Glycogenin is Dispensable for Glycogen Synthesis in Human Muscle, and Glycogenin Deficiency Causes Polyglucosan Storage

Glycogenin is considered to be an essential primer for glycogen biosynthesis. Nevertheless, patients with glycogenin-1 deficiency due to biallelic GYG1 (NM_004130.3) mutations can store glycogen in muscle. Glycogenin-2 has been suggested as an alternative primer for glycogen synthesis in patients with glycogenin-1 deficiency. OBJECTIVE: The objective of this article is to investigate the importance of glycogenin-1 and glycogenin-2 for glycogen synthesis in skeletal and cardiac muscle. DESIGN, SETTING, AND PATIENTS: Glycogenin-1 and glycogenin-2 expression was analyzed by Western blot, mass spectrometry, and immunohistochemistry in liver, heart, and skeletal muscle from controls and in skeletal and cardiac muscle from patients with glycogenin-1 deficiency. RESULTS: Glycogenin-1 and glycogenin-2 both were found to be expressed in the liver, but only glycogenin-1 was identified in heart and skeletal muscle from controls. In patients with truncating GYG1 mutations, neither glycogenin-1 nor glycogenin-2 was expressed in skeletal muscle. However, nonfunctional glycogenin-1 but not glycogenin-2 was identified in cardiac muscle from patients with cardiomyopathy due to GYG1 missense mutations. By immunohistochemistry, the mutated glycogenin-1 colocalized with the storage of glycogen and polyglucosan in cardiomyocytes. CONCLUSIONS: Glycogen can be synthesized in the absence of glycogenin, and glycogenin-1 deficiency is not compensated for by upregulation of functional glycogenin-2. Absence of glycogenin-1 leads to the focal accumulation of glycogen and polyglucosan in skeletal muscle fibers. Expression of mutated glycogenin-1 in the heart is deleterious, and it leads to storage of abnormal glycogen and cardiomyopathy

COMP (Cartilage Oligomeric Matrix Protein) Neoepitope A Novel Biomarker to Identify Symptomatic Carotid Stenosis

Objective:COMP (cartilage oligomeric matrix protein) is abundantly expressed in the cardiovascular system, cartilage, and atherosclerotic plaques. We investigated if the total COMP (COMPtotal) and COMP neoepitope (COMPneo) with other cardiovascular markers and clinical parameters could identify symptomatic carotid stenosis.Approach and Results:Blood samples were collected from patients with symptomatic carotid stenosis (stenosis, n=50), patients with stroke without carotid stenosis but small plaques (plaque, n=50), and control subjects (n=50). COMPtotal and COMPneo were measured using an ELISA. Ninety-two cardiovascular disease markers were measured by the Olink CVD kit. The presence of native COMP and COMPneo was determined by immunohistochemistry. The concentration of COMPneo was higher and COMPtotal was lower in the stenosis group. When the concentration was compared between the stenosis and control groups, IL-1ra (interleukin-1 receptor antagonist protein), IL6 (interleukin-6), REN (Renin), MMP1 (matrix metalloproteinase-1), TRAIL-R2 (tumor necrosis factor-related apoptosis-inducing ligand receptor 2), ITGB1BP2 (integrin beta 1 binding protein 2), and COMPneo were predictive of stenosis. Conversely, KLK6 (kallikrein-6), COMPtotal, NEMO (nuclear factor-kappa-B essential modulator), SRC (Proto-oncogene tyrosine-protein kinase Src), SIRT2 (SIR2-like protein), CD40 (cluster of differentiation 40), TF (tissue factor), MP (myoglobin), and RAGE (receptor for advanced glycation end-products) were predictive of the control group. Model reproducibility was good with the receiver operating characteristic plot area under the curve being 0.86. When comparing the plaque group and stenosis group, COMPneo, GAL (galanin), and PTX3 (pentraxin-related protein PTX3) were predictive of stenosis. Model reproducibility was excellent (receiver operating characteristic plot area under the curve 0.92). COMPneo was detected in smooth muscle-, endothelial-, and foam-cells in carotid stenosis.Conclusions:Degradation of COMP may be associated with atherosclerosis progression and generation of a specific COMP fragment-COMPneo. This may represent a novel biomarker that together with COMPtotal and other risk-markers could be used to identify symptomatic carotid stenosis

Identification and characterization of progenitor populations in the human adult heart

