Cardiomyocyte proliferation and heart regeneration in adult Xenopus tropicalis evidenced by a transgenic reporter line

Generation and characterization of Tg(mlc2:H2C) reporter line

To label cardiomyocyte nucleus in X. tropicalis, we generated a transgenic plasmid (pMlc2-H2B-mCherry) that expresses the H2B-mCherry (H2C) fusion red fluorescent protein¹⁶ specifically in cardiomyocyte nuclei, which was driven by a 3 kb fragment of Xenopus mlc2 promoter¹⁴ (Fig. 1a). The F0 funders successfully passed their transgenes to the F1 generation, which were determined by cardiac-specific expression of mCherry in live tadpoles (Fig. 1b, Supplementary Video 1). Confocal microscope image (Fig. 1c) and video (Supplementary Video 2) revealed that the reporter gene mCherry specifically expressed in cardiomyocyte nuclei in F1 transgenic X. tropicalis tadpole. Transgene is also specifically expressed in heart of adult F1 X. tropicalis line (Fig. 1d). The cardiomyocyte nucleus-specific expression of mCherry reporter gene in ventricle and atrium was further determined in the adult F1 generation by confocal microscope (Fig. 1e). These data suggest that we successfully generated the heritable transgenic reporter line Tg(mlc2:H2C) which can be used to label all cardiomyocytes in the whole heart.

a Schematic representation of the transgenic plasmid pMlc2-H2B-mCherry which harboring the expression cassette of H2B-mCherry (H2C) fusion protein under control of Xenopus mlc2 promoter. b Whole-mount bright-field (upper), epifluorescence (middle), and merged (lower) images showing mCherry expression in Wild-type (WT, left) and F1 Tg(mlc2:H2C) (right) tadpoles. Arrows indicate heart location. c Representative image of the living heart in F1 Tg(mlc2:H2C) tadpole. Red color means mCherry expression specifically in cardiomyocyte nuclei. d Whole-mount bright-field (upper) and epifluorescence (lower) images showing mCherry expression in the adult hearts from WT (left) and F1 Tg(mlc2:H2C) (right) frogs. e Immunostained images for mCherry (red) expression in the adult heart from F1 Tg(mlc2:H2C) frog. DAPI was used as a nuclear stain (blue). Left panel, whole cardiac section in low-magnification. Right panels, high-magnification images of the atrial (upper) and ventricular (lower) regions white boxed in the whole cardiac section. This figure was created by Photoshop Image 12 software using our own data in the work.

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To evaluate the impact of reporter gene on heart development and function in X. tropicalis, we compared the expression of cardiac development-related genes (tbx5, tbx20, tnni3, tnnt2, hand1, hand2, bmp4, fbrsl1) between wild-type and transgenic tadpoles. Our data showed that there were no significant differences in the expressions of these cardiac development-related genes in transgenic line compared with that in wild-type tadpoles (Supplementary Figure 1a, b). Moreover, heart growth and cardiac contraction function in adult frogs were comparable between wild-type and transgenic line animals (Supplementary Figure 1c, d). In addition, the transgenic line was survived and heritable. Taken together, these findings suggest that the continued expression of H2B-mCherry specifically in cardiomyocyte nuclei has no significant impacts on heart development and function in X. tropicalis.

Comparation of the accuracy of cardiomyocyte identification by traditional staining and genetic labeling in adult X. tropicalis

