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Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart.

Malliaras K, Zhang Y, Seinfeld J, Galang G, Tseliou E, Cheng K, Sun B, Aminzadeh M, Marbán E - EMBO Mol Med (2013)

Bottom Line: After MI, new cardiomyocytes arise from both progenitors as well as pre-existing cardiomyocytes.Transplantation of CDCs upregulates host cardiomyocyte cycling and recruitment of endogenous progenitors, while boosting heart function and increasing viable myocardium.The observed phenomena cannot be explained by cardiomyocyte polyploidization, bi/multinucleation, cell fusion or DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Cedars-Sinai Heart Institute, Los Angeles, CA, USA.

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Related in: MedlinePlus

Cycling resident cardiomyocytes in the peri-infarct areaA–C. BrdU+/GFP+ resident cardiomyocytes in the peri-infarct area are indicated by yellow arrows in (A–C) and orange arrows in (C). Purple arrows in (A) show BrdU+/GFP− cardiomyocytes. Z-stacks in (C) reveal that the BrdU+ nuclei belong to cardiomyocytes.D. Quantification of the rates of cycling endogenous cardiomyocytes by immunohistochemistry reveals that ∼90% of GFP+/αSA+/BrdU+ cardiomyocytes were located in the peri-infarct area after MI and CDC therapy (*p < 0.05 compared to remote, #p < 0.05 compared to sham, ∧p < 0.05 compared to MI, n = 3–5/group). One-way ANOVA followed by LSD post hoc test and independent samples t-test were used for statistical analysis (MI vs sham p = 0.039; CDCs vs sham p < 0.001; CDCs vs MI p < 0.001; MI peri-infarct vs remote p = 0.008; CDCs peri-infarct vs remote p = 0.001; all other p = ns).E. Immunohistochemistry reveals that BrdU+ resident cardiomyocytes (yellow arrows) are connected with gap junctions (orange arrows) to neighbouring BrdU− cardiomyocytes.F. Vessel density was similar in areas of the border zone that contained BrdU+/GFP+ resident cardiomyocytes (yellow arrows; images on the right are high power images of insets on left) compared to areas that did not contain BrdU+ cardiomyocytes (n = 3/group). Independent samples t-test was used for statistical analysis.G. After ex vivo retrograde perfusion with Celltracker RED dye the percentage of Celltracker RED+ cardiomyocytes did not differ between BrdU+ and BrdU− GFP+ cardiomyocytes, verifying that cycling myocytes are normally perfused (n = 3/group). Independent samples t-test was used for statistical analysis. All error bars represent SDs. Scale bars: 20 µm (A,F), 10 µm (B,C,E,F[high power],G).
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fig05: Cycling resident cardiomyocytes in the peri-infarct areaA–C. BrdU+/GFP+ resident cardiomyocytes in the peri-infarct area are indicated by yellow arrows in (A–C) and orange arrows in (C). Purple arrows in (A) show BrdU+/GFP− cardiomyocytes. Z-stacks in (C) reveal that the BrdU+ nuclei belong to cardiomyocytes.D. Quantification of the rates of cycling endogenous cardiomyocytes by immunohistochemistry reveals that ∼90% of GFP+/αSA+/BrdU+ cardiomyocytes were located in the peri-infarct area after MI and CDC therapy (*p < 0.05 compared to remote, #p < 0.05 compared to sham, ∧p < 0.05 compared to MI, n = 3–5/group). One-way ANOVA followed by LSD post hoc test and independent samples t-test were used for statistical analysis (MI vs sham p = 0.039; CDCs vs sham p < 0.001; CDCs vs MI p < 0.001; MI peri-infarct vs remote p = 0.008; CDCs peri-infarct vs remote p = 0.001; all other p = ns).E. Immunohistochemistry reveals that BrdU+ resident cardiomyocytes (yellow arrows) are connected with gap junctions (orange arrows) to neighbouring BrdU− cardiomyocytes.F. Vessel density was similar in areas of the border zone that contained BrdU+/GFP+ resident cardiomyocytes (yellow arrows; images on the right are high power images of insets on left) compared to areas that did not contain BrdU+ cardiomyocytes (n = 3/group). Independent samples t-test was used for statistical analysis.G. After ex vivo retrograde perfusion with Celltracker RED dye the percentage of Celltracker RED+ cardiomyocytes did not differ between BrdU+ and BrdU− GFP+ cardiomyocytes, verifying that cycling myocytes are normally perfused (n = 3/group). Independent samples t-test was used for statistical analysis. All error bars represent SDs. Scale bars: 20 µm (A,F), 10 µm (B,C,E,F[high power],G).

