Nonclinical evaluation of chronic cardiac contractility modulation on 3D human engineered cardiac tissues.

INTRODUCTION: Cardiac contractility modulation (CCM) is a medical device-based therapy delivering non-excitatory electrical stimulations to the heart to enhance cardiac function in heart failure (HF) patients. The lack of human in vitro tools to assess CCM hinders our understanding of CCM mechanisms of action. Here, we introduce a novel chronic (i.e., 2-day) in vitro CCM assay to evaluate the effects of CCM in a human 3D microphysiological system consisting of engineered cardiac tissues (ECTs). METHODS: Cryopreserved human induced pluripotent stem cell-derived cardiomyocytes were used to generate 3D ECTs. The ECTs were cultured, incorporating human primary ventricular cardiac fibroblasts and a fibrin-based gel. Electrical stimulation was applied using two separate pulse generators for the CCM group and control group. Contractile properties and intracellular calcium were measured, and a cardiac gene quantitative PCR screen was conducted. RESULTS: Chronic CCM increased contraction amplitude and duration, enhanced intracellular calcium transient amplitude, and altered gene expression related to HF (i.e., natriuretic peptide B, NPPB) and excitation-contraction coupling (i.e., sodium-calcium exchanger, SLC8). CONCLUSION: These data represent the first study of chronic CCM in a 3D ECT model, providing a nonclinical tool to assess the effects of cardiac electrophysiology medical device signals complementing in vivo animal studies. The methodology established a standardized 3D ECT-based in vitro testbed for chronic CCM, allowing evaluation of physiological and molecular effects on human cardiac tissues.

Structural maturation of myofilaments in engineered 3D cardiac microtissues characterized using small angle x-ray scattering.

Understanding the structural and functional development of human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is essential to engineering cardiac tissue that enables pharmaceutical testing, modeling diseases, and designing therapies. Here we use a method not commonly applied to biological materials, small angle x-ray scattering, to characterize the structural development of hiPSC-CMs within three-dimensional engineered tissues during their preliminary stages of maturation. An x-ray scattering experimental method enables the reliable characterization of the cardiomyocyte myofilament spacing with maturation time. The myofilament lattice spacing monotonically decreases as the tissue matures from its initial post-seeding state over the span of 10 days. Visualization of the spacing at a grid of positions in the tissue provides an approach to characterizing the maturation and organization of cardiomyocyte myofilaments and has the potential to help elucidate mechanisms of pathophysiology, and disease progression, thereby stimulating new biological hypotheses in stem cell engineering.

Filamin C Cardiomyopathy Variants Cause Protein and Lysosome Accumulation.

[Figure: see text].

SARS-CoV-2 Disrupts Proximal Elements in the JAK-STAT Pathway.

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19. IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.

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.

Geometry and length control of 3D engineered heart tissues using direct laser writing.

Geometry and mechanical characteristics of the environment surrounding the Engineered Heart Tissues (EHT) affect their structure and function. Here, we employed a 3D tissue culture platform fabricated using two-photon direct laser writing with a high degree of accuracy to control parameters that are relevant to EHT maturation. Using this platform, we first explore the effects of geometry based on two distinct shapes: a rectangular seeding well with two attachment sites, and a stadium-like seeding well with six attachment sites that are placed symmetrically along hemicylindrical membranes. The former geometry promotes uniaxial contraction of the tissues; the latter additionally induces diagonal fiber alignment. We systematically increase the length of the seeding wells for both configurations and observe a positive correlation between fiber alignment at the center of the EHTs and tissue length. With increasing length, an undesirable thinning and "necking" also emerge, leading to the failure of longer tissues over time. In the second step, we optimize the stiffness of the seeding wells and modify some of the attachment sites of the platform and the seeding parameters to achieve tissue stability for each length and geometry. Furthermore, we use the platform for electrical pacing and calcium imaging to evaluate the functional dynamics of EHTs as a function of frequency.

Probing the subcellular nanostructure of engineered human cardiomyocytes in 3D tissue.

The structural and functional maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is essential for pharmaceutical testing, disease modeling, and ultimately therapeutic use. Multicellular 3D-tissue platforms have improved the functional maturation of hiPSC-CMs, but probing cardiac contractile properties in a 3D environment remains challenging, especially at depth and in live tissues. Using small-angle X-ray scattering (SAXS) imaging, we show that hiPSC-CMs matured and examined in a 3D environment exhibit a periodic spatial arrangement of the myofilament lattice, which has not been previously detected in hiPSC-CMs. The contractile force is found to correlate with both the scattering intensity (R 2 = 0.44) and lattice spacing (R 2 = 0.46). The scattering intensity also correlates with lattice spacing (R 2 = 0.81), suggestive of lower noise in our structural measurement than in the functional measurement. Notably, we observed decreased myofilament ordering in tissues with a myofilament mutation known to lead to hypertrophic cardiomyopathy (HCM). Our results highlight the progress of human cardiac tissue engineering and enable unprecedented study of structural maturation in hiPSC-CMs.

Plakophilin-2 truncating variants impair cardiac contractility by disrupting sarcomere stability and organization.

Progressive loss of cardiac systolic function in arrhythmogenic cardiomyopathy (ACM) has recently gained attention as an important clinical consideration in managing the disease. However, the mechanisms leading to reduction in cardiac contractility are poorly defined. Here, we use CRISPR gene editing to generate human induced pluripotent stem cells (iPSCs) that harbor plakophilin-2 truncating variants (PKP2tv), the most prevalent ACM-linked mutations. The PKP2tv iPSC­derived cardiomyocytes are shown to have aberrant action potentials and reduced systolic function in cardiac microtissues, recapitulating both the electrical and mechanical pathologies reported in ACM. By combining cell micropatterning with traction force microscopy and live imaging, we found that PKP2tvs impair cardiac tissue contractility by destabilizing cell-cell junctions and in turn disrupting sarcomere stability and organization. These findings highlight the interplay between cell-cell adhesions and sarcomeres required for stabilizing cardiomyocyte structure and function and suggest fundamental pathogenic mechanisms that may be shared among different types of cardiomyopathies.

SARS-CoV-2 desensitizes host cells to interferon through inhibition of the JAK-STAT pathway.

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we performed global proteomic analysis of the virus-host interface in a newly established panel of phenotypically diverse, SARS-CoV-2-infectable human cell lines representing different body organs. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cell lines and primary-like cardiomyocytes, and found that several pathway components were targeted by SARS-CoV-2 leading to cellular desensitization to interferon. These findings indicate that the suppression of interferon signaling is a mechanism widely used by SARS-CoV-2 in diverse tissues to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19.

Quantification of a single score (1+) in the Modified Ashworth Scale (MAS), a clinical assessment of spasticity.

The Modified Ashworth Scale (MAS) is an assessment that is often used by clinicians to grade spasticity in the affected limbs of stroke survivors. The MAS is a function of the angle at which the clinician perceives a resistance to stretch and/or a `catch' during a passive joint rotation. The qualitative nature of the assessment in combination with the low resolution of the scale could result in varied grouping of spastic patients, even for a single score. The objective of this pilot study was to develop a method for the quantification of the MAS, which could provide greater resolution and could eventually guide better informed therapeutic interventions. The MAS assessment at the elbow joint for four stroke survivors with the same clinical MAS score of 1+ was performed by a clinician and quantified using signals from surface electromyography (EMG) and an electrogoniometer. The subjects were tested on both the affected and contralateral upper limbs. The findings from this study show a varied set of signal outputs across four stroke survivors, all graded at 1+. The quantification provides insight as to the mechanisms underlying the passive resistance.