Heme oxygenase-1 induction enhances cell survival and restores contractility to unvascularized three-dimensional adult cardiomyocyte grafts implanted in vivo

Abstract

Autologous adult cardiomyocytes are not utilized for heart repair strategies because of their rapid apoptosis after implantation. We examined whether induction of heme oxygenase-1 (HO-1), a mediator of preconditioning, could enhance early postimplant myocyte survival. Three-dimensional 5×5 mm patches of full-thickness adult murine atrial wall, including cardiomyocytes, capillary networks, and extracellular matrix, were cultured with or without HO-1 inducer cobalt protoporphyrin (CoPP), or the HO-1 inhibitor, tin protoporphyrin (SnPP), or both. Patches were then implanted subcutaneously. Freshly procured atrial wall patches implanted without preculturing served as additional controls. By 14 days postimplant, graft cardiomyocyte content was significantly greater in CoPP-treated patches than in either control group (p<0.02). Adult cardiomyocytes did not contract in culture or immediately after implantation. However, by 14 days postimplant, spontaneous contraction had recovered in 47% of CoPP-treated patches, but in only 6% of precultured patches without CoPP, 0% of SnPP-treated patches, and 0% of uncultured patches (p<0.03). CoPP-treated adult cardiomyocyte patches were also observed to remodel spontaneously into endothelial-lined chambers that pumped nonclotting blood. These findings demonstrate that adult cardiomyocytes have more plasticity and capacity for functional recovery than previously recognized and could have application as an autologous cardiomyocyte source for tissue engineering.

FIG. 1.

Western blot and IHC of atrial appendage wall after 3 days of in vitro organ culture. Adding CoPP to the culture medium on day 0 induces HO-1 protein production. Inhibitor SnPP does not inhibit HO-1 protein production (but instead sterically inhibits HO-1 enzymatic activity). Culturing full-thickness atrial wall in the absence of CoPP results in low levels of endogenous HO-1 production, whereas no HO-1 is seen in freshly procured atrial wall that has not been cultured. On IHC, CoPP treatment results in widespread HO-1 expression (brown DAB staining) in cardiomyocytes, endothelial cells, endocardium, and epicardium. Atrial walls cultured without CoPP develop low levels (lighter staining) of HO-1 in cardiomyocytes, with patchy areas of darker staining in central cardiomyocytes. No HO-1 staining is seen in freshly procured, uncultured atrial tissue. Magnification bars=100 μm. IHC, immunohistochemistry; CoPP, cobalt protoporphyrin; SnPP, tin protoporphyrin; HO-1, heme oxygenase-1.

FIG. 2.

Cardiomyocyte volume in atrial grafts at 14 days after in vivo implantation in different pretreatment groups. *p<0.001 versus the two control groups, analysis of variance. ANP, atrial natriuretic peptide.

FIG. 3.

Examples of subcutaneous atrial grafts in vivo at 14 days postimplant, with IHC for ANP (brown DAB staining) to demonstrate cardiomyocyte content. Black dotted lines indicate graft margins, but are not those actually used to calculate graft volumes. (A) Graft pretreated with CoPP prior to implantation; (B) graft precultured prior to implantation without CoPP; (C) graft implanted with neither preculture nor CoPP treatment. Magnification bars=200 μM. Color images available online at www.liebertonline.com/tea

FIG. 4.

Chamber formation seen in cross-sections of an atrial graft at 14 days after subcutaneous implantation. Atrial cardiomyocytes are stained by antibodies to both sarcomeric myosin (mAb MF-20) and ANP. Endothelial-lined lumens (L) are filled with red blood cells without clot formation. Numerous smaller vessels, also filled with red blood cells, are seen in the chamber walls (V). Magnification bars=100 μm. Color images available online at www.liebertonline.com/tea

FIG. 5.

Troponin T staining at 7 days after in vivo implantation. On IHC, troponin T staining is seen in some CoPP-treated patches at 7 days (A) and early sarcomeric structures are occasionally seen (B), whereas no troponin T is seen at 7 days in patches without CoPP pretreatment (C).