Connexin43 ablation in foetal atrial myocytes decreases electrical coupling, partner connexins, and sodium current

Aims Remodelling and regional gradients in expression of connexins (Cx) are thought to contribute to atrial electrical dysfunction and atrial fibrillation. We assessed the effect of interaction between Cx43, Cx40, and Cx45 on atrial cell-to-cell coupling and inward Na current (INa) in engineered pairs of atrial myocytes derived from wild-type mice (Cx43+/+) and mice with genetic ablation of Cx43 (Cx43−/−). Methods and results Cell pairs were engineered by microcontact printing from atrial Cx43+/+ and Cx43−/− murine myocytes (1 day before birth, 3-5 days in culture). Dual and single voltage clamp were used to measure intercellular electrical conductance, gj, and its dependence on transjunctional voltage, Vj, single gap junction channel conductances, and INa. 3D reconstructions of Cx43, Cx40, and Cx45 immunosignals in gap junctions were made from confocal slices. Full genetic Cx43 ablation produced a decrease in immunosignals of Cx40 to 62 ± 10% (mean ± SE; n= 17) and Cx45 to 66 ± 8% (n= 16). Gj decreased from 80 ± 9 nS (Cx43+/+, n= 17) to 24 ± 2 nS (Cx43−/−, n= 35). Single channel analysis showed a shift in the main peak of the channel histogram from 49 ± 1.7 nS (Cx43+/+) to 67 ± 1.8 nS (Cx43−/−) with a second minor peak appearing at 27 ± 1.5 pS. The dependence of gj on Vj decreased with Cx43 ablation. Importantly, peak INa decreased from −350 ± 44 pA/pF (Cx43+/+) to −154 ± 28 pA/pF (Cx43−/−). Conclusions The dependence of Cx40, Cx45, and INa on Cx43 expression indicates a complex interaction between connexins and INa in the atrial intercalated discs that is likely to be of relevance for arrhythmogenesi

A tissue-engineered jellyfish with biomimetic propulsion

Reverse engineering of biological form and function requires hierarchical design over several orders of space and time. Recent advances in the mechanistic understanding of biosynthetic compound materials, computer-aided design approaches in molecular synthetic biology and traditional soft robotics, and increasing aptitude in generating structural and chemical microenvironments that promote cellular self-organization have enhanced the ability to recapitulate such hierarchical architecture in engineered biological systems. Here we combined these capabilities in a systematic design strategy to reverse engineer a muscular pump. We report the construction of a freely swimming jellyfish from chemically dissociated rat tissue and silicone polymer as a proof of concept. The constructs, termed 'medusoids', were designed with computer simulations and experiments to match key determinants of jellyfish propulsion and feeding performance by quantitatively mimicking structural design, stroke kinematics and animal-fluid interactions. The combination of the engineering design algorithm with quantitative benchmarks of physiological performance suggests that our strategy is broadly applicable to reverse engineering of muscular organs or simple life forms that pump to survive

Modeling the mitochondrial cardiomyopathy of Barth syndrome with iPSC and heart-on-chip technologies

Studying monogenic mitochondrial cardiomyopathies may yield insights into mitochondrial roles in cardiac development and disease. Here, we combine patient-derived and genetically engineered iPSCs with tissue engineering to elucidate the pathophysiology underlying the cardiomyopathy of Barth syndrome (BTHS), a mitochondrial disorder caused by mutation of the gene Tafazzin (TAZ). Using BTHS iPSC-derived cardiomyocytes (iPSC-CMs), we defined metabolic, structural, and functional abnormalities associated with TAZ mutation. BTHS iPSC-CMs assembled sparse and irregular sarcomeres, and engineered BTHS “heart on chip” tissues contracted weakly. Gene replacement and genome editing demonstrated that TAZ mutation is necessary and sufficient for these phenotypes. Sarcomere assembly and myocardial contraction abnormalities occurred in the context of normal whole cell ATP levels. Excess levels of reactive oxygen species mechanistically linked TAZ mutation to impaired cardiomyocyte function. Our study provides new insights into the pathogenesis of Barth syndrome, suggests new treatment strategies, and advances iPSC-based in vitro modeling of cardiomyopathy

Mechanotransduction: the role of mechanical stress, myocyte shape, and cytoskeletal architecture on cardiac function

