Heart ventricular cells of neonatal rats in a culture: DNA synthesis, ultrastructure and localization of atrial natriuretic peptide

We determined the optimal conditions suitable for expanding cardiac cells in vitro for their future use in experimental transplantation into injured myocardium of adult animals. Ventricular cardiac cells were isolated enzymatically from 2-3 day-old rats and cultured at different cell densities within 5-7 days to 4 weeks. Mixed cultures of muscle and non-muscle cells were examined by light autoradiography, electron microscopy, and immunogold method. The best results were obtained at a density of 3 x 10(5) cells/ml in the medium, consisting of 90% DMEM and 10% fetal calf serum, during 5-7 days of cultivation. In such cultures myocytes made 62.5 +/- 7.9%. After a 24 h incubation with 3H-thymidine, 22.0 +/- 2.2% of myocytes were labeled. Muscle cells contact with each other and with non-muscle cells, contain myofibrils, contract and display atrial natriuretic peptide (ANP)-like immunoreactivity.     

Regeneration and DNA synthesis in cultures of rat heart ventricles after damage to the cellular layer

By means of the 3H-thymidine radioautography method replicative activity of myocytes and other cells in a dense layer of cellular culture of the traumatized ventricular myocardium has been studied in newborn (2-3-day-old) rats after lesion of the myocardium. Proliferation of the non-muscular cells is sharply suppressed after the cellular layer formation: their labelling index (LI) is 5.7 +/- 2.2% on the 8th day. Simultaneously, LI of unicellular myocytes is 25 +/- 6%, and that of binuclear ones, specific for the myocardium of mature animals is 6.4 +/- 2.9%. That demonstrates autonomy of DNA synthesis in myocytes from its suppression in the surrounding non-muscular cells. For 1-2 days after trauma intensive migration of fibroblasts into the wound is observed; they are often oriented perpendicularly to the edges of the cellular layer. There is an activation of DNA synthesis in the non-muscular cells in the wound area (in 20 h after the lesion LI is 73.4 +/- 1.7%) and in the layer edge directed to the wound LI is 23.9 +/- 0.0%, while in the depth of the zone situating close to the wound their LI is 7.8 +/- 2.4%. The replicative activity of mononuclear cardiomyocytes in the zone mentioned increases very weakly (LI 32 +/- 5%), and LI of binuclear ones is practically unchanged (6.1 +/- 2.3%). Karyo-kinetic activity is estimated by amount of binuclear cardiomyocytes and in the zone near the wound, in comparison to that in an intact layer, it does not change (35 +/- 3% and 34.4 +/- 2.5%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in binucleation and cellular dimensions of rat left atrial myocytes after induced left ventricular infarction

The left atrium of young rats has previously been demonstrated to respond with DNA synthesis and binucleation 11 days after left ventricular infarction. This investigation was designed to examine the hypertrophic response of the left atrial myocyte of the rat at 20 and 60 days after ventricular infarction. Male Sprague-Dawley rats were subjected to left coronary artery ligation (CAL) or sham operation. Following enzymatic separation, left atrial myocytes were examined at 20 and 60 days postoperation for number of nuclei and cellular dimensions (cell length, width and area, and nuclear area). Results demonstrated that the level of binucleation at 20 days (77.3%) and 60 days (71.3%) was nearly twice that observed in sham-operated animals, which were 33.1% binucleated at 20 days and 43.5% binucleated at 60 days. In both mononucleated and binucleated myocytes, the mean lengths, widths, and cell areas from CAL hearts were significantly greater than those of corresponding sham-operated animals. In all cases, these values were larger in binucleated myocytes than in mononucleated cells. The mean area of CAL cells was approximately twice that of sham-operated myocytes. With regard to mean lengths and widths, although both were greater in the CAL animals, there was a decrease in length and increase in width between 20 and 60 days in the CAL group. Mean nuclear areas were significantly greater in CAL myocytes than in those from the sham-operated group. These increases in nuclear number and cellular dimensions of the atrial myocyte are prominent features of the response to the stress imposed by left ventricular infarction.     

