pH-Responsive Titratable Inotropic Performance of Histidine-Modified Cardiac Troponin I

AbstractCardiac troponin I (cTnI) functions as the molecular switch of the thin filament. Studies have shown that a histidine button engineered into cTnI (cTnI A164H) specifically enhances inotropic function in the context of numerous pathophysiological challenges. To gain mechanistic insight into the basis of this finding, we analyzed histidine ionization states in vitro by studying the myofilament biophysics of amino acid substitutions that act as constitutive chemical mimetics of altered histidine ionization. We also assessed the role of histidine-modified cTnI in silico by means of molecular dynamics simulations. A functional in vitro analysis of myocytes at baseline (pH 7.4) indicated similar cellular contractile function and myofilament calcium sensitivity between myocytes expressing wild-type (WT) cTnI and cTnI A164H, whereas the A164R variant showed increased myofilament calcium sensitivity. Under acidic conditions, compared with WT myocytes, the myocytes expressing cTnI A164H maintained a contractile performance similar to that observed for the constitutively protonated cTnI A164R variant. Molecular dynamics simulations showed similar intermolecular atomic contacts between the WT and the deprotonated cTnI A164H variant. In contrast, simulations of protonated cTnI A164H showed various potential structural configurations, one of which included a salt bridge between His-164 of cTnI and Glu-19 of cTnC. This salt bridge was recapitulated in simulations of the cTnI A164R variant. These data suggest that differential histidine ionization may be necessary for cTnI A164H to act as a molecular sensor capable of modulating sarcomere performance in response to changes in the cytosolic milieu

Transmembrane protein 88: A Wnt regulatory protein that specifies cardiomyocyte development

Genetic regulation of the cell fate transition from lateral plate mesoderm to the specification of cardiomyocytes requires suppression of Wnt/β-catenin signaling, but the mechanism for this is not well understood. By analyzing gene expression and chromatin dynamics during directed differentiation of human embryonic stem cells (hESCs), we identified a suppressor of Wnt/β-catenin signaling, transmembrane protein 88 (TMEM88), as a potential regulator of cardiovascular progenitor cell (CVP) specification. During the transition from mesoderm to the CVP, TMEM88 has a chromatin signature of genes that mediate cell fate decisions, and its expression is highly upregulated in advance of key cardiac transcription factors in vitro and in vivo. In early zebrafish embryos, tmem88a is expressed broadly in the lateral plate mesoderm, including the bilateral heart fields. Short hairpin RNA targeting of TMEM88 during hESC cardiac differentiation increases Wnt/β-catenin signaling, confirming its role as a suppressor of this pathway. TMEM88 knockdown has no effect on NKX2.5 or GATA4 expression, but 80% of genes most highly induced during CVP development have reduced expression, suggesting adoption of a new cell fate. In support of this, analysis of later stage cell differentiation showed that TMEM88 knockdown inhibits cardiomyocyte differentiation and promotes endothelial differentiation. Taken together, TMEM88 is crucial for heart development and acts downstream of GATA factors in the pre-cardiac mesoderm to specify lineage commitment of cardiomyocyte development through inhibition of Wnt/β-catenin signaling

Dynamic chromatin organization and regulatory interactions in human endothelial cell differentiation

Vascular endothelial cells are a mesoderm-derived lineage with many essential functions, including angiogenesis and coagulation. The gene-regulatory mechanisms underpinning endothelial specialization are largely unknown, as are the roles of chromatin organization in regulating endothelial cell transcription. To investigate the relationships between chromatin organization and gene expression, we induced endothelial cell differentiation from human pluripotent stem cells and performed Hi-C and RNA-sequencing assays at specific time points. Long-range intrachromosomal contacts increase over the course of differentiation, accompanied by widespread heteroeuchromatic compartment transitions that are tightly associated with transcription. Dynamic topologically associating domain boundaries strengthen and converge on an endothelial cell state, and function to regulate gene expression. Chromatin pairwise point interactions (DNA loops) increase in frequency during differentiation and are linked to the expression of genes essential to vascular biology. Chromatin dynamics guide transcription in endothelial cell development and promote the divergence of endothelial cells from cardiomyocytes

