Regulation of sarcomere formation and function in the healthy heart requires a titin intronic enhancer.

Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure. To understand mechanisms that regulate abundant cardiomyocyte TTN expression we characterized highly conserved intron 1 sequences that exhibited dynamic changes in chromatin accessibility during differentiation of human cardiomyocytes from induced pluripotent stem cells (hiPSC-CMs). Homozygous deletion of these sequences in mice caused embryonic lethality while heterozygous mice demonstrated allele-specific reduction in Ttn expression.

Induced pluripotent stem cell-derived cardiomyocyte in vitro models: benchmarking progress and ongoing challenges.

Recent innovations in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSCs) have unlocked a viable path to creating in vitro cardiac models. Currently, hiPSC-derived cardiomyocytes (hiPSC-CMs) remain immature, leading many in the field to explore approaches to enhance cell and tissue maturation. Here, we systematically analyzed 300 studies using hiPSC-CM models to determine common fabrication, maturation and assessment techniques used to evaluate cardiomyocyte functionality and maturity and compiled the data into an open-access database.

MicroBundlePillarTrack: A Python package for automated segmentation, tracking, and analysis of pillar deflection in cardiac microbundles.

Movies of human induced pluripotent stem cell (hiPSC)-derived engineered cardiac tissue (microbundles) contain abundant information about structural and functional maturity. However, extracting these data in a reproducible and high-throughput manner remains a major challenge. Furthermore, it is not straightforward to make direct quantitative comparisons across the multiple in vitro experimental platforms employed to fabricate these tissues.

MicroBundlePillarTrack: A Python package for automated segmentation, tracking, and analysis of pillar deflection in cardiac microbundles.

Movies of human induced pluripotent stem cell (hiPSC)-derived engineered cardiac tissue (microbundles) contain abundant information about structural and functional maturity. However, extracting these data in a reproducible and high-throughput manner remains a major challenge. Furthermore, it is not straightforward to make direct quantitative comparisons across the multiple experimental platforms employed to fabricate these 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.

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.

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.

Multi-Omics Profiling of Hypertrophic Cardiomyopathy Reveals Altered Mechanisms in Mitochondrial Dynamics and Excitation-Contraction Coupling.

Hypertrophic cardiomyopathy is one of the most common inherited cardiomyopathies and a leading cause of sudden cardiac death in young adults. Despite profound insights into the genetics, there is imperfect correlation between mutation and clinical prognosis, suggesting complex molecular cascades driving pathogenesis. To investigate this, we performed an integrated quantitative multi-omics (proteomic, phosphoproteomic, and metabolomic) analysis to illuminate the early and direct consequences of mutations in myosin heavy chain in engineered human induced pluripotent stem-cell-derived cardiomyocytes relative to late-stage disease using patient myectomies.

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 (tv), the most prevalent ACM-linked mutations.

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.

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.

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.

Genetic Studies of Hypertrophic Cardiomyopathy in Singaporeans Identify Variants in and That Are Common in Chinese Patients.

Background: To assess the genetic architecture of hypertrophic cardiomyopathy (HCM) in patients of predominantly Chinese ancestry.

Methods: We sequenced HCM disease genes in Singaporean patients (n=224) and Singaporean controls (n=3634), compared findings with additional populations and White HCM cohorts (n=6179), and performed in vitro functional studies.

Results: Singaporean HCM patients had significantly fewer confidently interpreted HCM disease variants (pathogenic/likely pathogenic: 18%, <0.

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.

Functional Properties of Engineered Heart Slices Incorporating Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for cardiac studies, but their structural and functional immaturity precludes their use as faithful models of adult myocardium. Here we describe engineered heart slices (EHS), preparations of decellularized porcine myocardium repopulated with hiPSC-CMs that exhibit structural and functional improvements over standard culture. EHS exhibited multicellular, aligned bundles of elongated CMs with organized sarcomeres, positive inotropic responses to isoproterenol, anisotropic conduction of action potentials, and electrophysiological functionality for more than 200 days.

SarcTrack.

Rationale: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in combination with CRISPR/Cas9 genome editing provide unparalleled opportunities to study cardiac biology and disease. However, sarcomeres, the fundamental units of myocyte contraction, are immature and nonlinear in hiPSC-CMs, which technically challenge accurate functional interrogation of contractile parameters in beating cells. Furthermore, existing analysis methods are relatively low-throughput, indirectly assess contractility, or only assess well-aligned sarcomeres found in mature cardiac tissues.

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.