The mechanical interplay between contractility and mechanosensing in striated muscles is of fundamental importance for tissue morphogenesis, load adaptation, and disease progression, but remains poorly understood. In this study, we investigate the dependence of contractile force generation of cardiac and skeletal muscle on environmental stiffness. Using in vitro engineered muscle micro-tissues that are attached to flexible elastic pillars, we vary the stiffness of the microenvironment over three orders of magnitude and study its effect on contractility. We find that the active contractile force upon electrical stimulation of both cardiac and skeletal micro-tissues increases with environmental stiffness according to a strong power-law relationship. To explore the role of adhesion-mediated mechanotransduction processes, we deplete the focal adhesion protein β-parvin in skeletal micro-tissues. This reduces the absolute contractile force but leaves the mechanoresponsiveness unaffected. Our findings highlight the influence of external stiffness on the adaptive behavior of muscle tissue and shed light on the complex mechanoadaptation processes in striated muscle.### Competing Interest StatementThe authors have declared no competing interest.

3D-bioprinting is a promising technology to produce human tissues as drug screening tool or for organ repair. However, direct printing of living cells has proven difficult. Here, we present a method to directly 3D-bioprint human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes embedded in a collagen-hyaluronic acid ink generating centimeter-sized functional ring- and ventricle-shaped cardiac tissues in an accurate and reproducible manner. The printed tissues contained hiPSC-derived cardiomyocytes with well-organized sarcomeres and exhibited spontaneous and regular contractions, which persisted for several months and were able to contract against passive resistance. Importantly, beating frequencies of the printed cardiac tissues could be modulated by pharmacological stimulation. This approach opens up new possibilities for generating complex functional cardiac tissues as models for advanced drug screening or as tissue grafts for organ repair or replacement.

During bioprinting, cells are suspended in a viscous bioink and extruded under pressure through small diameter printing needles. The combination of high pressure and small needle diameter exposes cells to considerable shear stress, which can lead to cell damage and death. Approaches to monitor and control shear stress-induced cell damage are currently not well established. To visualize the effects of printing-induced shear stress on plasma membrane integrity, we add FM 1-43 to the bioink, a styryl dye that becomes fluorescent when bound to lipid membranes, such as the cellular plasma membrane. Upon plasma membrane disruption, the dye enters the cell and also stains intracellular membranes. Extrusion of alginate-suspended NIH/3T3 cells through a 200 µm printing needle led to an increased FM 1-43 incorporation at high pressure, demonstrating that typical shear stresses during bioprinting can transiently damage the plasma membrane. Cell imaging in a microfluidic channel confirmed that FM 1-43 incorporation is caused by cell strain. Notably, high printing pressure also impaired cell survival in bioprinting experiments. Using cell types of different stiffnesses, we find that shear stress-induced cell strain, FM 1-43 incorporation and cell death were reduced in stiffer compared to softer cell types and demonstrate that cell damage and death correlate with shear stress-induced cell deformation. Importantly, supplementation of the suspension medium with physiological concentrations of CaCl2 greatly reduced shear stress-induced cell damage and death but not cell deformation. As the sudden influx of calcium ions is known to induce rapid cellular vesicle exocytosis and subsequent actin polymerization in the cell cortex, we hypothesize that calcium supplementation facilitates the rapid resealing of plasma membrane damage sites. We recommend that bioinks should be routinely supplemented with physiological concentrations of calcium ions to reduce shear stress-induced cell damage and death during extrusion bioprinting.

