Open access peer-reviewed chapter

The Cellular Stress Response Interactome and Extracellular Matrix Cross-Talk during Fibrosis: A Stressed Extra-Matrix Affair

Written By

Maryada Sharma, Kavita Kaushal, Sanjay Singh Rawat, Manjul Muraleedharan, Seema Chhabra, Nipun Verma, Anupam Mittal, Ajay Bahl, Madhu Khullar, Anurag Ramavat and Naresh K. Panda

Submitted: 13 July 2020 Reviewed: 19 November 2020 Published: 17 February 2021

DOI: 10.5772/intechopen.95066

From the Edited Volume

Extracellular Matrix - Developments and Therapeutics

Edited by Rama Sashank Madhurapantula, Joseph Orgel P.R.O. and Zvi Loewy

Chapter metrics overview

631 Chapter Downloads

View Full Metrics

Abstract

Diverse internal and external pathologic stimuli can trigger cellular stress response pathways (CSRPs) that are usually counteracted by intrinsic homeostatic machinery, which responds to stress by initiating complex signaling mechanisms to eliminate either the stressor or the damaged cells. There is growing evidence that CSRPs can have context-dependent homeostatic or pathologic functions that may result in tissue fibrosis under persistence of stress. CSRPs can drive intercellular communications through exosomes (trafficking and secretory pathway determinants) secreted in response to stress-induced proteostasis rebalancing. The injured tissue environment upon sensing the stress turns on a precisely orchestrated network of immune responses by regulating cytokine-chemokine production, recruitment of immune cells, and modulating fibrogenic niche and extracellular matrix (ECM) cross-talk during fibrotic pathologies like cardiac fibrosis, liver fibrosis, laryngotracheal stenosis, systemic scleroderma, interstitial lung disease and inflammatory bowel disease. Immunostimulatory RNAs (like double stranded RNAs) generated through deregulated RNA processing pathways along with RNA binding proteins (RBPs) of RNA helicase (RNA sensors) family are emerging as important components of immune response pathways during sterile inflammation. The paradigm-shift in RNA metabolism associated interactome has begun to offer new therapeutic windows by unravelling the novel RBPs and splicing factors in context of developmental and fibrotic pathways. We would like to review emerging regulatory nodes and their interaction with CSRPs, and tissue remodeling with major focus on cardiac fibrosis, and inflammatory responses underlying upper airway fibrosis.

Keywords

  • extracellular matrix
  • homeostasis
  • tissue repair
  • fibrosis
  • cellular stress
  • RNA binding proteins
  • RNA interactome

1. Introduction

Fibrosis is an inherent reparative response invoked to restore tissue integrity following a pathologic insult, which metamorphoses into a devastating pathology culminating in scars due to self-catenating heralding inflammatory loops. Regeneration is a fundamental biological process initiated to orderly replace the damaged tissues, however, deregulation of chronic inflammation, growth factor-receptor cross-talk, intra-intercellular communication, and various extracellular matrix proteins eventually result in an aberrant wound healing response marked by fibrosis or scarring. Excessive scarring can obliterate tissue architecture, culminating in organ failure and death. Therefore, lack of coordination and synchronization in molecular and cellular events that guide “regeneration” results in “degeneration” of the affected organ. Fibroproliferative disorders are widely occurring and include pulmonary fibrosis, systemic sclerosis, liver cirrhosis, cardiovascular disease, progressive kidney disease, corneal scarring, proliferative vitreoretinopathy and posterior capsular opacification. Aberrant tissue remodeling marked by fibrosis is also implicated in cancer metastasis and chronic graft rejection in transplant recipients. The obvious deprecating impacts of fibrosis are enormous deterrents to patients; moreover, the disease has failed to meet the required treatments till date. Lack of availability of desired therapeutic interventions is majorly due to an incomplete understanding of the mechanism of the disease, therefore, gaining insights into the mechanistic pathways of fibrosis would facilitate improved therapeutic approaches to target novel mediators besides the cryptic or altered ECM components as previously reported by our group [1, 2, 3, 4, 5].

TGF-β is a central node in driving fibrotic pathways and other diseases, however, targeting TGF-β pathways has not met desirable clinical success, perhaps due to the incomplete mechanistic information on its role in development and pathology. Therefore, gaining insights into regulation of TGF-β during normal development and pathology could facilitate recognition of alternate target-pathways that may spare or minimally perturb the role of TGF-β in physiology. An interesting and relatively less explored theme is involvement and regulation of TGF-β isoforms in fetal wound healing that is marked by absence of scar. Intriguingly, there are common mediators and unifying pathways that underlie tissue repair, homeostasis and fibrosis in diverse organs; therefore, harnessing the potential non-fibrotic themes from scarless wound healing might add to the understanding of challenging fibrotic disorders. A systematic and meticulous re-assessment and re-evaluation of the role of mediators of scarlessly healing wounds might offer a reasonably potential tool to be manipulated to prevent fibrosis and decode the invisible lines dividing the “homeostasis-tissue repair-fibrosis continuum”.

Interestingly, there are few physiological paradigms where wounds heal scarlessly or with minimal scarring, for instance the wounds in the early gestation fetus and in the oral mucosa of mammals heal without scar [6] the transition from scarless to scarred healing occurs in humans during late weeks of gestation [7]. A recent study showed that dermal fibroblasts with a scarring phenotype when transplanted into oral mucosa ended up generating more scar-like connective tissue compared with oral mucosal fibroblasts transplanted into the dermis [8]. The oral mucosal fibroblasts were shown to possess a higher baseline production capacity of several ECM-associated proteins than the skin fibroblasts, except type III collagen, which could be possibly attributed to a more favorable wound healing in oral mucosa [9]. Healthy endometrium heals scarlessly and is suggestive of regenerative healing and can be paralleled to fetal-like scarless healing responses that are also seen in the buccal mucosa of the oral cavity. Endometrial repair involves highly orchestrated cross-stalk in stromal, epithelial, vascular, and immune cells and presents a remarkable epitome of healing that involves over 400 cycles of resolution of inflammation, angiogenesis, tissue remodeling, and formation of new tissue without any residual scarring [10]. Recently, neutrophil gelatinase-associated lipocalin, follistatin like-1, chemokine ligand-20, and secretory leukocyte protease inhibitor were identified as important signatures in menstrual fluid that were proposed to facilitate scar-free repair [11]. Endometrial stromal cells were shown to exhibit distinct phenotypic and immunomodulatory profiles and displayed lack of HLA class II that was proposed to drive their physiological roles in tissue repair and immune tolerance during pregnancy [12].

Psoriasis represents a unique form of “scarless-like or hyper-regenerative” wound healing marked by nonscarring, inflammatory, and hyperproliferative tissue repair responses. An amazing aspect about the psoriatic lesions is the fact that with appropriate therapy the complex skin lesions can be reverted back to healthy appearing skin, with little if any evidence of altered changes in the epidermis and dermis. Psoriatic plaques are exciting conundrums as they do not go to fibrosis even amidst heralding auto-inflammatory loops [13]. An interesting common mediator oncofetal fibronectin extra domain B (Fn-EDB), has been reported to be prominent in psoriatic lesions and wound healing in fetal tissue [14]. Interestingly, psoriatic plaques despite being vulnerable to infections, do not tend to get infected because of the presence of massive antimicrobial peptides like LL37. Psoriasis pathogenesis involves strong polymorphonuclear neutrophil (PMN) infiltration and high levels of the PMN associated antimicrobial peptide, LL37. Psoriasis is marked by self-reactive inflammatory loops of innate immune responses, which trigger subsequent adaptive immune responses against auto-antigens like LL-37, ADAMTSL5, and HNRNPA1, with LL-37 and HNRNPA1 having RNA-binding properties. The phenomenon of self-RNA sensing by nucleic acid sensors [15, 16, 17] is central to autoinflammatory and autoimmune diseases like psoriasis, however, the role of RNA-binding proteins LL37 and HNRNPA1 (the proven autoantigens) in contributing to inflammatory loops remains largely unexplored. In a psoriatic mice model study excessive polyamine generation was shown to facilitate self-RNA sensing by immune cells [18] independent of RBPs, however, a recent study has implicated the role of neutrophil extracellular trap (NET)-associated RNA and LL37 (RBP) in self-amplifying inflammation in psoriasis [19]. Herster et al., highlight an unappreciated yet potential axis involving neutrophils, LL37 (RBP-like) and surprisingly, RNA that are abundant in psoriatic as opposed to healthy skin; suggesting a novel role of NET-derived RNA-RBP (LL37) complexes in self-propagating inflammatory loops. Host defence peptides or antimicrobial peptides like LL37 can have immunomodulatory protective [20] or pathological roles. The dual roles are proposed to be linked to post-translational modifications of peptides by citrullination or carbamylation that may depend on the disease context and result in altered ability of antimicrobial peptides to bind nucleic acids, thereby compromising their immunomodulatory potential (reviewed in [21]). Since RNA binding proteins (like LL37 and HNRNPA1) are emerging as potential molecules that can rewire inflammatory circuits depending on the pathological context, and several RBPs are also known to regulate developmental and fibrotic pathways by interacting with spliceosome machinery and acting as trans- regulators of RNA processing machinery [22], we would like to discuss their role in driving ECM remodeling in context of cardiac fibrosis with particular focus on RBM20, a cardiac-specific RBP that is emerging as a global regulator of cardiac development and disease.

Advertisement

2. Cardiac stress induced wound healing, repair and fibrosis: patch up, break up or a stressed extra-matrix affair

Plethora of extrinsic or intrinsic stressors (persistent hypertension, myocardial infarction, neurohormonal deregulation, hypoxia, ischemia-reperfusion, pressure over load, drug toxicity, mechanical stretch, radiation, etc.) can result in fibrosis and heart failure. The cardiac myocardium is a mosaic of diverse cell types with overrepresentation of cardiomyocytes and fibroblasts, and moderately populating endothelial cells, vessel smooth muscle cells, and immune cells like tissue resident macrophages. The absolute proportions of the cellular components are not determined; however, lineage tracing studies have identified the above inmates in the myocardium of healthy heart. Cardiac wound healing following a stress-induced injury (resulting in development of contraction bands, mitochondrial calcification and membrane disruption) involves a sequalae of pro-inflammatory, anti-inflammatory, and reparative events and majorly resulting in cardiomyocyte death and functional decline. Concomitantly, the neighboring fibroblasts in the myocardial niche act as central nodes to drive aberrant wound healing, ECM (extracellular matrix) remodeling and fibrosis. The continuum of tissue homeostasis, repair, and fibrosis is not appreciably understood, however, the inherent plasticity of fibroblasts and existence of pro- and anti- inflammatory, and pro-fibrogenic polarizing fibroblast phenotypes suggests a cardinal role of these cells in cardiac regeneration and repair. The most challenging aspect of cardiac remodeling is the recreation and restoration of qualitatively (structurally and functionally), and quantitatively compliant ECM, which is pro-regenerative or anti-fibrotic. It is known that extensive ECM deposition in the myocardial infarct can result in arrhythmias, however repopulating the native ECM following destruction is an absolute requirement for maintaining optimal tension in a highly contractile organ like heart. Fibrosis is projected to be the major player behind compromised myocardial compliance resulting in altered contraction-coupling events, reduced ventricular filling, decreased cardiac output and arrhythmias. End-stage heart failure and cardiac arrhythmia happen to associate with fibrosis, which is triggered as a compensatory response to counteract tissue damage, however, perpetuating cycles of stress and inflammation may result in decompensatory fibrosis and organ-failure during late-stage pathologies marked by recalcitrant accentuating cellular stress and catenating chronic inflammatory loops. Cardiac fibrosis is common in several cardiac diseases including atrial fibrillation, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and heart failure with preserved ejection fraction. Aberrant extracellular matrix remodeling can be present in myocardial ischemia and infarction as seen in ischemic (imbalanced oxygen supply and demand) heart diseases caused by atherosclerosis of the epicardial coronary arteries, and nonischemic heart diseases like aortic stenosis, diabetic cardiomyopathy, hypertensive heart disease, and hypertrophic cardiomyopathy, in which myocardial interstitial fibrosis results in adverse ventricular remodeling. Hypertrophying of ventricular cardiomyocytes is an initial adaptive response to compensate for cardiac over load and restore normalization of cardiac output by balancing ventricular wall stress, however, persistence of stress/stressors eventually culminates in cardiomyocyte death, fibroblast activation into myofibroblast, deposition of aberrant ECM, interstitial fibrosis, and adverse cardiac remodeling [23, 24].

