PMC

(A) Representative bioluminescence images of a single EHM loop ex vivo (left) and post-implantation up to 85 days (right). (B,C) EHMs showed no significant decline in viability between day 14 and 85 (n=6, P=0.33). (D) Implantation of EHM onto immunocompetent rat hearts (Sprague Dawley, SD) led to almost complete signal loss within 7 days (dashed red line, n=2). However, when Tacrolimus was administered twice daily (7.5 mg/kg/day), the BLI signal was comparable to that seen in nude rats, indicating that effective immune suppression was achieved. (E) Effective immune suppression in SD rats enabled graft survival comparable to nude rats (SD+Tac n=5 vs. nude n=6; P=0.88). Average radiance expressed as 10⁶ photons/second/cm²/steradian.

(A–D) One month after implantation EHM, grafts were found covering the scar area or border zone on H&E or Masson’s trichrome (M–T) sections. EHMs were typically separated from the host by a small layer of fibrotic tissue. (E) Grafts staining positive for human beta integrin 1 (β-Integ) could be found for all EHM hearts assessed. Grafts were perfused by host vessels (CD31) that grew into the graft (maximum intensity projection of a 30 μm section). (F) Grafts consisted primarily of human CMs staining positive for beta myosin heavy chain (MyHc), but there were few cell junctions that stained positive for connexin 43 (Con43). (G) While clearly striated sarcomeres could be found (cardiac troponin T, cTnT; alpha sarcomeric actin, α-act) there were few signs of myofibril alignment. (H) EHMs showed few infiltrating macrophages (CD68) indicating stable grafts. Scale bars A,C: 5 mm; B,D: 1 mm; E,H: 100 μm; F,G: 50 μm.

(A,B) Photographs of EHM on individual stretchers for mechanical maturation and on a plate prior to force testing. (C) Fluorescence microscopy image of an EHM illustrating the distribution of cardiomyocytes (alpha actinin, α-act) across its cross-section with slightly higher concentration along the outer boundaries. (D) Two days of shipment did not change cell viability (n=3, P=0.83), cardiomyocyte content (n=3, P=0.86), or active force generation (n=3, P=0.87). (E) Cardiac-like tissue organization of cardiomyocytes (human cardiac troponin T, cTnT) was found in some areas of EHMs particular towards the outer boundaries. (F) Most of the EHM cross sections showed cardiomyocyte (β myosin heavy chain, MyHc) alignment in band-like structures along the principle strain axis with low levels of connexin 43 (Con43) expression. (G) Band-like cell arrangement is further illustrated by a maximum intensity projection of a 250 μm thick imaging volume from the center of an EHM loop. Scale bars B: 5 mm; C: 1 mm; E,F: 50 μm; G: 100 μm.

(A–C) The top row shows representative 2-chamber long-axis views at end-diastole (ED) one day prior to and 28 days after EHM implantation or sham surgery (control group). The middle row shows the same hearts at end-systole (ES) while the bottom row shows corresponding late gadolinium enhancement images (LGE) at mid-ventricular level. (D,E) End-diastolic volumes increased in all groups but were not significantly different (P=0.35). End-systolic volumes also increased in all groups, but the control group mas most pronounced compared to all other groups (control n=12 vs. EHM n=14; P=0.05). (F,G) Ejection fractions declined in all groups. The control group showed the highest relative decline compared to EHM implantation groups (P=0.03); however, there was no difference between EHMs or irradiated EHMs that did not contain viable CMs (P=0.19), indicating that beneficial effects on remodelling may not be directly mediated by grafted cells. (H,I,J) No significant difference was observed for changes in scar size over time (P=0.32). (K) There was a trend toward increase in diastolic dysfunction (E′/A′ <1) for hearts receiving irradiated EHM loops while a small decline was observed for the other groups; however, this did not reach statistical significance (P=0.12). Scale bars A–C: 5 mm.

(A,E) A high blood vessel density (CD31, maximum intensity projection of a 30 μm section) was found in human grafts (human beta 1 integrin, β-Integ) at 110 and 220 days after implantation. (B,F) Connexin (connexion 43, Con43) expression could be detected in 110 day old grafts (B), but grafts which were implanted for 220 days showed a more mature expression pattern with connexin localised at cell interfaces (F). Sarcomere alignment also increased with time in human CMs (beta myosin heavy chain, β-MyHc). (C–D) Long-term engraftment led to better sarcomere alignment and more mature sarcomeres (alpha sarcomeric actinin, α-act; cardiac troponin T, cTnT) spanning the entire width of CMs. (D,H) Only a small number of human cells showed signs of an active cell cycle (Ki67) after 110 or 220 days of engraftment. (I) Interestingly, the 220-day-old grafts contained small foci where CMs showed a glycogen-rich, hamartoma-like phenotype (H&E). (J,K) These hamartoma-like cells were of human origin (human nuclear antigen, hNA) and stained positive for smooth muscle actin (SMA) as well as cardiac markers (α-act, cTnT), with some showing organised sarcomeres. (L) None of the hamartoma-like cardiomyocytes was in an active state of the cell cycle (Ki67, negative). Scale bars A,E,I: 100 μm; B–D, F–H, J–L: 50 μm.