after uteroplacental insufficiency: implications for the preterm neonate

A reduced complement of cardiomyocytes in early life can adversely affect life-long cardiac functional reserve. In the present study, using a cross-fostering approach in rats, we examined the contributions of the prenatal and postnatal environments in the programming of cardiomyocyte growth. Rat dams underwent either bilateral uterine vessel ligation (Restricted) or sham surgery (Control) on day 18 of gestation. One day after birth, Control and Restricted pups were cross-fostered onto Control (normal lactation) or Restricted (impaired lactation due to impaired mammary gland formation) mothers. In male offspring, genes involved in cardiomyocyte differentiation, proliferation, hypertrophy and apoptosis were examined at gestational day 20 and postnatal days 1 and 7 to assess effects on cardiomyocyte growth. At postnatal day 7 cardiomyocyte number was determined stereologically. Offspring were examined at age 6 mo for evidence of hypertension and pathological cardiac gene expression. There was an increase in Igf1 and Igf2 mRNA expression in hearts of Restricted pups at gestational day 20. At postnatal day 7, Agtr1a and Agtr1b mRNA expression as well as Bcl2 and Cmyc were elevated in all hearts from offspring that were prenatally or postnatally growth restricted. There was a significant reduction (-29%) in cardiomyocyte number in the Restricted-on-Restricted group. Importantly, this deficit was prevented by optimization of postnatal nutrition (in the Restricted-on-Control group). At 6 mo, blood pressure was significantly elevated in the Restricted-on-Restricted group, but there was no difference in expression of the cardiac hypertrophy, remodeling or angiogenic genes across groups. In conclusion, the findings reveal a critical developmental window, when cardiomyocytes are still proliferating, whereby improved neonatal nutrition has the capacity to restore cardiomyocyte number to normal levels. These findings are of particular relevance to the preterm infant who is born at a time when cardiomyocytes are immature and still dividing.