Leukemia Inhibitory Factor Enhances Endogenous Cardiomyocyte Regeneration after Myocardial Infarction

<div><p>Cardiac stem cells or precursor cells regenerate cardiomyocytes; however, the mechanism underlying this effect remains unclear. We generated CreLacZ mice in which more than 99.9% of the cardiomyocytes in the left ventricular field were positive for 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) staining immediately after tamoxifen injection. Three months after myocardial infarction (MI), the MI mice had more X-gal-negative (newly generated) cells than the control mice (3.04 ± 0.38/mm², MI; 0.47 ± 0.16/mm², sham; p < 0.05). The cardiac side population (CSP) cell fraction contained label-retaining cells, which differentiated into X-gal-negative cardiomyocytes after MI. We injected a leukemia inhibitory factor (LIF)-expression construct at the time of MI and identified a significant functional improvement in the LIF-treated group. At 1 month after MI, in the MI border and scar area, the LIF-injected mice had 31.41 ± 5.83 X-gal-negative cardiomyocytes/mm², whereas the control mice had 12.34 ± 2.56 X-gal-negative cardiomyocytes/mm² (p < 0.05). Using 5-ethynyl-2'-deoxyurinide (EdU) administration after MI, the percentages of EdU-positive CSP cells in the LIF-treated and control mice were 29.4 ± 2.7% and 10.6 ± 3.7%, respectively, which suggests that LIF influenced CSP proliferation. Moreover, LIF activated the Janus kinase (JAK)signal transducer and activator of transcription (STAT), mitogen-activated protein kinase/extracellular signal-regulated (MEK)extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)–AKT pathways in CSPs <i>in vivo</i> and <i>in vitro</i>. The enhanced green fluorescent protein (EGFP)-bone marrow-chimeric CreLacZ mouse results indicated that LIF did not stimulate cardiogenesis via circulating bone marrow-derived cells during the 4 weeks following MI. Thus, LIF stimulates, in part, stem cell-derived cardiomyocyte regeneration by activating cardiac stem or precursor cells. This approach may represent a novel therapeutic strategy for cardiogenesis.</p></div

X-gal-negative Newly Formed Cardiomyocytes Originate from LRCs, Which Consisted of CSCs or CPCs.

<p>(A) A CPC co-expressing Sca-1 and GATA4. In the two left panels, the arrowhead indicates a Sca-1 (green) and GATA4 (red) double-positive CPC surrounded by a basement membrane of cardiomyocytes (laminin in blue). The GATA4 signal co-localized with DAPI nuclear staining (blue) in the two right panels. Arrows indicate Sca-1-positive capillaries. Scale bar, 10 μm. (B) Partial overlap of LRCs and CPCs. The hearts of 10-week-old mice that were administered BrdU during the fetal period were immunohistochemically examined. Some anti-sarcomeric α-actinin (SA-actinin)-negative and BrdU-positive LRCs were Nkx2.5-positive (arrowhead, upper panels) or pGATA4-positive (arrowhead, lower panels). Some Nkx2.5-positive (arrow, upper panels) or pGATA4-positive (arrow, lower panels) cardiomyocytes retained BrdU. Nuclei were stained with DAPI. Scale bar, 10 μm. (C) Partial overlap of LRCs and CSCs. Cardiac side population cells (CSPs) were isolated from the hearts of 10-week-old mice that had been administered BrdU during the fetal period. BrdU-positive and multi-drug-resistant protein 1(MDR1)-positive CSPs were identified (arrowheads). Scale bar, 10 μm. (D) X-gal-negative newly formed cardiomyocytes originate from LRCs. A white arrowhead indicates SA-actinin-positive and X-gal-negative LRC-derived newly formed cardiomyocytes. An arrow indicates SA-actinin- and X-gal-positive pre-existing cardiomyocytes. Scale bar, 10 μm. Note that the nuclei of both cell types retained BrdU because of their quiescence after birth. Two yellow arrowheads indicate SA-actinin-positive and X-gal-negative cardiomyocytes, the ancestral CSCs or CPCs of which circumvented the BrdU labeling because of their quiescence. (E) Cre-mediated recombination does not occur in CSPs after tamoxifen treatment. X-gal staining of the cardiomyocyte fraction (right) and CSP cell fraction (left) from a tamoxifen-treated mouse. Arrowheads indicate X-gal-negative CSPs. An arrow indicates X-gal-positive cardiomyocytes. β-galactosidase mRNA expression in cardiomyocyte suspension and sorted CSP fraction from tamoxifen-treated and -untreated mice. Scale bar, 10 μm.</p

LIF Increases the Number of X-gal-negative Newly Formed Cardiomyocytes and the Frequency of BrdU Incorporation.

