Improved profile of bad phosphorylation and caspase 3 activation after blood versus crystalloid cardioplegia.

BACKGROUND

Expression of Bcl-2 family proteins and activation of terminal caspase 3 are important for ischemia-reperfusion-induced apoptosis. Bad and Bax are pro-apoptotic proteins, whereas, phosphorylation of Bad inhibits its binding to and inactivation of anti-apoptotic Bcl-2. Thus, decreases in phospho-Bad would be proapoptotic. We investigated if blood (BCP) or crystalloid cardioplegia (CCP) differentially affects apoptosis gene-related proteins.

METHODS

Rabbit hearts were perfused with Krebs-Henseleit buffer (KHB) on a Langendorff apparatus. Control hearts (n = 6) were perfused for 90 minutes without cardioplegic ischemia. In the other two groups, hearts were arrested for 30 minutes (37 degrees C) with BCP (n = 6) or with CCP (n = 6) administered continuously (1.5 mL/min). The hearts were reperfused for 30 minutes with KHB. Left ventricle (LV) performance was measured before cardioplegic arrest and at 30 minutes of reperfusion. In vitro relaxation responses of precontracted microvessels (100-180 microm) were obtained in a pressurized no-flow state. Total and activated or phosphorylated caspase 3, Bcl-2, Bad, and Bax were measured by quantitative immunoblotting using specific antibodies.

RESULTS

Blood cardioplegia significantly improved the recovery of LV developed pressure compared to CCP (p < 0.05). The endothelium-dependent relaxation in response to adenosine 5'-diphosphate was greater after BCP than after CCP (59.9 +/- 4% vs 26.9 +/- 6%, respectively; p < 0.05). There were no differences in total protein levels of caspase 3, Bcl-2, Bad, and Bax between the groups. Both BCP and CCP increased caspase 3 activity as compared with controls, but CCP caused more activation of caspase 3 than BCP (6.2 +/- 0.7 fold vs 3.1 +/- 0.4, p < 0.05). Both BCP and CCP induced phosphorylation of Bad at Ser(112), but BCP caused greater phosphorylation of Bad (3.5 +/- 0.2 fold vs 2.0 +/- 0.12 fold, respectively, p < 0.05) than CCP.

CONCLUSIONS

Blood cardioplegia is superior to CCP in inhibiting the activation of caspase 3 and in increasing phospho-Bad. These actions of BCP were associated with improved LV function and endothelium-dependent relaxation of coronary microvessels. These results may provide molecular mechanisms by which to improve myocardial protection during cardiac surgery.