Cardiomyocyte apoptosis in ischaemia-reperfusion due to the exogenous oxidants at the time of reperfusion Ana-Maria Rosca*, Camelia Matei*, Emanuel Dragan* and Alexandrina Burlacu 1 * ,{ * Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’ of the Romanian Academy, Bucharest, Romania { ‘Petru Poni’ Institute of Macromolecular Chemistry, Iasi, Romania Abstract Various studies performed on different models have demonstrated that apoptosis occurs in ischaemic-reperfused myocardium in vivo; however, the individual contribution of ischaemia and reperfusion to CMC (cardiomyocyte) apoptosis remains uncertain. We have determined the main inducer of CMC apoptosis in ischaemia-reperfusion by exposing CMCs to either 30 min ischaemia followed by reperfusion or to 25-OH-cholesterol (25-hydroxycholesterol) for 1–3 days. Both ischaemia-reperfusion and exogenous oxidants increased the Bax/Bcl-2 ratio, a favourable effect for the apoptotic process. However, apoptosis was not observed in ischaemic CMCs in the absence of reperfusion. Moreover, reperfusion after 30 min ischaemia did not make an important contribution to CMC apoptosis in culture in terms of caspase 3 activation. In contrast, 25-OH-cholesterol promoted CMC apoptosis by a caspase 3-dependent mechanism that involved the transcriptional activation of the pro-apoptotic protein, Bax and post-translational degradation of the anti-apoptotic protein, Bcl-2. From these results, we conclude that CMC apoptosis is not induced by ischaemia per se, but by the oxidants from the surrounding environment at the time of reperfusion. These exogenous oxidants exacerbate the alterations induced by ischaemia and complete the apoptotic process at the time when ATP and glucose levels are restored. Keywords: 25-hydroxycholesterol; apoptosis; Bcl-2; cardiomyocyte; ischaemia-reperfusion; oxidative stress 1. Introduction In vitro and in vivo investigations have provided compelling evidence that CMCs (cardiomyocytes) have a high-energy requirement for their contractile function, which makes the myocardium very sensitive to ischemia (Stanley et al., 2005; Santos et al., 2011). Thus, cell death occurs in myocardial infarction following ischaemia/reperfusion injury in both human and animal studies, but the relative importance of apoptotic cell death induced by ischaemia versus reperfusion remains contro- versial (Fliss and Gattinger, 1996; Zhao et al., 2000; Dorn, 2009). Numerous factors contribute to the initiation of cell death during ischaemia, e.g. oxygen loss, tissue acidosis, generation of ROS (reactive oxygen species), loss of calcium homoeostasis and DNA oxidative lesions. Their effects result in significant necrotic areas, which are directly corroborated to the ischaemia intensity and extent (Garcia-Dorado and Ruiz-Meana, 2000; Santos et al., 2011). Through seemingly opposing insults to ischaemia, reperfu- sion exacerbates the damage initiated by ischaemia, inducing further injury that culminates in apoptosis of cardiac myocytes viable immediately before myocardial reperfusion (Piper et al., 1998; Garcia-Dorado et al., 2009). Unfortunately, the real contribution of apoptosis to CMC death has been generally grossly underestimated due to the prompt clearance of apoptotic cells by phagocytosis (Fulda et al., 2010). These observations have raised questions whether ischaemia or reperfusion is the major inducer of CMC apoptosis, and whether therapeutic strategies can be developed to slow the progression of heart failure by targeting the apoptotic process. In this regard, two mechanisms have been proposed as contributing to the programmed cell death in ischaemic-reperfused myocar- dium: apoptosis is triggered in CMCs by the ischaemic injury in an energy-depleted state and is completed after reperfusion; or apoptosis is initiated by exogenous apoptotic-promoting factors originating from blood or non-myocytes in ischaemic-reperfused tissue. If the latter is the case, it seems reasonable to expect that future therapies will succeed in inhibiting apoptosis at the time of reperfusion by interfering with specific stages in a multi-stage pathway when the process can still be reversed. The purpose of this study was to determine the individual contribution of the ischaemia, reperfusion and exogenous oxidants to the CMC apoptosis, and thus establish the major contributor to the progression of heart failure in myocardial ischaemia-reperfusion. 2. Materials and methods 2.1. Isolation of CMC CMCs were obtained by trypsin digestion of 1–3 day-old rat hearts. The rats were housed and used in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 82-23, revised 1996) and all experiments were approved by 1 To whom correspondence should be addressed (email sanda.burlacu@icbp.ro). Abbreviations: CMC, cardiomyocyte; DEVD-AFC, Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HBSS, Hank’s balanced saline solution; HIF, hypoxia-inducible factor; NAC, N-acetyl-L-cysteine; NCMC, non-CMC; 25-OH-cholesterol, 25-hydroxycholesterol; ROS, reactive oxygen species; RT, reverse transcription. Cell Biol. Int. (2012) 36, 1207–1215 (Printed in Great Britain) Research Article E The Author(s) Journal compilation E 2012 International Federation for Cell Biology Volume 36 (12) N pages 1207–1215 N doi:10.1042/CBI20120080 N www.cellbiolint.org 1207