Variability of the cardiomyocyte ploidy in normal human hearts.

We have performed cytophotometry for DNA in isolated myocytes of the left ventricle from 16 men, aged 19-39 years, who died from various non-cardiac or pulmonary causes. The mean ploidy of myocytes varied from 3.2-3.9 c to 6.6-7.3 c in different layers of the anterior wall of the left ventricle (where c is the haploid DNA content measured by cytophotometry in Feulgen-stained preparations). There was no correlation between the layers. The percentage of binuclear cells varied from 25 to 86% and correlated in every layer with the mean ploidy value of the whole myocyte population. Approximate calculation of total ploidy revealed low values in the ventricles of some individuals, and high values in others. Averaging the values for all the hearts studied obscures this variation. Mean myocyte ploidy in different layers of the anterior wall was similar: in the external layer it was 5.1 +/- 0.3 c, in the middle layer 5.5 +/- 0.3 c and in the inner layer 4.8 +/- 0.4 c. The mean percentage of binuclear myocytes in these three layers was also similar, being 61 +/- 3%, 63 +/- 4% and 54 +/- 5%, respectively. Myocyte ploidy in tissue from the posterior wall of the left ventricle also varied, but was always higher than for the same layer of the anterior wall in the same ventricle. We propose that high or low myocyte ploidy, as well as different proportions of mono- and binucleate cells, can be a factor affecting the course and result of cardiac pathology in the absence of any changes of myocyte genome determined during early ontogenesis and representing a stable characteristic of the individual.     

Stem cells do not play a significant role in repopulation of adult human cardiomyocytes

Currently, there are two points of view on the ability of adult human heart to regenerate. One of them holds that the myocardium has a poor ability to regenerate. According to the other, the myocardium can rapidly regenerate due to the presence of resident stem cells in it. The purpose of this study was to test these hypotheses by investigating the distribution of cardiomyocytes by size and ploidy in human beings of different age. Using cytofluorometry and interferometry, we determined the dry weight, volume, and ploidy of myocytes isolated from the left ventricle of a normal heart of 12 men at the age of 20–30 (n = 7) and 40–50 (n = 5) years. The mean dry weight of cardiomyocytes was 6906 ± 182 pg (10^–12 g) in the 20- to 30-yearold men and 9126 ± 263 pg in 40- to 50-year-old men; the myocyte volume was 55250 ± 1457 and 73005 ± 2106 µm³, respectively. Cells with volumes intermediate between the cells at the stage of “dividing myocytes” and mature myocytes were absent. The number of cardiomyocytes in the left ventricle was (3.18 ± 0.05) × 10⁹ in the 20–30-year-old age group and (2.06 ± 0.6) × 10⁹ in the 40–50-year-old group. The largest subset (41.3%) of the myocyte population was represented by mononuclear cells with tetraploid nuclei. The proportion of myocytes of different ploidy classes and their mean ploidy did not change in the range of 20–50 years. On the basis on these data, we concluded that stem cells do not play a significant role in restoring the number of lost myocytes. Hypertrophy of myocytes caused by the increase in their cytoplasm is the main mechanism of compensation of the function of the left ventricle of the heart in aging human beings.     

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     

The ratio in the DNA content and protein mass of human cardiomyocytes

DNA and total protein content were measured in human cardiac myocytes using cytophotometry of Feulgen-naphthol yellow stained cells isolated from the left ventricle. The percentage of DNA classes was 2%, 32%, 53%, 12% and less than 0.5% for diploids, tetraploids (both 4c and 2c X 2), octaploids (8c, 4c X 2 and 2c X 4), hexadecaploids (16c, 8c X 2 and 4c X 4) and 32c cells, respectively. Binuclear cells comprised about 65% of the myocytes; the main class was 4c X 2. A discrepancy between total protein content and gene dose has been pointed out. DNA level ratio in a number of myocytes of different ploidy were 2:4:8:16:32; the same ratio for total protein content was 2:3.5:6:11.4:25.5, respectively.     