Traditionally, the heart has been regarded as a non-regenerative organ. During the last 10 years, this notion has been challenged. By 14C measurements, it was calculated that at the age of 50, about 45% of all cardiomyocytes had formed after birth. An endogenous population of progenitor cells in the heart has been suggested as the source of this regeneration. Until now, most studies have however been conducted in animal models which may not fully reflect the human situation. The overall aim of this thesis was to add to our knowledge of the identity, distribution and function of endogenous progenitor cells in the human adult heart. In paper I, a small population of C-kit+ cells was identified, that could be sub-divided based on expression of the hematopoietic marker CD45. The C-kit+CD45+ population was determined to be of mast cell phenotype whereas the C-kit+CD45- population expressed endothelial associated markers. Differentiation assays showed further endothelial maturation but no evidence of cardiac differentiation. In paper II, heterogeneity within the C-kit+CD45- population was further investigated by single cell qPCR. The results indicated that while most of the C-kit+CD45- cells were committed to the endothelial lineage, a minor portion of them could represent cardiac progenitors. In paper III, Side Population (SP) cells were identified in the left atrium. The SP phenotype was linked to the MDR1 protein. On gene expression level, the SP cells expressed high levels of MDR1 as well as stem cell associated genes C-KIT and OCT-4. Furthermore, the SP could be subdivided based on expression of the hematopoietic marker CD45. The CD45- SP cells had an endothelial profile while the CD45+ SP cells were neither committed to the endothelial, nor the cardiomyogenic lineage. In paper IV, expression of SSEA-1, 3 and 4 was investigated. All SSEAs were expressed at variable levels. The SSEA-1+ population was determined to be of hematopoietic origin. Of the SSEA-4+ cells, some co-expressed CD34. In right atrium, the SSEA-4+CD34- population displayed a high expression of cardiomyocyte genes. By immunohistochemistry, SSEA-4+ cells were identified both within and outside the myocardium. In conclusions, in the present thesis, three different cell populations with characteristics were isolated from human cardiac biopsy material. One C-kit+CD45- population that consisted of both endothelial and cardiac committed progenitors. SP cells where the CD45- fraction showed evidence of endothelial commitment and SSEA-4+CD34- cells that showed signs of cardiac commitment

Supplemental Table 1

 Supplemental Table 1</p

Supplemental Tables Differential Gene Expression

 Supplemental Tables Differential Gene Expression </p

Supplemental material, Cytoscape files

Supplemental material, Cytoscape files based on Gene Set Enrichment Analysis (GSEA) using the gene ontology: biological processes annotation set. </p

RNA sequencing raw count file and sample phenotypic information.

 RNA sequencing raw count file and sample phenotypic information.  </p

Supplemental Figures S1-S6

Supplemental Figures S1-S6 </p

Neither Notch1 expression nor cellular size correlate with mesenchymal stem cell properties of adult articular chondrocytes.

BACKGROUND: Tissue repair is thought to be regulated by progenitor cells, which in other tissues are characterized by their Notch1 expression or small cellular size. Here we studied if these traits affect the chondrogenic potential and are markers for multipotent progenitor cell populations in adult articular cartilage. METHODS: Directly isolated articular chondrocytes were sorted with regard to their Notch1 expression or cellular size. Their colony forming efficiency (CFE) and their potential to differentiate towards adipogenic, osteogenic and chondrogenic lineages were investigated. The different sorted populations were also expanded in monolayer and analyzed in the same manner as the directly isolated cells. RESULTS: No differences in CFE or adipogenic, osteogenic and chondrogenic potentials were detected among the sorted populations. Expanded cells displayed a higher osteochondral potential than directly isolated cells. CONCLUSION: Cellular size or Notch1 expression is not per se a specific marker for mesenchymal progenitor cells in adult articular cartilage. Monolayer-expanded adult chondrocytes contain a larger mesenchymal progenitor cell-like population than directly isolated cells, highly likely as a result of dedifferentiation. If there are resident Notch1-positive cells or cells of a specific size in adult articular cartilage with functional features of progenitor cells, the population consists of only a very small number of cells

Hypoxic cardiac fibroblasts from failing human hearts decrease cardiomyocyte beating frequency in an ALOX15 dependent manner.

A common denominator for patients with heart failure is the correlation between elevated serum levels of proinflammatory cytokines and adverse clinical outcomes. Furthermore, lipoxygenase-induced inflammation is reportedly involved in the pathology of heart failure. Cardiac fibroblasts, which are abundant in cardiac tissue, are known to be activated by inflammation. We previously showed high expression of the lipoxygenase arachidonate 15 lipoxygenase (ALOX15), which catalyzes the conversion of arachidonic acid to 15-hydroxy eicosatetraenoic acid (15-HETE), in ischemic cardiac tissue. The exact roles of ALOX15 and 15-HETE in the pathogenesis of heart failure are however unknown. Biopsies were collected from all chambers of explanted failing human hearts from heart transplantation patients, as well as from the left ventricles from organ donors not suffering from chronic heart failure. Biopsies from the left ventricles underwent quantitative immunohistochemical analysis for ALOX15/B. Gene expression of ALOX enzymes, as well as 15-HETE levels, were examined in cardiac fibroblasts which had been cultured in either hypoxic or normoxic conditions after isolation from failing hearts. After the addition of fibroblast supernatants to human induced pluripotent stem cell-derived cardiomyocytes, intracellular calcium concentrations were measured to examine the effect of paracrine signaling on cardiomyocyte beating frequency. While ALOX15 and ALOX15B were expressed throughout failing hearts as well as in hearts from organ donors, ALOX15 was expressed at significantly higher levels in donor hearts. Hypoxia resulted in a significant increase in gene and protein expression of ALOX15 and ALOX15B in fibroblasts isolated from the different chambers of failing hearts. Finally, preconditioned medium from hypoxic fibroblasts decreased the beating frequency of human cardiomyocytes derived from induced pluripotent stem cells in an ALOX15-dependent manner. In summary, our results demonstrate that ALOX15/B signaling by hypoxic cardiac fibroblasts may play an important role in ischemic cardiomyopathy, by decreasing cardiomyocyte beating frequency