Methodological aspects of flow cytometric analysis of DNA polyploidy in human heart tissue

Summary The purpose of the study was to investigate the possibilities of flow cytometry (FCM) for the analysis of DNA polyploidy in human heart tissue. Suspensions of single nuclei were prepared with the detergenttrypsin procedure and stained with propidium iodide. A mathematical correction procedure was developed to correct for background and clumping. For diploid model populations of chicken and trout red blood cells this correction reduced artifactual fractions in the FCM DNA profile to less than 0.5%, indicating that interference by background and clumping was almost completely overcome by this correction procedure. FCM DNA profiles were obtained from 12 hypertrophic and 7 normal human adult hearts. Clear differences were found between the DNA profiles from the normal and the hypertrophic hearts, the latter showing a higher degree of polyploidization. From the corrected DNA profiles, six different polyploidization parameters were computed, all of which showed a significant correlation with at least three out of four different parameters for heart hypertrophy. FCM appears to be a reliable method for the measurement of polyploidization in heart tissue, provided background and clumping are corrected for.In honour of Prof. P. van Duijn     

Ploidies of cardiomyocytes in human myocardial hypertrophy]

DNA cytophotometry has been performed in ventricular cardiomyocytes of hypertrophic human hearts. In the cases of hypertrophy in adults (generalized atherosclerosis, postinfarct scars), polyploidy expression did not exceed the limits of normal variability developed during childhood. In the cases of hypertrophy caused by congenital heart defects, high polyploidy has been revealed (the mean level 20c and more, where c is haploid DNA content), which considerably exceeded the upper limit of normal variability (approximately 10c). Our hypothesis has confirmed that heart hypertrophy in adults proceeds in conditions of stable genome rather than due to redundant polyploidization of the ventricular myocytes. The same idea assumes enhanced polyploidization of the myocytes in childhood in humans with congenital heart diseases.     

Bleomycin-induced DNA damage and repair in Xenopus laevis and Xenopus tropicalis

Microgel cell electrophoresis has been used with various species to measure breakage of DNA and DNA repair following exposure to the radiomimetic antibiotic, bleomycin. With humans, a high degree of DNA damage is considered to be predictive of cancer susceptibility. Non-isogeneic Xenopus laevis, the South African clawed toad, rarely develop spontaneous or induced cancers. Here, we investigate bleomycin-induced DNA damage and repair in splenic lymphocytes of this species to test consistency with cancer predictability. As X. laevis is pseudotetraploid in nature, while Xenopus tropicalis is diploid, we additionally explore the effect of polyploidy on DNA damage and repair in these vertebrates. The results show that higher doses of bleomycin are required to induce comparable levels of DNA damage in both Xenopus species, than in humans. X. tropicalis, the diploid, is more bleomycin-sensitive than is X. laevis. Additionally, repair rates of damaged DNA of X. laevis lymphocytes are more rapid than those of X. tropicalis, although both are hours slower than human leukocytes. While no data exist on cancer susceptibility in X. tropicalis, the results suggest greater susceptibility to cancer than X. laevis, but less than in humans. Thus, polyploidy serves as a protection against DNA damage and allows more rapid repair.     

Methodological aspects of flow cytometric analysis of DNA polyploidy in human heart tissue

The purpose of the study was to investigate the possibilities of flow cytometry (FCM) for the analysis of DNA polyploidy in human heart tissue. Suspensions of single nuclei were prepared with the detergent-trypsin procedure and stained with propidium iodide. A mathematical correction procedure was developed to correct for background and clumping. For diploid model populations of chicken and trout red blood cells this correction reduced artifactual fractions in the FCM DNA profile to less than 0.5%, indicating that interference by background and clumping was almost completely overcome by this correction procedure. FCM DNA profiles were obtained from 12 hypertrophic and 7 normal human adult hearts. Clear differences were found between DNA profiles from the normal and the hypertrophic hearts, the latter showing a higher degree of polyploidization. From the corrected DNA profiles, six different polyploidization parameters were computed, all of which showed a significant correlation with at least three out of four different parameters for heart hypertrophy. FCM appears to be a reliable method for the measurement of polyploidization in heart tissue, provided background and clumping are corrected for.     