Fibroblasts are key players in the secretion, deposition, organization and regulation of ECM turnover, however, their phenotypic heterogeneity, functional diversity, and attendant signalling pathways that modulate fibrotic over regenerative repair in cardiac diseases remain largely indecisive [25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37]. The sphingosine 1-phosphate (S1P) signaling pathway is a hot spot in research for fibrotic diseases involving lung, liver, and heart [29], and improved understanding of its cross-talk with fibroblasts is in progress as the attendant pathways are still controversial. Therefore, improved understanding of the molecular mechanisms and cross-talk underlying fibroblast activation and cardiomyocyte death are central for restoring cardiac homeostasis, designing novel regenerative approaches and developing anti-fibrotic therapies. Given the close association underlying tissue repair and fibrosis, it is intuitive that the balanced participation of common mediators can drive regeneration as opposed to skewed responses that may result in fibrosis. However, what are these checks and balances and how do they determine differential outcomes (homeostasis, repair or fibrosis) is the major challenge in the field of treating fibrosis or regenerating cardiac tissues in vitro. Delineating the subtle mechanisms and cellular, molecular, and extracellular matrix players implicated in regenerative and non-regenerative hearts can provide insights into the homeostasis-repair-fibrosis continuum, which remains the most vexing challenge in developing successful regenerative/anti-fibrotic approaches for fibrotic disorders. Further, the refractory response of cardiomyocytes to complete cell-cycle progression through mitosis limits their self-renewal, therefore, cardiomyogenic approaches to treat heart failure remain practically intractable [38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48].

Advertisement

3. Abortive cellular homeostasis rebalancing in cardiac fibrosis: a tragedy behind the broken heart

Cardiomyocytes being post-mitotic senesce with age and create cellular stress induced pool of biological waste over the course of ageing favoring late onset of fibrotic cardiomyopathies (acquired and inherited). Therefore, ageing and pro-fibrotic gene inheritance serve as additional “self-contained spontaneous stressors” that can impair the cellular homeostatic autophagic machinery, which is indispensable for cardiac tissue repair and proteostasis rebalancing [49, 50, 51, 52]. Regulation of autophagy pathways are strongly implicated in liver, lung, heart, kidney and cystic fibrosis, which suggests that autophagy is a potential target for the treatment of chronic multiorgan fibrotic diseases involving aberrant extracellular remodeling [53]. The divergent homeostatic pathways converge to counter cellular stress or clear the stressor by intersecting and coordinating with precision, therefore, the intersection points, interacting partners and regulatory nodes are under extensive research. Molecular and cellular players driving homeostasis rebalancing in diverse diseases including fibrosis are continuing to emerge and recently reported for endoplasmic and mitochondrial stress pathway networks [54, 55, 56]. Mitochondria and endoplasmic reticulum (ER) in cardiomyocytes are overrepresented organelles to co-ordinate the increased metabolic demands and maintain active calcium stores for smooth flow. Endoplasmic reticulum and mitochondria quality control circuits are also integral to cardiac function, and deregulation of these pathways are strongly implicated in cardiac diseases including heart failure [57, 58, 59, 60, 61, 62, 63]. ECM remodeling is emerging to coincide with metabolic rewiring in cardiomyocytes and matrix-guided control of mitochondrial function in cardiomyocytes is seen as a potential therapeutic target in cardiac fibrosis, repair, regeneration and tissue engineering [64]. A recent trend in increasing ribosome profiling studies has stratified quality control checks that provide additional fidelity by clearance of defective messenger RNAs under ribosome associated quality control [65]. A genetic locus for cardiac hypertrophy that has been associated with alterations in stoichiometric translation rates of sarcomeric proteins has recently been defined [66]. Pro-fibrotic translationally regulated genes underlying cardiac fibrosis with proline (amino acid rich in collagen) codon usage promoted collagen synthesis emphasizing the importance of translational rates that may be heightened in failing hearts that select for codon biasing for profibrotic genes [67].

Importantly, the stress pathways gradually converge, overlap and cross-talk with RNA metabolic, and sterile inflammatory pathways through processing, secretion, and regulation of DAMPS (danger associated molecular patterns) generated in response to heralding stress and inflammation. DAMPs include diverse endogenous host-derived molecules (extracellular ATP, histones, HMGB1 chaperons, etc.), which can be sensed by innate immune receptors [17] owing to their cellular/extracellular mis-localization, stress induced modification or overexpression related conformational anomalies. DAMPs are primarily released by damaged and dying cells to facilitate sterile inflammation, which is important for tissue repair and regeneration, however, if left unchecked they can result in of numerous inflammatory diseases, metabolic disorders, neurodegenerative diseases, autoimmune diseases, cancer and fibrosis. Recently TGF-β has been proposed as an inducible DAMP that activates mechanotransducing pathways resulting in self-perpetuating loops leading to activation of myofibroblasts in diverse pathologies including cardiac fibrosis. Sarcomere integrity and rhythmic efficiency amidst high protein turnover, multiple protein-protein interactions, cyclic contractions and relaxations, and diverse “stress-stimuli” such as pressure overload, metabolic alterations, oxidative stress, hypoxia, ischemia-reperfusion, mechanical stress etc. is remarkable. However, it is also highly vulnerable to succumbing to these multiple stressors that can result in generation of DAMPs leading to inflammation, fibrosis and heart failure. The myocardium cell types express DAMP- sensing receptors and are proficient to respond immediately to stress and damage. Therefore, efficient quality control mechanism regulating cardiac homeostasis are indispensable to sarcomere maintenance and dynamic adaptation to stress [68, 69, 70, 71, 72].

Advertisement

4. Inflammatory networking in cardiac fibrosis: a heart on (in) flame

It is becoming increasingly recognized that the regenerative ability is not completely reliant on genetic makeup, environmental conditions or evolutionary hierarchies but the nature and extent of the immune responses to cardiac injury equally play important role in governing regenerative and non-regenerative modes of wound healing [73]. Importantly, cellular stress pathways cross- talk with inflammatory pathways that actively participate in restoring tissue homeostasis and overactivation of the inflammatory mediators can result in cardiomyocyte death and fibrosis. Macrophages are crucial for tissue homeostasis, following injury, circulating monocytes give rise to proinflammatory macrophages through activation mediated by DAMPs or cytokine secretion. Macrophages may contribute to cardiac fibrotic remodelling through secretion of TNF-α, IL-1β, IL-10, TGF-β and growth factors [74]. Studies have shown that Gata6 expressing macrophages can regulate cardiac fibrosis [75], CX3CR1+ and CCR2+ resident macrophages may positively or negatively regulate cardiac fibrosis, respectively following injury [76, 77]. Mast cells exist in low density in heart tissue, however, following an injury mast cells infiltrate heart tissue [78]. DAMPs may trigger degranulation of mast cells that leads to the release of inflammatory mediators including tryptase, chymase, TNF-α, and IL-1β [79]. Tryptase and chymase activate a potent fibrogenic mediator i.e. TGF-β (that promote myofibroblast differentiation and collagen production). Mast cells also produce PDGF-A and FGF2, which positively regulate fibrosis [80, 81]. However, studies have shown that mast cells can also produce IL-10 (anti-inflammatory agent) that is a negative regulator of fibrosis [82, 83]. Dendritic cells (DCs) play important role in initiating an adaptive immune response in post-injury cardiac remodelling. Studies have shown that DCs infiltrate cardiac tissue following an injury, specifically CD11+ DCs (bone marrow derived) are held crucial for cardiac homeostasis. Deficiency of CD11+ DCs following a cardiac injury may result in enhanced fibrosis [84]. Another study had shown that deletion of cardiac CD103+ DCs resulted in increased fibrosis [85]. Adaptive T and B lymphocytes are also central to cardiac inflammation as B and T cells infiltrate cardiac tissue following injury. There are different subsets of T cells including: CD4+, CD8+, CD73+ and Tregs. CD4+ cells have been reported to produce proinflammatory and fibrotic cytokines like IFN-α, following injury [86], while CD73 expressing T cells reduced fibrosis [87]. B cells secreted proinflammatory cytokines like IL-1β, IL-6 and TNF have been positively associated with fibrosis [88]. Neutrophils are known to regulate fibrosis in context dependent manner, however, neutrophil-derived extracellular traps (NETs) are becoming increasingly implicated in fibrotic pathologies including cardiac fibrosis. NETs have recently gained attention in chronic inflammatory, autoimmune and fibrotic settings including cystic fibrosis, interstitial lung disease, thromboinflammation, hypertrophic cardiomyopathy and liver fibrosis (reviewed in [89, 90, 91, 92, 93, 94, 95]). Notably, NETs have been reported to be associated with disease-specific bioactive proteins loaded onto them [96]. Intriguingly, emerging clinical and experimental studies indicate that neutrophils are able to release intrinsically and qualitatively different NETs decorated with disease-specific bioactive proteins dictated by diseased inflammatory environment containing tissue factor, IL-1β, IL-17, and LL37, suggesting systemic inflammation driven transcriptional-reprogramming in circulating neutrophils, which triggers de novo expression of disease-specific protein fingerprints that are extracellularly delivered through generation of NETs [97] and references therein, these exciting findings implicate NETs as potential anti-fibrotic targets. The non-immune cells of myocardial niche also participate in inflammatory responses, e.g. cardiomyocytes can generate pro-inflammatory mediators leading to profibrotic TGF-β and IGF-1 signalling [98, 99]. Endothelial cells can serve as both positive and negative regulator of fibrosis by generating profibrotic mediators like TGF-β, FGFs, or endothelin-1 [100] and undergoing endothelial to mesenchymal transition [101]. Endothelial cells express HIF-1 (hypoxia inducible factor) that can have anti-fibrotic effects [102] endothelial CXC chemokine Interferon-gamma-inducible protein (IP)-10/CXCL10, is also an anti-fibrotic molecule [103, 104].

The key observations that reflected elevated circulating proinflammatory cytokines in heart failure with reduced ejection fractions pumped the research into exploring role of immune system in heart failure pathogenesis. If inflammation is the cause or result of heart failure is still debatable, however, the developments in understanding the roles of innate and adaptive immune cells in heart failure are in active progress to identify heart failure patients who can have a cardio-inflammatory phenotype and can receive prospective anti-inflammatory and immunomodulatory regimens. The CANTOS trial with anti- IL-1β antibody canakinumab indicated decreased hospitalization rates in certain group of heart failure patients [105], these findings have renewed the interest in decoding cardio-inflammatory pathways for therapeutic targeting. Sensing of DAMPs can trigger non-cellular and cellular effectors in including IL-1, IL-6, IL-8, TNF, chemokines, complement system, inflammasome assembly, and activation of neutrophils, monocytes, macrophage innate immune cells that further engage the adaptive immune arm to trigger inflammatory loops [106]. Leukocyte dependent regulation of cardiac fibrosis is an ongoing area; however, it stays controversial and warrants further studies to exploit leukocyte plasticity and heterogeneity in cardiac fibrosis therapeutics [107]. Recent demonstration of engineered T cells or the CAR T-cell therapy directed against activated fibroblast specific antigen has sparked new hopes to existing limited clinical interventions and therapies in fibrotic heart failure [108, 109]. A recent study has demonstrated that macrophages expressing Mertk immune receptor in the heart supports cardiomyocyte health by phagocytosing exopher particles ejected from stressed cardiomyocytes harboring defective mitochondria. Mertk facilitated defective elimination of mitochondria from the myocardial tissue and prevented activation of the inflammasome, autophagy, metabolic stress, and ventricular dysfunction [110]. IL-11 signaling is also implicated in cardiac and cardiorenal fibrosis, however further studies will better indicate its precise role in driving pathogenesis [111]. A cross-talk between RNA processing pathways and immune- and non-immune cells through diverse mediators in context of cardiac fibrosis has been depicted in Figure 1.

Figure 1.

RNA processing and modifications and their link to inflammation in cardiac fibrosis: (a) m6A Methylation, (b) Uridylation, (c) Editing, (d) Alternative Splicing, (e) Immunostimulatory RNA may result in Interferon and cytokines production. (f) Interaction between immune and non-immune cells during fibrotic remodeling. Diverse RNA processing pathways can result in generation of immunostimulatory RNAs that can trigger inflammatory cascade.

Advertisement

5. Messenger RNA regulatory networks may modulate cellular stress and inflammation driving cardiac fibrosis: a message to the heart still in the outbox

The global co- and post- transcriptional mechanisms implicated in cardiac fibrosis are not well established, however, emerging studies are geared at extracting the subtle communications to identify intersection points between the cellular stress regulating pathways, regulatory non-coding RNAs, RNA metabolism intermediates/mediators and cardiac RNA binding proteins (RBFox, HuR, MBNL2, PUM2, QKI, CELF1, MBNL1, PTBP1) in context of cardiac diseases including fibrosis [66, 112, 113, 114, 115, 116, 117, 118, 119, 120]. A recent study implicated NUP155 subdomain hotspot with enriched allelic variants of the gene that suggests important role of RNA metabolism in cardiac disease and development [121].

