Brenda Russell

Heart failure contributes to Duchenne muscular dystrophy (DMD), which arises from mutations that ablate dystrophin, rendering the plasma membrane prone to disruption. Cardiomyocyte membrane breakdown in patients with DMD yields a serum injury profile similar to other types of myocardial injury with the release of creatine kinase and troponin isoforms. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are highly useful but can be improved. We generated hiPSC-CMs from a patient with DMD and subjected these cells to equibiaxial mechanical strain to mimic in vivo stress. Compared to healthy cells, DMD hiPSC-CMs demonstrated greater susceptibility to equibiaxial strain after 2 h at 10% strain. We generated an aptamer-based profile of proteins released from hiPSC-CMs both at rest and subjected to strain and identified a strong correlation in the mechanical stress-induced proteome from hiPSC-CMs and serum from patients with DMD. We exposed hiPSC-CMs to recombinant annexin A6, a protein resealing agent, and found reduced biomarker release in DMD and control hiPSC-CMs subjected to strain. Thus, the application of mechanical strain to hiPSC-CMs produces a model that reflects an in vivo injury profile, providing a platform to assess pharmacologic intervention.

Loss of myocardial mass in a neonatal rat cardiomyocyte culture is studied to determine whether there is a distinguishable cellular response based on the origin of mechano‐signals. The approach herein compares the sarcomeric assembly and disassembly processes in heart cells by imposing mechano‐signals at the interface with the extracellular matrix (extrinsic) and at the level of the myofilaments (intrinsic). Experiments compared the effects of imposed internal (inside/out) and external (outside/in) loading and unloading on modifications in neonatal rat cardiomyocytes. Unloading of the cellular substrate by myosin inhibition (1 μm mavacamten), or cessation of cyclic strain (1 Hz, 10% strain) after preconditioning, led to significant disassembly of sarcomeric α‐actinin by 6 h. In myosin inhibition, this was accompanied by redistribution of intracellular poly‐ubiquitin K48 to the cellular periphery relative to the poly‐ubiquitin K48 reservoir at the I‐band. Moreover, loading and unloading of the cellular substrate led to a three‐fold increase in post‐translational modifications (PTMs) when compared to the myosin‐specific activation or inhibition. Specifically, phosphorylation increased with loading while ubiquitination increased with unloading, which may involve extracellular signal‐regulated kinase 1/2 and focal adhesion kinase activation. The identified PTMs, including ubiquitination, acetylation, and phosphorylation, are proposed to modify internal domains in α‐actinin to increase its propensity to bind F‐actin. These results demonstrate a link between mechanical feedback and sarcomere protein homeostasis via PTMs of α‐actinin that exemplify how cardiomyocytes exhibit differential responses to the origin of force. The implications of sarcomere regulation governed by PTMs of α‐actinin are discussed with respect to cardiac atrophy and heart failure.

Age‐related wild‐type transthyretin amyloidosis (wtATTR) is characterized by systemic deposition of amyloidogenic fibrils of misfolded transthyretin (TTR) in the connective tissue of many organs. In the heart, this leads to age‐related heart failure with preserved ejection fraction (HFpEF). The hypothesis tested is that TTR deposited in vitro disrupts cardiac myocyte cell‐to‐cell and cell‐to‐matrix adhesion complexes, resulting in altered calcium handling, force generation, and sarcomeric disorganization. Human iPSC‐derived cardiomyocytes and neonatal rat ventricular myocytes (NRVMs), when grown on TTR‐coated polymeric substrata mimicking the stiffness of the healthy human myocardium (10 kPa), had decreased contraction and relaxation velocities as well as decreased force production measured using traction force microscopy. Both NRVMs and adult mouse atrial cardiomyocytes had altered calcium kinetics with prolonged transients when cultured on TTR fibril‐coated substrates. Furthermore, NRVMs grown on stiff (~GPa), flat or microgrooved substrates coated with TTR fibrils exhibited significantly decreased intercellular electrical coupling as shown by FRAP dynamics of cells loaded with the gap junction‐permeable dye calcein‐AM, along with decreased gap junction content as determined by quantitative connexin 43 staining. Significant sarcomeric disorganization and loss of sarcomere content, with increased ubiquitin localization to the sarcomere, were seen in NRVMs on various TTR fibril‐coated substrata. TTR presence decreased intercellular mechanical junctions as evidenced by quantitative immunofluorescence staining of N‐cadherin and vinculin. Current therapies for wtATTR are cost‐prohibitive and only slow the disease progression; therefore, better understanding of cardiomyocyte maladaptation induced by TTR amyloid may identify novel therapeutic targets.