Mentions: Immunocytochemistry of isolated cells revealed that cycling (BrdU+ or Ki67+ or H3P+) GFP+/αSA+ cardiomyocytes were smaller (Fig 4A–E) and more often mononucleated (Figs 4A–C and 9A–C), compared to non-cycling (BrdU−, or Ki67− or H3P−) GFP+/αSA+ cardiomyocytes, consistent with previous findings (Chen et al, 2007). Flow cytometric analysis confirmed that cycling endogenous cardiomyocytes (BrdU+/GFP+ cells) were smaller (decreased time of flight, decreased forward scatter area) and less granular/complex (decreased side scatter area) compared to non-cycling endogenous cardiomyocytes (BrdU−/GFP+; Supporting Information Fig 4). Tissue immunohistochemistry revealed that ∼90% of cycling resident cardiomyocytes after MI and CDC therapy were located in the peri-infarct area [Fig 5A–D and F; defined as the area within one low-power field from the edges of the scar, but not including the scar itself (Supporting Information Fig 5)]. Furthermore, BrdU+ cardiomyocytes appear to be structurally integrated with the surrounding myocardium, as they are connected by gap junctions to neighbouring non-cycling myocytes (Fig 5E). Within the border zone, vessel densities were comparable in areas adjacent to BrdU+ cardiomyocytes as compared to those remote from BrdU+ cardiomyocytes (Fig 5F). To unequivocally show that cycling myocytes are normally perfused, we performed ex vivo retrograde perfusion of hearts (obtained from BrdU-pulsed bitransgenic mice) with a fluorescent dye (Celltracker RED), followed by enzymatic dissociation. The vast majority (29 of 30) BrdU+ cardiomyocytes were positive for Celltracker RED; the percentage of Celltracker RED+ cardiomyocytes did not differ between BrdU+ and BrdU− GFP+ cardiomyocytes (Fig 5G). Given that access to infused Celltracker RED is haematogenous, we conclude that cycling myocytes are normally perfused.


Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart.

Malliaras K, Zhang Y, Seinfeld J, Galang G, Tseliou E, Cheng K, Sun B, Aminzadeh M, Marbán E - EMBO Mol Med (2013)