Stem cell based regenerative approaches have found limited applicability in clinical translation to treat fibrosis. This ignited the research targeted at identifying cell- free secretory molecules that could not only have potential anti-fibrotic/regenerative potential to ameliorate fibrosis but characterization of these molecular players would also facilitate understanding of mechanisms underlying pathogenesis of fibrosis. In this context, bioactive vesicles/exosomes have been extensively investigated to explore the role of regulatory RNAs (generated through RNA processing pathways) in delivering pro- or anti- fibrotic outcomes, besides non-coding RNAs are also extensively studied in context of fibrosis independent of their exosomal/vesicular loading [120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141]. The cardiac inflammatory circuits also tend to converge at the regulatory networks controlled by RNA processing, metabolism and surveillance pathways. Particularly, the post- transcriptional regulation of cytokines to stabilize mRNA, determine the strength of proinflammatory pathways. Altered expression of AU rich (ARE) or GU rich (GRE) elements in cytokine and cytokine pathway intermediate transcripts impairs mRNA decay and can result in heightened immune responses as seen in diseased states [142]. Immunostimulatory RNAs (like double stranded RNAs) generated through deregulated RNA processing pathways along with RNA binding proteins of RNA helicase (RNA sensors) family are emerging as important components of immune response pathways during sterile inflammation that involves DAMP sensing [15, 16]. Mitochondrial quality control pathways intersecting with the endosomal compartments and lysosomes are recently reported to favor generation and release of mitochondrial-derived vesicles in former condition [143], further offering discernible biologically stable lipid vesicles that may help investigate how secreted cargos can impact tissue repair and homeostasis or trigger fibrosis, and can be extended to establishment of liquid biopsies for studying the progression of fibrotic diseases or alternatively these vesicles may serve as therapeutic tools like exosome-derived non coding RNAs [121, 125, 127, 128, 138, 139, 141, 144] circulating microRNAs (miRNAs) and tissue resident miRNAs play paradoxical role both as anti-fibrotic [145, 146, 147, 148] and pro-fibrotic [149, 150, 151].

Advertisement

6. Harnessing RNA metabolic pathways in cardiac development and disease: getting to core of the heart

The cardiac output is tightly tuned to the functional outputs of the cardiac transcriptome or faithful expression of cardiac-specific genes. It is becoming evident that cellular processes (that are linked to generation of RNA variants) including alternative splicing, RNA editing, epitranscriptomic modifications like methylation, and alternative polyadenylation [152] have a major role in shaping the cardiac adaptive responses [119]. The advent of high throughput NGS sequencing has revealed striking diversity in RNA species/variants/isoforms that have revolutionized the field of RNA biology by informing on the codes of burgeoning RNA inventory, which has now been exploited in context of functional relevance of the neo-RNA entities in context of physiological and pathological outcomes. Expanding information and identification of genetic markers for heterogeneous complex diseases like heart failure has made it appreciably evident that cardiac development and differentiation cues are under tight regulation of splicing events [153], and mis-splicing of certain genes like titin (TTN) that is implicated in contractility and mechanosensation can result in adverse cardiac extracellular remodeling and fibrosis. Interestingly, single cell RNA sequencing of cochlear hair cells recently documented unappreciated complexity in splicing diversity and isoform abundance underlying biology of hearing and deafness, and reported sorcin (a key player in cardiac excitation-contraction) as a top hit in cochlear outer hair cells [154]. These exciting findings reflect potential shared mechanosensory targets that could result in co-manifestation of heterogenous genetic disorders like heart failure and hearing loss, which involve electrical conductance and contraction events (it remains to be explored if sorcin may have isoformic pattern of expression). It is already known that cardiac arrhythmias are a feature of Jervell and Lange-Nielsen syndrome (JLNS), an autosomal recessive disorder associated with congenital profound sensorineural hearing loss arising from homozygous or compound heterozygous mutations in either KCNQ1 or KCNE1 subunits of potassium ion channel conducting the slow component of the delayed rectifier current [155, 156].

Constitutive RNA splicing primarily involves the spliceosome machinery acting at the splice sites, however, alternative splicing may differ in its mechanism of action by engaging further cis- elements (regions within pre mRNA besides 5’/3’ splice sites) like enhancers of exon/intron splicing [157]; the cis-elements are further acted upon by trans- acting modulators that constitute a family of RNA binding proteins (RBPs) with RNA binding motif (RBM), which further regulate negatively (repress) or positively (activate) the splice site selection. The activators include serine/arginine-domain–containing (SR) proteins and SR-related factors that dictate binding and assembly of the spliceosome complex and decide differential inclusion of exons in the mature transcript [158]. In contrast, the repressor trans-elements include the family of heterogenous nuclear ribonucleoproteins (hnRNPs) that tend to suppress splice site recognition [159, 160]. Therefore, alternative splicing events diversify the overall repository of functional and/or regulatory genes by inclusion/exclusion of exon, intron retention, alternative 5’ or 3’ splicing, and mutually exclusive exon utilization [119, 152], and at the same time splicing diversity may reflect pathological vulnerabilities dictated by the inherited variants that offer isoformic switching.

Advertisement

7. RBM20 interactome in cardiac development and disease: determining the soft- or hard- heartedness

Cardiac diseases including cardiomyopathy and arrhythmia are long known to be regulated by isoformic pattern of protein expression for genes including titin [161], CAMK2D, LDB3 and CACNA1C [162, 163]. Titin isoform-switching mechanisms at RNA (alternative splicing) and protein (post-translational modification) levels, which direct titin-based passive tension tuning remained largely elusive [164, 165, 166, 167, 168, 169]. RNA binding protein RBM20, which is a splicing related factor was known to steer various aspects of cardiac function by regulating genes involved in biomechanics (TTN and TPM1), ion homeostasis and electrical activity (CAMK2D and CACNA1C) and signal transduction (CAMK2D and SPEN). Titin is the best exemplified target of RBM20 and TTN mutations are vastly implicated in cardiomyopathy [170, 171], and cardiovascular diseases [164, 172, 173, 174]. However, the mechanisms underlying alternative splicing of titin and role of thyroid hormone and insulin signaling in regulating it were nearly correlative [175, 176, 177, 178] as the splice factors regulating alternative splicing in titin remained undetermined until early this decade. The role of post-transcriptional regulation in cardiac function and pathogenesis of human heart failure gained impetus following a pioneer study [179] on RNA binding motif (RBM20) protein, which has now emerged as a global regulator of cardiac alternative splicing isoformic switch in protein titin. RBM20 was found to be predominantly expressed in striated muscle, with maximum expression in the heart and its deficiency in rats was reported to resemble the pathophysiology of genetic dilated cardiomyopathy (fibrotic remodeling in heart). The RBM20-null rats exhibited increased subendocardial fibrosis with age and this effect was accompanied by electrical abnormalities and sudden death. The reduced activity of RBM20 (due to mutations/variants) resulted in altered isoform expression of genes central to biomechanics, electrophysiology and signal transduction culminating in cardiomyopathy, fibrosis, arrhythmia and sudden death [179]. Following these seminal findings that delineated the role of RBM20 driven splicing in titin isoforms, the ribonucleoprotein RBM20 has now paralled the role of titin in regulating structural and functional characteristics of cardiac development and disease. Cardiac-specific splicing events are attributed to RBM20, and recent studies with defective RBM20 variants have been shown to be associated with cardiac transcript variants resulting in cardiomyopathy including DCM (dilated cardiomyopathy) [180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190].

Structurally, RBM20 is a 1227 amino acid long protein with a leucine-rich N-terminal domain, zinc finger (ZnF) domain 1, RNA recognition motif (RRM) followed by mutational hotspot arginine-serine (RS) rich domain, glutamate (E) rich and ZnF2 regions located towards C-terminal. RRM and RS regions, and phosphorylation within RS region, are reported to be crucial for nuclear localization [179, 191, 192], RRM is important for binding to “UCUU” RNA sequence that dictates RBM20 binding to target genes. Several mutations are reported in RS region (predominantly the RSRSP stretch, amino acids 634-638) that likely disrupt its nuclear localization and hence splicing of target genes like titin, subsequently resulting in adverse cardiac modelling and familial dilated cardiomyopathy with associated fibrosis [148, 183, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203]. Compared to nuclear localization regions, the structural components contributing to splicing activity of RBM20 towards its targets are not fully explored, the near C-terminal E region has shown to have some contributions to splicing though [179, 204]. RBM20 is known to interact with spliceosome complex subunits U1 and U2 (small nuclear ribonucleic particles) and U2 related proteins like U2AF6 and U2AF35 [205]. The inventory of RBM20 regulated cardiac pre-mRNAs is dynamic and includes the following validated genes in human and rat- titin (TTN), calcium voltage gated channel subunit α1 C (CACNA1C), calcium/calmodulin dependent protein kinase II delta and gamma (CAMK2D and CAMK2G), formin homology 2 domain containing 3 (FHOD3), Lim domain binding 3 (LDB3), Lim domain only protein 7 (LMO7), muscular-enriched A type laminin-interacting protein (MLIP), PDZ and LIM domain 3 (PDLIM3), reticulon 4 (RTN4), ryanodine receptor 2 (RyR2), SH3 domain containing kinase binding protein 1 (SH3KBP1), sorbin and SH3 domain containing protein (SORBS1), and triadin (TRDN) [153, 179, 205, 206, 207, 208].

Titin is an integral sarcomere protein responsible for maintaining passive elasticity in heart, structurally it is organized into modular structure with immunoglobulin-like (Ig), fibronectin type III (FnIII), proline (P), glutamate (E), valine (V), and lysine (K) containing highly elastic I-band region. The N-terminal domain of titin anchors it to the sarcomeric Z-band and C-terminal domain embeds it into M-band. The A-band maintains rigidity during contraction by binding to myosin. Titin’s structural integrity is central to normal cardiac function, maintaining passive tension and driving length-dependent activation/Frank-Starling effect. Besides providing mechanical properties, titin stretching also participates in cellular signaling that facilitates cardiomyocyte growth and might be implicated in chronic myocardium remodeling, hypertrophy and fibrosis. Cardiomyopathy patients with mutations in titin gene further demonstrate the contribution of titin to systolic and diastolic heart failure. Systolic dysfunction underlies dilated cardiomyopathy (dilation of left ventricle) and hypertrophic cardiomyopathy (myocardial hypertrophy and ventricular thickening). Diastolic dysfunction is a hallmark of restrictive cardiomyopathy with preserved contractile force, however, abnormal relaxation during diastole results in decreased cardiac output due to inappropriate ventricle filling. Truncation mutations in titin gene cause dilated cardiomyopathy through diverse pathways that involve haploinsufficiency, activation of mTOR energy sensor pathways and increased metabolic stress (recently reviewed in [209]). Human induced pluripotent stem cell (hiPSCs) culture models aimed at generating cardiomyocytes from titin mutation carrying patients depict disorganized sarcomeric array, contraction disability and impaired force generation, however, similar extent of sarcomeric damage and myofibril contraction impairment has not been recapitulated in human studies or biopsied cardiomyocytes from titin variant patients. Therefore, it is alternatively proposed that titin variants may operate through creating a metabolic stress that could impair cardiac function independent of mutation sites by altering RNA metabolism pathways triggering non-sense mRNA decay (NMD) of abnormal titin variants and development of DCM phenotype. The cardiac metabolism could switch to branched chain amino acid pathway in place of fatty acid metabolism, deregulation of mTOR and autophagy pathways [210, 211, 212, 213, 214, 215, 216, 217, 218].