Polyploidy: significance for cardiomyocyte function and heart aerobic capacity

Somatic polyploidy, defined as genome multiplication, was found in all differentiated mammalian tissues. The highest level of such a polyploidy was found in the myocardium. This phenomenon was shown to be associated with changes in the pattern of gene expression. Hence, polyploidization may create cells with new physiology. The effect of polyploidy on the heart function has never been studied. The aim of the present study was to investigate the effect of polyploidy on cardiomyocyte functioning and heart aerobic capacity. DNA and the total protein content, nucleolar activity reflecting the rate of rRNA synthesis and, consequently, ribosome biogenesis, were measured in ventricular myocytes isolated from the human and from 21 mammalian species by image cytometry and microscopic morphometry. The total protein content was estimated after staining slides with naphtol-yellow dye. For measurement of DNA and nucleolar area, staining with Hoechst and AgNO3 was applied. Cardiac aerobic capacity was evaluated by the heart mass to body mass ratio. A negative correlation between the heart index and the average cell ploidy was revealed (r = -0.79; P < 0.0001). The average genome number per myocyte was registered to be higher by approximately 35% in the sedentary mammals, with the heart index about 0.4% from body mass, than in the athletes with heart index about 0.6% of body mass. Polyploidization was shown to be associated with a sharp decrease in the protein/DNA ratio in cardiomyocytes. As a result, cardiomyocytes in the athletic mammals with poorly polyploid hearts have much higher protein content per genome than do cells in the sedentary species with highly polyploid hearts. Surprisingly, despite decreased protein/DNA ratio, the nucleolar area per genome significantly increased with polyploidization, indicating the imbalance between the cellular protein content and the rate of ribosome biogenesis. Such an imbalance should obviously impair cardiac function, because the additional genomes take some valuable space and biological resources from the cell, which could have been otherwise directed to the maintenance of cardiomyocyte contractile machinery. It is generally accepted that somatic polyploidy is associated with oxidative stress and energetic starvation. Thus, we suppose that additional genomes may serve for cardiomyocyte protection from oxidative damage in the hearts.     

Effect of exercise training on aortic collagen content in spontaneously hypertensive rats (SHR)

We investigated the effects of exercise training on the amount of aortic collagen and systolic blood pressure in spontaneously hypertensive rats (SHR). Ten-week old SHR were trained either by forced treadmill running (26.8 m X min-1 -1 h X day-1, five times a week, 0% incline) or by voluntary running in revolving wheels (7,800 m X day-1 at peak) for 8 weeks. Succinate dehydrogenase (SDH) activity measured as a marker of an endurance training effect was 13% higher (P less than 0.01) in the soleus of forced-exercised animals than in that of sedentary ones. (6.56 +/- 0.17 mumol X g-1 X min-1; mean +/- SEM), whereas SDH activity in that of voluntarily-exercised group was found to be at the same level as in sedentary animals. The systolic blood pressure after training increased by 26.4 in sedentary, 21.1 in voluntarily-exercised, and 33.9 mm Hg in forced-exercised rats, when compared with the value of each group at the beginning of the training program. A significant difference was observed in the increment of blood pressure only between the voluntarily- and forced-exercised groups (P less than 0.05). The amount of aortic collagen in voluntarily-trained rats (96.5 +/- 2.0 mg X g tissue-1, 39.8 +/- 0.7 mg X 100 mg protein-1) was significantly less than that in forced-trained rats (P less than 0.05). These results suggest that voluntary, mild exercise training may be more effective in the reduction of collagen accumulation in the aorta associated with the suppression of blood pressure increase than forced, vigorous exercise training in SHR.     

