Objective Cold blood cardioplegia (CBC) has been advocated as an advancement in myocardial protection during cardiopulmonary bypass (CPB) and cardioplegic arrest (CA) leading to decreased postoperative morbidity. Bcl-2, Bad, and caspase 3 are intermediates within the apoptosis cascade, which is activated following ischemia-reperfusion (IR)-induced myocardial injury. We studied the expression and modification of these apoptosis intermediates due to cardioplegia and CPB in humans within cardiac and skeletal muscle.
Methods Right atrial and skeletal muscle was harvested from cardiac surgical patients (N = 6) before and after CPB, CBC, and mild hypothermia. Total and modified (cleaved/phosphorylated) caspase 3, Bcl-2, and Bad were measured by quantitative immunoblotting using specific antibodies. Microarray gene expression analysis was carried out using Affymetrix U95 GeneChip following RNA isolation. Activity of terminal caspase 3 was assessed by fluorometric assay. TUNEL assay was performed on atrial sections to identify cells undergoing apoptosis. Two-tailed paired t-test was used for statistical analysis.
Results In response to CPB, skeletal muscle samples did not show changes in total or modified apoptosis protein levels in response to CPB. In myocardial tissue, CBC significantly increased phosphorylation or cleavage of Bcl-2, Bad, or caspase 3, while there was no significant change in total protein levels. Bcl-2 (Ser70) and Bad (Ser112) phosphorylation were increased by 2.35 ± 0.40 fold and 1.64 ± 0.25 fold, respectively (p < .05), while caspase 3 activity was increased 1.50 ± 0.14 fold (p < .05) after cardioplegic IR. The number of apoptotic cells in atrial tissue using TUNEL staining was increased following CBC/CA. Microarray analysis did not reveal any significant differential gene expression for the genes studied.
Conclusion Cold blood cardioplegic arrest triggers the modification/activation balance of both proapoptotic (caspase 3) and antiapoptotic (phospho-Bad and phospho-Bcl-2) proteins. This change is specific to myocardium as apoptosis cascade is not significantly altered following CPB in peripheral skeletal muscle. Moreover, protein activation rather than total protein levels may be the primary indicator of apoptosis induction in myocardium.
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