Xenopus tropicalis is an ideal model for regenerative research as many of its organs possess a remarkable regenerative capacity. Here, we present a method for constructing a heart injury model in X. tropicalis via apex resection.
It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. Previous research has demonstrated the regenerative ability of the heart of the adult Xenopus tropicalis, an anuran amphibian with a diploid genome and a close evolutionary relationship with mammals. Additionally, studies have shown that following ventricular apex resection, the heart can regenerate without scarring in X. tropicalis. Consequently, these previous results suggest that X. tropicalis is an appropriate alternative vertebrate model for the study of adult heart regeneration. A surgical model of cardiac regeneration in the adult X. tropicalis is presented herein. Briefly, the frogs were anesthetized and fixed; then, a small incision was made with iridectomy scissors, penetrating the skin and pericardium. Gentle pressure was applied to the ventricle, and the apex of the ventricle was then cut out with scissors. Cardiac injury and regeneration were confirmed by histology at 7-30 days post resection (dpr). This protocol established an apical resection model in adult X. tropicalis, which can be employed to elucidate the mechanisms of adult heart regeneration.
Heart failure has been a leading cause of mortality worldwide in recent years. Since 2000, the number of deaths due to heart failure has been increasing over time. More than 9 million people died from cardiomyopathy in 2019, which accounted for 16% of the total number of mortalities globally1. Due to the loss of the regenerative capacity of the heart in adult mammals, in some cases, there are not enough cardiomyocytes to maintain the contraction functions in the heart, which affects heart function and contributes to abnormal ventricular remodeling and heart failure2,3,4. Indeed, in mammals, the heart has the poorest regenerative capacity compared to the other organs, such as the liver, lungs, intestines, bladder, bone, and skin. As the aging of the world's population becomes a global megatrend, the challenges we face with heart disease will intensify5.
Elucidating the mechanisms of cardiac regeneration may have significant implications for therapies for ischemic heart disease. Reports have revealed that the hearts of neonatal mice have a regenerative capacity after apex resection6. Nevertheless, this regenerative capacity is lost after 7 days of age7. Studies have demonstrated that adult mammalian hearts are unable to regenerate because their capacity for cardiomyocyte proliferation has diminished8,9. However, the hearts of lower vertebrates possess a powerful regenerative capacity after injury. For instance, zebrafish10, X. tropicalis11, Xenopus laevis12, newt13, and axolotl14 are capable of complete regeneration after apex resection. Additionally, the other parts of the bodies of some lower vertebrates can also undergo complete regeneration, such as the limbs of newts and the tails, lenses, and arms of tropical clawed frogs4,15,16.
Establishing cardiac injury models is the first step to elucidating the mechanisms underlying cardiac regeneration and has great significance in regenerative research. Researchers have developed various methods to build cardiac injury models, including stabbing, contusing, genetic ablation, cryoinjury, and infarction5,6.
Cryoinjury, myocardial infarction (MI), and apex resection are widely used for inducing cardiac injury, and the type of injury may have substantial effects on the following regeneration of cardiomyocytes6. Depending on the surgical technique, the heart's response to regeneration could vary. Cryoinjury causes massive cell death and produces fibrotic scars in the hearts of zebrafish17, thus creating a model that resembles mammalian infarction. Apical resection is performed by cutting away a part of the ventricular tissues, which has been done in zebrafish10 and X. tropicalis11, without causing permanent scars. This study performed apical resection, which is a simpler operation and requires fewer surgical instruments than cryoinjury. Using lineage-tracing analysis, a previous study demonstrated that cardiac regeneration is related to the proliferation of cardiomyocytes that pre-exist in the hearts of the mouse6 and zebrafish18, but no reports exist for amphibians. Hence, the model of apex resection in X. tropicalis plays an important role in elucidating the mechanisms underlying regenerative responses.
All the experimental protocols related to X. tropicalis were approved by the Jinan University Animal Care Committee.