Number and mass of the myocytes of the mouse heart

The mean number of cardiomyocytes is constant in the heart ventricles of 1, 1.5-2, 3-4, 5-6 and 12-month old mice. However, differences in the myocyte number among mice of the same age and with similar heart weight may reach 30%. There are only small differences in the mean ploidy among these mice. The mean protein content in the myocytes correlates to the ventricle weight. In some mice, however, no correlation was observed between the myocyte and ventricle weights, or between the calculated dry and wet weights of myocytes.     

Myocyte polyploidy in human cardiac pathology]

With general atherosclerosis, the ploidy of left ventricle myocytes in the hearts of patients that underwent infarction corresponds to the norm variation irrespective of the ventricle and heart weights. At heart diseases the myocyte nucleus ploidy is often much higher than the norm variability both in hypertrophied ventricles and in those with normal weight. An additional polyploidization is suggested that may occur at some natural ontogenetic periods of human development (in the childhood) during heart diseases both innate or spontaneously appearing at the particular time. Unlike, the myocardial hypertrophy in adults does not stimulate myocyte polyploidy.     

Genome multiplication in cardiomyocytes of fast- and slow-growing mice

The number of myocytes and the percentage of cells with a high degree of ploidy increased in the heart ventricles of fast-growing mice compared with slow-growing ones. The mean incidence of octa- and hexadecaploid (by summary DNA content) myocytes was 7% in the slow-growing and 23% in the fast-growing, weaned mice. In these groups, the total myocyte number varied by 20%. There were 43% more myocyte genomes in the heart ventricles of the fast-growing mice than in those of the slow-growing mice. The same differences in cell number and ploidy persist in 90-day-old mice in spite of feeding ad libitum after weaning.     

Peculiarities of liver regeneration in Chinese hamster <Emphasis Type="Italic">Cricetulus griseus</Emphasis>

With the aid of cytofluorimetry and interference microscopy, the ploidy level and the hepatocyte ploidy class distribution were studied and the dry mass of hepatocytes was measured in hepatocytes in liver of Chinese hamsters Cricetulus griseus and of Balb/c mice before and one month after partial hepatectomy. The mean ploidy level in hepatocytes of the Chinese hamster normal liver amounted to 2.35 ± 0.03 c. The modal class was mononuclear hepatocytes with diploid nuclei (82.4 ± 1.3%). The mean dry mass of hepatocytes amounted to 605.2 ± 4.8 pg. In the process of liver regeneration in the Chinese hamsters, the ratio of ploidy classes and the hepatocyte dry mass did not change. After a similar liver resection in the mice, a significant polyploidization of liver parenchyma occurred. The mean ploidy level in hepatocytes rose by 32%. Instead of 4cx2-hepatocytes, the modal class became mononuclear octaploid cells the relative portion of which increased, on average, by five times. The portion of binuclear hepatocytes with octaploid nuclei in mouse liver rose by more than five times. Thus, in the Chinese hamsters Cricetulus griseus, unlike mice, regeneration of liver occurred exclusively at the expense of proliferation of hepatocytes.     

Electromechanical coupling in cardiomyocytes from transmural layers of guinea pig left ventricle

The electrical and mechanical activity of heart ventricle cardiomyocytes is known to vary depending on the spatial location of cells in the wall, in particular, transmurally from the sub-endocardial layer to the sub-epicardial one. To investigate intracellular mechanisms of the functional heterogeneity of cardiomyocytes we developed mathematical models of the electromechanical coupling in cardiomyocytes from different transmural layers across the left ventricle (LV) wall of guinea pig. It is shown that the mechanisms of both direct linkages and feedback in the electromechanical coupling contribute to differences in both the shape and duration of action potential, and speed characteristics of contraction between isolated cardiac myocytes from the sub-endocardial and sub-epicardial layers.     

