MiRNA-21: a key to controlling the cardiac fibroblast compartment?

Myocardial ischaemia is a condition in which the coronary blood flow to the heart is reduced, which results in deficient oxygen and nutrients supply to the heart. In the clinical setting, reperfusion of the ischaemic heart is achieved by primary angioplasty, a process that, if induced early, rescues a large portion of the ischaemic myocardium. However, this is often obscured by adverse processes such as myocardial stunning injury and cardiac remodelling, i.e. ventricular hypertrophy, interstitial fibrosis, contractile dysfunction, inflammation, and chamber dilatation.1 Both cardiomyocytes and non-myocytes are affected, and at the molecular level, multiple extra- and intracellular signalling systems are involved, orchestrating a multitude of factors that determine the final phenotypic manifestation of the injury and the disease prognosis.2 In the acute phase of myocardial ischaemia–reperfusion (I/R), activation of matrix metalloproteinases (MMPs) allows degradation of fibrillar collagen and cleavage of the basement membrane surrounding necrotic myocytes—a prerequisite for the invasion and proliferation of cells, e.g. fibroblasts, inflammatory, and vascular cells—ensuring tissue repair and scar formation.3 It is of major interest for the clinical arena to understand these processes in order to identify novel therapeutic targets for the prevention of ventricular injury and maladaptive remodelling and dysfunction in response to I/R. In this regard, the study by Roy et al.4 opens a novel aspect by demonstrating that microRNA-21 (miR-21) is upregulated in cardiac fibroblasts after I/R injury and there plays potentially important roles in regulating major signalling pathways and their downstream effectors. MicroRNAs (miRNAs) are short regulatory RNAs that act as negative regulators of gene expression by inhibiting mRNA translation or by promoting mRNA degradation.5 It is estimated that more than 1000 miRNAs are encoded by the human genome, and evidence shows that many are regulated in a tissue- and cell-type-specific manner.6 Several methods are available to selectively target miRNAs in vivo and in vitro using antisense viral vectors or chemically modified antisense oligonucleotides, providing the basis for novel therapeutic strategies.7,8 Experimental evidence is rapidly accumulating, which demonstrates that miRNAs constitute a new mode of governing pathophysiological mechanisms in cardiac hypertrophy and heart failure.9,10 Initially, the main focus of miRNAs in cardiac pathophysiology was put on their role in cardiomyocytes. Meanwhile, interesting aspects of miRNA-mediated effects have emerged in the non-myocyte fraction of the heart. For example, da Costa Martins et al.11 generated an inducible cardiomyocyte-specific Dicer depletion that provoked ventricular enlargement and severe histopathology marked by fibrosis, inflammation, and cardiomyocyte disarray in the adult heart. Interestingly, many miRNAs remained unchanged or were even upregulated in the myocardium of this transgenic mouse model, suggesting that they mainly derived from non-cardiomyocytes in terms of a secondary fashion response to the remodelling processes.11 Along the same line, Thum et al.12 recently reported on the intriguing finding that miR-21 is upregulated selectively in fibroblasts of the pressure-overloaded heart, but not in cardiomyocytes. Upregulation of miR-21 in cardiac fibroblasts targeted the sprouty homologue 1 (Spry1), thereby indirectly enhancing the activity of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signalling pathway, which in turn promoted fibroblast survival and growth factor secretion. As a result, interstitial fibrosis was augmented and, in a paracrine way, growth factors released from fibroblasts enhanced cardiomyocyte hypertrophy.12 The present study by Roy et al.,4 using laser capture microdissection, reports the induction of miR-21 specifically in the infarct zone of hearts subjected to I/R. As a direct target of miR-21 in fibroblasts, the authors identified phosphatase and tension homologue (PTEN), a widely expressed lipid and protein phosphatase, that negatively regulates PI3K-Akt signalling as well as other signalling pathways and affects cellular features such as survival, growth, metabolism, protein synthesis, and secretory activity.13 The miR-21-dependent suppression of PTEN led to a significant induction of PI3K and elevated downstream Akt activity. The expression of MMP-2 increased subsequently to Akt activation, shedding new light on the regulation of MMPs at