The heart is viscoelastic and exhibits both viscous and elastic behavior with deformation. Cardiac viscoelasticity influences heart function by regulating the volume of blood that can fill, and subsequently be pumped from, the cardiac chambers. Tissue viscoelasticity can also influence cellular functions, motivating the need to measure and model viscoelasticity from the cellular to the organ scale under healthy and disease conditions. Here, we review current protocols, instrumentation, and results from cardiac viscoelastic measurements from the organ to the subcellular level. Since viscoelasticity is regulated by tissue structure and composition, we describe what is known about the viscoelasticity of intracellular and extracellular proteins, cardiac cells, and cardiac tissue, as well as how changes in these proteins with disease progression may influence cardiac viscoelasticity. Finally, we discuss the outlook for the field, including recommendations for standardizing reports of cardiac viscoelastic measurements to increase their utility for biomaterials design for tissue engineering, cardiovascular modeling, and diagnosis.

The feedback between mechanical and chemical signals plays a key role in controlling many biological processes and collective cell behavior. Here we focus on the emergence of spatiotemporal density waves in a one-dimensional "cell train." Combining a minimal theoretical model with observations in an in vitro experimental system of MDCK epithelial cells confined to a linear pattern, we examine the spontaneous oscillations driven by the feedback between myosin activation and mechanical deformations and their effect on the response of the tissue to externally applied deformations. We show that the nature and frequency of spontaneous oscillations is controlled by the size of the cell train, with a transition from size-dependent standing waves to intrinsic spontaneous waves at the natural frequency of the tissue. The response to external boundary perturbations exhibit a resonance at this natural frequency, providing a possible venue for inferring the mechanochemical couplings that control the tissue behavior from rheological experiments.

Determining the pathogenicity of hypertrophic cardiomyopathy–associated mutations in the β-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 MYH7 WT/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.

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.

The sense of touch is conferred by the conjoint function of somatosensory neurons and skin cells. These cells meet across a gap filled by a basal lamina, an ancient structure found in metazoans. Using Caenorhabditis elegans, we investigate the composition and ultrastructure of the extracellular matrix at the epidermis and touch receptor neuron (TRN) interface. We show that membrane-matrix complexes containing laminin, nidogen, and the MEC-4 mechano-electrical transduction channel reside at this interface and are central to proper touch sensation. Interestingly, the dimensions and spacing of these complexes correspond with the discontinuous beam-like extracellular matrix structures observed in serial-section transmission electron micrographs. These complexes fail to coalesce in touch-insensitive extracellular matrix mutants and in dissociated neurons. Loss of nidogen reduces the density of mechanoreceptor complexes and the amplitude of the touch-evoked currents they carry. Thus, neuron-epithelium cell interfaces are instrumental in mechanosensory complex assembly and function. Unlike the basal lamina ensheathing the pharynx and body wall muscle, nidogen recruitment to the puncta along TRNs is not dependent upon laminin binding. MEC-4, but not laminin or nidogen, is destabilized by point mutations in the C-terminal Kunitz domain of the extracellular matrix component, MEC-1. These findings imply that somatosensory neurons secrete proteins that actively repurpose the basal lamina to generate special-purpose mechanosensory complexes responsible for vibrotactile sensing.