RBM20 cardiomyopathy has high penetrance and correlates with increased rates of heart failure, arrhythmias, and sudden cardiac death, new insights into RBM20 cardiomyopathy are extensively discussed recently [190]. Given the large size of titin (near 300kb) it is known to undergo extensive splicing events and yield several titin isoforms with cardiac N2B and N2BA to be the best characterized. N2B (shorter and stiff isoform) and N2BA (longer and pliant isoform) are adult cardiac isoforms of titin that regulate passive stiffness in the heart, and this is attributed to their structural dissimilarity in the highly elastic I-band region. Alternative splicing variants of titin during cardiac development keeps selecting for the shorter and stiffer isoform N2B in course of fetal to adult cardiac development, and physiologically N2B is overexpressed as compared to pliant N2BA isoform. However, aberrant expression patterns of titin isoforms resulting in altered ratios of N2B and N2BA are associated with cardiac diseases including cardiomyopathy with fibrosis and heart failure. RBM20 is shown to facilitate exon skipping events thereby selecting for shorter and stiffer forms of titin over development. In animal models, RBM20 homozygous mutations show increased ratio of N2BA/N2B (mirroring DCM phenotype), induced expression of RBM20 in RBM null mice decreases this ratio, however, intermediate effects (titin length, passive tension, sarcomere length) are seen in heterozygous mutations, indicating quantitative modulations of RBM20 as potential therapeutic approach to treat cardiac diseases [153, 179, 183, 194, 219]. RBM20 mutations in human patients result in severe inherited early onset DCM, manifesting even early on in younger patients with sudden death [190, 220]. Patient-specific stem cell based hiPSC culture models or CRISPR/CAS9 gene editing tools have been exploited to measure the effect of RBM20 point mutations in cardiomyocytes and alterations in sarcomere length, calcium handling, electrical coupling have been reported. The human iPSCs containing RBM20 mutations offer great tractable and tunable system to model cardiomyopathy in vitro and investigate potential signaling pathways contributing to the pro-fibrotic phenotypes [189, 221, 222]. The paradigm-shift in RNA metabolism associated interactome has begun to offer new therapeutic windows by unravelling the novel RNA binding proteins and splicing factors in context of cardiac development and fibrotic cardiomyopathies [119]. Biogenesis of regulatory non-coding RNAs i.e. microRNA, long noncoding RNA, and circular RNA, and their role in cardiac fibrosis, and RBM20 mediated alternative splicing of titin pre-mRNA is shown in Figure 2.

Figure 2.

Biogenesis of regulatory non-coding RNAs i.e. microRNA, long noncoding RNA and circular RNA and their role in cardiac fibrosis (a-c). Fate of aberrant RNA transcripts (d). RBM20 dependent splicing of Titin pre-mRNA resulting in formation of Titin isoforms which regulate cardiac development and fibrosis. Abbreviations: miRNA: microRNA; lnRNA: long non-coding RNA; cirRNA: circular RNA; NMD: nonsense-mediated decay; NSD: nonstop-mediated decay; NGD: no-go decay

We briefly discuss the inflammatory networks in upper airway fibrotic diseases like laryngotracheal stenosis and subglottic stenosis that are relatively less explored in terms of mechanisms of fibrotic pathways, however, these pathologies need special attention as they might affect increasing number of patients given the current COVID-19 pandemic. Recent reports show that COVID-19 critically ill patients need mechanical ventilation, and many of these patients who need prolonged ventilation need surgical tracheostomy that is implicated in development of upper airway fibrosis.

Advertisement

8. Laryngotracheal stenosis: the pathogenesis and inflammatory pathways

Laryngotracheal stenosis (LTS) is an abnormal wound healing process of laryngotracheal mucosal inflammation, wound healing and scar formation. LTS is a fibrotic disease leading to pathologic narrowing of the larynx, subglottis, and trachea (the upper airway). There can be multiple etiologies to LTS, ranging from intubation injury (iatrogenic), radiation, autoimmune disease, to idiopathic [223]. The early stages of LTS are marked by dysphonia and communication difficulties that can develop into life-threatening progressive dyspnea leading to the airway compromise [224]. The most common form of LTS is the iatrogenic LTS (iLTS) caused by regional hypoxic and ischaemic pressure (stress) induced necrosis of the airway following prolonged intubation or tracheostomy [225]. The possible pathophysiology behind iatrogenic LTS is the surpassing of the pressure exerted by the cuff while prolonged intubations to that of the mucosal capillary perfusion pressure (approx. 35 mmHg), which results in ischemia, inflammation of the mucosa, and subsequent fibrotic strictures [226, 227]. The airway is primarily formed of 3 sets of cell types including epithelial cells, the fibroblasts, and the resident immune cells. The cross-talk of fibroblasts, immune cells and inflammatory cytokines participates in the development and propagation of LTS.

Advertisement

9. Inflammatory networks in the pathogenesis of LTS

TGFβ-SMAD2/3 cascade has been implicated in LTS and TGF-β antagonists have shown to attenuate fibrosis, however, TGF-β3 isoform has been reported to have antifibrotic response in LTS healing by significantly decreasing the inflammation and collagen deposition [228, 229], indicating opposing roles for isoforms of TGF-β. Hypoxia induced expression of IL-6 plays an important role in the pathogenesis of LTS, the pressure exerted by the endotracheal tube cuff causes hypoxic and ischemic necrosis of the laryngotracheal mucosal tissue leading to inflammation and scarring marked by increased expression of IL-6, α-SMA & collagen, importantly IL-6 and myofibroblasts were also increased in an ex-vivo culture of healthy tracheal fibroblasts cultured under hypoxic conditions [230]. Possible role of B or T-cells in the formation of granulation tissue has also been been suggested [231]. Increased expression of profibrogenic Th2 cytokine IL-4 was seen in the brush biopsy samples of LTS scar and it stimulated fibroblast activation and excessive collagen formation in the LTS wound [225]. The expression of another Th2 cytokine IL-13 appeared to follow the same expression pattern of IL-4 and resulted in excessive fibrosis [232]. In contrast to Th2 cytokines, the Th1 cytokine IFN-γ inhibited fibrosis in LTS patients. Subsequent studies have shown the impact of IFN-γ on the LTS fibroblasts as significant decrease in levels of collagen and TGF-β expression was reported in the IFN-γ treated human LTS-derived fibroblasts compared to the untreated LTS-derived fibroblasts and normal laryngotracheal fibroblasts [226]. Dysregulated functioning of macrophages is related to fibroproliferative LTS [232], a prolonged cytokine signalling in the form of IL-4/IL-13 by Th2 cells can also contribute to impairment of macrophages by switching the non-fibroproliferative “classically activated” M1 macrophages into the fibroproliferative “alternatively activated” M2 macrophages [232]. Mice LTS model of chemical and mechanical injuries showed increased expression of M2 cell surface marker CD206 [224]. Inflammatory cytokine expression study in iLTS and autoimmune LTS patients demonstrated elevated levels of the macrophage growth factor granulocyte macrophage colony-stimulating factor (GM-CSF) and M2 cytokine IL-10 than that in controls [233]. Fibroblasts are the mesenchymal cells which are not terminally differentiated and rest in inactive state, under homeostasis. In their inactive but normal state they localise to the subepithelial layer of the airway tissue and provide for the biochemical and mechanical support to the tissue [234]. However, studies have reported increased ECM production and migration, and reduced contraction of iLTS fibroblasts [235], moreover, studies involving use of beta-aminopropionitrile (βAPN), an inhibitor of collagen cross-linking also demonstrated enhanced profibrotic features (overexpression of collagen I and II) in iLTS-derived fibroblasts [236], metabolically they exhibited enhanced glycolysis to oxidative phosphorylation ratio as see in proliferative cancer cells, justifying highly proliferative nature of iLTS-derived fibroblasts [237]. Emerging studies are reporting genetic link to LTS pathology, suggesting alternative treatment approaches to cure this fibrotic pathology. A functional single nucleotide polymorphism of TGF-β1 located in a negative regulatory element of its promoter was associated with the iatrogenic LTS. The data identified protective and susceptible genetic loci in patients undergoing endotracheal intubation. Another study, focused on 3 candidate genes encoding the innate immune receptor CD14, matrix metalloproteinase-1 (MMP-1), and the cytokine transforming growth factor β1 (TGF- β1). Reported association between MMP-1 and susceptibility to iLTS following intubation merits further investigation in a lager patient cohort [238].

Advertisement

10. Subglottic stenosis: a complex interplay between inflammation and fibrosis

Subglottic stenosis (SGS) is a relatively less explored fibrotic pathology in terms of mechanistic insights on inflammatory pathways. Researchers have gathered information to some extent by evaluating the changes that occur in the airway as a result of obliterative bronchiolitis [239], which shares the same fibrotic features of subglottic stenosis as studied in a murine model with emphasis on cytokines such as IL-1β, TGF-β and prostaglandin PGE2 [240]. SGS is accompanied by an acute and an exaggerated inflammatory response that triggers a shift in the cellular and molecular components in the healing wound in favor of more fibroblastic etiology [235]. SGS has numerous potential etiologies of which the most common cause in adults and children alike is prolonged endotracheal intubation. With developments in intensive care and associated intubations, it is natural that iatrogenic stenosis will become a major factor affecting post ICU quality of life. Animal and human studies have shown an upregulation of inflammatory markers in stenotic tissues. Patient factors like increased BMI, diabetes mellitus and chronic laryngopharyngeal reflux have also been implicated as causative factors for development of subglottic stenosis [241]. Cytokines like IL-1β, IL -10, TNF α, IFN γ and GM-CSF have shown significant increase in subglottic stenosis specimens [226]. Enhanced expression of profibrotic growth factors and cytokines like TGF-β, PDGF, IL-1, and Prostaglandin E2 was seen in patients of healing laryngeal lesions [242]. Expression of matrix metalloproteinases (MMPs), α-SMA, SMADs, IL-1 continued to rise 3 weeks beyond the initial insult [231]. Therefore, it appears that SGS undergoes an aberrant healing response as cytokines corresponding to various stages of wound healing process including inflammation (IL-1, TGFβ), proliferation (SMADs, TGFβ), and maturation (MMPs, αSMAs) are reported to present in pathogenesis of SGS. Idiopathic subglottic stenosis (iSGS) also has an inflammatory network evident from various studies suggesting the role of γδ T cells in IL-17A dependent tissue inflammation and airway remodeling in iSGS. Further studies delineating the role of RNA biology pathways might open up viable therapeutic options for this devastating pathology.

11. Future perspectives and summary

We are encouraged to chase the role of RBPs in homeostasis-tissue-repair-fibrosis continuum based on our recent preliminary findings, where we report for the first time an in vitro model that rigorously recapitulates proteolytic stress (as encountered in fibrotic pathologies) induced stress granule (SG) biomolecular condensate-like proteome signatures [243]. Dynamic phase separated membraneless organelles including SGs are induced upon varied stress-stimuli (infectious or non-infectious) and are implicated in spatiotemporal control of various cellular functions including formation of signalling complexes, clustering of vesicles, sorting and trafficking of cargo [244]. Phase separated biomolecular condensates are becoming increasingly linked to developmental and pathological pathways [245, 246, 247, 248, 249]. We proposed proteases as novel stressors that can have diverse outcomes when present at varying concentrations (protease-antiprotease balance is crucial for driving tissue repair or fibrotic phenotypes). We observed heightened ribonucleoproteins (RNPs), spliceosome machinery, regulatory RNA generating proteins, and RNA binding proteins (RBPs) in our high-throughput proteomics data [243]. The formation of SGs like proteome was concomitant to translational halt in majority of proteins, sparing few essential cytoprotective proteins including exosome biogenesis and secretory pathway proteins that undergo synthesis despite stress environ. We hypothesize that the unique stress-associated proteins that represent “stress-essentialome” might get packaged and enriched into exosome vesicles in addition to the hitchhiking of SG regulatory RNAs, RBPs, RNPs and cytoprotective proteins onto the exosomal carriers leading to conglomeration of unique disease/stressor-specific cargos in exosome silos. Translational halt would result in polysome run off and dissociation of ribosome and translating mRNAs that would partition into the stress granules, therefore, RNA and polysome profiling of stressed cells in addition to exosome cargo profiling might offer valuable information on pathologic transcripts, associated regulatory RNAs (miRNAs, lncRNA) and translating ribosome composition besides the proteome signatures, thereby offering new dimensions to investigate stress-induced regenerative or fibrotic responses and the role of RNA processing machinery and RBPs in driving these triggers. It might offer identification of cell or tissue specific splicing factors and an opportunity to attempt rescuing splicing related alterations inherited in genome (pathogenic variants), to switch for native isoforms. In addition, stress-induced secretion of extracellular vesicles/exosomes could offer novel therapeutic opportunities by developing liquid biopsies in fibrotic diseases (a less explored area), which may yield meaningful information and help predict progression of the disease in suitable disease-specific in vitro models. Exosomes are relatively stable, can avoid background noise, can be easily detected from blood and their molecular analysis may decipher pathobiology of disease, fibrotic liver derived exosomes (in mice) showed increased CCN2; decreased Twist1, miR-214 than control mice [250]. Circulating exosomes from mice with alcohol related liver disease when transmitted to normal mice resulted in pro-inflammatory/fibrogenic liver phenotype [251]. MiR-125 has also been found to be upregulated in serum from patients with cirrhosis than controls [252]. Multiple studies in-vitro and in mice have revealed pathologic micro-RNAs associated with liver injury, liver fibrosis and liver malignancy [250]. However, major challenge in translating bench to bedside knowledge include reliable standardization and characterization protocols, and validation in a well-characterized patient population. Therefore, we are interested in deciphering the molecular information stored in exosomes of patients with liver cirrhosis using standardized protocol and their correlation with key events like death, sepsis, organ failures. Our proposed model of stress- induced reshaping of RNA metabolic pathways that are coupled to mitochondrial alterations and exosome biosynthesis is shown in Figure 3.

