Distinct phenotypes induced by acute hypoxia and TGF-ß1 in human adult cardiac fibroblasts.

Myocardial infarction (MI) causes hypoxic injury to downstream myocardial tissue, which initiates a wound healing response that replaces injured myocardial tissue with a scar. Wound healing is a complex process that consists of multiple phases, in which many different stimuli induce cardiac fibroblasts to differentiate into myofibroblasts and deposit new matrix. While this process is necessary to replace necrotic tissue, excessive and unresolved fibrosis is common post-MI and correlated with heart failure. Therefore, defining how cardiac fibroblast phenotypes are distinctly regulated by stimuli that are prevalent in the post-MI microenvironment, such as hypoxia and transforming growth factor-beta (TGF-ß), is essential for understanding and ultimately mitigating pathological fibrosis. In this study, we acutely treated primary human adult cardiac fibroblasts with TGF-ß1 or hypoxia and then characterized their phenotype through immunofluorescence, quantitative RT-PCR, and proteomic analysis. We found that fibroblasts responded to low oxygen with increased localization of hypoxia inducible factor 1 (HIF-1) to the nuclei after 4h, which was followed by increased gene expression of vascular endothelial growth factor A (VEGFA), a known target of HIF-1, by 24h. Both TGF-ß1 and hypoxia inhibited proliferation after 24h. TGF-ß1 treatment also upregulated various fibrotic pathways. In contrast, hypoxia caused a reduction in several protein synthesis pathways, including collagen biosynthesis. Collectively, these data suggest that TGF-ß1, but not acute hypoxia, robustly induces the differentiation of human cardiac fibroblasts into myofibroblasts. Discerning the overlapping and distinctive outcomes of TGF-ß1 and hypoxia treatment is important for elucidating their roles in fibrotic remodeling post-MI and provides insight into potential therapeutic targets.

Hypoxic-Normoxic Crosstalk Activates Pro-Inflammatory Signaling in Human Cardiac Fibroblasts and Myocytes in a Post-Infarct Myocardium on a Chip.

Myocardial infarctions locally deprive myocardium of oxygenated blood and cause immediate cardiac myocyte necrosis. Irreparable myocardium is then replaced with a scar through a dynamic repair process that is an interplay between hypoxic cells of the infarct zone and normoxic cells of adjacent healthy myocardium. In many cases, unresolved inflammation or fibrosis occurs for reasons that are incompletely understood, increasing the risk of heart failure. Crosstalk between hypoxic and normoxic cardiac cells is hypothesized to regulate mechanisms of repair after a myocardial infarction. To test this hypothesis, microfluidic devices are fabricated on 3D printed templates for co-culturing hypoxic and normoxic cardiac cells. This system demonstrates that hypoxia drives human cardiac fibroblasts toward glycolysis and a pro-fibrotic phenotype, similar to the anti-inflammatory phase of wound healing. Co-culture with normoxic fibroblasts uniquely upregulates pro-inflammatory signaling in hypoxic fibroblasts, including increased secretion of tumor necrosis factor alpha (TNF-α). In co-culture with hypoxic fibroblasts, normoxic human induced pluripotent stem cell (hiPSC)-derived cardiac myocytes also increase pro-inflammatory signaling, including upregulation of interleukin 6 (IL-6) family signaling pathway and increased expression of IL-6 receptor. Together, these data suggest that crosstalk between hypoxic fibroblasts and normoxic cardiac cells uniquely activates phenotypes that resemble the initial pro-inflammatory phase of post-infarct wound healing.

User-friendly microfluidic system reveals native-like morphological and transcriptomic phenotypes induced by shear stress in proximal tubule epithelium.

Drug-induced nephrotoxicity is a leading cause of drug attrition, partly due to the limited relevance of pre-clinical models of the proximal tubule. Culturing proximal tubule epithelial cells (PTECs) under fluid flow to mimic physiological shear stress has been shown to improve select phenotypes, but existing flow systems are expensive and difficult to implement by non-experts in microfluidics. Here, we designed and fabricated an accessible and modular flow system for culturing PTECs under physiological shear stress, which induced native-like cuboidal morphology, downregulated pathways associated with hypoxia, stress, and injury, and upregulated xenobiotic metabolism pathways. We also compared the expression profiles of shear-dependent genes in our in vitro PTEC tissues to that of ex vivo proximal tubules and observed stronger clustering between ex vivo proximal tubules and PTECs under physiological shear stress relative to PTECs under negligible shear stress. Together, these data illustrate the utility of our user-friendly flow system and highlight the role of shear stress in promoting native-like morphological and transcriptomic phenotypes in PTECs in vitro, which is critical for developing more relevant pre-clinical models of the proximal tubule for drug screening or disease modeling.

A myocardial infarct border-zone-on-a-chip demonstrates distinct regulation of cardiac tissue function by an oxygen gradient.

