Human-Engineered Atrial Tissue for Studying Atrial Fibrillation.

This chapter details the generation of atrial fibrin-based engineered heart tissue (EHT) in standard 24-well format as a 3D model for the human atrium. Compared to 2D cultivation, human-induced pluripotent stem cells (hiPSCs)-derived atrial cardiomyocytes demonstrated a higher degree of maturation in 3D format. Furthermore, we have demonstrated in previous work that the model displayed atrial characteristics in terms of contraction and gene expression patterns, electrophysiology, and pharmacological response. Here, we describe how to embed atrial cardiomyocytes differentiated from hiPSCs in a fibrin hydrogel to form atrial EHT attached to elastic silicone posts, allowing auxotonic contraction. In addition, we describe how force and other contractility parameters can be derived from these beating atrial EHTs by video-optical monitoring. The presented atrial EHT model is suitable to study chamber-specific mechanisms, drug effects and to serve for disease modeling.

Correction: Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue.

[This corrects the article DOI: 10.1371/journal.pone.0145937.].

Blinded Contractility Analysis in hiPSC-Cardiomyocytes in Engineered Heart Tissue Format: Comparison With Human Atrial Trabeculae.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) may serve as a new assay for drug testing in a human context, but their validity particularly for the evaluation of inotropic drug effects remains unclear. In this blinded analysis, we compared the effects of 10 indicator compounds with known inotropic effects in electrically stimulated (1.5 Hz) hiPSC-CM-derived 3-dimensional engineered heart tissue (EHT) and human atrial trabeculae (hAT). Human EHTs were prepared from iCell hiPSC-CM, hAT obtained at routine heart surgery. Mean intra-batch variation coefficient in baseline force measurement was 17% for EHT and 49% for hAT. The PDE-inhibitor milrinone did not affect EHT contraction force, but increased force in hAT. Citalopram (selective serotonin reuptake inhibitor), nifedipine (LTCC-blocker) and lidocaine (Na+ channel-blocker) had negative inotropic effects on EHT and hAT. Formoterol (beta-2 agonist) had positive lusitropic but no inotropic effect in EHT, and positive clinotropic, lusitropic, and inotropic effects in hAT. Tacrolimus (calcineurin-inhibitor) had a negative inotropic effect in EHTs, but no effect in hAT. Digoxin (Na+-K+-ATPase-inhibitor) showed a positive inotropic effect only in EHTs, but no effect in hAT probably due to short incubation time. Ryanodine (ryanodine receptor-inhibitor) reduced contraction force in both models. Rolipram and acetylsalicylic acid showed noninterpretable results in hAT. Contraction amplitude and kinetics were more stable over time and less variable in hiPSC-EHTs than hAT. HiPSC-EHT faithfully detected cAMP-dependent and -independent positive and negative inotropic effects, but limited beta-2 adrenergic or PDE3 effects, compatible with an immature CM phenotype.

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

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.

Human Engineered Heart Tissue: Analysis of Contractile Force.

Analyzing contractile force, the most important and best understood function of cardiomyocytes in vivo is not established in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). This study describes the generation of 3D, strip-format, force-generating engineered heart tissues (EHT) from hiPSC-CM and their physiological and pharmacological properties. CM were differentiated from hiPSC by a growth factor-based three-stage protocol. EHTs were generated and analyzed histologically and functionally. HiPSC-CM in EHTs showed well-developed sarcomeric organization and alignment, and frequent mitochondria. Systematic contractility analysis (26 concentration-response curves) reveals that EHTs replicated canonical response to physiological and pharmacological regulators of inotropy, membrane- and calcium-clock mediators of pacemaking, modulators of ion-channel currents, and proarrhythmic compounds with unprecedented precision. The analysis demonstrates a high degree of similarity between hiPSC-CM in EHT format and native human heart tissue, indicating that human EHTs are useful for preclinical drug testing and disease modeling.

