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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

Isolation of resident cardiomyocytesStudy schematic. A tamoxifen-inducible cardiomyocyte-specific genetic fate mapping approach was employed, utilizing the MerCreMEr/ZEG mouse strain.Fluorescence-activated cell sorting of enzymatically dispersed myocardial cell preparations obtained from 4-OH Tamoxifen pulsed and non-pulsed bitransgenic (control) mice. Cells were initially gated on the basis of size (Forward scatter; FSC) and granularity (Side scatter; SSC) and subsequently sorted based on expression of GFP, a specific marker for endogenous cardiomyocytes. Boxes denote the boundaries of sorted populations.Flow cytometric analysis for assessment of purity of FACS-sorted GFP+ cardiomyocytes. Markers include troponin T, troponin I, and α-sarcomeric actinin (for cardiomyocytes), CD90 (for mesenchymal cells and fibroblasts), α-smooth muscle actin (α-SMA) (for smooth muscle cells), CD31 (for endothelial cells), c-Kit and Sca-1 (for progenitor cells).
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fig01: Isolation of resident cardiomyocytesStudy schematic. A tamoxifen-inducible cardiomyocyte-specific genetic fate mapping approach was employed, utilizing the MerCreMEr/ZEG mouse strain.Fluorescence-activated cell sorting of enzymatically dispersed myocardial cell preparations obtained from 4-OH Tamoxifen pulsed and non-pulsed bitransgenic (control) mice. Cells were initially gated on the basis of size (Forward scatter; FSC) and granularity (Side scatter; SSC) and subsequently sorted based on expression of GFP, a specific marker for endogenous cardiomyocytes. Boxes denote the boundaries of sorted populations.Flow cytometric analysis for assessment of purity of FACS-sorted GFP+ cardiomyocytes. Markers include troponin T, troponin I, and α-sarcomeric actinin (for cardiomyocytes), CD90 (for mesenchymal cells and fibroblasts), α-smooth muscle actin (α-SMA) (for smooth muscle cells), CD31 (for endothelial cells), c-Kit and Sca-1 (for progenitor cells).

Mentions: We used an inducible fate mapping approach which provides efficient and highly specific cardiomyocyte labelling (Hsieh et al, 2007; Loffredo et al, 2011; Zhang et al, 2010). Bitransgenic mice were generated by crossbreeding female transgenic B6129-Tg(Myh6-cre/Esr1)1 Jmk/J (hereafter referred to as MerCreMer) mice with male B6.Cg-Tg(ACTB-Bgeo/GFP)21Lbe/J (hereafter referred to as ZEG) reporter mice. MerCreMer mice carry a fusion transgene of Cre recombinase flanked by Mer (mutated estrogen receptor ligand binding domains), driven by the cardiac α-myosin heavy chain promoter (encoded by Myh6); thus the Cre recombinase activity is tamoxifen-sensitive and cardiomyocyte-specific. ZEG reporter mice carry a lacZ transgene flanked by LoxP sites, followed by stop codons and then the GFP gene; therefore, upon excision of the LoxP sites and stop codons mediated by Cre recombinase activity, the reporter switches to GFP (driven by the β-actin promoter), permanently and specifically marking resident cardiomyocytes and their progeny as GFP+. No spontaneous expression of GFP was detected in non-tamoxifen-pulsed bitransgenic animals [confirming previous studies reporting very low rates of leakage in MerCreMer/ZEG mice (Hsieh et al, 2007)], while pulsed mice exhibited robust GFP expression (Fig 1B, Supporting Information Fig 1).


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)

Isolation of resident cardiomyocytesStudy schematic. A tamoxifen-inducible cardiomyocyte-specific genetic fate mapping approach was employed, utilizing the MerCreMEr/ZEG mouse strain.Fluorescence-activated cell sorting of enzymatically dispersed myocardial cell preparations obtained from 4-OH Tamoxifen pulsed and non-pulsed bitransgenic (control) mice. Cells were initially gated on the basis of size (Forward scatter; FSC) and granularity (Side scatter; SSC) and subsequently sorted based on expression of GFP, a specific marker for endogenous cardiomyocytes. Boxes denote the boundaries of sorted populations.Flow cytometric analysis for assessment of purity of FACS-sorted GFP+ cardiomyocytes. Markers include troponin T, troponin I, and α-sarcomeric actinin (for cardiomyocytes), CD90 (for mesenchymal cells and fibroblasts), α-smooth muscle actin (α-SMA) (for smooth muscle cells), CD31 (for endothelial cells), c-Kit and Sca-1 (for progenitor cells).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569637&req=5

fig01: Isolation of resident cardiomyocytesStudy schematic. A tamoxifen-inducible cardiomyocyte-specific genetic fate mapping approach was employed, utilizing the MerCreMEr/ZEG mouse strain.Fluorescence-activated cell sorting of enzymatically dispersed myocardial cell preparations obtained from 4-OH Tamoxifen pulsed and non-pulsed bitransgenic (control) mice. Cells were initially gated on the basis of size (Forward scatter; FSC) and granularity (Side scatter; SSC) and subsequently sorted based on expression of GFP, a specific marker for endogenous cardiomyocytes. Boxes denote the boundaries of sorted populations.Flow cytometric analysis for assessment of purity of FACS-sorted GFP+ cardiomyocytes. Markers include troponin T, troponin I, and α-sarcomeric actinin (for cardiomyocytes), CD90 (for mesenchymal cells and fibroblasts), α-smooth muscle actin (α-SMA) (for smooth muscle cells), CD31 (for endothelial cells), c-Kit and Sca-1 (for progenitor cells).
Mentions: We used an inducible fate mapping approach which provides efficient and highly specific cardiomyocyte labelling (Hsieh et al, 2007; Loffredo et al, 2011; Zhang et al, 2010). Bitransgenic mice were generated by crossbreeding female transgenic B6129-Tg(Myh6-cre/Esr1)1 Jmk/J (hereafter referred to as MerCreMer) mice with male B6.Cg-Tg(ACTB-Bgeo/GFP)21Lbe/J (hereafter referred to as ZEG) reporter mice. MerCreMer mice carry a fusion transgene of Cre recombinase flanked by Mer (mutated estrogen receptor ligand binding domains), driven by the cardiac α-myosin heavy chain promoter (encoded by Myh6); thus the Cre recombinase activity is tamoxifen-sensitive and cardiomyocyte-specific. ZEG reporter mice carry a lacZ transgene flanked by LoxP sites, followed by stop codons and then the GFP gene; therefore, upon excision of the LoxP sites and stop codons mediated by Cre recombinase activity, the reporter switches to GFP (driven by the β-actin promoter), permanently and specifically marking resident cardiomyocytes and their progeny as GFP+. No spontaneous expression of GFP was detected in non-tamoxifen-pulsed bitransgenic animals [confirming previous studies reporting very low rates of leakage in MerCreMer/ZEG mice (Hsieh et al, 2007)], while pulsed mice exhibited robust GFP expression (Fig 1B, Supporting Information Fig 1).

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