Figure 3.

Proposed model for cellular stress induced biological condensates that can regulate homeostasis, tissue repair and fibrosis. A cellular-stress induced reshaping of RNA processing machinery by generation of biomolecular condensates, and their coupling to mitochondrial and exosomal pathways. The stress-coupled exosomes are proposed to carry pathologic cargo which can be exploited to develop liquid biopsies in the context of fibrotic disorders. The expression of cardiac-specific RBPs in our model can be utilized to develop cardiac regenerative approaches in vitro or to study the role of RBPs in cardiac fibrosis. RNPs: ribonucleoproteins: RBPs: RNA binding proteins; CBC: cap binding complex; EJC: exon junction complex; SERBP1: SERPINE1 mRNA-binding protein 1; PAI-1: Plasminogen activator inhibitor-1; ISG: Interferon stimulated gene; PRKRA: Protein kinase, interferon-inducible double stranded RNA dependent activator; ADAR: Adenosine deaminases acting on RNA; EF2AK2: Eukaryotic Translation Initiation Factor 2-alpha Kinase 2; RED color: upregulated proteins; MT: mitochondrion; CPT: Carnitine palmitoyl-transferase; MPC: mitochondrial pyruvate carrier; PDC: Pyruvate dehydrogenase complex; ACAD: Acyl-CoA dehydrogenase; ACAA2: acetyl-Coenzyme A acyltransferase 2; BCKDHA: branched-chain alpha-keto acid dehydrogenase; NDUFS: NADH-ubiquinone oxidoreductase subunit; SDHAF: Succinate dehydrogenase complex assembly factor 1; UQCRB: Ubiquinol-cytochrome c reductase binding protein; SCO: synthesis of cytochrome c oxidase; TOM: translocase of the outer membrane; TIM: translocase of the inner membrane; ETC: electron transport chain. DAMPs- danger associated molecular patterns.

Acknowledgments

MS thanks financial support from PGIMER grant: No.71/2-Edu-16/1098, and SERB funded grants-SERB/CRG/2019/006745, and SPR/2019/001447. KK thanks SERB/CRG/2019/006745 for providing Junior Research Fellowship.