Human fetal ventricular cardiomyocytes in vitro: proliferation and differentiation

In this study, in the primary cell culture of human fetal cardiomyocytes proliferation of myocytes combines with their differentiation. The cells were isolated enzymatically from 19-22 week-old human fetuses and cultured for 14 days. DNA synthesis, ultrastructure and presence of atrial natriuretic peptide (ANP) were examined. In 7 day-old culture, the myocytes make about 60%, in 14 day-old culture--about 50%. Myocytes synthesize DNA and divide mitotically. After a 24 h incubation with 3H-thymidine in 7 day-old culture 1.8 +/- 0.5% of muscle and 25.2 +/- 11.7% of non-muscle cells are labeled, in 14 day-old culture--2.5 +/- 0.5 and 8.1 +/- 1.7% of cells are labeled, respectively. In 7 and 14 day-old cultures the degree of redifferentiation of contractile apparatus in myocytes varies from scattered actin and myosin filaments surrounded by ribosomes to differentiating myofibrils with distinct sarcomeres and Z-discs. Single electron-dense granules, morphologically similar to secretory atrial granules, display ANP-immunoreactivity. Thus, human fetal ventricular cardiomyocytes in cell culture proliferate, differentiate and synthesize ANP for 14 days; this is indicative of vitality of these cells.     

The atrial proliferative response following partial ventricular amputation in the heart of the adult newt. A light and electron microscopic autoradiographic study

This investigation characterizes the atrial proliferative response following partial ventricular amputation in adult newts. Newts processed for light microscopic autoradiography were given either a single injection (SI) of ³H-thymidine 1 hr before fixation and killed at intervals up to 25 days after ventricular wounding or were given six injections (MU), one every 12 hr, and fixed at intervals up to 21 days. Atria processed for EM autoradiography (EMA) were removed 1 hr after injection and 15 days after wounding. Mitotic (MI) and thymidine-labeling indices (TI) were calculated for the epicardium, subepicardial CT and myocardium of both atria. Sham-operated and unoperated animals served as controls. There was no localization of labeled or mitotic cells within the atria of SI or MU animals (P > 0.16) for any cell type. MI and TI for the epicardial and CT cells did not differ from sham-operated controls (P > 0.35). A maximum TI of 6.4% and MI of 0.4% was observed in the atrial myocardium of SI animals on day 15. A maximum TI of 13.8 and 5.9% was observed for the left and right atrial myocardium, respectively, of MI animals on day 12. EMA confirmed that atrial myocytes were engaging in mitosis and DNA synthesis.     

Mononucleated and binucleated cardiomyocytes in left atrium and pulmonary vein have different electrical activity and calcium dynamics

Cardiomyocytes consist of single- and bi-nucleus myocytes. However, the electrophysiological characteristics of mononucleated and binucleated myocytes have never been elucidated. Left atrium (LA) and pulmonary veins (PVs) are important substrate and initiators of atrial fibrillation. The purposes of this study were to evaluate the electrical properties and calcium homeostasis in mononucleated and binucleated cardiomyocytes in the LA and PVs. A whole-cell clamp, fluo-4 fluorescence, and immunocytostaining were used to investigated mononucleated and binucleated cardiomyocytes in the LA and PVs. Both mononucleated PV and LA cardiomyocytes had more positive resting membrane potential than respective PV and LA binucleated cardiomyocytes. Additionally, mononucleated PV cardiomyocytes (n = 36) had faster beating rates (2.1 ± 0.2 Hz versus 1.0 ± 0.2 Hz, P < 0.05) than binucleated (n = 10) PV cardiomyocytes. The PV (n = 18) and LA (n = 15) mononucleated cardiomyocytes had larger [Ca2?](i) transients (F/F? 1.64 ± 0.09 versus 1.20 ± 0.03 IU, P < 0.05; 1.52 ± 0.06 versus 1.19 ± 0.05 IU, P < 0.05) than the binucleated PV (n = 10) and LA (n = 10) cardiomyocytes. The immunostaining showed that mononucleated cardiomyocytes had lower Kir 2.3 and higher ryanodine receptor densities than did binucleated cardiomyocytes both in the PV and LA. In conclusions, mononucleated PV and LA cardiomyocytes contain distinctive electrophysiological characteristics with a higher arrhythmogenic activity, which indicates that cell nucleus number may potentially determine the electrical activity and calcium handling in cardiomyocytes.     

