J Clin Invest.
Retention and functional integration of PCMs after intramyocardial injection.
(A and B) Immunofluorescent labeling of α-actinin (red, A) and connexin43 (red, B) in adult ventricular mouse heart tissue 3 weeks after injection of PCMs (EGFP, green; nuclei, blue). (C–F) Two-photon laser scanning microscopy of intracellular Ca2+ transients in adult mouse hearts after injection of PCMs: 2D-scan (C) and line-scan (D) images of stimulated (3 Hz) and spontaneous Ca2+ transients. Arrow 1, EGFP-positive cell; arrow 2, EGFP-negative cell; the dotted line indicates the location of the line scan. Bands of increased rhod-2 fluorescence intensity reflect AP-induced Ca2+ transients. (E) Plots of rhod-2 and GFP line-scan data in the EGFP-expressing cardiomyocyte 1 and the GFP-negative (native) cardiomyocyte 2 as a function of time. (F) Superimposed tracings of AP-evoked changes in rhod-2 fluorescence as a function of time from cardiomyocytes 1 (green) and 2 (red). For each cell, the relative changes in fluorescence were normalized such that 0 represents the prestimulus fluorescence intensity (F0), and 1 represents the peak fluorescence intensity. Additional immunofluorescence analyses confirmed the maturity of engrafted PCMs: A3 line; EGFP-positive; (G) α-actinin (red); (H) actin (red); (I) troponin (red); nuclei (blue; DAPI). Scale bars: (A) 100 μm, (B, C, G–I) 20 μm.