References

  1. 1. M. Sharma, A Tiwari, S. Sharma, P. Bhoria, V. Gupta, A. Gupta, and M. Luthra-Guptasarma (2013) Fibrotic remodeling of the extracellular matrix through a novel (engineered, dual-function) antibody reactive to a cryptic epitope on the N-terminal 30 kDa fragment of fibronectin. PLOS one 8 (7): e69343
  2. 2. M. Sharma, A Tiwari, S. Sharma, R. Bansal, V. Gupta, A. Gupta, and M. Luthra-Guptasarma (2014) Pathologic vitreous causes cell line-derived (but not donor-derived) retinal pigment epithelial cells to display PVR-like features in culture. Clinical and Experimental Ophthalmology; doi: 10.1111/ceo.12307
  3. 3. A. Tiwari, R. Kumar, J. Ram, M. Sharma* and M. Luthra-Guptasarma* (2016) Control of fibrotic changes through the synergistic effects of anti-fibronectin antibody and an RGDS-tagged form of the same antibody. Scientific Reports 6:30872
  4. 4. Brij Bhushan Mehta, Anil Tiwari, Saniya Sharma, Ashu Shukla, M.Sharma, Rakesh K Vasishta, Ramesh Sen, Aman Sharma, Manni Luthra-Guptasarma (2018). Amelioration of collagen antibody induced arthritis in mice by an antibody directed against the fibronectin type III repeats of tenascin-C. International Immunopharmacology 58, 15-23
  5. 5. Santra M, Sharma M, Katoch D, Jain S, Saikia UN, Dogra MR, Luthra-Guptasarma M. Induction of posterior vitreous detachment (PVD) by non-enzymatic reagents targeting vitreous collagen liquefaction as well as vitreoretinal adhesion. Sci Rep. 2020 Jul 16;10(1):12083. doi: 10.1038/s41598-020-69093-w
  6. 6. Marshall CD, Hu MS, Leavitt T, Barnes LA, Lorenz HP, Longaker MT. Cutaneous Scarring: Basic Science, Current Treatments, and Future Directions. Adv Wound Care (New Rochelle). 2018;7(2):29-45. doi: 10.1089/wound.2016.0696kin
  7. 7. Karppinen SM, Heljasvaara R, Gullberg D, Tasanen K, Pihlajaniemi T. Toward understanding scarless skin wound healing and pathological scarring. F1000Res. 2019;8:F1000 Faculty Rev-787. Published 2019 Jun 5. doi:10.12688/f1000research.18293.1
  8. 8. Nikoloudaki G, Creber K, Hamilton DW. Wound healing and fibrosis: a contrasting role for periostin in skin and the oral mucosa. Am J Physiol Cell Physiol. 2020 Jun 1;318(6):C1065-C1077. doi: 10.1152/ajpcell.00035.2020. Epub 2020 Apr 8. PMID: 32267719; PMCID: PMC7311745
  9. 9. Hara-Saito, Y., Kato, H., Saito, N. et al. Distinct differences in hypoxic responses between human oral mucosa and skin fibroblasts in a 3D collagen matrix. In Vitro Cell Dev.Biol.-Animal 2020; 56, 452-479 (2020). https://doi.org/10.1007/s11626-020-00458-1
  10. 10. Critchley HO, Maybin JA, Armstrong GM, Williams AR. Physiology of the Endometrium and Regulation of Menstruation. Physiological reviews. 2020 Jul 1;100(3):1149-79
  11. 11. Evans J, Infusini G, McGovern J, Cuttle L, Webb A, Nebl T, Milla L, Kimble R, Kempf M, Andrews CJ, Leavesley D. Menstrual fluid factors facilitate tissue repair: identification and functional action in endometrial and skin repair. The FASEB Journal. 2019 Jan;33(1):584-605
  12. 12. Queckboerner S, Von Grothusen C, Boggavarapu NR, Davies LC, Gemzell-Danielsson K. Stromal Heterogeneity in the Proliferative Endometrial Functionalis-A single-cell approach. BioRxiv. 2020 Jan 1
  13. 13. Nickoloff BJ, Bonish BK, Marble DJ, Schriedel KA, DiPietro LA, Gordon KB, Lingen MW. Lessons learned from psoriatic plaques concerning mechanisms of tissue repair, remodeling, and inflammation. J Investig Dermatol Symp Proc. 2006 Sep;11(1):16-29. doi: 10.1038/sj.jidsymp.5650010. PMID: 17069007
  14. 14. Georgescu SR, Tampa M, Caruntu C, et al. Advances in Understanding the Immunological Pathways in Psoriasis. Int J Mol Sci
  15. 15. Liu G, Gack MU. Distinct and Orchestrated Functions of RNA Sensors in Innate Immunity. Immunity. 2020 Jul 14;53(1):26-42
  16. 16. McWhirter SM, Jefferies CA. Nucleic Acid Sensors as Therapeutic Targets for Human Disease. Immunity. 2020 Jul 14;53(1):78-97
  17. 17. Gong T, Liu L, Jiang W, Zhou R. DAMP-sensing receptors in sterile inflammation and inflammatory diseases. Nature Reviews Immunology. 2019 Sep 26:1-8
  18. 18. Lou F, Sun Y, Xu Z, Niu L, Wang Z, Deng S, Liu Z, Zhou H, Bai J, Yin Q, Cai X. Excessive Polyamine Generation in Keratinocytes Promotes Self-RNA Sensing by Dendritic Cells in Psoriasis. Immunity. 2020 Jul 14;53(1):204-16
  19. 19. Herster F, Bittner Z, Archer NK, Dickhöfer S, Eisel D, Eigenbrod T, Knorpp T, Schneiderhan-Marra N, Löffler MW, Kalbacher H, Vierbuchen T. Neutrophil extracellular trap-associated RNA and LL37 enable self-amplifying inflammation in psoriasis. Nature communications. 2020 Jan 8;11(1):1-3
  20. 20. Alford MA, Baquir B, Santana FL, Haney EF, Hancock RE. Cathelicidin Host Defense Peptides and Inflammatory Signaling: Striking a Balance. Frontiers in Microbiology. 2020 Aug 27;11:1902
  21. 21. Liang W, Diana J. The Dual Role of Antimicrobial Peptides in Autoimmunity. Frontiers in Immunology. 2020 Sep 2;11:2077
  22. 22. Gao C, Wang Y. mRNA Metabolism in Cardiac Development and Disease: Life After Transcription. Physiological Reviews. 2020 Apr 1;100(2):673-94
  23. 23. Frangogiannis NG. Cardiac fibrosis: cell biological mechanisms, molecular pathways and therapeutic opportunities. Molecular aspects of medicine. 2019 Feb 1;65:70-99
  24. 24. Frangogiannis NG, Kovacic JC. Extracellular Matrix in Ischemic Heart Disease, Part 4/4: JACC Focus Seminar. Journal of the American College of Cardiology. 2020 May 5;75(17):2219-35
  25. 25. Daseke II MJ, Tenkorang MA, Chalise U, Konfrst SR, Lindsey ML. Cardiac Fibroblast Activation during Myocardial Infarction Wound Healing: Fibroblast polarization after MI. Matrix Biology. 2020 May 21
  26. 26. Delaunay M, Osman H, Kaiser S, Diviani D. The Role of Cyclic AMP Signaling in Cardiac Fibrosis. Cells. 2020 Jan;9(1):69
  27. 27. González A, López B, Ravassa S, San José G, Díez J. Reprint of “The complex dynamics of myocardial interstitial fibrosis in heart failure. Focus on collagen cross-linking”. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2020 Mar 1;1867(3):118521
  28. 28. Kingma JG. Myocardial Infarction: Perspectives on Cardiac Regeneration and Cardiac Remote Conditioning Interventions to Limit Cellular Injury. World Journal of Cardiovascular Diseases. 2020 Apr 22;10(04):188
  29. 29. Wang E, He X, Zeng M. The role of S1P and the related signaling pathway in the development of tissue fibrosis. Frontiers in Pharmacology. 2019 Jan 8;9:1504
  30. 30. Ceccato TL, Starbuck RB, Hall JK, Walker CJ, Brown TE, Killgore JP, Anseth KS, Leinwand LA. Defining the Cardiac Fibroblast Secretome in a Fibrotic Microenvironment. Journal of the American Heart Association. 2020 Sep 12:e017025
  31. 31. Humeres C, Frangogiannis NG. Fibroblasts in the infarcted, remodeling, and failing heart. JACC: Basic to Translational Science. 2019 Jun 1;4(3):449-67
  32. 32. Nwabuo CC, Vasan RS. Pathophysiology of hypertensive heart disease: beyond left ventricular hypertrophy. Current Hypertension Reports. 2020 Feb 1;22(2):11
  33. 33. Wingard MC, Frasier CR, Singh M, Singh K. Heart failure and diabetes: role of ATM. Current Opinion in Pharmacology. 2020 Oct 1;54:27-35
  34. 34. Angelini A, Trial J, Ortiz-Urbina J, Cieslik KA. Mechanosensing dysregulation in the fibroblast: a hallmark of the aging heart. Ageing Research Reviews. 2020 Aug 23:101150
  35. 35. DeLeon-Pennell KY, Barker TH, Lindsey ML. Fibroblasts: The arbiters of extracellular matrix remodeling. Matrix Biology. 2020 Jun 3
  36. 36. Cojan-Minzat BO, Zlibut A, Agoston-Coldea L. Non-ischemic dilated cardiomyopathy and cardiac fibrosis. Heart Failure Reviews. 2020 Mar 13:1-21
  37. 37. Czubryt MP. Cardiac Fibroblast to Myofibroblast Phenotype Conversion—An Unexploited Therapeutic Target. Journal of cardiovascular development and disease. 2019 Sep;6(3):28
  38. 38. Frangogiannis NG. Transforming growth factor–β in tissue fibrosis. Journal of Experimental Medicine. 2020 Mar 2;217(3)
  39. 39. Hanna A, Frangogiannis NG. The role of the TGF-beta superfamily in myocardial infarction. Frontiers in cardiovascular medicine. 2019;6:140
  40. 40. Hortells L, Johansen AK, Yutzey KE. Cardiac Fibroblasts and the Extracellular Matrix in Regenerative and Nonregenerative Hearts. Journal of cardiovascular development and disease. 2019 Sep;6(3):29
  41. 41. Balbi C, Costa A, Barile L, Bollini S. Message in a bottle: upgrading cardiac repair into rejuvenation. Cells. 2020 Mar;9(3):724
  42. 42. Broughton KM, Sussman MA. Adult cardiomyocyte cell cycle detour: off-ramp to quiescent destinations. Trends in Endocrinology & Metabolism. 2019 Aug 1;30(8):557-67
  43. 43. Pagano F, Picchio V, Chimenti I, Sordano A, De Falco E, Peruzzi M, Miraldi F, Cavarretta E, Zoccai GB, Sciarretta S, Frati G. On the Road to Regeneration:“Tools” and “Routes” Towards Efficient Cardiac Cell Therapy for Ischemic Cardiomyopathy. Current cardiology reports. 2019 Nov 1;21(11):133
  44. 44. Dronkers E, Wauters MM, Goumans MJ, Smits AM. Epicardial TGFβ and BMP Signaling in Cardiac Regeneration: What Lesson Can We Learn from the Developing Heart?. Biomolecules. 2020 Mar;10(3):404
  45. 45. Marchianò S, Bertero A, Murry CE. Learn from your elders: developmental biology lessons to guide maturation of stem cell-derived cardiomyocytes. Pediatric Cardiology. 2019 Oct 1;40(7):1367-87
  46. 46. Leitolis A, Robert AW, Pereira IT, Correa A, Stimamiglio MA. Cardiomyogenesis modeling using pluripotent stem cells: the role of microenvironmental signaling. Frontiers in cell and developmental biology. 2019;7:164
  47. 47. Guo Y, Pu WT. Cardiomyocyte maturation: new phase in development. Circulation Research. 2020 Apr 10;126(8):1086-106
  48. 48. Israeli Y, Gabalski M, Ball K, Wasserman A, Zou J, Ni G, Zhou C, Aguirre A. Generation of Heart Organoids Modeling Early Human Cardiac Development Under Defined Conditions. Available at SSRN 3654622. 2020 Jan 1
  49. 49. Abdellatif M, Sedej S, Carmona-Gutierrez D, Madeo F, Kroemer G. Autophagy in cardiovascular aging. Circulation research. 2018 Sep 14;123(7):803-24
  50. 50. Abdellatif M, Ljubojevic-Holzer S, Madeo F, Sedej S. Autophagy in cardiovascular health and disease. Prog Mol Biol Transl Sci. 2020 May 12;172:87-106
  51. 51. Islas-Carbajal MC, Rincón-Sánchez AR, Nava-Valdivia CA, Charles-Niño CL. The Importance of Autophagy and Proteostasis in Metabolic Cardiomyopathy. InCardiovascular Risk Factors in Pathology 2020 Jun 18. IntechOpen
  52. 52. Packer M. Autophagy stimulation and intracellular sodium reduction as mediators of the cardioprotective effect of sodium–glucose cotransporter 2 inhibitors. European Journal of Heart Failure. 2020 Apr;22(4):618-28
  53. 53. Li Y, Liu R, Wu J, Li X. Self-eating: friend or foe? The emerging role of autophagy in fibrotic diseases. Theranostics. 2020;10(18):7993
  54. 54. Zhao J, Qi YF, Yu YR. STAT3, a key regulator in liver fibrosis. Annals of Hepatology. 2020 Jul 21
  55. 55. Grandjean JM, Madhavan A, Cech L, Seguinot BO, Paxman RJ, Smith E, Scampavia L, Powers ET, Cooley CB, Plate L, Spicer TP. Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming. Nature Chemical Biology. 2020 Jul 20:1-0
  56. 56. Chareyron I, Wall C, Thevenet J, Santo-Domingo J, Wiederkehr A. Cellular stress is a prerequisite for glucose-induced mitochondrial matrix alkalinization in pancreatic β-cells. Molecular and cellular endocrinology. 2019 Feb 5;481:71-83
  57. 57. Belmadani S, Matrougui K. Broken heart: A matter of the endoplasmic reticulum stress bad management?. World journal of cardiology. 2019 Jun 26;11(6):159
  58. 58. Quiles JM, Gustafsson ÅB. Mitochondrial Quality Control and Cellular Proteostasis: Two Sides of the Same Coin. Frontiers in Physiology. 2020;11
  59. 59. Ajoolabady A, Aslkhodapasandhokmabad H, Aghanejad A, Zhang Y, Ren J. Mitophagy Receptors and Mediators: Therapeutic Targets in the Management of Cardiovascular Ageing. Ageing Research Reviews. 2020 Jul 22:101129
  60. 60. Rogers RG, Ciullo A, Marbán E, Ibrahim AG. Extracellular Vesicles as Therapeutic Agents for Cardiac Fibrosis. Frontiers in Physiology. 2020;11:479
  61. 61. Zhu B, Zhang L, Liang C, Liu B, Pan X, Wang Y, Zhang Y, Zhang Y, Xie W, Yan B, Liu F. Stem cell-derived exosomes prevent aging-induced cardiac dysfunction through a novel exosome/lncRNA MALAT1/NF-κB/TNF-α signaling pathway. Oxidative Medicine and Cellular Longevity. 2019 Apr 8;2019
  62. 62. Svaguša T, Martinić M, Martinić M, Kovačević L, Šepac A, Miličić D, Bulum J, Starčević B, Sirotković-Skerlev M, Seiwerth F, Kulić A. Mitochondrial unfolded protein response, mitophagy and other mitochondrial quality control mechanisms in heart disease and aged heart. Croatian Medical Journal. 2020 Apr;61(2):126
  63. 63. Lin R, Kerkelä R. Regulatory Mechanisms of Mitochondrial Function and Cardiac Aging. International Journal of Molecular Sciences. 2020 Jan;21(4):1359
