Histochemistry and Cell Biology https://doi.org/10.1007/s00418-018-1686-5 ORIGINAL PAPER A heart for fibrillin: spatial arrangement in adult wild-type murine myocardial tissue Felke Steijns1 · Jolanda van Hengel2 · Patrick Sips1 · Julie De Backer1,3 · Marjolijn Renard1 Accepted: 5 June 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract Fibrillins are major constituents of microfibrils, which are essential components of the extracellular matrix of connective tissues where they contribute to the tissue homeostasis. Although it is known that microfibrils are abundantly expressed in the left ventricle of the heart, limited data are available about the presence of microfibrils in the other parts of the myo- cardial tissue and whether there are age or sex-related differences in the spatial arrangement of the microfibrils. This basic knowledge is essential to better understand the impact of fibrillin-1 pathogenic variants on the myocardial tissue as seen in Marfan related cardiomyopathy. We performed histological analyses on wild-type male and female murine myocardial tissue collected at different time-points (1, 3 and 6 months). Fibrillin-1 and -2 immunofluorescence stainings were performed on cross-sections at the level of the apex, the mid-ventricles and the atria. In addition, other myocardial matrix components such as collagen and elastin were also investigated. Fibrillin-1 presented as long fibres in the apex, mid-ventricles and atria. The spatial arrangement differed between the investigated regions, but not between age groups or sexes. Collagen had a similar broad spatial arrangement to that of fibrillin-1, whereas elastic fibres were primarily present in the atria and the vessels. In contrast to fibrillin-1, limited amounts of fibrillin-2 were observed. Fibrillin-rich fibres contribute to the architecture of the myocardial tissue in a region-dependent manner in wild-type murine hearts. This knowledge is helpful for future experimental set-ups of studies evaluating the impact of fibrillin-1 pathogenic variants on the myocardial tissue. Keywords Microfibrils · Fibrillin-1 · Cardiac tissue · Spatial arrangement · Myocardial ECM Introduction (Purslow 2008). The perimysium is a layer of connective tissue associated with bundles of cardiac myocytes thereby The myocardial extracellular matrix (ECM) represents a providing a laminar architecture of muscle layers. The complex three-dimensional network of various macromol- endomysium surrounds and interconnects individual car- ecules in close interaction with each other and neighbour- diac myocytes to neighbouring cells and capillaries. This ing cardiac myocytes. The myocardial ECM can be subdi- close interaction between the myocardial ECM and the car- vided into two areas: the perimysium and the endomysium diac myocytes allows transmission of a plethora of signals from the extracellular environment to the cardiac myocyte, thereby influencing the cellular morphology and function Electronic supplementary material The online version of this (Borg et al. 1996). In addition, in response to local cell sig- article (https ://doi.org/10.1007/s0041 8-018-1686-5) contains nals the myocardial ECM also changes continuously, creat- supplementary material, which is available to authorized users. ing a dynamic responsive entity (Pope et al. 2008). Coordi- * Felke Steijns nated interplay between the signals originating from both the [email protected] myocardial ECM and the cardiac myocytes is crucial for a normal cardiac function (Pope et al. 2008). 1 Center for Medical Genetics, Ghent University Hospital, An important component of the myocardial ECM is the Ghent, Belgium protein fibrillin-1 (Bouzeghrane et al. 2005; Vracko et al. 2 Department of Basic Medical Sciences, Faculty of Medicine 1990; Lockhart et al. 2011). Previously, Bouzeghrane et al. and Health Sciences, Ghent University, Ghent, Belgium demonstrated by means of immunohistochemistry that fibril- 3 Department of Cardiology, Ghent University Hospital, lin-1 is abundantly present in the ventricular myocardium of Ghent, Belgium Vol.:(0123456789)1 3 Histochemistry and Cell Biology male wild-type adult rats. Furthermore, they reported a sig- with clinical manifestations mainly in the ocular, skeletal nificant increase in fibrillin-1 synthesis in male rats with car- and cardiovascular system (Campens et al. 2015; De Backer diac fibrosis and suggested that fibrillin-1 serves an impor- et al. 2006; De Backer 2009). Patients with Marfan syn- tant role in cardiac tissue repair (Bouzeghrane et al. 2005). drome are prone to develop intrinsic cardiomyopathy, how- Vracko et al. localized high amounts of fibrillin-1, also by ever, the exact underlying disease mechanism is yet to be means of immunohistochemistry, at the