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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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The magnitude of the observed DNA synthesis cannot be explained away by confounding factorsA–C. Immunocytochemistry of enzymatically dissociated myocytes reveals that cycling (BrdU+, Ki67+, H3P+) GFP+ and GFP− myocytes are more often mononucleated compared to non-cycling myocytes, thus excluding a prominent role of bi/multinucleation (*p < 0.05 compared to non-cycling cells, n = 4/group). One-way ANOVA followed by LSD post hoc test (A) and independent samples t-test (B,C) were used for statistical analysis (A: sham: BrdU+/GFP+vs BrdU−p < 0.001, BrdU+/GFP−vs BrdU−p < 0.001; MI: BrdU+/GFP+vs BrdU−p < 0.001, BrdU+/GFP−vs BrdU−p < 0.001; CDCs: BrdU+/GFP+vs BrdU−p < 0.001, BrdU+/GFP−vs BrdU−p < 0.001; B: sham: KI67+/GFP+vs Ki67−p < 0.001; MI: Ki67+/GFP+vs Ki67−p < 0.001; CDCs: Ki67+/GFP+vs Ki67−p = 0.001; C: sham: H3P+/GFP+vs H3P−p < 0.001; MI H3P+/GFP+vs H3P−p < 0.001; CDCs: H3P+/GFP+ vs H3P−p < 0.001; all other p = ns).D. Nuclei isolated form FACS-sorted GFP+ and GFP− cardiomyocytes were stained for TnI. Only TnI+ nuclei were examined.E. Flow cytometric analysis of isolated TnI+ nuclei from GFP+ and GFP− FACS-sorted cardiomyocytes reveals that 87–90% of TnI+/BrdU+ nuclei are diploid, thus excluding a prominent role of bi/multinucleation (n = 4/group) Numbers in flow cytometry plots indicate averages for groups.F. Ploidy distributions did not differ significantly between BrdU+/TnI+ nuclei (obtained from GFP+ or GFP− cardiomyocytes) and BrdU−/TnI+ nuclei (n = 4/group). One-way ANOVA followed by LSD post hoc test was used for statistical analysis.G. Percentages of BrdU+/TnI+ nuclei are similar to those obtained from flow cytometric analysis of whole cells (*p < 0.05 compared to nuclei from GFP+ cardiomyocytes; #p < 0.05 compared to MI and sham, ∧p < 0.05 compared to sham; n = 4/group). One-way ANOVA followed by LSD post hoc test and independent samples t-test were used for statistical analysis (GFP+: MI vs sham: p = 0.018, CDCs vs sham p < 0.001, CDCs vs MI p < 0.001; GFP−: MI vs sham: p < 0.001, CDCs vs sham p < 0.001, CDCs vs MI p < 0.001; GFP+vs GFP− MI p < 0.001, GFP+vs GFP− CDCs p < 0.001; all other p = ns). All error bars represent SDs.H. DNA was extracted from GFP+ and GFP− FACS-sorted cells obtained from female infarcted hearts injected with male CDCs. qPCR experiments using the male-specific SRY gene as a target resulted in no amplification, revealing no detectable fusion of exogenous CDCs with endogenous cardiomyocytes (n = 3).
