Cheryl R. Killingsworth

Plasma concentrations of inflammatory cytokines rise rapidly during ECMO-related SIRS due to the release of preformed stores in the intestine

Extracorporeal membrane oxygenation (ECMO) is a life-saving support system used in neonates and young children with severe cardiorespiratory failure. Although ECMO has reduced mortality in these critically ill patients, almost all patients treated with ECMO develop a systemic inflammatory response syndrome (SIRS) characterized by a ‘cytokine storm’, leukocyte activation, and multisystem organ dysfunction. We used a neonatal porcine model of ECMO to investigate whether rising plasma concentrations of inflammatory cytokines during ECMO reflect de novo synthesis of these mediators in inflamed tissues, and therefore, can be used to assess the severity of ECMO-related SIRS. Previously healthy piglets (3-week-old) were subjected to venoarterial ECMO for up to 8 h. SIRS was assessed by histopathological analysis, measurement of neutrophil activation (flow cytometry), plasma cytokine concentrations (enzyme immunoassays), and tissue expression of inflammatory genes (PCR/western blots). Mast cell degranulation was investigated by measurement of plasma tryptase activity. Porcine neonatal ECMO was associated with systemic inflammatory changes similar to those seen in human neonates. Tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) concentrations rose rapidly during the first 2 h of ECMO, faster than the tissue expression of these cytokines. ECMO was associated with increased plasma mast cell tryptase activity, indicating that increased plasma concentrations of inflammatory cytokines during ECMO may result from mast cell degranulation and associated release of preformed cytokines stored in mast cells. TNF-α and IL-8 concentrations rose faster in plasma than in the peripheral tissues during ECMO, indicating that rising plasma levels of these cytokines immediately after the initiation of ECMO may not reflect increasing tissue synthesis of these cytokines. Mobilization of preformed cellular stores of inflammatory cytokines such as in mucosal mast cells may have an important pathophysiological role in ECMO-related SIRS.

Activation Patterns of Purkinje Fibers During Long-Duration Ventricular Fibrillation in an Isolated Canine Heart Model

Background— The roles of Purkinje fibers (PFs) and focal wave fronts, if any, in the maintenance of ventricular fibrillation (VF) are unknown. If PFs are involved in VF maintenance, it should be possible to map wave fronts propagating from PFs into the working ventricular myocardium during VF. If wave fronts ever arise focally during VF, it should be possible to map them appearing de novo. Methods and Results— Six canine hearts were isolated, and the left main coronary artery was cannulated and perfused. The left ventricular cavity was exposed, which allowed direct endocardial mapping of the anterior papillary muscle insertion. Nonperfused VF was induced, and 6 segments of data, each 5 seconds long, were analyzed during 10 minutes of VF. During 36 segments of data that were analyzed, 1018 PF or focal wave fronts of activation were identified. In 534 wave fronts, activation was mapped propagating from working ventricular myocardium to PF. In 142 wave fronts, activation was mapped propagating from PF to working ventricular myocardium. In 342 wave fronts, activation was mapped arising focally. More than 1 of these 3 patterns could occur in the same wave front. Conclusions— PFs are highly active throughout the first 10 minutes of VF. In addition to retrograde propagation from the working ventricular myocardium to PFs, antegrade propagation occurs from PFs to working ventricular myocardium, which suggests PFs are important in VF maintenance. Prior plunge needle recordings in dogs indicate activation propagates from the endocardium toward the epicardium after 1 minute of VF, which suggests that focal sites on the endocardium may represent foci and not breakthrough. If so, in addition to reentry, abnormal automaticity or triggered activity may also occur during VF.

Chemical ablation of the Purkinje system causes early termination and activation rate slowing of long-duration ventricular fibrillation in dogs