Formation of tetraploid nuclei in regenerating rat liver]

The proliferation of binucleated cells in the liver of young Wistar rats after partial (2/3) hepatectomy was studied by means of autoradiography and cytophotometry. The analysis of the kinetics of 3H-thymidine labelled cells has shown that both the bi- and mononucleated cells proceed through the mitotic cycle and enter mitosis simultaneously. The nuclei of 2nX2 cells enter prophase simultaneously but fuse during metaphase, so that the subsequent division results in the formation of mononucleated tetraploid cells.     

DNA synthesis in the mono- and binuclear cells of regenerating rat liver after x-ray irradiation

The effect of X-irradiation on the dynamics of DNA synthesis during the S-period in bi- and mononucleated of regenerating rat liver was studied autoradiographically and microphotometrically. Rats were treated with X-rays at doses 3.84 X 10(-2), 15.48 X 10(-2), and 30.96 X 10(-2) Kl/kg 23 hours after a partial hepatectomy, and were sacrificed one hour after irradiation. In the control liver the rate of DNA synthesis was the lowest at the beginning of the S-period and the highest at the last quarter of this period in both mono- and binucleated cells. The irradiation results in the inhibition of DNA synthesis mainly at the end of the S-period depending on doses employed. This inhibition was the same in bi- and mononucleated cells. In addition, the increase of correlation of the 3H-thymidine incorporation rate and DNA content was found between nuclei of binucleated cells after irradiation.     

DNA synthesis in rat heart cells after injury and the regeneration of myocardia

The regenerative responses of the myocardia of post-natal rats of different age groups (1, 2, 3 and 4 weeks old) to an injury made by a clinical electricator were studied. DNA synthesis and the ultrastructural organization of the cardiac myocytes of the injured myocardia were examined for an evaluation of the potential for regeneration of the developing myocardia. The maximum labeling index of cardiac myocytes was observed in 1-week-old rats showing 8% labeled myocytes 3 days after injury as opposed to 3.2, 2.2 and 0.2% indices in 2-, 3- and 4-week-old rats respectively, 3 days after injury. In subsequent days after injury the labeling indices declined considerably in all age group hearts, and attained values less than 1% labeled myocytes 30 days after injury with the lowest labeling index in the oldest age group heart. When DNA synthesis in uninjured myocardial tissue adjacent to the injured tissue was examined, it was found to be significantly lower than it was in the injured tissue. However, both injured and adjacent uninjured tissue attained a peak in the labeling indices 3 days after injury, with the exception of 3- and 4-week-old uninjured tissue. The overall incorporation of ³H-thymidine into the DNA of heart cells as revealed by scintillation counts showed that the rate of incorporation of the isotope in younger hearts was significantly higher than in the older hearts. Non-muscle cells contributed significantly to the rise of scintillation counts in hearts of all age groups.Ultrastructural analyses of 1- to 4-week-old hearts showed that 24 hr after injury, injured areas of myocardia were heavily crowded with macrophages that surrounded damaged myocytes. Later on, fibroblasts and other non-muscle cells predominated the injury sites along with fibrous connective tissue. Scattered regenerating cardiac myocytes were frequently observed in the injury sites of 1- and 2-week-old hearts 3 days after injury. Myocytes were rare in the corresponding regions of 3- and 4-week-old hearts. Instead abundant non-muscle cells and fibrous connective tissue were predominant. In the fourth and final week of this study, the repaired areas of myocardia in 1- and 2-week-old rats contained more myocytes than those of the 3- and 4-week-old rats, and the repaired zone of the 1-week-old heart contained more myocytes than the repaired areas of the other age groups. These findings suggest that the mammalian myocardia possess an age-dependent potential for regeneration that involves the healing of injury sites with contractile and connective tissues.     

Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells.