Protein mass in human cardiomyocytes does not correspond to gene content

Lack of proportionality between DNA and protein content has been revealed in the human cardiac myocytes. The proportion 2:4:8:16 was observed in DNA of di-, tetra-, octa- and hexadeca myocytes while the protein content of the same cells was 2:3.5:5.2:7.2 in the inner layer, 2:3.5:6.5:8.9 in the central layer and 2:3.1:5.6:9.1 in the outer layer of the normal left ventricle. The protein content of myocytes of the same ploidy was higher in the inner layer than in other ones.     

Ploidy in the hearts of elderly patients

The DNA-ploidy of the left and right ventricular wall in 48 patients aged 80-98 years was investigated by scanning cytophotometry. The great variation in nuclear ploidy previously described in the heart muscle was shown to persist during the ninth decade. In addition, the percentage of nuclei with more than 2C DNA content in both ventricles correlated with the degree of coronary stenosis, and in the right ventricle with the presence and severity of pulmonary emphysema, and with the duration of digitalis therapy where this drug was given.     

Similarities and differences in the growth of the heart in situ and in transplants

Growth and ploidy of rat ventricular myocytes were studied during development in situ and in grafts (1 day old rat ventricle transplanted under kidney capsule of syngenic adult animals). Both in situ and in the transplants polyploidization occurred on days 4-14 of postnatal life, and the modal group of myocytes was represented by binucleate diploid (2c x 2) cells. Minor quantities of 4c, 4c x 2, 8c and 2c x 4 myocytes were detected as well. In ventricles of 14 and 28 days old rats and in the transplants of the corresponding age the portion of polyploid myocytes was 90-96% and 32-63% respectively. The intensity of postmitotic myocyte transplants was decreased as compared with in situ development, and cells that exit proliferation cycle did not grow until day 14. The data on thymidine label dilution suggest that diploid myocytes of the transplant can divide two or three times but the majority of labeled diploids divided only once. Labeled 2c x 2 myocytes originated from the first, and less frequently, the second cell generation or resulted from initial acytokinetic mitosis. Mononucleate tetraploids 4c originated from 2c x 2 and mostly from 2c cells. Octaploids were formed after 3d or 4th labeled mitosis. The conclusion about cardiac myocyte polyploidization as an intrinsic developmental program is supported, implying the programming of onset, mode, duration and termination of polyploidization and its prolongation during early postnatal life.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variability of water content and of depth profiles of global fallout 137Cs in grassland soils and the resulting external gamma-dose rates

¹³⁷Cs from global fallout of nuclear weapon testings in the 1950s and 1960s was determined in successive layers (0–30 cm) of eight undisturbed grassland soils in Bavaria, Germany. The maximum activity concentration was found in soil layers between 4 and 15 cm below the surface. Using the vertical distribution of the cesium activity, which varied considerably from site to site, the mean residence half-time of ¹³⁷Cs from global fallout in each soil layer was evaluated with a compartment model. These values ranged from 1.0 to 6.3 years/cm. The mean residence half-time averaged over all soil layers and all sites was 2.7±1.4 years/cm and, thus, about twice the corresponding residence half-time of the Chernobyl-derived ¹³⁷Cs as determined in the same soil layers (also in 1993). The dose rate of the external gamma-radiation due to ¹³⁷Cs from global fallout in the soil determined from the depth distributions varied between 0.34 and 0.57 (mean: 0.45±0.07) nGy/h per kBq/m². The effect of soil water content on the dose rate was studied by considering four states of the soil, from water content zero to complete water saturation of the total pore volume. It was shown that the difference between the dose rates at the permanent wilting point and the field capacity, which both represent the most relevant water contents of soils, was only 10% of the dose rate at the permanent wilting point for all sites. Received: 8 September 1997 / Accepted in revised form: 15 December 1997     