1. Surgery
2. Surgical recovery
3. Detection of the repair condition after cardiac injury
The hearts were collected at 0 dpr, 7 dpr, 14 dpr, and 30 dpr. The morphological analysis revealed that the blood clot caused by the heart injury disappeared at 30 dpr (Figure 2). At the same time, the appearance of the hearts at 30 dpr in the resection group was similar to that of the hearts in the sham operation group; there were no obvious wounds (Figure 2). After apical resection, a blood clot formed and sealed the wound in the ventricle, as observed by H&E (Figure 3) and Masson's trichrome staining (Figure 4). Within 14 dpr, the clot gradually disappeared and was replaced by fibrin (Figure 4). From 14 dpr to 30 dpr, the myocardium incrementally replaced the fibrin and repaired the damaged ventricle apex (Figure 4). The histological analysis showed no difference between the sham and resected hearts at 30 dpr (Figure 3 and Figure 4). This morphological and histological analysis revealed that adult X. tropicalis could repair and regenerate the injured ventricular apex by 30 dpr.
Figure 1: Surgical procedure. (A) Representative image of an exposed heart covered by the pericardium. (B) Representative image of a heart in the systolic phase, during which the pericardium was peeled off; the dashed line indicates the site of the apical resection of the heart. (C) Representative image of the apex resection of the heart. (D) Representative image of the skin suture after surgery. Scale bars = 1 mm. Please click here to view a larger version of this figure.
Figure 2: Morphological analysis of X. tropicalis hearts from 0 dpr to 30 dpr. (A) Representative images of hearts from the sham (left) and apical resection groups (right) at 0 dpr, with the apex of the heart significantly absent in the resection group. (B,C) Representative images of hearts from the sham (left) versus resection groups (right) at 7-14 dpr, with many blood clots appearing in the hearts of the resection group at 7 dpr. At 14 dpr, significant regeneration is seen at the apical resection site, but the edges of the apex of the heart are not even. (D) Representative image of hearts from the sham (left) versus resection groups (right) at 30 dpr; the apex of the resected heart has almost entirely regenerated. Scale bars = 1 mm. Abbreviation: dpr = days post resection. Please click here to view a larger version of this figure.
Figure 3: Histological analysis of Xenopus tropicalis hearts from 0 dpr to 30 dpr. Representative images of H&E staining using hearts from the sham and resection groups at 0 dpr to 30 dpr. (A) Representative image of H&E staining using hearts from the sham group, with dashed lines indicating the approximate amputation planes. (B) Representative image of H&E staining using hearts from the resection group at 0 dpr. (C–E) Representative images of H&E staining using hearts from the resection group at 7-30 dpr, with large amounts of red blood cells accumulated within the wound at 7 dpr (arrows). A mass of fibrin has appeared in the resection site at 14 dpr (arrowheads). The hearts have regenerated completely without scars at 30 dpr. Scale bar = 500 µm. Abbreviation: dpr = days post resection. Please click here to view a larger version of this figure.
Figure 4: Myocardial fibrosis analysis of X. tropicalis hearts from 0 dpr to 30 dpr. Representative images of Masson's staining of hearts from the sham and resection groups at 0 dpr to 30 dpr. (A) Representative image of Masson's staining of hearts from the sham group, with dashed lines indicating the approximate amputation planes. (B) Representative image of Masson's staining of hearts from the resection group at 0 dpr. (C–E) Representative images of Masson's staining of hearts from the resection group at 7-30 dpr, with large amounts of red blood cells accumulated within the wound at 7 dpr (arrows). A mass of fibrin has appeared in the resection site at 14 dpr (arrowheads). The hearts have regenerated completely without scars at 30 dpr. Scale bar = 500 µm. Abbreviation: dpr = days post resection. Please click here to view a larger version of this figure.