Comparison of the effects of a truncating and a missense MYBPC3 mutation on contractile parameters of engineered heart tissue.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. The most frequently mutated gene is MYBPC3, encoding cardiac myosin-binding protein-C (cMyBP-C). We compared the pathomechanisms of a truncating mutation (c.2373_2374insG) and a missense mutation (c.1591G>C) in MYBPC3 in engineered heart tissue (EHT). EHTs enable to study the direct effects of mutants without interference of secondary disease-related changes. EHTs were generated from Mybpc3-targeted knock-out (KO) and wild-type (WT) mouse cardiac cells. MYBPC3 WT and mutants were expressed in KO EHTs via adeno-associated virus. KO EHTs displayed higher maximal force and sensitivity to external [Ca(2+)] than WT EHTs. Expression of WT-Mybpc3 at MOI-100 resulted in ~73% cMyBP-C level but did not prevent the KO phenotype, whereas MOI-300 resulted in ≥95% cMyBP-C level and prevented the KO phenotype. Expression of the truncating or missense mutation (MOI-300) or their combination with WT (MOI-150 each), mimicking the homozygous or heterozygous disease state, respectively, failed to restore force to WT level. Immunofluorescence analysis revealed correct incorporation of WT and missense, but not of truncated cMyBP-C in the sarcomere. In conclusion, this study provides evidence in KO EHTs that i) haploinsufficiency affects EHT contractile function if WT cMyBP-C protein levels are ≤73%, ii) missense or truncating mutations, but not WT do not fully restore the disease phenotype and have different pathogenic mechanisms, e.g. sarcomere poisoning for the missense mutation, iii) the direct impact of (newly identified) MYBPC3 gene variants can be evaluated.

Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue.

INTRODUCTION: Left ventricular dysfunction is a frequent and potentially severe side effect of many tyrosine kinase inhibitors (TKI). The mode of toxicity is not identified, but may include impairment of mitochondrial or sarcomeric function, autophagy or angiogenesis, either as an on-target or off-target mechanism. METHODS AND RESULTS: We studied concentration-response curves and time courses for nine TKIs in three-dimensional, force generating engineered heart tissue (EHT) from neonatal rat heart cells. We detected a concentration- and time-dependent decline in contractile force for gefitinib, lapatinib, sunitinib, imatinib, sorafenib, vandetanib and lestaurtinib and no decline in contractile force for erlotinib and dasatinib after 96 hours of incubation. The decline in contractile force was associated with an impairment of autophagy (LC3 Western blot) and appearance of autophagolysosomes (transmission electron microscopy). CONCLUSION: This study demonstrates the feasibility to study TKI-mediated force effects in EHTs and identifies an association between a decline in contractility and inhibition of autophagic flux.

Human engineered heart tissue as a model system for drug testing.

Drug development is time- and cost-intensive and, despite extensive efforts, still hampered by the limited value of current preclinical test systems to predict side effects, including proarrhythmic and cardiotoxic effects in clinical practice. Part of the problem may be related to species-dependent differences in cardiomyocyte biology. Therefore, the event of readily available human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) has raised hopes that this human test bed could improve preclinical safety pharmacology as well as drug discovery approaches. However, hiPSC-CM are immature and exhibit peculiarities in terms of ion channel function, gene expression, structural organization and functional responses to drugs that limit their present usefulness. Current efforts are thus directed towards improving hiPSC-CM maturity and high-content readouts. Culturing hiPSC-CM as 3-dimensional engineered heart tissue (EHT) improves CM maturity and anisotropy and, in a 24-well format using silicone racks, enables automated, multiplexed high content readout of contractile function. This review summarizes the principal technology and focuses on advantages and disadvantages of this technology and its potential for preclinical drug screening.

Immunobiology of fibrin-based engineered heart tissue.

