Connie J. Wagner

Glucocorticoids reduce ischemia-reperfusion-induced myocardial apoptosis in immature hearts

BACKGROUND Transient myocardial dysfunction often occurs after ischemia-reperfusion with immature myocardium appearing particularly susceptible. Neutrophil adhesion and activation contribute to ischemia-reperfusion injury after cardiopulmonary bypass (CPB), possibly resulting in cell death. The hypothesis was that glucocorticoids could prevent reperfusion-induced myocardial dysfunction by blunting leukocyte-mediated injury. METHODS Neonatal piglets were cooled with CPB followed by 2 hours of circulatory arrest. Animals were rewarmed, removed from CPB, and allowed to recover for 2 hours. Methylprednisolone (60 mg/kg) was administered in the CPB priming solution to one group (intraoperative glucocorticoids). In another group (preoperative glucocorticoids), 30 mg/kg methylprednisolone was administered 6 hours before CPB in addition to the intraoperative dose (30 mg/kg). Control animals received no glucocorticoids. RESULTS Apoptotic myocardial cells measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and caspase-3 activity were reduced in animals administered glucocorticoids compared with controls (p < 0.05). Animals receiving either intraoperative or preoperative glucocorticoids had 0.10 +/- 0.07 and 0.13 +/- 0.05 apoptotic cells per high-power field, respectively, whereas 0.33 +/- 0.15 apoptotic cells were detected with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling in control animals. Glucocorticoid administration reduced myocardial intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 mRNA expression compared with control piglets. Maximum rate of increase of left ventricular pressure was 62% +/- 9% of baseline in control animals at 120 minutes of recovery compared with 96% +/- 6% and 95% +/- 10% of baseline in animals receiving intraoperative and preoperative glucocorticoids, respectively (p < 0.05). CONCLUSIONS The reduction of neutrophil adhesion and activation proteins in neonatal myocardium was associated with less apoptotic cell death after glucocorticoid administration. The blunting of apoptosis in glucocorticoid-treated animals was also associated with improved recovery of left ventricular systolic function in neonatal animals after CPB and circulatory arrest. Glucocorticoid attenuation of myocardial apoptosis might have important implications for maintaining long-term ventricular function after ischemia and reperfusion.

Glucocorticoids Preserve Calpastatin and Troponin I during Cardiopulmonary Bypass in Immature Pigs

Degradation of troponin I (TnI) by calpain occurs with myocardial stunning in ischemia-reperfusion injury. Glucocorticoids attenuate myocardial ischemia-reperfusion injury, but their effect on TnI degradation is unknown. A piglet model was used to test the hypotheses that cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA) are associated with TnI degradation and that TnI alterations could be prevented by glucocorticoid treatment. Piglets were cooled to 18°C, subjected to 2 h of circulatory arrest, rewarmed to 37°C, and allowed to recover for 2 h. Methylprednisolone was administered 6 h before surgery (3 0 mg/kg) and at initiation of CPB (30 mg/kg). The untreated group received saline. Left ventricular tissue was collected after recovery and analyzed by Western blot for TnI, calpain, and calpastatin (the natural inhibitor of calpain). CPB/DHCA animals had 27.4 ± 0.2% of total detected TnI present in degraded form. Glucocorticoid treatment significantly decreased the percentage of degraded TnI (12.0 ± 0.1%, p < 0.05). Calpain I and calpain II increased after CPB/DHCA compared with non-CPB/DHCA controls (p < 0.05), with or without glucocorticoid treatment. Calpastatin significantly decreased in untreated CPB/DHCA animals compared with non-CPB/DHCA controls (p < 0.05), but levels were preserved by glucocorticoids. Glucocorticoids were associated with preservation of maximum rate of increase of left ventricular pressure at 95 ± 10% of baseline, whereas maximum rate of increase of left ventricular pressure decreased to 62 ± 12% of baseline without steroids. TnI degradation occurs after CPB/DHCA in neonatal pigs. Reduction in reperfusion injury by glucocorticoids may depend partly on preservation of calpastatin activity and intact TnI.

Glucocorticoids reduce cardiac dysfunction after cardiopulmonary bypass and circulatory arrest in neonatal piglets.

Objective The hypotheses were that glucocorticoid administration could improve ventricular recovery by reducing cardiopulmonary bypass (CPB)-induced inflammatory response and that presurgical administration might be more effective than intraoperative dosing. Design Animal case study. Subjects Crossbred piglets (5–7 kg). Interventions Piglets were cooled with CPB, followed by 120 mins of deep hypothermic circulatory arrest (DHCA). Animals were rewarmed to 38°C, removed from CPB, and maintained for 120 mins. Methylprednisolone (60 mg/kg) was administered in the CPB pump prime (intraoperative glucocorticoid [intraop GC]) or 6 hrs before CPB (30 mg/kg) in addition to the intraoperative dose (30 mg/kg; pre- and intraop GC). Controls (no GC) received saline. Measurements and Main Results In no GC, left ventricle (LV) positive change in pressure in time (+dP/dt) (mm Hg/sec) had a mean ± sd of 1555 ± 194 at baseline vs. 958 ± 463 at 120 mins after CPB, p = .01). LV +dP/dt was maintained in glucocorticoid-treated animals (1262 ± 229 at baseline vs. 1212 ± 386 in intraop GC and 1471 ± 118 vs. 1393 ± 374 in pre- and intraop GC). Glucocorticoids reduced myocardial interleukin-6 messenger RNA expression, measured by ribonuclease protection assay, at 120 mins after CPB compared with animals receiving saline (p < .05), although interleukin-6 plasma and LV protein concentrations were not affected. Interleukin-10 myocardial protein concentrations were elevated after CPB-DHCA with higher concentrations in glucocorticoid-treated animals (p < .05). Glucocorticoid treatment maintained myocardial concentrations of the inhibitor of nuclear factor-&kgr;B in the cytosol and decreased nuclear factor-&kgr;B concentrations detected in the nucleus in a DNA/protein interaction array. Conclusions Glucocorticoids improved recovery of LV systolic function in neonatal animals undergoing CPB-DHCA. Animals receiving glucocorticoids before CPB had better postoperative oxygen delivery than those receiving only intraoperative treatment. Maintenance of cardiac function after glucocorticoids might be due, in part, to alterations in the balance of pro- and anti-inflammatory proteins, possibly through nuclear factor-&kgr;B-dependent pathways.

