DNA and sex chromatin content in nuclei of conductive system and working myocytes of normal and hypertrophied human heart.

DNA content was measured by means of Feulgen cytophotometry in myocyte nuclei of sinoatrial und atrioventricular nodes, Purkinje cells, left and right atria and ventricles from normal and hypertrophied human hearts both on 12 microns sections and on squash preparations of myocardial tissue. In contrast to ordinary atrial and ventricular myocytes of normal and hypertrophied adult hearts, which contain mostly polyploid nuclei, the DNA content in 74 to 88.5% of myocyte nuclei from sinoatrial and atrioventricular nodes does not exceed 2 c. Unlike nodal myocytes in Purkinje cells of hypertrophied adult heart there are 97 to 99.5% of polyploid nuclei, 49 to 88% of them displaying octaploid and higher DNA values. Myocytes of highly hypertrophied atria contain nuclei of considerably higher ploidy level than those of normal atria. Percentage of binucleated cells show a 4-fold increase in hypertrophied atria compared with normal ones. The total area of nucleoli per nucleus is proportional to the degree of ploidy. Number of sex chromatin bodies counted simultaneously with DNA measurements on sections of atrioventricular node and on squash preparations of atria and ventricles is correlated with ploidy level. Judging by correlations between the number of sex chromatin bodies and DNA content in nuclei, on one hand, and between the total area of nucleoli per nucleus and the degree of ploidy, on the other, the mitotic endoreduplication (true polyploidization) is responsible for the increased DNA content in nuclei of conductive system, atrium and ventricle.

Proliferation and biosynthetic activities of myocytes from conductive system and working myocardium of the developing mouse heart. Light microscopic autoradiographic study.

Incorporation of 3H-thymidine, 3H-uridine and 3H-leucine into myocytes and their mitotic activity have been investigated in different compartments of the mouse heart conductive system (CS) and working myocardium (WM) at pre- and postnatal stages of development. On 15th and 18th d of embryogenesis, 3H-thymidine pulse and cumulative labelling indices (LI) of myocytes in the sinoatrial node (SAN), atrioventricular node (AVN) and His bundle (HB) were found to be 4 to 6 times lower than in WM (0.001 less than or equal to P less than or equal to 0.01). On 3rd to 9th d after birth LI remained decreased (0.001 less than or equal to P less than or equal to 0.05) in SAN while replicative activity of myocytes in AVN and HB approached that of both ventricular and atrial WM. Since 13th d after birth LI did not exceed 1% in all the compartments studied. In the prenatal period LI in SAN were higher than in AVN P less than or equal to 0.01), whereas in the postnatal one it was just the opposite (0.001 less than or equal to P less than or equal to 0.05). During cardiogenesis change of mitotic indices in WM and CS correlates with that of LI. Duration of cell cycle and its periods (G2, G2 + 1/2 M, S) estimated by curves of labeled mitoses, double-labelling technique with 14C-thymidine and 3H-thymidine and label "dilution" method differ slightly in myocytes from WM and CS. The latter incorporate 3H-uridine much less intensively at most stages of heart development compared with WM. At perinatal stages of cardiogenesis 3H-leucine labels CS myocytes somewhat weaker than WM ones until 8th d after birth when the labelling intensities in all cell types become similar. It can be concluded that on the whole CS myocytes from the developing mouse heart appear to be less active than those of WM concerning replication, transcription, and translation processes.

Striated myocytes and atrial specific granules in the pulmonary veins of chronically infarcted rat hearts.

The ultrastructure of the striated myocytes in the pulmonary myocardium of the Wistar rat, was studied following left ventricular infarction (LVI) for 4 days and 4 weeks, respectively. Ischemia was provided by ligating of the left coronary artery which successively produces left ventricular dysfunction and congestion of the left atrium and pulmonary veins. In general, the striated myocytes of the extrapulmonary veins of the control groups displayed ultrastructural characteristics similar to those of the left atrium. However, in the former, transverse tubules commonly occurred making interior couplings with the SR at the I-band levels. Also, between striated myocytes of the intrapulmonary veins of the controls nexus-like junctions of about 2 micron length were observed. Further, in the same groups atrial specific granules (ASG), including A-granules and B-granules, were present in about 20%-30% of the striated myocytes of the extrapulmonary veins as shown in single sections. The ASG tended to decrease by number in areas approaching the lung hilus and were not observed in the intrapulmonary veins of the controls. After 4 days of ventricular ischemia granules also appeared in the intrapulmonary veins. They were mostly seen in single and randomly spread in the cytoplasm of a limited number of cells, i.e., in between 0% and 5% of the myocytes as seen in single sections. Four days of LVI produced only minor cellular injuries to the extrapulmonary and intrapulmonary venous wall, while more severe lesions appeared in both areas following 4 weeks of LVI. Such lesions were preferably localized to the adluminal surface and consisted mainly of Z-band and sarcomere anomalies, vacuolization of the cytoplasm and mitochondrial swelling.

DNA synthesis in myocytes from different myocardial compartments of young rats in norm, after experimental infarction and in vitro.

