Metastable Atrial State Underlies the Primary Genetic Substrate for MYL4 Mutation-Associated Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common clinical arrhythmia and is associated with heart failure, stroke, and increased mortality. The myocardial substrate for AF is poorly understood because of limited access to primary human tissue and mechanistic questions around existing in vitro or in vivo models. METHODS: Using an MYH6:mCherry knock-in reporter line, we developed a protocol to generate and highly purify human pluripotent stem cell-derived cardiomyocytes displaying physiological and molecular characteristics of atrial cells. We modeled human MYL4 mutants, one of the few definitive genetic causes of AF. To explore non-cell-autonomous components of AF substrate, we also created a zebrafish Myl4 knockout model, which exhibited molecular, cellular, and physiologic abnormalities that parallel those in humans bearing the cognate mutations. RESULTS: There was evidence of increased retinoic acid signaling in both human embryonic stem cells and zebrafish mutant models, as well as abnormal expression and localization of cytoskeletal proteins, and loss of intracellular nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide + hydrogen. To identify potentially druggable proximate mechanisms, we performed a chemical suppressor screen integrating multiple human cellular and zebrafish in vivo endpoints. This screen identified Cx43 (connexin 43) hemichannel blockade as a robust suppressor of the abnormal phenotypes in both models of MYL4 (myosin light chain 4)-related atrial cardiomyopathy. Immunofluorescence and coimmunoprecipitation studies revealed an interaction between MYL4 and Cx43 with altered localization of Cx43 hemichannels to the lateral membrane in MYL4 mutants, as well as in atrial biopsies from unselected forms of human AF. The membrane fraction from MYL4-/- human embryonic stem cell derived atrial cells demonstrated increased phospho-Cx43, which was further accentuated by retinoic acid treatment and by the presence of risk alleles at the Pitx2 locus. PKC (protein kinase C) was induced by retinoic acid, and PKC inhibition also rescued the abnormal phenotypes in the atrial cardiomyopathy models. CONCLUSIONS: These data establish a mechanistic link between the transcriptional, metabolic and electrical pathways previously implicated in AF substrate and suggest novel avenues for the prevention or therapy of this common arrhythmia.

In Vivo Subcellular Mass Spectrometry Enables Proteo-Metabolomic Single-Cell Systems Biology in a Chordate Embryo Developing to a Normally Behaving Tadpole (X. laevis)*.

We report the development of in vivo subcellular high-resolution mass spectrometry (HRMS) for proteo-metabolomic molecular systems biology in complex tissues. With light microscopy, we identified the left-dorsal and left-ventral animal cells in cleavage-stage non-sentient Xenopus laevis embryos. Using precision-translated fabricated microcapillaries, the subcellular content of each cell was double-probed, each time swiftly (<5 s/event) aspirating <5 % of cell volume (≈10 nL). The proteins and metabolites were analyzed by home-built ultrasensitive capillary electrophoresis electrospray ionization employing orbitrap or time-of-flight HRMS. Label-free detection of ≈150 metabolites (57 identified) and 738 proteins found proteo-metabolomic networks with differential quantitative activities between the cell types. With spatially and temporally scalable sampling, the technology preserved the integrity of the analyzed cells, the neighboring cells, and the embryo. 95 % of the analyzed embryos developed into sentient tadpoles that were indistinguishable from their wild-type siblings based on anatomy and visual function in a background color preference assay.

Single-Cell Analyses Reveal Diverse Mechanisms of Resistance to EGFR Tyrosine Kinase Inhibitors in Lung Cancer.

Tumor heterogeneity underlies resistance to tyrosine kinase inhibitors (TKI) in lung cancers harboring EGFR mutations. Previous evidence suggested that subsets of preexisting resistant cells are selected by EGFR-TKI treatment, or alternatively, that diverse acquired resistance mechanisms emerge from drug-tolerant persister (DTP) cells. Many studies have used bulk tumor specimens or subcloned resistant cell lines to identify resistance mechanism. However, intratumoral heterogeneity can result in divergent responses to therapies, requiring additional approaches to reveal the complete spectrum of resistance mechanisms. Using EGFR-TKI-resistant cell models and clinical specimens, we performed single-cell RNA-seq and single-cell ATAC-seq analyses to define the transcriptional and epigenetic landscape of parental cells, DTPs, and tumor cells in a fully resistant state. In addition to AURKA, VIM, and AXL, which are all known to induce EGFR-TKI resistance, CD74 was identified as a novel gene that plays a critical role in the drug-tolerant state. In vitro and in vivo experiments demonstrated that CD74 upregulation confers resistance to the EGFR-TKI osimertinib and blocks apoptosis, enabling tumor regrowth. Overall, this study provides new insight into the mechanisms underlying resistance to EGFR-TKIs. SIGNIFICANCE: Single-cell analyses identify diverse mechanisms of resistance as well as the state of tolerant cells that give rise to resistance to EGFR tyrosine kinase inhibitors.

Metabolic Comparison of Dorsal versus Ventral Cells Directly in the Live 8-cell Frog Embryo by Microprobe Single-cell CE-ESI-MS.

Single-cell mass spectrometry (MS) empowers the characterization of metabolomic changes as cells differentiate to different tissues during early embryogenesis. Using whole-cell dissection and capillary electrophoresis electrospray ionization (CE-ESI) MS, we recently uncovered metabolic cell-to-cell differences in the 8- and 16-cell embryo of the South African clawed frog (Xenopus laevis), raising the question whether metabolic cell heterogeneity is also detectable across the dorsal-ventral axis of the 8-cell embryo. Here, we tested this hypothesis directly in the live embryo by quantifying single-cell metabolism between the left dorsal-animal (D1L) and left ventral-animal (V1L) cell pairs in the same embryo using microprobe single-cell CE-ESI-MS in the positive ion mode. After quantifying ~70 molecular features, including 52 identified metabolites, that were reproducibly detected in both cells among n = 5 different embryos, we employed supervised multivariate data analysis based on partial least squares discriminant analysis (PLSDA) to compare metabolism between the cell types. Statistical analysis revealed that asparagine, glycine betaine, and a yet-unidentified molecule were statistically significantly enriched in the D1L cell compared to V1L (p < 0.05 and fold change ≥ 1.5). These results demonstrate that cells derived from the same hemisphere (animal pole) harbor different metabolic activity along the dorsal-ventral axis as early as the 8-cell stage. Apart from providing new evidence of metabolic cell heterogeneity during early embryogenesis, this study demonstrates that microprobe single-cell CE-ESI-MS enables the analysis of multiple single cells in the same live vertebrate embryo.