SUMMARY Proliferating nonmyocardial cells (NMCs) complicate primary heart cultures and may influence myocardial cell (MC) differentiation. In cultures from the day-old rat ventricle, we validated a method to arrest this proliferation; and we quantitated cross-striated cells and the chronotropic response to isoproterenol to assess MC differentiation. MCs were cultured at single cell density using an improved method. By 4 days, all cells could be identified as MCs or NMCs. In cultures treated for 3 days with bromodeoxyuridine (BrdU), 0.1 miw, serial cell counts were unchanged through 11 days. Among 50,000 cells from 110 cultures, 75-80% were MCs. In control cultures without BrdU, NMC density was 3-and 6-fold that in BrdU-treated cultures at days 5 and 8, respectively (P < 0.01), although the MCs were not overgrown. The MCs did not proliferate in either culture. The proportion of living MCs with cross-striations was similar in treated and control cultures at day 5 (72.6% vs. 69.9%, P > 0.05) but was lower in controls at day 8 (86.3% vs. 50.4%, P < 0.01). A sensitive (ED 50 10-100 pM), specific chronotropic response to L-isoproterenol was present in both treated and control cultures, but the maximum response was only 20-30% as great in controls on days 4 and 8 (P < 0.01). Baseline beating rates were the same. The MCs had well-differentiated ultrastructure, including a T system. By autoradiography, a maximum 18.4% of MCs had nuclear incorporation of 3 H-BrdU at day 8. Media conditioned by NMCs or by the control cultures did not change the cross-striations or isoproterenol response of BrdU-treated cultures. Thus, in a new culture preparation with few and stable NMCs, morphological and functional MC characteristics were different from those of MCs in cultures with proliferating NMCs. We suggest that an MC-NMC interaction can alter MC properties and that this effect should be considered in studies of primary rat heart cultures. The pure, stable, well-differentiated MCs in BrdUtreated cultures will be useful for studying MC growth, repair, and other time-dependent phenomena.

SUMMARY. The role of hormones and other humoral factors in the regulation of myocardial hypertrophy has been difficult to evaluate. We asked whether myocardial cell hypertrophy could be demonstrated in cultures from the day-old rat ventricle and evaluated the effect of serum concentration and catecholamines on the growth process. Two single-cell preparations were used: serumsupplemented, bromodeoxyurdine-treated cultures and serum-free cultures with transferrin and insulin. Both preparations were characterized by myocardial cell predominance (about 75-80% of total cells) and constant cell numbers. Myocardial cell size was documented by photomicroscopy and quantified by volume (microscopic diameter of suspended cells), surface area (planimetry of attached cells), and total cell protein concentration (Lowry method and cell counts). Growth was also evaluated in pure nonmyocardial cell cultures. In cultures with 5% (vol/vol) serum, myocardial cell size increased 2-to 3-fold over 11 days in culture. Final volume, surface area, and protein concentration were about 3000 jum 3 /cell, 5000 /nmVcell, and 1500 pg/cell, respectively. Serum had a dose-related effect on myocardial cell hypertrophy; myocardial cell size increased about 4-fold when serum concentration was increased from 0% to 5% or 10%. Cells maintained in serum-free medium with transferrin and insulin (each 10 fig/ml) did not hypertrophy, but did remain responsive to the growth-promoting activity of serum. Chronic exposure to isoproterenol or norepinephrine (1 /IM) significantly stimulated myocardial cell hypertrophy. This stimulation was dose-related, was not blocked by equimolar propranolol, was not associated with a sustained chronotropic effect, and was more pronounced in the serum-free preparation. In pure cultures of nonproliferating (bromodeoxyuridine-treated) nonmyocardial cells, cell size also increased with time in culture, but variation in serum concentration and addition of norepinephrine had no significant effect on cell size. Myocardial cell hypertrophy occurs in culture and is regulated by variations in the culture medium, including serum, with its contained hormones and growth factors, and catecholamines. The culture preparation can be used to explore the regulation of myocardial cell hypertrophy by nonhemodynamic factors. (Ore Res 51: 787-801, 1982) VERY little is known about the role of hormones or other humoral factors in myocardial hypertrophy (Cohen, 1974). Experiments in vivo are complicated by hemodynamic, hormonal, and neural reflexes elicited by endocrine ablations or hormone treatments. In vitro models such as the isolated perfused heart or the heart in organ culture offer greater experimental control, but growth does not occur in these preparations (Morkin, 1974;Wildenthal et al., 1976). Cell cultures have been widely used to study the regulation of cell proliferation, and it has recently been proposed (Clark and Zak, 1981;Frelin, 1980;Speicher et al., 1981) that heart cells in primary culture may be a useful model system for stud...

This work demonstrates for the first time that TNF-alpha may act as a protector of embryos exposed to teratogenic stress. One possible mechanism may be restoration of NF-kappaB activity in embryonic cells surviving the teratogenic insult.

It is known that programmed cell death (apoptosis) is an important physiological determinant of embryonic development. In parallel, it may be one of the major events involved in induced teratogenesis. The present study was designated to evaluate to what extent is apoptosis involved in the formation of some final abnormalities induced by cyclophosphamide (CP) in ICR mice. The level of apoptosis in limbs, tail, liver, and whole embryo was assessed 24 h after administration of various doses of CP (day 12 of pregnancy) by flow cytometric analysis and by DNA fragmentation assay. In parallel, the rate of limb and tail malformations, resorptions, and growth retardation induced by various doses of CP was evaluated in animals sacrificed on day 19 of pregnancy using routine teratological methods. A striking correlation between the rate of CP-induced apoptosis in limb and tail cells and the severity of limb and tail anomalies was found after administration of CP ranging from 10 to 40 mg/kg. Thus, the percent of apoptotic cells collected from limbs and tails increased from 18 to 78%. In parallel, the severity of limb and tail anomalies increased from digit anomalies to amely and from crooked to short or absent tail. CP-induced embryolethality and fetal growth retardation also correlated with the level of apoptosis in cells collected from whole embryos but to a lesser extent. These results claim that CP-induced apoptosis is one of the inevitable events in the pathway leading to the formation of CP-induced abnormalities and also suggest that the extent of the involvement of apoptosis in the formation of different types of final abnormalities, may be different.

Our results suggest a possible role for the apoptotic process in mechanisms mediating pregnancy loss and indicate an involvement of p53 and bcl-2 in its regulation.