Myocyte DNA synthesis with aging: Correlation with ventricular loading in rats

To determine whether the detrimental mechanical and anatomical changes that occur biventricularly with aging are associated with activation of DNA synthesis, flow cytometric analysis was performed on myocyte nuclei prepared from the left and right ventricles of rats at 4, 12, 20, and 29 months of age. Heart weight increased significantly with age, and this growth adaptation was associated with the development of left ventricular failure and right ventricular dysfunction. These phenomena were coupled with marked elevations in diastolic wall stress and increases in the percentage of myocyte nuclei in S+G₂M in both ventricles. Linear regression analyses revealed a direct correlation between the fraction of myocytes that entered the cell cycle and diastolic pressure and wall stress. An inverse relation was found between the percentage of myocyte nuclei in S+G₂M and +dP/dt and systolic wall stress. Thus the depression of hemodynamic performance coupled with alterations in the loading conditions contributes, at least in part, to increased DNA synthesis in cardiac myocytes with age. © 1993 Wiley-Liss, Inc.     

Cyclic strain induces proliferation of cultured embryonic heart cells

Summary Embryonic heart cells undergo cyclic strain as the developing heart circulates blood to the embryo. Cyclic strain may have an important regulatory role in formation of the adult structure. This study examines the feasibility of a computerized cell-stretching device for applying strain to embryonic cardiocytes to allow measurement of the cellular response. A primary coculture of myocytes and a secondary culture of nonmyocytes from stage-31 (7 d) embryonic chick hearts were grown on collagen-coated membranes that were subsequently strained at 2 Hz to 20% maximal radial strain. After 24 h, total cell number increased by 37±6% in myocyte cocultures and by 26±6% in nonmyocyte cultures over unstrained controls. Lactate dehydrogenase and apoptosis assays showed no significant differences in cell viabilities between strained and unstrained cells. After 2 h strain, bromodeoxyuridine incorporation was 38±1.2% versus 19±0.2% (P<0.01) in strained versus unstrained myocyte cocultures, and 35±2.1% versus 16±0.2% (P=0.01) in nonmyocyte cultures. MF20 antibody labeling and periodic acid-Schiff (PAS) staining estimated the number of myocytes in strained wells as 50–67% larger than in control wells. Tyrosine phosphorylation may play a role in the cellular response to strain, as Western blot analysis showed an increase in tyrosine phosphorylation of two proteins with approximate molecular weights of 63 and 150 kDa within 2 min of strain. The results of this study indicate that embryonic chick cardiocytes can be cultured in an active mechanical environment without significant detachment and damage and that increased proliferation may be a primary response to strain.     

Theoretical and experimental study of growth and remodeling in the developing heart

A theoretical model is presented for growth and remodeling in the developing embryonic heart. The model is a thick-walled tube composed of two layers of orthotropic pseudoelastic material. The analysis includes stress and strain dependent volumetric growth, with changes in material properties specified to reflect the evolving structure of the heart wall. For use in model validation, experimental measurements of ventricular opening angles are reported for 3–4-day old chick embryos under control and pressure overload conditions. Owing to changes in residual stress in the overloaded heart, the opening angle decreased from 31 ± 10° to −8 ± 12° (mean ± SD) within 12 h and then increased slightly. The opening angle at 12 h was significantly less than the control value. With an appropriate choice of parameters, the model yields reasonable agreement with these and other published opening angle data, as well as with temporal changes in lumen radius, wall thickness, epicardial strains, and pressure–volume curves during development before and after birth. Received: 26 November 2001 / Accepted: 21 December 2001     

Changes in the ultrastructure and DNA and protein content in human atrial myocytes in cardiac hyperfunction due to mitral valve defects

Biopsies from human right auricles were obtained during open heart surgery, prior to valve replacement, from six patients (aged from 20 to 49 years) with rheumatic heart disease. DNA and the total protein contents were measured in isolated myocytes by means of the two wave-length scanning cytophotometry after the double Feulgen and Naphthol yellow S staining procedure. In all the biopsies polyploid hypertrophied myocytes predominate. The hypertrophic, nondegenerated cells and the cells with degenerative changes of varying severity (in the first place, changes of contractile apparatus and membranes) are present. The highest degree of cell ploidy occurs in patients of functional class IV according to the New York Heart Association classification, 72 to 98% of cells displaying octaploid and higher DNA values. With the increase in ploidy of myocytes in series 2c----4c----8c----16c----32c----64c the protein content increases only as 2.0----3.0----5.8----7.8----13.0----16.8. Neither direct correlation between the ploidy level and the degree of cell degeneration, no inverse correlation between the degree of degeneration and the value of ejection fraction was observed.     

