Towards a Tissue-Engineered Contractile Fontan-Conduit: The Fate of Cardiac Myocytes in the Subpulmonary Circulation

Abstract

The long-term outcome of patients with single ventricles improved over time, but remains poor compared to other congenital heart lesions with biventricular circulation. Main cause for this unfavourable outcome is the unphysiological hemodynamic of the Fontan circulation, such as subnormal systemic cardiac output and increased systemic-venous pressure. To overcome this limitation, we are developing the concept of a contractile extracardiac Fontan-tunnel. In this study, we evaluated the survival and structural development of a tissue-engineered conduit under in vivo conditions. Engineered heart tissue was generated from ventricular heart cells of neonatal Wistar rats, fibrinogen and thrombin. Engineered heart tissues started beating around day 8 in vitro and remained contractile in vivo throughout the experiment. After culture for 14 days constructs were implanted around the right superior vena cava of Wistar rats (n = 12). Animals were euthanized after 7, 14, 28 and 56 days postoperatively. Hematoxylin and eosin staining showed cardiomyocytes arranged in thick bundles within the engineered heart tissue-conduit. Immunostaining of sarcomeric actin, alpha-actin and connexin 43 revealed a well -developed cardiac myocyte structure. Magnetic resonance imaging (d14, n = 3) revealed no constriction or stenosis of the superior vena cava by the constructs. Engineered heart tissues survive and contract for extended periods after implantation around the superior vena cava of rats. Generation of larger constructs is warranted to evaluate functional benefits of a contractile Fontan-conduit.

Fig 1. The concept of a subpulmonary „neo-ventricle”from engineered heart tissue.

A: Extracardiac tunnel for the treatment of children with univentricular hearts. Commonly, a non-contractile GoreTex-conduit is used to bypass blood from the inferior caval vein to the right pulmonary artery. B: Our group aims for a contractile, valved conduit made from engineered heart tissue (EHT) to propel blood actively through the lungs and avoid endorgan damage in the long-term.

Fig 2. Generation of a simple contractile conduit from EHT.

The illustration shows the construction of a simple „neo-ventricle”out of engineered heart tissue. Two lamellar EHTs were wrapped around the right SVC of adult Wistar rats. The ends were sutured using a single stitch of nylon suture.

Fig 3. Hematoxylin and eosin (HE) staining of “neo-ventricle” 56 days after implantation around the superior caval vein (SVC) of rats.

A: Engineered heart tissue organized around the SVC. Thick bundles of cross-striated cardiomycytes are shown above the smooth muscle of the native vessel wall. The arrow indicates a suture with surrounding lymphocytic infiltration. B: Magnification of detail in left panel. Cross-striation can be seen in the highly organized cardiomyocytes within the contractile conduit. The arrow indicates red blood cells in a capillary. Scale bar: 50 μm.

Fig 4. Cardiac morphology of EHT-conduit 28 days after grafting.

A+D: Connexin 43 (red), an important gap junction protein indicating functional syncytium of the grafted cells is shown. Scale bar: 20 μm. B+E: CD31 staining (green) revealed dense vascularization. Vessels displayed a small diameter compatible with capillaries. Alpha-actin staining (red) showed dense bundles of cross-striated cardiomyocytes around the SVC. C+F: DAPI (blue) labeling of cells before implantation allowed for clear identification of grafted cells after harvest of the specimen. Scale bar: 50 μm. G: Overview of depicted (A-F) sarcomeric details showing the circumferential alignment of the „neo-ventricle“. Scale bar: 100 μm.

Fig 5. Sarcomeric integrity of implanted EHTs after 14 and 28 days in vivo.

A+B: Cross-striation of cardiomyocytes in DAPI-positive areas is shown. Scale bar: 20 μm.

Fig 6. Cine MRI 14 days after implantation of EHTs around the SVC of rats.

A: Approximately 1 mm above the right atrium two stripes of EHT were implanted around the SVC (box). No stenosis or constriction of the vein could be detected in the treatment group. B: The box shows the SVC of a Sham operated animal 14 days after the procedure. Scale bars: 1 cm.