Field Guide to Traction Force Microscopy.

Introduction: Traction force microscopy (TFM) is a widely used technique to measure cell contractility on compliant substrates that mimic the stiffness of human tissues. For every step in a TFM workflow, users make choices which impact the quantitative results, yet many times the rationales and consequences for making these decisions are unclear. We have found few papers which show the complete experimental and mathematical steps of TFM, thus obfuscating the full effects of these decisions on the final output. Methods: Therefore, we present this "Field Guide" with the goal to explain the mathematical basis of common TFM methods to practitioners in an accessible way. We specifically focus on how errors propagate in TFM workflows given specific experimental design and analytical choices. Results: We cover important assumptions and considerations in TFM substrate manufacturing, substrate mechanical properties, imaging techniques, image processing methods, approaches and parameters used in calculating traction stress, and data-reporting strategies. Conclusions: By presenting a conceptual review and analysis of TFM-focused research articles published over the last two decades, we provide researchers in the field with a better understanding of their options to make more informed choices when creating TFM workflows depending on the type of cell being studied. With this review, we aim to empower experimentalists to quantify cell contractility with confidence. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-024-00801-6.

Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration.

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the ß-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

Improved Cardiac Performance and Decreased Arrhythmia in Hypertrophic Cardiomyopathy With Non-ß-Blocking R-Enantiomer Carvedilol.

BACKGROUND: Hypercontractility and arrhythmia are key pathophysiologic features of hypertrophic cardiomyopathy (HCM), the most common inherited heart disease. ß-Adrenergic receptor antagonists (ß-blockers) are the first-line therapy for HCM. However, ß-blockers commonly selected for this disease are often poorly tolerated in patients, where heart-rate reduction and noncardiac effects can lead to reduced cardiac output and fatigue. Mavacamten, myosin ATPase inhibitor recently approved by the US Food and Drug Administration, has demonstrated the ability to ameliorate hypercontractility without lowering heart rate, but its benefits are so far limited to patients with left ventricular (LV) outflow tract obstruction, and its effect on arrhythmia is unknown. METHODS: We screened 21 ß-blockers for their impact on myocyte contractility and evaluated the antiarrhythmic properties of the most promising drug in a ventricular myocyte arrhythmia model. We then examined its in vivo effect on LV function by hemodynamic pressure-volume loop analysis. The efficacy of the drug was tested in vitro and in vivo compared with current therapeutic options (metoprolol, verapamil, and mavacamten) for HCM in an established mouse model of HCM (Myh6R403Q/+ and induced pluripotent stem cell (iPSC)-derived cardiomyocytes from patients with HCM (MYH7R403Q/+). RESULTS: We identified that carvedilol, a ß-blocker not commonly used in HCM, suppresses contractile function and arrhythmia by inhibiting RyR2 (ryanodine receptor type 2). Unlike metoprolol (a ß1-blocker), carvedilol markedly reduced LV contractility through RyR2 inhibition, while maintaining stroke volume through α1-adrenergic receptor inhibition in vivo. Clinically available carvedilol is a racemic mixture, and the R-enantiomer, devoid of ß-blocking effect, retains the ability to inhibit both α1-receptor and RyR2, thereby suppressing contractile function and arrhythmias without lowering heart rate and cardiac output. In Myh6R403Q/+ mice, R-carvedilol normalized hyperdynamic contraction, suppressed arrhythmia, and increased cardiac output better than metoprolol, verapamil, and mavacamten. The ability of R-carvedilol to suppress contractile function was well retained in MYH7R403Q/+ iPSC-derived cardiomyocytes. CONCLUSIONS: R-enantiomer carvedilol attenuates hyperdynamic contraction, suppresses arrhythmia, and at the same time, improves cardiac output without lowering heart rate by dual blockade of α1-adrenergic receptor and RyR2 in mouse and human models of HCM. This combination of therapeutic effects is unique among current therapeutic options for HCM and may particularly benefit patients without LV outflow tract obstruction.

Multi-scale models reveal hypertrophic cardiomyopathy MYH7 G256E mutation drives hypercontractility and elevated mitochondrial respiration.

