Subramanian Sundaram

The contractile activity of cardiomyocytes (CMs) critical to heart function emerges from the collective shortening of sarcomeres. However, how these sarcomeric forces are transmitted within CMs during this process remains poorly understood. Traction force microscopy has been used to measure overall forces exerted by CMs, but it falls short in providing insights into which specific proteins within sarcomeres transmit and whether cell shape influences forces within each sarcomere. Here, we aimed to characterize force generation on α-actinin-2, a z-disk protein that crosslinks anti-parallel actin filaments from adjacent sarcomeres and transmits force within a cell. By incorporating a Förster resonance energy transfer (FRET)-based molecular tension sensor in α-actinin-2, we measured contraction-induced deformation of the α-actinin-2 sensor in human-induced pluripotent stem cell-derived cardiomyocytes cultured on rectangular and circular adhesive patterns. We observed α-actinin-2 localized within sarcomeres, and actinin-2 loading correlated with sarcomere maturation and organization. α-actinin-2 tension increased in contracting rectangular-shaped cells, but not in circular cells. Moreover, the increase in tension was only observed in rectangular cardiomyocytes that were cultured for 5 days, and not after 24 h. Interestingly, the spread of FRET index values was increased in both rectangular and circular cells after 5 days in culture, compared to cells that were kept for 24 h in culture. Together, these data suggest that cell shape and maturation modulates tension on a load-bearing sarcomeric protein, α-actinin-2, and highlights the importance of characterizing tension across sarcomeric structures to understand cardiomyocyte contractile activity.

The health and function of microvascular beds are dramatically impacted by the mechanical forces that they experience due to fluid flow. These fluid flow‐generated forces are challenging to measure directly and are typically calculated from experimental flow data. However, current computational fluid dynamics (CFD) models either employ truncated 2D models or overlook the presence of extraluminal flows within the interstitial space between vessels that result from the permeability of the endothelium lining the vessels, which are crucial components affecting flow dynamics. To address this, we present a bottom‐up modeling approach that assesses fluid flow in 3D‐engineered vessel networks featuring an endothelial lining and interstitial space. Using image processing algorithms to segment 3D confocal image stacks from engineered capillary networks, we reconstructed a 3D computational model of the networks. We incorporated vascular permeability and matrix porosity values to model the contributions of the endothelial lining and interstitial spaces to the flow dynamics in the networks. Simulations suggest that including the endothelial monolayer and the interstitium significantly affects the predicted flow magnitude in the vessels and flow profiles in the interstitium. To demonstrate the importance of these factors, we showed experimentally and computationally that while cytokine (IL‐1β) treatment did not affect the network architecture, it significantly increased vessel permeability and resulted in a dramatic decrease in wall shear stresses and flow velocities intraluminally within the networks. In conclusion, this framework offers a robust methodology for studying flow dynamics in 3D in vitro vessel networks, enhancing our understanding of vascular physiology and pathology.

Natural tissues are composed of diverse cells and extracellular materials whose arrangements across several length scales—from subcellular lengths¹ (micrometre) to the organ scale² (centimetre)—regulate biological functions. Tissue-fabrication methods have progressed to large constructs, for example, through stereolithography³ and nozzle-based bioprinting^4,5, and subcellular resolution through subtractive photoablation^6,7,8. However, additive bioprinting struggles with sub-nozzle/voxel features⁹ and photoablation is restricted to small volumes by prohibitive heat generation and time¹⁰. Building across several length scales with temperature-sensitive, water-based soft biological matter has emerged as a critical challenge, leaving large classes of biological motifs—such as multiscalar vascular trees with varying calibres—inaccessible with present technologies^11,12. Here we use gallium-based engineered sacrificial capillary pumps for evacuation (ESCAPE) during moulding to generate multiscalar structures in soft natural hydrogels, achieving both cellular-scale (<10 µm) and millimetre-scale features. Decoupling the biomaterial of interest from the process of constructing the geometry allows non-biocompatible tools to create the initial geometry. As an exemplar, we fabricated branched, cell-laden vascular trees in collagen, spanning approximately 300-µm arterioles down to the microvasculature (roughly ten times smaller). The same approach can micropattern the inner surface of vascular walls with topographical cues to orient cells in 3D and engineer fine structures such as vascular malformations. ESCAPE moulding enables the fabrication of multiscalar forms in soft biomaterials, paving the way for a wide range of tissue architectures that were previously inaccessible in vitro.

Using two-photon direct laser writing, we developed a versatile platform to generate, scale, and study hiPSC-derived engineered heart tissues (EHTs) in various geometries, with the goal of promoting fiber alignment and maturation of the EHTs.

