To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.Generation of cardiovascular cells from human pluripotent stem cells (hPSCs) in culture could provide a powerful model system for investigating cellular interactions and molecular regulators that govern the specification, commitment and maturation of these lineages, as well as a unique and unlimited source of human cardiomyocytes for drug testing and regenerative medicine strategies [1][2][3][4] . Translating this potential into practice, however, will depend on the development of technologies that enable the reproducible generation of highly enriched populations of cardiomyocytes, as contaminating cell types could affect drug Reprints and permissions information is available online at

Defective mobilization of Ca2؉ by cardiomyocytes can lead to cardiac insufficiency, but the causative mechanisms leading to congestive heart failure (HF) remain unclear. In the present study we performed exhaustive global proteomics surveys of cardiac ventricle isolated from a mouse model of cardiomyopathy overexpressing a phospholamban mutant, R9C (PLN-R9C), and exhibiting impaired Ca 2؉ handling and death at 24 weeks and compared them with normal control littermates. The relative expression patterns of 6190 high confidence proteins were monitored by shotgun tandem mass spectrometry at 8, 16, and 24 weeks of disease progression. Significant differential abundance of 593 proteins was detected. These proteins mapped to select biological pathways such as endoplasmic reticulum stress response, cytoskeletal remodeling, and apoptosis and included known biomarkers of HF (e.g. brain natriuretic peptide/atrial natriuretic factor and angiotensin-converting enzyme) and other indicators of presymptomatic functional impairment. These altered proteomic profiles were concordant with cognate mRNA patterns recorded in parallel using high density mRNA microarrays, and top candidates were validated by RT-PCR and Western blotting. Mapping of our highest ranked proteins against a human diseased explant and to available data sets indicated that many of these proteins could serve as markers of disease. Indeed we showed that several of these proteins are detectable in mouse and human plasma and display differential abundance in the plasma of diseased mice and affected patients. These results offer a systems-wide perspective of the dynamic maladaptions associated with impaired Ca

Summary The advent of reprogramming and its impact on stem cell biology has renewed interest in lineage restriction in mammalian embryos, the source of embryonic (ES), epiblast (EpiSC), trophoblast (TS), and extraembryonic endoderm (XEN) stem cell lineages. Isolation of specific cell types during stem cell differentiation and reprogramming, and also directly from embryos, is a major technical challenge because few cell-surface proteins are known that can distinguish each cell type. We provide a large-scale proteomic resource of cell-surface proteins for the four embryo-derived stem cell lines. We validated 27 antibodies against lineage-specific cell-surface markers, which enabled investigation of specific cell populations during ES-EpiSC reprogramming and ES-to-XEN differentiation. Identified markers also allowed prospective isolation and characterization of viable lineage progenitors from blastocysts by flow cytometry. These results provide a comprehensive stem cell proteomic resource and enable new approaches to interrogate the mechanisms that regulate cell fate specification.

We have developed a method for creating C3A liver spheroids and demonstrated cellular polarisation, zonation as well as increased liver-specific functionality and more predictive toxicological response compared to standard 2D liver models.

Malignant hyperthermia susceptibility (MHS) is a subclinical pharmacogenetic disorder caused by an impairment of skeletal muscle calcium homeostasis in response to triggering agents. While in vitro contracture testing (IVCT) is the gold standard for defining MHS, molecular analysis is increasingly used to diagnosis MHS. Mutations associated with MHS have been reported in two genes: RYR1 and CACNA1S. Mutations in RYR1 are also responsible for central core disease (CCD), a myopathy that can be associated with a positive IVCT response. We report here the results of correlation studies performed with molecular, pharmacological, histological, and functional data obtained in 175 families (referred to as confirmed (129) or potential (46) MHS families). Extensive molecular analysis allowed us to identify a variant in 60% of the confirmed MHS families, and resulted in the characterization of 11 new variants in the RYR1 gene. Most mutations clustered to MH1 and MH2 domains of RYR1. Functional analysis allowed us to assign a causative role for seven MHS mutations that we propose to add to the panel of MHS mutations used for genetic testing. The use of genetic data to determine MHS status led to a 99.5% sensitivity for IVCT. IVCT-positive/mutation-negative diagnoses were analyzed not only in terms of specificity for IVCT, but also to assess the presence of a second MHS trait in families, and the genetic heterogeneity of the disease. Histological analyses revealed the presence of cores in more than 20% of muscle biopsies originating from 242 genotyped and tested MHS patients who did not present with clinical symptoms. This indicates that these patients must be considered as MHS patients with cores, and are clearly differentiated from CCD patients who have been tested positive for MHS.