The popular method to determine cell proliferation is nuclear staining of cell cycle markers including Ki67, PCNA, pH3, as well as nuclear incorporation of EdU¹⁰. Although the ideal method for the evaluation of cardiomyocyte proliferation during heart regeneration is to label cardiomyocyte nuclei using antibody against Mef2c or PCM-1¹⁷, some previous studies combined cell proliferating marker staining with whole-cell staining of cardiomyocytes using antibodies against cardiac contractile proteins such as cardiac troponin T (cTnT)^4,18,19, α-actinin^20,21,22, and myosin heavy chain (MHC)^5,23,24. However, the whole-cell staining strategy is not rigorous because the proliferating non-cardiomyocyte nucleus alongside or overlapped with cardiac muscle cells may be included as proliferating cardiomyocytes. To evaluate the accuracy of traditional whole-cell staining of cardiomyocytes in adult X. tropicalis, the adult heart from F1 Tg(mlc2:H2C) line was firstly subjected to whole-cell staining of cardiomyocytes using the cryosection with common thickness (5 μm). Double staining of α-actinin (green) and mCherry (red) revealed the similar distribution patterns in the whole cardiac section (Fig. 2a). As expected, the high-magnification image of confocal microscopy showed that α-actinin expressed in whole cells, but mCherry expressed in nuclei (Fig. 2b). Although most mCherry-positive nuclei were completely and closely surrounded by α-actinin-positive cytoplasm (mCherry⁺α-actinin⁺), the mCherry⁺α-actinin^– cell population (arrow) was detected, in which α-actinin-positive cytoplasm only partially surrounded the mCherry-positive nuclei. Moreover, some α-actinin-positive cells never expressed mCherry in nuclei (mCherry^–α-actinin⁺, arrowhead) (Fig. 2c, d). The different spatial location of mCherry-positive nuclei and α-actinin-positive cytoplasm in these three cell populations were further confirmed by Z-stack confocal images (Fig. 2e–g). To quantify the ratio of these three cell populations, over 200 sections from sixteen adult hearts of Tg(mlc2:H2C) reporter line were examined. We found that the numbers of mCherry⁺α-actinin⁺ cardiomyocytes significantly lower than that of mCherry⁺ cardiomyocytes (Fig. 2h, i). Quantitatively, 84.23 ± 0.84% and 15.77 ± 0.84% cardiomyocytes were mCherry⁺α-actinin⁺ and mCherry⁺α-actinin^– in Tg(mlc2:H2C) hearts, respectively (Fig. 2j). Moreover, 1.96 ± 0.25% cardiomyocytes were detected to be mCherry^–α-actinin⁺ in in Tg(mlc2:H2C) hearts (Fig. 2k).

a Immunostaining for mCherry (red) and α-actinin (green) expression in a whole cardiac section from the adult heart of F1 Tg(mlc2:H2C) frog using cryosections with common thickness (5 μm). DAPI was used as a nuclear stain (blue). b Magnified immunostaining image of the non-apical region white boxed in the whole cardiac section. c Magnified immunostaining image of the white boxed region in figure B showing that most mCherry-positive nuclei are completely and closely surrounded by α-actinin-positive cytoplasm (mCherry⁺α-actinin⁺). Arrow denotes the mCherry⁺α-actinin^– cell in which α-actinin-positive cytoplasm only partially surrounds the mCherry-positive nucleus. Arrowhead denotes the α-actinin-positive cell which never expressed mCherry in nucleus (mCherry^–α-actinin⁺ cell). d Single- and double-channel fluorescence images of figure C for α-actinin (green), mCherry (red), DAPI (blue), and mCherry/DAPI (pink) expression. e–g Representative Z-stack confocal images of mCherry⁺α-actinin⁺ (e), mCherry⁺α-actinin^– (f), and mCherry^–α-actinin⁺ (g) cells in figure c. h, i Quantification of mCherry⁺ and mCherry⁺α-actinin⁺ cell numbers in adult hearts of F1 Tg(mlc2:H2C) frogs. Data are presented as mean ± SEM (n = ~200 sections from 16 hearts for (h), n = 16 hearts for i). *p < 0.05, ****p < 0.0001 (Student’s t test). j Quantification of mCherry⁺α-actinin⁺ and mCherry⁺α-actinin^– cells percentages in adult hearts of F1 Tg(mlc2:H2C) frogs (mean ± SEM, n = 16 hearts). k Quantification of mCherry^–α-actinin⁺ cell percentage in adult hearts of F1 Tg(mlc2:H2C) frogs (mean ± SEM, n = 16 hearts). l–o Representative images (l) and quantification of mCherry⁺, mCherry⁺α-actinin⁺, and mCherry⁺α-actinin^– cells (m–o) using thicker cryosections (10 μm). Data are presented as mean ± SEM (n = ~100 sections from 9 hearts for m, n = 9 hearts for n, o). Ns denotes no significant difference (Student’s t test).

