Mechanism And Application Research Of Myocardial Protection Of HTK Solution In Premature Heart During Cardiopulmonary Bypass

General design of the studyHTK (Histidine-tryptophane-ketoglutarate) solution developed by Bretschneider has been widely used to preserve various grafts since1975. It also has been used as a cardioplegic or lung protective solution. HTK solution provides a single dose, long-time protection to the heart. To adult patients, HTK gives equally good myocardial protection as cold blood cardioplegia (CBC) during cardiac surgery. Two studies of Meta-analysis have suggested that blood cardioplegia enhances the early cardiac function postoperatively, while crystalloid cardioplegia is helpful to improve the long-term outcome of the patients.There are many kinds of cardioplegic solution used to pediatric patients in different cardiac centers around the world. Whether blood cardioplegia is superior to crystalloid cardioplegia remains controversial. Angeli et al. reported that serum TnT level on postoperative1day was less than3mg/ml in neonates who used HTK solution during arterial switch operation (ASO). Recently, Bojan et al. found that postoperative serum biochemical markers in HTK group was higher than that in warm blood cardioplegia group in a retrospective study including218neonates undergoing ASO, however, the30-day mortality did not differ between the two groups. Our group had investigated118cases and found that lower ventricular fibrillation after declamping and lower mortality were observed in HTK group compared with St. Thomas group. HTK solution is validity to be applied to long-term ischemic heart in children undergoing congenital heart surgery. To date, no randomized controlled clinical trials have been carried out to compare the superiority between HTK and blood cardioplegia in pediatric patients.In recent years, with the increasing number of younger patients who undergo complex congenital heart surgery, the choice of cardioplegia to the immature heart with prolonged cross-clamp time has become a key issue in clinical practice. There is no cardioplegia designed for children in our country, HTK solution has been used to pediatric patients with complex heart surgery in Fuwai hospital. However, Whether or not HTK is the optimal cardioplegia in infants/neonates needs to be further investigated.The first part of the study was designed to compare the myocardial protection of HTK solution and CBC to neonatal heart in a piglet model undergoing cardiopulmonary bypass (CPB). The model mimics clinical procedure in infants with cardiac surgery in terms of anesthesia and CPB management, including anesthetics, blood gas management, perfusion techniques. All animals were placed on deep hypothermic CPB. The aortic cross clamping (ACC) time was2hours, and the experimental endpoint was at2hours after weaning from CPB. By using enzyme-linked immunosorbent assays, immunohistochemistry, light and electronic microscopy, the blood and tissue samples were examined. From different aspects, including myocardial metabolism, histopathology, and ultrastructural changes, the comparative myocardial protection between HTK and CBC was performed.The second part of the study is based on that oxidative stress was the most common pathological myocardial damage in dead infants after congenital heart surgery in recent study and the HTK’s antioxidant capacity is limited compared to the blood cardioplegia. We hypothesized that adding Ebselen targeted to mitochondria to the HTK can give better myocardial protection to the premature heart. The piglet model of CPB was similar to that in the first part. The ACC time was2hours, and myocardial samples were collected at2h after off-CPB. By using electronic microscopy, western blot, PCR, the protective mechanism of HTK added by Ebselen was examined compared to the HTK solution.The third part of this study was carried out to observe the effect of HTK solution on the recovery of normal cardiac rhythm in pediatric patients undergoing complex congenital heart surgery. The correlation between recovery time of normal rhythm and postoperative short-term outcomes were examined. We also investigated retrospectively322cases, in order to determine the relationship between the hyponatremia, lower plasma osmolarity induced by HTK and postoperative cerebral morbidity. Part I Comparative myocardial protection between HTK solution and cold blood cardioplegia in immature heart during cardiopulmonary bypassObjective The optimal myocardial protection strategy for newborns/infants undergoing prolonged cardiac arrest during congenital heart surgery remains controversial. Which is the better option between HTK or blood cardioplegia during neonatal cardiopulmonary bypass is unclear. The purpose of this study was to compare myocardial