α-Crystallin B (cryAB) is the most abundant small heat shock protein in cardiomyocytes (CMs) and has been shown to have potent antiapoptotic properties. Because the mechanism by which cryAB prevents apoptosis has not been fully characterized, we examined its protective effects at the cellular level by silencing cryAB in mouse neonatal CMs using lentivector-mediated transduction of short hairpin RNAs. Subcellular fractionation of whole hearts showed that cryAB is cytosolic under control conditions, and after H(2)O(2) exposure, it translocates to the mitochondria. Phosphorylated cryAB (PcryAB) is mainly associated with the mitochondria, and any residual cytosolic PcryAB translocates to the mitochondria after H(2)O(2) exposure. H(2)O(2) exposure caused increases in cryAB and PcryAB levels, and cryAB silencing resulted in increased levels of apoptosis after exposure to H(2)O(2). Coimmunoprecipitation assays revealed an apparent interaction of both cryAB and PcryAB with mitochondrial voltage-dependent anion channels (VDAC), translocase of outer mitochondrial membranes 20 kDa (TOM 20), caspase 3, and caspase 12 in mouse cardiac tissue. Our results are consistent with the conclusion that the cardioprotective effects of cryAB are mediated by its translocation from the cytosol to the mitochondria under conditions of oxidative stress and that cryAB interactions with VDAC, TOM 20, caspase 3, and caspase 12 may be part of its protective mechanism.

The ryanodine receptor (RyR) is a large, homotetrameric sarcoplasmic reticulum membrane protein that is essential for Ca 2+ cycling in both skeletal and cardiac muscle. Genetic mutations in RyR1 are associated with severe conditions including malignant hyperthermia (MH) and central core disease. One phosphorylation site (Ser 2843) has been identified in a segment of RyR1 flanked by two RyR motifs, which are found exclusively in all RyR isoforms as closely associated tandem (or paired) motifs, and are named after the protein itself. These motifs also contain six known MH mutations. In this study, we designed, expressed and purified the tandem RyR motifs, and show that this domain contains a putative binding site for the Ca 2+ /calmodulin-dependent protein kinase b isoform. We present a 2.2 Å resolution crystal structure of the RyR domain revealing a two-fold, symmetric, extended four-helix bundle stabilized by a b sheet. Using mathematical modelling, we fit our crystal structure within a tetrameric electron microscopy (EM) structure of native RyR1, and propose that this domain is localized in the RyR clamp region, which is absent in its cousin protein inositol 1,4,5-trisphosphate receptor. DatabaseThe crystal structure of the RyR1 phosphorylation domain (amino acid residues 2734-2940) has been submitted to the Protein Data Bank under accession number 3RQR. Structured digital abstractRyR1 C3 physically interacts with CaMKIIb by pull down (View interaction) RyR1 C3 binds to CaMKIIb by pull down (View interaction) CaMKIIb physically interacts with RyR1 C3 by anti tag coimmunoprecipitation (View Interaction: 1, 2)Abbreviations EM, electron microscopy; CaMK, calcium/calmodulin-dependent protein kinase; GFP, green fluorescent protein; IP 3 , inositol 1,4,5-trisphosphate; IP 3 R, inositol 1,4,5-trisphosphate receptor; IP 3 R-SD-IBC, IP 3 R-suppressor domain-IP 3 binding core; MH, malignant hyperthermia; RyR1, ryanodine receptor 1.

High resolution peptide separation is pivotal for successful shot-gun proteomics. The need for capable techniques propels invention and improvement of ever more sophisticated approaches. Recently, Agilent Technologies has introduced the OFFGEL fractionator, which conducts peptide separation by isoelectric focusing in an off-gel setup. This platform has been shown to accomplish high resolution of peptides for diverse sample types, yielding valuable advantages over comparable separation techniques. In this study, we deliver the first comparison of the newly emerging OFFGEL approach to the well-established on-line MudPIT platform. Samples from a membrane-enriched fraction isolated from murine C2C12 cells were subjected to replicate analysis by OFFGEL (12 fractions, pH 3 -10) followed by RP-LC-MS/MS or 12-step on-line MudPIT. OFFGEL analyses yielded 1398 proteins (identified by 10,269 peptides) while 1428 proteins (11,078 peptides) were detected with the MudPIT approach. Thus, our data shows that both platforms produce highly comparable results in terms of protein/peptide identifications and reproducibility for the sample type analyzed. We achieve more accurate peptide focusing after OFFGEL fractionation with 88 % of all peptides binned to a single fraction, as compared to 61 % of peptides detected in only one step in MudPIT analyses. Our study suggests that both platforms are equally capable of high quality peptide separation of a sample with medium complexity, rendering them comparably valuable for comprehensive proteomic analyses.