Cardiac tissue engineering is a promising strategy to generate human cardiac tissues for modeling cardiac diseases, screening for therapeutic drugs, and repairing the injured heart. Yet, several issues remain to be resolved including the generation of tissues with high cardiomyocyte density. Here, it is shown that the integration of the glycogen synthase kinase-3β inhibitor CHIR99021 in collagen I hydrogels promotes proliferation of human-induced pluripotent stem cell-derived (hiPSC) cardiomyocytes post-fabrication improving contractility of and calcium flow in engineered 3D cardiac microtissues. CHIR99021 has no effect on the gelation kinetics or the mechanical properties of collagen I hydrogels. Analysis of cell density and proliferation based on Ki-67 staining indicates that integration of CHIR99021 together with external CHIR99021 stimulation increases hiPSC-cardiomyocyte number by ≈2-fold within 7 d post-fabrication. Analysis of the contractility of engineered cardiac tissues after another 3 d in the absence of external CHIR99021 shows that CHIR99021-induced hiPSC-cardiomyocyte proliferation results in synchronized calcium flow, rhythmic beating, increased speed of contraction and contraction amplitude, and reduced peak-to-peak time. The CHIR99021-stimulated engineered cardiac microtissues exhibit spontaneous rhythmic contractions for at least 35 d. Collectively, the data demonstrate the potential of induced cardiomyocyte proliferation to enhance engineered cardiac microtissues by increasing cardiomyocyte density.

Abstract Background Cardiac tissue engineering is an effective strategy to generate tissues for drug testing and disease modelling as well as for cardiac repair. Tissues produced by casting show good functionality and advanced maturation, but do not replicate the native tissue architecture and hierarchy. Additive manufacturing technologies, such as 3D bioprinting, enable the generation of hierarchically structured tissues with complex geometries. This technology has been used previously to generate models of the heart. However, these approaches either showed limited tissue functionality or required a two-step procedure using a structural and a cell-laden bioink. Purpose Here, we aimed to develop a collagen-based bioink, which enables direct 3D-bioprinting of hiPSC-derived cardiomyocytes and supports the formation of functional cardiac tissue. Methods To generate cardiac tissues, a commercial pneumatic extrusion bioprinter with custom modifications to enable passive cooling of the bioink was used. Gelatin/gum arabic microparticles were obtained through complex coacervation, compacted by centrifugation and utilized as support bath. Cardiomyocytes were differentiated in 2D monolayer and expanded by CHIR99021-treatment and regular passaging. Cells were encapsulated in a rat collagen-I based bioink and printed into support bath prior to gelation. After bioink gelation at 37°C, support bath was removed, and constructs cultivated free-floating for up to 30 days. Results We printed ring-shaped cardiac tissues measuring 5 x 5 x 1 mm, which remained stable over the course of cultivation. First contractions were observed after three days, which increased in magnitude and synchronized across the tissue with prolonged culture. HiPSC-cardiomyocytes displayed striated sarcomeres and were responsive to pharmacological stimulation. In addition, using two distinct bioinks, multi-layered constructs were generated. Conclusion 3D-bioprinting is a promising tool to generate engineered cardiac tissues with complex geometries and improved functionality through designed hierarchy. Our collagen-based bioink and associated printing strategy enables the fabrication of Collagen-based contractile cardiac tissues in a direct manner. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Contractions of printed cardiac tissue

Materials made of recombinant spider silk proteins are promising candidates for cardiac tissue engineering, and their suitability has so far been investigated utilizing primary rat cardiomyocytes. Herein, we expanded the tool box of available spider silk variants and demonstrated for the first time that human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes attach, contract, and respond to pharmacological treatment using phenylephrine and verapamil on explicit spider silk films. The hiPSC-cardiomyocytes contracted for at least 14 days on films made of positively charged engineered Araneus diadematus fibroin 4 (eADF4(κ16)) and three different arginyl-glycyl-aspartic acid (RGD)-tagged spider silk variants (positively or negatively charged and uncharged). Notably, hiPSC-cardiomyocytes exhibited different morphologies depending on the spider silk variant used, with less spreading and being smaller on films made of eADF4(κ16) than on RGD-tagged spider silk films. These results indicate that spider silk engineering is a powerful tool to provide new materials suitable for hiPSC-based cardiac tissue engineering.