UNLABELLED: Different tissue-engineering approaches have been developed to induce and promote cardiac regeneration; however, the impact of the immune system and its responses to the various scaffold components of the engineered grafts remains unclear. Fibrin-based engineered heart tissue (EHT) was generated from neonatal Lewis (Lew) rat heart cells and transplanted onto the left ventricular surface of three different rat strains: syngeneic Lew, allogeneic Brown Norway, and immunodeficient Rowett Nude rats. Interferon spot frequency assay results showed similar degrees of systemic immune activation in the syngeneic and allogeneic groups, whereas no systemic immune response was detectable in the immunodeficient group (p < .001 vs. syngeneic and allogeneic). Histological analysis revealed much higher local infiltration of CD3- and CD68-positive cells in syngeneic and allogeneic rats than in immunodeficient animals. Enzyme-linked immunospot and immunofluorescence experiments revealed matrix-directed TH1-based rejection in syngeneic recipients without collateral impairment of heart cell survival. Bioluminescence imaging was used for in vivo longitudinal monitoring of transplanted luciferase-positive EHT constructs. Survival was documented in syngeneic and immunodeficient recipients for a period of up to 110 days after transplant, whereas in the allogeneic setting, graft survival was limited to only 14 ± 1 days. EHT strategies using autologous cells are promising approaches for cardiac repair applications. Although fibrin-based scaffold components elicited an immune response in our studies, syngeneic cells carried in the EHT were relatively unaffected. SIGNIFICANCE: An initial insight into immunological consequences after transplantation of engineered heart tissue was gained through this study. Most important, this study was able to demonstrate cell survival despite rejection of matrix components. Generation of syngeneic human engineered heart tissue, possibly using human induced pluripotent stem cell technology with subsequent directed rejection of matrix components, may be a potential future approach to replace diseased myocardium.

Effects of proarrhythmic drugs on relaxation time and beating pattern in rat engineered heart tissue.

The assessment of proarrhythmic risks of drugs remains challenging. To evaluate the suitability of rat engineered heart tissue (EHT) for detecting proarrhythmic effects. We monitored drug effects on spontaneous contractile activity and, in selected cases, on action potentials (sharp microelectrode) and Ca2+ transients (Fura-2) and contraction under electrical pacing. The Ito-blocker inhibitor 4-aminopyridine increased action potential duration and T2 and caused aftercontractions, which were abolished by inhibitors of ryanodine receptors (RyR2; JTV-519) or sodium calcium exchanger (NCX; SEA0400). 77 Drugs were then tested at 1-10-100× free therapeutic plasma concentrations (FTPC): Inhibitors of IKr, IKs, Ito, antiarrhythmics (8), drugs withdrawn from market for torsades des pointes arrhythmias (TdP, 5), drugs with measurable (7) or isolated TdP incidence (13), drugs considered safe (14), 28 new chemical entities (NCE). Inhibitors of IKr or IKs had no effect alone, but substantially prolonged relaxation time (T2) when combined at high concentration. 15/33 drugs associated with TdP and 6/14 drugs considered non-torsadogenic (cibenzoline, diltiazem, ebastine, ketoconazole, moxifloxacin, and phenytoin) induced concentration-dependent T2 prolongations (10-100× FTPC). Bepridil, desipramine, imipramine, thioridazine, and erythromycin induced irregular beating. Three NCE prolonged T2, one reduced force. Drugs inhibiting repolarization prolong relaxation in rat EHTs and cause aftercontractions involving RyR2 and NCX. Insensitivity to IKr inhibitors makes rat EHTs unsuitable as general proarrhythmia screen, but favors detection of effects on Ito, IKs + Ito or IKs + IKr. Screening a large panel of drugs suggests that effects on these currents, in addition to IKr, are more common than anticipated.

Generation of strip-format fibrin-based engineered heart tissue (EHT).

This protocol describes a method for casting fibrin-based engineered heart tissue (EHT) in standard 24-well culture dishes. In principle, a hydrogel tissue engineering method requires cardiomyocytes, a liquid matrix that forms a gel, a casting mold, and a device that keeps the developing tissue in place. This protocol refers to neonatal rat heart cells as the cell source; the matrix of choice is fibrin, and the tissues are generated in rectangular agarose-casting molds (12 × 3 × 3 mm) prepared in standard 24-well cell culture dishes, in which a pair of flexible silicone posts is suspended from above. A master mix of freshly isolated cells, medium, fibrinogen, and thrombin is pipetted into the casting mold and, over a period of 2 h, polymerizes and forms a fibrin cell block around two silicone posts. Silicone racks holding four pairs of silicone posts each are used to transfer the fresh fibrin cell blocks into new 24-well dishes with culture medium. Without further handling, the cells start to remodel the fibrin gel, form contacts with each other, elongate, and condense the gel to approximately » of the initial volume. Spontaneous and rhythmic contractions start after 1 week. EHTs are viable and relatively stable for several weeks in this format and can be subjected to repeated measurements of contractile function and final morphological and molecular analyses.

Functional improvement and maturation of rat and human engineered heart tissue by chronic electrical stimulation.

Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT.

