Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. METHODS: We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. RESULTS: Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. CONCLUSIONS: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.

Mechanical confinement via a PEG/Collagen interpenetrating network inhibits behavior characteristic of malignant cells in the triple negative breast cancer cell line MDA.MB.231.

To decouple the effects of collagen fiber density and network mechanics on cancer cell behavior, we describe a highly tunable in vitro 3D interpenetrating network (IPN) consisting of a primary fibrillar collagen network reinforced by a secondary visible light-mediated thiol-ene poly(ethylene glycol) (PEG) network. This PEG/Collagen IPN platform is cytocompatible, inherently bioactive via native cellular adhesion sites, and mechanically tunable over several orders of magnitude-mimicking both healthy and cancerous breast tissue. Furthermore, we use the PEG/Collagen IPN platform to investigate the effect of mechanical confinement on cancer cell behavior as it is hypothesized that cells within tumors that have yet to invade into the surrounding tissue experience mechanical confinement. We find that mechanical confinement via the IPN impairs behavior characteristic of malignant cells (i.e., viability, proliferation, and cellular motility) in the triple negative breast cancer cell line MDA.MB.231, and is more effective than removal of soluble growth signals. The PEG/Collagen IPN platform is a useful tool for studying mechanotransductive signaling pathways and motivates further investigation into the role of mechanical confinement in cancer progression. STATEMENT OF SIGNIFICANCE: In this study, we have developed, optimized, and applied a novel 3D in vitro cell culture platform composed of an interpenetrating network (IPN) that is both mechanically tunable and inherently bioactive. The IPN consists of a primary fibrillar collagen type-1 network reinforced by a secondary thiol-ene poly(ethylene glycol) (PEG) network. The IPNs are formed via a novel strategy in which cell-laden collagen gels are formed first, and soluble PEG monomers are added later and crosslinked via visible light. This approach ensures that the collagen gels contain a fibrillar architecture similar to the collagen architecture present in vivo. We applied our IPN platform to study the effect of mechanical confinement on cancer cell behavior and found that it inhibits malignant-like behavior.

Electrospun Nanofiber Scaffolds and Their Hydrogel Composites for the Engineering and Regeneration of Soft Tissues.

Electrospinning has emerged as a simple, elegant, and scalable technique that can be used to fabricate polymeric nanofibers. Pure polymers as well as blends and composites of both natural and synthetic ones have been successfully electrospun into nanofiber matrices for many biomedical applications. Tissue-engineered medical implants, such as polymeric nanofiber scaffolds, are potential alternatives to autografts and allografts, which are short in supply and carry risks of disease transmission. These scaffolds have been used to engineer various soft tissues, including connective tissues, such as skin, ligament, and tendon, as well as nonconnective ones, such as vascular, muscle, and neural tissue. Electrospun nanofiber matrices show morphological similarities to the natural extracellular matrix (ECM), characterized by ultrafine continuous fibers, high surface-to-volume ratios, high porosities, and variable pore-size distributions. The physiochemical properties of nanofiber matrices can be controlled by manipulating electrospinning parameters so that they meet the requirements of a specific application.Nanostructured implants show improved biological performance over bulk materials in aspects of cellular infiltration and in vivo integration, taking advantage of unique quantum, physical, and atomic properties. Furthermore, the topographies of such scaffolds has been shown to dictate cellular attachment, migration, proliferation, and differentiation, which are critical in engineering complex functional tissues with improved biocompatibility and functional performance. This chapter discusses the use of the electrospinning technique in the fabrication of polymer nanofiber scaffolds utilized for the regeneration of soft tissues. Selected scaffolds will be seeded with human mesenchymal stem cells (hMSCs), imaged using scanning electron and confocal microscopy, and then evaluated for their mechanical properties as well as their abilities to promote cell adhesion, proliferation , migration, and differentiation.