The presence of desmin was characterized in cultured rat and bovine satellite cells and its potential usefulness as a marker for identifying satellite cells in vitro was evaluated. In primary cultures, positive immunohistochemical staining for desmin and skeletal muscle myosin was observed in rat and bovine myotubes. A small number of mononucleated cells (20% of rat satellite cells and 5% of bovine satellite cells) were myosin-positive, indicative of post-mitotic differentiated myocytes. In bovine satellite cell cultures 13% of the mononucleated cells were desmin-positive, while 84% of the mononucleated cells in rat satellite cell cultures were desmin-positive. Rat satellite cell mass cultures and bovine satellite cell clonal density cultures were pulsed with 3H-thymidine, and autoradiographic data revealed that greater than 94% of dividing rat cells were desmin-positive, suggesting that desmin is synthesized in proliferating rat satellite cells. However, no desmin was seen in cells that incorporated labeled thymidine in bovine satellite cell clones. Analysis of clonal density cultures revealed that only 14% of the mononucleated cells in bovine satellite cell colonies were desmin-positive, whereas 98% of the cells in rat satellite cell colonies were desmin-positive. Fibroblast colonies from both species were desmin-negative. In order to further examine the relationship between satellite cell differentiation and desmin expression, 5-bromo-2'-deoxyuridine (BrdU) was added to culture medium at the time of plating to inhibit differentiation. Fusion was inhibited in rat and bovine cultures, and cells continued to divide. Very few desmin-positive cells were found in bovine cultures, but greater than 90% of the cells in rat cultures stained positive for desmin. The presence of desmin and sarcomeric myosin was also evaluated in regenerating rat tibialis anterior five days after bupivacaine injection. In regenerating areas of the muscle many desmin-positive cells were present, and only a few cells stained positive for skeletal muscle myosin. Application of desmin staining to rat satellite cell growth assays indicated that rat satellite cells cultured in serum-containing medium were contaminated with fibroblasts at levels that ranged from approximately 5% in 24 hr cultures to 15% in mature cultures. In defined medium 4 day cultures contain approximately 95% to 98% desmin-positive satellite cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

A cytophotometric and karyometric study of the cardiac muscle cells of young rats exposed to intermittent high altitude hypoxia

Thirty-day-old rats were exposed to intermittent high altitude in barochamber (7000 m, 4 h per day, 4 exposures) and number of mono- and binucleated myocytes as well as DNA content per nucleus were determined in the right ventricular myocardium. The data indicate enhancement of mitotic division of mono- and binuclear diploid cells in hypoxic animals. Simultaneously, an increase in mononuclear and binuclear tetraploid cells occurred; the latter is probably due to DNA synthesis not followed by mitosis of some binuclear diploid cells.     

Proliferative processes in the myocardium in different parts of the heart during the creation of experimental myocardial infarct of the left ventricle in 2- and 3-week-old rats

As a result of 30 times repeated injections of 3H-thymidine (3HTdr) to neonate rats, beginning from days 13 or 21 post partum, ca. 20 and 10% of myonuclei in the left and right atria were labeled, respectively, while in both ventricles cumulative labeling of myocytes was nearly ten times lower. In rats of the same age with experimental infarction of the left ventricular myocardium the number of myonuclei labeled after 30-fold 3HTdr injections increased in atria up to 40-50%, in perinecrotic myofibers of the left ventricles up to 8-11%, and in myofibers of the left and right ventricle located far from the necrotic foci up to 3-4 and 2-3%, respectively. In some of rats subendocardial and/or subepicardial layers of the surviving left ventricular myocardium contained up to 15-35% of labeled myonuclei. Thus, in neonatal rats the extent of DNA synthesis reactivation in the nuclei of cardiomyocytes, the majority of which have recently completed normal ontogenetic proliferation, is, on the whole, of the same order as found in similar experiments on adult rats (Rumiantsev, Kassem, 1976; Oberpriller et al., 1984). However, still immature ventricular myocytes of neonatal rats resume mitotic cycle easier than those of adult animals which is evidenced not only by higher numbers of 3HTdr labeled myonuclei in subepicardial and subendocardial ventricular myocardia of some rats, but even more by reactivation of DNA synthesis in a limited fraction (2-3%) of the whole population of non-perinecrotic myocytes in both ventricles. Besides, reactive proliferation of cardiomyocytes in the atria of neonate rats, unlike in adults, starts on day 3 rather than on day 5 after infarction is induced. In the atria of neonatal rats polyploidization of myonuclei at later postinfarction stages is less pronounced than in adult rats which may be accounted for by formation of individual daughter nuclei during acytokinetic mitoses or, more seldom, by completion of cytotomy.     