After a myocardial infarction, the boundary between the injured, hypoxic tissue and the adjacent viable, normoxic tissue, known as the border zone, is characterized by an oxygen gradient. Yet, the impact of an oxygen gradient on cardiac tissue function is poorly understood, largely due to limitations of existing experimental models. Here, we engineered a microphysiological system to controllably expose engineered cardiac tissue to an oxygen gradient that mimics the border zone and measured the effects of the gradient on electromechanical function and the transcriptome. The gradient delayed calcium release, reuptake, and propagation; decreased diastolic and peak systolic stress; and increased expression of inflammatory cascades that are hallmarks of myocardial infarction. These changes were distinct from those observed in tissues exposed to uniform normoxia or hypoxia, demonstrating distinct regulation of cardiac tissue phenotypes by an oxygen gradient. Our border-zone-on-a-chip model advances functional and mechanistic insight into oxygen-dependent cardiac tissue pathophysiology.

Engineering the Cellular Microenvironment of Post-infarct Myocardium on a Chip.

Myocardial infarctions are one of the most common forms of cardiac injury and death worldwide. Infarctions cause immediate necrosis in a localized region of the myocardium, which is followed by a repair process with inflammatory, proliferative, and maturation phases. This repair process culminates in the formation of scar tissue, which often leads to heart failure in the months or years after the initial injury. In each reparative phase, the infarct microenvironment is characterized by distinct biochemical, physical, and mechanical features, such as inflammatory cytokine production, localized hypoxia, and tissue stiffening, which likely each contribute to physiological and pathological tissue remodeling by mechanisms that are incompletely understood. Traditionally, simplified two-dimensional cell culture systems or animal models have been implemented to elucidate basic pathophysiological mechanisms or predict drug responses following myocardial infarction. However, these conventional approaches offer limited spatiotemporal control over relevant features of the post-infarct cellular microenvironment. To address these gaps, Organ on a Chip models of post-infarct myocardium have recently emerged as new paradigms for dissecting the highly complex, heterogeneous, and dynamic post-infarct microenvironment. In this review, we describe recent Organ on a Chip models of post-infarct myocardium, including their limitations and future opportunities in disease modeling and drug screening.

Contact photolithography-free integration of patterned and semi-transparent indium tin oxide stimulation electrodes into polydimethylsiloxane-based heart-on-a-chip devices for streamlining physiological recordings.

Controlled electrical stimulation is essential for evaluating the physiology of cardiac tissues engineered in heart-on-a-chip devices. However, existing stimulation techniques, such as external platinum electrodes or opaque microelectrode arrays patterned on glass substrates, have limited throughput, reproducibility, or compatibility with other desirable features of heart-on-a-chip systems, such as the use of tunable culture substrates, imaging accessibility, or enclosure in a microfluidic device. In this study, indium tin oxide (ITO), a conductive, semi-transparent, and biocompatible material, was deposited onto glass and polydimethylsiloxane (PDMS)-coated coverslips as parallel or point stimulation electrodes using laser-cut tape masks. ITO caused substrate discoloration but did not prevent brightfield imaging. ITO-patterned substrates were microcontact printed with arrayed lines of fibronectin and seeded with neonatal rat ventricular myocytes, which assembled into aligned cardiac tissues. ITO deposited as parallel or point electrodes was connected to an external stimulator and used to successfully stimulate micropatterned cardiac tissues to generate calcium transients or propagating calcium waves, respectively. ITO electrodes were also integrated into the cantilever-based muscular thin film (MTF) assay to stimulate and quantify the contraction of micropatterned cardiac tissues. To demonstrate the potential for multiple ITO electrodes to be integrated into larger, multiplexed systems, two sets of ITO electrodes were deposited onto a single substrate and used to stimulate the contraction of distinct micropatterned cardiac tissues independently. Collectively, these approaches for integrating ITO electrodes into heart-on-a-chip devices are relatively facile, modular, and scalable and could have diverse applications in microphysiological systems of excitable tissues.

Mitochondrial division inhibitor 1 (mdivi-1) increases oxidative capacity and contractile stress generated by engineered skeletal muscle.

In skeletal muscle fibers, mitochondria are densely packed adjacent to myofibrils because adenosine triphosphate (ATP) is needed to fuel sarcomere shortening. However, despite this close physical and biochemical relationship, the effects of mitochondrial dynamics on skeletal muscle contractility are poorly understood. In this study, we analyzed the effects of Mitochondrial Division Inhibitor 1 (mdivi-1), an inhibitor of mitochondrial fission, on the structure and function of both mitochondria and myofibrils in skeletal muscle tissues engineered on micromolded gelatin hydrogels. Treatment with mdivi-1 did not alter myotube morphology, but did increase the mitochondrial turbidity and oxidative capacity, consistent with reduced mitochondrial fission. Mdivi-1 also significantly increased basal, twitch, and tetanus stresses, as measured using the Muscular Thin Film (MTF) assay. Finally, mdivi-1 increased sarcomere length, potentially due to mdivi-1-induced changes in mitochondrial volume and compression of myofibrils. Together, these results suggest that mdivi-1 increases contractile stress generation, which may be caused by an increase in maximal respiration and/or sarcomere length due to increased volume of individual mitochondria. These data reinforce that mitochondria have both biochemical and biomechanical roles in skeletal muscle and that mitochondrial dynamics can be manipulated to alter muscle contractility.