Modulation of nuclear factor-kappaB improves cardiac dysfunction associated with cardiopulmonary bypass and deep hypothermic circulatory arrest.

Objective:The hypothesis is that partial nuclear factor-kappaB (NF-&kgr;B) inhibition can alleviate cardiopulmonary dysfunction associated with ischemia and reperfusion injury following cardiopulmonary bypass and deep hypothermic circulatory arrest (CPB/DHCA) in a pediatric model. Design:Animal case study. Subjects:Two-week-old piglets (5–7 kg). Interventions:Piglets received 100 &mgr;g/kg of SN50, a peptide inhibitor of NF-&kgr;B translocation and activation, 1 hour before CPB. The control group received saline. Animals were cooled to 18°C with CPB, the piglets were in DHCA for 120 minutes, and the piglets were then rewarmed on CPB to 38°C and maintained for 120 minutes after CPB/DHCA. Measurements:Sonomicrometry and pressure catheters collected hemodynamic data. Transmural left and right ventricular tissues were obtained at the terminal time point for determination of NF-&kgr;B activity by enzyme-linked immunosorbent assay. Data are expressed as mean ± sd. Main Points:Oxygen delivery was maintained at 76 ± 13 mL/min at baseline and 75 ± 5 mL/min at 120 minutes after CPB/DHCA (p = 0.75) in SN50-treated animals vs. 99 ± 26 mL/min at baseline and 63 ± 20 mL/min at 120 minutes in the untreated group (p = 0.0001). Pulmonary vascular resistance (dynes·sec−1·cm−5) increased from 124 ± 59 at baseline to 369 ± 104 at 120 minutes in the untreated piglets (p = 0.001) compared with SN50-treated animals (100 ± 24 at baseline and 169 ± 88 at 120 minutes, p = 0.1). NF-&kgr;B activity was reduced by 74% in left ventricles of SN50-treated compared with SN50-untreated animals (p < 0.001). Plasma endothelin-1 (pg/mL), an important vasoconstrictor regulated by NF-&kgr;B, increased from 2.1 ± 0.4 to 14.2 ± 5.7 in untreated animals (p = 0.004) but was elevated to only 4.5 ± 2 with SN50 treatment (p = 0.005). Conclusions:Improvement of cardiopulmonary function after ischemia/reperfusion was associated with the reduction of NF-&kgr;B activity in piglet hearts. Maintenance of systemic oxygen delivery and alleviation of pulmonary hypertension after CPB/DHCA in piglets administered SN50, possibly through a reduction of circulating endothelin-1, suggest that selective inhibition of NF-&kgr;B activity may reduce ischemia and reperfusion injury after pediatric cardiac surgery.

Glucocorticoids Improve Calcium Cycling in Cardiac Myocytes after Cardiopulmonary Bypass

BACKGROUND Glucocorticoids can reduce myocardial dysfunction associated with ischemia and reperfusion injury following cardiopulmonary bypass (CPB) and circulatory arrest. The hypothesis was that maintenance of cardiac function after CPB with methylprednisolone therapy results, in part, from preservation of myocyte calcium cycling. METHODS Piglets (5-7 kg) underwent CPB and 120 min of hypothermic circulatory arrest with (CPB-GC) or without (CPB) methylprednisolone (30 mgkg(-1)) administered 6h before and at CPB. Controls (No-CPB) did not undergo CPB or receive glucocorticoids (n=6 per treatment). Myocardial function was monitored in vivo for 120 min after CPB. Calcium cycling was analyzed using rapid line-scan confocal microscopy in isolated, fluo-3-AM-loaded cardiac myocytes. Phospholamban phosphorylation and sarco(endo)plasmic reticulum calcium-ATPase (SERCA2a) protein levels were determined by immunoblotting of myocardium collected 120 min after CPB. Calpain activation in myocardium was measured by fluorometric assay. RESULTS Preload recruitable stroke work in vivo 120 min after reperfusion decreased from baseline in CPB (47.4±12 versus 26.4±8.3 slope of the regression line, P<0.05), but was not different in CPB-GC (41±8.1 versus 37.6±2.2, P=0.7). In myocytes isolated from piglets, total calcium transient time remained unaltered in CPB-GC (368±52.5 ms) compared with controls (434.5±35.3 ms; P=0.07), but was prolonged in CPB myocytes (632±83.4 ms; P<0.01). Calcium transient amplitude was blunted in myocytes from CPB (757±168 nM) compared with controls (1127±126 nM, P<0.05) but was maintained in CPB-GC (1021±155 nM, P>0.05). Activation of calpain after CPB was reduced with glucocorticoids. Phospholamban phosphorylation and SERCA2a protein levels in myocardium were decreased in CPB compared with No-CPB and CPB-GC (P<0.05). CONCLUSIONS The glucocorticoid-mediated improvement in myocardial function after CPB might be due, in part, to prevention of calpain activation and maintenance of cardiac myocyte calcium cycling.