4. Discussions:
4.4 Collagen processing HSP47 is an important regulator of heart muscle formation
4.4.3 Defective ECM Collagen type I secretion and organisation impairs
As fibrillar collagen plays a critical role in maintaining cardiac shape, size and function (Porter and Turner, 2009), we next investigated how defective collagen processing HSP47 influences the cardio-instructive properties of the ECM. We firstly demonstrated that purified mESC-CMs cultured on WT MEF-derived ECM displayed a mature CM phenotype with advanced sarcomeric organization and morphological features with regular cross striations and primarily rod shaped bodies, which are orientated along the collagen type I microfibrillar structure (Figure 31). Interestingly, CMs exhibited an impaired phenotype when cultured on collagen defective HSP47 KO MEF-derived ECMs, displayed by a rounded, flat, immature morphology with
4. Discussions
The structure and orientation of ECM fibrillar collagen has shown to be important in determining CM structure. Mechanically aligned collagen fibrils have the capacity to elongate muscle cells along its parrellel axis (Vandenburgh and Karlisch, 1989).
Furthermore, in vitro studies have demonstrated that microcontact printing parallel lines of ECM proteins on 2D substrates induce CM alignment (Pong et al., 2011;
Feinberg et al., 2012; Grosberg et al., 2011). Alignment of CMs is important for both electromechanical coupling and contractile force generation (Black et al., 2009;
Chung et al., 2007) and it can also promote differentiation and maturation of cardiac progenitor cells (Domian et al., 2009). Further immunohistochemical stainings for cardiac gap junction protein Cx43 and Cx45 would provide further information into possible differences in electrical coupling of CMs on collagen-defective ECMs.
Due to FBs depositing dense ECM networks of insoluble proteins (after 11 days) in culture, we tested several different published protocols in attempt to solubilise and quantity of ECM protein deposited on plates (Beacham et al., 2007; DeQuach et al., 2010; Harvey et al., 2013 Turoverova et al., 2009). None were able to aid in successful homogeneous solubilisation of FB-derived ECMs. We are thus unable to clearly distinguish whether this impaired CM phenotype is a consequence of the immature unprocessed collagen depostion, or due to the insufficient amount of total collagen deposited from HSP47 KO MEFs. Future experiments using higher concentrations of SDS (≥3 %
)
may aid in successful solubilization of ECM. In addition, the stiffness of the myocardial ECM enviroment may also be important for CM maturation and function (Majkut et al., 2013; Young and Engler, 2011).Additional studies to assess the biophyisal properties (stiffness) of these ECMs produced from these MEFs would provide us further information of how defective collagen processing in FBs can influence the mechanical enviroment of the ECM, and CM structure and function. From these obervations, we can however conclude that defective collagen processing and secretion from HSP47 KO MEFs impairs the ECM cardio-instructive properties in guiding immature CMs towards a mature phenotype.
We next investigated the influence of defective ECM collagen type I in cardiac tissue formation and maturation utilizing our 3D EHM model of heart muscle development.
We found that HSP47 KO MEF-supplemented EHMs demonstrate a lower degree of
4. Discussions
maturation (Figure 32), which is characterized by the failure to form compact mature functional tissue. These tissues exhibited an impaired CM phenotype, with disorganized sarcomeric structures, immature rounded morphology and failed to form tightly pack, dense, aligned CM muscle bundles. Furthermore, these EHMs failed to develop contractile force which is representative of a impaired functional syncytium.
This obervation may likely be due to reduced Itgb1 expression and impairment of mechanical signalling in HSP47 KO MEFs (Figure 33), which may explain for why these tissues failed to condense. We did not observe a significant difference in ITGB1 at protein level (n=3 group), however further experiments with more replicates/group may elimate variability and demonstrate signficant differences. The defective ECM collagen type I derived from HSP47KO MEFs (observed in 2D culture) may furthermore impair CM structure and organisation within the tissue. It would also be critical to assess whether ER stress is induced within these tissues, by assessing markers of ER stress at both protein and mRNA levels. We could speculate that this reduction in Itgb1 transcripts could be due to defective protein trafficking and ER stress induction in HSP47 KO MEFs. This however requires further investigation. In addition, further experiments to determine whether this impaired EHM phenotype could be rescued by transiently expressing HSP47 into HSP47 KO cells via transfection with a mouse HSP47 expression vector (Ishida et al., 2006; Nagai et al., 2000), would provide additional evidence that HSP47 in FBs is critical for EHM formation and maturation.
Although the role of HSP47 in the myocardium has currently not been extensively studied, the influence of HSP47 inhibition in cardiac remodelling after myocardial infarction has been previously investigated (Hagiwara et al., 2011). This study demonstrated that HSP47 antisense-oligonucleotides could reduce ligated left anterior descending (LAD) coronary artery-induced myocardial infarction in rats. Rats with ligated LAD coronary artery exhibited an upregulation of HSP47 expression, remodelling of the left ventricle, and cardiac dysfunction. In contrast, rats with ligated LAD coronary artery treated with antisense oligonucleotides had significantly reduced HSP47 expression, cardiac remodelling, and improved cardiac function (Hagiwara et al., 2011). HSP47 has been closely related to the development of many different types
4. Discussions
cytokine TGF-β can induce HSP47 expression (Sasaki et al., 2002). In addition, the microRNA-29 (miR-29) family act as regulators in cardiac fibrosis (van Rooij et al., 2008) and have been recently suggested to target and down regulate HSP47 expression (Zhang et al., 2014; Zhu et al., 2015; Zhao et al., 2014; Yamamoto et al., 2013).
Another more recent study has demonstrated that administration of the statin Atorvastatin, ameliorated cardiac fibrosis and improved left ventricular diastolic function in a rat model of hypertensive diastolic heart failure while reducing the expression of HSP47 in fibrotic hearts and cFBs (Akahori et al., 2014). In addition to mouse models, previous studies have revealed that the SERPINH1 is one of the genes responsible for recessive OI in both human (Christiansen et al., 2010) and dog (Lindert et al., 2015; Drogemuller et al., 2009). It has been recently reported that patients with IO have increased risk of heart disease and in particular valve diseases, which include dilation of the aorta, compared to healthy control patients (Ashournia et al., 2015; Karamifar et al., 2013; Rudunovic et al., 2011). Taken together, these data along with our findings demonstrate the importance of collagen processing HSP47 as a key regulator of ECM homeostasis, which can thus impact cardiac remodelling and function.