Endocardial mapping has suggested that Purkinje fibers may play a role in the maintenance of long-duration ventricular fibrillation (LDVF). To determine the influence of Purkinje fibers on LDVF, we chemically ablated the Purkinje system with Lugol solution and recorded endocardial and transmural activation during LDVF. Dog hearts were isolated and perfused, and the ventricular endocardium was exposed and treated with Lugol solution (n = 6) or normal Tyrode solution as a control (n = 6). The left anterior papillary muscle endocardium was mapped with a 504-electrode (21 x 24) plaque with electrodes spaced 1 mm apart. Transmural activation was recorded with a six-electrode plunge needle on each side of the plaque. Ventricular fibrillation (VF) was induced, and perfusion was halted. LDVF spontaneously terminated sooner in Lugol-ablated hearts than in control hearts (4.9 +/- 1.5 vs. 9.2 +/- 3.2 min, P = 0.01). After termination of VF, both the control and Lugol hearts were typically excitable, but only short episodes of VF could be reinduced. Endocardial activation rates were similar during the first 2 min of LDVF for Lugol-ablated and control hearts but were significantly slower in Lugol hearts by 3 min. In control hearts, the endocardium activated more rapidly than the epicardium after 4 min of LDVF with wave fronts propagating most often from the endocardium to epicardium. No difference in transmural activation rate or wave front direction was observed in Lugol hearts. Ablation of the subendocardium hastens VF spontaneous termination and alters VF activation sequences, suggesting that Purkinje fibers are important in the maintenance of LDVF.

Impact of Myocardial Ischemia and Reperfusion on Ventricular Defibrillation Patterns, Energy Requirements, and Detection of Recovery

Background—Shocks that have defibrillated spontaneous ventricular fibrillation (VF) during acute ischemia or reperfusion may seem to have failed if VF recurs before the ECG amplifier recovers after shock. This could explain why the defibrillation threshold (DFT) for spontaneous VF appears markedly higher than for electrically induced VF. Methods and Results—The DFT for electrically induced VF (E-DFT) was determined in 15 pigs before ischemia, followed by left anterior ascending or left circumflex artery occlusion. VF was electrically induced 20 minutes after occlusion, followed 5 minutes later by reperfusion. Whether spontaneous or electrically induced, VF during occlusion or reperfusion was treated with up to 3 shocks at 1.5×E-DFT. If all 3 shocks failed, shock strength was increased. Thirty minutes after reperfusion, the other artery was occluded and the protocol was repeated. Defibrillation was considered successful if postshock sinus/idioventricular rhythm was present for ≥30 seconds. VF recurring within 30 seconds after the shock was considered immediate or delayed if the first postshock activation complex in a rapidly restored ECG recording was VF or sinus/idioventricular rhythm, respectively. Defibrillation efficacy at 1.5×E-DFT was significantly higher for electrically induced ischemic VF (76%) than for spontaneous VF (31%). The incidence of delayed recurrence after electrically induced nonischemic (3%) or ischemic (20%) VF was significantly lower than after spontaneous VF (75%). Mean VF recurrence time after spontaneous VF was 4.6±5.3 seconds. Conclusions—Spontaneous VF can be halted by a shock but then quickly restart before a standard ECG amplifier has recovered from postshock saturation, making it appear that the shock failed.

Defibrillation threshold and cardiac responses using an external biphasic defibrillator with pediatric and adult adhesive patches in pediatric-sized piglets

Before recommendations for using an automatic external defibrillator on pediatric patients can be made, a protocol for the energy of a biphasic waveform energy dosing needs to be determined that will allow ventricular defibrillation of 8 year olds while causing only a minimal amount of cardiac damage to infants. Pediatric- and adult-sized electrode patches were alternately applied to 10 isoflurane-anesthetized piglets weighing 3.8-20.1 kg to approximate the body weights of newborns to children < 8 years old. The defibrillation threshold (DFT) was determined for biphasic truncated exponential waveform shocks. Additional shocks, varying from the DFT to 360 Joules (J), were delivered during sinus rhythm or following 30 s of ventricular fibrillation (VF). The DFT was 2.4+/-0.81 and 2.1+/-0.65 J/kg for pediatric and adult patches, respectively (P = N.S.). The change in left ventricular (LV) dP/dt from baseline as a function of shock strength was significantly different at 1 and 10 s after shocks of increasing energy that were delivered in sinus rhythm, and 1, 10, 20, and 30 s after defibrillation shocks. There was no significant difference in LV dP/dt with increasing shock energy at 60 s with either patch size. The time to return of sinus rhythm, ST-segment deviation, and cardiac output were also not significantly different from baseline 60 s following shocks of up to 360 J delivered during sinus rhythm or VF with either patch. The same amount of energy delivered with a biphasic external defibrillator successfully defibrillated VF whether adult or pediatric patches were used. Cardiac rhythm and hemodynamic variables were unaltered at 60 s after shocks delivered at energies of up to 360 J. These data suggest that there is a substantial safety margin above a DFT strength shock for this biphasic waveform in piglets.

