An engineered human cardiac tissue model reveals contributions of systemic lupus erythematosus autoantibodies to myocardial injury.

Systemic lupus erythematosus (SLE) is a highly heterogenous autoimmune disease that affects multiple organs, including the heart. The mechanisms by which myocardial injury develops in SLE, however, remain poorly understood. Here we engineered human cardiac tissues and cultured them with IgG fractions containing autoantibodies from SLE patients with and without myocardial involvement. We observed unique binding patterns of IgG from two patient subgroups: (i) patients with severe myocardial inflammation exhibited enhanced binding to apoptotic cells within cardiac tissues subjected to stress, and (ii) patients with systolic dysfunction exhibited enhanced binding to the surfaces of viable cardiomyocytes. Functional assays and RNA sequencing (RNA-seq) revealed that IgGs from patients with systolic dysfunction exerted direct effects on engineered tissues in the absence of immune cells, altering tissue cellular composition, respiration and calcium handling. Autoantibody target characterization by phage immunoprecipitation sequencing (PhIP-seq) confirmed distinctive IgG profiles between patient subgroups. By coupling IgG profiling with cell surface protein analyses, we identified four pathogenic autoantibody candidates that may directly alter the function of cells within the myocardium. Taken together, these observations provide insights into the cellular processes of myocardial injury in SLE that have the potential to improve patient risk stratification and inform the development of novel therapeutic strategies.

Engineered cardiac tissue model of restrictive cardiomyopathy for drug discovery.

Restrictive cardiomyopathy (RCM) is defined as increased myocardial stiffness and impaired diastolic relaxation leading to elevated ventricular filling pressures. Human variants in filamin C (FLNC) are linked to a variety of cardiomyopathies, and in this study, we investigate an in-frame deletion (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp) in a patient with RCM. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with this variant display impaired relaxation and reduced calcium kinetics in 2D culture when compared with a CRISPR-Cas9-corrected isogenic control line. Similarly, mutant engineered cardiac tissues (ECTs) demonstrate increased passive tension and impaired relaxation velocity compared with isogenic controls. High-throughput small-molecule screening identifies phosphodiesterase 3 (PDE3) inhibition by trequinsin as a potential therapy to improve cardiomyocyte relaxation in this genotype. Together, these data demonstrate an engineered cardiac tissue model of RCM and establish the translational potential of this precision medicine approach to identify therapeutics targeting myocardial relaxation.

Pathological remodeling of distal lung matrix in end-stage cystic fibrosis patients.

BACKGROUND: Manifestations of cystic fibrosis, although well-characterized in the proximal airways, are understudied in the distal lung. Characterization of the cystic fibrosis lung 'matrisome' (matrix proteome) has not been previously described, and could help identify biomarkers and inform therapeutic strategies. METHODS: We performed liquid chromatography-mass spectrometry, gene ontology analysis, and multi-modal imaging, including histology, immunofluorescence, and electron microscopy for a comprehensive evaluation of distal human lung extracellular matrix (matrix) structure and composition in end-stage cystic fibrosis. RESULTS: Quantitative proteomic profiling identified sixty-eight (68) matrix constituents with significantly altered expression in end-stage cystic fibrosis. Over 90% of significantly different matrix peptides detected, including structural and basement membrane proteins, were expressed at lower levels in cystic fibrosis. However, the total abundance of matrix in cystic fibrosis lungs was not significantly different from control lungs, suggesting that cystic fibrosis leads to loss of diversity among lung matrix proteins rather than an absolute loss of matrix. Visualization of distal lung matrix via immunofluorescence and electron microscopy revealed pathological remodeling of distal lung tissue architecture and loss of alveolar basement membrane, consistent with significantly altered pathways identified by gene ontology analysis. CONCLUSIONS: Dysregulation of matrix organization and aberrant wound healing pathways are associated with loss of matrix protein diversity and obliteration of distal lung tissue structure in end-stage cystic fibrosis. While many therapeutics aim to functionally restore defective cystic fibrosis transmembrane conductance regulator (CFTR), drugs that target dysregulated matrix pathways may serve as adjunct interventions to support lung recovery.

The influence of hypoxia and IFN-γ on the proteome and metabolome of therapeutic mesenchymal stem cells.

Over the past 15 years, mesenchymal stem cells (MSCs) have been assessed for their capacity to suppress inflammation and promote tissue repair. Regardless of whether the cells are primed (exposed to instructive cues) before administration, their phenotype will respond to environmental signals present in the pathophysiological setting being treated. Since hypoxia and inflammation coexist in the settings of acute injury and chronic disease we sought to explore how the proteome and metabolome of MSCs changes when cells were exposed to 48 h of 1% oxygen, interferon gamma (IFN-γ), or both cues together. We specifically focused on changes in cell metabolism, immune modulation, extracellular matrix secretion and modification, and survival capacity. IFN-γ promoted expression of anti-pathogenic proteins and induced MSCs to limit inflammation and fibrosis while promoting their own survival. Hypoxia instead led to cell adaptation to low oxygen, including upregulation of proteins involved in anaerobic metabolism, autophagy, angiogenesis, and cell migration. While dual priming resulted in additive effects, we also found many instances of synergy. These data lend insight to how MSCs may behave after administration to a patient and suggest how priming cells beforehand could improve their therapeutic capacity.

Clonally related methicillin-resistant Staphylococcus aureus isolated from short-finned pilot whales (Globicephala macrorhynchus), human volunteers, and a bayfront cetacean rehabilitation facility.

In May of 2011, a live mass stranding of 26 short-finned pilot whales (Globicephala macrorhynchus) occurred in the lower Florida Keys. Five surviving whales were transferred from the original stranding site to a nearby marine mammal rehabilitation facility where they were constantly attended to by a team of volunteers. Bacteria cultured during the routine clinical care of the whales and necropsy of a deceased whale included methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). In order to investigate potential sources or reservoirs of MSSA and MRSA, samples were obtained from human volunteers, whales, seawater, and sand from multiple sites at the facility, nearby recreational beaches, and a canal. Samples were collected on 3 days. The second collection day was 2 weeks after the first, and the third collection day was 2 months after the last animal was removed from the facility. MRSA and MSSA were isolated on each day from the facility when animals and volunteers were present. MSSA was found at an adjacent beach on all three collection days. Isolates were characterized by utilizing a combination of quantitative real-time PCR to determine the presence of mecA and genes associated with virulence, staphylococcal protein A typing, staphylococcal cassette chromosome mec typing, multilocus sequence typing, and pulsed field gel electrophoresis (PFGE). Using these methods, clonally related MRSA were isolated from multiple environmental locations as well as from humans and animals. Non-identical but genetically similar MSSA and MRSA were also identified from distinct sources within this sample pool. PFGE indicated that the majority of MRSA isolates were clonally related to the prototype human strain USA300. These studies support the notion that S. aureus may be shed into an environment by humans or pilot whales and subsequently colonize or infect exposed new hosts.