Granulocyte colony-stimulating factor treatment plus dipeptidylpeptidase-IV inhibition augments myocardial regeneration in mice expressing cyclin D2 in adult cardiomyocytes

Abstract

Aims: Although pharmacological interventions that mobilize stem cells and enhance their homing to damaged tissue can limit adverse post-myocardial infarction (MI) remodelling, cardiomyocyte renewal with this approach is limited. While experimental cell cycle induction can promote cardiomyocyte renewal following MI, this process must compete with the more rapid processes of scar formation and adverse remodelling. The current study tested the hypothesis that the combination of enhanced stem cell mobilization/homing and cardiomyocyte cell cycle induction would result in increased myocardial renewal in injured hearts.

Methods and results: Myocardial infarction was induced by coronary artery ligation in adult MHC-cycD2 transgenic mice (which exhibit constitutive cardiomyocyte cell cycle activity) and their non-transgenic littermates. Mice were then treated with saline or with granulocyte colony-stimulating factor (G-CSF) plus the dipeptidylpeptidase-IV (DPP-IV) inhibitor Diprotin A (DipA) for 7 days. Infarct thickness and cardiomyocyte number/infarct/section were significantly improved in MHC-cycD2 mice with G-CSF plus DipA treatment when compared with MHC-cycD2 transgene expression or G-CSF plus DipA treatment alone. Echocardiographic analyses revealed that stem cell mobilization/homing and cardiomyocyte cell cycle activation had an additive effect on functional recovery.

Conclusion: These data strongly suggest that G-CSF plus DPP-IV inhibition, combined with cardiomyocyte cell cycle activation, leads to enhanced myocardial regeneration following MI. The data are also consistent with the notion that altering adverse post-injury remodelling renders the myocardium more permissive for cardiomyocyte repopulation.

Figure 1

Infarct size at 60 days post-myocardial infarctionMI. (A) Representative images of haematoxylin and eosin-stained transverse sections. Bar = 500 µm. (B) Infarct size (%, mean ± SEM) for: (1) saline-treated NON-TXG mice, (2) G-CSF plus DipA-treated NON-TXG mice, (3) saline-treated MHC-cycD2 mice, and (4) G-CSF plus DipA-treated MHC-cycD2 mice. *P< 0.05 vs. saline/NON-TXG.

Figure 2

Left ventricular infarct wall thickness at 60 days post-myocardial infarction. (A) Representative photomicrographs of Masson's trichrome-stained sections. Bar = 500 µm. (B) Infarct thickness (mm, mean ± SEM) for: (1) saline-treated NON-TXG mice, (2) G-CSF plus DipA-treated NON-TXG mice, (3) saline-treated MHC-cycD2 mice, and (4) G-CSF plus DipA-treated MHC-cycD2 mice.*P < 0.05 vs. saline/NON-TXG; ^ΨP< 0.05 vs. G-CSF plus DipA/NON-TXG; ^†P< 0.05 vs. saline/MHC-cycD2.

Figure 3

Peri-infarct vessel density at 60 days post-myocardial infarction. (A) Representative photomicrographs of Masson's trichrome-stained sections. Bar = 20 µm. (B) Infarct vessel area (%, mean ± SEM) for: (1) saline-treated NON-TXG mice, (2) G-CSF plus DipA-treated NON-TXG mice, (3) saline-treated MHC-cycD2 mice, and (4) G-CSF plus DipA-treated MHC-cycD2 mice. *P < 0.05 vs. saline/NON-TXG; ^†P< 0.05 vs. saline/MHC-cycD2.

Figure 4

Infarct and infarct border zone cardiomyocyte ³H-Thy-labelling at 7 days post-myocardial infarction. (A) Representative photomicrographs of autoradiograms of X-GAL-stained sections. Arrows indicate cardiomyocyte DNA synthesis (as evidenced by silver grains over blue, β-galactosidase-positive nuclei); arrowheads indicate non-cardiomyocyte DNA synthesis (as evidenced by silver grains over non-blue nuclei). Bar = 20 µm in all panels. (B) Cardiomyocyte ³H-Thy-labelling index (%, mean ± SEM) for: (1) saline-treated NON-TXG mice, (2) G-CSF plus DipA-treated NON-TXG mice, (3) saline-treated MHC-cycD2 mice, and (4) G-CSF plus DipA-treated MHC-cycD2 mice. *P < 0.05 vs. saline/NON-TXG; ^ΨP< 0.05 vs. G-CSF plus DipA/NON-TXG.

Figure 5

Cardiomyocyte content at 60 days post-myocardial infarction. (A) Representative photomicrographs of Masson's trichrome-stained sections. Bar = 50 µm. (B) Cardiomyocyte number/infarct/section at 60 days post-myocardial infarction: (1) saline-treated NON-TXG mice, (2) G-CSF plus DipA-treated NON-TXG mice, (3) saline-treated MHC-cycD2 mice, and (4) G-CSF plus DipA-treated MHC-cycD2 mice. *P < 0.05 vs. saline/NON-TXG; ^ΨP< 0.05 vs. G-CSF plus DipA/NON-TXG; ^†P< 0.05 vs. saline/MHC-cycD2.

Figure 6

Cardiac function at 60 days post-myocardial infarction. (A) Representative echocardiograms. (B) Ejection fraction (EF%, mean ± SEM) for: (1) saline-treated NON-TXG mice, (2) G-CSF plus DipA-treated NON-TXG mice, (3) saline-treated MHC-cycD2 mice, and (4) G-CSF plus DipA-treated MHC-cycD2 mice. *P < 0.05 vs. saline/NON-TXG; ^ΨP< 0.05 vs. G-CSF plus DipA/NON-TXG; ^†P< 0.05 vs. saline/MHC-cycD2. Ejection fraction in non-infarcted, untreated NON-TXG adult mice was 87 ± 1.1.