Mechanotransduction refers to the conversion of mechanical forces into biochemical or electrical signals that initiate structural and functional remodeling in cells and tissues. The heart is a kinetic organ whose form changes considerably during development and disease, requiring cardiac myocytes to be mechanically durable and capable of fusing a variety of environmental signals on different time scales. During physiological growth, myocytes adaptively remodel to mechanical loads. Pathological stimuli can induce maladaptive remodeling. In both of these conditions, the cytoskeleton plays a pivotal role in both sensing mechanical stress and mediating structural remodeling and functional responses within the myocyte.Engineering and Applied Science

Characterization of Gelatin Hydrogels Cross-Linked with Microbial Transglutaminase as Engineered Skeletal Muscle Substrates

Engineered in vitro models of skeletal muscle are essential for efficiently screening drug safety and efficacy. However, conventional culture substrates poorly replicate physical features of native muscle and do not support long-term culture, which limits tissue maturity. Micromolded gelatin hydrogels cross-linked with microbial transglutaminase (gelatin-MTG hydrogels) have previously been shown to induce C21C2 myotube alignment and improve culture longevity. However, several properties of gelatin-MTG hydrogels have not been systematically characterized, such as changes in elastic modulus during incubation in culture-like conditions and their ability to support sarcomere maturation. In this study, various gelatin-MTG hydrogels were fabricated and incubated in ambient or culture-like conditions. Elastic modulus, mass, and transmittance were measured over a one- or two-week period. Compared to hydrogels in phosphate buffered saline (PBS) or ambient air, hydrogels in Dulbecco’s Modified Eagle Medium (DMEM) and 5% CO2 demonstrated the most stable elastic modulus. A subset of gelatin-MTG hydrogels was micromolded and seeded with C2C12 or primary chick myoblasts, which aligned and fused into multinucleated myotubes with relatively mature sarcomeres. These data are important for fabricating gelatin-MTG hydrogels with predictable and stable mechanical properties and highlight their advantages as culture substrates for engineering relatively mature and stable muscle tissues

Ensembles of engineered cardiac tissues for physiological and pharmacological study: Heart on a chip

Traditionally, muscle physiology experiments require multiple tissue samples to obtain morphometric, electrophysiological, and contractility data. Furthermore, these experiments are commonly completed one at a time on cover slips of single cells, isotropic monolayers, or in isolated muscle strips. In all of these cases, variability of the samples hinders quantitative comparisons among experimental groups. Here, we report the design of a “heart on a chip” that exploits muscular thin film technology – biohybrid constructs of an engineered, anisotropic ventricular myocardium on an elastomeric thin film – to measure contractility, combined with a quantification of action potential propagation, and cytoskeletal architecture in multiple tissues in the same experiment. We report techniques for real-time data collection and analysis during pharmacological intervention. The chip is an efficient means of measuring structure-function relationships in constructs that replicate the hierarchical tissue architectures of laminar cardiac muscle.Engineering and Applied Science

Modeling Patient-Specific Muscular Dystrophy Phenotypes and Therapeutic Responses in Reprogrammed Myotubes Engineered on Micromolded Gelatin Hydrogels.

In vitro models of patient-derived muscle allow for more efficient development of genetic medicines for the muscular dystrophies, which often present mutation-specific pathologies. One popular strategy to generate patient-specific myotubes involves reprogramming dermal fibroblasts to a muscle lineage through MyoD induction. However, creating physiologically relevant, reproducible tissues exhibiting multinucleated, aligned myotubes with organized striations is dependent on the introduction of physicochemical cues that mimic the native muscle microenvironment. Here, we engineered patient-specific control and dystrophic muscle tissues in vitro by culturing and differentiating MyoD-directly reprogrammed fibroblasts isolated from one healthy control subject, three patients with Duchenne muscular dystrophy (DMD), and two Limb Girdle 2A/R1 (LGMD2A/R1) patients on micromolded gelatin hydrogels. Engineered DMD and LGMD2A/R1 tissues demonstrated varying levels of defects in α-actinin expression and organization relative to control, depending on the mutation. In genetically relevant DMD tissues amenable to mRNA reframing by targeting exon 44 or 45 exclusion, exposure to exon skipping antisense oligonucleotides modestly increased myotube coverage and alignment and rescued dystrophin protein expression. These findings highlight the value of engineered culture substrates in guiding the organization of reprogrammed patient fibroblasts into aligned muscle tissues, thereby extending their value as tools for exploration and dissection of the cellular and molecular basis of genetic muscle defects, rescue, and repair