Loss of plasma membrane integrity, complement response and formation of reactive oxygen species during early myocardial ischemia/reperfusion
Introduction
Loss of plasma membrane integrity (LPMI), a hallmark of necrotic cell death, has been studied extensively in cell culture. However, in vivo studies are relatively few due to the lack of effective methods for LPMI identification in vivo. Necrosis of cardiomyocytes occurs in myocardial ischemia, and typically, myocardial necrosis (infarction) is determined histologically using 2,3,5-triphenyltetrazolium chloride (TTC). TTC enters all cells and is reduced to red-color 1,3,5-triphenylformazan by endogenous dehydrogenases/cofactors when cells are intact. In necrotic cells, when reperfusion following ischemia is extensive, the enzymes/cofactors have been washed out and TTC remains colorless, defining infarction. Since inadequate reperfusion underestimates the extent of infarction (Birnbaum et al., 1997, Ito et al., 1997), effective TTC staining requires at least 3-h reperfusion in the coronary artery occlusion model (Birnbaum et al., 1997). The need for adequate reperfusion limits the use of TTC when focusing on events early in reperfusion. In addition, TTC-stained tissue is not suitable for detection of other markers of injury, obviating a valuable experimental option and requiring study of additional animals.
Propidium iodide (PI), described in three studies using in vivo rodent myocardial ischemia/reperfusion (I/R) models (Ito et al., 1997, Weinbrenner et al., 2004, Wolff et al., 2000), provides an alternative approach for identifying LPMI. PI enters necrotic, but not intact, cells after compromise of plasma membrane integrity, intercalating with DNA to produce red fluorescence. PI can be introduced in vivo and does not require extensive reperfusion. In addition, we recognized that PI-stained tissues can be analyzed for concurrent pathological events.
In the present study, we extend previous PI-based assessments of LPMI occurring during I/R injury (Ito et al., 1997, Weinbrenner et al., 2004, Wolff et al., 2000) by (i) evaluating the early temporal development of LPMI in a murine myocardial I/R model, (ii) working with PI-stained tissue, documenting the innate inflammatory response as denoted by deposition of complement C3, the central molecule in complement pathways associated with myocardial infarction in clinical and animal studies (Weisman et al., 1990), and (iii) using superoxide dismutase 1 (SOD1) and catalase transgenic mice, evaluating the time-dependent contribution of reactive oxygen species (ROS) to necrosis following myocardial I/R.
The ability of PI to assess LPMI early in reperfusion provided an opportunity to re-examine conflicting results using TCC to study the effects of over-expressing or administering SOD1 on reperfusion injury. While several animal studies found that SOD1 could reduce infarction in both regional (Gross et al., 1986, Hangaishi et al., 2001, Kanamasa et al., 2001) and global heart ischemia (Ambrosio and Flaherty, 1992, Nishikawa et al., 1991, Otani et al., 1986, Wang et al., 1998), others found that it failed to protect against infarction in various ischemia models (Gallagher et al., 1986, Jones et al., 2003, Klein et al., 1988, Matsuda et al., 1991, Nejima et al., 1989, Ooiwa et al., 1991, Patel et al., 1990, Przyklenk and Kloner, 1989, Richard et al., 1988, Uraizee et al., 1987, Watanabe et al., 1993). Downey et al. (1991) suggested that SOD1 might interfere with the ability of TTC to differentiate between living and dead cells, with the result that TTC produced an artifactually reduced infarct size in SOD1-treated animals (Shirato et al., 1989).
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Mouse model of myocardial I/R injury
The WT (C57BL/6) mouse strain was obtained from the Jackson Laboratory (Bar Harbor, ME). Transgenic mice over-expressing human SOD1 or catalase and their WT littermates were generously provided by Dr. Arlan Richardson's group (Chen et al., 2003). All mice were maintained at the Division of Laboratory Animal Resources of SUNY Downstate Medical Center. Genotyping was provided by GeneTyper (New York, NY) using established PCR protocols (Chen et al., 2003, Wessels et al., 1995). All mice were used
LPMI occurred early in myocardial reperfusion
LPMI was studied in 7 groups of WT (C57BL/6) mice (Fig. 1a). Group (i) was sham-operated; groups (ii)–(vii) were subjected to 1 h of LAD occlusion followed by 0, 1, 2, 3, 6 or 24 h of reperfusion, respectively (n = 5 mice/group/time point, except for the 24-h group where n = 10).
Since the one-way ANOVA test for % of at-risk tissue with LPMI of all groups showed statistical significance (P < 0.01), Levene's test of the homogeneity of variances was applied and indicated P < 0.01. Therefore, the Dunnett T3
Discussion
In a murine cardiac I/R model, using in vivo injection of PI to detect LPMI and BFMs to detect ischemic tissue (at risk for necrosis), a significant percent of at-risk tissue with LPMI (33 ± 5%) was detected at the earliest time investigated, 1 h of reperfusion (Fig. 1a). This level of LPMI was maintained through 24-h reperfusion, indicating that the histological boundary of tissue with LPMI was essentially established by the first hour of reperfusion. This is a significant new result, not
Acknowledgements
The authors thank Dr. James Cottrell for continued support and Javi Balroop, Oghomwen Shaka-Idusuyi, EunHee Ko and Danielle Green for technical assistance. We also thank Drs. Arlan Richardson and Holly Van Remmen for providing SOD1 and catalase tg mice. The research was funded in part by NIH grant 1R21HL088527(MZ) and a SUNY-Downstate Dean's Award (MZ).
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These authors contributed equally to this paper.