Apical resection, which involves the surgical amputation of the apex of the heart, has been described in zebrafish and mice6,18; however, this has not been described in X. tropicalis. This report describes a credible model of cardiac injury and demonstrates that the heart of adult X. tropicalis can fully regenerate after apical resection without scarring. However, some shortcomings need to be improved, and certain details need attention.
Although apical resection can establish a heart injury model, ensuring the same extent of excision of the heart is challenging due to the diversity of the frogs, which have different heart sizes. To ensure that hearts with apexes of the same size are resected, it is necessary to select frogs of the same size, and the operator must practice extensively prior to the experiment.
Second, the ability to accurately and quickly locate and expose the heart is critical to the procedure’s success, as the process of finding the heart can result in the puncture of the adjacent blood vessels and cause bleeding, thus impeding the procedure’s progress or even causing failure. The duration of anesthesia should not be too short or too long. If it is too short, the frog may wake up before the operation is complete, and if this process is too long, it is likely to lead to the death of the frog. As mentioned in protocol sections 2-5, the suturing must be done cautiously. To ensure that the ratio of the amount of apex resection compared to the whole heart is about the same across frogs, the heart weight (HW) and surface area with or without resection are analyzed20, and a small portion of the heart apex is resected to ensure chamber exposure6.
Cryoinjury, MI, and apical resection are the three most widely used methods to induce cardiac injury in zebrafish and mice6,18,21. The limitation of this article is that only the third method was used, meaning this work does not provide any information about the different regenerative responses after injury with the other two methods6. Cryoinjury results in the death of a large number of cells in ~20% of the ventricular wall, similar to mammalian infarcts21. MI, which is also called coronary artery occlusion, has higher survival rates in X. tropicalis in comparison to apical resection in mice6 but is more difficult to achieve in X. tropicalis because the hearts of tropical clawed frogs are smaller than those of mice. The X. tropicalis apical resection method established in this study is simpler to perform and has a high postoperative survival rate.
The apical resection developed in this report will be a significant method for investigating the molecular mechanisms of regeneration in the hearts of X. tropicalis. A previous study also demonstrated that Fosl1 is indispensable for cardiac regeneration in X. tropicalis following apical resection20. This model of apical resection heart injury in X. tropicalis can be used for learning more about the molecular mechanisms underlying cardiac regeneration to promote the study of the genes and molecular mechanisms related to human cardiac regeneration.
The authors have nothing to disclose.
This work was supported by grants from the National Key R&D Program of China (2016YFE0204700), the National Natural Science Foundation of China (82070257, 81770240), and the Research Grant of Key Laboratory of Regenerative Medicine, Ministry of Education, Jinan University (ZSYXM202004 and ZSYXM202104), China.
Acetic acid | GHTECH | 64-19-7-500ml | |
Acid Alcohol Fast Differentiation Solution | Beyotime | C0163M | |
Acid Fuchsin | aladdin | A104916 | |
Alcohol Soluble Eosin Y Stainin Solution | Servicebio | G1001-500ML | |
BioReagent | Beyotime | ST2600-100g | |
Ethanol absolute | Guangzhou Chemical Reagent Factory | HB15-GR-0.5L | |
Hematoxylin Stain Solution | Servicebio | G1004-500ML | |
Neutral balsam | Solarbio | G8590 | |
Operating Scissors | Prosperich | HC-JZ-YK-Z-10cm | |
Paraffins | Leica | 39601095 | |
Para-formaldehyde Fixative | Servicebio | G1101-500ML | |
Phosphate Buffered Saline (PBS) powder | Servicebio | G0002-2L | |
Phosphomolybdic acid hydrate | Macklin | P815551 | |
Stereo microscope | Leica | ||
surgical forceps | ChangZhou | zfq-11-btjw | |
Surgical Suture | HUAYON | 18-5140 | |
Tricaine | Macklin | ||
Xylene | Guangzhou Chemical Reagent Factory | IC02-AR-0.5L |