We compared the effects of STH solution, histidine‒tryptophan‒ketoglutarate stopping solution, and the non-stopping of the heart on the fetal sheep myocardium. Myocardial protection in the fetal sheep undergoing extracorporeal circulation was significantly better with the non-stop beating than when the beating was stopped with the STH or HTK solution. Compared with the STH arrest solution, histidine‒tryptophan‒ketoglutarate stopping solution had a significantly lower release of the markers of myocardial damage once the beating was stopped, and resulted in less apoptosis.
Research on the theory of fetal extracorporeal circulation began in 1984, with attempts at extracorporeal circulation in fetal sheep first reported by Slate et al. at the University of California . Based on many experiments on fetal sheep and a thorough understanding of fetal sheep physiology, a consensus has been reached on the following points regarding the fetal sheep extracorporeal circulation technique. First, fetal extracorporeal circulation needs to be performed at room temperature; second, the extracorporeal circulation needs to be transferred at a high flow rate; third, the transfer needs pulsatile perfusion to mimic fetal physiological perfusion; fourth, the fetal blood volume is low, and the tubing needs low prefilling during extracorporeal circulation. In the 21st century, the pathophysiological changes in fetal sheep after extracorporeal circulation and, most importantly, the preservation of fetal cardiac function have been studied in greater depth. The introduction of minimally invasive and robotic techniques in cardiac surgery, the development of fetal anesthesia concepts, advances in extracorporeal circulation technology and improvement in extracorporeal circulation equipment have made fetal cardiac surgery possible in the 21st century.
Nevertheless, concerns regarding myocardial protection have been a major hindrance, and the perioperative myocardial injury and postoperative cardiac dysfunction are yet to be addressed . Because of the fetus's unique characteristics, the need for the protection of immature fetal myocardium is much higher than that in the adults. There are significant differences between the immature and mature myocardium in terms of function, ultrastructure, energy metabolism, and tolerance of ischemia and hypoxia. The suitability of adult cardioprotective solutions for immature myocardium is unclear. In order to meet the high level of fetal surgical refinement required, myocardial protective fluid arrest needs to be effective and prolonged. At present, the protection of immature myocardium is still being explored . Thus, the differences and similarities between immature and mature myocardium should be the starting point for studying fetal myocardial protection.
Comparison of the cardiac arrest effects of the two stopping fluids
When the blood flow in the coronary vessels ceases because the heart has stopped beating, cardiomyocytes are no longer supplied with blood and oxygen from the coronary arteries, resulting in the cessation of cardiomyocyte aerobic metabolism and a decrease in energy production. Cardiomyocytes still need to be supplied with energy to survive, but their metabolism of energy-producing material is lower: when they stop contracting, their metabolic rate is only one-tenth that of the beating state . During prolonged ischemia, the key to the reduction of myocardial injury and the maintenance of cardiac function is the availability of sufficient energy substrates, and the stopping or preserving fluids currently in clinical use do not contain the energy substrates. Therefore, to minimize the energy consumption of cardiomyocytes during the infusion of cardiac arrest fluid, the metabolism, energy and physical integrity of cardiomyocytes are preserved by minimizing the duration of irrigation and arrest. The commonly used clinical cardioprotective solution, St. Thomas stopping solution, is a hyperkalemic arrest solution that effectively reduces myocardial ischemia/reperfusion injury. However, it does not provide complete protection, and some patients may experience reversible myocardial hypocontractility or myocardial tonic depression.
The blood-bearing pacing solution can carry oxygen and metabolic substrates and has certain acid-base buffering and oxygen free-radical scavenging ability. However, because of the many erythrocytes in the blood-bearing pacing fluid, its viscosity increases significantly at low temperatures, which affects its effective and uniform perfusion to the myocardium . In addition, CPB-activated leukocytes, which are among the major contributors to the CPB-induced inflammatory response syndrome, are also responsible for myocardial ischemia/reperfusion injury . Our experiments were performed at room temperature (28°C), and the STH solution and HTK stopping solution were used to perfuse the heart downstream. During the room temperature perfusion, the stopping speed is faster with the STH solution and slower with HTK solution. Because the STH solution contains a high concentration of potassium ions, it is possible to stop the heart quickly. After the infusion of STH solution, the myocardial cell membrane is rapidly depolarized due to its high potassium level reaching above the threshold potential of fast sodium channels, resulting in inactivation of these channels and failure to form the ascending branch of the action potential, resulting in rapid cardiac arrest. In contrast, the reason for the slower stopping rate of the HTK stopping solution compared to the St. Thomas stopping solution is its significantly lower concentration of potassium ions. Cardioprotection of fetal cardiomyocytes involves the induction of rapid cardiac arrest, and the STH solution has advantages in this regard. However, the overall protection of cardiomyocytes in fetal sheep should be analyzed further.