Regional appearance of atrial natriuretic peptide in the ventricles of infarcted rat hearts

The appearance of atrial natriuretic peptide (ANP) in the ventricular myocardium was investigated in rat hearts subjected to severe left ventricular infarction. The left coronary artery was ligated for 1, 2, 3, 4 and 6 days and for 3 weeks, and the tissue was prepared for microscopic examination of immunoreactive ANP and for electron microscopy. In the normal and sham-operated hearts, and in hearts subjected to 1 day of coronary ligation, ANP immunoreactivity was restricted to a few ventricular myocytes of the conduction system. Following 2–3 days of coronary ligation, ANP immunoreactivity was detected in the viable myocardium of the lateral border of the infarct and in a few layers of viable cardiac myocytes located in the subendocardial areas of the ischemic left free ventricular wall. Further, during the following days and after 3 weeks of coronary ligation, a gradient of specific labeling was commonly seen across the lateral border area of the infarct. Thus, the strongest immunoreactivities were present in the cardiac myocytes located adjacent to the non-contracting myocardium. Electron microscopic examination of the immunoreactive cardiac myocytes confirmed the presence of electron-dense specific granules within these cells. The present findings suggest that the increased regional production of ANP within the ventricular myocardium is induced by increased mechanical stretch of the cardiac myocytes, and that this might contribute to the increased release of ANP in myocardial infarction.     

Cumulative indices of DNA synthesizing myocytes in different compartments of the working myocardium and conductive system of the rat’s heart muscle following extensive left ventricle infarction

Ten successive³H-thymidine injections at 12h intervals (which is a little shorter than the adult heart myocyte S phase) were performed for labeling of the majority of cardiac myocytes synthesizing DNA at any moment of such a 5 days experiment. In the hearts of control unoperated rats ten-fold repeated³H-thymidine administration results in labeling of 2–3% myocyte nuclei, in both atria, ca. 1% of the specialized muscle cell nuclei in the atrioventricular conductive system, only occasional muscle cells being labeled in the working ventricular myocardium. When ten successive³H-thymidine injections were made between the 5th and 10th days following extended left ventricle infarction, the percentage of labeled myocytes in left and right atria reaches, respectively, 51.4±4.4% and 34.7±3.6%. In the left ventricle labeled muscle nuclei are accumulated predominantly (9.3±2.1%) within the thin subepicardial layer of the surviving myofibers, while myofibers located in other perinecrotic areas contained only 1.3±0.5% labeled muscle nuclei. The number of these nuclei in the atrioventricular system remains at the level observed in control hearts (up to 2%), approaching closely the zero level in the working myocardium of both the ventricles and interventricular septum, located at the considerable distance from the infarcted region. When similar experiments with ten-fold repeated³H-thymidine injections were performed between 15th and 20th post-infarction days the number of labeled myocyte nuclei was found to be reduced 4–6 times in atria, being changed rather a little in the perinecrotic ventricular myocardium and in the specialized myocardium of the atrioventricular system. Some possible reasons of the observed differences in the proliferative behaviour of cardiac myocytes in terms of their topology and/or specialization are discussed     

Ultrastructure of the DNA-synthesizing cells of the sinoatrial node in mouse embryos according to electron microscopic autoradiographic data

By means of electron microscopic autoradiography with 3H-thymidine a study was made of the differentiation degree of DNA synthesizing muscle cells in the sinoatrial node (SAN) of the heart conductive system of the 18 day old mouse embryos. Clear myocytes (CM), predominating in the SAN at this stage, are irregular in shape, with interdigitating protrusions. Nuclei are clear, spherical or ellipsoidal. One hour following 3H-thymidine injection, about 6% of CM display labeled nuclei; this index is considerably lower than in working ventricular myocardium. Like unlabeled myocytes, CM being in phase S contain sparse, randomly located thin myofibrilles. In some areas of the sarcoplasm, only myofilament bundles and Z-disk material can be seen. The number of CM myofibrilles is always considerably less than in the working ventricular myocytes. Accumulations of intermediate (8--11 nm) filaments are present. Mitochondria with a few cristae are not numerous. The sarcoplasmic reticulum and Golgi apparatus being relatively well developed, multivesicular bodies, centrioles, and occasional cilia are often seen. Near the centrioles (basal bodies), striated filamentous bundles are found sometimes showing periodic dense lines separated by 50--70 nm. Specialized contacts between CM are rare, being presented only by desmosomes and primitive intercalated discs. Besides CM, sparse small dark cells occur filled with myofibrilles and mitochondria. In the peripheral regions of the node "transitional" cells are seen. The SAN of the 18 day old embryo mouse heart grown due to proliferation of CM with a poorly developed myofibrillar apparatus.     

