Characteristics of death of neonatal rat cardiomyocytes following hypoxia or hypoxia-reoxygenation: the association of apoptosis and cell membrane disintegrity

Abstract

Irreversibly injured cardiomyocytes are positive for terminal deoxynucleotidyl transferase nick end-labeling (TUNEL), making it controversial as to whether TUNEL-positive cardiomyocytes induced by hypoxia-reoxygenation are apoptotic (secondarily necrotic) or primarily necrotic. We investigated the relationship between plasma membrane integrity and DNA fragmentation in hypoxic-reoxygenated cardiomyocytes. Cardiomyocytes were prepared from neonatal rat heart and exposed to hypoxia. The plasma membrane integrity was assessed by propidium iodide (PI) staining. The mode of DNA fragmentation was assessed by TUNEL and in situ polymerase chain reaction ligation assay. Furthermore, caspase-3 activity was measured in hypoxic-reoxygenated cardiomyocytes. Reoxygenation for 24 h after 3-8 h of hypoxia increased TUNEL positivity. However, the appearance of PI-positivity preceded that of TUNEL at various time points following reoxygenation. In contrast, TUNEL-positive but PI-negative cells were rarely found. In the hypoxic-reoxygenated cells, caspase-3 activity was increased, and PI- and TUNEL-positive cardiomyocytes possessed a sufficient number of double-strand DNA breaks with single-base 3'-OH terminals. In cardiomyocytes subjected to hypoxia-reoxygenation, the appearance of TUNEL positivity was delayed in comparison to membrane disintegrity, but in these cells caspase-3 has been activated and the mode of DNA fragmentation was apoptosis-specific. Thus, hypoxia-reoxygenation induces apoptosis associated with cell membrane disintegrity in cardiomyocytes.

Attenuation of Ischemic Myocardial Injury and Dysfunction by Cardiac Fibroblast-Derived Factor(s)

“…Nonadherent cells were collected as cardiomyocytes, and incubated in gelatin -coated 24 -well culture plates at 1×10 5 cells/mL. Cells were maintained with Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 50 U/mL penicillin G, 50 µg/mL streptomycin, and 10 −5 mol/L cytosine arabinoside (Ara -C) to eliminate any contaminated non -cardiomyocytes 12) . After a 48 -hour incubation with Ara -C, the original culture media was exchanged for media without Ara -C. Sarcomeric α -actin antibody (DAKO, Japan) staining showed that the isolated cardiomyocytes were 95% pure.…”

Differences in left ventricular cardiomyocyte loss induced by chronic intermittent hypoxia between spontaneously hypertensive and Wistar–Kyoto rats

“…This condition, called chronic intermittent hypoxia (CIH) [2,3], causes injury to cardiomyocytes similar to that caused by ischaemia-reperfusion injury [4]. Recent studies have suggested that CIH-induced cell damage may be related to the increase in intracellular reactive oxygen species (ROS) generated during reoxygenation after hypoxia [2,5,6]. Moreover, CIH may cause lipid peroxidation [7], protein oxidation and DNA damage [8] and attenuation of antioxidant enzyme capacity, thus reducing cardiomyocyte numbers by cell death [3].…”

“…Since the accumulation of ROS from CIH [2,3,5,6] and hypertension [10,16] is a major cause of cardiomyocyte death, we postulated that patients with both OSA and hypertension have higher ROS levels due to an additive effect, compared to patients with either condition alone. To further investigate the influence of combined OSA and hypertension on cardiomyocyte death, we performed an animal experiment that simulated the physiology of cardiomyocyte death in patients with OSA who had longstanding hypertension.…”

Balance between hypertrophic and hypoxic stimulus in caspase-3 activation during rat heart development

“…For example, under hypoxia some cells are irreversibly damaged and die, whereas others can adapt to the stressful environment and survive. Another variable is the different response to hypoxia displayed by in vitro or in vivo models, since it has been demonstrated that in vivo acidosis, reoxygenation or reperfusion, but not hypoxia alone, are strong stimuli for the induction of apoptosis (Bishopric et al 1999, Jung et al 2001, Matsuoka et al 2002. Thus intermittent exposure to hypoxia of pregnant rats has been shown to determine significant effects on fetal growth, i.e.…”