The Extracellular Matrix
The cardiac ECM is a complex architectural network consisting of a variety of proteins, including fibronectin, fibrillin, periostin, more than 28 different types of collagen, glycoproteins (e.g. thrombospondins, secreted protein acidic and rich in cysteine, tenascins), proteoglycans (e.g. versican, syndecans, biglycan), and glycosaminoglycans (e.g. hyaluronan, heparan sulphate), creating strength and plasticity.35,36 Fibronectin is a multi-domain protein that interacts with proteoglycans and collagen to mediate cellular function. Periostin interacts with other components of the ECM, as well as with fibroblasts, playing a role in their differentiation. Collagen is crucial for maintaining the elasticity and integrity of the heart. Glycoproteins, proteoglycans and glycosaminoglycans are upregulated on cardiac injury and control key processes in the remodelling of myocardium, such as inflammation, fibrosis, and angiogenesis. The ECM is able to store and release a number of growth factors, chemokines and cytokines.4,35 When tissue is injured, there is an increased expression of MMPs and increased degradation of the ECM to promote healing and scar formation. Excessive degradation is blocked by endogenous TIMPs. In fact, there is a delicate balance between MMPs and TIMPs that contributes to the regulation of the cardiac remodelling process37.
Cardiac Cell–cell and Cell–ECM Communications
Cardiomyocyte to Cardiomyocyte Communication
There are many routes of CM–CM communication, including the secretion of autocrine factors and direct contact via gap junctions and adhesion complexes. Autocrine cross-talk is carried out by myocyte- secreted factors that include leptin, hepatocyte growth factor, endothelin-1 and FGF and TGF- family members.29 Gap junctions allow CM-CM communication via the exchange of ions and small solutes. In the myocardium, gap junction proteins of the connexin family have been shown to play a crucial role in determining impulse conduction and the heart morphogenesis.38 Adhesion-complex communications include intracellular signalling cascades that are triggered by cell–cell or cell–ECM engagement of specific proteins in these complexes. This type of communication may alter CM responses to growth factors leading to myocardial hypertrophy.29
Another factor released by the CM upon acute myocardial infarction is tumour necrosis factor-alpha (TNF-alpha).39 TNF-alpha has been found to be mainly released via hypoxia-inducible factor 1-alpha- mediated non-classical secretory pathways, involving release of vesicles containing a membrane variant of TNF. The identified vesicles were suggested to be exosomes and, when obtained from hypoxic CM, could trigger cell death in other CM.40,41
Cardiac Fibroblast to Myocyte Communication
CM and CF are spatially intermingled in the myocardium with virtually every CM bordering one or more CF. Bidirectional communication between CF and CM can be mediated by paracrine signals, direct cell- cell interactions and indirect interaction via ECM.42
Both CF and CM secrete many different chemokines, cytokines, growth factors and other soluble agents that play a key role in cardiac physiology and pathophysiology.42 Connective tissue growth factor (CTGF), which is induced by TGF- and expressed in both CM and CF, has been associated specifically with CF proliferation and ECM production in the setting of myocardial fibrosis.43,44 CTGF expression is negatively regulated by two cardiac microRNAs (miRNAs), miR-133 and miR-30.45 miRNAs are non-coding RNAs that regulate messenger RNA (mRNA) translation or degradation. They can be actively secreted or passively leaked from a cell to act either on an adjacent cell or on distant cells along the vasculature.46 While miR-30 is expressed in both CF and CM, miR-133 is expressed specifically in CM. miR-133-knockout mice develop excessive fibrosis and HF while miR-133 knockdown also causes cardiac hypertrophy with impaired cardiac function.47,48 CF also secrete interleukin 33, which cross-regulates CM in vitro, reduces pressure-overload hypertrophy and fibrosis, and improves cardiac function and survival after myocardial infarction in vivo.49,50 Interleukin 6 (IL-6) induces cell proliferation, protects cells from apoptosis, promotes ECM turnover, and causes cardiac hypertrophy.51 It is of note that interactions between CM and CF markedly promote the secretion of IL-6, as shown by increased levels in conditioned medium from CM-CF co-cultures compared with CM or CF only cultures, indicating that communication between the two cell types is required for the secretion of some paracrine factors.52,53 Platelet-derived growth factor (PDGF) has been shown to play an important role in cardiac fibrosis and angiogenesis via its binding to protein tyrosine kinase receptors. The latter control fibroblast proliferation and migration and are also linked to ECM deposition.4 In transgenic mice, PDGF expression can be largely enhanced, leading to dilated cardiomyopathy and HF.54 IGF-1 secretion by fibroblasts also plays a key role in mediating the adaptive response of myocardium to pressure overload.55 Moreover, paracrine signals released from CF may affect the expression and function of ion channels and gap junctions in CM.56,57,58,59 Apart from the conventional exocytosis of paracrine factors, there is evidence that a pannexin- based mechanism exists in the myocardium. Pannexins were found to form functional channels in single membranes that can make possible paracrine intercellular communication by releasing ATP and other small molecules from the cytoplasm to the extracellular space.42
CM and CF are able to communicate electrically through connexin- mediated gap junctional connections.4 CF are not electrically excitable, but their membrane contains ion channels.60,61,62 Without coupling to CM, CF operate as passive electrical insulators.63 However, when coupled to CM, they can affect action-potential characteristics and conduction velocity in CM.42,64 CM–CF coupling has also been shown to alter intercellular calcium cycling alternans, which could play an additional role in arrhythmogenesis in fibrotic heart tissue.65 Another mechanism for mechanical coupling between CM and CF is through adherens junctions and the cadherin–catenin complex at their core.42 Cadherins are transmembrane receptors that bind adjacent cells, link intracellularly to actin and intermediate filaments via catenins and facilitate bidirectional transmission of cytoskeletal tension between cells.66,67 Cadherin staining has been detected between co-cultured CM and myofibroblasts.68,69 It has been shown that TGF –activated myofibroblasts can exert tonic contractile forces on CM and slow electric propagation as a result of increased mechanosensitive channel activation.69 A more recently described route of cell–cell cross talk that permits intercellular communication over longer distances is via membrane nanotubes. It has been demonstrated that organelles and cytoplasmic proteins exchange and calcium signal propagation occurred from CM to CF and vice versa through long, thin membrane nanotubular structures containing actin and microtubules.70,71,72