Personalized in vitro Extracellular Matrix Models of Collagen VI-Related Muscular Dystrophies

Abstract

Collagen VI-related dystrophies (COL6-RDs) are a group of rare congenital neuromuscular dystrophies that represent a continuum of overlapping clinical phenotypes that go from the milder Bethlem myopathy (BM) to the severe Ullrich congenital muscular dystrophy, for which there is no effective treatment. Mutations in one of the three Collagen VI genes alter the incorporation of this protein into the extracellular matrix (ECM), affecting the assembly and the structural integrity of the whole fibrillar network. Clinical hallmarks of COL6-RDs are secondary to the ECM disruption and include muscle weakness, proximal joint contractures, and distal hyperlaxity. Although some traits have been identified in patients' ECMs, a correlation between the ECM features and the clinical phenotype has not been established, mainly due to the lack of predictive and reliable models of the pathology. Herein, we engineered a new personalized pre-clinical model of COL6-RDs using cell-derived matrices (CDMs) technology to better recapitulate the complexity of the native scenario. We found that CDMs from COL6-RD patients presented alterations in ECM structure and composition, showing a significantly decreased Collagen VI secretion, especially in the more severe phenotypes, and a decrease in Fibrillin-1 inclusion. Next, we examined the Collagen VI-mediated deposition of Fibronectin in the ECM, finding a higher alignment, length, width, and straightness than in patients with COL6-RDs. Overall, these results indicate that CDMs models are promising tools to explore the alterations that arise in the composition and fibrillar architecture due to mutations in Collagen VI genes, especially in early stages of matrix organization. Ultimately, CDMs derived from COL6-RD patients may become relevant pre-clinical models, which may help identifying novel biomarkers to be employed in the clinics and to investigate novel therapeutic targets and treatments.

Keywords: Collagen VI related muscular dystrophy; decellularisation; extracellular matrix; in vitro model; patient-derived ECMs.

FIGURE 1

Preparation and application of CDMs. (A) Confluent cultures of fibroblasts were stimulated with AA for 8 days to promote the secretion of a dense fibrillar ECM rich in collagen. On the right, representative confocal images show cell staining at day 1 and 8 of culture (prior decellularization). Cell nuclei were stained with Hoechst. The complexity and interconnection of secreted fibrils increased over time. (B) The ECM was evaluated by immunostaining before and after decellularization. After decellularization no cellular structures such as cell nuclei (stained with Hoechst) and cytoskeleton (F-actin) were observed. Scale bars: 100 µm.

FIGURE 2

COL6 deposition in the CDMs. (A) Representative confocal images of COL6 in the CDMs obtained from patients with COL6-RDs and healthy donors (controls). Scale bar: 50 µm. (B) Quantification of the mean fluorescence intensity of COL6. 13 ≥ n ≥ 46, (C) Quantification of the number of fibrils. 13 ≥ n ≥ 46, (D) fibril alignment. 13 ≥ n ≥ 44, and (E) fibril length. 3413 ≥ n ≥ 18011, in CDMs through computational analysis, showing a significant decrease for patients with COL6-RDs. Three microscope fields of view were analyzed per replicate of at least 3 replicates per donor. Results are the mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001. “a.u.”: arbitrary units.

FIGURE 3

Analysis of FN fibrils distribution and morphology in the CDMs. (A) Representative images showing the immunostained FN fibrils obtained in the CDMs from healthy donors (controls) and from patients with COL6-RDs. Scale bar: 20 µm. (B,C) Quantification of FN fibril length. 4270 ≥ n ≥ 9042, width. 4815 ≥ n ≥ 9928, and straightness. 4654 ≥ n ≥ 9633, and (D) Quantification of fibrils alignment in CDMs. 11 ≥ n ≥ 94, through computational analysis. Two microscope fields of view were analyzed per replicate of at least 2 replicates per donor. Results are the mean ± SEM. ns = non-significant, ****p < 0.0001.

FIGURE 4

FBN1 deposition in CDMs. (A) Representative confocal images of FBN1 immunostaining in CDMs from healthy donors (controls) and patients with COL6-RDs. FBN1 immunolabelling show distinct motifs of secretion. (B) Representative images showing the co-localization of FBN1 with FN fibrils. Scale bars: 50 µm. (C) Quantification of the mean fluorescence intensity of FBN1 immunostaining. 11 ≥ n ≥ 86, showing its decrease for patients with COL6-RDs. (D) Quantification of FN fibril length. 4964 ≥ n ≥ 46439, width. 5497 ≥ n ≥ 50922, alignment. 11 ≥ n ≥ 87, and straightness. 5345 ≥ n ≥ 49168 in CDMs through computational analysis. Three microscope fields of view were analyzed per replicate of at least 3 replicates per donor. Results are the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.