<p>(A) Representative images of X-gal-negative cardiomyocytes in PBS- and LIF-treated CreLacZ mice. X-gal staining (left) and immunofluorescence images (right; SA-actinin, green; laminin, red; nuclei were stained with DAPI, blue) are shown. Arrows indicate X-gal-negative cardiomyocytes. Scale bar, 20 μm. (B) Number of X-gal-negative cardiomyocytes in the MI remote area (closed bar) and the MI area (open bar) in the PBS- and LIF-treated mice after MI. Asterisks indicate significant differences between two groups. *p < 0.05. (C) Frequencies of BrdU-positive X-gal-negative cardiomyocytes among all X-gal-negative cardiomyocytes in the MI remote area (closed bar) and the MI area (open bar) in the PBS- and LIF-treated mice after MI. Asterisks indicate significant differences between two groups. *p < 0.05. Three pairs of adjacent heart sections were examined per mouse. Data indicate the mean of five mice. (D) Representative images of a pair of adjacent sections. Left panels were stained with SA-actinin (green), laminin (red), DAPI (blue), and X-gal. Right panels were stained with BrdU (green), DAPI (blue) and X-gal. Top panels represent a group of X-gal-negative cardiomyocytes in the MI area of a LIF-treated mouse. The enlarged images of a region of interest (white square) are shown. Two X-gal-negative cardiomyocytes (arrowheads in the left panels) and the corresponding BrdU-positive nuclei (arrowheads in the right panels) are shown. Scale bar, 20 μm.</p

LIF Increases the Number of Proliferative CPCs.

<p>(A) Proliferative CPCs in the border of an MI heart. An arrow indicates a BrdU-positive (green), Nkx2.5-positive (red), and SA-actinin-negative (white) CPC. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (B) Number of total CPCs (closed bar) and BrdU-positive CPCs (open bar) in the PBS- and LIF-treated mice after MI. Asterisks indicate significant differences between two groups. *p < 0.05. (C) LIF-injected or PBS-injected mice were sacrificed at 1 week after MI. An arrow in the left two panels indicates the Nkx2.5-positive (red) SA-actinin-negative (green) cells. The serial adjacent sections in the right two panels indicate that the same cell (arrow) was also Ki67-positive (green) and SA-actinin-negative (red). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. (D) Number of Ki67 and Nkx2.5 double-positive cells per area in the PBS- and LIF-treated mice after MI.</p

Efficiency and Specificity of Tamoxifen-induced β-galactosidase Expression in Cardiomyocytes in CreLacZ Mice.

<p>(A) Generation of CreLacZ mice. CAG-CAT-LacZ mice were crossbred with the MerCreMer strain in which tamoxifen-inducible Cre recombinase expression protein was driven by the α-myosin heavy chain promoter. (B) β-galactosidase expression in cardiomyocytes detected by X-gal staining in a tamoxifen-treated CreLacZ mouse (left) and non-treated mouse (right). The bottom panels show immunofluorescence images of the same samples co-stained with sarcomeric α-actinin (SA-actinin) in green and laminin in red. Scale bar, 20 μm. (C) Frequency of X-gal-positive cardiomyocytes in the LV with or without tamoxifen administration (n = 1833 cardiomyocytes pooled from three sections from three mice and n = 1538 cardiomyocytes pooled from two sections from two mice, respectively). The mice were examined immediately after completion of tamoxifen treatment. (D) Number of X-gal-negative cardiomyocytes per area in CreLacZ mice at 1 day (n = 5), 6 months (n = 5), and 1 year (n = 4) after tamoxifen treatment. The Kruskal–Wallis test showed no significant difference among the three groups.</p