Effect of sparteine sulfate on insulin secretion in normal men

This study aimed at evaluating the influence of sparteine sulfate either upon basal plasma glucose and insulin or glucose-induced insulin secretion in normal man. Thirteen overnight fasted volunteers took part in this study; five of them were submitted to sparteine sulfate bolus (15 mg in 10 ml of saline solution) followed by a slow infusion (90 mg/100 ml X 60 min) and eight subjects underwent two different glucose pulses (20 gr. i.v.) in absence or in presence of sparteine, infused as described above. In basal conditions, along with sparteine infusion, plasma glucose showed a progressive and significant decrease (P less than 0.0001) and plasma insulin was significantly higher from min 10 to 120' (P less than 0.0005-0.001). Even during the glucose-induced insulin secretion, in the presence of sparteine infusion, plasma glucose levels were significantly lower while plasma insulin levels were significantly higher when compared to those observed after glucose alone. The acute insulin response (AIR) was 42 +/- 10 microU/ml after glucose alone vs 67 +/- 9 microU/ml after glucose plus sparteine (P less than 0.05). Total insulinemic areas were significantly different being 1410 +/- 190 vs 2250 +/- 310 microU/ml/min (P less than 0.001) during glucose and glucose plus sparteine infusion, respectively. This study thereby, demonstrates that in normal man sparteine sulfate, administrated by intravenous infusion, is able to increase either basal or glucose-induced insulin secretion.     

Circulating lipoprotein cholesterol concentrations in the summer-active ground squirrel (Spermophilus lateralis): a comparison with those in humans and rabbits.

1. The concentrations of total cholesterol (free cholesterol plus cholesteryl ester) in the sera and in two lipoprotein fractions of golden-mantled ground squirrels (Spermophilus lateralis) were measured and compared to those found in humans and New Zealand White rabbits (Oryctolagus cuniculus). 2. Squirrels showed significantly higher concentrations of total serum cholesterol (TSC; P less than 0.0005), high density lipoprotein cholesterol (HDL-C; P less than 0.0005), and very low density plus low density lipoprotein cholesterol (VLDL + LDL-C; P less than 0.0005) than those in rabbits. 3. Squirrels had significantly higher TSC (P less than 0.0005) and HDL-C (P less than 0.0005) concentrations than did humans. 4. Squirrels additionally exhibited significantly lower TSC/HDL-C ratios than did rabbits (P less than 0.005) or humans (P less than 0.0005). 5. The significant differences in lipoprotein metabolism observed in this study between the active hibernator and non-hibernators, may reflect the marked biochemical and physiological adjustments hibernating species make throughout their circannual cycle.     

Preterm infants: ventilation and P100 changes with CO2 and inspiratory resistive loading

The ventilatory effects of inspiratory flow-resistive loading and increased chemical drive were measured in ten neonates during progressive hypercapnia in control and loaded states. Hypercapnia (mean increase PCO2 = 15-20) resulted from inspiring 8% CO2 in room air and inspiratory loading by a flow-resistive load = 100 cmH2O X l-1) X s. Hypercapnia produced an increase in group minute ventilation secondary to increasing tidal volumes and breathing frequencies. Loading shifted the minute ventilation-CO2 response to the right, and slopes decreased significantly (P less than 0.05) consequent to a significant decrease in the frequency-CO2 slopes (P less than 0.05), which became negative in four of the ten subjects. Mouth pressure measured at 100 ms after onset of inspiratory effort (P100) occlusion pressure-CO2 slopes measured in five subjects showed no significant increase with load application. Resistive loading produced significant increases in inspiratory time (P less than 0.02) and the inspiratory time/total breath time ratio (P less than 0.01). Airway occlusion elicited the Hering-Breuer reflex, with a significant increase in inspiratory time-to-total breath time ratio (P less than 0.01). The results show that the inspiratory resistive load produced ventilatory compromise in newborns and insufficient compensatory augmentation of central drive.     