level of the papillary unravelled (Alpendurada et al. 2010; Campens et al. 2015; muscle of both female and male adult patients obtained after Cook et al. 2014; Hetzer et al. 2016; De Backer 2009). Our transplantation for ischemic heart disease. As the papillary group associated a reduced fibrillin-1 deposition in the myo- muscle contains many anchorage points between the cardiac cardial tissue with a deterioration of the left ventricular func- myocytes and the collagen fibres of the chordae tendineae, tion in 6 months old mice harbouring a missense mutation the abundance of fibrillin-1 in this specific area, therefore, (C1039G/+) in the Fbn1 gene (Campens et al. 2015). This suggests that fibrillin-1 serves an important role in trans- implies that the microfibrillar network serves an important mitting forces from the ECM to cardiac myocytes (Vracko role in the mechanical properties of the myocardium. Moreo- et al. 1990). ver, based on the study of Cook et al. performed in hypo- In humans, three fibrillin genes (FBN1, -2 and -3) encod- morphic Fbn1 mutant mgR/mgR mice presenting a severe ing fibrillin-1, -2 and -3, respectively, have been identified form of Marfan syndrome, fibrillin-1 is a force-transmitting (Cain et al. 2006; Sakai et al. 2016). Fibrillin-1 is expressed molecule crucial for the mechanosignaling in the mamma- from gastrulation throughout postnatal life (Sakai et al. lian heart (Cook et al. 2014). 2016; Cain et al. 2006; Ramirez and Pereira 1999). Whereas, In this study, we evaluated the spatial arrangement of fibrillin-2 is mainly expressed during foetal development fibrillins throughout the entire wild-type murine myocar- with only limited expression in postnatal tissues (Zhang dial tissue by means of (immuno)histochemistry. Male and et al. 1994). Fibrillin-3 is exclusively expressed in foetal female mice were studied at the ages of 1, 3 and 6 months, tissue (Sakai et al. 2016; Cain et al. 2006). In mice, there based on the time span of our previous study (Campens et al. are only two Fbn genes, Fbn1 and Fbn2. The Fbn3 gene 2015). In addition, collagen and elastin staining was per- has been inactivated in the mouse genome during evolution formed as reference. (Corson et al. 2004). Fibrillins are the principal components of the 10–12 nm diameter extracellular matrix microfibrils (Cain et al. 2006; Materials and methods Hubmacher et al. 2008; Sakai et al. 1986, 2016; Sherratt et al. 2001). Microfibrils are highly conserved macromol- Mice ecules distributed in all dynamic connective tissues, either associated with elastin in elastic fibres or as elastin-free Male and female wild-type C57BL/6J mice, aged 1, 3 and 6 microfibrils in non-elastic tissues subjected to repeated months, were used for this study (n = 3 mice/time point/sex). mechanical stress (Jensen and Handford 2016; Sherratt et al. All applicable international, national and/or institutional 2001; Ramirez and Pereira 1999). guidelines for the care and use of animals were followed. To date, although fibrillin-1 dysfunction has been linked All procedures performed in this study involving animals to myocardial disease, the exact spatial arrangement of were in accordance with the ethical standards of the Ethics fibrillin-1 in the myocardial tissue remains largely unknown Committee of the Ghent University Hospital (permit num- (Campens et al. 2015; De Backer et al. 2006). The few stud- ber: ECD17/07). ies that have been published on this subject specifically focus on the left ventricle or papillary muscle (Bouzeghrane Immunofluorescence and histological staining et al. 2005; Vracko et al. 1990). Moreover, the studies are limited to one time point and do not compare both sexes; Mice were sacrificed by means ofCO 2-inhalation (1.0 L/ therefore, they might have overlooked potential age- and/ min). Subsequently, a thoracotomy was performed and the or sex-dependent differences in the spatial arrangement of heart was flushed with 1x phosphate buffered saline (PBS) fibrillin-1 in the myocardial tissue. and dissected. Next, the hearts were fixed for 20 h in 4% Knowledge of the spatial arrangement of fibrillin-1 paraformaldehyde (PFA). Following fixation, the hearts throughout the entire normal murine myocardial tissue is were dehydrated and embedded in paraffin per routine his- essential for future experimental set-ups of studies evalu- tological procedure (Leica TP1020 tissue processor, Leica ating the impact of pathological changes associated with biosystems). Five µm thick sections were cut at the level of fibrillin-1 pathogenic variants on the myocardial tissue. the apex, mid-ventricles and atria as presented in Online Pathogenic variants in FBN1 cause Marfan syndrome, an Resource 1 (Microm HM355S, Thermo Scientific). Addi- autosomal dominantly