Cardiac extracellular matrix proteomics: Challenges, techniques, and clinical implications

Chang, Chia Wei; Dalgliesh, Ailsa J.; López, Javier E.; Griffiths, Leigh G.

doi:10.1002/prca.201500030

Cited by 54 publications

(41 citation statements)

References 63 publications

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“…However, studies have shown successful decellularization using SDS without damaging the components of the ECM (Garrigues et al, ; He & Callanan, ; Oberwallner et al, ; Sullivan et al, ). Furthermore, a thorough proteomic study of the ECM components and the cellular secretome would act as validation for the PCR results and would give a better understanding of which proteins lead to the improved adhesive properties (Chang, Dalgliesh, Lopez, et al, ; Hathout, ). Further analysis on how the ECM was incorporated into the electrospun fibers is required.…”

Section: Discussionmentioning

confidence: 99%

Hybrid cardiovascular sourced extracellular matrix scaffolds as possible platforms for vascular tissue engineering

Reid

Callanan

2019

J Biomed Mater Res

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The aim when designing a scaffold is to provide a supportive microenvironment for the native cells, which is generally achieved by structurally and biochemically imitating the native tissue. Decellularized extracellular matrix (ECM) possesses the mechanical and biochemical cues designed to promote native cell survival. However, when decellularized and reprocessed, the ECM loses its cell supporting mechanical integrity and architecture. Herein, we propose dissolving the ECM into a polymer/solvent solution and electrospinning it into a fibrous sheet, thus harnessing the biochemical cues from the ECM and the mechanical integrity of the polymer. Bovine aorta and myocardium were selected as ECM sources. Decellularization was achieved using sodium dodecyl sulfate (SDS), and the ECM was combined with polycaprolactone and hexafluoro‐2‐propanol for electrospinning. The scaffolds were seeded with human umbilical vein endothelial cells (HUVECs). The study found that the inclusion of aorta ECM increased the scaffold's wettability and subsequently lead to increased HUVEC adherence and proliferation. Interestingly, the inclusion of myocardium ECM had no effect on wettability or cell viability. Furthermore, gene expression and mechanical changes were noted with the addition of ECM. The results from this study show the vast potential of electrospun ECM/polymer bioscaffolds and their use in tissue engineering.

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Section: Discussionmentioning

confidence: 99%

Hybrid cardiovascular sourced extracellular matrix scaffolds as possible platforms for vascular tissue engineering

Reid

Callanan

2019

J Biomed Mater Res

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“…However, the mechanical micro-environment likely accounts for only a portion of the short-range maturational cues. The cardiac extracellular matrix (ECM) is a complex assembly of glycosaminoglycans, glycoproteins, and adhesive proteoglycans that have both signaling and mechanical functions [54]. Cardiomyocytes grown on decellularized heart ECM showed greater maturation as measured by protein expression than cells grown on mechanically similar liver ECM [55].…”

Section: Discussionmentioning

confidence: 99%

Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencing

et al. 2017

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Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly for tissue repair in humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, and electrophysiology similar to that of embryonic cardiomyocytes. We grew hiPSC-CM in patterned islands of different sizes and shapes, and measured the effect of island geometry on action potential waveform and calcium dynamics using optical recordings of voltage and calcium from 970 islands of different sizes. hiPSC-CM in larger islands showed electrical and calcium dynamics indicative of greater functional maturity. We then compared transcriptional signatures of the small and large islands against a developmental time course of cardiac differentiation. Although island size had little effect on expression of most genes whose levels differed between hiPSC-CM and adult primary CM, we identified a subset of genes for which island size drove the majority (58%) of the changes associated with functional maturation. Finally, we patterned hiPSC-CM on islands with a variety of shapes to probe the relative contributions of soluble factors, electrical coupling, and direct cell-cell contacts to the functional maturation. Collectively, our data show that optical electrophysiology is a powerful tool for assaying hiPSC-CM maturation, and that island size powerfully drives activation of a subset of genes involved in cardiac maturation.

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“…Normalizing protein content to MC numbers and/or types should maximize the confidence that future proteomic analysis in cardiac growth is representative of variable MCs changes in size. LP-FACS purified MCs also offer a new entry point for cell-specific analysis into relationship between cellular organization and function, as well as variations in normal cardiac tissues 50 at the level of individual cells.…”

Section: Discussionmentioning

confidence: 99%

Novel large-particle FACS purification of adult ventricular myocytes reveals accumulation of myosin and actin disproportionate to cell size and proteome in normal post-weaning development

López

Sharma

Ávila

et al. 2017

Journal of Molecular and Cellular Cardiology

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Rationale Quantifying cellular proteins in ventricular myocytes (MCs) is challenging due to tissue heterogeneity and the variety of cell sizes in the heart. In post-weaning cardiac ontogeny, rod-shaped MCs make up the majority of the cardiac mass while remaining a minority of cardiac cells in number. Current biochemical analyses of cardiac proteins do not correlate well the content of MC-specific proteins to cell type or size in normally developing tissue. Objective To develop a new MC-specific large-particle fluorescent-activated cell sorting (LP-FACS) strategy for the purification of adult rod-shaped MCs. This approach is developed to enable growth-scaled measurements per-cell of the MC proteome and sarcomeric protein (i.e. myosin heavy chain (MyHC) and alpha-actin (α-actin)) content. Methods and Results Individual cardiac cells were isolated from 21-94 days old mice. An LP-FACS jet-in-air system with a 200-μm nozzle was defined by the first time to purify adult MCs. Cell-type specific immunophenotyping and sorting yielded ≥95% purity of adult MCs independently of cell morphology and size. This approach excluded other cell types and tissue contaminants from further analysis. MC proteome, MyHC and α-actin proteins were measured in linear biochemical assays normalized to cell numbers. Using the allometric coefficient α, we scaled the MC-specific rate of protein accumulation to growth post-weaning. MC-specific volumes (α=1.02) and global protein accumulation (α=0.94) were proportional (i.e. isometric) to body mass. In contrast, MyHC and α-actin accumulated at a much greater rate (i.e. hyperallometric) than body mass (α= 1.79 and 2.19 respectively) and MC volumes (α= 1.76 and 1.45 respectively). Conclusion Changes in MC proteome and cell volumes measured in LP-FACS purified MCs are proportional to body mass post-weaning. Oppositely, MyHC and α-actin are concentrated more rapidly than what would be expected from MC proteome accumulation, cell enlargement, or animal growth alone. LP-FACS provides a new standard for adult MC purification and an approach to scale the biochemical content of specific proteins or group of proteins per cell in enlarging MCs.

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Cardiac extracellular matrix proteomics: Challenges, techniques, and clinical implications

Cited by 54 publications

References 63 publications

Hybrid cardiovascular sourced extracellular matrix scaffolds as possible platforms for vascular tissue engineering

Hybrid cardiovascular sourced extracellular matrix scaffolds as possible platforms for vascular tissue engineering

Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencing

Novel large-particle FACS purification of adult ventricular myocytes reveals accumulation of myosin and actin disproportionate to cell size and proteome in normal post-weaning development

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