Wheat germ agglutinin staining as a suitable method for detection and quantification of fibrosis in cardiac tissue after myocardial infarction

Abstract

<jats:p>The quantification of fibrotic tissue is an important task in the analysis of cardiac remodeling. The use of established fibrosis staining techniques is limited on frozen cardiac tissue sections due to a reduced color contrast compared to paraffin embedded sections. We therefore used FITC-labeled wheat germ agglutinin (WGA), which marks fibrotic tissue in comparable quality as the established picrosirius red (SR) staining, for the staining of post myocardial infarction scar tissue. The fibrosis amount was quantified in a histogram-based approach using the non-commercial image processing program ImageJ. Our results clearly demonstrate that WGA-FITC is a suitable marker for cardiac fibrosis in frozen tissue sections. In combination with the histogram-based analysis, this new quantification approach is i) easy and fast to perform; ii) suitable for raw frozen tissue sections; and iii) allows the use of additional antibodies in co-immunostaining. </jats:p>

Transverse tubule remodelling: a cellular pathology driven by both sides of the plasmalemma?

“…Figure adapted from Crossman et al (2015) characterises fibrosis (Segura et al 2014), indicating that fibroblasts could be involved in t-tubule remodelling. Fibroblasts are also the likely source of collagen VI, as evidenced by the increased numbers of collagen VI-positive fibroblasts we found in human dilated heart failure (Crossman et al 2017) and is in agreement with a cell culture study of skeletal muscle that only found collagen VI protein and mRNA in fibroblasts and not in other cell types (Zou et al 2008). Particularly interesting is the identification of collagen VI-positive fibroblast filopodia within the lumen of some of the enlarged t-tubules in heart failure, supporting a likely role of fibroblasts in either maintaining or remodelling t-tubules from within the tubule lumen.…”

“…Assessment of tissue levels of collagen VI showed a change of distribution from a predominately basement membrane labelling pattern to one dominated by increased interstitial labelling reminiscent of fibrillar collagen fibrosis. Subsequent, confocal and superresolution microscopy work then identified increased fibrillar collagens, type I and III, within the t-tubular lumen in heart failure (Crossman et al 2017). These data are highly suggestive that increases of collagen could be involved in the aberrant t-tubule remodelling that occurs in heart failure.…”

“…3, demonstrates not only a disrupted t-tubule organisation but significantly increased diameter of t-tubules in the failing heart, consistent with findings in human and mouse heart failure. However, it should be noted that the increases in diameter of mouse and rat ttubules in heart failure (10% and 15%, respectively) are relatively modest compared to the t-tubule diameter change from 294 nm to 436 nm (or 48% increase) that occurs in human heart failure (Crossman et al 2017). The smaller diameter of ttubules in rodents may restrict the extent that collagen can accumulate within the lumen.…”

“…To identify which glycoproteins are bound by WGA, protein blots of human heart were probed with fluorescently labelled WGA (Crossman et al 2017) (Fig. 2).…”

“…Consistent with this proposal, the MDX mouse that has no dystrophin has disrupted cellular cytoskeleton (Viola et al 2014) and disrupted calcium release (Cheng et al 2012). In addition, the vinculin-talin-integrin system could likely have a role in t-tubule remodelling, given its linkage to the actin cytoskeleton (Ziegler et al 2008) and the promiscuity of integrin (depending on the receptor sub-type) to bind to different ECM components, including laminin, fibronectin and collagens, including types I, III, IV and VI (Ross and Borg 2001;Tulla et al 2001), which have all been shown to be within the t-tubules (Kostin et al 1998;Crossman et al 2017). These observations highlight that any number of ECM proteins could participate in either mechanical or chemical communications which could determine the local t-tubule structure.…”