Short-Acting β-Adrenergic Antagonist Esmolol Given at Reperfusion Improves Survival After Prolonged Ventricular Fibrillation

Background—High catecholamine concentrations are cytotoxic to cardiac myocytes. We hypothesized that myocardial interstitial catecholamine levels are greatly elevated immediately after long-duration ventricular fibrillation (VF), defibrillation, and reperfusion and that the short-acting β-antagonist esmolol administered at reperfusion would protect against this catecholamine surge and improve survival. Methods and Results—In part 1 of this study, catecholamines from myocardial interstitial fluid (ISF) and aortic and coronary sinus plasma were quantified by use of 3H-labeled radioenzymatic assay in 8 open-chest, anesthetized pigs. Eight minutes of electrically induced VF was followed by internal defibrillation and reperfusion. By 4 minutes of VF, ISF norepinephrine increased significantly, from 1.3± 0.3 to 7.4± 2.4 ng/mL. Epinephrine increased significantly, from 0.4± 0.2 to 1.5± 0.7 ng/mL. ISF norepinephrine and epinephrine peaked at 219.2± 92.1 and 63.7± 25.1 ng/mL after defibrillation and reperfusion and decreased significantly to 12.2± 3.5 and 6.7± 3.1 ng/mL 23 minutes after defibrillation. Transcardiac catecholamine changes were similar. In part 2, 8 minutes of VF was followed by external defibrillation in anesthetized, closed-chest pigs. Animals received 1.0 mg/kg esmolol (n= 8) or saline (n= 8) intravenously at the start of cardiopulmonary resuscitation (CPR). Advanced cardiac life support, including CPR and epinephrine, was delivered to both groups. Esmolol before reperfusion improved return of spontaneous circulation and 4-hour survival (7/8 versus 3/8 survivors, χ2P < 0.05). Conclusions—Transcardiac and ISF norepinephrine and epinephrine levels are briefly massively elevated after 8 minutes of VF, defibrillation, and reperfusion. A short-acting β-antagonist administered immediately after defibrillation improves return of spontaneous circulation and 4-hour survival after this prolonged VF.

Low Intensity Shear Stress Increases Endothelial ELR+ CXC Chemokine Production via a Focal Adhesion Kinase-p38β MAPK-NF-κB Pathway

CXC chemokines with a glutamate-leucine-arginine (ELR) tripeptide motif (ELR+ CXC chemokines) play an important role in leukocyte trafficking into the tissues. For reasons that are not well elucidated, circulating leukocytes are recruited into the tissues mainly in small vessels such as capillaries and venules. Because ELR+ CXC chemokines are important mediators of endothelial-leukocyte interaction, we compared chemokine expression by microvascular and aortic endothelium to investigate whether differences in chemokine expression by various endothelial types could, at least partially, explain the microvascular localization of endothelial-leukocyte interaction. Both in vitro and in vivo models indicate that ELR+ CXC chemokine expression is higher in microvascular endothelium than in aortic endothelial cells. These differences can be explained on the basis of the preferential activation of endothelial chemokine production by low intensity shear stress. Low shear activated endothelial ELR+ CXC chemokine production via cell surface heparan sulfates, β3-integrins, focal adhesion kinase, the mitogen-activated protein kinase p38β, mitogen- and stress-associated protein kinase-1, and the transcription factor.

Biphasic waveform external defibrillation thresholds for spontaneous ventricular fibrillation secondary to acute ischemia

