Influence of the chronic nitric oxide synthesis inhibition on cardiomyocytes number.

This work analyzes the relationship between the number of viable cells and alteration of the cardiomyocytes growth response capacity of the hypertensive rat myocardium. Hypertension was induced in Wistar rats by means of nitric oxide synthesis blockade using NG-nitro-L-arginine methyl ester (L-NAME). L-NAME (12 mg/kg per day) was given to animals in drinking water ad lib for 15 weeks. Proliferating cell nuclear antigen (PCNA) protein expression and the disector method were used to evaluate the proliferation capacity of the cardiomyocytes and its numerical density alteration (Nv[m]), respectively. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and monoclonal antibody to single-stranded DNA were two methods that detected the process of the apoptotic cell death. The association of the p53 expression with the apoptosis was investigated using anti-p53 antibody. The heart weight, body weight, and heart weight/body weight ratio of the control rats increased 114%, 77%, and 22%, respectively, and the Nv[m] decreased 60% (P<0.0001) relative to the L-NAME rats. The cardiomyocytes did not present PCNA labeling, indicating the absence of cellular proliferation. The decline of the Nv[m] was also associated with apoptotic cell death in the myocardium of the hypertensive rats. A p53-dependent pathway seems to mediate the programmed cell death in this model of hypertension.

Myocardial repair with long-term and low-dose administration of a nitric oxide synthesis inhibitor. Myofibroblasts, type III collagen and fibronectin.

OBJECTIVE: To study the healing process of the myocardium in hypertensive rats undergoing inhibition of nitric oxide synthesis. METHODS: Two groups of animals were studied: one received L-NAME, 12 mg/kg/day, and the other was a control group. The presence of type III collagen, fibronectin, and alpha-smooth muscle actin-positive cells was assessed by immunohistochemistry. RESULTS: Fibronectin was seen in both early and late lesions, while type III collagen was seen mainly in areas of incomplete healing, situated among myocytes and around the intramyocardial branches of the coronary arteries. Areas representing early and late lesions showed a population of spindle-shaped cells. Immunohistochemistry showed that these cells were positive for alpha-smooth muscle actin. CONCLUSION: In the myocardium of hypertensive rats, the alpha-smooth muscle actin-positive cells are related to the accumulation of type III collagen and fibronectin in the areas of myocardial damage.

Quantitative study of the myocardium in human embryos.

We studied six human embryos of the second gestational month (postsomitic period, from stages 15 to 23). They were fixed in 4% formaldehyde and serially sectioned. Stereological determinations were made from the compact layer of the ventricular myocardium: a) volume density of the myocardial parts: myocytes (Vv[myocyte]) and interstitium (Vv[interstitium]), b) numerical density of the myocytes (Nv[myocyte] mm3) calculated from six optical disector pairs per embryo, c) total number of myocytes (N[myocyte]), d) volume of the myocytes (V[myocyte] micron 3). In embryos from stages 15 to 19 the quantities of the myocytes and interstitium remained practically unchanged (no statistical difference was found). However, the volume of the ventricular myocardium mass increased more than 5 times during this period. Comparing embryos of stages 15 and 23, the mean value of the Nv[myocyte] decreased about 30 per cent, while N[myocyte] increased almost 2,000 per cent in the same period. Simultaneously, the volume of the ventricular myocardial mass increased almost 30 times, and Vv[myocyte] and Vv[interstitium] showed a small increase in the myocyte component (about 20 per cent), with a decrease of the interstitial component (about 70 per cent). In the early post-somitic period the human myocardium has a relatively small number of small myocytes, in the late post-somitic period it is composed of large and relatively abundant cardiac myocytes. The conspicuous increase in the ventricular myocardial volume observed in stage 23 seems not to be related to the increase in the interstitial portion of the myocardium. These arguments suggest both the enlargement and the division of the cardiac myocytes during the post-somitic period.

Stereology of the myocardium in embryos, fetuses and neonates of the rat.

We studied with quantitative methods the myocardium of 32 specimens of rats divided into three age groups: embryos, fetuses and neonates. Days of gestation were counted from the day following an overnight mating that was considered day 1 of gestation and only one animal per litter was used. The hearts were fixed in Bouin's fixative, sectioned and stained by routine methods. Stereological determinations were made on ventricular myocardium: (1) volume density of the myocytes [Vv(myocyte)] and interstitium [Vv(interstitium)], and (2) numerical density of the myocytes [Nv(myocyte) 1/mm3] calculated from fifteen optical dissector pairs per specimen. The total number of cardiac myocytes [N(myocyte)] was calculated as the product of Nv(myocyte) and the cardiac volume. The Nv(myocyte) increased from embryos to neonates, differences between embryos and fetuses and between embryos and neonates were statistically significant. The Vv(myocyte) increased from embryos to neonates (from 80.0 to 94.0%). During this period the Vv(interstitium) decreased from 21.0 to 5.5%. Differences of the Vv(myocyte) and Vv(interstitium) were significant comparing embryos with neonates and comparing fetuses with neonates. The N(myocyte) is roughly (mean +/- standard error of the mean) 9,297 +/- 487 in fetuses and 38,438 +/- 612 in neonates. This represents an increase of about 3.1 times from fetuses to neonates while the cardiac weight increased about 2.2 times in the same period. Coefficients of error for the Nv(myocyte) estimates averaged about 7.3%, for the Vv(myocyte) estimates averaged about 1.8%, and for the Vv(interstitium) estimates averaged about 9.1%. These results suggest a high mitotic activity in the rat myocardium during prenatal life and after birth.