Mitochondrial calcium uniporter stabilization preserves energetic homeostasis during Complex I impairment.

Calcium entering mitochondria potently stimulates ATP synthesis. Increases in calcium preserve energy synthesis in cardiomyopathies caused by mitochondrial dysfunction, and occur due to enhanced activity of the mitochondrial calcium uniporter channel. The signaling mechanism that mediates this compensatory increase remains unknown. Here, we find that increases in the uniporter are due to impairment in Complex I of the electron transport chain. In normal physiology, Complex I promotes uniporter degradation via an interaction with the uniporter pore-forming subunit, a process we term Complex I-induced protein turnover. When Complex I dysfunction ensues, contact with the uniporter is inhibited, preventing degradation, and leading to a build-up in functional channels. Preventing uniporter activity leads to early demise in Complex I-deficient animals. Conversely, enhancing uniporter stability rescues survival and function in Complex I deficiency. Taken together, our data identify a fundamental pathway producing compensatory increases in calcium influx during Complex I impairment.

The history and geographic distribution of a KCNQ1 atrial fibrillation risk allele.

The genetic architecture of atrial fibrillation (AF) encompasses low impact, common genetic variants and high impact, rare variants. Here, we characterize a high impact AF-susceptibility allele, KCNQ1 R231H, and describe its transcontinental geographic distribution and history. Induced pluripotent stem cell-derived cardiomyocytes procured from risk allele carriers exhibit abbreviated action potential duration, consistent with a gain-of-function effect. Using identity-by-descent (IBD) networks, we estimate the broad- and fine-scale population ancestry of risk allele carriers and their relatives. Analysis of ancestral migration routes reveals ancestors who inhabited Denmark in the 1700s, migrated to the Northeastern United States in the early 1800s, and traveled across the Midwest to arrive in Utah in the late 1800s. IBD/coalescent-based allele dating analysis reveals a relatively recent origin of the AF risk allele (~5000 years). Thus, our approach broadens the scope of study for disease susceptibility alleles to the context of human migration and ancestral origins.

A Functional Assay for Sick Sinus Syndrome Genetic Variants.

BACKGROUND/AIMS: Congenital Sick Sinus Syndrome (SSS) is a disorder associated with sudden cardiac death due to severe bradycardia and prolonged pauses. Mutations in HCN4, the gene encoding inward Na+/K+ current (If), have been described as a cause of congenital SSS. The objective of this study is to develop an SSS model in embryonic zebrafish, and use zebrafish as a moderate-throughput assay to functionally characterize HCN4 variants. METHODS: To determine the function of hcn4 in zebrafish, embryos were either bathed in the If -specific blocker (ZD-7288), or endogenous hcn4 expression was knocked down using splice-blocking morpholinos. To assess whether the zebrafish model discriminates benign from pathogenic variants, we tested four HCN4 mutations known to cause human SSS and four variants of unknown significance (VUS). RESULTS: Pharmacological blockade and knockdown of hcn4 in zebrafish phenocopied human SSS, displaying bradycardia and cardiac pauses in intact embryos and explanted hearts. The zebrafish assay correctly identified all disease-causing variants. Of the VUS, the assay predicted 2 as benign and 2 as hypomorphic variants. CONCLUSIONS: We conclude that our embryonic zebrafish assay is a novel and effective tool to functionally characterize human HCN4 variants, which can be translated into important clinical prognostic information.

A near-infrared fluorescent voltage-sensitive dye allows for moderate-throughput electrophysiological analyses of human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM)-based assays are emerging as a promising tool for the in vitro preclinical screening of QT interval-prolonging side effects of drugs in development. A major impediment to the widespread use of human iPSC-CM assays is the low throughput of the currently available electrophysiological tools. To test the precision and applicability of the near-infrared fluorescent voltage-sensitive dye 1-(4-sulfanatobutyl)-4-{ß[2-(di-n-butylamino)-6-naphthyl]butadienyl}quinolinium betaine (di-4-ANBDQBS) for moderate-throughput electrophysiological analyses, we compared simultaneous transmembrane voltage and optical action potential (AP) recordings in human iPSC-CM loaded with di-4-ANBDQBS. Optical AP recordings tracked transmembrane voltage with high precision, generating nearly identical values for AP duration (AP durations at 10%, 50%, and 90% repolarization). Human iPSC-CMs tolerated repeated laser exposure, with stable optical AP parameters recorded over a 30-min study period. Optical AP recordings appropriately tracked changes in repolarization induced by pharmacological manipulation. Finally, di-4-ANBDQBS allowed for moderate-throughput analyses, increasing throughput >10-fold over the traditional patch-clamp technique. We conclude that the voltage-sensitive dye di-4-ANBDQBS allows for high-precision optical AP measurements that markedly increase the throughput for electrophysiological characterization of human iPSC-CMs.

Overexpression of Snai3 suppresses lymphoid- and enhances myeloid-cell differentiation.

The altered expression of transcription factors in hematopoietic stem cells and their subsequent lineages can alter the development of lymphoid and myeloid lineages. The role of the transcriptional repressor Snai3 protein in the derivation of cells of the hemato-poietic system was investigated. Snai3 is expressed in terminal T-cell and myeloid lineages, therefore, we chose to determine if expressing Snai3 in the early stages of hematopoietic development would influence cell-lineage determination. Expression of Snai3 by retroviral transduction of hematopoietic stem cells using bone marrow chimera studies demonstrated a block in lymphoid-cell development and enhanced expansion of myeloid-lineage cells. Analysis of Snai3-expressing hematopoietic precursor cells showed normal numbers of immature cells, but a block in the development of cells committed to lymphoid lineages. These data indicate that the overexpression of Snai3 does alter bone marrow cell development and that the identification of genes whose expression is altered by the presence of Snai3 would aid in our understanding of these developmental pathways.