All cells sense force and build their cytoskeleton to optimize function. How is this achieved? Two major systems are involved. The first is that load deforms specific protein structures in a proportional and orientation-dependent manner. The second is post-translational modification of proteins as a consequence of signaling pathway activation. These two processes work together in a complex way so that local subcellular assembly as well as overall cell function are controlled. This review discusses many cell types but focuses on striated muscle. Detailed information is provided on how load deforms the structure of proteins in the focal adhesions and filaments, using α-actinin, vinculin, talin, focal adhesion kinase, LIM domain-containing proteins, filamin, myosin, titin, and telethonin as examples. Second messenger signals arising from external triggers are distributed throughout the cell causing post-translational or chemical modifications of protein structures, with the actin capping protein CapZ and troponin as examples. There are numerous unanswered questions of how mechanical and chemical signals are integrated by muscle proteins to regulate sarcomere structure and function yet to be studied. Therefore, more research is needed to see how external triggers are integrated with local tension generated within the cell. Nonetheless, maintenance of tension in the sarcomere is the essential and dominant mechanism, leading to the well-known phrase in exercise physiology: “use it or lose it.”

Transthyretin (TTR) cardiac amyloidosis involves deposition of fibrils of misfolded TTR in the aging human heart, leading to cardiac dysfunction and heart failure. Our novel in vitro studies show that TTR fibrils alter primary cardiac fibroblast cytoskeletal and nuclear structure and focal adhesion formation. Furthermore, both fibrillar and tetrameric TTR significantly increased cellular migration velocity and caused upregulation of inflammatory genes determined by transcriptomic RNA and protein analysis. These findings may suggest new therapeutic approaches.

A transduced mechanical signal arriving at its destination in muscle alters sarcomeric structure and function. A major question addressed is how muscle mass and tension generation are optimized to match actual performance demands so that little energy is wasted. Three cases for improved energy efficiency are examined: the troponin complex for tuning force production, control of the myosin heads in a resting state, and the Z-disc proteins for sarcomere assembly. On arrival, the regulation of protein complexes is often controlled by post-translational modification (PTM), of which the most common are phosphorylation by kinases, deacetylation by histone deacetylases and ubiquitination by E3 ligases. Another branch of signals acts not through peptide covalent bonding but via ligand interactions (e.g. Ca²⁺ and phosphoinositide binding). The myosin head and the regulation of its binding to actin by the troponin complex is the best and earliest example of signal destinations that modify myofibrillar contractility. PTMs in the troponin complex regulate both the efficiency of the contractile function to match physiologic demand for work, and muscle mass via protein degradation. The regulation of sarcomere assembly by integration of incoming signaling pathways causing the same PTMs or ligand binding are discussed in response to mechanical loading and unloading by the Z-disc proteins CapZ, α-actinin, telethonin, titin N-termini, and others. Many human mutations that lead to cardiomyopathy and heart disease occur in the proteins discussed above, which often occur at their PTM or ligand binding sites.

Cells interact intimately with complex microdomains in their extracellular matrix (ECM) and maintain a delicate balance of mechanical forces through mechanosensitive cellular components. Tissue injury results in acute degradation of the ECM and disruption of cell-ECM contacts, manifesting in loss of cytoskeletal tension, leading to pathological cell transformation and the onset of disease. Recently, microscale hydrogel constructs have been developed to provide cells with microdomains to form focal adhesion binding sites, which enable restoration of cytoskeletal tension. These synthetic anchors can recapitulate the complex 3D architecture of the native ECM to provide microtopographical cues. The mechanical deformation of proteins at the cell surface can activate signaling cascades to modulate downstream gene-level transcription, making this a unique materials-based approach for reprogramming cell behavior. An overview of the mechanisms underlying these mechanosensitive interactions in fibroblasts, stem and other cell types is provided to review their effects on cellular reprogramming. Recent investigations on the fabrication, functionalization and implementation of these materials and microtopographical features for drug testing and therapeutic applications are discussed.

