The main objective of this study was to present an in-depth biochemical, hemodynamic and electrophysiological characterization of our isolated ex-vivo slaughterhouse heart experiments in the PhysioHeart™ platform. Initially, the resuscitated porcine hearts showed physiological metabolic, electrical and hemodynamic activities. However, electrophysiological and hemodynamic cardiac functions gradually diminished due to the initiation of waste product intoxication, reduction of essential nutrients, ion imbalances, cardiac necrosis and, most likely lastly, reperfusion injury and inflammation. On one hand, we conclude that the variability observed in the baseline pump function is a consequence of the ‘random’ selection of the slaughterhouse animals and the harvesting techniques. On the other hand, the superimposed progressive diminishment in cardiac function is concluded to be a result of the isolated slaughterhouse heart pathophysiology. Namely, the observed loss of function is associated with an increased level of metabolites and electrolytes, declining nutrients, a gradual loss of tissue integrity with edema and cell death which we believe is a result of the lack of hepatic and nephrological plasma clearance in the isolated heart setting. Figure 12 and Table 3 summarize these results, which resemble the deterioration of the heart function in a multi-organ failure situation. These observations support the use of plasma clearance interventions and support the working hypothesis that isolated hearts should be treated, as far as possible, as heart-and-organ failure environment.
The limited duration of acceptable performance during the isolated heart experiments highlights that the isolated working heart needs to be in an environment that resembles the in-vivo physiology to avoid loss of its morphological and functional integrity. The slaughterhouse pigs used in this study were in general good health and were examined by a veterinarian prior to slaughtering. However, previous research has shown that domestication, selective breeding, scarce physical activity and improved feeding efficiency lead to morphological abnormalities in slaughterhouse-derived porcine hearts [25]; this was observed in our specimens as hypertrophic cardiomyopathy. Also, the baseline blood measurements revealed elevated levels in damage markers (i.e. CK, ASAT, LDH, troponin and myoglobin). As previous research has shown, elevated damage markers were most likely caused by the limited heart capacity observed in farm animals due to an intensive selection pressure and high stress during regrouping, transport and slaughtering [26]. The baseline measurements also revealed high ion concentrations and hyperosmolarity in blood; as previously reported by Heinze and Mitchell [27], this was probably a consequence of water accumulation in the intra- and inter-cellular space caused by the electrical stunning. Moreover, it is believed that the hyperkalemia observed at baseline was a consequence of the rapid drop in pH produced by the slaughtering; this drop in pH is known to lead to a cellular intake of H+ and release of K+ as a physiological process of pH balancing [28]. Electrical stunning also produces muscle contraction, which leads to hypoglycemia, hyperlactatemia, elevated creatinine levels and hyperammonemia [29]. These contractions can further lead to acidosis (low pH) and hyperlactatemia, an effect that has previously been reported during epileptic seizures when the muscles suffer from hypoxia [30, 31].
For each heart experiment, blood from different pigs was collected immediately after exsanguination and stored for about 2 h until preparation for reperfusion. Generally, pooling blood leads to transfusion reaction in humans, but the particular characteristics of the porcine hematopoietic system make porcine blood pooling less harmful as it causes transfusion reaction only in very rare cases [32]. However, the storage lesion of erythrocytes, during which glucose is consumed, levels of 2,3-diphosphoglycerate (DPG) and ATP decrease, and ammonia and potassium levels increase [33,34,35], is most likely contributing to the pathological blood values observed already at baseline.
Despite the quick harvesting process, warm cardiac ischemia is still expected to occur and to cause cardiac nutrient deficiency, hypoxia, acidosis and necrosis. It is expected that these processes will continue to damage the tissue during the cold storage period. Finally, these already stressed, hypertrophic hearts, were stored in a St. Thomas solution 2, a hypooncotic solution that promotes the influx of water through the endothelial layer into the intracellular space; this causes a further risk of cardiac edema [36, 37]. A more complex composed cardioplegic solutions like Custodiol [38], Somah [39], Celsior [40, 41] or UWS [41], could be of favor during hypothermic storage of slaughterhouse hearts. However, the use of a more complex solution also requires a careful consideration of price and advantages, which are currently under evaluation.
