Reference glycan structure libraries of primary human cardiomyocytes and pluripotent stem cell-derived cardiomyocytes reveal cell-type and culture stage-specific glycan phenotypes

AbstractCell surface glycoproteins play critical roles in maintaining cardiac structure and function in health and disease and the glycan-moiety attached to the protein is critical for proper protein folding, stability and signaling. However, despite mounting evidence that glycan structures are key modulators of heart function and must be considered when developing cardiac biomarkers, we currently do not have a comprehensive view of the glycans present in the normal human heart. In the current study, we used porous graphitized carbon liquid chromatography interfaced with mass spectrometry (PGC-LC-MS) to generate glycan structure libraries for primary human heart tissue homogenate, cardiomyocytes (CM) enriched from human heart tissue, and human induced pluripotent stem cell derived CM (hiPSC-CM). Altogether, we established the first reference structure libraries of the cardiac glycome containing 265 N- and O-glycans. Comparing the N-glycome of CM enriched from primary heart tissue to that of heart tissue homogenate, 21 structures significantly differed, and the high mannose class is increased in enriched CM. Moreover, by comparing primary CM to hiPSC-CM collected during 20-100 days of differentiation, dynamic changes in the glycan profile throughout in vitro differentiation were observed and differences between primary and hiPSC-CM were revealed. Namely, >30% of the N-glycome significantly changed across these time-points of differentiation and only 23% of the N-glycan structures were shared between hiPSC-CM and primary CM. These observations are an important complement to current genomic, transcriptomic, and proteomic profiling and reveal new considerations for the use and interpretation of hiPSC-CM models for studies of human development, disease, and drug testing. Finally, these data are expected to support future regenerative medicine efforts by informing targets for evaluating the immunogenic potential of hiPSC-CM and harnessing differences between immature, proliferative hiPSC-CM and adult primary CM.

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Abstract MP143: Harnessing Glycomics to Understand Cardiac Biology and Disease

Circulation Research ◽

10.1161/res.127.suppl_1.mp143 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Christopher Ashwood ◽

Linda Berg Luecke ◽

Rebekah L Gundry

Keyword(s):

Human Heart ◽

Heart Function ◽

Heart Tissue ◽

Cardiac Biomarkers ◽

Tissue Homogenate ◽

Glycan Structure ◽

Cell Type ◽

Cell Type Specific ◽

Human Heart Tissue

Cell surface glycoproteins play critical roles in maintaining cardiac structure and function, and the glycan-moiety attached to a protein is critical for proper protein folding, stability, and signaling. Despite mounting evidence that glycan structures are key modulators of heart function and must be considered when developing cardiac biomarkers, we currently do not have a comprehensive view of the glycans present in the normal human heart. Here, we used an innovative mass spectrometry approach to generate the first glycan structure libraries for primary human heart tissue, cardiomyocytes (CM) enriched from human heart tissue, and human induced pluripotent stem cell derived CM (hiPSC-CM), containing >260 N- and O- glycans. Comparing the glycome of CM enriched from primary heart tissue to that of heart tissue homogenate, 21 structures significantly differed, and the high mannose class is increased in enriched CM. Moreover, >30% of the glycome significantly changed across 20-100 days of in vitro differentiation, and only 23% of the N -glycan structures were shared between hiPSC-CM and primary CM. Overall, these observations are an important complement to genomic, transcriptomic, and proteomic profiling and reveal new considerations for the use and interpretation of hiPSC-CM models for studies of human development, disease, and drug testing. These data are also expected to aid in the evaluation of the immunogenic potential of hiPSC-CM for transplantation. Finally, harnessing differences observed between immature, proliferative hiPSC-CM and adult primary CM may be exploited to drive in vitro differentiation towards a more mature phenotype. Building on these data, current efforts are underway to develop chamber- and cell-type specific views ( e.g. cardiomyocytes, fibroblasts) of the glycome in the healthy and failing human heart. Such analyses provide a key link to understand the role glycosylation plays in cell-type specific functions and cardiac disease. The structural differences observed here, either among cell types or stages of differentiation, require complex regulation of multiple enzymes in the biosynthetic pathway, and therefore would be challenging to measure with antibody arrays, RNAseq, or proteomics. Therefore, continued application of structure-based glycomics approaches, such as the method used here, will be essential for elucidating the roles that glycans and glycoproteins play during developmental and disease processes in the human heart.