protection using HTK and cold blood cardioplegia in a neonatal piglet model.Materials and Methods Fifteen piglets were randomized to three groups:the control group (C group, n=5), a single dose of HTK group (HTK group, n=5) and muti-dose cold blood cardioplegia group (cardioplegia:blood=1:1, CBC group, n=5). Animals in the two experimental groups were placed on hypothermic CPB, after which the ascending aorta was clamped for2h. The control animals underwent normothermic CPB without cardiac arrest. Serum troponin-T, troponin-I, CK-MB levels were measured before CPB and2h after weaning from CPB. eNOS and VEGF content in coronary vessels and iNOS, TNF-a, IL-1β and ATP content in myocardium were assayed. TUNEL positive myocytes were counted. Myocardial histopathology was examined by light microscopy. Mitochondrial structural damages were assessed by electronic microscopy. Results Transfusion requirement in the CBC group was significantly more than that in the HTK group (p<0.01). Immediately after declamping, both the difference between arterial and coronary sinus blood lactate concentrations and the oxygen extraction did not differ between the two experimental groups(p>0.05). At2h after weaning from CPB, rise in serum TnT, TnI and CK-MB levels showed no significant differences between the three groups (p>0.05). There were no differences in eNOS, VEGF, iNOS, TNF-α and IL-1β content in myocardium as well as histopathological score between the HTK group and the CBC group(p>0.05). However, mitochondrial score under electronic microscopy in HTK group was higher than that in the CBC group (p=0.045), and myocardial ATP content in the HTK group was lower than that in the control group (p=0.017). Moreover, TUNEL positive cells in the HTK group increased compared to the CBC group(p=0.037).Conclusion Compared with cold blood cardioplegia, there are no differences in myocardial metabolism, biochemical markers, endothelial activation and early inflammatory reaction and histopathological injury between the HTK group and the CBC group. However, mitochondrial injury in the HTK group shows more damages than that in the CBC group, and there are increased apoptotic cells and trend of reduced ATP content in myocardium in the HTK group. Less transfusion requirement is needed in the HTK group than that in the CBC group. HTK solution can be used as a good alternative cardioplegia to CBC for the neonatal heart within2hours of cardiac arrest. Part II Myocardial protection of HTK added by Ebselen on immature heart during cardiopulmonary bypassObjective Modified HTK solution giving better myocardial protection to mature heart has been documented. However, the role of HTK added by antioxidant Ebselen on immature heart is unknown. The purpose of this study was to compare myocardial protection using Histidine-tryptophan-ketoglutarate (HTK) added by Ebselen and HTK in a neonatal piglet model.Materials and Methods Fifteen piglets were randomized to three groups:the control group (C group, n=5), a single dose of HTK group (HTK group, n=5), a single dose of HTK added by Ebselen (10nM) group (HTK+E group, n=5). Animals in the two experimental groups were placed on hypothermic cardiopulmonary bypass, after which the ascending aorta was clamped for2h. The control animals underwent normothermic CPB without cardiac arrest. Oxidative stress biomarkers, antioxidant activity, and mitochondrial structures were assessed. Myocardial ATP content was measured. TUNEL positive myocytes were also counted. The release of cytochrome c and the expression of Bax, Bcl-2and HSP72in myocardium were examined by using western blotting. The expression of HSP72mRNA was also detected by RT-PCR.Results Transfusion requirement was no significant differences between the HTK group and the HTK+E group (p<O.01). HTK+E group showed increased superoxide dismutase (SOD) content and higher Mn-SOD activity(p=0.021and p=0.020) compared with the HTK group. Increased MDA in myocardium in the HTK group was observed compared to the control group (p=0.038). Meanwhile, myocardial TUNEL positive cells and the release of cytochrome c were reduced in the HTK+E group as compared to the HTK goup (p=0.045and p=0.010, respectively). The Bax/Bcl-2ratio in the HTK group were significant higher than those in the control group (p=0.024and p=0.028, respectively). The expression of HSP72protein and mRNA increased in the HTK+E group when compared to the HTK group (p=0.039and p=0.035, respectively). There was positive correlation between the HSP72and Mn-SOD content(r=0.581,p=0.023). Mitochondrial score under electronic microscopy in the HTK+E group was lower than that in the HTK group (p=0.047). Myocardial ATP content in the HTK group was lower than that in the control group (P=0.011).Conclusion Reduced myocardial oxidative stress and