Despite causing 1 in 3 deaths worldwide, cardiovascular disease and the heart have only recently been studied in women. Contrary to previous belief, the female heart is not just a smaller replica of a male heart. Almost all heart diseases show sexual differences in risk, treatment, and symptoms. Even with these differences there is a lack of female models in animal and cellular studies. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are a powerful human model to test baseline and stressed sex differences at the cellular level. We hypothesized that using stressors that mimic disease conditions (drugs, substrate stiffness) in multiple male and female hiPSC-CMs will result in differential sex-based responses. We ran Traction Force Microscopy on 3 male (n = 424 cells, 3 batches) and 3 female (n = 318 cells, 3 batches) cell lines on 10 kPa polyacrylamide hydrogels. Male cells had 42% higher twitch force (p = 8.8E-6) with 25% higher contraction (p = 1.77E-5) and relaxation (p = 0.0006) velocities. However, female cells had a larger spread area (823 +/- 17.32 µm 2 vs. 756 +/- 21.64 µm 2 , p = 0.0018). To validate hiPSC-CMs as a model we compared preliminary RNA-seq to publicly available Heart Cell Atlas sc/snRNA-seq data sets of primary ventricular cardiomyocytes. They had similar differentially expressed genes (DEGs) as our hiPSC-CMs. Furthermore, DEGs had significantly enriched gene sets including a 25-fold enrichment in hypertrophic cardiomyopathy (FDR = 1.6E-7), 23-fold in dilated cardiomyopathy (FDR = 1.6E-7), and 16-fold in cardiac muscle contraction (FDR = 6.3E-4). We analyzed stiffness by differentiating and culturing hiPSC-CMs on 10 kPa and 100 kPa stiffness hydrogels. Our RNA-seq analysis at day 10 and 30 after differentiation found 7000 DEGs at 10 kPa while only 556 DEGs at 100 kPa. We looked hypercontractility caused stres and performed RNA-seq on day 60 wild type and hypercontractile mutant hiPSC-CMs. The wild type was dosed with positive inotrope levosimendan to induce hypercontractility while the hypercontractile mutant was dosed with negative inotrope verapamil to lower contractility. Hypercontractility had significant upregulation of the cellular response to stress including ER stress, stress-activated MAPK cascades, stress fibers, and cardiac muscle hypertrophy in response to stress. These differences can identify possible gene targets for preventing cardiovascular disease and inform personalized medicine.

The heart is a dynamic pump whose function is influenced by its mechanical properties. The viscoelastic properties of the heart, i.e., its ability to exhibit both elastic and viscous characteristics upon deformation, influence cardiac function. Viscoelastic properties change during heart failure (HF), but direct measurements of failing and non-failing myocardial tissue stress relaxation under constant displacement are lacking. Further, how consequences of tissue remodeling, such as fibrosis and fat accumulation, alter the stress relaxation remains unknown. To address this gap, we conducted stress relaxation tests on porcine myocardial tissue to establish baseline properties of cardiac tissue. We found porcine myocardial tissue to be fast relaxing, characterized by stress relaxation tests on both a rheometer and microindenter. We then measured human left ventricle (LV) epicardium and endocardium tissue from non-failing, ischemic HF and non-ischemic HF patients by microindentation. Analyzing by patient groups, we found that ischemic HF samples had slower stress relaxation than non-failing endocardium. Categorizing the data by stress relaxation times, we found that slower stress relaxing tissues were correlated with increased collagen deposition and increased α-smooth muscle actin (α-SMA) stress fibers, a marker of fibrosis and cardiac fibroblast activation, respectively. In the epicardium, analyzing by patient groups, we found that ischemic HF had faster stress relaxation than non-ischemic HF and non-failing. When categorizing by stress relaxation times, we found that faster stress relaxation correlated with Oil Red O staining, a marker for adipose tissue. These data show that changes in stress relaxation vary across the different layers of the heart during ischemic versus non-ischemic HF. These findings reveal how the viscoelasticity of the heart changes, which will lead to better modeling of cardiac mechanics for in vitro and in silico HF models.

