Layered implantation of myoblast sheets attenuates adverse cardiac remodeling of the infarcted heart Naosumi Sekiya, MD, Goro Matsumiya, MD, PhD, Shigeru.

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Layered implantation of myoblast sheets attenuates adverse cardiac remodeling of the infarcted heart Naosumi Sekiya, MD, Goro Matsumiya, MD, PhD, Shigeru Miyagawa, MD, PhD, Atsuhiro Saito, PhD, Tatsuya Shimizu, MD, PhD, Teruo Okano, PhD, Naomasa Kawaguchi, PhD, Nariaki Matsuura, MD, PhD, Yoshiki Sawa, MD, PhD The Journal of Thoracic and Cardiovascular Surgery Volume 138, Issue 4, Pages 985-993 (October 2009) DOI: 10.1016/j.jtcvs.2009.02.004 Copyright © 2009 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Cultured myoblasts and cross section of a sheet. Almost all cultured cells were actin-positive (A), and roughly 40% to 50% of them were desmin-positive muscle lineage cells (B). DAPI (4,6-diamino-2-phenylindole) staining of nuclei (C), and merged image (D) (A–D; magnification ×10, bar = 100 μm). Toluidine blue staining of cross section of sheet-constructed cells (E) (bar = 100 μm). The Journal of Thoracic and Cardiovascular Surgery 2009 138, 985-993DOI: (10.1016/j.jtcvs.2009.02.004) Copyright © 2009 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Cardiac function after layered myoblast sheet implantation. Echocardiographic measurements of rat heart (A–C). Baseline represents 2 weeks after left anterior descending coronary artery ligation. The pressure-volume study was performed at 8 weeks after treatment to estimate the systolic function of rat heart (D). ∗P < .05 versus sham group. †P < .05 versus S1 group. #P < .05 versus S3 group. EF, Ejection fraction; FS, fractional shortening; EDA, end-diastolic area; ESPVR, end-systolic pressure-volume relationship. The Journal of Thoracic and Cardiovascular Surgery 2009 138, 985-993DOI: (10.1016/j.jtcvs.2009.02.004) Copyright © 2009 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Histologic findings of implanted host hearts. Macroscopic (×20) view of anterior wall of hearts (hematoxylin and eosin stain): sham, A; S1, B; S3, C; S5, D (bar = 200 μm). Sections of infarcted regions were stained with antibody to von Willebrand factor (factor VIII): sham, E; S1l F; S3, G; S5, H (bar = 100 μm). Sirius-red staining of myocardium of noninfarcted regions: sham, I; S1, J; S3, K; S5, L (bar = 100 μm). Periodic acid–Schiff–stained myocardium of noninfarcted regions: sham, M; S1, N; S3, O; S5, P (bar = 50 μm). The Journal of Thoracic and Cardiovascular Surgery 2009 138, 985-993DOI: (10.1016/j.jtcvs.2009.02.004) Copyright © 2009 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Quantitative assessment of histologic findings. A, Assessment of anterior wall thickness after implantation. B, Assessment of vascular density in the infarcted area of host hearts vascular density. C, Quantitative results for fibrotic change in the left ventricle. D, Cardiomyocyte short-axis diameters were measured as described in Methods (n = 5 for each group: A–D, 10 randomly chosen fields per section: B–D). ∗P < .05 versus sham group. †P < .05 versus S1 group. #P <. 05 versus S3 group. The Journal of Thoracic and Cardiovascular Surgery 2009 138, 985-993DOI: (10.1016/j.jtcvs.2009.02.004) Copyright © 2009 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 Relative expression of growth factors (A–C). Relative levels of mRNA transcripts were measured by the real-time quantitative RT-PCR (n = 10 for each of the four groups, S1, S3, S5, and sham). The average copy number of gene transcripts in each sample was normalized to that for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).∗P < .05 versus sham group. †P < .05 versus S1 group. HGF, Hepatocyte growth factor; VEGF, vascular endothelial growth factor; SDF-1, stromal-derived factor-1. The Journal of Thoracic and Cardiovascular Surgery 2009 138, 985-993DOI: (10.1016/j.jtcvs.2009.02.004) Copyright © 2009 The American Association for Thoracic Surgery Terms and Conditions

Figure 6 Elastic fibers were constructed after implantation, with expression of mRNA. Elastic fibers were increased in sheets and host infarcted area in the S5 and S3 groups (S5, A and B; S3, C and D). There were few elastic fibers in the S1 and sham groups (S1, E; sham, F). Relative expression of mRNA of tropoelastin (G). ∗P < .05 versus sham group. †P < .05 versus S1 group. The Journal of Thoracic and Cardiovascular Surgery 2009 138, 985-993DOI: (10.1016/j.jtcvs.2009.02.004) Copyright © 2009 The American Association for Thoracic Surgery Terms and Conditions