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The high number of mCherry⁺α-actinin^– cells (15.77% of mCherry⁺ cells) might result from the misplacement of nucleus and cytoplasm of cardiomyocytes induced by cardiac sectioning due to the big size of myofibrils. To resolve the potential issue, we further performed this experiment using much thicker sections (10 μm). Indeed, there was no significant differences in the numbers of mCherry⁺α-actinin⁺ populations compared with that of mCherry⁺ cells, when used the thicker sections (Fig. 2l–n). Moreover, our new data using thicker sections showed an increased population of mCherry⁺α-actinin⁺ cells (96.92 ± 1.56% of total mCherry⁺ cells) and a decreased population of mCherry⁺α-actinin^– cells (3.08 ± 1.55% of total mCherry⁺ cells) in the adult heart of Tg(mlc2:H2C) transgenic line (Fig. 2o), when compared with the thin sections (5 μm) (Fig. 2j). These findings reveal that the transgenic reporter line is really reliable to specifically identify cardiomyocytes. In addition, these data also imply that the accuracy of traditional whole-cell staining of cardiomyocytes is largely dependent on imaging depth.

Subsequently, the cryosection with common thickness (5 μm) of the adult heart from F1 Tg(mlc2:H2C) line was subjected to nuclear staining of cardiomyocytes using antibody against mammalian Mef2c. Nuclear double staining of Mef2c and mCherry showed the similar distribution patterns of Mef2C and mCherry in the whole cardiac section of X. tropicalis heart (Supplementary Figure 2a). The high-magnification image showed that all mCherry and most Mef2c were expressed in nuclei in cardiac section of F1 Tg(mlc2:H2C) line (Supplementary Figure 2b). Although most mCherry⁺ nuclei were co-labeled by Mef2c (mCherry⁺Mef2c⁺), many Mef2c signals were detected outside the cell nucleus (trigon) and in mCherry^– nuclei (arrowhead), which indicating the non-specificity of Mef2c for cardiomyocyte nuclei in X. tropicalis (Supplementary Figure 2c, d). Z-stack confocal images further determined the individual spatial localization of mCherry⁺ and/or Mef2c⁺ nuclei in these different cell populations (Supplementary Figure 2e–g). Quantitatively, there was no significant differences between the numbers of mCherry⁺ and mCherry⁺Mef2c⁺ cells after examination of over 150 sections from eleven hearts of F1 Tg(mlc2:H2C) line (Supplementary Figure 2h, i). Moreover, the percentages of mCherry⁺Mef2c⁺ and mCherry⁺Mef2c^– nuclei were 94.79 ± 0.62% and 5.21 ± 0.62% in Tg(mlc2:H2C) hearts, respectively (Supplementary Figure 2j). However, the percentage of mCherry⁺Mef2c⁺ cell population was significantly higher than that of mCherry⁺α-actinin⁺ cells (5 μm sections) in whole-cell staining of cardiomyocytes (Supplementary Figure 2k).

Transgenic reporter line confirms the newly forming myocardium in adult X. tropicalis hearts upon injury

We next examined the restoration of ventricular myocardium in adult F1 Tg(mlc2:H2C) line upon apical resection injury. Adult transgenic frogs were subjected to apical resection injury as previously reported^8,9. Morphological analysis of heart revealed the progressive restoration of the injured apex within 30 days post-resection (dpr). A large blood clot was firstly detected in the apex at 1 dpr. The gradual resorption of the apical blood clot and its replacement by normal myocardium were observed in the subsequent time points. By 30 dpr, the blood clot completely disappeared as determined by morphological analysis (Fig. 3a). Through the detection of mCherry fluorescence in Tg(mlc2:H2C) line, we found that mCherry was largely present in the adult ventricle. However, after resection of the ventricular apex, mlc2-driven mCherry was absent in the areas of removed apical tissues (arrow), which was gradually present along with apical blood clot resorption and myocardium restoration (asterisk) during a period from 0 to 30 dpr (Fig. 3b). These findings suggest that the adult Tg(mlc2:H2C) frog can restore the injured apex within 30 days, in the similar manner of the wild-type X. tropicalis as previously reported^8,9. In consistent with these results, there was no significant difference in heart weight (HW), body weight (BW), and HW/BW ratio between the sham and the resected Tg(mlc2:H2C) frogs at 30 dpr (Fig. 3c). As expected, although the systolic properties of beating heart at 7 and 14 dpr were significantly lower than that in sham group, which was restored to a normal level at 30 dpr (Fig. 3d).

