Ken Suzuki, MD, PhD, Nigel J. Brand, PhD, Sean Allen, PhD, Mahboob A

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Overexpression of connexin 43 in skeletal myoblasts: Relevance to cell transplantation to the heart Ken Suzuki, MD, PhD, Nigel J. Brand, PhD, Sean Allen, PhD, Mahboob A. Khan, PhD, Aldo O. Farrell, Bari Murtuza, FRCS, Reida El Oakley, FRCS, Magdi H. Yacoub, FRS The Journal of Thoracic and Cardiovascular Surgery Volume 122, Issue 4, Pages 759-766 (October 2001) DOI: 10.1067/mtc.2001.116210 Copyright © 2001 American Association for Thoracic Surgery Terms and Conditions

Fig. 1 Diagram of gap junction and differences among cardiomyocytes, skeletal myoblasts, and skeletal myotubes. Gap junction is composed of connexons that are made of connexins, regulating intercellular passage of molecules, including inorganic ions and second messengers. Skeletal muscle cells have several important differences in cellular characteristics from cardiomyocytes. The Journal of Thoracic and Cardiovascular Surgery 2001 122, 759-766DOI: (10.1067/mtc.2001.116210) Copyright © 2001 American Association for Thoracic Surgery Terms and Conditions

Fig. 2 Cx43 overexpression. A, Six clones of L6 skeletal myoblasts genetically engineered to overexpress Cx43, named Cx43(8), Cx43(11), Cx43(15), Cx43(16), Cx43(19), Cx43(21), were generated by means of gene transfection and clonal selection. B, Control cells were transfected with pCI-neo vector only. Whereas Cx43 expression in the control cells was downregulated to a minimum level after differentiation, Cx43(15) and Cx43(19) cells continued to express significantly larger amounts of Cx43 protein throughout the period. Data are shown as relative levels of Cx43, assigning a value of 1.0 to the band density from day 1 sample of control-transfected L6 cells.*P <.05 versus control-transfected L6 cells (n = 5 in each point). Values are expressed as means ± SEM. The Journal of Thoracic and Cardiovascular Surgery 2001 122, 759-766DOI: (10.1067/mtc.2001.116210) Copyright © 2001 American Association for Thoracic Surgery Terms and Conditions

Fig. 3 Immunocytochemical staining for Cx43. A, Immunocytochemical study with anti-Cx43 monoclonal antibody demonstrated low-level Cx43 expression (shown by green fluorescence) in cultures of control myoblasts. B, In contrast, Cx43(19) displayed obvious Cx43 overexpression (green fluorescence) on the cell surfaces. Cell nuclei are stained orange by propidium iodide. Bar = 20μm. The Journal of Thoracic and Cardiovascular Surgery 2001 122, 759-766DOI: (10.1067/mtc.2001.116210) Copyright © 2001 American Association for Thoracic Surgery Terms and Conditions

Fig. 4 Intercellular communication. Scrape loading with Lucifier yellow dye demonstrated that the dye spread more quickly and widely throughout the cell monolayer up to 7 or 9 cell diameters away from the scrape cut in Cx43(19) cells (D) compared with control cells (C). A and B are pictures of reverse-phase contrast microscopic observation of the corresponding fields. Microinjection of 6-CF into single cells showed that injected dye spread to cells well beyond immediate neighbors in Cx43-transfected cells, reaching cells up to 7 cell diameters away (F), whereas dye permeated only into the immediate neighbors in control L6 cells (E).Arrows show microinjected cells with dye. Bar for A, B, C, and D = 250 μm. Bar for E and F = 20 μm. The Journal of Thoracic and Cardiovascular Surgery 2001 122, 759-766DOI: (10.1067/mtc.2001.116210) Copyright © 2001 American Association for Thoracic Surgery Terms and Conditions

Fig. 5 Myogenic differentiation and proliferation. A and B, Cells were fed with growth medium for the first 2 days and thereafter with differentiation medium. Morphologically, an enhancement of fusion and differentiation into multinucleated myotubes is evident in Cx43(19) at day 4 by comparison with control myoblasts Bar = 100μm. C, CK activity was significantly higher in Cx43(15) and Cx43(19) myoblasts than in control myoblasts. D, Cell proliferation after plating of 1% 104 cells was significantly more rapid in control cells than in Cx43(15) and Cx43(19) cells. *P <.05 versus control-transfected L6 cells (n = 5 for CK measurement and n = 6 for proliferation in each point). Values are expressed as means ± SEM. The Journal of Thoracic and Cardiovascular Surgery 2001 122, 759-766DOI: (10.1067/mtc.2001.116210) Copyright © 2001 American Association for Thoracic Surgery Terms and Conditions