Abstract 3877: The Magnitude and Temporal Dependence of Apoptosis Early After Myocardial Ischemia with or without Reperfusion

Abstract

It has been hoped and conjectured that in addition to improving the balance between myocardial oxygen supply and demand amelioration of apoptosis could preserve jeopardized ischemic myocardium destined to undergo necrosis (cell death). Assessment of apoptosis in experimental animals has been based generally on TUNEL assays. Unfortunately, TUNEL positivity occurs with oncotic, mitotic, and autophagic, as well as apoptotic cell death. Accordingly, we characterized the extent of apoptosis after transient or persistent ischemia with highly specific methods. Coronary artery occlusion with and without subsequent revascularization was induced in three groups of 10 week old C57BL/6 mice: those subjected to 1 hr or 4 hr transient ligation followed by 24 hr of reperfusion (1HTLR24H, 4HTLR24H); or 24 hr persistent ligation (24HPL). Apoptosis was quantified throughout the LV by TUNEL, single stranded DNA (ssDNA), and caspase 3 immunohistochemistry, electron microscopy (EM), and caspase 3 and 8 activities assessed biochemically. TUNEL staining markedly exceeded and did not correlate with ssDNA, caspase 3 staining, or apoptosis defined by EM in any group. It was lowest in the 1HTLR24H group (averaging 38.6 of cells ± 3.1% [SEM], n = 9) greater in the 4HTLR24H group (48.5 ± 3.1%, n = 9), and significantly greater than both in the 24HPL group (67.2 ± 4.3%, n = 9, p < 0.01). ssDNA staining was minimal and similar in all three groups and significantly less than TUNEL staining (p < 0.01) (1HTLR24H, 1.0 ± 0.2%, n = 9; 4HTLR24H, 0.8 ± 0.1%, n = 9; 24HPL, 2.9 ± 1.6%, n = 9). Caspase 3 activity/mg protein (1HTLR24H, 15.3 ± 12, n = 3; 4HTLR24H, 58.5 ± 4, n = 3; 24HPL, 56.5 ± 10, n = 3) was similar to that in normal hearts (53.2 ± 11, n = 4), as was caspase 8 activity. No cleaved caspase 3 was seen immunohistochemically, and only rare, definitively identified apoptotic cells were seen by EM in hearts from any group. Apoptosis is de minimus early after transitory or persistent ischemia contrary to its overestimated, large extent as judged from TUNEL. Thus, antiapoptotic interventions per se are not likely to preserve substantial amounts of jeopardized ischemic myocardium early after ischemic insults.