Toshiyuki Mitsui

Mechanical cues encoded in the extracellular microenvironment play essential roles in regulating cardiac rhythm and tissue-level coordination; however, systematic interrogation of when mechanical inputs are most effective remains technically challenging. While substrate stiffness and cyclic stretch are widely used to condition cardiomyocytes in vitro, most platforms lack the temporal precision required to probe phase-dependent mechanosensitivity during spontaneous beating. Here, we report a biomaterials-based mechanical stimulation platform that enables real-time, phase-specific delivery of localized stretch to autonomously beating cardiomyocyte aggregates cultured on soft polydimethylsiloxane (PDMS) substrates. By integrating live imaging-based beat detection with piezo-driven microscale actuation, mechanical stimuli were triggered at defined fractions of the intrinsic interbeat interval (IBI), allowing direct comparison of in-phase and out-of-phase mechanical perturbations. Using one-dimensional cardiomyocyte aggregates as a model system, we show that randomly timed mechanical stimulation rarely altered intrinsic rhythm (∼0.3% of beats). In contrast, phase-locked stimulation applied near the end of the contraction–relaxation cycle (∼90% of the IBI) reproducibly induced transient IBI shortening, reversed contraction-wave propagation, and shifted pacemaker activity toward the site of stimulation. Mechanical stimulation delivered at midcycle (∼50% of the IBI) produced minimal effects. Displacement-resolved analysis revealed that phase-specific stimulation redistributed mechanical output within the aggregate: contraction amplitude was maintained or enhanced near the stimulation site while being attenuated in distal regions. These results demonstrate that developing cardiac tissues exhibit narrow temporal windows of mechanical susceptibility and robust resistance to perturbation outside these phases. This platform provides a versatile biomaterials-enabled framework for dissecting dynamic mechano-electrical feedback, optimizing mechanical conditioning strategies, and engineering cardiac tissues with controllable rhythmic properties.

The diversification of life is driven by the acquisition of new phenotypic traits, known as evolutionary novelties. While some genetic mechanisms underlying such traits have been identified, the role of physical properties of cellular and extracellular components remains largely unexplored. Here, we show that the evolution and development of the bulbus arteriosus (BA)—a teleost-specific, smooth muscle-rich heart outflow tract—is regulated by the mechanical properties of its extracellular environment. Specifically, we demonstrate that the teleost-specific extracellular matrix gene elastin b confers the uniquely low stiffness of the BA. In contrast to the BA, the homologous organ in nonteleost actinopterygians, the conus arteriosus (CA), and the ventricle are predominantly composed of cardiomyocytes and exhibit higher extracellular stiffness. Loss of elastin b function by knockdown or knockout leads to ectopic cardiomyocyte formation in the BA, accompanied by increased extracellular stiffness comparable to that of the ventricle. Furthermore, artificial stiffening of the BA extracellular environment is sufficient to induce ectopic cardiomyocyte differentiation. Taken together, these findings demonstrate that extracellular stiffness governs cell fate determination and highlight its role in the emergence of evolutionary novelties in the teleost heart.

Cardiac fibroblasts play a pivotal role in heart tissue dynamics, responding to mechanical and interfacial cues in their microenvironment. This study examines how substrate stiffness and surface condition influence the motility and morphology of chicken embryonic cardiac fibroblasts cultured on collagen-coated polydimethylsiloxane (PDMS) substrates with approximate elastic moduli of ~10 kPa and ~400 kPa, representing mechanically soft and stiff conditions, respectively. The PDMS surfaces were used without plasma or UV/ozone treatment, retaining their native hydrophobic character without surface oxidation. This absence of surface activation resulted in weak cell-substrate adhesion, leading to delayed spreading and frequent partial detachment during migration. Using time-lapse phase-contrast microscopy, we observed irregular motility cycles characterized by contraction-driven retraction, followed by partial detachment and re-adhesion. On soft substrates, fibroblasts exhibited longer motility cycles and left trailing edges upon retraction, while on stiff substrates, cells remained rounded and displayed less polarized motion. Quantitative analysis revealed that the duration of static phases and the degree of cell elongation varied with substrate stiffness but were also strongly influenced by adhesion instability due to unoxidized PDMS surfaces. These findings clarify the roles of substrate stiffness and surface adhesion in regulating fibroblast motility, and demonstrate that non-oxidized PDMS surfaces—though less adhesive—can preserve a rounded, less-spread cell morphology. Rather than promoting full spreading, such conditions may maintain fibroblasts in a mechanically less engaged or undifferentiated state. This work provides insight into how surface condition and mechanics together shape fibroblast behavior, with implications for mechanobiology and in vitro model design using non-activated biomaterials.