the level of post-transcriptional modification. MMPs are best known for their actions in extracellular matrix (ECM) remodelling, but evidence is accumulating, especially for MMP-2, that they may also play an important role intracellularly, particularly in response to I/R injury of the heart.14 Myocardial stunning in response to I/R involves the generation of ONOO−, a feature that is thought to activate MMPs and inactivate their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs).14 It is therefore hypothesized that an imbalance between TIMPs and MMPs in the heart may be one of the contributing factors to acute I/R injury.14 MMP-2 is expressed ubiquitously in cardiac cells, including cardiomyocytes, endothelial cells, and fibroblasts.14 In experimental I/R, it has been shown that there is a positive correlation between increasing duration of ischaemia, enhanced release of MMP-2, and a reduction in cardiac function during reperfusion.14 Although the cellular source of MMP-2 release is not precisely known, MMP inhibitors such as o-phenanthroline, doxycycline, or a neutralizing MMP-2 antibody functionally protected hearts from stunning injury in experimental models.15 To date, it is not clear whether MMP-2 directly facilitates this decrease in cardiac function, but proteolytic degradation of contractile proteins (troponin I) and/or matrix degradation may be important intracellular targets of MMP-2 in cardiomyocytes. Interestingly, selective MMP inhibition failed to show a treatment benefit after 90 days of myocardial infarction follow-up in a clinical trial (PREMIER).16 Notably, another experimental study revealed higher mortality rates and adverse left ventricular remodelling after prolonged MMP inhibition,17 suggesting that global MMP inhibition may not be beneficial. Indeed, one could speculate that there are important cell-specific and timely regulated processes of MMPs to exert beneficial effects, while exuberant and prolonged MMP expression may provoke impairment of the structural integrity of the heart and facilitate inflammatory processes.18 In addition to the temporal profile of MMP activity, the cell-type-specific contribution of MMPs for remodelling of the injured heart is another important matter of investigation. In this regard, the miR-21-dependent expression of MMP-2 in the study by Roy et al.4 was specifically detected in cardiac fibroblasts. This fibroblast-associated regulation of miR-21 under pathophysiological conditions again strikingly matches with and supports the findings by Thum et al. that revealed a key role for this particular miRNA in cardiac fibroblasts under pathophysiological stress.12,19 Knowledge about cell-type specificity of dysregulated miRNAs, such as suggested for miR-21, under a given pathophysiological condition would help to optimize strategies of targeted delivery of miRNAs or their inhibitors (antagomirs) to the cell-type or tissue of interest for systemic application in terms of a therapeutic approach. This could, for example, be achieved by tagging specific antibodies or surface ligands together with protamine or other positive charges that would allow a receptor-mediated uptake.20 In addition to enhanced effectiveness, these modifications would certainly minimize undesirable secondary effects and increase the safety. Thus, in view of the failure of systemic MMP blockade in the ischaemic heart in clinical trials, it will be highly interesting to evaluate the functional aspects of cell-type-specific, miR-21-mediated regulation of MMP-2 in the fibroblast compartment after I/R or other cardiac diseases. Whether the upregulation of miR-21 in cardiac fibroblasts and its subsequent consequences for proliferation and ECM constitution directly result from stress execution on fibroblasts, or whether it is, at least partly, based on signals [e.g. tumour growth factor (TGF)-β] from adjacent cells such as cardiomyocytes or immunocompetent cells21 is still elusive and awaits further investigation. In this regard, identifying putative intercellular pathways and paracrine features that may orchestrate miRNA expression in neighbouring cells, e.g. through induction or suppression of cytokines and growth factors, will be one of the future challenges in this area. Although miRNA research in the cardiovascular field is still in the early stages of development, it is exciting to observe the promising experimental approaches and the steadily improving delivery technologies, which let potential miRNA-based therapeutic and diagnostic utilization move closer and closer. We thank Tina Selle for fruitful discussions.