Therefore, the here above described ‘slaughterhouse-associated’ adverse effects should not be ignored when comparing the results with the carefully removed heart. These effects result in an increased chance for a reduced preservation, loss of cardiac tissue and function of the slaughterhouse porcine hearts. Despites these limitations one can learn from this pig heart the following:
Biomarkers and electrolytes
Immediately after cardiac resuscitation, an increase in potassium and magnesium in blood is observed. This is probably due to the washout of the cardioplegic solution from the coronary system. This solution, which is administered during harvesting, contains potassium and magnesium at 16 mmol/L to ensure cardiac arrest during storage. Figure 6g illustrates this wash out on the example of magnesium which experiences its largest increase in the first hour.
Throughout the experiment, we observe a rise in cardiac injury markers caused by reperfusion injury [42] and inflammatory responses of leukocytes and platelets. It remains speculative, but possible causes for the increasing markers may be heterogeneous cardioplegia delivery to the myocardium, harvest-related thrombosis, air emboli, and/or hypertrophic myocardium. These circumstances vary amongst hearts and therefore result in the observed fluctuating necrosis marker concentrations [43], initial cardiac outputs and pump functions of slaughterhouse-based hearts.
Hypertrophic hearts are known to be more vulnerable to ischemia and reperfusion injury [44] due to dilated epicardial coronaries, reduced capillary density and vascular dilatation reserve, which reduces the diffusion of nutrients and oxygen [45] and could potentially negatively influence the cardiac arrest. The presence of dilated, hyperemic coronaries in beating pig hearts revived in the PhysioHeart™ platform has been recently confirmed by Schampaert, van ‘t Veer [21] who associated the hyperemia to an endothelial response to the organ harvest and preparation. However, whether the hyperemic circulation is related to these preparation processes or a hypertrophy-related impairment to pharmacological and physiological stimulation, as other works suggest [46, 47], is still not fully understood.
The acidic environment during cardiac storage reduced the pH of the blood pool after cardiac resuscitation. The pH balancing with sodium bicarbonate led to an increase of sodium and reduction of ionized calcium [48] which was then counter balanced with calcium chloride administration resulting in constant rise of sodium and chloride in the blood. Besides these processes, the revived hearts showed a physiological aerobic cardiac metabolism, supported by free fatty acid uptake and lactate as well as glucose metabolism similar to previous reports [40, 49,50,51]. The constant rise of ammonia also confirms an amino acid catabolism.
However, as the cardiac hemoperfusion progresses, essential cardiac nutrients like free fatty acids decrease and toxic waste products like ammonia increase; this is known to cause edema and to disturb oxidative phosphorylation in the mitochondria [52]. This could explain the increasing lactate values and gain of heart weight of more than 20% at the end of the experiments.
The rise of plasma free hemoglobin in our study was not significant. However, in only one experiment free hemoglobin passed 0.08 mmol/L, which occurred already from the beginning of the experiment. That could have resulted from pre-experimental blood handling. We identified the centrifugal pump as the source with the highest risk to induce hemolysis. Finally, the static concentrations of albumin, triglycerides, urea, creatinine, calcitriol but also potassium exclude the possibility that the rise of electrolytes could arise from evaporation of free water in our system.
Epicardial electrical activity during the working mode
Electrical measurements showed physiological electrical activities of hearts revived in the PhysioHeart™ platform and during the working mode. This can be appreciated in the activation patterns presented in Fig. 10 and Fig. 11, which show unaltered electrical conduction pathways with no observable conduction block or ischemic effects in the areas of interest. Also, WPV restitution (i.e. a decrease in wave propagation velocity as the pacing frequency is increased) was observed and presented in Table 2. The analysis of restitution effects is central in the early detection of arrhythmia and in testing anti-arrhythmic drugs and devices; consequently, observing restitution effects in the PhysioHeart™ platform enables its use to investigate these phenomena within the scope of normothermic perfusion.
Although normal physiological behavior was observed during the working mode, all PhysioHeart™ experiments showed abnormally high sodium, potassium and ionized calcium concentrations in blood. These concentrations increased as the experiment progressed, this is evident from Fig. 6. The abnormally high sodium concentration translated, as observed in Table 2 and as supported by previous research [53], in high wave propagation velocity. Abnormally fast depolarization waves could induce arrhythmias because they may cause re-entrant waves or conduction block. High ionized calcium concentration in blood has also been shown to be related to longer action potentials [54] and abnormal membrane excitability [53]. Also, the observed hyperkalemia is known to cause elevated resting membrane potentials and reduced cellular excitability [55] and, consequently, arrhythmia such as atrial fibrillation or ventricular tachycardia. The use of insulin in our experiments may have support these effects as insulin leads to a dose-dependent influx of potassium into the cells [56]. This last fact was also evident because, in some experiments, stimulation protocols induced arrhythmias when pacing higher than sinus rhythm.
