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Abstract Macrophages hold vital roles in immune defense, wound healing, and tissue homeostasis, and have the exquisite ability to sense and respond to dynamically changing cues in their microenvironment. Much of our understanding of their behavior has been derived from studies performed using in vitro culture systems, in which the cell environment can be precisely controlled. Recent advances in miniaturized culture platforms also offer the ability to recapitulate some features of the in vivo environment and analyze cellular responses at the single‐cell level. Since macrophages are sensitive to their surrounding environments, the specific conditions in both macro‐ and micro‐scale cultures likely contribute to observed responses. In this study, we investigate how the presence of neighboring cells influence macrophage activation following proinflammatory stimulation in both bulk and micro‐scale culture. We found that in bulk cultures, higher seeding density negatively regulated the average TNF‐α secretion from individual macrophages in response to inflammatory agonists, and this effect was partially caused by the reduced cell‐to‐media volume ratio. In contrast, studies conducted using microwells to isolate single cells and groups of cells revealed that increasing numbers of cells positively influences their inflammatory activation, suggesting that the absolute cell numbers in the system may be important. In addition, a single inflammatory cell enhanced the inflammatory state of a small group of cells. Overall, this work helps to better understand how variations of macroscopic and microscopic culture environments influence studies in macrophage biology and provides insight into how the presence of neighboring cells and the soluble environment influences macrophage activation. 

Cardiac adaptation to hypoxic injury is regulated by dynamic interactions between cardiomyocytes and macrophages, yet the impacts of immune phenotypes on cardiac structure and contractility remain poorly understood. To address this, we developed the immuno-heart on a chip, a novel in vitro platform to investigate cardiomyocyte–macrophage interactions under normoxic and hypoxic conditions. By integrating neonatal rat ventricular myocytes (NRVMs) and bone marrow-derived macrophages—polarized to pro-inflammatory (M1) or pro-healing (M2/M2*) phenotypes—we elucidated the dual protective and detrimental roles macrophages play in modulating cardiomyocyte cytoskeletal architecture and contractility. Pro-inflammatory stimulation reduced cardiomyocyte structural metrics (z-line length, fraction, and integrity) in normoxic co-cultures. Under hypoxia, M1-stimulated NRVM monocultures exhibited declines in cytoskeletal organization—quantified by actin and z-line orientational order parameters. Relative to monocultures, M1-stimulated co-cultures attenuated hypoxia-induced active stress declines but produced weaker normoxic stresses. In contrast, pro-healing stimulation improved normoxic z-line metrics and preserved post-hypoxia cytoskeletal organization but reduced normoxic contractility. Notably, M2-stimulated macrophages restored normoxic contractility and preserved post-hypoxia systolic stress, albeit with increased diastolic stress. RNAseq analysis of M2-stimulated co-cultures identified upregulated structural and immune pathways driving these hypoxia-induced changes. Cytokine profiles revealed stimulation-specific and density-dependent tumor necrosis factor-alpha and interleukin-10 secretion patterns. Together, these findings quantitatively link clinically relevant macrophage phenotypes and cytokines to distinct changes in cardiac structure and contractility, offering mechanistic insights into immune modulation of hypoxia-induced dysfunction. Moreover, the immuno-heart on a chip represents an innovative framework to guide the development of future therapies that integrate immune and cardiac targets to enhance patient outcomes. 

Understanding muscle contraction mechanisms is a standing challenge, and one of the approaches has been to create models of the sarcomere–the basic contractile unit of striated muscle. While these models have been successful in elucidating many aspects of muscle contraction, they fall short in explaining the energetics of functional phenomena, such as rigor, and in particular, their dependence on the concentrations of the biomolecules involved in the cross-bridge cycle. Our hypothesis posits that the stochastic time delay between ATP adsorption and ADP/Pi release in the cross-bridge cycle necessitates a modeling approach where the rates of these two reaction steps are controlled by two independent parts of the total free energy change of the hydrolysis reaction. To test this hypothesis, we built a two-filament, stochastic-mechanical half-sarcomere model that separates the energetic roles of ATP and ADP/Pi in the cross-bridge cycle’s free energy landscape. Our results clearly demonstrate that there is a nontrivial dependence of the cross-bridge cycle’s kinetics on the independent concentrations of ATP, ADP, and Pi. The simplicity of the proposed model allows for analytical solutions of the more basic systems, which provide novel insight into the dominant mechanisms driving some of the experimentally observed contractile phenomena.