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fig09: The magnitude of the observed DNA synthesis cannot be explained away by confounding factorsA–C. Immunocytochemistry of enzymatically dissociated myocytes reveals that cycling (BrdU+, Ki67+, H3P+) GFP+ and GFP− myocytes are more often mononucleated compared to non-cycling myocytes, thus excluding a prominent role of bi/multinucleation (*p < 0.05 compared to non-cycling cells, n = 4/group). One-way ANOVA followed by LSD post hoc test (A) and independent samples t-test (B,C) were used for statistical analysis (A: sham: BrdU+/GFP+vs BrdU−p < 0.001, BrdU+/GFP−vs BrdU−p < 0.001; MI: BrdU+/GFP+vs BrdU−p < 0.001, BrdU+/GFP−vs BrdU−p < 0.001; CDCs: BrdU+/GFP+vs BrdU−p < 0.001, BrdU+/GFP−vs BrdU−p < 0.001; B: sham: KI67+/GFP+vs Ki67−p < 0.001; MI: Ki67+/GFP+vs Ki67−p < 0.001; CDCs: Ki67+/GFP+vs Ki67−p = 0.001; C: sham: H3P+/GFP+vs H3P−p < 0.001; MI H3P+/GFP+vs H3P−p < 0.001; CDCs: H3P+/GFP+ vs H3P−p < 0.001; all other p = ns).D. Nuclei isolated form FACS-sorted GFP+ and GFP− cardiomyocytes were stained for TnI. Only TnI+ nuclei were examined.E. Flow cytometric analysis of isolated TnI+ nuclei from GFP+ and GFP− FACS-sorted cardiomyocytes reveals that 87–90% of TnI+/BrdU+ nuclei are diploid, thus excluding a prominent role of bi/multinucleation (n = 4/group) Numbers in flow cytometry plots indicate averages for groups.F. Ploidy distributions did not differ significantly between BrdU+/TnI+ nuclei (obtained from GFP+ or GFP− cardiomyocytes) and BrdU−/TnI+ nuclei (n = 4/group). One-way ANOVA followed by LSD post hoc test was used for statistical analysis.G. Percentages of BrdU+/TnI+ nuclei are similar to those obtained from flow cytometric analysis of whole cells (*p < 0.05 compared to nuclei from GFP+ cardiomyocytes; #p < 0.05 compared to MI and sham, ∧p < 0.05 compared to sham; n = 4/group). One-way ANOVA followed by LSD post hoc test and independent samples t-test were used for statistical analysis (GFP+: MI vs sham: p = 0.018, CDCs vs sham p < 0.001, CDCs vs MI p < 0.001; GFP−: MI vs sham: p < 0.001, CDCs vs sham p < 0.001, CDCs vs MI p < 0.001; GFP+vs GFP− MI p < 0.001, GFP+vs GFP− CDCs p < 0.001; all other p = ns). All error bars represent SDs.H. DNA was extracted from GFP+ and GFP− FACS-sorted cells obtained from female infarcted hearts injected with male CDCs. qPCR experiments using the male-specific SRY gene as a target resulted in no amplification, revealing no detectable fusion of exogenous CDCs with endogenous cardiomyocytes (n = 3).

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)

The magnitude of the observed DNA synthesis cannot be explained away by confounding factorsA–C. Immunocytochemistry of enzymatically dissociated myocytes reveals that cycling (BrdU+, Ki67+, H3P+) GFP+ and GFP− myocytes are more often mononucleated compared to non-cycling myocytes, thus excluding a prominent role of bi/multinucleation (*p < 0.05 compared to non-cycling cells, n = 4/group). One-way ANOVA followed by LSD post hoc test (A) and independent samples t-test (B,C) were used for statistical analysis (A: sham: BrdU+/GFP+vs BrdU−p < 0.001, BrdU+/GFP−vs BrdU−p < 0.001; MI: BrdU+/GFP+vs BrdU−p < 0.001, BrdU+/GFP−vs BrdU−p < 0.001; CDCs: BrdU+/GFP+vs BrdU−p < 0.001, BrdU+/GFP−vs BrdU−p < 0.001; B: sham: KI67+/GFP+vs Ki67−p < 0.001; MI: Ki67+/GFP+vs Ki67−p < 0.001; CDCs: Ki67+/GFP+vs Ki67−p = 0.001; C: sham: H3P+/GFP+vs H3P−p < 0.001; MI H3P+/GFP+vs H3P−p < 0.001; CDCs: H3P+/GFP+ vs H3P−p < 0.001; all other p = ns).D. Nuclei isolated form FACS-sorted GFP+ and GFP− cardiomyocytes were stained for TnI. Only TnI+ nuclei were examined.E. Flow cytometric analysis of isolated TnI+ nuclei from GFP+ and GFP− FACS-sorted cardiomyocytes reveals that 87–90% of TnI+/BrdU+ nuclei are diploid, thus excluding a prominent role of bi/multinucleation (n = 4/group) Numbers in flow cytometry plots indicate averages for groups.F. Ploidy distributions did not differ significantly between BrdU+/TnI+ nuclei (obtained from GFP+ or GFP− cardiomyocytes) and BrdU−/TnI+ nuclei (n = 4/group). One-way ANOVA followed by LSD post hoc test was used for statistical analysis.G. Percentages of BrdU+/TnI+ nuclei are similar to those obtained from flow cytometric analysis of whole cells (*p < 0.05 compared to nuclei from GFP+ cardiomyocytes; #p < 0.05 compared to MI and sham, ∧p < 0.05 compared to sham; n = 4/group). One-way ANOVA followed by LSD post hoc test and independent samples t-test were used for statistical analysis (GFP+: MI vs sham: p = 0.018, CDCs vs sham p < 0.001, CDCs vs MI p < 0.001; GFP−: MI vs sham: p < 0.001, CDCs vs sham p < 0.001, CDCs vs MI p < 0.001; GFP+vs GFP− MI p < 0.001, GFP+vs GFP− CDCs p < 0.001; all other p = ns). All error bars represent SDs.H. DNA was extracted from GFP+ and GFP− FACS-sorted cells obtained from female infarcted hearts injected with male CDCs. qPCR experiments using the male-specific SRY gene as a target resulted in no amplification, revealing no detectable fusion of exogenous CDCs with endogenous cardiomyocytes (n = 3).