a Representative bright-field images of adult hearts from F1 Tg(mlc2:H2C) frogs at the indicated time points after apical resection injury. b Representative epifluorescence images of adult hearts from F1 Tg(mlc2:H2C) frogs at the indicated time points after apical resection injury. Arrow marks the removed apex. Asterisk marks the restored apex. c Quantification of heart weight (HW), body weight (BW), and HW/BW ratio of frogs in sham-operated and 30 dpr groups. Data are presented as mean ± SEM (n = 3 ~ 10 frogs). Ns, no significant difference (Student’s t test). d Quantification of heart rate at 30 dpr indicating the restoration of normal systolic function of regenerated hearts. Data are presented as mean ± SEM (n = 3 ~ 10 frogs). *p < 0.05, ***p < 0.001 versus control, ns denotes no significant difference (one-way ANOVA test). e Schematic for quantification of ventricular cardiomyocyte numbers and ventricular volumes. f Representative images of adult hearts at the indicated time points after apical resection injury. g, h Quantification of ventricular cardiomyocyte numbers (g) and ventricular volumes (h) at the indicated time points. Data are presented as mean ± SEM (n = 8 ~ 10 hearts per group). *p < 0.05, **p < 0.01, ***p < 0.001 versus control, ns denotes no significant difference (one-way ANOVA test). This figure was created by Photoshop Image 12 software using our own data in the work.

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To explore whether the restored apex results from the hypertrophy of cardiomyocytes located in the resection plane, we firstly assessed the size of cardiomyocytes in apex and revealed that there were no significant differences between sham and resected groups at 7 and 30 dpr (Supplementary Figure 3a, b). Moreover, no differences in the expression of hypertrophic genes in ventricles at 7 and 30 dpr compared with sham-operated group (Supplementary Figure 3c). These data imply that the restored apex might not result from cardiomyocyte hypertrophy. To further elucidate whether apex restoration is contributed by the newly formed cardiomyocytes, we subsequently quantified total cardiomyocyte numbers in the entire ventricles and ventricular volumes covered by mCherry-positive cells, using an unbiased stereological reconstruction method as described previously²⁵ with some modification (Fig. 3e). We found that ventricular cardiomyocyte numbers were significantly lower at 1 dpr compared with sham-operated group, whereas resection injury-induced loss of cardiomyocytes were restored to the levels of pre-resection by 30 dpr (Fig. 3f, g). In line with cardiomyocyte numbers, apical resection-induced loss of ventricular volumes covered by mCherry-positive cells at 1 dpr were significantly restored by 30 dpr (Fig. 3h). Importantly, there were no significant differences in total cardiomyocyte numbers and ventricular volumes between 30 dpr and sham groups (Fig. 3g, h). Taken together, these findings from the reporter line genetically revealed the newly forming myocardium in adult X. tropicalis upon apical resection injury.

Transgenic reporter line verifies cardiomyocyte proliferation during heart regeneration in adult X. tropicalis