Following 30 repeated 3H-thymidine (3HTdr) injections to rats beginning from days 13 and 21 after birth ca. 20 and 10% of labeled nuclei were observed in the left and right atria, respectively, while in both ventricles cumulative labeling of myocytes was nearly ten times lower. In rats of the same age with experimental myocardial infarction of the left ventricle the number of myonuclei labeled after 30-fold 3HTdr injections increased in atria up to 40-50%, in the perinecrotic area of the left ventricle up to 8-11% and in myofibers of the left and right ventricles located far from the necrotic focus up to 3-4 and 2-3%, respectively. In some of the rats subendocardial and/or subepicardial layers of the surviving left ventricular tissue contained up to 15-35% of labeled myonuclei. Thus, the reproduction capacity of ventricular myocytes in neonates is significantly higher than in adults. Reactive proliferation of cardiomyocytes in the atria of young rats, as opposed to adults, starts on day 3 rather than day 5 after infarction, however the DNA synthesis reactivation is of the same order as found in similar experiments on adult rats. The cultivation in vitro does not eliminate the specificity of the atrial myocytes behavior, their proliferation intensity being 5-7 times higher than that of ventricular ones. Active DNA synthesis and subsequent acytokinetic mitoses of atrial myocytes lead to a significant increase in the number of binucleated cells both after infarction and in vitro.

Ultrastructure of DNA-synthesizing and mitotically dividing myocytes in sinoatrial node of mouse embryonal heart.

Unlike the cells of the working myocardium (WM), myocytes from the sinoatrial node (SAN) of the hearts of 16- to 18-day-old mouse embryos are small and 'pale'. They contain few poorly organized myofibrils and no specific granules. The frequency of myocytes labeled with 3H-thymidine or dividing mitotically is 3-4 times lower in the SAN compared to the WM. The ultrastructure of myofibrils and other cytoplasmic organelles in DNA-synthesizing myocytes of the SAN remains practically the same as in most of its muscle cells not labeled with 3H-thymidine. However in prometaphase Z-discs disappear and myofibrils break into free myofilament bundles. Intercalated discs and desmosomes persist in all mitotic phases. As a result of cytotomy, two daughter myocytes appear, while occasional acytokinetic mitoses give rise to binucleated cells of the SAN.

Ultrastructure of muscle fibers and cells synthesizing DNA in lymph hearts of developing frogs and chick embryos.

The ultrastructure of striated muscle fibers and 3H-thymidine (3HTdr)-labeled cells adjacent to them in the lymph hearts of larvae of Rana temporaria, yearling frogs, and 9- to 13-day-old chick embryos was studied by use of electron-microscopic autoradiography. A comparatively high level of differentiation of lymph-heart muscle fibers was observed not only in yearling frogs but also in larvae. Myosatellites occurred in all stages of development. No mitoses were found in muscle fibers. In 9- to 13-day-old chick embryos the myofibers of lymph hearts were somewhat less differentiated than those of the larvae and yearling frogs. Differentiating sarcomeres were often seen in the sarcoplasm of myofibers of chick embryos. The analysis of the ultrastructure of 3HTdr-incorporating cells shows that 2-4 h after the single 3HTdr administration only mononucleated cells devoid of myofilaments are commonly labeled both in tadpoles and chick embryos. When fixation is postponed by 48-70 h, myonuclei frequently become labeled. Thus, the data obtained support the evidence that proliferation and differentiation processes in the developing muscle tissue of the lymph heart of both species studied are mutually exclusive, similar to the situation in differentiating vertebrate skeletal muscle.

DNA synthesis in atrial myocytes of rats with aortic stenosis.

Indices of labeled myonuclei have been determined in hypertrophying hearts of adult Wistar rats by autoradiography after single-pulse or repeated [3H]thymidine administration. After single [3H]thymidine injections, only 1.36 +/- 0.66 and 1.32 +/- 0.87% labeled myonuclei were observed in the left and right atria, respectively. In the experiments with multiple [3H]thymidine administration, the first injection of this precursor was given on the seventh day after aortic constriction; thereafter, 30 or 42 injections of [3H]thymidine were given at 12-hr intervals up to the fourth postoperation week. Following 30 repeated [3H]thymidine injections, 29.75 +/- 4.65 and 16.78 +/- 3.33% labeled myonuclei were visible in left and right atrial muscle cells, respectively. The cumulative labeling index for left atrium myocytes clearly correlates (r = 0.65-0.73) with an increase in the weight of the heart. Increase in heart weight to more than 160% of controls corresponds to [3H]thymidine labeling of 38.06 +/- +/- and 21.67 +/- 4.16% in left and right atrial myocytes, respectively, whereas in hearts weighing less than 140% of controls, [3H]thymidine labels only 8.20 +/- 1.93% in the left atrium and 3.94 +/- 1.57% in the right one. In the ventricles, cumulative indices of myonuclear labeling do not exceed 0.217 +/- 0.11% even in hearts weighing nearly 180% of controls. Cumulative frequencies of labeling for AV system myocytes are almost ten times higher (1.97 +/- 0.38). These results, together with our data concerning mycocardial infarction (27-29,31), make it necessary to reconsider the role of cardiomyocyte hyperplasia in different experimental and pathological conditions, paying special attention to the proliferative behavior of the atrial muscle cells. DNA synthesis in atrial myocytes seems to be stimulated by heart hyperfunction.

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 successive 3H-thymidine injections at 12 h 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 repeated 3H-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 successive 3H-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 repeated 3H-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.