Single-cell RNA-seq of human induced pluripotent stem cells reveals cellular heterogeneity and cell state transitions between subpopulations

Heterogeneity of cell states represented in pluripotent cultures have not been described at the transcriptional level. Since gene expression is highly heterogeneous between cells, single-cell RNA sequencing can be used to identify how individual pluripotent cells function. Here, we present results from the analysis of single-cell RNA sequencing data from 18,787 individual WTC CRISPRi human induced pluripotent stem cells. We developed an unsupervised clustering method, and through this identified four subpopulations distinguishable on the basis of their pluripotent state including: a core pluripotent population (48.3%), proliferative (47.8%), early-primed for differentiation (2.8%) and late-primed for differentiation (1.1%). For each subpopulation we were able to identify the genes and pathways that define differences in pluripotent cell states. Our method identified four discrete predictor gene sets comprised of 165 unique genes that denote the specific pluripotency states; and using these sets, we developed a multigenic machine learning prediction method to accurately classify single cells into each of the subpopulations. Compared against a set of established pluripotency markers, our method increases prediction accuracy by 10%, specificity by 20%, and explains a substantially larger proportion of deviance (up to 3-fold) from the prediction model. Finally, we developed an innovative method to predict cells transitioning between subpopulations, and support our conclusions with results from two orthogonal pseudotime trajectory methods

Genotype-free demultiplexing of pooled single-cell RNA-seq.

A variety of methods have been developed to demultiplex pooled samples in a single cell RNA sequencing (scRNA-seq) experiment which either require hashtag barcodes or sample genotypes prior to pooling. We introduce scSplit which utilizes genetic differences inferred from scRNA-seq data alone to demultiplex pooled samples. scSplit also enables mapping clusters to original samples. Using simulated, merged, and pooled multi-individual datasets, we show that scSplit prediction is highly concordant with demuxlet predictions and is highly consistent with the known truth in cell-hashing dataset. scSplit is ideally suited to samples without external genotype information and is available at: https://github.com/jon-xu/scSplit

Combined Mesenchymal Stromal Cell Therapy and ECMO in ARDS:A Controlled Experimental Study in Sheep

Rationale: Mesenchymal stromal cell (MSC) therapy is a promising intervention for acute respiratory distress syndrome (ARDS), although trials to date have not investigated its use alongside extracorporeal membrane oxygenation (ECMO). Recent preclinical studies have suggested that combining these interventions may attenuate the efficacy of ECMO. Objectives: To determine the safety and efficacy of MSC therapy in a model of ARDS and ECMO. Methods: ARDS was induced in 14 sheep, after which they were established on venovenous ECMO. Subsequently, they received either endobronchial induced pluripotent stem cell-derived human MSCs (hMSCs) (n = 7) or cell-free carrier vehicle (vehicle control; n = 7). During ECMO, a low VT ventilation strategy was employed in addition to protocolized hemodynamic support. Animals were monitored and supported for 24 hours. Lung tissue, bronchoalveolar fluid, and plasma were analyzed, in addition to continuous respiratory and hemodynamic monitoring. Measurements and Main Results: The administration of hMSCs did not improve oxygenation (PaO2/FIO2 mean difference =2146mmHg; P= 0.076) or pulmonary function.However, histological evidence of lung injury(lung injuryscoremeandifference=20.07;P=0.04) and BALIL-8 were reduced. In addition, hMSC-treated animals had a significantly lower cumulative requirement for vasopressor. Despite endobronchial administration, animals treated with hMSCs had a significant elevation in transmembrane oxygenator pressure gradients. Thiswas accompanied by more pulmonary artery thromboses and adherent hMSCs found on explanted oxygenator fibers. Conclusions: Endobronchial hMSC therapy in an ovine model of ARDS and ECMO can impair membrane oxygenator function and does not improve oxygenation. These data do not recommend the safe use of hMSCs during venovenous ECMO. </p