Interpretation of field and LEAP potentials recorded from cardiomyocyte monolayers.

Multielectrode arrays (MEAs) are the method of choice for electrophysiological characterization of cardiomyocyte monolayers. The field potentials recorded using an MEA are like extracellular electrograms recorded from the myocardium using conventional electrodes. Nevertheless, different criteria are used to interpret field potentials and extracellular electrograms, which hamper correct interpretation and translation to the patient. To validate the criteria for interpretation of field potentials, we used neonatal rat cardiomyocytes to generate monolayers. We recorded field potentials using an MEA and simultaneously recorded action potentials using sharp microelectrodes. In parallel, we recreated our experimental setting in silico and performed simulations. We show that the amplitude of the local RS complex of a field potential correlated with conduction velocity in silico but not in vitro. The peak time of the T wave in field potentials exhibited a strong correlation with APD90 while the steepest upslope correlated well with APD50. However, this relationship only holds when the T wave displayed a biphasic pattern. Next, we simulated local extracellular action potentials (LEAPs). The shape of the LEAP differed markedly from the shape of the local action potential, but the final duration of the LEAP coincided with APD90. Criteria for interpretation of extracellular electrograms should be applied to field potentials. This will provide a strong basis for the analysis of heterogeneity in conduction velocity and repolarization in cultured monolayers of cardiomyocytes. Finally, a LEAP is not a recording of the local action potential but is generated by intracellular current provided by neighboring cardiomyocytes and is superior to field potential duration in estimating APD90.NEW & NOTEWORTHY We present a physiological basis for the interpretation of multielectrode array-derived, extracellular, electrical signals.

Molecular and electrophysiological evaluation of human cardiomyocyte subtypes to facilitate generation of composite cardiac models.

Paucity of physiologically relevant cardiac models has limited the widespread application of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes in drug development. Here, we performed comprehensive characterization of hiPSC-derived cardiomyocyte subtypes from 2D and 3D cultures and established a novel 3D model to study impulse initiation and propagation. Directed differentiation approaches were used to generate sinoatrial nodal (SANCM), atrial (ACM) and ventricular cardiomyocytes (VCM). Single cell RNA sequencing established that the protocols yield distinct cell populations in line with expected identities, which was also confirmed by electrophysiological characterization. In 3D EHT cultures of all subtypes, we observed prominent expression of stretch-responsive genes such as NPPA. Response to rate modulating drugs noradrenaline, carbachol and ivabradine were comparable in single cells and EHTs. Differences in the speed of impulse propagation between the subtypes were more pronounced in EHTs compared with 2D monolayers owing to a progressive increase in conduction velocities in atrial and ventricular cardiomyocytes, in line with a more mature phenotype. In a novel binary EHT model of pacemaker-atrial interface, the SANCM end of the tissue consistently paced the EHTs under baseline conditions, which was inhibited by ivabradine. Taken together, our data provide comprehensive insights into molecular and electrophysiological properties of hiPSC-derived cardiomyocyte subtypes, facilitating the creation of next generation composite cardiac models for drug discovery, disease modeling and cell-based regenerative therapies.

A single cell transcriptional roadmap of human pacemaker cell differentiation.

Each heartbeat is triggered by the sinoatrial node (SAN), the primary pacemaker of the heart. Studies in animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into 'transitional', 'tail', and 'head' subtypes. However, the underlying molecular mechanisms, especially of human pacemaker cell development, are poorly understood. Here, we performed single cell RNA sequencing (scRNA-seq) and trajectory inference on human induced pluripotent stem cells (hiPSCs) differentiating to SAN-like cardiomyocytes (SANCMs) to construct a roadmap of transcriptional changes and lineage decisions. In differentiated SANCM, we identified distinct clusters that closely resemble different subpopulations of the in vivo SAN. Moreover, the presence of a side population of proepicardial cells suggested their shared ontogeny with SANCM, as also reported in vivo. Our results demonstrate that the divergence of SANCM and proepicardial lineages is determined by WNT signaling. Furthermore, we uncovered roles for TGFß and WNT signaling in the branching of transitional and head SANCM subtypes, respectively. These findings provide new insights into the molecular processes involved in human pacemaker cell differentiation, opening new avenues for complex disease modeling in vitro and inform approaches for cell therapy-based regeneration of the SAN.

