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. 2012 Dec;4(12):966-77.
doi: 10.18632/aging.100526.

Complete cardiac regeneration in a mouse model of myocardial infarction

Affiliations

Complete cardiac regeneration in a mouse model of myocardial infarction

Bernhard Johannes Haubner et al. Aging (Albany NY). 2012 Dec.

Abstract

Cardiac remodeling and subsequent heart failure remain critical issues after myocardial infarction despite improved treatment and reperfusion strategies. Recently, complete cardiac regeneration has been demonstrated in fish and newborn mice following resection of the cardiac apex. However, it remained entirely unclear whether the mammalian heart can also completely regenerate following a complex cardiac ischemic injury. We established a protocol to induce a severe heart attack in one-day-old mice using left anterior descending artery (LAD) ligation. LAD ligation triggered substantial cardiac injury in the left ventricle defined by Caspase 3 activation and massive cell death. Ischemia-induced cardiomyocyte death was also visible on day 4 after LAD ligation. Remarkably, 7 days after the initial ischemic insult, we observed complete cardiac regeneration without any signs of tissue damage or scarring. This tissue regeneration translated into long-term normal heart functions as assessed by echocardiography. In contrast, LAD ligations in 7-day-old mice resulted in extensive scarring comparable to adult mice, indicating that the regenerative capacity for complete cardiac healing after heart attacks can be traced to the first week after birth. RNAseq analyses of hearts on day 1, day 3, and day 10 and comparing LAD-ligated and sham-operated mice surprisingly revealed a transcriptional programme of major changes in genes mediating mitosis and cell division between days 1, 3 and 10 postnatally and a very limited set of genes, including genes regulating cell cycle and extracellular matrix synthesis, being differentially regulated in the regenerating hearts. We present for the first time a mammalian model of complete cardiac regeneration following a severe ischemic cardiac injury. This novel model system provides the unique opportunity to uncover molecular and cellular pathways that can induce cardiac regeneration after ischemic injury, findings that one day could be translated to human heart attack patients.

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Conflict of interest statement

The authors of this manuscript have no conflict of interests to declare.