Using the whole-cell staining strategy, it has been reported that cardiomyocyte proliferation is vital to heart regeneration in adult X. tropicalis^8,9. However, this conclusion might be contentious due to the defects of traditional whole-cell staining method and the non-specificity of antibodies. Thus, this potential issue should be clarified by more reliable genetic evidence. To further define the proliferation of cardiomyocytes during heart regeneration in adult X. tropicalis, we rigorously evaluated the proliferative capacity of cardiomyocytes using the Tg(mlc2:H2C) transgenic line. Ki67 is widely used as a general proliferation marker duo to its specific expression in the cell cycle stages of S, G2, and M in actively cycling cells¹⁰. We first examined cardiomyocyte proliferation in the apex of adult Tg(mlc2:H2C) transgenic frogs using colocalization of Ki67 with mCherry. In sham-operated apex, almost no mCherry⁺Ki67⁺ nuclei were detected. However, increased numbers of mCherry⁺Ki67⁺ nuclei were observed near the resection plane at 3-30 dpr when compared with sham group. The maximal level of mCherry⁺Ki67⁺ nuclei (26.58 ± 2.36%) was detected at 7 dpr, which was significantly decreased in the later period of regeneration at 14 and 30 dpr (Fig. 4a, b). By capturing the large-scale and Z-stack images of mCherry⁺Ki67⁺ nuclei, we observed the representative proliferating responses of cardiomyocytes in the apex at 7 and 30 dpr (Fig. 4c).

a Immunostaining for mCherry (red) and Ki67 (green) expression in the apex of adult hearts from F1 Tg(mlc2:H2C) frogs at the indicated time points after resection. DAPI was used as a nuclear stain (blue). b Quantification of mCherry⁺Ki67⁺ cells in ventricular apex during heart regeneration within 30 days. Data are presented as mean ± SEM (n = 3 hearts for sham, 5 ~ 8 hearts for injured groups). *p < 0.05, ****p < 0.0001 versus sham. ^###p < 0.001, ^####p < 0.0001 (one-way ANOVA test). c Representative images of mCherry⁺Ki67⁺ cells in the whole apical regions at 7 and 30 dpr. Magnified Z-stack confocal images of mCherry⁺Ki67⁺ cells are shown in right panel. d Immunostaining for mCherry (red) and pH3 (green, antibody from CST, #9701) expression in the apex of adult hearts from F1 Tg(mlc2:H2C) frogs at the indicated time points after resection. DAPI was used as a nuclear stain (blue). e Quantification of mCherry⁺pH3⁺ cells in ventricular apex during heart regeneration within 30 days. Data are presented as mean ± SEM (n = 3 hearts for sham, 5 hearts for injured groups). **p < 0.01, ****p < 0.0001 versus sham. ^#p < 0.05, ^####p < 0.0001 (one-way ANOVA test). f Representative images of mCherry⁺pH3⁺ cells in the whole apical regions at 7 and 30 dpr. Magnified confocal images of mCherry⁺pH3⁺ cells are shown in right panel.

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It has been demonstrated that cardiomyocytes can go through cell cycle phases but stop and not complete it, which may lead to false-positive results when using general proliferation markers expressed in early phases of cell cycle²⁶. Therefore, the later cell cycle marker phospho-Histone H3 (pH3) was further used in this study. In agreement with Ki67 staining, immunofluorescent staining used the antibody against pH3 (CST, #3377) showed that apical resection injury greatly promoted cardiomyocyte proliferation in apex as demonstrated by the increased percentages of mCherry⁺pH3⁺ nuclei at 3-30 dpr (Supplementary Figure 4a, b). Consistently, the maximal level of pH3⁺mCherry⁺ nuclei (20.29 ± 2.15%) was detected at 7 dpr, which was remarkably decreased in the later period of regeneration at 30 dpr (Supplementary Figure 4a, b). The representative proliferating responses of cardiomyocytes in the apex at 7 and 30 dpr were confirmed by large-scale and Z-stack imaging of mCherry⁺pH3⁺ nuclei (Supplementary Figure 4c, d). Given that pH3 is a later marker of cell cycle, the number of pH3-positive cardiomyocytes should be much lower than that of Ki67-positive cardiomyocytes. The inappropriate high numbers of pH3-positive cardiomyocytes (20.29 ± 2.15%) observed above may result from the non-specific staining of pH3 antibody. Indeed, the reactivity of the above used antibody against pH3 (CST, #3377) includes zebrafish and mammalians rather than Xenopus. We think that this should be the main reason for the high numbers of pH3-positive cardiomyocytes in above observation. To resolve the potential problem, we further performed this experiment using a new antibody against pH3 (CST, #9701), which was predicted to react with Xenopus due to that the antigen sequence used to produce this antibody shares 100% sequence homology with Xenopus. As expected, staining used the new antibody against pH3 (CST, #9701) showed a much lower percentage of pH3-positive cardiomyocytes during adult heart regeneration in the transgenic line Tg(mlc2:H2C) (Fig. 4d–f) when compared with the original antibody (CST, #3377) (Supplementary Figure 4). Importantly, the variation trends of pH3-positive cardiomyocytes during heart regeneration were very similar between these two antibodies against pH3 (Fig. 4d–f and Supplementary Figure 4). These findings suggest that selection of mammalian antibodies for Xenopus studies should be very carefully because of the limitation of Xenopus-specific antibodies. Taken Ki67 and pH3 staining results together, our results from transgenic reporter line rigorously confirmed that apical resection injury indeed induces cardiomyocyte proliferation in adult X. tropicalis.