apoptosis, as well as better preserved myocardial mitochondrial structure were observed in the HTK+E group, and the release of cytochrome c decreased in the HTK+E group compared with the HTK group. Moreover, increased expression of HSP72in the HTK+E group suggests improved antioxidant defense. HTK solution added by Ebselen provides better myocardial protection to HTK solution for the neonatal heart with equivalent transfusion requirement. Therefore, HTK+E solution would be a better alternative cardioplegia to blood cardioplegic solution for the immature heart. Part III The effect of delayed recovery of normal rhythm and hyponatremia induced by HTK solution on short-term outcomes in children undergoing congenital heart surgeryObjective The effect of HTK-induced delayed cardiac rhythm restoration and hyponatremia on postoperative outcome of children undergoing congenital heart surgery remains unclear. This retrospective investigation was designed to determine whether or not delayed rhythm restoration and hyponatremia induced by HTK solution influence the short-term outcome of pediatric patients after cardiac surgery.Methods Group Ⅰ:From June to December in2012, there were169pediatric patients who underwent selective complex congenital heart surgery with CPB. HTK solution was used as cardioplegia during operations in all children. The cardiac resuscitation after declamping was observed. The correlation between recovery time of normal heart rate/rhythm and perioperative factors were analyzed. The risk factors of using pacemaker after surgery were also determined by Logistic regression. Group Ⅱ:From Febrary to December in2012, HTK solution was applied to322children with congenital heart disease during cardiac surgery. Perioperative factors, including age, gender, weight, surgical type, prime volume, HTK volume, ventricular fibrillation after declamping, antegrade cerebral perfusion duration, lowest nasopharynx temperature, ACC and CPB duration, change of blood sodium concentrations above15mmol/L, highest sodium concentration, lowest sodium concentration, highest glucose, lowest glucose, using pacemaker after surgery, accumulated thoracic drainage on postoperative1day, mechanical ventilation time, intensive care unit lengths of stay, were used to analyze the risk factors of postoperative cerebral morbidity such as dysphoria and seizure.Results Group I:The incidence of ventricular fibrillation after declamping was significantly higher in children above3-year-old than that in infants. The recovery time of normal rhythm is positively correlated to cardiothoracic ratio, ACC and CPB time, postoperative24h milrinone dose, and ICU length of stay in infants undergoing congenital heart surgery, respectively. Moreover, the recovery time is negatively correlated to nasopharynx temperature at aortic declamping. Surgical type (TGA), milrinone dose, and adrenaline dose were independent factors of using pacemaker after surgery in infants. Group II:the incidence of hyponatremia was71.9%(220/306),≤125mmol/L was4.2%,≤130mmol/L was18%,≤135mmol/L was49.7%. Compared with the baseline levels, sodium concentrations decreased at early after aortic clamping (p=0.000). Sodium concentrations at PICU and postoperative12h significantly higher than that before operation (p=0.000). The incidence of postoperative sodium concentrations>146mmol/L was38.9%(119/306),≥150mmol/L was10.1%(31/306). Change of plasma osmolarity is consistent with the changes in sodium concentrations. However, osmolarity was lower than normal values during and immediately after CPB, and returned to normal levels after entering PICU. The incidence of seizure and dysphoria were0.9%(3/306) and18%(55/306), respectively. The highest sodium concentration was the independent factor of postoperative brain morbidity.Conclusion Larger cardiothoracic ratio, longer ACC and CPB duration, and lower nasopharynx temperature make the restoration of normal cardiac rhythm prolonged in infants undergoing congenital heart surgery. There are correlations between the recovery time of normal rhythm and postoperative milrinone dose or ICU length of stay, suggesting that elevated temperature at declamping may be helpful to improve the early outcome of the pediatric patients. Atrioventricular block and delayed rhythm restoration induced by HTK are not the independent factors predicting the use of pacemaker in children after cardiac surgery. However, for some surgical types such as TAPVC and TECD, ICU length of stay in children with atrioventricular block is longer than that in children without cardiac block. Postoperative hypernatremia is associated with the incidence of seize and dysphoria in pediatric patients. Moreover, plasma sodium concentrations at postoperative12h have predictive values in the incidence of brain morbidity.