Abstract Background/Introduction Cardiac tissue engineering is a promising strategy to generate human cardiac tissues for modelling cardiac diseases, screening for therapeutic drugs, and repairing the injured heart. Yet, several issues remain to be resolved including the generation of tissues with high cardiomyocyte density. Purpose Determining the effects of the induction of human-induced pluripotent stem cell-derived (hiPSC) cardiomyocyte proliferation post-fabrication. Methods hiPSCs were differentiated into cardiomyocytes, embedded with or without CHIR990121 at three concentrations in a collagen pre-gel, and cast. The engineered cardiac tissues were then cultured in the absence or presence of CHIR99021 for up to 35 days. Hydrogels and engineered cardiac tissues were analysed utilizing rheology and assays to determine viability, proliferation, calcium flow, and contractility. Results Here, we show that the integration of CHIR99021 in collagen I hydrogels promotes proliferation of hiPSC-cardiomyocytes post-fabrication improving contractility of and calcium flow in engineered cardiac tissues. Presence of CHIR99021 has no effect on the gelation kinetic or the mechanical properties of collagen I hydrogels. Analysis of cell density and proliferation based on Ki-67 staining indicates that integration of CHIR99021 together with external CHIR99021 stimulation increases hiPSC-cardiomyocyte number by ∼2-fold within 7 days post-fabrication. Analysis of the contractility of engineered cardiac tissues after another 3 days in the absence of external CHIR99021 shows that CHIR99021-induced hiPSC-cardiomyocyte proliferation results in synchronized calcium flow, rhythmic beating, increases speed of contraction and contraction amplitude, and reduces peak-to-peak time. The CHIR99021-stimulated engineered cardiac tissues exhibited spontaneous rhythmic contractions for at least 35 days. Conclusion Collectively, our data demonstrate the potential of induced cardiomyocyte proliferation to enhance engineered cardiac tissues by increasing cardiomyocyte density and reducing arrhythmia. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Human pluripotent stem cells (hPSCs) hold great promise for applications in cell therapy and drug screening in the cardiovascular field. Bone morphogenetic protein 4 (BMP4) is key for early cardiac mesoderm induction in hPSC and subsequent cardiomyocyte derivation. Small-molecular BMP4 mimetics may help to standardize cardiomyocyte derivation from hPSCs. Based on observations that chalcones can stimulate BMP4 signaling pathways, we hypothesized their utility in cardiac mesoderm induction. To test this, we set up a two-tiered screening strategy, (1) for directed differentiation of hPSCs with commercially available chalcones (4'-hydroxychalcone [4'HC] and Isoliquiritigen) and 24 newly synthesized chalcone derivatives, and (2) a functional screen to assess the propensity of the obtained cardiomyocytes to self-organize into contractile engineered human myocardium (EHM). We identified 4'HC, 4-fluoro-4'-methoxychalcone, and 4-fluoro-4'-hydroxychalcone as similarly effective in cardiac mesoderm induction, but only 4'HC as an effective replacement for BMP4 in the derivation of contractile EHM-forming cardiomyocytes.

Significance: Heart failure is among the leading causes of morbidity worldwide with a 5-year mortality rate of ∼50%. Therefore, major efforts are invested to reduce heart damage upon injury or maintain and at best restore heart function. Recent Advances: In clinical trials, acellular constructs succeeded in improving cardiac function by stabilizing the infarcted heart. In addition, strategies utilizing stem-cell-derived cardiomyocytes have been developed to improve heart function postmyocardial infarction in small and large animal models. These strategies range from injection of cell-laden hydrogels to unstructured hydrogel-based and complex biofabricated cardiac patches. Importantly, novel methods have been developed to promote differentiation of stem-cell-derived cardiomyocytes to prevascularized cardiac patches. Critical Issues: Despite substantial progress in vascularization strategies for heart-on-the-chip technologies, little advance has been made in generating vascularized cardiac patches with clinically relevant dimensions. In addition, proper electrical coupling between engineered and host tissue to prevent and/or eliminate arrhythmia remains an unresolved issue. Finally, despite advanced approaches to include hierarchical structures in cardiac tissues, engineered tissues do not generate forces in the range of native adult cardiac tissue. Future Directions: It involves utilizing novel materials and advancing biofabrication strategies to generate prevascularized three-dimensional multicellular constructs of clinical relevant size; inclusion of hierarchical structures, electroconductive materials, and biologically active factors to enhance cardiomyocyte differentiation for optimized force generation and vascularization; optimization of bioreactor strategies for tissue maturation. Antioxid. Redox Signal. 35, 143-162.

