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2.
Cell Rep ; 17(12): 3292-3304, 2016 12 20.
Article in English | MEDLINE | ID: mdl-28009297

ABSTRACT

AMP-activated protein kinase (AMPK) is a metabolic enzyme that can be activated by nutrient stress or genetic mutations. Missense mutations in the regulatory subunit, PRKAG2, activate AMPK and cause left ventricular hypertrophy, glycogen accumulation, and ventricular pre-excitation. Using human iPS cell models combined with three-dimensional cardiac microtissues, we show that activating PRKAG2 mutations increase microtissue twitch force by enhancing myocyte survival. Integrating RNA sequencing with metabolomics, PRKAG2 mutations that activate AMPK remodeled global metabolism by regulating RNA transcripts to favor glycogen storage and oxidative metabolism instead of glycolysis. As in patients with PRKAG2 cardiomyopathy, iPS cell and mouse models are protected from cardiac fibrosis, and we define a crosstalk between AMPK and post-transcriptional regulation of TGFß isoform signaling that has implications in fibrotic forms of cardiomyopathy. Our results establish critical connections among metabolic sensing, myocyte survival, and TGFß signaling.


Subject(s)
AMP-Activated Protein Kinases/genetics , Cardiomyopathies/genetics , Hypertrophy, Left Ventricular/genetics , Transforming Growth Factor beta1/genetics , AMP-Activated Protein Kinases/metabolism , Animals , Cardiomyopathies/metabolism , Cardiomyopathies/pathology , Cell Survival/genetics , Glycogen/metabolism , Humans , Hypertrophy, Left Ventricular/metabolism , Hypertrophy, Left Ventricular/pathology , Induced Pluripotent Stem Cells/transplantation , Metabolome/genetics , Mice , Muscle Cells/metabolism , Muscle Cells/pathology , Mutation, Missense , Sequence Analysis, RNA , Signal Transduction , Tissue Engineering/methods , Transforming Growth Factor beta1/metabolism
3.
Development ; 142(5): 846-57, 2015 Mar 01.
Article in English | MEDLINE | ID: mdl-25633351

ABSTRACT

The transcriptional profiles of cardiac cells derived from murine embryos and from mouse embryonic stem cells (mESCs) have primarily been studied within a cell population. However, the characterization of gene expression in these cells at a single-cell level might demonstrate unique variations that cannot be appreciated within a cell pool. In this study, we aimed to establish a single-cell quantitative PCR platform and perform side-by-side comparison between cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) derived from mESCs and mouse embryos. We first generated a reference map for cardiovascular single cells through quantifying lineage-defining genes for CPCs, CMs, smooth muscle cells (SMCs), endothelial cells (EDCs), fibroblasts and mESCs. This panel was then applied against single embryonic day 10.5 heart cells to demonstrate its ability to identify each endocardial cell and chamber-specific CM. In addition, we compared the gene expression profile of embryo- and mESC-derived CPCs and CMs at different developmental stages and showed that mESC-derived CMs are phenotypically similar to embryo-derived CMs up to the neonatal stage. Furthermore, we showed that single-cell expression assays coupled with time-lapse microscopy can resolve the identity and the lineage relationships between progenies of single cultured CPCs. With this approach, we found that mESC-derived Nkx2-5(+) CPCs preferentially become SMCs or CMs, whereas single embryo-derived Nkx2-5(+) CPCs represent two phenotypically distinct subpopulations that can become either EDCs or CMs. These results demonstrate that multiplex gene expression analysis in single cells is a powerful tool for examining the unique behaviors of individual embryo- or mESC-derived cardiac cells.


Subject(s)
Embryo, Mammalian/cytology , Embryonic Stem Cells/cytology , Animals , Cell Differentiation/physiology , Cell Lineage , Cells, Cultured , Embryoid Bodies/cytology , Endothelial Cells/cytology , Female , Fibroblasts/cytology , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/metabolism , Mice , Myocytes, Cardiac/cytology , Myocytes, Smooth Muscle/cytology , Pregnancy , Transcription Factors/metabolism
4.
PLoS One ; 9(12): e113775, 2014.
Article in English | MEDLINE | ID: mdl-25436607

ABSTRACT

Vertebrate heart development is strictly regulated by temporal and spatial expression of growth and transcription factors (TFs). We analyzed nine TFs, selected by in silico analysis of an Nkx2.5 enhancer, for their ability to transactivate the respective enhancer element that drives, specifically, expression of genes in cardiac progenitor cells (CPCs). Mzf1 showed significant activity in reporter assays and bound directly to the Nkx2.5 cardiac enhancer (Nkx2.5 CE) during murine ES cell differentiation. While Mzf1 is established as a hematopoietic TF, its ability to regulate cardiogenesis is completely unknown. Mzf1 expression was significantly enriched in CPCs from in vitro differentiated ES cells and in mouse embryonic hearts. To examine the effect of Mzf1 overexpression on CPC formation, we generated a double transgenic, inducible, tetOMzf1-Nkx2.5 CE eGFP ES line. During in vitro differentiation an early and continuous Mzf1 overexpression inhibited CPC formation and cardiac gene expression. A late Mzf1 overexpression, coincident with a second physiological peak of Mzf1 expression, resulted in enhanced cardiogenesis. These findings implicate a novel, temporal-specific role of Mzf1 in embryonic heart development. Thereby we add another piece of puzzle in understanding the complex mechanisms of vertebrate cardiac development and progenitor cell differentiation. Consequently, this knowledge will be of critical importance to guide efficient cardiac regenerative strategies and to gain further insights into the molecular basis of congenital heart malformations.


Subject(s)
Enhancer Elements, Genetic , Heart/embryology , Homeodomain Proteins/genetics , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Transcription Factors/genetics , Animals , Cell Differentiation , Cell Line , Cells, Cultured , Computer Simulation , Embryonic Stem Cells/cytology , Gene Expression Regulation, Developmental , HEK293 Cells , Homeobox Protein Nkx-2.5 , Humans , Mice , Mice, Transgenic
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