Cardiomyocyte proliferation contributes to heart regeneration in adult Tg(mlc2:H2C) transgenic frogs

Finally, we assessed the cell cycle entry of cardiomyocytes in adult Tg(mlc2:H2C) transgenic frogs by measuring the nuclear incorporation of EdU, an efficient marker of DNA synthesis. To determine the proliferating cardiomyocytes during the whole period of heart regeneration within 30 dpr, EdU was injected once every three days for 30 days to label all proliferating cardiomyocytes during the whole period of cardiac regeneration (Fig. 5a). After injection, almost no mCherry⁺EdU⁺ nuclei were detected in the apex of sham-operated hearts. However, representative mCherry⁺EdU⁺ nuclei were observed from 3 to 30 dpr, and the percentages of mCherry⁺EdU⁺ nuclei were significantly increased at 7-30 dpr when compared with sham group. As expected, the percentage of mCherry⁺EdU⁺ nuclei at 30 dpr (28.30 ± 3.52%) was higher than that at 7 dpr (18.40 ± 2.36%) due to the accumulation of proliferating cardiomyocytes during the period from 7 to 30 dpr (Fig. 5b, c). Whole cardiac sections showed that the distributions of EdU-psotive mCherry signals were enriched in the apexes at 7 and 30 dpr, respectively (Fig. 5d). These expression patterns were further confirmed by the high-magnification images of apexes, which showed that proliferating cells including EdU⁺ cardiomyocytes were located near the resection plane (Fig. 5e). The EdU⁺ cardiomyocytes in apex were further verified by Z-stack confocal images at 7 and 30 dpr, respectively (Fig. 5f). These findings indicate that the newly forming myocardium in apex might result, at least in part, from the proliferating cardiomyocytes in adult X. tropicalis upon resection injury.

a Schematic of EdU multiple injection experiment designed to label the proliferating cardiomyocytes in adult Tg(mlc2:H2C) transgenic X. tropicalis line during heart regeneration within 30 days. b Immunostaining for mCherry (red) and EdU (green) expression in the apex of adult hearts from F1 Tg(mlc2:H2C) frogs at the indicated time points after resection. DAPI was used as a nuclear stain (blue). c Quantification of mCherry⁺EdU⁺ cells in ventricular apex during heart regeneration within 30 days. Data are presented as mean ± SEM (n = 5 ~ 8 hearts per group). **p < 0.01, ***p < 0.001, ****p < 0.0001 versus sham (one-way ANOVA test). ^#p < 0.05 (Student’s t test). d Immunostaining for mCherry (red) and EdU (green) expression in a whole cardiac section from the adult heart of F1 Tg(mlc2:H2C) frog at 7 and 30 dpr. DAPI was used as a nuclear stain (blue). e Representative images of mCherry⁺EdU⁺ cells in the apical region at 7 and 30 dpr.^. f Magnified Z-stack confocal images of mCherry⁺EdU⁺ cells white boxed in figure e. This figure was created by Photoshop Image 12 software using our own data in the work.

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• Source: https://www.nature.com/articles/s41536-024-00384-w