  64. 64. Lyra-Leite DM, Andres AM, Cho N, Petersen AP, Ariyasinghe NR, Kim SS, Gottlieb RA, McCain ML. Matrix-guided control of mitochondrial function in cardiac myocytes. Acta biomaterialia. 2019 Oct 1;97:281-95
  65. 65. Hickey KL, Dickson K, Cogan JZ, Replogle JM, Schoof M, D’Orazio KN, Sinha NK, Hussmann JA, Jost M, Frost A, Green R. GIGYF2 and 4ehp inhibit translation initiation of defective messenger RNAs to assist Ribosome-Associated quality control. Molecular cell. 2020 Jul 28
  66. 66. Witte F, Ruiz-Orera J, Mattioli CC, Blachut S, Adami E, Schulz JF, Schneider-Lunitz V, Hummel O, Patone G, Mücke MB, Šilhavý J. Trans control of cardiac mRNA translation in a protein length-dependent fashion. bioRxiv. 2020 Jan 1
  67. 67. Wu J, Subbaiah KC, Xie LH, Jiang F, Khor ES, Mickelsen D, Myers JR, Tang WH, Yao P. Glutamyl-Prolyl-tRNA Synthetase Regulates Proline-Rich Pro-Fibrotic Protein Synthesis During Cardiac Fibrosis. Circulation Research. 2020 Aug 28;127(6):827-46
  68. 68. Zheng M, Jacob J, Hung SH, Wang J. The Hippo Pathway in Cardiac Regeneration and Homeostasis: New Perspectives for Cell-Free Therapy in the Injured Heart. Biomolecules. 2020 Jul;10(7):1024
  69. 69. Land WG. Use of DAMPs and SAMPs as Therapeutic Targets or Therapeutics: A Note of Caution. Molecular Diagnosis & Therapy. 2020 Apr 4:1
  70. 70. Seclì L, Sorge M, Morotti A, Brancaccio M. Blocking Extracellular Chaperones to Improve Cardiac Regeneration. Frontiers in Bioengineering and Biotechnology. 2020 May 26;8:411
  71. 71. Takahashi T, Shishido T, Kinoshita D, Watanabe K, Toshima T, Sugai T, Narumi T, Otaki Y, Tamura H, Nishiyama S, Arimoto T. Cardiac nuclear high-mobility group box 1 ameliorates pathological cardiac hypertrophy by inhibiting DNA damage response. JACC: Basic to Translational Science. 2019 Apr 29;4(2):234-47
  72. 72. Liu FY, Fan D, Yang Z, Tang N, Guo Z, Ma SQ, Ma ZG, Wu HM, Deng W, Tang QZ. TLR9 is essential for HMGB1-mediated post-myocardial infarction tissue repair through affecting apoptosis, cardiac healing, and angiogenesis. Cell death & disease. 2019 Jun 17;10(7):1-6
  73. 73. Lai SL, Marín-Juez R, Stainier DY. Immune responses in cardiac repair and regeneration: a comparative point of view. Cellular and Molecular Life Sciences. 2019 Apr 15;76(7):1365-80
  74. 74. Frangogiannis NG. Cardiac fibrosis: cell biological mechanisms, molecular pathways and therapeutic opportunities. Molecular aspects of medicine. 2019 Feb 1;65:70-99
  75. 75. Deniset JF, Belke D, Lee WY, Jorch SK, Deppermann C, Hassanabad AF, Turnbull JD, Teng G, Rozich I, Hudspeth K, Kanno Y. Gata6+ pericardial cavity macrophages relocate to the injured heart and prevent cardiac fibrosis. Immunity. 2019 Jul 16;51(1):131-40
  76. 76. Liao X, Shen Y, Zhang R, Sugi K, Vasudevan NT, Alaiti MA, Sweet DR, Zhou L, Qing Y, Gerson SL, Fu C. Distinct roles of resident and nonresident macrophages in nonischemic cardiomyopathy. Proceedings of the National Academy of Sciences. 2018 May 15;115(20):E4661-9
  77. 77. Ma Y, Mouton AJ, Lindsey ML. Cardiac macrophage biology in the steady-state heart, the aging heart, and following myocardial infarction. Translational Research. 2018 Jan 1;191:15-28
  78. 78. Ngkelo A, Richart A, Kirk JA, Bonnin P, Vilar J, Lemitre M, Marck P, Branchereau M, Le Gall S, Renault N, Guerin C. Mast cells regulate myofilament calcium sensitization and heart function after myocardial infarction. Journal of Experimental Medicine. 2016 Jun 27;213(7):1353-74
  79. 79. Legere SA, Haidl ID, Légaré JF, Marshall JS. Mast cells in cardiac fibrosis: new insights suggest opportunities for intervention. Frontiers in immunology. 2019 Mar 28;10:580
  80. 80. Liao CH, Akazawa H, Tamagawa M, Ito K, Yasuda N, Kudo Y, Yamamoto R, Ozasa Y, Fujimoto M, Wang P, Nakauchi H. Cardiac mast cells cause atrial fibrillation through PDGF-A–mediated fibrosis in pressure-overloaded mouse hearts. The Journal of clinical investigation. 2010 Jan 4;120(1):242-53
  81. 81. Wernersson S, Pejler G. Mast cell secretory granules: armed for battle. Nature Reviews Immunology. 2014 Jul;14(7):478-94
  82. 82. Krishnamurthy P, Rajasingh J, Lambers E, Qin G, Losordo DW, Kishore R. IL-10 inhibits inflammation and attenuates left ventricular remodeling after myocardial infarction via activation of STAT3 and suppression of HuR. Circulation research. 2009 Jan 30;104(2):e9-18
  83. 83. Verma SK, Garikipati VN, Krishnamurthy P, Schumacher SM, Grisanti LA, Cimini M, Cheng Z, Khan M, Yue Y, Benedict C, Truongcao MM. Interleukin-10 inhibits bone marrow fibroblast progenitor cell–mediated cardiac fibrosis in pressure-overloaded myocardium. Circulation. 2017 Sep 5;136(10):940-53
  84. 84. Anzai A, Anzai T, Nagai S, Maekawa Y, Naito K, Kaneko H, Sugano Y, Takahashi T, Abe H, Mochizuki S, Sano M. Regulatory role of dendritic cells in postinfarction healing and left ventricular remodeling. Circulation. 2012 Mar 13;125(10):1234-45
  85. 85. Clemente-Casares X, Hosseinzadeh S, Barbu I, Dick SA, Macklin JA, Wang Y, Momen A, Kantores C, Aronoff L, Farno M, Lucas TM. A CD103+ conventional dendritic cell surveillance system prevents development of overt heart failure during subclinical viral myocarditis. Immunity. 2017 Nov 21;47(5):974-89
  86. 86. Bansal SS, Ismahil MA, Goel M, Patel B, Hamid T, Rokosh G, Prabhu SD. Activated T lymphocytes are essential drivers of pathological remodeling in ischemic heart failure. Circulation: Heart Failure. 2017 Mar;10(3):e003688
  87. 87. Borg N, Alter C, Görldt N, Jacoby C, Ding Z, Steckel B, Quast C, Bönner F, Friebe D, Temme S, Flögel U. CD73 on T cells orchestrates cardiac wound healing after myocardial infarction by purinergic metabolic reprogramming. Circulation. 2017 Jul 18;136(3):297-313
  88. 88. Cordero-Reyes AM, Youker KA, Trevino AR, Celis R, Hamilton DJ, Flores-Arredondo JH, Orrego CM, Bhimaraj A, Estep JD, Torre-Amione G. Full expression of cardiomyopathy is partly dependent on B-cells: a pathway that involves cytokine activation, immunoglobulin deposition, and activation of apoptosis. Journal of the American Heart Association. 2016 Jan 14;5(1):e002484
  89. 89. Frangou E, Vassilopoulos D, Boletis J, Boumpas DT. An emerging role of neutrophils and NETosis in chronic inflammation and fibrosis in systemic lupus erythematosus (SLE) and ANCA-associated vasculitides (AAV): implications for the pathogenesis and treatment. Autoimmunity reviews. 2019 Aug 1;18(8):751-60
  90. 90. Mutua V, Gershwin LJ. A Review of Neutrophil Extracellular Traps (NETs) in Disease: Potential Anti-NETs Therapeutics. Clinical Reviews in Allergy & Immunology. 2020 Aug 1:1-8
  91. 91. Sollberger G, Tilley DO, Zychlinsky A. Neutrophil extracellular traps: the biology of chromatin externalization. Developmental cell. 2018 Mar 12;44(5):542-53
  92. 92. Neubert E, Meyer D, Kruss S, Erpenbeck L. The power from within–understanding the driving forces of neutrophil extracellular trap formation. Journal of Cell Science. 2020 Mar 1;133(5)
  93. 93. Németh T, Sperandio M, Mócsai A. Neutrophils as emerging therapeutic targets. Nature Reviews Drug Discovery. 2020 Jan 22:1-23
  94. 94. Becker RC, Owens AP, Sadayappan S. Tissue-level inflammation and ventricular remodeling in hypertrophic cardiomyopathy. Journal of thrombosis and thrombolysis. 2020 Feb 1;49(2):177-83
  95. 95. Fousert E, Toes R, Desai J. Neutrophil Extracellular Traps (NETs) Take the Central Stage in Driving Autoimmune Responses. Cells. 2020 Apr;9(4):915
  96. 96. Frangou E, Vassilopoulos D, Boletis J, Boumpas DT. An emerging role of neutrophils and NETosis in chronic inflammation and fibrosis in systemic lupus erythematosus (SLE) and ANCA-associated vasculitides (AAV): implications for the pathogenesis and treatment. Autoimmunity reviews. 2019 Aug 1;18(8):751-60
  97. 97. Stakos D, Skendros P, Konstantinides S, Ritis K. Traps N'Clots: NET-Mediated Thrombosis and Related Diseases. Thrombosis and haemostasis. 2020 Mar;120(03):373-83
  98. 98. Koitabashi N, Danner T, Zaiman AL, Pinto YM, Rowell J, Mankowski J, Zhang D, Nakamura T, Takimoto E, Kass DA. Pivotal role of cardiomyocyte TGF-β signaling in the murine pathological response to sustained pressure overload. The Journal of clinical investigation. 2011 Jun 1;121(6):2301-12
  99. 99. Ock S, Lee WS, Ahn J, Kim HM, Kang H, Kim HS, Jo D, Abel ED, Lee TJ, Kim J. Deletion of IGF-1 receptors in cardiomyocytes attenuates cardiac aging in male mice. Endocrinology. 2016 Jan 1;157(1):336-45
  100. 100. Adiarto S, Heiden S, Vignon-Zellweger N, Nakayama K, Yagi K, Yanagisawa M, Emoto N. ET-1 from endothelial cells is required for complete angiotensin II-induced cardiac fibrosis and hypertrophy. Life sciences. 2012 Oct 15;91(13-14):651-7
  101. 101. Aisagbonhi O, Rai M, Ryzhov S, Atria N, Feoktistov I, Hatzopoulos AK. Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition. Disease models & mechanisms. 2011 Jul 1;4(4):469-83
  102. 102. Wei H, Bedja D, Koitabashi N, Xing D, Chen J, Fox-Talbot K, Rouf R, Chen S, Steenbergen C, Harmon JW, Dietz HC. Endothelial expression of hypoxia-inducible factor 1 protects the murine heart and aorta from pressure overload by suppression of TGF-β signaling. Proceedings of the National Academy of Sciences. 2012 Apr 3;109(14):E841-50
  103. 103. Frangogiannis NG, Mendoza LH, Lewallen M, Michael LH, Smith CW, Entman ML. Induction and suppression of interferon-inducible protein (IP)-10 in reperfused myocardial infarcts may regulate angiogenesis. The FASEB Journal. 2001 Jun;15(8):1428-30
  104. 104. Bujak M, Dobaczewski M, Gonzalez-Quesada C, Xia Y, Leucker T, Zymek P, Veeranna V, Tager AM, Luster AD, Frangogiannis NG. Induction of the CXC chemokine interferon-γ-inducible protein 10 regulates the reparative response following myocardial infarction. Circulation research. 2009 Nov 6;105(10):973-83
  105. 105. Everett BM, MacFadyen JG, Thuren T, Libby P, Glynn RJ, Ridker PM. Inhibition of interleukin-1β and reduction in atherothrombotic cardiovascular Events in the CANTOS trial. Journal of the American College of Cardiology. 2020 Oct 6
  106. 106. Adamo L, Rocha-Resende C, Prabhu SD, Mann DL. Reappraising the role of inflammation in heart failure. Nature Reviews Cardiology. 2020 Jan 22:1-7
  107. 107. Okyere AD, Tilley DG. Leukocyte-Dependent Regulation of Cardiac Fibrosis. Frontiers in Physiology. 2020 Apr 8;11:301
  108. 108. Aghajanian H, Kimura T, Rurik JG, Hancock AS, Leibowitz MS, Li L, Scholler J, Monslow J, Lo A, Han W, Wang T. Targeting cardiac fibrosis with engineered T cells. Nature. 2019 Sep;573(7774):430-3
  109. 109. Vagnozzi RJ, Johansen AK, Molkentin JD. CARdiac Immunotherapy: T Cells Engineered to Treat the Fibrotic Heart. Molecular Therapy. 2019 Nov 6;27(11):1869-71
  110. 110. Nicolás-Ávila JA, Lechuga-Vieco AV, Esteban-Martínez L, Sánchez-Díaz M, Díaz-García E, Santiago DJ, Rubio-Ponce A, Li JL, Balachander A, Quintana JA, Martínez-de-Mena R. A network of macrophages supports mitochondrial homeostasis in the heart. Cell. 2020 Sep 15
  111. 111. Corden B, Lim WW, Song W, Chen X, Ko NS, Su L, Tee NG, Adami E, Schafer S, Cook SA. Therapeutic Targeting of Interleukin-11 Signalling Reduces Pressure Overload–Induced Cardiac Fibrosis in Mice. Journal of Cardiovascular Translational Research. 2020 Jun 26:1-7
  112. 112. Hao K, Lei W, Wu H, Wu J, Yang Z, Yan S, Lu XA, Li J, Xia X, Han X, Deng W. LncRNA-Safe contributes to cardiac fibrosis through Safe-Sfrp2-HuR complex in mouse myocardial infarction. Theranostics. 2019;9(24):7282
  113. 113. Govindappa PK, Patil M, Garikipati VN, Verma SK, Saheera S, Narasimhan G, Zhu W, Kishore R, Zhang J, Krishnamurthy P. Targeting exosome-associated human antigen R attenuates fibrosis and inflammation in diabetic heart. The FASEB Journal. 2020 Feb;34(2):2238-51
  114. 114. Chothani S, Schäfer S, Adami E, Viswanathan S, Widjaja AA, Langley SR, Tan J, Wang M, Quaife NM, Jian Pua C, D’Agostino G. Widespread translational control of fibrosis in the human heart by RNA-binding proteins. Circulation. 2019 Sep 10;140(11):937-51
  115. 115. Cao Y, Liu C, Wang Q, Wang W, Tao E, Wan L. Pum2 mediates Sirt1 mRNA decay and exacerbates hypoxia/reoxygenation-induced cardiomyocyte apoptosis. Experimental Cell Research. 2020 May 8:112058
  116. 116. Ji X, Ding W, Xu T, Zheng X, Zhang J, Liu M, Liu G, Wang J. MicroRNA-31-5p attenuates doxorubicin-induced cardiotoxicity via quaking and circular RNA Pan3. Journal of Molecular and Cellular Cardiology. 2020 Mar 3
  117. 117. Yan F, Liu R, Zhuang X, Li R, Shi H, Gao X. Salidroside Attenuates Doxorubicin-Induced Cardiac Dysfunction Partially Through Activation of QKI/FoxO1 Pathway. Journal of Cardiovascular Translational Research. 2020 Jul 16:1-0
  118. 118. Xia W, Zou C, Chen H, Xie C, Hou M. Immune checkpoint inhibitor induces cardiac injury through polarizing macrophages via modulating microRNA-34a/Kruppel-like factor 4 signaling. Cell death & disease. 2020 Jul 24;11(7):1-5