Titin Circular RNAs Create a Back-Splice Motif Essential for SRSF10 Splicing.

BACKGROUND: TTN (Titin), the largest protein in humans, forms the molecular spring that spans half of the sarcomere to provide passive elasticity to the cardiomyocyte. Mutations that disrupt the TTN transcript are the most frequent cause of hereditary heart failure. We showed before that TTN produces a class of circular RNAs (circRNAs) that depend on RBM20 to be formed. In this study, we show that the back-splice junction formed by this class of circRNAs creates a unique motif that binds SRSF10 to enable it to regulate splicing. Furthermore, we show that one of these circRNAs (cTTN1) distorts both localization of and splicing by RBM20. METHODS: We calculated genetic constraint of the identified motif in 125 748 exomes collected from the gnomAD database. Furthermore, we focused on the highest expressed RBM20-dependent circRNA in the human heart, which we named cTTN1. We used shRNAs directed to the back-splice junction to induce selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Human genetics suggests reduced genetic tolerance of the generated motif, indicating that mutations in this motif might lead to disease. RNA immunoprecipitation confirmed binding of circRNAs with this motif to SRSF10. Selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes induced structural abnormalities, apoptosis, and reduced contractile force in engineered heart tissue. In line with its SRSF10 binding, loss of cTTN1 caused abnormal splicing of important cardiomyocyte SRSF10 targets such as MEF2A and CASQ2. Strikingly, loss of cTTN1 also caused abnormal splicing of TTN itself. Mechanistically, we show that loss of cTTN1 distorts both localization of and splicing by RBM20. CONCLUSIONS: We demonstrate that circRNAs formed from the TTN transcript are essential for normal splicing of key muscle genes by enabling splice regulators RBM20 and SRSF10. This shows that the TTN transcript also has regulatory roles, besides its well-known signaling and structural function. In addition, we demonstrate that the specific sequence created by the back-splice junction of these circRNAs has important functions. This highlights the existence of functionally important sequences that cannot be recognized as such in the human genome but provides an as-yet unrecognized source for functional sequence variation.

Aliphatic Chain Modification of Collagen Type I: Development of Elastomeric, Compliant, and Suturable Scaffolds.

Collagen type I is one of the most suitable natural biomaterials for constructing tissue-engineering scaffolds. Despite their biocompositional similarities to physiological tissues, these scaffolds lack host specific and matching mechanical properties. While it is possible to enhance their stiffness by cross-linking, it often compromises their abilities to expand or strain under minimal stress, that is, compliance (inverse of stiffness). Here, we report a simple, inexpensive, cross-linking- and elastin-free collagen-based material composition for developing elastomeric scaffolds that are highly compliant, soft yet strong, and suturable, therefore, clinically attractive. Our strategy utilizes room-temperature modification of collagen type I scaffolds with linear aliphatic chains of various lengths (C7-C18). In particular, dodecenylsuccinic anhydride (size: C12, DDSA) modified scaffolds elongated up to 400% of its initial length compared to only ∼20% for collagen-control within the applied tensile stress of 0.2 MPa without breaking. Furthermore, the suture retention strength value increased to 60 g-force from 30 g-force for collagen control. We confirmed that the C12-modified material remained structurally stable at the physiological temperature (37 °C) with a tan δ value of ∼0.3, similar to collagen control; however, tan δ increased sharply for C12-modified collagen above 42 °C, compared to 59 °C for collagen control. To understand the mechanism of hyperextensibility, we studied the morphology of the resultant material by transmission electron microscopy (TEM), which showed an altered microstructure of C12-modified collagen scaffolds. While the partially C12-modified sample had a mixture of typical collagen type I triple helix and diffused gelatinized random coil-like configuration, the fully modified samples showed thick wrinkled and entangled ribbon-like microstructures, which was different than that of thermally denatured gelatin. We further confirmed that the resultant material allowed cell growth in vitro and in vivo in a subcutaneous mouse model.