Controlling cellular shape with micropatterning extracellular matrix (ECM) proteins on hydrogels has been shown to improve the reproducibility of the cell structure, enhancing our ability to collect statistics on single-cell behaviors. Patterning methods have advanced efforts in developing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a promising human model for studies of the heart structure, function, and disease. Patterned single hiPSC-CMs have exhibited phenotypes closer to mature, primary CMs across several metrics, including sarcomere alignment and contractility, area and aspect ratio, and force production. Micropatterning of hiPSC-CM pairs has shown further improvement of hiPSC-CM contractility compared to patterning single cells, suggesting that CM-CM interactions improve hiPSC-CM function. However, whether patterning single hiPSC-CMs on a protein associated with CM-CM adhesion, like N-cadherin, can drive similar enhancement of the hiPSC-CM structure and function has not been tested. To address this, we developed a novel dual-protein patterning process featuring covalent binding of proteins at the hydrogel surface to ensure robust force transfer and force sensing. The patterns comprised rectangular laminin islands for attachment across the majority of the cell area, with N-cadherin "end caps" to imitate CM-CM adherens junctions. We used this method to geometrically control single-cell CMs on deformable hydrogels suitable for traction force microscopy (TFM) to observe cellular dynamics. We seeded α-actinin::GFP-tagged hiPSC-CMs on dual-protein patterned hydrogels and verified the interaction between hiPSC-CMs and N-cadherin end caps via immunofluorescent staining. We found that hiPSC-CMs on dual-protein patterns exhibited higher cell area and contractility in the direction of sarcomere organization than those on laminin-only patterns but no difference in sarcomere organization or total force production. This work demonstrates a method for covalent patterning of multiple proteins on polyacrylamide hydrogels for mechanobiological studies. However, we conclude that N-cadherin only modestly improves single-cell patterned hiPSC-CM models and is not sufficient to elicit increases in contractility observed in hiPSC-CM pairs.

Epithelial cell migration is critical in regulating wound healing and tissue development. The epithelial microenvironment is incredibly dynamic, subjected to mechanical cues including cyclic stretch. While cyclic cell stretching platforms have revealed responses of the epithelium such as cell reorientation and gap formation, few studies have investigated the long-term effects of cyclic stretch on cell migration. We measured the migratory response of the epithelium to a range of physiologically relevant frequencies and stretch. We integrated our experimental approach with high-throughput cell segmentation to discover a relationship between changes in cell morphology and migration as a function of cyclic stretch. Our results indicate that lower stretch frequencies (i.e., 0.1 Hz) arrest epithelial migration, accompanied by cell reorientation and high cell shape solidity. We found that this response is also accompanied by increased recruitment of vinculin to cell-cell contacts, and this recruitment is necessary to arrest cell movements. This work demonstrates a critical role for frequency dependence in epithelial response to mechanical stretch. These results confirm the mechanosensitive nature of vinculin within the adherens junction, but independently reveal a novel mechanism of low frequency stress response in supporting epithelial integrity by arresting cell migration.

Hypertrophy Cardiomyopathy (HCM) is the most prevalent hereditary cardiovascular disease - affecting >1:500 individuals. Advanced forms of HCM clinically present with hypercontractility, hypertrophy and fibrosis. Several single-point mutations in b-myosin heavy chain (MYH7) have been associated with HCM and increased contractility at the organ level. Different MYH7 mutations have resulted in increased, decreased, or unchanged force production at the molecular level. Yet, how these molecular kinetics link to cell and tissue pathogenesis remains unclear. The Hippo Pathway, specifically its effector molecule YAP, has been demonstrated to be reactivated in pathological hypertrophic growth. We hypothesized that changes in force production (intrinsically or extrinsically) directly alter the homeostatic mechano-signaling of the Hippo pathway through changes in stresses on the nucleus. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we asked whether homeostatic mechanical signaling through the canonical growth regulator, YAP, is altered 1) by changes in the biomechanics of HCM mutant cardiomyocytes and 2) by alterations in the mechanical environment. We use genetically edited hiPSC-CM with point mutations in MYH7 associated with HCM, and their matched controls, combined with micropatterned traction force microscopy substrates to confirm the hypercontractile phenotype in MYH7 mutants. We next modulate contractility in healthy and disease hiPSC-CMs by treatment with positive and negative inotropic drugs and demonstrate a correlative relationship between contractility and YAP activity. We further demonstrate the activation of YAP in both HCM mutants and healthy hiPSC-CMs treated with contractility modulators is through enhanced nuclear deformation. We conclude that the overactivation of YAP, possibly initiated and driven by hypercontractility, correlates with excessive CCN2 secretion (connective tissue growth factor), enhancing cardiac fibroblast/myofibroblast transition and production of known hypertrophic signaling molecule TGFβ. Our study suggests YAP being an indirect player in the initiation of hypertrophic growth and fibrosis in HCM. Our results provide new insights into HCM progression and bring forth a testbed for therapeutic options in treating HCM.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in vitro models to study the mechanisms underlying cardiomyopathies and cardiotoxicity. Quantification of the contractile function in single hiPSC-CMs at high-throughput and over time is essential to disentangle how cellular mechanisms affect heart function. Here, we present CONTRAX, an open-access, versatile, and streamlined pipeline for quantitative tracking of the contractile dynamics of single hiPSC-CMs over time. Three software modules enable: parameter-based identification of single hiPSC-CMs; automated video acquisition of >200 cells/hour; and contractility measurements via traction force microscopy. We analyze >4,500 hiPSC-CMs over time in the same cells under orthogonal conditions of culture media and substrate stiffnesses; +/- drug treatment; +/- cardiac mutations. Using undirected clustering, we reveal converging maturation patterns, quantifiable drug response to Mavacamten and significant deficiencies in hiPSC-CMs with disease mutations. CONTRAX empowers researchers with a potent quantitative approach to develop cardiac therapies.

