Cell cycle reentry of ventricular and atrial cardiomyocytes and cells within the epicardium following amputation of the ventricular apex in the axolotl, Amblystoma mexicanum: confocal microscopic immunofluorescent image analysis of bromodeoxyuridine-labeled nuclei

Abstract

To understand how to reinitiate cell division in adult human myocardium, a heart regeneration model was examined in the amphibian axolotl salamander, Amblystoma mexicanum. The ventricular apex was surgically amputated and resected for 3 weeks. At 14 days of recovery, the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) was injected intraperitoneally to identify cell types undergoing S-phase by indirect immunofluorescence using primary anti-BrdU antibodies. This is the first report showing a concentrated area of cell division in the ventricle and cells throughout the atrial epicardium by confocal microscopic image analysis in response to wounding of the ventricle. Tissues coimmunostained with anti-BrdU and sarcomeric myosin-specific MF20 antibodies showed 12.8 +/- 4.10% of myocytes contained BrdU(+) nuclei in a 75 microm to 750 microm zone in the ventricular myocardium subjacent to the amputation plane. BrdU(+) nuclei also were present in newly formed ventricular epicardium that surround dividing myocytes, and in epicardial mesothelium (74.3 +/- 6.36 %) and connective tissue (44.9 +/- 13.31%) cells distal to the wound. Unexpectedly, immunofluorescent BrdU(+) nuclei were observed in isolated atrial myocytes (13.9 +/- 1.45%) and in uninjured atrial epicardial mesothelium (64.3 +/- 1.55%) and connective tissue (29.4 +/- 5.50%). No BrdU(+) nuclei were present in cardiomyocytes or epicardium from sham-operated and BrdU-treated controls. These results suggest that renewed cell division is a specific response to wounding of the ventricle. Additionally, release of a growth factor may be responsible for the specific localized mitotic ventricular cardiomyocyte response adjacent to the wound, and the more generalized atrial proliferative response distal to the amputation.