Genetic manipulation of CCN2/CTGF unveils cell‐specific ECM‐remodeling effects in injured skeletal muscle

Abstract

In skeletal muscle, extracellular matrix (ECM) remodeling can either support the complete regeneration of injured muscle or facilitate pathologic fibrosis and muscle degeneration. Muscular dystrophy (MD) is a group of genetic disorders that results in a progressive decline in muscle function and is characterized by the abundant deposition of fibrotic tissue. Unlike acute injury, where ECM remodeling is acute and transient, in MD, remodeling persists until fibrosis obstructs the regenerative efforts of diseased muscles. Thus, understanding how ECM is deposited and organized is critical in the context of muscle repair. Connective tissue growth factor (CTGF or CCN2) is a matricellular protein expressed by multiple cell types in response to tissue injury. Although used as a general marker of fibrosis, the cell type-dependent role of CTGF in dystrophic muscle has not been elucidated. To address this question, a conditional Ctgf myofiber and fibroblast-knockout mouse lines were generated and crossed to a dystrophic background. Only myofiber-selective inhibition of CTGF protected δ-sarcoglycan-null ( Sgcd) mice from the dystrophic phenotype, and it did so by affecting collagen organization in a way that allowed for improvements in dystrophic muscle regeneration and function. To confirm that muscle-specific CTGF functions to mediate collagen organization, we generated mice with transgenic muscle-specific overexpression of CTGF. Again, genetic modulation of CTGF in muscle was not sufficient to drive fibrosis, but altered collagen content and organization after injury. Our results show that the myofibers are critical mediators of the deleterious effects associated with CTGF in MD and acutely injured skeletal muscle.-Petrosino, J. M., Leask, A., Accornero, F. Genetic manipulation of CCN2/CTGF unveils cell-specific ECM-remodeling effects in injured skeletal muscle.

Mineralocorticoid receptor antagonists improve membrane integrity independent of muscle force in muscular dystrophy

“…Despite there being no difference in immune response and the known contribution of the immune response on downstream fibrosis, MRcko/mdx 8-week-old quadriceps muscles did show a lower percentage of tightly packaged collagen relative to total collagen compared to Cre−/mdx. Tightly packaged collagen networks can create barriers that block the migration of muscle precursor cells to the site of damage, which inhibits muscle regeneration (47). Less tightly packaged collagen supports that the MRcko/mdx 8-week-old quadriceps muscles were less stiff and had more potential to regenerate.…”

“…These data suggest that either the myofiber MR is not working directly through immune cell signaling or immune cells have an effect on disease progression earlier than 8 weeks of age in mice. To further understand the interplay between the myofiber MR and fibrosis, we quantified the degree to which the collagen was packaged by visualizing picrosirius red staining of collagen with polarized light and the amount of total collagen infiltration (47). Quadriceps muscle from 8-week-old MRcko/mdx had a lower (P = 0.0061) percentage (29.2% ± 2.5%) of red, tightly packaged collagen relative to total collagen compared to Cre−/mdx (39.4% ± 1.8%; Fig.…”

“…The diaphragms stained for fibronectin were imaged on a Nikon (Melville, NY) Eclipse 800 microscope with the 10× objective with a SPOT RT slider digital camera software and composited using Adobe (San Jose, CA) Photoshop (CS6). For collagen analysis, sections were stained with picrosirius red (24901-500; Polysciences) according to the manufacturer's recommendations (47). The sections were imaged with the 4× objective with either brightfield for total collagen infiltration images or polarized light for collagen packaging images with a Zeiss Axioskop Widefield LM microscope and QImaging QCapture Pro software.…”

“…For the collagen infiltration data, the area of picrosirius redstained collagen was quantified relative to the total muscle area by a blinded individual as previously described in 8-week-old quadriceps (n = 7 Cre−/mdx [4M, 3F] and n = 7 MRcko/mdx [4M, 3F]) and 52-week-old diaphragm (n = 8 Cre−/mdx [4M, 4F] and n = 8 MRcko/mdx [2M, 6F]; 47). For analysis of collagen network compaction, polarized images of the picrosirius red-stained sections were analyzed and the amount of loosely (green) packaged and densely (red) packaged collagen were quantified relative to the total collagen area with the red-green-blue quantification feature in NIH (Bethesda, MD) ImageJ by a blinded individual as previously described (47).…”

The antagonistic effects and mechanisms of microRNA-26a action in hypertensive vascular remodeling

“…In vitro studies showed that miR-26a mimic treatment inhibited but miR-26a inhibitor treatment promoted Col I and III expression in AngII-induced VSMCs. CTGF belongs to matricellular protein expressed by various cells in response to stimuli and is an important molecule prominently implicated in increased deposition of ECM (Li et al , 2016;Petrosino et al , 2019). Our results showed that that CTGF is a downstream target gene of miR-26a and the level of miR-26a was inversely associated with CTGF expression both in vivo and in vitro, so miR-26a may diminish ECM deposition by targeting CTGF.…”