Muscarinic receptors and responses in intact embryonic chick atrial and ventricular heart cells

We have studied muscarinic acetylcholine (ACh) receptors in intact atrial and ventricular heart cells dissociated from 8-day chick embryos and maintained in sparse cell cultures. Two specific antagonists, [³H]quinuclidinyl benzilate (QNB) and [³H]N-methyl scopolamine (NMS), bind to surface sites with affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{s}\text{? 40}\text{and}\text{400}\text{p}\text{M}$, respectively). The concentration of [³H]QNB sites in ventricular cell cultures (460 fmole/mg protein) was comparable to the concentration of sites in atrial cultures (420 fmole/mg protein). The same result was obtained with [³H]NMS. Autoradiography following incubation in saturating concentrations of [³H]QNB shows that nearly all of the atrial and ventricular myocytes were labeled and that the distribution of grains over individual cells was uniform. The mean binding site density was 109/μm² for atrial cells 117/μm² for ventricular cells. In contrast to the antagonist binding results, microelectrode recordings from individual myocytes or from small clusters of cells showed that many more atrial myocytes (89%) were sensitive to 10^?4M carbachol than were ventricular myocytes (26%). Saline extract of embryonic brain tissue added to the culture medium did not alter the number or distribution of ligand binding sites but it produced a 2.6-fold increase in the number of carbachol-sensitive ventricular cells.     

Undifferentiated cells in the snail myocardium are capable of DNA synthesis and myodifferentiation

Cellular mechanisms of heart-muscle growth in the snail Achatina fulica have been studied using cytophotometry and electron microscopic autoradiography. Cytophotometric DNA measurements showed that the snail cardiomyocytes are mononucleated cells with diploid nuclei. Ultrastructural analysis of the snail myocardium revealed that, in addition to mature myocytes, it contains small roundish undifferentiated cells (UCs) and poorly differentiated muscle cells. EM autoradiography detected silver grains over the nuclei of UCs 2 h after injection of tritiated thymidine ([(3)H]Tdr), while the nuclei of both mature and poorly differentiated myocytes remained unlabeled. In EM autographs of the myocardial tissue fixed 14 days after [(3)H]Tdr administration, labeled myonuclei were evident, which may suggest some myodifferentiation of prelabeled UCs. Many labeled UCs persist for 14 days after a single [(3)H]Tdr injection, suggesting that not all UCs undergo myodifferentiation after passing through the cell cycle, and that those that do not can enter the next cycle. UCs in the snail myocardium presumably provide not only reserve but also stem cells for myocytes. Thus, the heart muscle of the adult snail consists of mononucleated diploid myocytes with blocked proliferative activity and a renewable population of precursor myogenic cells. The results obtained suggest that the growth of this muscle involves a myoblastic mechanism of myogenesis; this mechanism differs from that of vertebrate cardiac muscle growth, which is non-myoblastic-that is, based on proliferation or polyploidization of cardiomyocytes. Evolutionary aspects of cellular mechanisms of the heart-muscle growth are discussed.     

Myocyte enlargement, differentiation, and proliferation kinetics in the fetal sheep heart.

The generation of new myocytes is an essential process of in utero heart growth. Most, or all, cardiac myocytes lose their capacity for proliferation during the perinatal period through the process of terminal differentiation. An increasing number of studies focus on how experimental interventions affect cardiac myocyte growth in the fetal sheep. Nevertheless, fundamental questions about normal growth of the fetal heart remain unanswered. In this study, we determined that during the last third of gestation the hearts of fetal sheep grew primarily by four processes. 1) Myocyte proliferation contributed substantially to daily cardiac mass gain, and the number of cardiac myocytes continued to increase to term. 2) The (hitherto unrecognized) contribution to cardiac growth by the increase in myocyte size associated with the transition from mononucleation to binucleation (terminal differentiation) became considerable from approximately 115 days of gestational age (dGA) until term (145dGA). Because binucleation became the more frequent outcome of myocyte cell cycle activity after approximately 115dGA, the number of binucleated myocytes increased at the expense of the number of mononucleated myocytes. Both the interval between nuclear divisions and the duration of cell cycle activity in myocytes decreased substantially during this same period. Finally, cardiac growth was in part due to enlargement of 3) mononucleated and 4) binucleated myocytes, which grew in cross-sectional diameter but not length during the last third of gestation. These data on normal cardiac growth may enable a more detailed understanding of the consequences of experimental and pathological interventions in prenatal life.