Nuclear size of myocardial cells in end-stage cardiomyopathies

OBJECTIVE: To determine the alteration of nuclear size in myocardial cells and the relationship between nuclear size and DNA ploidy classes in normal and cardiomyopathic human hearts. STUDY DESIGN: The study group consisted of 46 hearts obtained at biopsy. These patients had undergone cardiac transplantation for intractable congestive heart failure (18 cases with ischemic cardiomyopathy and 28 cases with idiopathic dilated cardiomyopathy). Another 10 hearts were collected at autopsy and used as control hearts according to preautopsy, autopsy and histology criteria. One hundred fibroblasts and 200 myocytes were evaluated in each ventricle. The nuclear area and DNA content were estimated using image cytometry. RESULTS: End-stage ischemic and dilated cardiomyopathies were characterized by an increase in nuclear size of both the myocyte and nonmyocyte population. The nuclear area of interstitial cells increased about 30% in cardiomyopathic hearts. Augmentation of average nuclear area of myocytes was 1.2-fold in the ischemic group and about 1.5-fold in the dilated group as compared with the control group. Also, a tendency was found for the coefficient of variation of average nuclear area to decrease in the interstitial cell population and increased in the myocyte population in cardiomyopathic situations. Furthermore, the nuclear area of myocytes enlarged as augmentation of nuclear DNA content. The relative nuclear areas of myocytes can be presented as: 2c:4c:8c:16c :32c:64c = 1:1.65:2.75:4.60:7.25:9.18. CONCLUSION: The increase in nuclear size follows either one of two different processes: the first does not involve an increase in DNA content, whereas the second is concomitant with an incremental increase in DNA content. In the first instance, the enlargement of nuclear size is limited. In the second, augmentation of nuclear size can become very impressive. In end-stage ischemic and dilated cardiomyopathies, the nuclear growth of myocytes and interstitial cells may be due to different mechanisms. Enlargement of the nuclear area of myocytes represents a complex process, including simple nuclear hypertrophy, polyploidization and multinucleation. The main pattern of nuclear growth of interstitial cells is nuclear hypertrophy without an increase in DNA content.     

Canine bronchoconstriction, gas trapping, and hypoxia with methacholine

The effects of an intravenous methacholine infusion on cardiovascular-pulmonary function were measured in seven mongrel dogs (22.0 +/- 2.8 kg), anesthetized with chloralose and urethan and beta-adrenergically blocked with propranolol. In a volume-displacement plethysmograph, physiological measurements were made at base line and 25 min after establishing a methacholine infusion (0.1-1.0 mg X kg-1 X h-1). Methacholine significantly (P less than 0.05) increased airways resistance (1.9 +/- 0.8 to 8.2 +/- 2.9 cmH2O X l-1 X s), decreased static lung compliance (84.7 +/- 18.5 to 48.2 +/- 9.4 ml/cmH2O), depressed arterial PO2 (81 +/- 17 to 56 +/- 10 Torr), and lowered blood pressure (132 +/- 10 to 69 +/- 18 Torr) and cardiac output (5.7 +/- 1.9 to 4.1 +/- 1.2 l/min). These effects persisted during a further 80 min of methacholine infusion conducted in five of the animals. During the initial 25-min period of methacholine, the end-expired volume (volume-displacement Krogh spirometer) rose in all animals, indicating an increase in functional residual capacity from 997 +/- 115 to 1,623 +/- 259 ml (P less than 0.0005). Analysis of pulmonary pressure-volume curves revealed no change in total lung capacity but an increase in residual volume from 489 +/- 168 to 1,106 +/- 216 ml (P less than 0.001). Thus methacholine caused 617 ml of gas trapping, which was not detected by the Boyle's law principle, presumably because gas was trapped at high transpulmonary pressure. We suggest that intravenous methacholine-induced canine bronchoconstriction, which causes gas trapping and hypoxia, may be a useful animal model of clinical status asthmaticus.     

Treatment of autoimmune thrombocytopenic purpura with rhesus antibodies (anti-Rh0(D)]