Effects of carbenoxolone on heart rhythm, contractility and intracellular calcium in streptozotocin-induced diabetic rat

Cardiac dysfunction is a frequently reported complication of clinical and experimental diabetes mellitus. Streptozotocin (STZ) – induced diabetes in rat is associated with a variety of cardiac defects including disturbances to heart rhythm and prolonged time-course of cardiac muscle contraction and/or relaxation. The effects of carbenoxolone (CBX), a selective gap junction inhibitor, on heart rhythm and contractility in STZ-induced diabetic rat have been investigated. Heart rate was significantly (P < 0.05) reduced in Langendorff perfused spontaneously beating diabetic rat heart (171±12 BPM) compared to age-matched controls (229± 9 BPM) and further reduced by 10⁻⁵ M CBX in diabetic (20%) and in control (17%) hearts. Action potential durations (APDs), recorded on the epicardial surface of the left ventricle, were prolonged in paced (6 Hz) diabetic compared to control hearts. Perfusion of hearts with CBX caused further prolongation of APDs and to a greater extent in control compared to diabetic heart. Percentage prolongation at 70% from the peak of the action potential amplitude after CBX was 18% in diabetic compared to 48% in control heart. CBX had no significant effect on resting cell length or amplitude of ventricular myocyte shortening in diabetic or control rats. However, resting fura-2 ratio (indicator for intracellular Ca²⁺ concentration) and amplitude of the Ca²⁺ transient were significantly (P < 0.05) reduced by CBX in diabetic rats but not in controls. In conclusion the larger effects of CBX on APD in control ventricle and the normalizing effects of CBX on intracellular Ca²⁺ in ventricular myocytes from diabetic rat suggest that there may be alterations in gap junction electrophysiology in STZ-induced diabetic rat heart.     

How well do single samples reflect rotifer species diversity? A test based on interannual variation of rotifer communities in Big Bend National Park (Texas,USA)

Studies of rotifer community composition and dynamics often rely on limited sampling regimes. To determine how well species richness is reflected in these studies, we examined interannual variation of rotifer species richness and monogonont community structure from 10 aquatic systems comprising four habitat types—springs, rock pools (tinajas), former cattle tanks, and artificial ponds—in Big Bend National Park (Texas, USA). Planktonic, littoral, and benthic samples were collected from all sites at about the same date for each of five summers (2001–2005). Our survey yielded 15 monogonont families including 30 genera and 84 species. Two bdelloid taxa also were designated. Species richness varied widely among these four habitats: range, 1–32; mean (±1 SD), 11.2 ± 8.0. Total Species richness in the habitats also varied considerably: springs (54 taxa) > artificial ponds (35 taxa) > tinajas (19 taxa) > cattle tanks (15 taxa). Sessile species comprised ≈13% of the taxa in our samples. Species turnover indices (STI) of these systems indicate low overall relatedness: mean (±1 S.D.) = 85.2 ± 7.1%. The relative frequency of encounter of most taxa in the four systems was low, with 79 taxa (≈92%) having values ≤2.0%. Singleton rates were quite high, ranging from 46.7 to 71.4%, with an overall mean ≈65.1%. Most importantly, we found that both species richness and STI varied considerably among habitat type. Species richness varied by 2–10× between consecutive years and STI ranged from 64 to 89% over the entire study. Our results indicate that rotifer community composition fluctuates greatly over time, and that rotifer community structure may be more labile than is generally believed. Species richness and thus biodiversity may be dramatically underestimated using single sampling or short-term strategies that are often employed in studies of zooplankton community structure. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont & R. Rico-Martínez Advances in Rotifer Research