For characterization of the growth pattern of cardiac myocytes during posthatching development, cardiac myocytes were enzymatically isolated from the ventricles of 1-, 15-, 29-, and 42-day-old chickens for measurement of myocyte nucleation, length, width, volume, and number, and for immunolabeling of cytoskeletal proteins. Ventricular myocyte number increased 156% from day 1 to day 42. Average cell volume increased more than 400%, and myocytes lengthened 125%, but cell width only increased 53% during this period. All myocytes were mononucleated at day 1. At day 15, 18% of myocytes became binucleated with < 1% of myocytes containing more than two nuclei. Interestingly, binucleated myocytes were able to divide with two nuclei going through mitosis at the same time. As demonstrated by staining with tubulin and alpha-actinin antibodies, two mitotic spindles and two cleavage furrows were formed in dividing binucleated myocytes. At day 42, binucleated myocytes increased to 44% with 11% of myocytes containing more than two nuclei. Sarcomeric alpha-actinin was partially disassembled in prometaphase and was reorganized into regular Z lines of sarcomeres in telophase. Desmin was disassembled in prophase and was reassembled during late telophase. These results suggest that chicken myocytes undergo hypertrophy and continue to proliferate during posthatching maturation, although it is currently believed that myocytes of all vertebrates withdraw from the cell cycle shortly after birth. We provide direct evidence for the first time of in vivo myocyte division in 6-wk-old chicken hearts.
A relatively inexpensive, expeditious, new nonradioactive microsphere method for measuring regional myocardial blood flow (RMBF) was developed with unlabeled microspheres and a Coulter Channelyzer. To validate the efficiency of this method, hearts from rats were perfused ex vivo by retrograde aortic cannulation. Unlabeled microspheres of varying size were injected into a side arm in the aortic cannula or added to blood samples collected from the rats. Microspheres were then recovered from the cardiac tissue and blood samples. It was found that > 97% of perfused microspheres (diam > 9.4 microns) were retained in the myocardium and that 94.8 +/- 2.2% of the trapped microspheres were recovered and counted successfully using a Counter Channelyzer. The percent recovery of microspheres from 2- and 0.5-ml blood samples were 95.4 +/- 2.3 and 95.3 +/- 3.1%, respectively. Blood flow to the anterior and posterior halves of the ventricular free walls and septum were measured in six rats; excellent agreements were found between the results yielded by 10-, 15-, and 20-microns unlabeled microspheres injected simultaneously. The transmural flow gradients in the left ventricular free wall estimated by 10- and 15-microns spheres did not significantly differ from each other. Thus the method developed here provides a new alternative for measurement of RMBF, which currently allows at least three measurements for nontransmural gradient RMBF and at least two measurements for transmural gradient RMBF.
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