Mesenchymal stem cell transplantation improves regional cardiac remodeling following ovine infarction

Abstract

Progressive cardiac remodeling, including the myopathic process in the adjacent zone following myocardial infarction (MI), contributes greatly to the development of cardiac failure. Cardiomyoplasty using bone marrow-derived mesenchymal stem cells (MSCs) has been demonstrated to protect cardiomyocytes and/or repair damaged myocardium, leading to improved cardiac performance, but the therapeutic effects on cardiac remodeling are still under investigation. Here, we tested the hypothesis that MSCs could improve the pathological remodeling of the adjacent myocardium abutting the infarct. Allogeneic ovine MSCs were transplanted into the adjacent zone by intracardiac injection 4 hours after infarction. Results showed that remodeling and contractile strain alteration were reduced in the adjacent zone of the MSC-treated group. Cardiomyocyte hypertrophy was significantly attenuated with the normalization of the hypertrophy-related signaling proteins phosphatidylinositol 3-kinase α (PI3Kα), PI3Kγ, extracellular signal-regulated kinase (ERK), and phosphorylated ERK (p-ERK) in the adjacent zone of the MSC-treated group versus the MI-alone group. Moreover, the imbalance of the calcium-handling proteins sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA2a), phospholamban (PLB), and sodium/calcium exchanger type 1 (NCX-1) induced by MI was prevented by MSC transplantation, and more strikingly, the activity of SERCA2a and uptake of calcium were improved. In addition, the upregulation of the proapoptotic protein Bcl-xL/Bcl-2-associated death promoter (BAD) was normalized, as was phospho-Akt expression; there was less fibrosis, as revealed by staining for collagen; and the apoptosis of cardiomyocytes was significantly inhibited in the adjacent zone by MSC transplantation. Collectively, these data demonstrate that MSC implantation improved the remodeling in the region adjacent to the infarct after cardiac infarction in the ovine infarction model.

Figure 1.

Characterization of ovine mesenchymal stem cells (MSCs). MSCs at passage 4 were analyzed by flow cytometry for cell surface markers and by microscopy for morphology, and differentiation assays were performed. (A): Flow cytometric analysis of cell surface markers of the MSCs, showing that the MSCs displayed a high level of expression of the hyaluronan receptor CD44 and ALCAM CD166, displayed reactivity to CD105 and CD106, and lacked expression of the leukocyte marker CD45 and the hematopoietic marker CD34 (M1, gate range). (B–E): Differentiation of the MSCs (B) showed positive staining toward adipocytes (Oil Red O) (C), osteoblasts (alizarin red staining) (D), and chondrocytes (Alcian blue) (E).

Figure 2.

Effects of MSC transplantation on the remodeling and contractile strain in the adjacent and remote zones. (A): Photograph of the infarcted sheep heart on day 1 with the sonocrystals implanted. Solid arrow identifies initial left anterior descending ligation site. *, heart apex. (B): Alteration of cardiac strain was detected by the sonocrystals on the surface of the heart, showing the reduction in left ventricular remodeling strain (expansion) by MSC transplantation. (C, D): Alterations of contractile strain in the remote zone (C) and adjacent zone (D) showing the reduction of contractile strain alteration in the adjacent zone by MSC transplantation. Abbreviations: MI, myocardial infarction; MSC, mesenchymal stem cell.

Figure 3.

Attenuation of cardiomyocyte hypertrophy in the adjacent zone of the MSC-treated group. (A): Representative images of heart tissue cross-sections stained by hematoxylin and eosin in the adjacent and remote zones of the sham, MI, and MSC-treated groups. (B): Quantitative analysis of cardiomyocyte size shows that the hypertrophy of cardiomyocytes in the adjacent zone was significantly attenuated by MSC transplantation. *, p < .05 versus other groups. (C): Picrosirius red staining of heart sections for collagen deposition after 12 weeks. SR and SA represent two sections in sham animals that showed some staining. MR and MA are sections from the myocardial infarct, remote and adjacent; there was a clear increase in collagen staining in the adjacent region. MMR and MMA are sections from the remote and adjacent myocardial infarct regions in animals that received MSCs. Note that less collagen staining was found in the adjacent regions of animals that received MSCs. Abbreviations: MA, myocardial infarct adjacent; MI, myocardial infarction; MMA, MSC-treated myocardial infarct adjacent; MMR, MSC-treated myocardial infarct remote; MR, myocardial infarct remote; MSC, mesenchymal stem cell; SA, sham adjacent; SR, sham remote.

Figure 4.

Expression of hypertrophy-related signaling proteins in the adjacent and remote zones of the MI and MI+MSC groups, as well as the sham group, at 12 weeks. (A): Representative photomicrographs of Western blot, showing that the upregulation of PI3Kα, PI3Kγ, and p-ERK induced by myocardial infarction in the adjacent zone was normalized by MSC transplantation. (B–E): Quantitative analysis of PI3Kα (B), PI3Kγ (C), and p-ERK (D, E) quantified and confirmed their normalization after MSC delivery on the infarct adjacent tissue. *, p < .05. Abbreviations: ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MI, myocardial infarction; MSC, mesenchymal stem cell; p-ERK, phosphorylated extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase.

Figure 5.

Expression and activity of calcium-handling proteins in the adjacent and remote zones of the MI and MI+MSC groups at 12 weeks. (A, B): Representative photomicrographs of Western blots, showing that the upregulation of NCX-1 and downregulation of SERCA2a and PLB induced by myocardial infarction in the adjacent zone was normalized by MSC transplantation. (C–E): Quantitative analysis of SERCA2a, NCX-1, and PLB expression demonstrated normalization due to MSC delivery. (F, G): Effects of MSC transplantation on cardiac SERCA2a function, showing that the SERCA2a activity (F) and ⁴⁵Ca²⁺ uptake (G) were improved by MSC transplantation. *, p < .05. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MI, myocardial infarction; MSC, mesenchymal stem cell; NCX-1, sodium/calcium exchanger type 1; PLB, phospholamban; SERCA, sarcoplasmic reticulum Ca²⁺ adenosine triphosphatase (SERCA2a); SR, sarcoplasmic reticulum.

Figure 6.

Normalized expression of the apoptosis-related protein Bcl-xL/Bcl-2-associated death promoter (BAD) and representative photomicrographs of in situ detection of apoptotic cells using the TUNEL assay in the adjacent and remote zones of the MI and MI+MSC groups, as well as the sham group, at 12 weeks. (A, B): Representative photomicrographs of Western blot, showing that the upregulation of BAD induced by myocardial infarction in the adjacent zone was normalized by MSC transplantation. (C, D): Representative photomicrographs (×40) of the detection of apoptotic cells using the TUNEL assay, showing the significant inhibition of apoptosis in the adjacent zone by MSC transplantation. Arrows indicate TUNEL-positive apoptotic cells in the section. The expression of Akt and p-Akt was examined in the heart regions adjacent to and remote from the infarct (E, F). Although there was an increase in p-Akt in the adjacent zone of the MI animals, this either did not occur in the MI+MSC animals or was resolved by the time of sacrifice and analysis at 12 weeks. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MA, adjacent zone of the MI group; MI, myocardial infarction; MMA, adjacent zone of the MI+MSC-treated group; MMR, remote zone of the MI+MSC-treated group; MR, remote zone of the MI group (no MSCs); MSC, mesenchymal stem cell; p-Akt, phospho-Akt; PC, DNase-treated positive apoptosis control; SC, sham control; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.