Rationale: Over 200 mutations in the sarcomeric protein ß-myosin heavy chain (MYH7) have been linked to hypertrophic cardiomyopathy (HCM). However, different mutations in MYH7 lead to variable penetrance and clinical severity, and alter myosin function to varying degrees, making it difficult to determine genotype-phenotype relationships, especially when caused by rare gene variants such as the G256E mutation. Objective: This study aims to determine the effects of low penetrant MYH7 G256E mutation on myosin function. We hypothesize that the G256E mutation would alter myosin function, precipitating compensatory responses in cellular functions. Methods: We developed a collaborative pipeline to characterize myosin function at multiple scales (protein to myofibril to cell to tissue). We also used our previously published data on other mutations to compare the degree to which myosin function was altered. Results: At the protein level, the G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 50.9%, suggesting more myosins available for contraction. Myofibrils isolated from hiPSC-CMs CRISPR-edited with G256E (MYH7 WT/G256E ) generated greater tension, had faster tension development and slower early phase relaxation, suggesting altered myosin-actin crossbridge cycling kinetics. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. Single-cell transcriptomic and metabolic profiling demonstrated upregulation of mitochondrial genes and increased mitochondrial respiration, suggesting altered bioenergetics as an early feature of HCM. Conclusions: MYH7 G256E mutation causes structural instability in the transducer region, leading to hypercontractility across scales, perhaps from increased myosin recruitment and altered crossbridge cycling. Hypercontractile function of the mutant myosin was accompanied by increased mitochondrial respiration, while cellular hypertrophy was modest in the physiological stiffness environment. We believe that this multi-scale platform will be useful to elucidate genotype-phenotype relationships underlying other genetic cardiovascular diseases.

Engineering tools for quantifying and manipulating forces in epithelia.

The integrity of epithelia is maintained within dynamic mechanical environments during tissue development and homeostasis. Understanding how epithelial cells mechanosignal and respond collectively or individually is critical to providing insight into developmental and (patho)physiological processes. Yet, inferring or mimicking mechanical forces and downstream mechanical signaling as they occur in epithelia presents unique challenges. A variety of in vitro approaches have been used to dissect the role of mechanics in regulating epithelia organization. Here, we review approaches and results from research into how epithelial cells communicate through mechanical cues to maintain tissue organization and integrity. We summarize the unique advantages and disadvantages of various reduced-order model systems to guide researchers in choosing appropriate experimental systems. These model systems include 3D, 2D, and 1D micromanipulation methods, single cell studies, and noninvasive force inference and measurement techniques. We also highlight a number of in silico biophysical models that are informed by in vitro and in vivo observations. Together, a combination of theoretical and experimental models will aid future experiment designs and provide predictive insight into mechanically driven behaviors of epithelial dynamics.

Morphological control enables nanometer-scale dissection of cell-cell signaling complexes.

Protein micropatterning enables robust control of cell positioning on electron-microscopy substrates for cryogenic electron tomography (cryo-ET). However, the combination of regulated cell boundaries and the underlying electron-microscopy substrate (EM-grids) provides a poorly understood microenvironment for cell biology. Because substrate stiffness and morphology affect cellular behavior, we devised protocols to characterize the nanometer-scale details of the protein micropatterns on EM-grids by combining cryo-ET, atomic force microscopy, and scanning electron microscopy. Measuring force displacement characteristics of holey carbon EM-grids, we found that their effective spring constant is similar to physiological values expected from skin tissues. Despite their apparent smoothness at light-microscopy resolution, spatial boundaries of the protein micropatterns are irregular at nanometer scale. Our protein micropatterning workflow provides the means to steer both positioning and morphology of cell doublets to determine nanometer details of punctate adherens junctions. Our workflow serves as the foundation for studying the fundamental structural changes governing cell-cell signaling.

Independently paced Ca2+ oscillations in progenitor and differentiated cells in an ex vivo epithelial organ.

Cytosolic Ca2+ is a highly dynamic, tightly regulated and broadly conserved cellular signal. Ca2+ dynamics have been studied widely in cellular monocultures, yet organs in vivo comprise heterogeneous populations of stem and differentiated cells. Here, we examine Ca2+ dynamics in the adult Drosophila intestine, a self-renewing epithelial organ in which stem cells continuously produce daughters that differentiate into either enteroendocrine cells or enterocytes. Live imaging of whole organs ex vivo reveals that stem-cell daughters adopt strikingly distinct patterns of Ca2+ oscillations after differentiation: enteroendocrine cells exhibit single-cell Ca2+ oscillations, whereas enterocytes exhibit rhythmic, long-range Ca2+ waves. These multicellular waves do not propagate through immature progenitors (stem cells and enteroblasts), of which the oscillation frequency is approximately half that of enteroendocrine cells. Organ-scale inhibition of gap junctions eliminates Ca2+ oscillations in all cell types - even, intriguingly, in progenitor and enteroendocrine cells that are surrounded only by enterocytes. Our findings establish that cells adopt fate-specific modes of Ca2+ dynamics as they terminally differentiate and reveal that the oscillatory dynamics of different cell types in a single, coherent epithelium are paced independently.

