Nuclear-cytoplasmic interactions affecting DNA synthesis during induced cardiac muscle growth in the rat.

Nuclear-cytoplasmic interactions affecting DNA synthesis during induced cardiac muscle growth were examined in 29 to 46 day old rats. DNA synthesis was examined in vitro using isolated nuclei from rat heart and adult X. laevis spleen. Cytoplasmic extract (CE) was obtained from a 105 000 g supernatant of rat heart and fetal liver homogenates. To measure DNA synthesis we utilised DNA within the isolated quiescent nucleus as the template and measured the effect of CE on the incorporation of 3H-TTP into an acid precipitable product. In a homologous system of rat heart nuclei from weanling rats and CE from cardiac muscle undergoing induced growth, no stimulation of 3H-TTP incorporation was observed. Cardiac muscle CE however, did possess stimulatory factor(s) since quiescent X. laevis nuclei could be stimulated with the rat heart CE. Furthermore, CE from hearts undergoing induced growth had greater activity than extract from control hearts. While cardiac muscle nuclei were not stimulated by heart CE, they showed substantial stimulation by CE from fetal rat liver, which contains a large population of proliferating cells. Stimulation by fetal rat liver was greater with nuclei obtained from hearts undergoing induced growth than from control hearts. Stimulatory factor(s) in CE was distinct from DNA polymerase-alpha activity, as shown by separation of the two activities on a 5 to 15% glycerol gradient.

Myosin isozyme changes in the heart following constriction of the ascending aorta of a 25-day old rat.

When a constricting band is placed around the ascending aorta of young (25-day old) rats, all chambers of the heart eventually produce hypertrophy. Both the left and right ventricles show strong shifts toward an isozyme pattern in which V3 is predominant, similar to that seen in models where hypertrophy is induced in adult rats. The hypertrophied atria however, show no detectable change in the native myosin isozymes or the light chain subunits.

Non-coordinate expression of collagen mRNAs during carbon monoxide-induced cardiac hypertrophy.

Collagen gene expression during volume overload-induced cardiac hypertrophy was investigated in adult male rats. Hypertrophy of the left ventricle (22%) and right ventricle (37%) occurred following 27 days continuous exposure to 700 ppm carbon monoxide; hematocrit increased nearly 47%. To examine potential cellular and molecular control of restructuring in the heart, we investigated the expression of two specific procollagen mRNAs for collagen types which have different structural-functional roles [Type I (alpha-1) & Type IV]. Type I (interstitial) mRNA levels increased at least 100% relative to controls within 3 days of initial exposure to 700 ppm CO, then declined afterwards; type IV (basement membrane) mRNA levels increased more modestly at first, and increased further afterwards. The ratio of type I/type IV RNA's also increased initially, then later declined, with the greatest differences in the relative responses of type I and IV mRNAs seen in the right ventricle. These data suggest that types I and IV collagen mRNA expression is not coordinately expressed during this type of volume overload-induced hypertrophy in rat heart.

Differentiation of adult rat cardiac myocytes in cell culture.

Cardiac myocytes isolated from adult rat hearts were grown on laminin coated culture dishes for more than a month. During this time, the cells underwent a morphological transformation which has also been referred to by others as cell remodeling (Guo J-X, Jacobson SL, Brown DL: Cell Mot Cytoskeleton 1986;6:291-304). This results in a change in myocyte morphology from its typical in vivo cylindrical shape to one which is more pleiomorphic. Despite the long-term change in morphology, myocytes expressed for differing lengths of time several aspects of the adult phenotype as evidenced by the following: 1) maintenance of cylindrical shape and/or evident cross-striations for the first 24-48 hours in culture, 2) reappearance of cross-striations during the second week in culture, 3) little or no spontaneous contractility for the first 4 days in culture, 4) expression of only the V1 isoform of myosin for at least 7 days, and 5) altered myosin isoform expression in response to changes in environmental conditions. These factors taken together suggest that in culture the adult cardiac myocyte remains a highly differentiated cell (as opposed to possible dedifferentiation) and maintains many of its previous in vivo characteristics. Such highly differentiated adult cells should be suitable as an in vitro system for studying the direct cellular effects of factors which regulate growth and differentiation of the in vivo heart.

