Jodie Y. Duffy

Inhaled nitric oxide increases endothelin-1 levels: a potential cause of rebound pulmonary hypertension.

Objective Inhaled nitric oxide (iNO) is front-line therapy for pulmonary hypertension after repair of congenital heart disease. However, little clinical data exists regarding the effects of iNO on regulators of pulmonary vascular resistance. An imbalance between primary vasodilators, such as NO, and vasoconstrictors, such as endothelin-1 (ET-1), has been implicated in rebound pulmonary hypertension upon iNO withdrawal. The objective of this study was to determine whether iNO therapy alters plasma ET-1 levels. Design This is a prospective study involving pediatric and adult patients at risk for pulmonary hypertension. Setting Pediatric patients were in the cardiac intensive care unit and adult patients were in a tertiary-care hospital. Patients Group 1 included children with congenital heart disease requiring iNO for treatment of pulmonary hypertension after cardiopulmonary bypass (n = 15), group 2 was adults receiving iNO (n = 10), and group 3 included children at risk for pulmonary hypertension after bypass that did not require iNO (n = 8). Interventions Dosages of iNO were 2–60 ppm. The duration of therapy ranged from 23 to 188 hrs in group 1 and 29 to 108 hrs in group 2. Measurements and Main Results Arterial blood was obtained for the measurement of ET-1 levels before and during iNO therapy and 24 hrs after iNO withdrawal. Group 1 mean ET-1 levels increased to 127% of baseline by 12 hrs of iNO, remained elevated at 48 hrs (p < .05), then decreased to 71% of iNO levels 24 hrs after withdrawal (p < .01). Group 2 ET-1 levels increased to 147%, and 137% of baseline at 12 and 24 hrs of iNO therapy, then fell to 68% of baseline within 24 hrs of discontinuing iNO. ET-1 levels in group 3 decreased after surgery (p < .05). Conclusions These data suggest that iNO increased plasma ET-1 levels, which subsequently decreased when iNO was discontinued. Increased circulating ET-1 levels might contribute to rebound pulmonary hypertension upon iNO withdrawal.

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.

Cellular and molecular aspects of myocardial dysfunction.

Disruption of any one of a large number of balanced systems that maintain cardiomyocyte structure and function can cause myocardial dysfunction. Such disruption can occur either in response to acute stresses such as cardiac surgery with cardiopulmonary bypass and cross-clamping of the aorta or because of more chronic stresses resulting from factors such as genetic abnormalities, infection, or chronic ischemia. Several currently available therapies such as &bgr;-adrenergic receptor agonists and antagonists, phosphodiesterase inhibitors, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and other agents affect cardiomyocytes in ways that are more far reaching than initially appreciated when these agents were first introduced into clinical practice. As our knowledge and understanding of myocardial dysfunction increases, particularly in the neonatal and pediatric patient, we will be able to further target interventions to highly specific perturbations of cellular function and individual genetic variability.

Alterations in protein kinase C activity and processing during zinc-deficiency-induced cell death

Protein kinases C (PKCs) are a family of serine/threonine kinases that are critical for signal transduction pathways involved in growth, differentiation and cell death. All PKC isoforms have four conserved domains, C1-C4. The C1 domain contains cysteine-rich finger-like motifs, which bind two zinc atoms. The zinc-finger motifs modulate diacylglycerol binding; thus, intracellular zinc concentrations could influence the activity and localization of PKC family members. 3T3 cells were cultured in zinc-deficient or zinc-supplemented medium for up to 32 h. Cells cultured in zinc-deficient medium had decreased zinc content, lowered cytosolic classical PKC activity, increased caspase-3 processing and activity, and reduced cell number. Zinc-deficient cytosols had decreased activity and expression levels of PKC-alpha, whereas PKC-alpha phosphorylation was not altered. Inhibition of PKC-alpha with Gö6976 had no effect on cell number in the zinc-deficient group. Proteolysis of the novel PKC family member, PKC-delta, to its 40-kDa catalytic fragment occurred in cells cultured in the zinc-deficient medium. Occurrence of the PKC-delta fragment in mitochondria was co-incident with caspase-3 activation. Addition of the PKC-delta inhibitor, rottlerin, or zinc to deficient medium reduced or eliminated proteolysis of PKC-delta, activated caspase-3 and restored cell number. Inhibition of caspase-3 processing by Z-DQMD-FMK (Z-Asp-Gln-Met-Asp-fluoromethylketone) did not restore cell number in the zinc-deficient group, but resulted in processing of full-length PKC-delta to a 56-kDa fragment. These results support the concept that intracellular zinc concentrations influence PKC activity and processing, and that zinc-deficiency-induced apoptosis occurs in part through PKC-dependent pathways.