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fig09: The magnitude of the observed DNA synthesis cannot be explained away by confounding factorsA–C. Immunocytochemistry of enzymatically dissociated myocytes reveals that cycling (BrdU+, Ki67+, H3P+) GFP+ and GFP− myocytes are more often mononucleated compared to non-cycling myocytes, thus excluding a prominent role of bi/multinucleation (*p < 0.05 compared to non-cycling cells, n = 4/group). One-way ANOVA followed by LSD post hoc test (A) and independent samples t-test (B,C) were used for statistical analysis (A: sham: BrdU+/GFP+vs BrdU−p < 0.001, BrdU+/GFP−vs BrdU−p < 0.001; MI: BrdU+/GFP+vs BrdU−p < 0.001, BrdU+/GFP−vs BrdU−p < 0.001; CDCs: BrdU+/GFP+vs BrdU−p < 0.001, BrdU+/GFP−vs BrdU−p < 0.001; B: sham: KI67+/GFP+vs Ki67−p < 0.001; MI: Ki67+/GFP+vs Ki67−p < 0.001; CDCs: Ki67+/GFP+vs Ki67−p = 0.001; C: sham: H3P+/GFP+vs H3P−p < 0.001; MI H3P+/GFP+vs H3P−p < 0.001; CDCs: H3P+/GFP+ vs H3P−p < 0.001; all other p = ns).D. Nuclei isolated form FACS-sorted GFP+ and GFP− cardiomyocytes were stained for TnI. Only TnI+ nuclei were examined.E. Flow cytometric analysis of isolated TnI+ nuclei from GFP+ and GFP− FACS-sorted cardiomyocytes reveals that 87–90% of TnI+/BrdU+ nuclei are diploid, thus excluding a prominent role of bi/multinucleation (n = 4/group) Numbers in flow cytometry plots indicate averages for groups.F. Ploidy distributions did not differ significantly between BrdU+/TnI+ nuclei (obtained from GFP+ or GFP− cardiomyocytes) and BrdU−/TnI+ nuclei (n = 4/group). One-way ANOVA followed by LSD post hoc test was used for statistical analysis.G. Percentages of BrdU+/TnI+ nuclei are similar to those obtained from flow cytometric analysis of whole cells (*p < 0.05 compared to nuclei from GFP+ cardiomyocytes; #p < 0.05 compared to MI and sham, ∧p < 0.05 compared to sham; n = 4/group). One-way ANOVA followed by LSD post hoc test and independent samples t-test were used for statistical analysis (GFP+: MI vs sham: p = 0.018, CDCs vs sham p < 0.001, CDCs vs MI p < 0.001; GFP−: MI vs sham: p < 0.001, CDCs vs sham p < 0.001, CDCs vs MI p < 0.001; GFP+vs GFP− MI p < 0.001, GFP+vs GFP− CDCs p < 0.001; all other p = ns). All error bars represent SDs.H. DNA was extracted from GFP+ and GFP− FACS-sorted cells obtained from female infarcted hearts injected with male CDCs. qPCR experiments using the male-specific SRY gene as a target resulted in no amplification, revealing no detectable fusion of exogenous CDCs with endogenous cardiomyocytes (n = 3).
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