Controlling cellular shape with protein micropatterning can mimic physiological morphologies and has been shown to improve reproducibility, enhancing our ability to collect statistics on single-cell behaviors. It has also advanced efforts in developing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a promising human model for studies of heart structure and function. hiPSC-CMs have key physiological differences from primary human cardiomyocytes (CMs), including lower sarcomere alignment and contractility, smaller area and lower aspect ratio, and lower force production. Protein micropatterning has been demonstrated to make hiPSC-CMs behave more like primary human CMs across these metrics. However, these micropatterned models typically use only extracellular matrix (ECM) proteins and have not investigated whether providing a protein associated with CM-CM interactions, such as N-cadherin, further enhances hiPSC-CM structure and function. Here, we developed a novel dual-protein patterning process to geometrically control single-cell CM placement on deformable hydrogels suitable for traction force microscopy (TFM). The patterns were comprised of rectangular laminin islands for attachment across the majority of the cell area, with N-cadherin “end-caps” imitating cell-cell interactions. We first photopatterned two proteins on a glass coverslip using a two-step process with photomolecular adsorption of proteins. After both photopatterning steps were complete, we transferred the pattern from the coverslip to a physiologically relevant ∼10-kPa polyacrylamide hydrogel. We seeded α-actinin-tagged hiPSC-CMs on the dual-protein-patterned hydrogels and verified interaction between the hiPSC-CMs and the N-cadherin end-caps via immunofluorescent staining. We found hiPSC-CMs on dual-protein patterns have a higher cell area and contractility in the direction of sarcomere organization than those on laminin-only patterns, but no difference in sarcomere organization or force production. While N-cadherin modestly improves the single-cell patterned hiPSC-CM model, it is not sufficient to replicate the role of cell-cell contacts in CM development for in vitro hiPSC-CM systems.