Enrichment of functionally distinct mouse hematopoietic progenitor cell populations using CD62L.

The details of the bifurcation of the lymphoid and myeloid lineages following commitment by multipotent progenitor cells (MPP) remain a topic of controversy. We report that the surface glycoprotein CD62L can be characterized as a novel marker of this and other stages of early hematopoietic differentiation. Cell isolation and transplant studies demonstrated CD62L(neg/low) long-term hematopoietic stem cells and CD62L(high) MPP within the traditionally defined c-kit(pos)Lin(neg/low)Sca-1(pos) stem/progenitor cell population. Within the MPP population, previously defined as c-kit(pos)Lin(neg/low)Sca-1(pos)-Thy-1.1(neg)Flt3(pos), Sca-1 and CD62L resolved four populations and segregated Sca-1(high)CD62L(neg/low) MPP from Sca-1(high)CD62L(high) leukocyte-biased progenitors. Using a novel transplantation method that allows tracking of erythroid and platelet engraftment as an alternative to the classical method of in vitro colony formation, we characterized Sca-1(high)CD62L(neg/low) cells as MPP, based on transient engraftment of these lineages. These data establish CD62L as a useful tool in the study of early hematopoiesis and emphasize the power of trilineage-engraftment studies in establishing the lineage potential of MPP subsets.

Hematopoietic origin of pathological grooming in Hoxb8 mutant mice.

Mouse Hoxb8 mutants show unexpected behavior manifested by compulsive grooming and hair removal, similar to behavior in humans with the obsessive-compulsive disorder spectrum disorder trichotillomania. As Hox gene disruption often has pleiotropic effects, the root cause of this behavioral deficit was unclear. Here we report that, in the brain, Hoxb8 cell lineage exclusively labels bone marrow-derived microglia. Furthermore, transplantation of wild-type bone marrow into Hoxb8 mutant mice rescues their pathological phenotype. It has been suggested that the grooming dysfunction results from a nociceptive defect, also exhibited by Hoxb8 mutant mice. However, bone marrow transplant experiments and cell type-specific disruption of Hoxb8 reveal that these two phenotypes are separable, with the grooming phenotype derived from the hematopoietic lineage and the sensory defect derived from the spinal cord cells. Immunological dysfunctions have been associated with neuropsychiatric disorders, but the causative relationships are unclear. In this mouse, a distinct compulsive behavioral disorder is associated with mutant microglia.

The inhibition of T-cells proliferation by mouse mesenchymal stem cells through the induction of p16INK4A-cyclin D1/cdk4 and p21waf1, p27kip1-cyclin E/cdk2 pathways.

Mesenchymal stem cells (MSCs) have been shown to down-regulate T-cell responses. However, the mechanisms underlying remain unknown. In this study, we report that BALB/c bone marrow-derived MSCs inhibit the proliferation of allogeneic T-cells in mixed lymphocyte reactions (MLR), This inhibition is dependent on cell-cell contact, and do not induce apoptosis. Furthermore, cell-cycle analyses reveal that T-cells, in the presence of MSCs, are arrested in the G0/G1 phase through. The blockage of phosphorylation of retinoblastoma protein (Rb), mediated by the p16(INK4A)-cyclin D1/cdk4 complex and p21(waf1), p27(kip1)-cyclin E/cdk2 complex pathway. Our results suggest that MSCs may perform a crucial function in the maintenance of immune homeostasis, via direct regulation of the clonal expansion of activated T-cells. The novel T-cell regulatory mechanism exhibited by MSCs may prove useful in a variety of therapeutic applications.

Gradients of antigen expression and developmental potential in hematopoiesis.

Prospective isolation of hematopoietic stem and progenitor cell subsets depends upon the premise that expression of combinations of surface antigens reflects developmental potential. During the process of differentiation, however, the loss of antigens associated with stem cells and the concomitant gain of those associated with progenitor cells often occurs as a continuum rather than by discrete binary steps. Coupled with the fact that assay conditions can profoundly influence the developmental fates of prospectively isolated cells, gradients of antigen expression during differentiation have led to a variety of interpretations of lineage commitment in hematopoiesis.

Mouse models of hematopoietic engraftment: limitations of transgenic green fluorescent protein strains and a high-performance liquid chromatography approach to analysis of erythroid chimerism.

Transgenic mouse strains ubiquitously expressing green fluorescent protein (GFP) have enabled investigators to develop in vivo transplant models that can detect donor contributions to many different tissues. However, most GFP transgenics lack expression of the reporter in the erythroid lineage. We evaluated expression of GFP in the bone marrow of the OsbY01 transgenic mouse (B6-GFP) in the context of CD71 and TER-119 expression and found that GFP fluorescence is lost prior to the basophilic erythroblast stage of development. However, platelets in B6-GFP mice were found to be uniformly positive for GFP. We therefore used the GFP transgenic model in combination with allelic variants of CD45 and the hemoglobin beta (Hbb) chain to develop a model system that allows all blood lineages to be followed in a mouse model of bone marrow transplantation (BMT). To detect Hbb variant molecules, we developed a new protocol based on high-performance liquid chromatography that is sensitive and precise, allowing rapid and quantitative analysis of erythroid chimerism. Platelet and leukocyte engraftment were detected by flow cytometry. BMT into sublethally irradiated (4 Gy) recipients demonstrated the failure of B6-GFP-derived cells to engraft relative to B6-CD45(a)-derived cells, suggesting that an immune barrier may prevent efficient engraftment of the transgenic cells in a setting of minimal ablation. These results establish limitations in the use of transgenic GFP expression as a donor marker in transplantation models.