During embryonic development, cell behaviors need to be tightly regulated in time and space. Yet how the temporal and spatial regulations of cell behaviors are interconnected during embryonic development remains elusive. To address this, we turned to zebrafish gastrulation, the process whereby dynamic cell behaviors generate the three principal germ layers of the early embryo. Here, we show that Hoxb cluster genes are expressed in a temporally collinear manner at the blastoderm margin, where mesodermal and endodermal (mesendoderm) progenitor cells are specified and ingress to form mesendoderm/hypoblast. Functional analysis shows that these Hoxb genes regulate the timing of cell ingression: under- or overexpression of Hoxb genes perturb the timing of mesendoderm cell ingression and, consequently, the positioning of these cells along the forming anterior-posterior body axis after gastrulation. Finally, we found that Hoxb genes control the timing of mesendoderm ingression by regulating cellular bleb formation and cell surface fluctuations in the ingressing cells. Collectively, our findings suggest that Hoxb genes interconnect the temporal and spatial pattern of cell behaviors during zebrafish gastrulation by controlling cell surface fluctuations.

Cardiac fibroblasts play a pivotal role in heart tissue dynamics, responding to mechanical cues from their environment. This study examines how substrate elasticity influences the motility and shape of chicken embryonic cardiac fibroblasts on polydimethylsiloxane (PDMS) substrates of different stiffness (10 kPa and 400 kPa). Using time-lapse phase-contrast microscopy, we identified distinct motility cycles characterized by elongation, contraction, and retraction phases. On soft substrates, cells exhibited slower retraction speeds and longer motility cycles compared to those on hard substrates. Notably, cells on soft substrates left trailing edges upon retraction, leading to pronounced morphological oscillations, whereas cells on hard substrates adopted near-circular shapes with less coordinated motility. Quantitative analyses revealed that the duration of the static phase was significantly shorter on hard substrates, correlating with differences in cell shape parameters, including length, circularity, and area. These findings suggest that substrate elasticity modulates the balance between cell-substrate adhesion and contractile forces, influencing cell polarity and motility. Our work advances the understanding of how mechanical environments shape fibroblast dynamics, offering insights relevant to tissue engineering and fibrosis research. SIGNIFICANCE: Mechanical cues from the cellular microenvironment are known to regulate cell behavior, yet how substrate stiffness shapes the dynamic motility cycle of fibroblasts remains incompletely understood. Here, we use high-resolution time-lapse imaging to reveal that cardiac fibroblasts on soft (10 kPa) PDMS undergo polarized, oscillatory migration with prolonged static phases, while those on stiff (400 kPa) substrates exhibit rapid retraction and random-like displacement with reduced polarity. These findings highlight how substrate elasticity modulates the interplay between traction forces and adhesion stability, governing directionality and morphology. This work provides mechanobiological insights into fibroblast behavior with implications for designing biomaterials to guide tissue remodeling and fibrosis progression.

The diversification of life is driven by the acquisition of new phenotypic traits, known as evolutionary novelties. While some genetic mechanisms underlying such traits have been identified, the role of physical properties of cellular and extracellular components remains largely unexplored. Here, we show that the evolution and development of the bulbus arteriosus (BA)–a teleost-specific, smooth muscle-rich heart outflow tract–is regulated by the mechanical properties of its extracellular environment. Specifically, we demonstrate that the teleost-specific extracellular matrix gene elastin b confers the uniquely low stiffness of the BA. In contrast to the BA, the homologous organ in non-teleost actinopterygians, the conus arteriosus (CA), and the ventricle are predominantly composed of cardiomyocytes, and exhibit higher extracellular stiffness. Loss of elastin b function by knockdown or knockout leads to ectopic cardiomyocyte formation in the BA, accompanied by increased extracellular stiffness comparable to that of the ventricle. Furthermore, artificial stiffening of the BA extracellular environment is sufficient to induce ectopic cardiomyocyte differentiation. Taken together, these findings demonstrate that extracellular stiffness governs cell fate determination and highlight its role in the emergence of evolutionary novelties in the teleost heart. Significant Statement: The acquisition of new phenotypic traits is a key driver for a diversification of life. While genetic underpinnings of such traits have been well studied, the contribution of physical properties remains elusive. Here, we show that extracellular stiffness plays a key role in the evolution and development of the bulbus arteriosus (BA), a smooth muscle-based cardiac outflow tract unique to teleost hearts. We demonstrate that elastin b , a teleost-specific gene, confers characteristically low stiffness to the BA, which is required for proper cell fate determination. Notably, such low stiffness is absent in non-teleost fishes and in teleosts with impaired elastin b function, and artificial stiffening alters cell fate. Our findings highlight the importance of physical properties in driving evolutionary novelties.

