Differentiation of cardiomyocytes from human embryonic stem cells is accompanied by changes in the extracellular matrix production of versican and hyaluronan

Abstract

Proteoglycans and hyaluronan play critical roles in heart development. In this study, human embryonic stem cells (hESC) were used as a model to quantify the synthesis of proteoglycans and hyaluronan in hESC in the early stages of differentiation, and after directed differentiation into cardiomyocytes. We demonstrated that both hESC and cardiomyocyte cultures synthesize an extracellular matrix (ECM) enriched in proteoglycans and hyaluronan. During cardiomyocyte differentiation, total proteoglycan and hyaluronan decreased and the proportion of proteoglycans bearing heparan sulfate chains was reduced. Versican, a chondroitin sulfate proteoglycan, accumulated in hESC and cardiomyocyte cultures. Furthermore, versican synthesized by hESC contained more N- and O-linked oligosaccharide than versican from cardiomyocytes. Transcripts for the versican variants, V0, V1, V2, and V3, increased in cardiomyocytes compared to hESC, with V1 most abundant. Hyaluronan in hESC had lower molecular weight than hyaluronan from cardiomyocyte cultures. These changes were accompanied by an increase in HAS-1 and HAS-2 mRNA in cardiomyocyte cultures, with HAS-2 most abundant. Interestingly, HAS-3 was absent from the cardiomyocyte cultures, but expressed by hESC. These results indicate that human cardiomyocyte differentiation is accompanied by specific changes in the expression and accumulation of ECM components and suggest a role for versican and hyaluronan in this process.

Figure 1. Directed differrentiation of cardiomyocyte cultures results in a reduction of total proteoglycan synthesis

(A). hESC-CM preparations express the cardiac marker β-myosin heavy chain. This confocal image shows a representative directly differentiated hESC culture that has been immunostained with an antibody against the human striated muscle marker β-myosin heavy chain. Note that, as was typical for the preparations used elsewhere in this study, approximately 50% of the cells were immunoreactive for this marker. Scale bar = 50 micrometer, red - β-myosin heavy chain, blue – Hoechst dye nuclear stain. (B). Proteoglycan accumulation by hESC and cardiomyocyte cultures. After 24 h incubation, total accumulated [³⁵S]-sulfate-labeled proteoglycans (cell plus medium extracts, normalized to total protein), were reduced in differentiated cells. This experiment was repeated 3 times with similar results. **P < 0.01 compared to hESC.

Figure 2. Ion-exchange analysis of radiolabeled proteoglycans in hESC and cardiomyocyte cultures

[³⁵S]-sulfate-labeled proteoglycans in 8M urea buffer were applied to DEAE sepharose and eluted with a 0-0.8M NaCl gradient in 8M urea buffer. (A). hESC medium; (B). cardiomyocyte medium; (C). hESC cell layer; and (D). cardiomyocyte cell layer. This experiment was repeated 2 times with similar results.

Figure 3. Molecular sieve chromatography of radiolabeled proteoglycans synthesized by hESC and cardiomyocyte cultures

[³⁵S]-sulfate-labeled proteoglycans were characterized by size exclusion chromatography using Sepharose CL-2B. (A). hESC medium (closed circles) and cardiomyocyte medium (open circles). (B). hESC cell layer (closed circles) and cardiomyocyte cell layer (open circles). Samples were applied on the basis of equal protein in A and B. Panels C through F depict column profiles before and after chondroitin ABC lyase digestion, results are expressed as percent of total DPM per column. (C). hESC medium; (D). hESC cell layer; (E). cardiomyocyte medium; and (F). cardiomyocyte cell layer. Triangles indicate enzyme digestion. Bars indicate area used to calculate percent composition. This experiment was repeated 3 times with similar results.

Figure 4. Characterization of radiolabeled proteoglycans by SDS-PAGE

[³⁵S]-sulfate-labeled proteoglycans were subjected to SDS-PAGE. (A). Intact proteoglycans. (B). Proteoglycans were digested with chondroitin ABC lyase (Ch'ase), Heparinase I, II, and III (Hep'ase) or the combination of all four enzymes. This experiment was repeated 3 times with similar results. Data shown is from a representative experiment. Each lane contains material from a separate well.

Figure 5. Identification of proteoglycan core proteins

(A). [³⁵S]-methionine labeled proteoglycans were digested with chondroitin ABC lyase (Ch'ase), Heparinase I, II, and III (Hep'ase) or the combination of all four enzymes and applied using an equal amount of radioactivity to 4-12% gradient SDS-PAGE. (B). Western analysis for versican after Ch'ase digestion. Gels were loaded with equal amounts of protein. (C). Western analysis of media from hESC or cardiomyocytes incubated with Ch'ase, or Ch'ase plus the total deglycosylation mixture to completely remove all N- and O-linked carbohydrates from glycoproteins. The protein content of lanes in C was not normalized. Lanes: 1 – hESC; 2 – cardiomyocyte cultures; 3 – Ch'ase plus buffer; and 4 – Ch'ase mixture, exchanged into water, plus the deglycosylation enzymes and buffer. Arrows indicate versican after Ch'ase: solid arrows – cardiomyocyte versican; open arrows – hESC versican; fine arrows – cardiomyocyte or hESC versican after Ch'ase digestion and total deglycosylation. Experiments shown in 5A and B were repeated 3 times with similar results. The experiment shown in 5C was repeated 3 times, but with a single batch of conditioned medium.

Figure 6. Versican splice variant and ADAMTS mRNA expression by QRTPCR

Relative copy numbers of (A). versican splice variants, V0, V1, V2, V3, and (B). versicanases, ADAMTS1, 4, and 5 were determined by QRTPCR and normalized to the mRNA copy number for 18S ribosomal protein. This experiment was repeated 3 times with similar results. *P < 0.05, **P < 0.01 compared to hESC.

Figure 7. Alterations in hyaluronan production which accompany differentiation of cardiomyocytes

(A). Hyaluronan accumulation by hESC and cardiomyocytes. After 24 h incubation with fresh medium, hyaluronan content was determined in the total cultures and normalized to total cell layer protein. This experiment was repeated 4 times with similar results. *P < 0.05 compared to hESC. (B - C). Molecular sieve analysis of hyaluronan synthesized by hESC and cardiomyocyte cultures. Cultures were incubated for 24 h in medium containing [³H]-glucosamine after which, cell layers and media were applied to Sepharose-S-1000 molecular sieve columns. Results show the distribution of hyaluronidase sensitive material. Open circles – cardiomyocytes, closed circles – ESC. This experiment was repeated 2 times with similar results. B - medium, C – cell layer.

Figure 8. HAS and HYAL mRNA expression by hESC and cardiomyocyte cultures

Differentiated and undifferentiated cultures were fed fresh medium and then harvested after 24 h for mRNA analysis. Levels of mRNA expression of (A). HAS- 1,-2 or- 3 or (B). HYALS 1 or 2 were determined by QRTPCR and expressed as mRNA copy number per 10⁵ 18S. This experiment was repeated 3 times with similar results. **P < 0.01 compared to control.