Muscle adaptation is a response to physiological demand elicited by changes in mechanical load, hormones, or metabolic stress. Cytoskeletal remodeling processes in many cell types are thought to be primarily regulated by thin filament formation due to actin-binding accessory proteins, such as the actin-capping protein. Here, we hypothesize that in muscle, the actin-capping protein (named CapZ) integrates signaling by a variety of pathways, including phosphorylation and phosphatidylinositol 4,5-bisphosphate (PIP2) binding, to regulate muscle fiber growth in response to mechanical load. To test this hypothesis, we assess mechanotransduction signaling that regulates muscle growth using neonatal rat ventricular myocytes cultured on substrates with the stiffness of the healthy myocardium (10 kPa), fibrotic myocardium (100 kPa), or glass. We investigate how PIP2 signaling affects CapZ using the PIP2 sequestering agent neomycin and the effect of PKC-mediated CapZ phosphorylation using the PKC-activating drug phorbol 12-myristate 13-acetate (PMA). Molecular simulations suggest that close interactions between PIP2 and the β-tentacle of CapZ are modified by phosphorylation at T267. Fluorescence recovery after photobleaching (FRAP) demonstrates that the kinetic binding constant of CapZ to sarcomeric thin filaments in living muscle cells increases with stiffness or PMA treatment but is diminished by PIP2 reduction. Furthermore, CapZ with a deletion of the β-tentacle that lacks the phosphorylation site T267 shows increased FRAP kinetics with lack of sensitivity to PMA treatment or PIP2 reduction. Förster resonance energy transfer (FRET) probes the molecular interactions between PIP2 and CapZ, which are decreased by PIP2 availability or by the β-tentacle truncation. These data suggest that CapZ is bound to actin tightly in the idle, locked state, with little phosphorylation or PIP2 binding. However, this tight binding is loosened in growth states triggered by mechanical stimuli such as substrate stiffness, which may have relevance to fibrotic heart disease.

The stiffness of the microenvironment surrounding a cell can result in cytoskeletal remodeling, leading to altered cell function and tissue macrostructure. In this study, we tuned the stiffness of the underlying substratum on which neonatal rat cardiomyocytes were grown in culture in order to mimic normal (10 kPa), pathological stiffness of fibrotic myocardium (100 kPa), and a non‐physiological extreme (glass). Cardiomyocytes were then challenged by beta adrenergic stimulation through isoproterenol treatment to investigate the response to acute work demand for cells grown on surfaces of varying stiffness. In particular, the PKCɛ signaling pathway and its role in actin assembly dynamics were examined. Significant changes in contractile metrics were seen for cardiomyocytes grown on different surfaces, but all cells responded to isoproterenol treatment, eventually reaching similar time to peak tension. In contrast, the assembly rate of actin was significantly higher on stiff surfaces, so that only cells grown on soft surfaces were able to respond to acute isoproterenol treatment. Förster Resonance Energy Transfer of immunofluorescence on the cytoskeletal fraction of cardiomyocytes confirmed that the molecular interaction of PKCɛ with the actin capping protein, CapZ, was very low on soft substrata, but significantly increased with isoproterenol treatment, or on stiff substrata. Therefore, the stiffness of the culture surface chosen for in vitro experiments might mask the normal signaling and affect the ability to translate basic science more effectively into human therapy.

Cell migration is regulated by several mechanotransduction pathways, which consist of sensing and converting mechanical microenvironmental cues to internal biochemical cellular signals, such as protein phosphorylation and lipid signaling. While there has been significant progress in understanding protein changes in the context of mechanotransduction, lipid signaling is more difficult to investigate. In this study, physical cues of stiffness (10, 100, 400 kPa, and glass), and microrod or micropost topography were manipulated in order to reprogram primary fibroblasts and assess the effects of lipid signaling on the actin cytoskeleton. In an in vitro wound closure assay, primary cardiac fibroblast migration velocity was significantly higher on soft polymeric substrata. Modulation of PIP2 availability through neomycin treatment nearly doubled migration velocity on 10 kPa substrata, with significant increases on all stiffnesses. The distance between focal adhesions and the lamellar membrane (using wortmannin treatment to increase PIP2 via PI3K inhibition) was significantly shortest compared to untreated fibroblasts grown on the same surface. PIP2 localized to the leading edge of migrating fibroblasts more prominently in neomycin‐treated cells. The membrane‐bound protein, lamellipodin, did not vary under any condition. Additionally, fifteen micron‐high micropost topography, which blocks migration, concentrates PIP2 near to the post. Actin dynamics within stress fibers, measured by fluorescence recovery after photobleaching, was not significantly different with stiffness, microtopography, nor with drug treatment. PIP2‐modulating drugs delivered from microrod structures also affected migration velocity. Thus, manipulation of the microenvironment and lipid signaling regulatory drugs might be beneficial in improving therapeutics geared toward wound healing.