These observations put in evidence the importance of, simultaneously, monitoring the ion concentrations in blood and the electrophysiological activity of the cardiac tissue. In particular, the use of electrode grids within normothermic perfusion platforms could enable the detection of localized ischemia and abnormal conduction patterns that could result in arrhythmia during transport. Moreover, the monitoring of the ion concentrations in blood would also enable to determine the causes of any unphysiological electrical behavior, which can result in fast action to prevent the decreased performance of the heart.
Achieving normal cardiac physiology during ex-vivo slaughterhouse heart perfusion
The PhysioHeart™ platform, with its starling resistor as preload and a standard four-element Windkessel model as afterload, generates flow patterns and pressure curves in the revived slaughterhouse hearts that are similar to those measured in humans [15]. For an average of 3 h, physiological and morphological cardiac characteristics, with normal electrical and metabolic activities, can be obtained without any corrective measures. Although not all blood values are physiological prior and during reperfusion, isolated beating slaughterhouse porcine hearts seem to tolerate these pathologies for a limited period. Therefore, it is inferred that the isolated working normothermic heart can be used as a baseline model to study cardiac intervention methods (LVADs, TAVI valve replacements, etc.).
These interventions may be unloading, moderate hypothermia, filtration of plasma for inflammatory components and metabolic waste, addition of nutrients and protective drugs. In view of ethical constraints regarding use of animals for short-term and uncertain-outcome experiments, the platform provides several benefits including availability, low cost, and no ethical objections.
In view of improved and prolonged preservation of the PhysioHeart™ model is the mitigation of the immune response of the pig blood. This can be achieved by separating lymphocytes and platelets, to obtain platelet and lymphocyte-poor blood in combination with administering inflammatory and autoimmune depressing drugs (i.e. dexamethasone, prednisone). The use of antibiotics and fungistatic medication would further serve to avoid infections. In addition, including anti-arrhythmic drugs in the platform will help mitigate the effects of high electrolyte concentrations in blood and, consequently, extend the time in the working mode.
We have identified that the PhyioHeart™ lacks hepatological and nephrological clearances and substance supplementations. In the upcoming future, the blood perfusate should be kept physiological and renewed constantly to maintain metabolomics and proteomic profiles and to remove toxins either with dialysis, new platelet-poor plasma [52] or a similar complex medium (i.e. HCO3, HEPES, inorganic salts, amino acids, carbohydrates, fatty acids, lipids, vitamins (Cernevit), trace elements, colloids and hormones for vasodilation like Milrinone). Dialysis and hemofiltration may be helpful by removing excess water, toxins and stabilizing the electrolyte and ion balance as the periods of arrhythmia observed in the PhysioHeart™ experiments were associated with high ion concentrations. Finally, these attempts are assumed to attenuate the loss of cardiac function in the platform and would lead to more standardized and improved experimentation.
Limitations of the current PhysioHeart™ experiments
Despite low animal costs, big animal isolated heart experiments remain costly. Therefore, the scope of this study was rather exploratory in order to determine the boundary conditions and attempts needed to prolong physiological cardiac perfusion in our model.
All the hearts had different initial left ventricular function and electrical activity, which result in a ‘normal variation’ that may affect small-number experiments. Therefore, it is important to standardize and optimize the harvest and mounting procedures to avoid outlying negative performance at the start of the experiment. An additional limitation is that the blood pool volumes between experiments were not equal could have influenced blood marker concentrations; a standard initial volume is required to ensure consistency across experimental acquisitions. Additionally, the priming volume for deairing the circuit was not equal between experiments and, most likely, biased the baseline measurements; in future experiments, the priming volume will be considered when analyzing baseline values. Finally, one may consider non-invasive epicardial monitoring such as echocardiography, stress-strain imaging and speckle imaging to detect early signs of ischemia that may result in corrective or supporting actions. Moreover, the use of square grids limits the electrophysiological study to a small region of the heart; the use of more sophisticated electrode arrays, capable of making whole-heart measurements, would enable a more comprehensive electrophysiological study. Finally, micro-puncture histology during and at the end of the experiments may be useful as a hard outcome parameter.