There is evidence that blockade of the reticuloendothelial system (RES) by sequestration of autologous red blood cells (RBC) leads to an elevation of platelet counts in immune thrombocytopenia. To substantiate this hypothesis, 10 Rh0(D)-positive adult patients (9 female, 1 male) with chronic autoimmune thrombocytopenic purpura (ITP) (1 to 21 years duration) were treated with low doses of intravenous IgG-anti-Rh0(D) (200 to 1,000 micrograms per dose; 300 to 3,600 micrograms per course; administration within 1 to 5 days). All patients improved clinically as indicated by cessation of bleeding. In eight out of ten patients there was a rise in platelet count. Platelet increments were excellent (greater than 100 X 10(9)/l) in one, good (50-100 X 10(9)/l) in three, fair (20-50 X 10(9)/1) in two and low (10-20 X 10(9)/1) in two patients. Splenectomized patients (N = 4) had a poorer response than non-splenectomized patients (N = 6) with mean increments of 16 X 10(9)/l (range 5-43 X 10(9)/l) versus 60 X 10(9)/l (range 10-110 X 10(9)/l). The increase in platelet counts persisted for seven to over 150 days. Transient and slight signs of haemolysis developed in seven out of ten patients (haemoglobin remained stable; increase of lactate dehydrogenase (greater than 250 IU/l) in four, decrease of haptoglobin (less than 60 mg/dl) in five patients). The direct antiglobulin test became positive in all cases due to IgG1 without complement fixation. We conclude that the interaction of antibody-coated RBC with macrophages (and, probably, other means of RBC alteration) is a feasible therapeutic approach in selected cases of ITP and related conditions.     

Detection of numerical chromosomal aberrations by flow cytometry: a novel process for identifying aneugenic agents

Aneuploidy plays a significant role in adverse human health conditions including birth defects, pregnancy wastage, and cancer. Currently, there is no screening method sufficiently validated that can be used routinely to identify aneugenic agents in vitro because most conventional test systems rely on the labor-intensive microscopic assessment of the aneuploid cell population. Our laboratory has recently developed a flow cytometry-based procedure for assessing numerical chromosomal aberrations in mitotic populations of lymphocytes on the basis of DNA content. Studies were conducted in 24 h treated human lymphocyte cultures to determine the sensitivity of this flow cytometry-based procedure to detect aneugenic agents. A comparison between the microscopic and the flow cytometry-based procedures for scoring polyploidy shows a strong agreement exists between the two methods. Treatments with two known aneugenic agents, griseofulvin, and paclitaxel (taxol), resulted in a dose-related increase in the mitotic index, aneuploidy, and polyploidy. In contrast, results from the treatments with two known clastogenic agents, mitomycin-C, and etoposide, show a dose-related decrease in the mitotic index with a slight increase in the frequency of hypodiploidy at concentrations that produce severe chromosomal breakage. There were no increases in hyperdiploidy and polyploidy observed. In conclusion, the reproducibility of the results obtained in this study indicates that this flow cytometry-based procedure for assessing numerical chromosomal effects in mitotic populations on the basis of DNA content is promising for the routine detection and characterization of aneugenic agents.     

Fibers of RecA protein and complexes of RecA protein and single-stranded phi X174 DNA as visualized by negative-stain electron microscopy

Monomers of purified RecA protein polymerize into helical fibers whose pitch is 7.2 nm to 7.5 nm and whose diameter is 11 nm. Either short (approximately 0.2 micron), single fibers, or bundles of aligned, longer fibers, can be formed preferentially, by varying the Mg2+ concentration. When RecA protein is bound to circular, single-stranded phi X174 DNA it forms helical fibers of different classes of contour lengths, ranging from 0.98 micron, depending upon the conditions of assembly. Two different helical pitches are found, one of 9.3 nm when the incubation buffer contains, besides the obligatory Mg2+, either ATP gamma S or ATP accompanied by single-strand binding protein, and one of 5.5 nm when the latter additives are omitted. Preformed fibers of the compact type can be converted to open ones of 9.3 nm pitch upon addition of ATP gamma S, even after the removal of unbound RecA. All signs of helicity are obliterated upon glutaraldehyde cross-linking except in those fibers whose assembly has been mediated by ATP gamma S. RecA protein and single-strand binding protein are competitively bound to single-stranded DNA. Composite complexes, however, are not encountered unless ATP gamma S is present. Otherwise, segments of DNA that are coated by one or the other protein are seen as separate regions. When the assembly of complexes of single-stranded DNA and RecA is mediated by single-strand binding protein and ATP, the axial separation between successive bases is 0 X 42 nm, somewhat greater than the axial distance between bases in one strand of duplex DNA in the B form. It is proposed that the bases of the single-stranded DNA in the complex are located near its inner surface, and that base-pairing with double-stranded DNA takes place following invasion of the central cavity of the complex.     