Automated analysis of contractile force and Ca2+ transients in engineered heart tissue.

Contraction and relaxation are fundamental aspects of cardiomyocyte functional biology. They reflect the response of the contractile machinery to the systolic increase and diastolic decrease of the cytoplasmic Ca(2+) concentration. The analysis of contractile function and Ca(2+) transients is therefore important to discriminate between myofilament responsiveness and changes in Ca(2+) homeostasis. This article describes an automated technology to perform sequential analysis of contractile force and Ca(2+) transients in up to 11 strip-format, fibrin-based rat, mouse, and human fura-2-loaded engineered heart tissues (EHTs) under perfusion and electrical stimulation. Measurements in EHTs under increasing concentrations of extracellular Ca(2+) and responses to isoprenaline and carbachol demonstrate that EHTs recapitulate basic principles of heart tissue functional biology. Ca(2+) concentration-response curves in rat, mouse, and human EHTs indicated different maximal twitch forces (0.22, 0.05, and 0.08 mN in rat, mouse, and human, respectively; P < 0.001) and different sensitivity to external Ca(2+) (EC50: 0.15, 0.39, and 1.05 mM Ca(2+) in rat, mouse, and human, respectively; P < 0.001) in the three groups. In contrast, no difference in myofilament Ca(2+) sensitivity was detected between skinned rat and human EHTs, suggesting that the difference in sensitivity to external Ca(2+) concentration is due to changes in Ca(2+) handling proteins. Finally, this study confirms that fura-2 has Ca(2+) buffering effects and is thereby changing the force response to extracellular Ca(2+).

Paradoxical effects on force generation after efficient ß1-adrenoceptor knockdown in reconstituted heart tissue.

Stimulation of myocardial ß(1)-adrenoceptors (AR) is a major mechanism that increases cardiac function. We investigated the functional consequences of genetic ß(1)-AR knockdown in three-dimensional engineered heart tissue (EHT). For ß(1)-AR knockdown, short interfering RNA (siRNA) sequences targeting specifically the ß(1)-AR (shB1) and a scrambled control (shCTR) were subcloned into a recombinant adeno-associated virus (AAV)-short hairpin RNA (shRNA) expression system. Transduction efficiency was ∼100%, and radioligand binding revealed 70% lower ß(1)-AR density in AAV6-shB1-transduced EHTs. Force measurements, performed over the culture period of 14 days, showed paradoxically higher force generation in AAV6-shB1 compared with shCTR under basal (0.19 ± 0.01 versus 0.13 ± 0.01 mN) and after ß-AR-stimulated conditions with isoprenaline (Δfractional shortening: 72 ± 5% versus 34 ± 4%). Large scale gene expression analysis revealed that AAV6-shCTR compared with nontransduced EHTs showed only few differentially regulated genes (<20), whereas AAV6-shB1 induced marked changes in gene expression (>250 genes), indicating that ß(1)-AR knockdown itself determines the outcome. None of the regulated genes pointed to obvious off-target effects to explain higher force generation. Moreover, compensational regulation of ß(2)-AR signaling or changes in prominent ß(1)-AR downstream targets could be ruled out. In summary, we show paradoxically higher force generation and isoprenaline responses after efficient ß(1)-AR knockdown in EHTs. Our findings 1) reveal an unexpected layer of complexity in gene regulation after specific ß(1)-AR knockdown rather than unspecific dysregulations through transcriptional interference, 2) challenge classic assumptions on the role of cardiac ß(1)-AR, and 3) may open up new avenues for ß-AR loss-of-function research in vivo.

In vitro perfusion of engineered heart tissue through endothelialized channels.

