Soluble miniagrin enhances contractile function of engineered skeletal muscle.

Neural agrin plays a pleiotropic role in skeletal muscle innervation and maturation, but its specific effects on the contractile function of aneural engineered muscle remain unknown. In this study, neonatal rat skeletal myoblasts cultured within 3-dimensional engineered muscle tissue constructs were treated with 10 nM soluble recombinant miniagrin and assessed using histological, biochemical, and functional assays. Depending on the treatment duration and onset time relative to the stage of myogenic differentiation, miniagrin was found to induce up to 1.7-fold increase in twitch and tetanus force amplitude. This effect was associated with the 2.3-fold up-regulation of dystrophin gene expression at 6 d after agrin removal and enhanced ACh receptor (AChR) cluster formation, but no change in cell number, expression of muscle myosin, or important aspects of intracellular Ca(2+) handling. In muscle constructs with endogenous ACh levels suppressed by the application of α-NETA, miniagrin increased AChR clustering and twitch force amplitude but failed to improve intracellular Ca(2+) handling and increase tetanus-to-twitch ratio. Overall, our studies suggest that besides its synaptogenic function that could promote integration of engineered muscle constructs in vivo, neural agrin can directly promote the contractile function of aneural engineered muscle via mechanisms distinct from those involving endogenous ACh.

Structure-related initiation of reentry by rapid pacing in monolayers of cardiac cells.

This study examines how a zigzag pattern of conduction, a form of structural heterogeneity frequently found in old or diseased hearts, affects the vulnerability to reentry during rapid pacing. A central rectangular island (8x4 mm) containing a predefined zigzag pattern was created in cultured isotropic monolayers of neonatal rat ventricular myocytes. Impulse propagation was optically mapped from 253 sites using voltage-sensitive dye and was anisotropic within the zigzag island. With increasing interval between neighboring transverse connections (a), relative to the distance between longitudinal strands (b), transverse conduction velocity (CV) decreased to 66+/-6%, 20+/-2%, and 15+/-2% of CV in the surrounding isotropic region, whereas longitudinal CV increased to 102+/-8%, 113+/-12%, and 131+/-23% for a:b ratios of 1:1, 1:5, and 1:9, respectively. During rapid pacing, propagation distal to the island was steered from the side of the island with more transverse connections ("dominant" side) toward the side with fewer connections ("weak" side). Increased asymmetry in the pattern accentuated this effect, and resulted in increased rate-dependent differences in CV on the 2 sides. Consequently, a functional obstacle formed on the weak side, followed by development of single loop reentry. The reentrant wave revolved around a line of block defined by the border of the island. Reentry chirality was determined by the weak side location, and the pacing rate needed to initiate reentry decreased with increased asymmetry in the pattern. In conclusion, reentry is readily induced by rapid pacing in confluent cardiac cell monolayers containing a central and asymmetric island of zigzag conduction.

Robust T-tubulation and maturation of cardiomyocytes using tissue-engineered epicardial mimetics.

Complex three-dimensional (3-D) heart structure is an important determinant of cardiac electrical and mechanical function. In this study, we set to develop a versatile tissue-engineered system that can promote important aspects of cardiac functional maturation and reproduce variations in myofiber directions present in native ventricular epicardium. We cultured neonatal rat cardiomyocytes within a 3-D hydrogel environment using microfabricated elastomeric molds with hexagonal posts. By varying individual post orientations along the directions derived from diffusion tensor magnetic resonance imaging (DTMRI) maps of human ventricle, we created large (2.5 × 2.5 cm(2)) 3-D cardiac tissue patches with cardiomyocyte alignment that replicated human epicardial fiber orientations. After 3 weeks of culture, the advanced structural and functional maturation of the engineered 3-D cardiac tissues compared to age-matched 2-D monolayers was evident from: 1) the presence of dense, aligned and electromechanically-coupled cardiomyocytes, quiescent fibroblasts, and interspersed capillary-like structures, 2) action potential propagation with near-adult conduction velocity and directional dependence on local cardiomyocyte orientation, and 3) robust formation of T-tubules aligned with Z-disks, co-localization of L-type Ca(2+) channels and ryanodine receptors, and accelerated Ca(2+) transient kinetics. This biomimetic tissue-engineered platform can enable systematic in vitro studies of cardiac structure-function relationships and promote the development of advanced tissue engineering strategies for cardiac repair and regeneration.

Local tissue geometry determines contractile force generation of engineered muscle networks.