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Reference glycan structure libraries of primary human cardiomyocytes and pluripotent stem cell-derived cardiomyocytes reveal cell-type and culture stage-specific glycan phenotypes

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2019.12.012 ◽

2020 ◽

Vol 139 ◽

pp. 33-46 ◽

Cited By ~ 2

Author(s):

Christopher Ashwood ◽

Matthew Waas ◽

Ranjuna Weerasekera ◽

Rebekah L. Gundry

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Glycan Structure ◽

Cell Type ◽

Human Cardiomyocytes ◽

Culture Stage

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Maturation of stem cell-derived human heart tissue by mimicking fetal heart rate

Future Cardiology ◽

10.2217/fca.13.71 ◽

2013 ◽

Vol 9 (6) ◽

pp. 751-754 ◽

Cited By ~ 6

Author(s):

Sara S Nunes ◽

Jason W Miklas ◽

Milica Radisic

Keyword(s):

Heart Rate ◽

Stem Cell ◽

Fetal Heart Rate ◽

Human Heart ◽

Fetal Heart ◽

Heart Tissue ◽

Human Heart Tissue

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Benefit of microwave-assisted acid hydrolysis of proteins for mass spectrometric profiling of the human heart tissue proteome

Rapid Communications in Mass Spectrometry ◽

10.1002/rcm.3125 ◽

2007 ◽

Vol 21 (16) ◽

pp. 2779-2783 ◽

Cited By ~ 5

Author(s):

Mulu Gebremedhin ◽

Hongying Zhong ◽

Shaohua Wang ◽

Michael Weinfeld ◽

Liang Li

Keyword(s):

Acid Hydrolysis ◽

Human Heart ◽

Heart Tissue ◽

Mass Spectrometric ◽

Microwave Assisted ◽

Hydrolysis Of ◽

Tissue Proteome ◽

Human Heart Tissue

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IMMUNOLOGIC RELATION OF STREPTOCOCCAL AND TISSUE ANTIGENS

Journal of Experimental Medicine ◽

10.1084/jem.119.4.651 ◽

1964 ◽

Vol 119 (4) ◽

pp. 651-666 ◽

Cited By ~ 109

Author(s):

Melvin H. Kaplan ◽

Kathryn H. Svec

Keyword(s):