Figures

Figure 1
Figure 1. LAD ligation in newborn mice results in massive cardiac damage
(A) Incisions into the left chest wall were used to open the chest cavity and irreversibly ligate the left anterior descending artery (LAD) in newborn mice. Representative images are shown. In Sham-operated mice the chest cavity was opened, without LAD ligation. (B-D) Massive cardiac damage 24 hours after the initial LAD ligation as demonstrated by H&E staining (B), immune-detection of cleaved Caspase 3 (cC3) (B), loss of TroponinT expression (negative = infarction area, asterisk) (B), and echocardiography (D). Arrows in B indicate infarction area. Data from sham operated newborn mice are shown as controls. In (B) and (B) images are representative of 5 mice analyzed. Data in (D) are shown as mean values +/− SEM (n=6 per group). Arrows in (D) indicate LVESD and LVEDD. Probability values were determined using a Mann-Whitney U Test.
Figure 2
Figure 2. Time-course of cardiac regeneration
(A) Time-course of regeneration. Whole hearts and magnifications at the infarction areas are shown on day P1.5, P4.5, P7.5, P12.5, P16.5, and P21.5 following LAD ligation of newborn mouse hearts at P0.5. Images are representative of 4 mice analyzed. Note the complete regeneration of the infarction zone starting ~ day P7.5. (B-C) Cardiac echocardiography of 3 months old mice that where either sham-operated on P0.5 or received LAD ligation on P0.5. Representative M-mode echocardiograms are shown in (B). Arrows show LVESD and LVEDD. Data in (C) show mean values +/− SEM of LVEDD, LVESD, and Fractional shortening (n = 4 per group). ns, not significant (Mann-Whitney U Test).
Figure 3
Figure 3. A time window for cardiac regeneration
Whole hearts and magnifications at the infarction areas are shown on day P28.5 following LAD ligation of newborn mice at P1.5 and LAD ligation in mice at P7.5 after birth. Images are representative of 6 mice analyzed. Note the complete regeneration of the infarction zone when mice received LAD-ligation on P1.5, whereas no regeneration was seen when mice were LAD-ligated on day P7.5.
Figure 4
Figure 4. Evidence for proliferation in cardiomyocytes
(A) Whole hearts and magnifications are shown for hearts LAD-ligated on P0.5 and harvested on P4.5. Sections were stained with H&E, immunoflourescently labeled for TroponinT and DAPI, and for the proliferation marker Ki67. Images are representative of 4 mice analyzed. Note the border zone of the infarction area (arrows) and the complete loss of TroponinT positive cardiomyocytes in the infarction zone (asterisk). (B) High magnification (63 fold) of a heart stained for TroponinT, phospho-Histone3 (Ser10, pH3), and DAPI after 4 days of LAD ligation (P0.5-P4.5). Note the double pH3 and TroponinT positive cardiomyocytes (arrows) at the border zone of the area of infarction (marked by X). (C) Whole hearts and magnifications are shown for hearts LAD-ligated on P0.5 and harvested on P7.5. Sections were stained using H&E and immunoflourescently labeled for TroponinT, DAPI, and BrdU as a marker for DNA replication. No residual TroponinT negative area is visible, indicating complete cardiac regeneration. Images are representative of 4 mice analyzed. (D) High magnification of a heart stained for TroponinT, BrdU, and DAPI after 7 days of LAD ligation (P0.5-P7.5). Arrows point at BrdU incoperation in TroponinT positive cardiomyocytes.
Figure 5
Figure 5. Fate mapping of cardiomyocytes that repair heart attacks
(A) Schematic representation of the lineage tracing experiment. Homozygous female Rosa26-loxPSTOPloxP-YFP reporter mice were crossed with male Myh6-MerCreMer mice. Tamoxifen (20mg/kg in peanut oil) was administered intraperitoneally into pregnant females at E19.5. Newborns were transferred to foster mothers immediately after birth. LAD ligation was performed on P1.5 and hearts were harvested 7 days later on P8.5. (B-C) No significant difference of YFP positive cardiomyocytes in the area at risk (left ventricular free wall = LV free wall) or in the remote myocardium, i.e. right ventricle (RV), between LAD-ligated and SHAM operated mice (n=4 per group). ns, not significant (Mann-Whitney U Test).
Figure 6
Figure 6. Differentially expressed genes in the left ventricle of postpartum P1, P3, and P10 mice
(A) Venn diagram of differentially expressed genes in the left ventricle of non-manipulated P1, P3 and P10 mice. Numbers of genes differentially expressed comparing P1 vs P3, P1 vs P10, and P3 vs P10 are indicated. (B) Heat map showing the progression of the 50 most differentially expressed genes in individual mice between P1, P3 and P10. Most transcripts progress in a linear, or approximately linear, fashion from P1 to P3 and P10 are shown. However, a small set of genes robustly shows an expression increase from P1 to P3 and then a decrease from P3 to P10, whereas another small gene set shows a decrease and then an increase. (C) Validation of RNA-seq results using qRT-PCR. Data indicate a strong correlation between RNAseq data and qPCR expression analyses for the majority of the validated genes accept Ddc, which could be caused by a poor reproducibility of this gene assay. (D) Reactome analysis of differentially expressed genes between P1, P3 and P10 mice showing that the most differentially expressed gene between these stages of the development belong to mitosis and cell cycle categories.
Figure 7
Figure 7. Differentially expressed genes between sham-operated and LAD-ligated mice left ventricles
(A) Venn diagram of differentially expressed genes in the left ventricle of non-manipulated, LAD-ligated and sham-operated mice, all from P10. The numbers of genes differentially expressed comparing non-manipulated P10 vs LAD-ligated hearts, non-manipulated P10 vs sham-operated heart, and LAD-ligated vs sham-operated hearts are indicated. (B) MA plots showing the distribution of gene expression plotted against log(fold change) for each gene in each pair-wise comparison of P10, LAD-ligated and sham-operated mice. Red dots indicate differentially expressed genes (Padj < 0.05), black dots indicate non-differentially expressed genes. (C) Reactome analysis of differentially expressed genes between sham-operated and LAD-ligated left ventricles at P10 revealing that the majority of these genes are annotated to the regulation of and mitosis cell cycle control as well as extracellular matrix and collagen pathways.

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