The switch from centrosomal microtubule-organizing centers (MTOCs) to non-centrosomal MTOCs during differentiation is poorly understood. Here, we identify AKAP6 as key component of the nuclear envelope MTOC. In rat cardiomyocytes, AKAP6 anchors centrosomal proteins to the nuclear envelope through its spectrin repeats, acting as an adaptor between nesprin-1α and Pcnt or AKAP9. In addition, AKAP6 and AKAP9 form a protein platform tethering the Golgi to the nucleus. Both Golgi and nuclear envelope exhibit MTOC activity utilizing either AKAP9, or Pcnt-AKAP9, respectively. AKAP6 is also required for formation and activity of the nuclear envelope MTOC in human osteoclasts. Moreover, ectopic expression of AKAP6 in epithelial cells is sufficient to recruit endogenous centrosomal proteins. Finally, AKAP6 is required for cardiomyocyte hypertrophy and osteoclast bone resorption activity. Collectively, we decipher the MTOC at the nuclear envelope as a bi-layered structure generating two pools of microtubules with AKAP6 as a key organizer.

Motile cilia serve vital functions in development, homeostasis and regeneration. We recently demonstrated that TAp73 is an essential transcriptional regulator of respiratory multiciliogenesis. Here, we show that TAp73 is expressed in multiciliated cells (MCCs) of diverse tissues. Analysis of TAp73 mutant animals revealed that TAp73 regulates Foxj1, Rfx2, Rfx3 , axonemal dyneins Dnali1 and Dnai1 , plays a pivotal role in the generation of MCCs in male and female reproductive ducts, and contributes to fertility. However, the function of MCCs in the brain appears to be preserved despite the loss of TAp73 , and robust activity of cilia-related networks is maintained in the absence of TAp73 . Notably, TAp73 loss leads to distinct changes in ciliogenic microRNAs: miR34bc expression is reduced, whereas the miR449 cluster is induced in diverse multiciliated epithelia. Among different MCCs, choroid plexus (CP) epithelial cells in the brain display prominent miR449 expression, whereas brain ventricles exhibit significant increase in miR449 levels along with an increase in the activity of ciliogenic E2F4/MCIDAS circuit in TAp73 mutant animals. Conversely, E2F4 induces robust transcriptional response from miR449 genomic regions. To address whether increased miR449 levels in the brain maintain the multiciliogenesis program in the absence of TAp73 , we deleted both TAp73 and miR449 in mice. Although loss of miR449 alone led to a mild ciliary defect in the CP, more pronounced ciliary defects and hydrocephalus were observed in the brain lacking both TAp73 and miR449 . In contrast, miR449 loss in other MCCs failed to enhance ciliary defects associated with TAp73 loss. Together, our study shows that, in addition to the airways, TAp73 is essential for generation of MCCs in male and female reproductive ducts, whereas miR449 and TAp73 complement each other to support multiciliogenesis and CP development in the brain.

Introduction: Vascularization remains one of the greatest yet unmet challenges in tissue engineering. When engineered tissues are scaled up to therapeutically relevant dimensions, their demand of oxygen and nutrients can no longer be met by diffusion. Thus, there is a need for perfusable vascular structures. Hypoxia-inducible factors (HIF) act as transcriptional oxygen sensors and regulate a multitude of genes involved in adaptive processes to hypoxia, including angiogenesis. Thus, targeting HIFs is a promising strategy to induce vascularization of engineered tissues.Areas covered: Here we review current vascularization strategies and summarize the present knowledge regarding activation of HIF signaling by ions, iron chelating agents, -Ketoglutarate (KG) analogues, and the lipid-lowering drug simvastatin to induce angiogenesis. Specifically, we focus on the incorporation of HIF-activating agents into biomaterials and scaffolds for controlled release.Expert opinion: Vascularization of tissue constructs through activation of upstream regulators of angiogenesis offers advantages but also suffers from drawbacks. HIFs can induce a complete angiogenic program; however, this program appears to be too slow to vascularize larger constructs before cell death occurs. It is therefore crucial that HIF-activation is combined with cell protective strategies and prevascularization techniques to obtain fully vascularized, vital tissues of therapeutically relevant dimensions.