Human Heart ◽

Streptococcal Infection ◽

Heart Tissue ◽

Antigenic Determinants ◽

Heat Inactivation ◽

Prolonged Storage ◽

Acute Glomerulonephritis ◽

Streptococcal Antigen ◽

Reactive Antibody ◽

Human Heart Tissue

Sera from patients with recent streptococcal infection or non-suppurative sequelae exhibit with variable frequency a precipitin reaction in agar gel with a partially purified streptococcal antigen which has been shown previously to be immunologically related to human heart tissue. This precipitin could be absorbed from sera with normal human heart tissue homogenates but not with homogenates of other organs. Demonstration of this cross-reaction by heart absorption was found dependent both upon the serologic properties of individual sera and the nature or state of purification of the streptococcal product employed as test antigen. Evidence was obtained of a close association of heart-related and non-heart-related antigenic determinants in partially purified preparations of the streptococcal antigen by both gel diffusion and immunoelectrophoresis. On immunoelectrophoretic analysis, cross-reactive antigen exhibited a more rapid mobility toward the anode than M protein. It was destroyed by digestion with trypsin, pepsin, and chymotrypsin. Based on specific absorption tests with a Type 5 and Type 19 strain, the antigen was localized to cell walls and to a lesser extent to cell membranes of these strains. Precipitating activity related to cross-reactive antibody was localized to the immunoglobulin zone in immunoelectrophoresis. Reactive sera showed diminution or loss of serological activity following heat inactivation at 56°C or after prolonged storage at 4°C. Sera containing cross-reactive precipitating antibody exhibited an immunofluorescent reaction with sarcolemma of cardiac myofibers, which was inhibited by streptococcal cross-reactive antigen. By this inhibition test, the immunofluorescent reaction related to cross-reactive antibody could be distinguished from that due to other heart-reactive factors. Antibody to streptococcal cross-reactive antigen defined by precipitation-absorption tests was observed in 24 per cent of patients with recent history of uncomplicated streptococcal infection and in the majority of patients with acute rheumatic fever, rheumatic heart disease, or acute glomerulonephritis. It was observed rarely in patients with non-streptococcal related disease. These data provide evidence that induction of cross-reactive autoantibody to heart in certain individuals is associated with streptococcal infection.

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Factors for C-Kit Expression in Cardiac Outgrowth Cells and Human Heart Tissue

International Heart Journal ◽

10.1536/ihj.16-559 ◽

2017 ◽

Vol 58 (6) ◽

pp. 962-968

Author(s):

Satoshi Matsushita ◽

Kazuo Minematsu ◽

Taira Yamamoto ◽

Hirotaka Inaba ◽

Kenji Kuwaki ◽

...

Keyword(s):

Human Heart ◽

Heart Tissue ◽

Human Heart Tissue

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Ion Channel Expression and Electrophysiology of Singular Human (Primary and Induced Pluripotent Stem Cell-Derived) Cardiomyocytes

Cells ◽

10.3390/cells10123370 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3370

Author(s):

Christina Schmid ◽

Najah Abi-Gerges ◽

Michael Georg Leitner ◽

Dietmar Zellner ◽

Georg Rast

Keyword(s):

Stem Cell ◽

Single Cell ◽

Pluripotent Stem Cell ◽

Quantitative Polymerase Chain Reaction ◽

Drug Effects ◽

Induced Pluripotent Stem Cell ◽

Ionic Currents ◽

Human Cardiomyocytes ◽

Ventricular Cells ◽

Induced Pluripotent

Subtype-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are promising tools, e.g., to assess the potential of drugs to cause chronotropic effects (nodal hiPSC-CMs), atrial fibrillation (atrial hiPSC-CMs), or ventricular arrhythmias (ventricular hiPSC-CMs). We used single-cell patch-clamp reverse transcriptase-quantitative polymerase chain reaction to clarify the composition of the iCell cardiomyocyte population (Fujifilm Cellular Dynamics, Madison, WI, USA) and to compare it with atrial and ventricular Pluricytes (Ncardia, Charleroi, Belgium) and primary human atrial and ventricular cardiomyocytes. The comparison of beating and non-beating iCell cardiomyocytes did not support the presence of true nodal, atrial, and ventricular cells in this hiPSC-CM population. The comparison of atrial and ventricular Pluricytes with primary human cardiomyocytes showed trends, indicating the potential to derive more subtype-specific hiPSC-CM models using appropriate differentiation protocols. Nevertheless, the single-cell phenotypes of the majority of the hiPSC-CMs showed a combination of attributes which may be interpreted as a mixture of traits of adult cardiomyocyte subtypes: (i) nodal: spontaneous action potentials and high HCN4 expression and (ii) non-nodal: prominent INa-driven fast inward current and high expression of SCN5A. This may hamper the interpretation of the drug effects on parameters depending on a combination of ionic currents, such as beat rate. However, the proven expression of specific ion channels supports the evaluation of the drug effects on ionic currents in a more realistic cardiomyocyte environment than in recombinant non-cardiomyocyte systems.

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