Chronic carbon monoxide poisoning: effects on performance of the isolated right ventricle.

Rats were exposed continuously for 21-35 days to 500 ppm carbon monoxide (37.4-39.2% HbCO). Aerobic resting force (RF)-active isometric twitch force (AF) curves of isolated right ventricle preparations were constructed at 27 degrees C. CO and control groups are nearly identical when expressed as percent of maximal AF over the range of RF used. There are no significant differences in AF either at the peak of the curve or at RF = 1 g. During 15 min of anoxia AF declines at both RF's used. Increased anoxic tolerance is apparent in all CO preparations when anoxic AF is plotted as percent of preanoxic AF. The effect is most marked at RF = 1 g, where time to one-half preanoxic AF is 12.6 +/- 0.9 vs.9.3 +/- 0.8 min for CO-exposed and controls, respectively. However, no significant differences in "absolute" AF are seen between the groups at either level of RF at any minute during anoxia. Aerobic recovery of CO preparations is markedly enhanced at 1 g RF. Anoxic tolerance and aerobic recovery differences, however, are smaller at the peak of the RF-AF curve. Effects on time to peak AF and maximal rate of rise of AF are not marked at either level of RF in CO preparations.

Structural differences in the subfragment 1 and rod portions of myosin isozymes from adult and developing rat skeletal muscles.

During development of fast contracting skeletal muscle in the rat hindleg, embryonic and neonatal forms of the myosin heavy chain are present prior to the accumulation of the adult fast type ( Whalen , R. G., Sell, S. M., Butler-Browne, G.S., Schwartz, K., Bouveret, P., and Pinset -H arstr öm, I. (1981) Nature (Lond.) 292, 805-809). Polypeptide mapping of the heavy chain subunit using partial proteolysis in the presence of sodium dodecyl sulfate has shown differences in the cleavage patterns for these various heavy chains. Using this technique, we have now examined subfragments, which represent functional domains, from several different myosin isozymes. The heavy chains of the S-1 subfragments containing either light chain 1 or light chain 3 are indistinguishable for the neonatal or fast myosin isozymes. We also isolated the S-1 fragments and the alpha-helical COOH-terminal half of the molecule (rod) from rat embryonic, neonatal, and adult fast and slow myosin, as well as myosin from cardiac ventricles. All of these S-1 and rod fragments were different, indicating that the previously reported differences among these different myosin heavy chain isozymes are located in both the S-1 and rod subfragments for all myosins examined. However, the polypeptide maps of neonatal and adult fast S-1 show clear similarities, as do the maps of slow and cardiac S-1. These similarities in the two pairs of polypeptide maps were confirmed by the results of immunoblotting experiments using antibodies to adult fast and to slow myosin.

Denervation of newborn rat muscle does not block the appearance of adult fast myosin heavy chain.

Several observations, both in vivo and in vitro, have indicated that the development and maturation of mammalian skeletal muscle fibres is influenced by nerve-muscle interactions. Morphological maturation of newly regenerated adult mouse muscle fibers in an organotypic nerve-muscle culture system depends on the presence of spinal cord neurones. Sciatic nerve transection in newborn rats has been shown to modify the development of the histochemical and contractile properties of the denervated muscles. In addition, neural influences are important for the appearance of certain of the myosin small subunits. It has been proposed that the nerve also controls the changes in myosin heavy chain isozymes appearing during development. One such transition occurs in rat muscle where the neonatal form of myosin heavy chain is replaced by the adult form during the second post-natal week. Here we demonstrate that innervation of the rat gastrocnemius muscle (a fast-contracting muscle in the adult) is not required for the appearance of the adult form of myosin heavy chain.

Differences in myosin isoform expression in the subepicardial and subendocardial myocardium during cardiac hypertrophy in the rat.