Hypertrophic cardiomyopathy (HCM) is characterized by hypercontractility, cardiomyocyte hypertrophy, myofibril disarray, and altered energetics. Despite the identification of >1000 mutations in sarcomeric proteins, one-third of which are in β-cardiac myosin (MYH7), the mechanism by which altered force at the level of the sarcomere is transduced into cellular hypertrophy and other phenotypes is still incompletely understood. Our biochemical studies reveal surprising heterogeneity in the impact of different MYH7 mutations on myosin force generation and ATPase activity with some mutations reducing activity at the molecular level. Using CRISPR-edited hiPSC-cardiomyocytes (hiPSC-CMs), we studied the impact of myosin HCM mutations on the generation of contractile forces, hypertrophy, myofibril organization, and mitochondrial function, and compared cellular phenotypes with the effects of the mutations on myosin biomechanics. Our studies reveal a critical role for alterations in the myosin super-relaxed state (SRX) as a mechanism for cellular hypercontractility. To validate the fidelity of our findings to human disease, we compared hiPSC-CM results to those in 26 septal myectomy samples from HCM patients and 13 donor heart controls. In both hiPSC-CMs and myectomy samples we observed fiber disarray, altered mitochondrial metabolism, and widening of the Z-disc. A genome-wide key driver analysis revealed altered expression of 52 Z-disc-related genes in patient samples; 24 Z-disc genes in hiPSC-CMs; with 14 Z-disc genes shared between the two platforms. Activation of calcineurin pro-hypertrophic signaling provides a potential link between Z-disc mechano-sensation of sarcomeric forces and downstream hypertrophic signaling, which we explore using a vinculin FRET-tension sensor to directly measure intracellular force at the Z-disc. Our multi-scale approach, combined with both patient samples and CRISPR-edited hiPSC-CMs, confirms the fidelity of our in vitro model in recapitulating human disease and increases the potential translation of our findings to develop new precision-based HCM therapies.

Heart disease is the leading cause of death in the US, and human cell models are needed to study how structure and function are related in heart health and disease. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are one promising cell model, but their immaturity (e.g., lower sarcomere alignment and contractility) relative to adult human CMs limits their physiological relevance. While controlling cellular shape with protein micropatterning has enhanced maturity of single-cell hiPSC-CMs, they lack a key driver in cell maturity: cell-cell contacts. Here, we used a novel dual-protein patterning process to geometrically control single single-cell cardiomyocytes while maintaining the influence of cell-cell interactions. We first photopatterned two proteins on a glass coverslip using a two-step process with photomolecular adsorption of proteins. A laminin rectangle served to mimic the cell-ECM interaction, which was flanked by N-cadherin caps to mimic cell-cell contacts. After both photopatterning steps were complete, we transferred the pattern from the coverslip to a physiologically relevant ∼10 kPa polyacrylamide hydrogel. We seeded α-actinin-tagged hiPSC-CMs (AICS line 75) on the dual-protein-patterned hydrogel and verified interaction between the hiPSC-CMs and the N-cadherin caps via immunofluorescent staining. We have imaged hiPSC-CMs on single- and dual-protein patterns to compare attachment, spread area, sarcomere alignment and length, and sarcomere contractility. In a preliminary experiment, one device with a 3:2 ratio of dual- to single-protein patterns had 70 hiPSC-CMs attached, 50 of which were patterned on dual-protein patterns and 20 on single-protein patterns. This dual-protein patterning method improves previous single-cell hiPSC-CM models by incorporating both CM-ECM and CM-CM interactions, while maintaining benefits of single-cell systems (e.g., studying subcellular structure and function relationships).

BACKGROUND: Hypercontractility and arrhythmia are key pathophysiologic features of hypertrophic cardiomyopathy (HCM), the most common inherited heart disease. β-Adrenergic receptor antagonists (β-blockers) are the first-line therapy for HCM. However, β-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 β-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 ( Myh6 R403Q /+ and induced pluripotent stem cell (iPSC)-derived cardiomyocytes from patients with HCM ( MYH7 R403Q/+ ). RESULTS: We identified that carvedilol, a β-blocker not commonly used in HCM, suppresses contractile function and arrhythmia by inhibiting RyR2 (ryanodine receptor type 2). Unlike metoprolol (a β 1 -blocker), carvedilol markedly reduced LV contractility through RyR2 inhibition, while maintaining stroke volume through α 1 -adrenergic receptor inhibition in vivo. Clinically available carvedilol is a racemic mixture, and the R-enantiomer, devoid of β-blocking effect, retains the ability to inhibit both α 1 -receptor and RyR2, thereby suppressing contractile function and arrhythmias without lowering heart rate and cardiac output. In Myh6 R403Q/+ 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 MYH7 R403Q/+ 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 α 1 -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.