OBJECTIVES The goal of this study was to determine if the defibrillation threshold (DFT) after spontaneous ventricular fibrillation (VF) secondary to acute ischemia differs from the DFT for electrically induced VF in the absence of ischemia in anesthetized, closed-chest dogs and pigs. BACKGROUND The efficacy of external defibrillators has been tested mainly in animals and humans using E-VF, yet external defibrillators are often used in patients to halt S-VF. METHODS Protocol 1: biphasic truncated exponential (BTE) waveform shocks were delivered through electrodes placed in an anterior-anterior (A-A) position (left and right lateral thorax) in nine dogs. After measuring the E-VF DFT, acute ischemia was induced with an angioplasty balloon in either the left anterior descending or left circumflex coronary artery, and the S-VF DFT was determined. Protocol 2: in a group of 12 pigs, the E-VF DFT and S-VF DFT were determined for electrodes in the A-A position and in the anterior-posterior position (A-P). Protocol 3: the E-VF DFT was determined in seven pigs. Then up to three shocks 1.5x the E-VF DFT were delivered to S-VF. If defibrillation did not occur, a step-up protocol was used until defibrillation occurred. RESULTS Protocol 1: the DFT for E-VF was 65 +/- 28 J (mean +/- SD) compared with 226 +/- 97 J for S-VF, p < 0.05. Protocol 2: the DFT was 152 +/- 58 J for E-VF and 315 +/- 123 J for S-VF for A-A electrodes. The DFT was 100 +/- 43 J for E-VF and 206 +/- 114 J for S-VF for A-P electrodes. Protocol 3: 11/37 shocks of strength 1.5x E-VF DFT (182 +/- 40 J) stopped the arrhythmia. The episodes of S-VF not halted by these shocks required energy levels of up to 400 J for defibrillation. CONCLUSIONS External defibrillation of S-VF induced by acute ischemia requires significantly more energy than VF induced by 60-Hz current in the absence of ischemia. A safety margin >1.5x the DFT for electrically induced VF may be necessary in BTE external defibrillators to defibrillate S-VF.

Improvement of Defibrillation Efficacy and Quantification of Activation Patterns During Ventricular Fibrillation in a Canine Heart Failure Model

Background —Little is known about the effects of heart failure (HF) on the defibrillation threshold (DFT) and the characteristics of activation during ventricular fibrillation (VF). Methods and Results —HF was induced by rapid right ventricular (RV) pacing for at least 3 weeks in 6 dogs. Another 6 dogs served as controls. Catheter defibrillation electrodes were placed in the RV apex, the superior vena cava, and the great cardiac vein (CV). An active can coupled to the superior vena cava electrode served as the return for the RV and CV electrodes. DFTs were determined before and during HF for a shock through the RV electrode with and without a smaller auxiliary shock through the CV electrode. VF activation patterns were recorded in HF and control animals from 21×24 unipolar electrodes spaced 2 mm apart on the ventricular epicardium. Using these recordings, we computed a number of quantitative VF descriptors. DFT was unchanged in the control dogs. DFT energy was increased 79% and 180% (with and without auxiliary shock, respectively) in HF compared with control dogs. During but not before HF, DFT energy was significantly lowered (21%) by addition of the auxiliary shock. The VF descriptors revealed marked VF differences between HF and control dogs. The differences suggest decreased excitability and an increased refractory period during HF. Most, but not all, descriptors indicate that VF was less complex during HF, suggesting that VF complexity is multifactorial and cannot be expressed by a scalar quantity. Conclusions —HF increases the DFT. This is partially reversed by an auxiliary shock. HF markedly changes VF activation patterns.

Chymase Inhibition Prevents Fibronectin and Myofibrillar Loss and Improves Cardiomyocyte Function and LV Torsion Angle in Dogs With Isolated Mitral Regurgitation

Background— The left ventricular (LV) dilatation of isolated mitral regurgitation (MR) is associated with an increase in chymase and a decrease in interstitial collagen and extracellular matrix. In addition to profibrotic effects, chymase has significant antifibrotic actions because it activates matrix metalloproteinases and kallikrein and degrades fibronectin. Thus, we hypothesize that chymase inhibitor (CI) will attenuate extracellular matrix loss and LV remodeling in MR. Methods and Results— We studied dogs with 4 months of untreated MR (MR; n=9) or MR treated with CI (MR+CI; n=8). Cine MRI demonstrated a >40% increase in LV end-diastolic volume in both groups, consistent with a failure of CI to improve a 25% decrease in interstitial collagen in MR. However, LV cardiomyocyte fractional shortening was decreased in MR versus normal dogs (3.71±0.24% versus 4.81±0.31%; P<0.05) and normalized in MR+CI dogs (4.85±0.44%). MRI with tissue tagging demonstrated an increase in LV torsion angle in MR+CI versus MR dogs. CI normalized the significant decrease in fibronectin and FAK phosphorylation and prevented cardiomyocyte myofibrillar degeneration in MR dogs. In addition, total titin and its stiffer isoform were increased in the LV epicardium and paralleled the changes in fibronectin and FAK phosphorylation in MR+CI dogs. Conclusions— These results suggest that chymase disrupts cell surface–fibronectin connections and FAK phosphorylation that can adversely affect cardiomyocyte myofibrillar structure and function. The greater effect of CI on epicardial versus endocardial titin and noncollagen cell surface proteins may be responsible for the increase in torsion angle in chronic MR.