Quantification of decellularized human myocardial matrix: A comparison of six patients

Abstract

Purpose: The purpose of this study was to characterize and quantitatively analyze human cardiac extracellular matrix (ECM) isolated from six different cadaveric donor hearts.

Experimental design: ECM was isolated by decellularization of six human cadaveric donor hearts and characterized by quantifying sulfated glycosaminoglycan content (sGAG) and via PAGE. The protein content was then quantified using ECM-targeted Quantitative conCATamers (QconCAT) by LC-SRM analysis using 83 stable isotope labeled (SIL) peptides representing 48 different proteins. Nontargeted global analysis was also implemented using LC-MS/MS.

Results: The sGAG content, PAGE, and QconCAT proteomics analysis showed significant variation between each of the six patient samples. The quantitative proteomics indicated that the majority of the protein content was composed of various fibrillar collagen components. Also, quantification of difficult to remove cellular proteins represented less than 1% of total protein content, which is very low for a decellularized biomaterial. Global proteomics identified over 200 distinct proteins present in the human cardiac ECM.

Conclusion and clinical relevance: In conclusion, quantification and characterization of human myocardial ECM showed significant patient-to-patient variability between the six investigated patients. This is an important outcome for the development of allogeneic derived biomaterials and for increasing our understanding of human myocardial ECM composition.

Keywords: Decellularization; Extracellular matrix; MS; QconCAT.

Figure 1. sGAG Quantification and PAGE on the Injectable Human Myocardial Matrices

The sGAG content of each ECM was quantified with a DMMB assay. The matrix samples were significantly different from one another (p<0.05). PAGE was used to visualize the molecular weight bands of the standard ladder in kDa (A), the rat tail type 1 collagen control (B) and for p1 (C), p2 (D), p3 (E), p4 (F), p5 (G), and p6 (H).

Figure 2. QconCAT Quantification of Injectable Human Myocardial Matrices

Pie chart represents the average percentage of total protein from six injectable human myocardial matrices as assayed by an ECM targeted QconCAT LC-SRM assay. Individual percentages of total protein from the matrices are shown by column. Proteins were consolidated into their respective functional gene ontology class for each graph. Values representing less than 2% are not labeled in the column chart.

Figure 3. PCA Analysis of Myocardial Matrices Grouped by Gelation

Loading plot (left) highlights the top 10 proteins that discriminate the two groups of samples based on principle component analysis (PCA) of absolute protein quantification between samples that gelled (p4 & p6) and samples that did not gel (p1-3, p5). Sample plot (right) portrays the ability to discriminate sample groupings based on PCA analysis.