Cycling resident cardiomyocytes in the peri-infarct areaA–C. BrdU+/GFP+ resident cardiomyocytes in the peri-infarct area are indicated by yellow arrows in (A–C) and orange arrows in (C). Purple arrows in (A) show BrdU+/GFP− cardiomyocytes. Z-stacks in (C) reveal that the BrdU+ nuclei belong to cardiomyocytes.D. Quantification of the rates of cycling endogenous cardiomyocytes by immunohistochemistry reveals that ∼90% of GFP+/αSA+/BrdU+ cardiomyocytes were located in the peri-infarct area after MI and CDC therapy (*p < 0.05 compared to remote, #p < 0.05 compared to sham, ∧p < 0.05 compared to MI, n = 3–5/group). One-way ANOVA followed by LSD post hoc test and independent samples t-test were used for statistical analysis (MI vs sham p = 0.039; CDCs vs sham p < 0.001; CDCs vs MI p < 0.001; MI peri-infarct vs remote p = 0.008; CDCs peri-infarct vs remote p = 0.001; all other p = ns).E. Immunohistochemistry reveals that BrdU+ resident cardiomyocytes (yellow arrows) are connected with gap junctions (orange arrows) to neighbouring BrdU− cardiomyocytes.F. Vessel density was similar in areas of the border zone that contained BrdU+/GFP+ resident cardiomyocytes (yellow arrows; images on the right are high power images of insets on left) compared to areas that did not contain BrdU+ cardiomyocytes (n = 3/group). Independent samples t-test was used for statistical analysis.G. After ex vivo retrograde perfusion with Celltracker RED dye the percentage of Celltracker RED+ cardiomyocytes did not differ between BrdU+ and BrdU− GFP+ cardiomyocytes, verifying that cycling myocytes are normally perfused (n = 3/group). Independent samples t-test was used for statistical analysis. All error bars represent SDs. Scale bars: 20 µm (A,F), 10 µm (B,C,E,F[high power],G).
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fig05: Cycling resident cardiomyocytes in the peri-infarct areaA–C. BrdU+/GFP+ resident cardiomyocytes in the peri-infarct area are indicated by yellow arrows in (A–C) and orange arrows in (C). Purple arrows in (A) show BrdU+/GFP− cardiomyocytes. Z-stacks in (C) reveal that the BrdU+ nuclei belong to cardiomyocytes.D. Quantification of the rates of cycling endogenous cardiomyocytes by immunohistochemistry reveals that ∼90% of GFP+/αSA+/BrdU+ cardiomyocytes were located in the peri-infarct area after MI and CDC therapy (*p < 0.05 compared to remote, #p < 0.05 compared to sham, ∧p < 0.05 compared to MI, n = 3–5/group). One-way ANOVA followed by LSD post hoc test and independent samples t-test were used for statistical analysis (MI vs sham p = 0.039; CDCs vs sham p < 0.001; CDCs vs MI p < 0.001; MI peri-infarct vs remote p = 0.008; CDCs peri-infarct vs remote p = 0.001; all other p = ns).E. Immunohistochemistry reveals that BrdU+ resident cardiomyocytes (yellow arrows) are connected with gap junctions (orange arrows) to neighbouring BrdU− cardiomyocytes.F. Vessel density was similar in areas of the border zone that contained BrdU+/GFP+ resident cardiomyocytes (yellow arrows; images on the right are high power images of insets on left) compared to areas that did not contain BrdU+ cardiomyocytes (n = 3/group). Independent samples t-test was used for statistical analysis.G. After ex vivo retrograde perfusion with Celltracker RED dye the percentage of Celltracker RED+ cardiomyocytes did not differ between BrdU+ and BrdU− GFP+ cardiomyocytes, verifying that cycling myocytes are normally perfused (n = 3/group). Independent samples t-test was used for statistical analysis. All error bars represent SDs. Scale bars: 20 µm (A,F), 10 µm (B,C,E,F[high power],G).
Mentions: Immunocytochemistry of isolated cells revealed that cycling (BrdU+ or Ki67+ or H3P+) GFP+/αSA+ cardiomyocytes were smaller (Fig 4A–E) and more often mononucleated (Figs 4A–C and 9A–C), compared to non-cycling (BrdU−, or Ki67− or H3P−) GFP+/αSA+ cardiomyocytes, consistent with previous findings (Chen et al, 2007). Flow cytometric analysis confirmed that cycling endogenous cardiomyocytes (BrdU+/GFP+ cells) were smaller (decreased time of flight, decreased forward scatter area) and less granular/complex (decreased side scatter area) compared to non-cycling endogenous cardiomyocytes (BrdU−/GFP+; Supporting Information Fig 4). Tissue immunohistochemistry revealed that ∼90% of cycling resident cardiomyocytes after MI and CDC therapy were located in the peri-infarct area [Fig 5A–D and F; defined as the area within one low-power field from the edges of the scar, but not including the scar itself (Supporting Information Fig 5)]. Furthermore, BrdU+ cardiomyocytes appear to be structurally integrated with the surrounding myocardium, as they are connected by gap junctions to neighbouring non-cycling myocytes (Fig 5E). Within the border zone, vessel densities were comparable in areas adjacent to BrdU+ cardiomyocytes as compared to those remote from BrdU+ cardiomyocytes (Fig 5F). To unequivocally show that cycling myocytes are normally perfused, we performed ex vivo retrograde perfusion of hearts (obtained from BrdU-pulsed bitransgenic mice) with a fluorescent dye (Celltracker RED), followed by enzymatic dissociation. The vast majority (29 of 30) BrdU+ cardiomyocytes were positive for Celltracker RED; the percentage of Celltracker RED+ cardiomyocytes did not differ between BrdU+ and BrdU− GFP+ cardiomyocytes (Fig 5G). Given that access to infused Celltracker RED is haematogenous, we conclude that cycling myocytes are normally perfused.

Bottom Line: After MI, new cardiomyocytes arise from both progenitors as well as pre-existing cardiomyocytes.Transplantation of CDCs upregulates host cardiomyocyte cycling and recruitment of endogenous progenitors, while boosting heart function and increasing viable myocardium.The observed phenomena cannot be explained by cardiomyocyte polyploidization, bi/multinucleation, cell fusion or DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Cedars-Sinai Heart Institute, Los Angeles, CA, USA.

Show MeSH
Related in: MedlinePlus