Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell–polymer constructs can be cultivated using isolated cells, 3‐dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well‐mixed conditions with rat heart cells at a high initial density ((6–8) × 106 cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm2). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05). Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2–4 times higher construct cellularity (p ≤ 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape. Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1–2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2–6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 580–589, 1999.

The objective of this study was to establish a three-dimensional (3-D) in vitro model system of cardiac muscle for electrophysiological studies. Primary neonatal rat ventricular cells containing lower or higher fractions of cardiac myocytes were cultured on polymeric scaffolds in bioreactors to form regular or enriched cardiac muscle constructs, respectively. After 1 wk, all constructs contained a peripheral tissue-like region (50–70 μm thick) in which differentiated cardiac myocytes were organized in multiple layers in a 3-D configuration. Indexes of cell size (protein/DNA) and metabolic activity (tetrazolium conversion/DNA) were similar for constructs and neonatal rat ventricles. Electrophysiological studies conducted using a linear array of extracellular electrodes showed that the peripheral region of constructs exhibited relatively homogeneous electrical properties and sustained macroscopically continuous impulse propagation on a centimeter-size scale. Electrophysiological properties of enriched constructs were superior to those of regular constructs but inferior to those of native ventricles. These results demonstrate that 3-D cardiac muscle constructs can be engineered with cardiac-specific structural and electrophysiological properties and used for in vitro impulse propagation studies.

The primary aim of this study was to relate molecular and structural properties of in vitro reconstructed cardiac muscle with its electrophysiological function using an in vitro model system based on neonatal rat cardiac myocytes, three-dimensional polymeric scaffolds, and bioreactors. After 1 wk of cultivation, we found that engineered cardiac muscle contained a 120- to 160-microm-thick peripheral region with cardiac myocytes that were electrically connected through gap junctions and sustained macroscopically continuous impulse propagation over a distance of 5 mm. Molecular, structural, and electrophysiological properties were found to be interrelated and depended on specific model system parameters such as the tissue culture substrate, bioreactor, and culture medium. Native tissue and the best experimental group (engineered cardiac muscle cultivated using laminin-coated scaffolds, rotating bioreactors, and low-serum medium) were comparable with respect to the conduction velocity of propagated electrical impulses and spatial distribution of connexin43. Furthermore, the structural and electrophysiological properties of the engineered cardiac muscle, such as cellularity, conduction velocity, maximum signal amplitude, capture rate, and excitation threshold, were significantly improved compared with our previous studies.

The congenital myopathies include a wide spectrum of clinically, histologically and genetically variable neuromuscular disorders many of which are caused by mutations in genes for sarcomeric proteins. Some congenital myopathy patients have a hypercontractile phenotype. Recent functional studies demonstrated that ACTA1 K326N and TPM2 ΔK7 mutations were associated with hypercontractility that could be explained by increased myofibrillar Ca(2+) sensitivity. A recent structure of the complex of actin and tropomyosin in the relaxed state showed that both these mutations are located in the actin-tropomyosin interface. Tropomyosin is an elongated molecule with a 7-fold repeated motif of around 40 amino acids corresponding to the 7 actin monomers it interacts with. Actin binds to tropomyosin electrostatically at two points, through Asp25 and through a cluster of amino acids that includes Lys326, mutated in the gain-of-function mutation. Asp25 interacts with tropomyosin K6, next to K7 that was mutated in the other gain-of-function mutation. We identified four tropomyosin motifs interacting with Asp25 (K6-K7, K48-K49, R90-R91 and R167-K168) and three E-E/D-K/R motifs interacting with Lys326 (E139, E181 and E218), and we predicted that the known skeletal myopathy mutations ΔK7, ΔK49, R91G, ΔE139, K168E and E181K would cause a gain of function. Tests by an in vitro motility assay confirmed that these mutations increased Ca(2+) sensitivity, while mutations not in these motifs (R167H, R244G) decreased Ca(2+) sensitivity. The work reported here explains the molecular mechanism for 6 out of 49 known disease-causing mutations in the TPM2 and TPM3 genes, derived from structural data of the actin-tropomyosin interface.

We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca2+-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca2+-sensitivity when compared with donor heart troponin and the Ca2+-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca2+-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca2+-sensitivity and was also completely uncoupled. Five TnT mutations (Δ14, Δ28 + 7, ΔE160, S179F and K273E) studied in recombinant troponin increased Ca2+-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca2+-sensitisation together with uncoupling in vitro is the usual response and both factors may contribute to the HCM phenotype. We also found that Epigallocatechin-3-gallate (EGCG) can restore coupling to all uncoupled HCM-causing TnT mutations. In fact the combination of Ca2+-desensitisation and re-coupling due to EGCG completely reverses both the abnormalities found in troponin with a TnT HCM mutation suggesting it may have therapeutic potential.