The coil–globule transition is a key phenomenon in polymer physics, where polymer chains shift between collapsed and extended states. This study investigates the relaxation dynamics of T4 DNA transitioning from an electrohydrodynamically compressed globule to a relaxed coil state in polyethylene glycol (PEG) solutions of varying molecular weights. Using an AC electric field to induce globule formation, we analyzed DNA expansion upon field cessation. Higher molecular weight PEG, particularly PEG 20000, markedly prolonged relaxation times and exhibited broader distributions compared to PEG 4000 and PEG 6000. In PEG 20000, exceeding the overlap concentration stabilized multi-core structures and delayed DNA relaxation. These dynamics diverged from simple exponential relaxation due to persistent entanglements and compressed regions. The findings highlight the significance of crowding effects and viscosity in influencing polymer dynamics, providing insights relevant to biological systems and synthetic applications, such as drug delivery and gene therapy.

SUMMARY: During embryonic development, cell behaviors need to be tightly regulated in time and space. Yet, how the temporal and spatial regulations of cell behaviors are interconnected during embryonic development remains elusive. To address this, we turned to zebrafish gastrulation, the process where dynamic cell behaviors generate the three principal germ layers of the early embryo. Here, we show that hoxb cluster genes are expressed in a temporally collinear manner at the blastoderm margin of the embryo to regulate the timing of mesoderm and endoderm (mesendoderm) cell ingression. Under- or over-expression of hoxb genes perturb the timing of mesendoderm cell ingression and, consequently, the positioning of these cells along the forming anterior-posterior body axis. Finally, we found that hoxb genes control the timing of mesendoderm ingression by regulating cell surface fluctuations. Thus, hoxb genes interconnect the temporal and spatial pattern of cell behavior in the early embryo by controlling cell surface fluctuations. Highlights: hoxb gene expression shows temporal collinearity at the blastoderm margin during zebrafish gastrulation. Temporal collinear expression of hoxb genes at the blastoderm margin regulates the timing of cell ingression and delineates spatial collinearity after gastrulation. hoxb genes regulate cell surface fluctuations and bleb formation at the blastoderm margin during cell ingression Graphical hoxb genes are expressed at the blastoderm margin in a temporally collinear manner and determine the timing of mesendoderm ingression by triggering dynamic cell surface fluctuations and cell blebbing. This results in a spatial collinearity of mesendoderm cell positioning along the anterior-posterior extent of the forming body axis with early ingressing cells being positioned more anteriorly than late ingressing cells.

The dynamic properties of the heart differ based on the regions that effectively circulate blood throughout the body with each heartbeat. These properties, including the inter-beat interval (IBI) of autonomous beat activity, are retained even in in vitro tissue fragments. However, details of beat dynamics have not been well analyzed, particularly at the sub-mm scale, although such dynamics of size are important for regenerative medicine and computational studies of the heart. We analyzed the beat dynamics in sub-mm tissue fragments from atria and ventricles of hearts obtained from chick embryos over a period of 40 h. The IBI and contraction speed differed by region and atrial fragments retained their values for a longer time. The major finding of this study is synchronization of these fragment pairs physically attached to each other. The probability of achieving this and the time required differ for regional pairs: atrium–atrium, ventricle–ventricle, or atrium–ventricle. Furthermore, the time required to achieve 1:1 synchronization does not depend on the proximity of initial IBI of paired fragments. Various interesting phenomena, such as 1:n synchronization and a reentrant-like beat sequence, are revealed during synchronization. Finally, our observation of fragment dynamics indicates that mechanical motion itself contributes to the synchronization of atria.