Yes Associated Protein (YAP) is a mechanosensitive transcriptional activator regulating a variety of cell fates, including proliferation and cell growth. YAP is activated (localized to the nucleus) in both physiological development (e.g., organ growth mediated by cell proliferation) and pathological heart diseases (e.g., organ growth mediated by cell enlargement). Cardiomyocytes (CMs) generate the mechanical work that powers each heartbeat, and each contraction shortens the CM and strains the nuclei.

Cardiomyocytes (CMs), the cells responsible for the contraction of the heart, experience mechanical forces such as active and resting tension as the cell contracts and relaxes. Active tension is generated by the contractile cytoskeleton, which is composed of actin-myosin arrays called sarcomeres. Resting tension is the tension that results when spring-like elements are stretched beyond their resting length, and this resting tension determines how forceful a contraction will be. The ability to investigate how resting tension is regulated has been a challenge due to the lack of a robust method to measure resting tension at the cell level. Previous methods involve stretching an individual cell and measuring the resulting resting tension; however, this method is low throughput and invasive. Single-cell Traction Force Microscopy (TFM) serves as a promising alternative method to measure resting tension due to the ability to measure the tension states of individual CMs with high throughput. We culture human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) on polyacrylamide (PA) gels with stiffness of healthy myocardium (10 kPa) patterned with islands of adhesive protein (Matrigel). We use TFM video analysis of beating cells to measure the traction forces when the hiPSC-CM is contracted and relaxed. We measure bead positions again after treatment with blebbistatin, and finally after the cell is dissociated from the gel. We calculate resting tension by calculating the TFM bead displacements from their positions under a relaxed cell to their positions after blebbistatin and after the cell is dissociated. To probe how resting tension is regulated by increased loading, we culture hiPSC-CMs on PA gels of increasing stiffness to mimic fibrotic tissue. These results provide insight into how the resting tension of a CM is regulated and how the CM adapts in response to pathophysiological conditions.

Doxorubicin (DOX) is a chemotherapy used to treat childhood and adult malignancies. However, despite being a cornerstone for oncological care, the use of DOX is limited due to dose-dependent cardiotoxicity, which can lead to contractile dysfunction and heart failure. The exact mechanism of DOX-induced cardiotoxicity is not fully understood. However, proposed mechanisms include sarcomere degradation and impairment of autophagy. To determine whether enhancing autophagy can restore contractile function, we treated µ-patterned single-cell human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), displaying sarcomere disarray and reduced contractile force post-DOX treatment with spermidine, an autophagy activator. The contractile force was quantified using traction force microscopy. There were five conditions (untreated control, DOX, maintenance medium post-DOX, spermidine post-DOX, and spermidine-only). We found that hiPSC-CMs treated with spermidine post-DOX produce 67% of the contractile force generated by control cells (0.22 µN vs. 0.33 µN, not significant) and significantly (0.22 µN vs. 0.11 µN, p < 0.0001) higher contractile force compared to cells in maintenance medium post-DOX. In the spermidine-only group, our results demonstrate that spermidine significantly (0.57 µN vs. 0.33 µN, p < 0.0001) increases contractile force compared to control. Spermidine-only treated cells were also bigger (spread area = ∼1716 µm2 vs. ∼1353 µm2, p < 0.00001) than the control and had a more robust and organized sarcomere network. Preliminary qPCR results suggest that spermidine upregulates genes that control sarcomere function (e.g., MYH7) and organization (e.g., ANKD1). These data demonstrate that spermidine can enhance contractile force post-DOX treatment and increase hiPSC-CMs size and sarcomere network. The data further suggests that sarcomere homeostasis is one of the mechanisms spermidine regulates in hiPSC-CMs. This work opens the door for developing therapies that target autophagy and sarcomere proteostasis to treat cardiac dysfunction.