Molecular indices of apoptosis after intermittent blood and crystalloid cardioplegia.

BACKGROUND: We investigated whether intermittent blood and crystalloid cardioplegia differentially affect myocardial apoptosis and apoptosis gene-related proteins. METHODS AND RESULTS: Rabbit hearts were perfused with Krebs-Henseleit buffer on a Langendorff apparatus. Control hearts (n=6) were perfused for 120 minutes without cardioplegic ischemia. Hearts were arrested for 60 minutes with warm (37 degrees C) crystalloid cardioplegia (iW-CCP) (n=8) or with warm blood cardioplegia (iW-BCP) (n=8) administered intermittently. In cold (0 to 4 degrees C) groups, hearts were arrested for 60 minutes with cold crystalloid cardioplegia (iC-CCP; n=8) or with cold blood cardioplegia (iC-BCP; n=6) administered intermittently. The hearts were reperfused for 30 minutes with Krebs-Henseleit buffer. iC-BCP significantly preserved the recovery of left ventricular and microvascular function compared with the other 3 experimental groups. There were no significant differences in total protein levels of caspase 3, Bcl-2, Bad, and Bax among the groups. iC-BCP significantly induced greater phosphorylation of Bad (5.6+/-0.8-fold) as compared with the other 3 groups (3.4+/-0.6-fold in iC-CCP, P<0.05; 2.5+/-0.3 in iW-BCP, P<0.05; and 1.4+/-0.2 in iW-CCP, P<0.01). iC-BCP induced less caspase 3 activation and apoptosis than the other 3 groups. CONCLUSIONS: iC-BCP is superior to the other cardioplegic solutions in increasing the phosphorylation of Bad, inhibiting the activation of caspase 3, and preventing apoptosis. These effects of iC-BCP were associated with preserved left ventricular function and endothelium-dependent relaxation of coronary microvessels.

Bradykinin preconditioning improves the profile of cell survival proteins and limits apoptosis after cardioplegic arrest.

BACKGROUND: We hypothesized that preconditioning the heart with bradykinin (BK) would improve the profile of antiapoptotic proteins and inhibit myocardial apoptosis. METHODS AND RESULTS: Eighteen rabbit hearts were retrogradely perfused with Krebs-Henseleit buffer (KHB). Six control hearts were perfused with KHB for 90 minutes without cardioplegia ischemia. Six hearts were arrested for 30 minutes (37 degrees C) with crystalloid cardioplegia (CCP). Six BK preconditioning (BKPC) hearts received a 10-minute coronary infusion of 10(-8) M BK-enriched KHB followed by a 5-minute recovery period and were then arrested for 30 minutes with CCP. The hearts were reperfused for 30 minutes with KHB. BKPC significantly improved the recovery of left ventricular pressure (73+/-5 versus 51+/-4 mm Hg; P<0.05) and reduced the percentage of myocardial apoptosis (3.4+/-0.3% versus 1.2+/-0.2%; P<0.05) as compared with CCP. There were no significant differences in total protein levels of caspase 3, Bcl-2, Bad, and Bax, among the groups. Both BKPC and CCP induced phosphorylation of Bad at Ser112, but the BKPC group had higher phosphorylated Bad than CCP (4.4+/-0.5 versus 2.0+/-0.3; P<0.05). Both BKPC and CCP alone increased caspase 3 cleavage and activity as compared with controls (P<0.05 and P<0.01, respectively), but BKPC caused less cleavage and activation of caspase 3 than CCP alone (P<0.05). CONCLUSIONS: BKPC increased Bad phosphorylation, inhibited caspase 3 activation, and limited myocardial apoptosis, which were associated with improvement of left-ventricular performance. These results identify novel molecular mechanisms underlying the protective effects of BKPC during cardiac surgery.

Aprotinin inhibits protease-dependent platelet aggregation and thrombosis.

BACKGROUND: Hemostatic effects of the protease inhibitor aprotinin in cardiac surgery are well described, and recent evidence suggests an antithrombotic mechanism of aprotinin through inhibition of thrombin-mediated platelet activation. We hypothesized that aprotinin provides hemostasis while reducing vascular thrombosis by attenuating protease-dependent platelet function. METHODS: Rabbits (3 to 4 kg) underwent carotid artery thrombosis induced by electrical current. Treatment animals (n = 8) received aprotinin by a 100,000-KIU bolus followed by a continuous infusion (25,000 KIU/h). Control animals (n = 8) received crystalloid solution. Thrombus weight and time to thrombotic occlusion were determined. Platelet aggregation was examined in response to protease-dependent (thrombin) and protease-independent (adenosine diphosphate, ADP) platelet agonists. Platelet thrombin protease-activated receptor (PAR) expression was analyzed by Western blot. Ear bleeding time and abdominal incisional bleeding were measured at baseline and serially. RESULTS: Thrombus weight was decreased by aprotinin (6.1 +/- 1.1 mg versus 10.8 +/- 1.5 mg, aprotinin versus control, p < 0.05). Time to thrombotic occlusion was prolonged in the aprotinin group (17.4 +/- 1.0 minutes versus 8.3 +/- 1.3 minutes, p < 0.001). Rabbit platelet expression of thrombin PARs was demonstrated by Western blot analysis, and was not altered by aprotinin therapy. Platelet aggregation due to thrombin was decreased by aprotinin therapy (59.2% +/- 3.0% versus 95.8% +/- 1.5%, p < 0.001), whereas protease-independent, ADP-induced platelet aggregation was unchanged with aprotinin. Incisional bleeding was not different between groups. In the aprotinin group, bleeding time was unchanged at baseline and then reduced for the duration of the experiment (35.0 +/- 4.7 seconds versus 76.8 +/- 6.4 seconds, p < 0.05). CONCLUSIONS: While providing hemostatic effects, aprotinin attenuates vascular thrombosis in part by inhibition of PAR activation, resulting in the prevention of thrombin-induced platelet aggregation.

Inhibition of the cardiac angiogenic response to exogenous vascular endothelial growth factor.

BACKGROUND: The angiogenic effects of vascular endothelial growth factor (VEGF) are mediated by the stimulation of endothelial nitric oxide synthase (eNOS) and nitric oxide release. Nitric oxide availability is decreased in patients with coronary disease, a possible explanation for the humble results of VEGF in clinical trials. We sought to examine the effects of exogenous VEGF in a model of endothelial dysfunction. METHODS: Miniswine fed either a regular (N = 6, group NORM) or hypercholesterolemic diet (N = 6, HICHOL) underwent ameroid placement on the circumflex artery. Three weeks later, baseline myocardial perfusion was assessed by microsphere injections, and all pigs were treated with VEGF. Four weeks later, microsphere injections were repeated and the hearts harvested. Endothelial-dependent coronary microvascular reactivity, and VEGF and eNOS expression were assessed. RESULTS: HICHOL pigs showed significant endothelial dysfunction in the ischemic territory. Post-treatment myocardial blood flow in the circumflex territory was significantly higher in the NORM compared to the HICHOL group. VEGF and eNOS levels were increased in the ischemic territory in the NORM group but decreased in the HICHOL group. CONCLUSIONS: The cardiac angiogenic response to VEGF was markedly inhibited in a hypercholesterolemia-induced porcine model of endothelial dysfunction. Coronary endothelial dysfunction could be an obstacle to the efficacy of clinical angiogenesis protocols and a putative therapeutic target.