In engineered heart tissues (EHT), oxygen and nutrient supply via mere diffusion is a likely factor limiting the thickness of cardiac muscle strands. Here, we report on a novel method to in vitro perfuse EHT through tubular channels. Adapting our previously published protocols, we expanded a miniaturized fibrin-based EHT-format to a larger six-well format with six flexible silicone posts holding each EHT (15×25×3 mm³). Thin dry alginate fibers (17×0.04×0.04 mm) were embedded into the cell-fibrin-thrombin mix and, after fibrin polymerization, dissolved by incubation in alginate lyase or sodium citrate. Oxygen concentrations were measured with a microsensor in 14-day-old EHTs (37°C, 21% oxygen) and ranged between 9% at the edges and 2% in the center of the tissue. Perfusion rapidly increased it to 10%-12% in the immediate vicinity of the microchannel. Continuous perfusion (20 µL/h, for 3 weeks) of the tubular lumina (100-500 µm) via hollow posts of the silicone rack increased mean dystrophin-positive cardiomyocyte density (36%±6% vs. 10%±3% of total cell number) and cross sectional area (73±2 vs. 48±1 µm²) in the central part of the tissue compared to nonperfused EHTs. The channels were populated by endothelial cells present in the reconstitution cell mix. In conclusion, we developed a novel approach to generate small tubular structures suitable for perfusion of spontaneously contracting and force-generating EHTs and showed that prolonged perfusion improved cardiac tissue structure.

Contractile abnormalities and altered drug response in engineered heart tissue from Mybpc3-targeted knock-in mice.

Myosin-binding protein C (Mybpc3)-targeted knock-in mice (KI) recapitulate typical aspects of human hypertrophic cardiomyopathy. We evaluated whether these functional alterations can be reproduced in engineered heart tissue (EHT) and yield novel mechanistic information on the function of cMyBP-C. EHTs were generated from cardiac cells of neonatal KI, heterozygous (HET) or wild-type controls (WT) and developed without apparent morphological differences. KI had 70% and HET 20% lower total cMyBP-C levels than WT, accompanied by elevated fetal gene expression. Under standard culture conditions and spontaneous beating, KI EHTs showed more frequent burst beating than WT and occasional tetanic contractions (14/96). Under electrical stimulation (6Hz, 37°C) KI EHTs exhibited shorter contraction and relaxation times and a twofold higher sensitivity to external [Ca(2+)]. Accordingly, the sensitivity to verapamil was 4-fold lower and the response to isoprenaline or the Ca(2+) sensitizer EMD 57033 2- to 4-fold smaller. The loss of EMD effect was verified in 6-week-old KI mice in vivo. HET EHTs were apparently normal under basal conditions, but showed similarly altered contractile responses to [Ca(2+)], verapamil, isoprenaline and EMD. In contrast, drug-induced changes in intracellular Ca(2+) transients (Fura-2) were essentially normal. In conclusion, the present findings in auxotonically contracting EHTs support the idea that cMyBP-C's normal role is to suppress force generation at low intracellular Ca(2+) and stabilize the power-stroke step of the cross bridge cycle. Pharmacological testing in EHT unmasked a disease phenotype in HET. The altered drug response may be clinically relevant.

Impact of ANKRD1 mutations associated with hypertrophic cardiomyopathy on contraction parameters of engineered heart tissue.

Hypertrophic cardiomyopathy (HCM) is a myocardial disease associated with mutations in sarcomeric genes. Three mutations were found in ANKRD1, encoding ankyrin repeat domain 1 (ANKRD1), a transcriptional co-factor located in the sarcomere. In the present study, we investigated whether expression of HCM-associated ANKRD1 mutations affects contraction parameters after gene transfer in engineered heart tissues (EHTs). EHTs were generated from neonatal rat heart cells and were transduced with adeno-associated virus encoding GFP or myc-tagged wild-type (WT) or mutant (P52A, T123M, or I280V) ANKRD1. Contraction parameters were analyzed from day 8 to day 16 of culture, and evaluated in the absence or presence of the proteasome inhibitor epoxomicin for 24 h. Under standard conditions, only WT- and T123M-ANKRD1 were correctly incorporated in the sarcomere. T123M-ANKRD1-transduced EHTs exhibited higher force and velocities of contraction and relaxation than WT- P52A- and I280V-ANKRD1 were highly unstable, not incorporated into the sarcomere, and did not induce contractile alterations. After epoxomicin treatment, P52A and I280V were both stabilized and incorporated into the sarcomere. I280V-transduced EHTs showed prolonged relaxation. These data suggest different impacts of ANKRD1 mutations on cardiomyocyte function: gain-of-function for T123M mutation under all conditions and dominant-negative effect for the I280V mutation which may come into play only when the proteasome is impaired.

Increased afterload induces pathological cardiac hypertrophy: a new in vitro model.