The field of skeletal muscle tissue engineering is currently hampered by the lack of methods to form large muscle constructs composed of dense, aligned, and mature myofibers and limited understanding of structure-function relationships in developing muscle tissues. In our previous studies, engineered muscle sheets with elliptical pores ("muscle networks") were fabricated by casting cells and fibrin gel inside elastomeric tissue molds with staggered hexagonal posts. In these networks, alignment of cells around the elliptical pores followed the local distribution of tissue strains that were generated by cell-mediated compaction of fibrin gel against the hexagonal posts. The goal of this study was to assess how systematic variations in pore elongation affect the morphology and contractile function of muscle networks. We found that in muscle networks with more elongated pores the force production of individual myofibers was not altered, but the myofiber alignment and efficiency of myofiber formation were significantly increased yielding an increase in the total contractile force despite a decrease in the total tissue volume. Beyond a certain pore length, increase in generated contractile force was mainly contributed by more efficient myofiber formation rather than enhanced myofiber alignment. Collectively, these studies show that changes in local tissue geometry can exert both direct structural and indirect myogenic effects on the functional output of engineered muscle. Different hydrogel formulations and pore geometries will be explored in the future to further augment contractile function of engineered muscle networks and promote their use for basic structure-function studies in vitro and, eventually, for efficient muscle repair in vivo.

The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle.

One of the obstacles to the potential clinical utility of bioengineered skeletal muscle is its limited force generation capacity. Since engineered muscle, unlike most native muscle tissue, is composed of relatively short myofibers, we hypothesized that, its force production and transmission would be profoundly influenced by cell-matrix interactions. To test this hypothesis, we systematically varied the matrix protein type (collagen I/fibrin/Matrigel) and concentration in engineered, hydrogel-based neonatal rat skeletal muscle bundles and assessed the resulting tissue structure, generation of contractile force, and intracellular Ca(2+) handling. After two weeks of culture, the muscle bundles consisted of highly aligned and cross-striated myofibers and exhibited standard force-length and force-frequency relationships achieving tetanus at 40 Hz. The use of 2 mg/ml fibrin (control) yielded isometric tetanus amplitude of 1.4 ± 0.3 mN as compared to 0.9 ± 0.4 mN measured in collagen I-based bundles. Higher fibrin and Matrigel concentrations synergistically yielded further increase in active force generation to 2.8 ± 0.5 mN without significantly affecting passive mechanical properties, tetanus-to-twitch ratio, and twitch kinetics. Optimized matrix composition yielded significant cellular hypertrophy (protein/DNA ratio = 11.4 ± 4.1 vs. 6.5 ± 1.9 µg/µg in control) and a prolonged Ca(2+) transient half-width (Ca(50) = 232.8 ± 33.3 vs. 101.7 ± 19.8 ms). The use of growth-factor-reduced Matrigel, instead of standard Matrigel did not alter the obtained results suggesting enhanced cell-matrix interactions rather than growth factor supplementation as an underlying cause for the measured increase in contractile force. In summary, biomaterial-based manipulation of cell-matrix interactions represents an important target for improving contractile force generation in engineered skeletal muscle.

Implantation of mouse embryonic stem cell-derived cardiac progenitor cells preserves function of infarcted murine hearts.

Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

Engineered skeletal muscle tissue networks with controllable architecture.

The engineering of functional skeletal muscle tissue substitutes holds promise for the treatment of various muscular diseases and injuries. However, no tissue fabrication technology currently exists for the generation of a relatively large and thick bioartificial muscle made of densely packed, uniformly aligned, and differentiated myofibers. In this study, we describe a versatile cell/hydrogel micromolding approach where polydimethylsiloxane (PDMS) molds containing an array of elongated posts were used to fabricate relatively large neonatal rat skeletal muscle tissue networks with reproducible and controllable architecture. By combining cell-mediated fibrin gel compaction and precise microfabrication of mold dimensions including the length and height of the PDMS posts, we were able to simultaneously support high cell viability, guide cell alignment along the microfabricated tissue pores, and reproducibly control the overall tissue porosity, size, and thickness. The interconnected muscle bundles within the porous tissue networks were composed of densely packed, aligned, and highly differentiated myofibers. The formed myofibers expressed myogenin, developed abundant cross-striations, and generated spontaneous tissue contractions at the macroscopic spatial scale. The proliferation of non-muscle cells was significantly reduced compared to monolayer cultures. The more complex muscle tissue architectures were fabricated by controlling the spatial distribution and direction of the PDMS posts.

Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues.

This protocol describes a cell/hydrogel molding method for precise and reproducible biomimetic fabrication of three-dimensional (3D) muscle tissue architectures in vitro. Using a high aspect ratio soft lithography technique, we fabricate polydimethylsiloxane (PDMS) molds containing arrays of mesoscopic posts with defined size, elongation and spacing. On cell/hydrogel molding, these posts serve to enhance the diffusion of nutrients to cells by introducing elliptical pores in the cell-laden hydrogels and to guide local 3D cell alignment by governing the spatial pattern of mechanical tension. Instead of ultraviolet or chemical cross-linking, this method utilizes natural hydrogel polymerization and topographically constrained cell-mediated gel compaction to create the desired 3D tissue structures. We apply this method to fabricate several square centimeter large, few hundred micron-thick bioartificial muscle tissues composed of viable, dense, uniformly aligned and highly differentiated cardiac or skeletal muscle fibers. The protocol takes 4-5 d to fabricate PDMS molds followed by 2 weeks of cell culture.