The effects of xeno-free cryopreservation on the contractile properties of human iPSC derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have advanced our ability to study the basic function of the heart and model cardiac diseases. Due to the complexities in stem cell culture and differentiation protocols, many researchers source their hiPSC-CMs from collaborators or commercial biobanks. Generally, the field has assumed the health of frozen cardiomyocytes is unchanged if the cells adhere to the substrate and commence beating. However, very few have investigated the effects of cryopreservation on hiPSC-CM's functional and transcriptional health at the cellular and molecular level. Here we review methods and challenges associated with cryopreservation, and examine the effects of cryopreservation on the functionality (contractility and calcium handling) and transcriptome of hiPSC-CMs from six healthy stem cell lines. Utilizing protein patterning methods to template physiological cell aspect ratios (7:1, length:width) in conjunction with polyacrylamide (PA) hydrogels, we measured changes in force generation and calcium handling of single hiPSC-CMs. We observed that cryopreservation altered the functionality and transcriptome of hiPSC-CMs towards larger sizes and contractile force as assessed by increased spread area and volume, single cell traction force microscopy and delayed calcium dynamics. hiPSC-CMs are broadly used for basic science research, regenerative medicine, and testing biological therapeutics. This study informs the design of experiments utilizing hiPSC-CMs to avoid confounding functional changes due to cryopreservation with other treatments.

Correction to: An Easy-to-Fabricate Cell Stretcher Reveals Density-Dependent Mechanical Regulation of Collective Cell Movements in Epithelia.

[This corrects the article DOI: 10.1007/s12195-021-000689-6.].

An Easy-to-Fabricate Cell Stretcher Reveals Density-Dependent Mechanical Regulation of Collective Cell Movements in Epithelia.

INTRODUCTION: Mechanical forces regulate many facets of cell and tissue biology. Studying the effects of forces on cells requires real-time observations of single- and multi-cell dynamics in tissue models during controlled external mechanical input. Many of the existing devices used to conduct these studies are costly and complicated to fabricate, which reduces the availability of these devices to many laboratories. METHODS: We show how to fabricate a simple, low-cost, uniaxial stretching device, with readily available materials and instruments that is compatible with high-resolution time-lapse microscopy of adherent cell monolayers. In addition, we show how to construct a pressure controller that induces a repeatable degree of stretch in monolayers, as well as a custom MATLAB code to quantify individual cell strains. RESULTS: As an application note using this device, we show that uniaxial stretch slows down cellular movements in a mammalian epithelial monolayer in a cell density-dependent manner. We demonstrate that the effect on cell movement involves the relocalization of myosin downstream of Rho-associated protein kinase (ROCK). CONCLUSIONS: This mechanical device provides a platform for broader involvement of engineers and biologists in this important area of cell and tissue biology. We used this device to demonstrate the mechanical regulation of collective cell movements in epithelia. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00689-6.

Wafer-Scale Patterning of Protein Templates for Hydrogel Fabrication.

Human-induced pluripotent stem cell-derived cardiomyocytes are a potentially unlimited cell source and promising patient-specific in vitro model of cardiac diseases. Yet, these cells are limited by immaturity and population heterogeneity. Current in vitro studies aiming at better understanding of the mechanical and chemical cues in the microenvironment that drive cellular maturation involve deformable materials and precise manipulation of the microenvironment with, for example, micropatterns. Such microenvironment manipulation most often involves microfabrication protocols which are time-consuming, require cleanroom facilities and photolithography expertise. Here, we present a method to increase the scale of the fabrication pipeline, thereby enabling large-batch generation of shelf-stable microenvironment protein templates on glass chips. This decreases fabrication time and allows for more flexibility in the subsequent steps, for example, in tuning the material properties and the selection of extracellular matrix or cell proteins. Further, the fabrication of deformable hydrogels has been optimized for compatibility with these templates, in addition to the templates being able to be used to acquire protein patterns directly on the glass chips. With our approach, we have successfully controlled the shapes of cardiomyocytes seeded on Matrigel-patterned hydrogels.

Hypertrophic cardiomyopathy ß-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state.

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in ß-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.

Increased tissue stiffness triggers contractile dysfunction and telomere shortening in dystrophic cardiomyocytes.

Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disease that is associated with severe progressive muscle degeneration culminating in death due to cardiorespiratory failure. We previously observed an unexpected proliferation-independent telomere shortening in cardiomyocytes of a DMD mouse model. Here, we provide mechanistic insights using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using traction force microscopy, we show that DMD hiPSC-CMs exhibit deficits in force generation on fibrotic-like bioengineered hydrogels, aberrant calcium handling, and increased reactive oxygen species levels. Furthermore, we observed a progressive post-mitotic telomere shortening in DMD hiPSC-CMs coincident with downregulation of shelterin complex, telomere capping proteins, and activation of the p53 DNA damage response. This telomere shortening is blocked by blebbistatin, which inhibits contraction in DMD cardiomyocytes. Our studies underscore the role of fibrotic stiffening in the etiology of DMD cardiomyopathy. In addition, our data indicate that telomere shortening is progressive, contraction dependent, and mechanosensitive, and suggest points of therapeutic intervention.