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.

Myocardial function after fetal cardiac bypass in an ovine model.

OBJECTIVE Fetal cardiac surgery might improve the prognosis of certain complex congenital heart defects that have significant associated mortality and morbidity in utero or after birth. An important step in translating fetal cardiac surgery is identifying potential mechanisms leading to myocardial dysfunction after bypass. The hypothesis was that fetal cardiac bypass results in myocardial dysfunction, possibly because of perturbation of calcium cycling and contractile proteins. METHODS Midterm sheep fetuses (n = 6) underwent 30 minutes of cardiac bypass and 120 minutes of monitoring after bypass. Sonomicrometric and pressure catheters inserted in the left and right ventricles measured myocardial function. Cardiac contractile and calcium cycling proteins, along with calpain, were analyzed by means of immunoblotting. RESULTS Preload recruitable stroke work (slope of the regression line) was reduced at 120 minutes after bypass (right ventricle: baseline vs 120 minutes after bypass, 38.6 ± 6.8 vs 20.4 ± 4.8 [P = .01]; left ventricle: 37 ± 7.3 vs 20.6 ± 3.9, respectively [P = .01]). Tau (in milliseconds), a measure of diastolic relaxation, was increased in both ventricles (right ventricle: baseline vs 120 minutes after bypass, 32.7 ± 4.5 vs 67.8 ± 9.4 [P < .01]); left ventricle: 26.1 ± 3.2 vs 63.2 ± 11.2, respectively [P = .01]). Cardiac output was lower and end-diastolic pressures were higher in the right ventricle, but not in the left ventricle, after bypass compared with baseline values. Right ventricular troponin I was degraded by increased calpain activity, and protein levels of sarco(endo)plasmic reticulum calcium ATPase were reduced in both ventricles. CONCLUSIONS Fetal cardiac bypass was associated with myocardial dysfunction and disruption of calcium cycling and contractile proteins. Minimizing myocardial dysfunction after cardiac bypass is important for successful fetal surgery to repair complex congenital heart defects.

Fetal Right Ventricular Myocardial Function Is Better Preserved by Fibrillatory Arrest During Fetal Cardiac Bypass

BACKGROUND Protection and preservation of fetal myocardial function are important for successful fetal intracardiac repair. Our objective was to determine fetal biventricular cardiac performance after two cardiac-arrest techniques. METHODS Three groups of midterm ovine fetuses underwent 90-minute bypass. A control group (no arrest shams, n = 3), and two groups that included 20 minutes of arrest, using fibrillatory (n = 3) or blood cardioplegia (n = 3), were compared. Blood cardioplegia consisted of 4:1 cold blood to crystalloid solution induction every 10 minutes, followed by a warm shot terminal dose before clamp removal. Myocardial function variables from biventricular intracardiac pressure catheters, and 3-axes cardiac sonomicrometry, fetal hemodynamics, and arterial blood gases were continuously recorded. Fetal myocardium was collected for troponin-I analysis at 90 minutes. Statistical analysis was by two-way analysis of variance for repeated measures. RESULTS Compared with sham, right ventricular myocardial contractility was reduced with plegia but not fibrillation at 90 minutes after arrest: dP/dt max (511 ± 347 vs 1208 ± 239, p < 0.01) and preload-recruitable stroke work (7.2 ± 8.5 vs 32.3 ± 14.6, p < 0.01). Right ventricular end diastolic pressure-volume relationship (ventricular stiffness) worsened by 90 minutes for plegia vs fibrillation (0.84 ± 0.18 vs 0.25 ± 0.16, p < 0.05). There were no differences in left ventricle performance between groups. Fetal heart rate increased in shams by 30 minutes after arrest compared with both arrest groups (p < 0.05). Right ventricular troponin-I degradation increased with plegia, but not fibrillation, compared with sham (p < 0.05). CONCLUSIONS In vivo, fetal right ventricular contractile function deteriorates with a common blood-plegia regimen. Fibrillatory arrest better preserves right ventricular function, the dominant ventricle in fetal life, for short arrest periods.