Tissue-Engineered Neo-Urinary Conduit from Decellularized Trachea.

Decellularized tissues have been increasingly popular for constructing scaffolds for tissue engineering applications due to their beneficial biological compositions and mechanical properties. It is therefore natural to consider decellularized trachea for construction of tissue-engineered trachea, as well as other tubular organs. A Neo-Urinary Conduit (NUC) is such a tubular organ that works as a passage for urine removal in bladder cancer patients who need a urinary diversion after their diseased bladder is removed. In this study, we report our findings on the feasibility of using a decellularized trachea for NUC applications. As a NUC scaffold, decellularized trachea provides benefits of having not only naturally occurring biological components but also having sufficient mechanical properties and structural integrity. We, therefore, decellularized rabbit trachea, evaluated its mechanical performance, and investigated its ability to support in vitro growth of human smooth muscle cells (hSMCs) and human urothelial cells (hUCs). The decellularized trachea had appropriate biomechanical properties with ultimate tensile strength of ∼0.34 MPa in longitudinal direction and ∼1.0 MPa in circumferential direction and resisted a radial burst pressure of >155 mm Hg. Cell morphology study by scanning electron microscopy further showed that hUCs grown on decellularized trachea adopted a typical flatten and interconnected network structure in the lumen of the scaffold, while they formed a round spherical shape and did not spread on the outer surfaces. SMCs, on the other hand, spread well throughout the scaffold. The gene expression analysis by real time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence studies further confirmed scaffold's ability to support long-term growth of hSMCs. Since uroepithelium has been shown to regenerate itself over time in vivo, these findings suggest that it is possible to construct a NUC from decellularized trachea without any preseeding of UCs. In future, we plan to translate decellularized trachea in a preclinical animal model and evaluate its biological performance.

Effect of reactions in small eddies on biomass gasification with eddy dissipation concept - Sub-grid scale reaction model.

Large-eddy simulation (LES) approach is used for gas turbulence, and eddy dissipation concept (EDC)-sub-grid scale (SGS) reaction model is employed for reactions in small eddies. The simulated gas molar fractions are in better agreement with experimental data with EDC-SGS reaction model. The effect of reactions in small eddies on biomass gasification is emphatically analyzed with EDC-SGS reaction model. The distributions of the SGS reaction rates which represent the reactions in small eddies with particles concentration and temperature are analyzed. The distributions of SGS reaction rates have the similar trend with those of total reactions rates and the values account for about 15% of the total reactions rates. The heterogeneous reaction rates with EDC-SGS reaction model are also improved during the biomass gasification process in bubbling fluidized bed.

Patterning of Fibroblast and Matrix Anisotropy within 3D Confinement is Driven by the Cytoskeleton.

Effects of 3D confinement on cellular growth and matrix assembly are important in tissue engineering, developmental biology, and regenerative medicine. Polydimethylsiloxane wells with varying anisotropy are microfabicated using soft-lithography. Microcontact printing of bovine serum albumin is used to block cell adhesion to surfaces between wells. The orientations of fibroblast stress fibers, microtubules, and fibronectin fibrils are examined 1 day after cell seeding using laser scanning confocal microscopy, and anisotropy is quantified using a custom autocorrelation analysis. Actin, microtubules, and fibronectin exhibit higher anisotropy coefficients for cells grown in rectangular wells with aspect ratios of 1:4 and 1:8, as compared to those in wells with lower aspect ratios or in square wells. The effects of disabling individual cytoskeletal components on fibroblast responses to anisotropy are then tested by applying actin or microtubule polymerization inhibitors, Rho kinase inhibitor, or by siRNA-mediated knockdown of AXL or cofilin-1. Latrunculin A decreases cytoskeletal and matrix anisotropy, nocodazole ablates both, and Y27632 mutes cellular polarity while decreasing matrix anisotropy. AXL siRNA knockdown has little effect, as does siRNA knockdown of cofilin-1. These data identify several specific cytoskeletal strategies as targets for the manipulation of anisotropy in 3D tissue constructs.