Branched epithelial tubule networks are a commonly found motif across many glands and organs, and how their forms evolve and are optimized in development and growth is not fully understood. Using the developing murine kidney as a model system, Prahl et al. study how the kidney tubular network optimally packs in the tubule tips (that increase in number but remain at the kidney surface through development) under the constraints of the organ surface area. Here, by analyzing whole-mount mouse kidneys at different developmental stages (E14 to E18), the authors argue that ureteric bud tubule networks can organize themselves simply through competition between crowding and repulsion of the tips - and does not require strict geometric control of the tips through morphogenetic programs. Through a series of experiments, the work demonstrates that actomyosin-based tubule tension (that acts radially) is a key factor driving the ordering of tips. In this state of radial tension, disrupting the basement membrane that holds these tips results in a radial retraction of the tips. This retraction is reduced with blebbistatin or the ROCK inhibitor Y-27632 and is enhanced by calyculin A (which increases actomyosin contractility). In addition, this work uses both physics-based modelling and soft material models to explain the trajectory of tip packing seen throughout development and how defective packing can result from abnormal physical forces. Physics-based modelling helps one explore the conditions required for the 6 packing phases including the optimal “vertical packing” phase (that is seen ~E16.5 onward). Broadly, this multipronged approach to studying the role of physical constraints and forces may perhaps help us understand how different organs are shaped.

Three-dimensional engineered heart tissues (EHTs) derived from human induced pluripotent stem cells (iPSCs) have become an important resource for both drug toxicity screening and research on heart disease. A key metric of EHT phenotype is the contractile (twitch) force with which the tissue spontaneously beats. It is well-known that cardiac muscle contractility – its ability to do mechanical work – depends on tissue prestrain (preload) and external resistance (afterload). Objectives: Here, we demonstrate a technique to control afterload while monitoring contractile force exerted by EHTs. Methods: We developed an apparatus that can regulate EHT boundary conditions using real-time feedback control. The system is comprised of a pair of piezoelectric actuators that can strain the scaffold and a microscope that can measure EHT force and length. Closed loop control allows dynamic regulation of effective EHT boundary stiffness. Results: When controlled to switch instantaneously from auxotonic to isometric boundary conditions, EHT twitch force immediately doubled. Changes in EHT twitch force as a function of effective boundary stiffness were characterized and compared to twitch force in auxotonic conditions. Conclusion: EHT contractility can be regulated dynamically through feedback control of effective boundary stiffness. Significance: The capacity to alter the mechanical boundary conditions of an engineered tissue dynamically offers a new way to probe tissue mechanics. This could be used to mimic afterload changes that occur naturally in disease, or to improve mechanical techniques for EHT maturation.

PKP 2 mutations lead to instability in cell-cell junctions and sarcomeres that impairs cardiac tissue contractility in ACM.

Rationale: Dominant heterozygous variants in filamin C ( FLNC ) cause diverse cardiomyopathies, although the underlying molecular mechanisms remain poorly understood. Objective: We aimed to define the molecular mechanisms by which FLNC variants altered human cardiomyocyte gene and protein expression, sarcomere structure, and contractile performance. Methods and Results: Using CRISPR/Cas9, we introduced FLNC variants into human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). We compared isogenic hiPSC-CMs with normal (wild-type), ablated expression ( FLNC ^−/− ), or haploinsufficiency ( FLNC ^+/− ) that causes dilated cardiomyopathy. We also studied a heterozygous in-frame deletion ( FLNC ^+/Δ7aa ) which did not affect FLNC expression but caused aggregate formation, similar to FLNC variants associated with hypertrophic cardiomyopathy. FLNC ^−/− hiPSC-CMs demonstrated profound sarcomere misassembly and reduced contractility. Although sarcomere formation and function were unaffected in FLNC ^{+/ −} and FLNC ^+/Δ7aa hiPSC-CMs, these heterozygous variants caused increases in lysosome content, enhancement of autophagic flux, and accumulation of FLNC-binding partners and Z-disc proteins. Conclusions: FLNC expression is required for sarcomere organization and physiological function. Variants that produce misfolded FLNC proteins cause the accumulation of FLNC and FLNC-binding partners which leads to increased lysosome expression and activation of autophagic pathways. Surprisingly, similar pathways were activated in FLNC haploinsufficient hiPSC-CMs, likely initiated by the loss of stoichiometric FLNC protein interactions and impaired turnover of proteins at the Z-disc. These results indicate that both FLNC haploinsufficient variants and variants that produce misfolded FLNC protein cause disease by similar proteotoxic mechanisms and indicate the therapeutic potential for augmenting protein degradative pathways to treat a wide range of FLNC -related cardiomyopathies.

Significance: Oscillators, widely found in nature, form the basis of a wide variety of actuating and signal processing mechanisms. While the ability to mimic arrays of oscillators and control their coupling is a central goal in the field of bioinspired soft materials, this has been difficult to achieve. Using Marangoni forces generated during spatially inhomogeneous illumination of photothermally responsive particles, we demonstrate a scheme for optically trapping arrays of particles at air–water interfaces that can incite a range of oscillatory and spinning behaviors. When multiple objects are arranged in proximity, they exhibit complex, collective behavior emerging from geometry-dependent interparticle coupling. This route to achieving collective motion is expected to open opportunities in the study of active materials.

Multimodal tactile sensors help robot hands accurately identify grasped objects by measuring thermal properties and contact loads.