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Transient and reversible deoxyribonucleic acid damage in human left ventricle under controlled ischemia and reperfusion

Gian G. Corbucci, MD^*, Cinzia Perrino, MD, Giuseppe Donato, MD, Antonio Ricchi, MD, Biagio Lettieri, MD^||, Giancarlo Troncone, MD^¶, Ciro Indolfi, MD^#, Massimo Chiariello, MD and Enrico V. Avvedimento, MD^¶,*

^* Division of Anesthesiology, University of Cagliari, Cagliari, Italy
Division of Cardiology, Naples, Italy
Department of Molecular and Cell Pathology, University Federico II, Naples, Italy
Division of Pathology, Catanzaro, Italy
^|| Division of Cardiology, University Magna Graecia, Catanzaro, Italy
^¶ Department of Cardiothoracic Surgery, Brotzu Hospital, Cagliari, Italy
^# Division of Anesthesiology, Second University of Naples, Naples, Italy

View larger version (24K): [in a new window]   Figure 1 Selective activation of extracellular signal-regulated kinase (ERK)1/2, Jun terminal kinase (JNK), and p38 in ischemic and reperfused hearts. Representative immunoblots of ERK1/2 and phospho-ERK1/2 (A), total and phospho-JNK (B), p38 and phospho-p38 (C) with relative densitometric and statistical analysis. All experiments were repeated at least 3 x and yielded similar results. Extracellular signal-regulated kinase activation was calculated as pERK/ERK ratio; JNK activation was calculated as pJNK/JNK ratio, and p38 activation was calculated as pp38/p38 ratio. *p < 0.01 vs. control (CON); #p < 0.05 vs. 20' and reperfusion (REP). Molecular weight markers are reported on the left of each panel.

View larger version (56K): [in a new window]   Figure 2 Mitochondrial stress in ischemic and reperfused hearts. (A) Representative electron microscopy sections taken from control (CON), 20', 58', and reperfusion (REP) left ventricular samples showing mitochondrial swelling (scale bars = 0.2 µm). We examined at least 10 fields/samples, counted at least two to three mitochondria/fields, and compared their median size to the scale bar indicated. In the CON samples, we did not find enlarged mitochondria (2 x the scale bar). (B) Biochemical determination of cytochrome C oxidase activity, ATP content, and malondialdehyde (MDA) levels on ventricular samples. For cytochrome C oxidase, *p < 0.01 vs. CON; for ATP levels, *p < 0.01 for ischemia and REP vs. CON and #p < 0.05 for REP vs. ischemia; for MDA, *p < 0.01 vs. CON. Abbreviations as defined in Figure 1.

View larger version (71K): [in a new window]   Figure 3 Massive TUNEL staining of ischemic and reperfused cardiomyocytes. Representative TUNEL and Haematoxylin Eosin (HE) staining of sections derived from control (CON), 20', 58', and reperfusion (REP) left ventricular samples (40 x magnification). The insets show a higher magnification of the same samples (80 x). TUNEL-positive cells were counted in at least 10 (20 x) fields/sample (CON, <1 ± 2%; ischemia 20', 30 ± 5%; ischemia 58', 50 ± 10%; reperfusion 20 ± 5%). Abbreviations as defined in Figure 1.

View larger version (29K): [in a new window]   Figure 4 Deoxyribonucleic acid damage responses in ischemic and reperfused hearts. Representative Western blots of caspase 3 (A), p53, p21WAF, and phosphoH2Ax levels (B), with relative densitometric evaluation and statistical analysis. All experiments were repeated at least 3 x, and differences observed were not due to different protein concentrations as all membranes were also probed with anti-extracellular signal-regulated kinase-1/2 antibodies (B, lower panel). For p53, p21WAF, and phospho-H2Ax, *p < 0.01 vs. control (CON). Molecular weight markers are reported on the left of each panel. (C) Proliferative cell nuclear antigen (PCNA) staining of control, ischemic (58'), and reperfused ventricular samples. Hystochemical staining with anti-PCNA monoclonal antibody was performed as described in the Methods section. Positive cells were counted in at least five (20 x) fields (control samples <0.5 ± 1.2%; ischemia 58 min: 10 ± 3%; reperfusion samples: 8 ± 2%). ADU = arbitrary densitometric units. Other abbreviations as defined in Figure 1.