We have investigated myosin isoform expression during progressive cardiac hypertrophy and the development of congestive heart failure in young male rats. Cardiac enlargement was produced by placing a constricting band (0.024-inch diameter) around the ascending aorta of 25-day-old animals, which resulted in progressively increased stenosis as the rat matured. A 57% and 77% cardiac hypertrophy was observed at 2 and 8 weeks, respectively, with signs of congestive failure at the latter time point. Myosin isoform expression was examined in the subendocardial and subepicardial myocardium of the left ventricle and the free wall of the right ventricle by use of native gel electrophoresis. The percentage of the V3 isoform increased dramatically in both ventricles. In the subendocardial myocardium of the left ventricle, expression of the V3 isoform increased (p less than or equal to 0.05) relative to the subepicardial myocardium at 2, 4, and 8 weeks (17.1% vs. 10.2%, 29.4% vs. 18%, and 46.6% vs. 36.2%). In addition to regional differences within a given transmural segment, we also observed a high degree of heterogeneity in myosin isoform expression throughout a given layer (particularly the subendocardial myocardium) when small (less than or equal to 10-15 mg) adjacent samples were examined. This variability illustrated a potential danger in interpretation of gel results obtained from a single small tissue sample. Thus, cardiac hypertrophy produced by pressure overload in 25-day-old rats resulted in significantly increased V3 myosin in both the left and right ventricles. Furthermore, within the hypertrophied left ventricle, the subendocardial myocardium contained a significantly greater percentage of V3 myosin than the subepicardial myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordinated changes in contractility, energetics, and isomyosins after aortic stenosis.

To investigate possible alterations of myocardial performance in young rats, cardiac hypertrophy was induced by stenosis of the ascending aorta (AS) in three groups of 25-day-old rats that were compared with three groups of sham-operated controls (C). The cardiac overload duration was 8-10 days, 1 mo, and 2 mo in groups 1, 2, and 3, respectively. Mechanics and energetics were studied in left ventricular papillary muscles, and determination of the V1 and V3 isomyosin pattern was achieved in the same papillary muscle. The majority of quantitative changes concerning the cardiac growth process, contractility, and isomyosin shifts occurred within 8-10 days of stenosis. At this point, the degree of left ventricular hypertrophy relative to C was 53 +/- 6%, whereas maximum unloaded shortening velocity (Vmax) decreased significantly (2.8 +/- 0.1 in C vs. 1.9 +/- 0.1 Lmax/s in AS), peak power output (Emax) decreased (1.8 +/- 0.3 in C vs. 0.6 +/- 0.1 in AS), and the curvature of Hill's hyperbola increased (1.3 +/- 0.4 in C vs. 2.0 +/- 0.7 in AS); moreover, the percent V1 isomyosin decreased significantly (98 +/- 1 in C vs. 51 +/- 3% in AS) and the percent V3 isomyosin increased significantly (2 +/- 1 in C vs. 26 +/- 2% in AS). Beyond 8-10 days of AS, additional changes in cardiac hypertrophy and in mechanical and biochemical parameters were less marked.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro differentiation of rabbit blastocyst cells.

Six- and seven-day post-coitus (p.c.) rabbit embryos have been cultured in an attempt to establish a trophectodermal cell line. Results indicate that cells with epithelial characteristics (i.e. positive staining for cytokeratin) will survive in culture until Passage 3. At that time a fibroblastlike cell becomes predominant. In addition, we have found that the presence of the inner cell mass is required for trophectodermal cells from 6-d p.c. embryos to attach to the collagen substrate. Culture of 7-d p.c. embryo explants often results in the development of cells that spontaneously contract. These cells stain positively for myosin, which indicates that they may be precardiac cells. Maximum diastolic potential was -59 +/- 1.2 mV and the threshold potential was -53 +/- 2.3 mV. Spontaneously contracting cells did not respond to atropine, acetylcholine, epinephrine, isoproterenol, or propranolol. Action potential seems to be a result of an inward calcium current, because the beating rate is decreased in a dose-related manner with the calcium channel blocker verapamil, whereas the voltage-sensitive sodium channel blocker tetrodotoxin was without effect.