3D Microwell Platforms for Control of Single Cell 3D Geometry and Intracellular Organization.

INTRODUCTION: Cell structure and migration is impacted by the mechanical properties and geometry of the cell adhesive environment. Most studies to date investigating the effects of 3D environments on cells have not controlled geometry at the single-cell level, making it difficult to understand the influence of 3D environmental cues on single cells. Here, we developed microwell platforms to investigate the effects of 2D vs. 3D geometries on single-cell F-actin and nuclear organization. METHODS: We used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment (3D/3D), 3D microwells with 2D adhesive areas at the bottom only (3D/2D), and flat 2D gels with 2D patterned adhesive areas (2D/2D). We measured geometric swelling and Young's modulus of the platforms. We then cultured C2C12 myoblasts on each platform and evaluated the effects of the engineered microenvironments on F-actin structure and nuclear shape. RESULTS: We tuned the mechanical characteristics of the microfabricated platforms by manipulating the gel formulation. Crosslinker ratio strongly influenced geometric swelling whereas total polymer content primarily affected Young's modulus. When comparing cells in these platforms, we found significant effects on F-actin and nuclear structures. Our analysis showed that a 3D/3D environment was necessary to increase actin and nuclear height. A 3D/2D environment was sufficient to increase actin alignment and nuclear aspect ratio compared to a 2D/2D environment. CONCLUSIONS: Using our novel polyacrylamide platforms, we were able to decouple the effects of 3D confinement and adhesive environment, finding that both influenced actin and nuclear structure.

CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes.

Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.

Silencing of MYH7 ameliorates disease phenotypes in human iPSC-cardiomyocytes.

Allele-specific RNA silencing has been shown to be an effective therapeutic treatment in a number of diseases, including neurodegenerative disorders. Studies of allele-specific silencing in hypertrophic cardiomyopathy (HCM) to date have focused on mouse models of disease. We here examine allele-specific silencing in a human-cell model of HCM. We investigate two methods of silencing, short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) silencing, using a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model. We used cellular micropatterning devices with traction force microscopy and automated video analysis to examine each strategy's effects on contractile defects underlying disease. We find that shRNA silencing ameliorates contractile phenotypes of disease, reducing disease-associated increases in cardiomyocyte velocity, force, and power. We find that ASO silencing, while better able to target and knockdown a specific disease-associated allele, showed more modest improvements in contractile phenotypes. These findings are the first exploration of allele-specific silencing in a human HCM model and provide a foundation for further exploration of silencing as a therapeutic treatment for MYH7-mutation-associated cardiomyopathy.

Micromechanobiology: Focusing on the Cardiac Cell-Substrate Interface.

Engineered, in vitro cardiac cell and tissue systems provide test beds for the study of cardiac development, cellular disease processes, and drug responses in a dish. Much effort has focused on improving the structure and function of engineered cardiomyocytes and heart tissues. However, these parameters depend critically on signaling through the cellular microenvironment in terms of ligand composition, matrix stiffness, and substrate mechanical properties-that is, matrix micromechanobiology. To facilitate improvements to in vitro microenvironment design, we review how cardiomyocytes and their microenvironment change during development and disease in terms of integrin expression and extracellular matrix (ECM) composition. We also discuss strategies used to bind proteins to common mechanobiology platforms and describe important differences in binding strength to the substrate. Finally, we review example biomaterial approaches designed to support and probe cell-ECM interactions of cardiomyocytes in vitro, as well as open questions and challenges.

Touch-induced mechanical strain in somatosensory neurons is independent of extracellular matrix mutations in Caenorhabditis elegans.

Cutaneous mechanosensory neurons are activated by mechanical loads applied to the skin, and these stimuli are proposed to generate mechanical strain within sensory neurons. Using a microfluidic device to deliver controlled stimuli to intact animals and large, immobile, and fluorescent protein-tagged mitochondria as fiducial markers in the touch receptor neurons (TRNs), we visualized and measured touch-induced mechanical strain in Caenorhabditis elegans worms. At steady state, touch stimuli sufficient to activate TRNs induce an average strain of 3.1% at the center of the actuator and this strain decays to near zero at the edges of the actuator. We also measured strain in animals carrying mutations affecting links between the extracellular matrix (ECM) and the TRNs but could not detect any differences in touch-induced mechanical strain between wild-type and mutant animals. Collectively, these results demonstrate that touching the skin induces local mechanical strain in intact animals and suggest that a fully intact ECM is not essential for transmitting mechanical strain from the skin to cutaneous mechanosensory neurons.