Somatic polyploidy in extraembryonic membranes of the snake,Elaphe guttata

Summary Video-densitometric DNA measurements of Feulgenstained tissues of 42 day old eggs of the corn snake,Elaphe g. guttata (Columbridae, Serpentes), revealed a basic DNA content of 2C=2.17 pg, with somatic polyploidy in the allantois, the chorioallontois, the yolk sac, and other extraembryonic membranes. The maximum value determined was 128C (in binucleate cells 2×128C) at the distal pole of the egg. This is the first report of somatic polyploidy in a snake, and one of the first in reptiles in general.     

Early ultrastructural changes in the myocardium following thyroxine-induced hypertrophy

The model of myocardial hypertrophy induced by thyroxine was studied with particular regard to the early ultrastructural changes in fractional volume of the mitochondria and myofibrils, and capillary distribution. Following injections of L-thyroxine (25 mg/kg IP) for 9 consecutive days, rats were sacrificed by vascular perfusion and cardiac tissue samples from the mid-wall zone of the left ventricle were processed routinely for electron microscopy. Heart weight/body weight ratios of thyroxine treated (T) rats showed a significant increase (P less than 0.001) over the ratios in control (C) rats. Likewise, the fractional volume of mitochondria (42%) was significantly increased (P less than 0.001) in the myocardium of T rats when compared with C rats (31%). However, the fractional volume of myofibrils was significantly decreased in the myocardium of T rats (P less than 0.001) and there was no significant difference between the hearts of T and C rats with respect to capillary luminal area/myocyte area. The mitochondria/myofibril ratio was increased in the hearts of T rats (0.82) over that found in control hearts (0.52). These results suggest that in the early stages of thyroxine-induced myocardial hypertrophy there is not an immediate increase in capillary area which may account for the ischemia and significant increase in mitochondrial volume which characterized myocardial hypertrophy in this model.     

Oxidative stress in the aging rat heart is reversed by dietary supplementation with (R)-(alpha)-lipoic acid.

Oxidative stress has been implicated as a causal factor in the aging process of the heart and other tissues. To determine the extent of age-related myocardial oxidative stress, oxidant production, antioxidant status, and oxidative DNA damage were measured in hearts of young (2 months) and old (28 months) male Fischer 344 rats. Cardiac myocytes isolated from old rats showed a nearly threefold increase in the rate of oxidant production compared to young rats, as measured by the rates of 2,7-dichlorofluorescin diacetate oxidation. Determination of myocardial antioxidant status revealed a significant twofold decline in the levels of ascorbic acid (P = 0.03), but not alpha-tocopherol. A significant age-related increase (P = 0.05) in steady-state levels of oxidative DNA damage was observed, as monitored by 8-oxo-2'-deoxyguanosine levels. To investigate whether dietary supplementation with (R)-alpha-lipoic acid (LA) was effective at reducing oxidative stress, young and old rats were fed an AIN-93M diet with or without 0.2% (w/w) LA for 2 wk before death. Cardiac myocytes from old, LA-supplemented rats exhibited a markedly lower rate of oxidant production that was no longer significantly different from that in cells from unsupplemented, young rats. Lipoic acid supplementation also restored myocardial ascorbic acid levels and reduced oxidative DNA damage. Our data indicate that the aging rat heart is under increased mitochondrial-induced oxidative stress, which is significantly attenuated by lipoic acid supplementation.     