Detection of myocardial injury
cTnI is a small molecular weight protein that is only found intracellularly and is released into the bloodstream when the cell membrane of cardiomyocytes is ruptured. cTnI is the most specific marker of myocardial injury . cTnI is also a sensitive marker, and has some diagnostic value if there is mild damage to cardiomyocytes .
In cardiomyocytes cTnI plays a major role as a linker between pro-tropomyosin and troponin to form the troponin-pro-tropomyosin complex. There is probably only a very small amount of cTnI in the cytoplasm of a normal organism, and a very small amount of cTnI can be used as an energy substrate during cardiomyocyte metabolism , primarily because the cell membrane is impermeable to cTnI. If a large amount of cTnI is detected in the blood, it indicates that the cardiomyocytes have been damaged, the cell membranes are ruptured, and cTnI from the cytoplasm has entered the body circulation.
During cardiomyocyte contraction, the main function of cTnT is to cooperate with cTnI in a regulatory role. cTnT also enters the humoral circulation after the cardiomyocyte damage, and the current assay requires 2‒3 hours to detect elevated cTnT levels, which can persist for up to 2 weeks after myocardial damage.
Another early indicator of myocardial injury is CKMB . CKMB is one of the many isoenzymes of creatine kinase, and CKMB is used as an indicator of the degree of myocardial damage because it is predominantly found in the myocardium, with minimal levels in other tissues. Therefore, the amount of CKMB in the circulation indicates the degree of myocardial cell damage.
CKMB, cTnI, and cTnT were measured as indicators of myocardial damage. In our study, the level of the indicators of the myocardial damage in the non-stop group was lower than that in the HTK and STH groups at all times, and the HTK group fared better than the STH group. The calcium-ion content in the HTK stopping solution is very low, which decreases the amount of calcium ions entering the cells during the ischemia/reperfusion and reduces the occurrence of calcium-ion overload. STH solution has a high concentration of potassium ions, which causes the coronary arteries to spasm and contract when the high potassium concentration of STH solution enters the perfused artery. During the re-entry process, the reperfused blood slows down the overall perfusion velocity due to the constriction of the coronary artery, ultimately causing the cardiac output to be less than before the perfusion . At the same time, high concentration of potassium ions reduces the difference between the potassium ion concentration inside and outside the cell membrane, resulting in a decrease in the negative membrane potential of the vascular endothelium, leading to an increase in vascular permeability, increased inflammation of cardiomyocytes and vascular thrombosis, and further aggravating ischemia and hypoxia in myocardial tissue .
During apoptosis, endogenous nucleic acid endonuclease cuts DNA in the nucleolus, exposing the cut end of the DNA, which can be identified using a TUNEL assay. The number of TUNEL-positive cells in the STH group was higher than that in the HTK group, indicating that the protective effect of HTK on cardiomyocytes was stronger than in the STH group.
Our study has some limitations. For example, it is difficult to obtain laboratory animals that are suitable for the conditions, resulting in a small sample size. The number of TUNEL-positive cells in the HTK group was higher than that in the non-stop group, indicating that the protective effect was still limited. In immature myocardium, damage due to the extracorporeal circulation often peaks at 6‒8 hours postoperatively, and the degree of apoptosis at 2 hours postoperatively is not necessarily representative of apoptosis at 6-8 hours postoperatively. False-positive results can also occur in TUNEL reactions. Moreover, there are genetic differences between human and sheep immature cardiomyocytes. Therefore, more experimental evidence is needed to fully evaluate the stopping solutions effects on the fetal myocardium.