Increased afterload results in 'pathological' cardiac hypertrophy, the most important risk factor for the development of heart failure. Current in vitro models fall short in deciphering the mechanisms of hypertrophy induced by afterload enhancement. The aim of this study was to develop an experimental model that allows investigating the impact of afterload enhancement (AE) on work-performing heart muscles in vitro. Fibrin-based engineered heart tissue (EHT) was cast between two hollow elastic silicone posts in a 24-well cell culture format. After 2 weeks, the posts were reinforced with metal braces, which markedly increased afterload of the spontaneously beating EHTs. Serum-free, triiodothyronine-, and hydrocortisone-supplemented medium conditions were established to prevent undefined serum effects. Control EHTs were handled identically without reinforcement. Endothelin-1 (ET-1)- or phenylephrine (PE)-stimulated EHTs served as positive control for hypertrophy. Cardiomyocytes in EHTs enlarged by 28.4 % under AE and to a similar extent by ET-1- or PE-stimulation (40.6 or 23.6 %), as determined by dystrophin staining. Cardiomyocyte hypertrophy was accompanied by activation of the fetal gene program, increased glucose consumption, and increased mRNA levels and extracellular deposition of collagen-1. Importantly, afterload-enhanced EHTs exhibited reduced contractile force and impaired diastolic relaxation directly after release of the metal braces. These deleterious effects of afterload enhancement were preventable by endothelin-A, but not endothelin-B receptor blockade. Sustained afterload enhancement of EHTs alone is sufficient to induce pathological cardiac remodeling with reduced contractile function and increased glucose consumption. The model will be useful to investigate novel therapeutic approaches in a simple and fast manner.

Physiological aspects of cardiac tissue engineering.

Cardiac tissue engineering aims at repairing the diseased heart and developing cardiac tissues for basic research and predictive toxicology applications. Since the first description of engineered heart tissue 15 years ago, major development steps were directed toward these three goals. Technical innovations led to improved three-dimensional cardiac tissue structure and near physiological contractile force development. Automation and standardization allow medium throughput screening. Larger constructs composed of many small engineered heart tissues or stacked cell sheet tissues were tested for cardiac repair and were associated with functional improvements in rats. Whether these approaches can be simply transferred to larger animals or the human patients remains to be tested. The availability of an unrestricted human cardiac myocyte cell source from human embryonic stem cells or human-induced pluripotent stem cells is a major breakthrough. This review summarizes current tissue engineering techniques with their strengths and limitations and possible future applications.

Complex interactions between human myoblasts and the surrounding 3D fibrin-based matrix.

Anchorage of muscle cells to the extracellular matrix is crucial for a range of fundamental biological processes including migration, survival and differentiation. Three-dimensional (3D) culture has been proposed to provide a more physiological in vitro model of muscle growth and differentiation than routine 2D cultures. However, muscle cell adhesion and cell-matrix interplay of engineered muscle tissue remain to be determined. We have characterized cell-matrix interactions in 3D muscle culture and analyzed their consequences on cell differentiation. Human myoblasts were embedded in a fibrin matrix cast between two posts, cultured until confluence, and then induced to differentiate. Myoblasts in 3D aligned along the longitudinal axis of the gel. They displayed actin stress fibers evenly distributed around the nucleus and a cortical mesh of thin actin filaments. Adhesion sites in 3D were smaller in size than in rigid 2D culture but expression of adhesion site proteins, including α5 integrin and vinculin, was higher in 3D compared with 2D (p<0.05). Myoblasts and myotubes in 3D exhibited thicker and ellipsoid nuclei instead of the thin disk-like shape of the nuclei in 2D (p<0.001). Differentiation kinetics were faster in 3D as demonstrated by higher mRNA concentrations of α-actinin and myosin. More important, the elastic modulus of engineered muscle tissues increased significantly from 3.5 ± 0.8 to 7.4 ± 4.7 kPa during proliferation (p<0.05) and reached 12.2 ± 6.0 kPa during differentiation (p<0.05), thus attesting the increase of matrix stiffness during proliferation and differentiation of the myocytes. In conclusion, we reported modulations of the adhesion complexes, the actin cytoskeleton and nuclear shape in 3D compared with routine 2D muscle culture. These findings point to complex interactions between muscle cells and the surrounding matrix with dynamic regulation of the cell-matrix stiffness.