Rate and time of DNA synthesis of human leukaemic blasts in bone marrow and peripheral blood

We have evaluated DNA synthesis rate (S rate) and time (Ts) and tritiated thymidine labelling index (LI) of peripheral blood (PB) and/or bone marrow (BM) leukaemic blasts (Bl) in nineteen cases of acute leukaemia (twelve non-lymphoblastic, AnLL, and seven lymphoblastic, ALL), in one case of non-Hodgkin's leukaemic lymphoma and in a case of plasma cell leukaemia. The LI of PB-Bl was significantly lower than that of BM-Bl (range 0.1-6.2% and 1.9-19.5%, respectively; P less than 0.01). The S rate was higher for PB-Bl than for BM-Bl (range 3.5-11.3 and 2.5-9.5 mol X 10(-18)/min; P less than 0.02) and the Ts of PB-Bl was shorter than that of BM-Bl (range 7.6-22.1 and 10.8-34.7 hr, respectively; P less than 0.02). In eight cases where S rates of both BM-Bl and PB-Bl were available, a linear correlation (r = 0.82; P less than 0.01) was found between the two parameters. This suggests that the DNA synthetic rate is a property of the leukaemic cell line in individual patients and differs from case to case. It further indicates that the environmental influences on the DNA synthesis rate in BM or PB are always of the same order of magnitude. From the results of this study we speculate that the DNA synthesis rate of leukaemic blasts is slowed down in the BM by environmental factors such as cell density.     

Right ventricular systolic pressure load alters myocyte maturation in fetal sheep

The effects of right ventricular (RV) systolic pressure (RVSP) load on fetal myocyte size and maturation were studied. Pulmonary artery (PA) pressure was increased by PA occlusion from mean 47.4 +/- 5.0 (+/-SD) to 71 +/- 13.6 mmHg (P < 0.0001) in eight RVSP-loaded near-term fetal sheep for 10 days. The maximal pressure generated by the RV with acute PA occlusion increased after RVSP load: 78 +/- 7 to 101 +/- 15 mmHg (P < 0.005). RVSP-load hearts were heavier (44.7 +/- 8.4 g) than five nonloaded hearts (31.8 +/- 0.2 g; P < 0.03); heart-to-body weight ratio (10.9 +/- 1.1 and 6.5 +/- 0.9 g/kg, respectively; P < 0.0001). RVSP-RV myocytes were longer (101.3 +/- 10.2 microm) than nonloaded RV myocytes (88.2 +/- 8.1 microm; P < 0. 02) and were more often binucleated (82 +/- 13%) than nonloaded myocytes (63 +/- 7%; P < 0.02). RVSP-loaded myocytes had less myofibrillar volume than did nonloaded hearts (44.1 +/- 4.4% and 56. 1 +/- 2.6%; P < 0.002). We conclude that RV systolic load 1) leads to RV myocyte enlargement, 2) has minor effects on left ventricular myocyte size, and 3) stimulates maturation (increased RV myocyte binucleation). Myocyte volume data suggest that RV systolic loading stimulates both hyperplastic and hypertrophic growth.     

Plural Breeding in the Splendid Fairy-wren,Malurus splendens (Aves: Maluridae), a Cooperative Breeder

Groups of the cooperatively breeding splendid fairy-wren Malurus splendens may include more than one female. Previously this species has been described as singular breeding (only one female breeds). This paper describes the occurrence of plural breeding (PB) groups in 10% of group years, in which two females had separate nests. In all cases, the secondary female (Y) was related to the primary breeding female (X) and was generally a 2-year old female which had helped in the group during the previous breeding season. Plural breeding was correlated with an increase in population density and in the number of female helpers; PB groups were larger than singular-breeding groups. In most cases, the X female was occupied with her own nest or offspring when the Y female began to nest, and there was no aggression between them. Which birds helped the Y female to feed at her nest depended on the time between the hatching of the two nests. If the interval was small, some group members helped at each nest; with longer intervals, the group members began to feed at the earlier nest, and the other female was left to raise her brood alone. Female helpers were very active in feeding at single nestings, and the cost to an X female of a Y female breeding was mainly a loss of this assistance. The success of individual X nests was not affected. Effects on productivity were slight, but fewer X females in PB groups raised second broods than did experienced singular breeding females. Y females were less productive than X females, but no less productive than singular breeding novice females without helpers. It is not known whether Y females copulated with primary or secondary males within their group, or with males from outside the group. Certainly, they did not form an observable pairing with any male in the group. Plural breeding occurred in a minority of group years in response to extrinsic conditions and the current demographic situation, and shows the extreme plasticity of the mating system in M. splendens.