  119. 119. Gao C, Wang Y. mRNA Metabolism in Cardiac Development and Disease: Life After Transcription. Physiological Reviews. 2020 Apr 1;100(2):673-94
  120. 120. Rogers RG, Ciullo A, Marbán E, Ibrahim AG. Extracellular Vesicles as Therapeutic Agents for Cardiac Fibrosis. Frontiers in Physiology. 2020;11:479
  121. 121. Leonard RJ, Preston CC, Gucwa ME, Afeworki Y, Selya AS, Faustino RS. Protein subdomain enrichment of NUP155 variants identify a novel predicted pathogenic hotspot. Frontiers in cardiovascular medicine. 2020 Feb 7;7:8
  122. 122. Mancuso T, Barone A, Salatino A, Molinaro C, Marino F, Scalise M, Torella M, De Angelis A, Urbanek K, Torella D, Cianflone E. Unravelling the Biology of Adult Cardiac Stem Cell-Derived Exosomes to Foster Endogenous Cardiac Regeneration and Repair. International Journal of Molecular Sciences. 2020 Jan;21(10):3725
  123. 123. Hocine HR, Brunel S, Chen Q, Giustiniani J, San Roman MJ, Ferrat YJ, Palacios I, de la Rosa O, Lombardo E, Bensussan A, Charron D. Extracellular vesicles released by allogeneic human cardiac stem/progenitor cells as part of their therapeutic benefit. Stem cells translational medicine. 2019 Sep;8(9):911-24
  124. 124. Liang B, He X, Zhao YX, Zhang XX, Gu N. Advances in Exosomes Derived from Different Cell Sources and Cardiovascular Diseases. BioMed Research International. 2020 Jul 7;2020
  125. 125. Cai L, Chao G, Li W, Zhu J, Li F, Qi B, Wei Y, Chen S, Zhou G, Lu X, Xu J. Activated CD4+ T cells-derived exosomal miR-142-3p boosts post-ischemic ventricular remodeling by activating myofibroblast. Aging (Albany NY). 2020 Apr 30;12(8):7380
  126. 126. Shanmuganathan M, Vughs J, Noseda M, Emanueli C. Exosomes: basic biology and technological advancements suggesting their potential as ischemic heart disease therapeutics. Frontiers in Physiology. 2018 Nov 19;9:1159
  127. 127. Ranjan P, Kumari R, Verma SK. Cardiac Fibroblasts and Cardiac Fibrosis: Precise Role of Exosomes. Frontiers in Cell and Developmental Biology. 2019;7
  128. 128. Tikhomirov R, Donnell BR, Catapano F, Faggian G, Gorelik J, Martelli F, Emanueli C. Exosomes: from potential culprits to new therapeutic promise in the setting of cardiac fibrosis. Cells. 2020 Mar;9(3):592
  129. 129. Zhang Z, Wan J, Liu X, Zhang W. Strategies and technologies for exploring long noncoding RNAs in heart failure. Biomedicine & Pharmacotherapy. 2020 Nov 1;131:110572
  130. 130. Wang Y, Liu B. Circular RNA in Diseased Heart. Cells. 2020 May;9(5):1240
  131. 131. Fioravanti A, Pirtoli L, Giordano A, Dotta F. Crosstalk between MicroRNA and Oxidative Stress in Physiology and Pathology
  132. 132. Chen C, Tang Y, Sun H, Lin X, Jiang B. The roles of long noncoding RNAs in myocardial pathophysiology. Bioscience Reports. 2019 Nov 29;39(11)
  133. 133. Chen G, Huang S, Song F, Zhou Y, He X. Lnc-Ang362 is a pro-fibrotic long non-coding RNA promoting cardiac fibrosis after myocardial infarction by suppressing Smad7. Archives of Biochemistry and Biophysics. 2020 Mar 30:108354
  134. 134. Liang H, Pan Z, Zhao X, Liu L, Sun J, Su X, Xu C, Zhou Y, Zhao D, Xu B, Li X. LncRNA PFL contributes to cardiac fibrosis by acting as a competing endogenous RNA of let-7d. Theranostics. 2018;8(4):1180
  135. 135. Wawrzyniak O, Zarębska Ż, Kuczyński K, Gotz-Więckowska A, Rolle K. Protein-Related Circular RNAs in Human Pathologies. Cells. 2020 Aug;9(8):1841
  136. 136. Lin R, Rahtu-Korpela L, Magga J, Ulvila J, Swan J, Kemppi A, Pakanen L, Porvari K, Huikuri H, Junttila J, Kerkelä R. miR-1468-3p promotes aging-related cardiac fibrosis. Molecular Therapy-Nucleic Acids. 2020 Apr 8
  137. 137. Li J, Cao LT, Liu HH, Yin XD, Wang J. Long non coding RNA H19: An emerging therapeutic target in fibrosing diseases. Autoimmunity. 2020 Jan 2;53(1):1-7
  138. 138. Wang X, Morelli MB, Matarese A, Sardu C, Santulli G. Cardiomyocyte-derived exosomal microRNA-92a mediates post-ischemic myofibroblast activation both in vitro and ex vivo. ESC heart failure. 2020 Feb;7(1):285-9
  139. 139. Henning RJ. Cardiovascular exosomes and MicroRNAs in cardiovascular physiology and pathophysiology. Journal of cardiovascular translational research. 2020 Jun 25:1-8
  140. 140. Ferrari S, Pesce M. Cell-Based Mechanosensation, Epigenetics, and Non-Coding RNAs in Progression of Cardiac Fibrosis. International Journal of Molecular Sciences. 2020 Jan;21(1):28
  141. 141. Morelli MB, Shu J, Sardu C, Matarese A, Santulli G. Cardiosomal microRNAs are essential in post-infarction myofibroblast phenoconversion. International journal of molecular sciences. 2020 Jan;21(1):201
  142. 142. Guo W, Zhu C, Yin Z, Wang Q, Sun M, Cao H, Greaser ML. Splicing factor RBM20 regulates transcriptional network of titin associated and calcium handling genes in the heart. International journal of biological sciences. 2018;14(4):369
  143. 143. Picca A, Guerra F, Calvani R, Marini F, Biancolillo A, Landi G, Beli R, Landi F, Bernabei R, Bentivoglio AR, Monaco MR. Mitochondrial signatures in circulating extracellular vesicles of older adults with Parkinson’s disease: Results from the EXosomes in PArkiNson’s Disease (EXPAND) study. Journal of clinical medicine. 2020 Feb;9(2):504
  144. 144. Lin R, Rahtu-Korpela L, Magga J, Ulvila J, Swan J, Kemppi A, Pakanen L, Porvari K, Huikuri H, Junttila J, Kerkelä R. miR-1468-3p promotes aging-related cardiac fibrosis. Molecular Therapy-Nucleic Acids. 2020 Apr 8
  145. 145. Zhou Y, Richards AM, Wang P. MicroRNA-221 is cardioprotective and anti-fibrotic in a rat model of myocardial infarction. Molecular Therapy-Nucleic Acids. 2019 Sep 6;17:185-97
  146. 146. Li L, Bounds KR, Chatterjee P, Gupta S. Micro RNA-130a, a Potential Antifibrotic Target in Cardiac Fibrosis. Journal of the American Heart Association. 2017 Nov 7;6(11):e006763
  147. 147. Du W, Liang H, Gao X, Li X, Zhang Y, Pan Z, Li C, Wang Y, Liu Y, Yuan W, Ma N. MicroRNA-328, a potential anti-fibrotic target in cardiac interstitial fibrosis. Cellular Physiology and Biochemistry. 2016;39(3):827-36
  148. 148. Zhao X, Wang K, Liao Y, Zeng Q, Li Y, Hu F, Liu Y, Meng K, Qian C, Zhang Q, Guan H. MicroRNA-101a inhibits cardiac fibrosis induced by hypoxia via targeting TGFβRI on cardiac fibroblasts. Cellular Physiology and Biochemistry. 2015;35(1):213-26
  149. 149. Liu X, Xu Y, Deng Y, Li H. MicroRNA-223 regulates cardiac fibrosis after myocardial infarction by targeting RASA1. Cellular Physiology and Biochemistry. 2018;46(4):1439-54
  150. 150. Huang W, Feng Y, Liang J, Yu H, Wang C, Wang B, Wang M, Jiang L, Meng W, Cai W, Medvedovic M. Loss of microRNA-128 promotes cardiomyocyte proliferation and heart regeneration. Nature communications. 2018 Feb 16;9(1):1-6
  151. 151. Nishiga M, Horie T, Kuwabara Y, Nagao K, Baba O, Nakao T, Nishino T, Hakuno D, Nakashima Y, Nishi H, Nakazeki F. MicroRNA-33 controls adaptive fibrotic response in the remodeling heart by preserving lipid raft cholesterol. Circulation research. 2017 Mar 3;120(5):835-47
  152. 152. Yang HD, Nam SW. Pathogenic diversity of RNA variants and RNA variation-associated factors in cancer development. Experimental & Molecular Medicine. 2020 Apr 28:1-2
  153. 153. Fochi S, Lorenzi P, Galasso M, Stefani C, Trabetti E, Zipeto D, Romanelli MG. The Emerging Role of the RBM20 and PTBP1 Ribonucleoproteins in Heart Development and Cardiovascular Diseases. Genes. 2020 Apr;11(4):402
  154. 154. Ranum PT, Goodwin AT, Yoshimura H, Kolbe DL, Walls WD, Koh JY, He DZ, Smith RJ. Insights into the biology of hearing and deafness revealed by single-cell RNA sequencing. Cell reports. 2019 Mar 12;26(11):3160-71
  155. 155. Zhang H, Zeitz MJ, Wang H, Niu B, Ge S, Li W, Cui J, Wang G, Qian G, Higgins MJ, Fan X. Long noncoding RNA-mediated intrachromosomal interactions promote imprinting at the Kcnq1 locus. Journal of Cell Biology. 2014 Jan 6;204(1):61-75
  156. 156. Wallace E, Howard L, Liu M, O’Brien T, Ward D, Shen S, Prendiville T. Long QT syndrome: genetics and future perspective. Pediatric cardiology. 2019 Aug 22:1-2
  157. 157. Lee Y, Rio DC. Mechanisms and regulation of alternative pre-mRNA splicing. Annual review of biochemistry. 2015 Jun 2;84:291-323
  158. 158. Obeng EA, Stewart C, Abdel-Wahab O. Altered RNA processing in cancer pathogenesis and therapy. Cancer discovery. 2019 Nov 1;9(11):1493-510
  159. 159. Shenasa H, Hertel KJ. Combinatorial regulation of alternative splicing. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms. 2019 Nov 1;1862(11-12):194392
  160. 160. Ule J, Blencowe BJ. Alternative splicing regulatory networks: functions, mechanisms, and evolution. Molecular cell. 2019 Oct 17;76(2):329-45
  161. 161. Gerull B, Atherton J, Geupel A, Sasse-Klaassen S, Heuser A, Frenneaux M, McNabb M, Granzier H, Labeit S, Thierfelder L. Identification of a novel frameshift mutation in the giant muscle filament titin in a large Australian family with dilated cardiomyopathy. Journal of molecular medicine. 2006 Jun 1;84(6):478-83
  162. 162. Cheng H, Zheng M, Peter AK, Kimura K, Li X, Ouyang K, Shen T, Cui L, Frank D, Dalton ND, Gu Y. Selective deletion of long but not short Cypher isoforms leads to late-onset dilated cardiomyopathy. Human molecular genetics. 2011 May 1;20(9):1751-62
  163. 163. Tang ZZ, Sharma S, Zheng S, Chawla G, Nikolic J, Black DL. Regulation of the mutually exclusive exons 8a and 8 in the CaV1. 2 calcium channel transcript by polypyrimidine tract-binding protein. Journal of Biological Chemistry. 2011 Mar 25;286(12):10007-16
  164. 164. Makarenko I, Opitz CA, Leake MC, Neagoe C, Kulke M, Gwathmey JK, Del Monte F, Hajjar RJ, Linke WA. Passive stiffness changes caused by upregulation of compliant titin isoforms in human dilated cardiomyopathy hearts. Circulation research. 2004 Oct 1;95(7):708-16
  165. 165. Lahmers S, Wu Y, Call DR, Labeit S, Granzier H. Developmental control of titin isoform expression and passive stiffness in fetal and neonatal myocardium. Circulation research. 2004 Mar 5;94(4):505-13
  166. 166. Opitz CA, Linke WA. Plasticity of cardiac titin/connectin in heart development. Journal of Muscle Research & Cell Motility. 2005 Dec 1;26(6-8):333-42
  167. 167. Warren CM, Krzesinski PR, Campbell KS, Moss RL, Greaser ML. Titin isoform changes in rat myocardium during development. Mechanisms of development. 2004 Nov 1;121(11):1301-12
  168. 168. Yamasaki R, Wu Y, McNabb M, Greaser M, Labeit S, Granzier H. Protein kinase A phosphorylates titin’s cardiac-specific N2B domain and reduces passive tension in rat cardiac myocytes. Circulation research. 2002 Jun 14;90(11):1181-8
  169. 169. Cazorla O, Freiburg A, Helmes M, Centner T, McNabb M, Wu Y, Trombitas K, Labeit S, Granzier H. Differential expression of cardiac titin isoforms and modulation of cellular stiffness. Circulation research. 2000 Jan 7;86(1):59-67
  170. 170. Itoh-Satoh M, Hayashi T, Nishi H, Koga Y, Arimura T, Koyanagi T, Takahashi M, Hohda S, Ueda K, Nouchi T, Hiroe M. Titin mutations as the molecular basis for dilated cardiomyopathy. Biochemical and biophysical research communications. 2002 Feb 22;291(2):385-93
  171. 171. Gerull B, Gramlich M, Atherton J, McNabb M, Trombitás K, Sasse-Klaassen S, Seidman JG, Seidman C, Granzier H, Labeit S, Frenneaux M. Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy. Nature genetics. 2002 Feb;30(2):201-4
  172. 172. Neagoe C, Kulke M, del Monte F, Gwathmey JK, de Tombe PP, Hajjar RJ, Linke WA. Titin isoform switch in ischemic human heart disease. Circulation. 2002 Sep 10;106(11):1333-41
  173. 173. Williams L, Howell N, Pagano D, Andreka P, Vertesaljai M, Pecor T, Frenneaux M, Granzier H. Titin isoform expression in aortic stenosis. Clinical Science. 2009 Sep 1;117(6):237-42
  174. 174. Chaturvedi RR, Herron T, Simmons R, Shore D, Kumar P, Sethia B, Chua F, Vassiliadis E, Kentish JC. Passive stiffness of myocardium from congenital heart disease and implications for diastole. Circulation. 2010 Mar 2;121(8):979
  175. 175. Wu Y, Peng J, Campbell KB, Labeit S, Granzier H. Hypothyroidism leads to increased collagen-based stiffness and re-expression of large cardiac titin isoforms with high compliance. Journal of molecular and cellular cardiology. 2007 Jan 1;42(1):186-95
  176. 176. Krüger M, Sachse C, Zimmermann WH, Eschenhagen T, Klede S, Linke WA. Thyroid hormone regulates developmental titin isoform transitions via the phosphatidylinositol-3-kinase/AKT pathway. Circulation research. 2008 Feb 29;102(4):439-47
  177. 177. Klein I, Ojamaa K. Thyroid hormone and the cardiovascular system. New England Journal of Medicine. 2001 Feb 15;344(7):501-9
  178. 178. Krüger M, Babicz K, von Frieling-Salewsky M, Linke WA. Insulin signaling regulates cardiac titin properties in heart development and diabetic cardiomyopathy. Journal of molecular and cellular cardiology. 2010 May 1;48(5):910-6
  179. 179. Guo W, Schafer S, Greaser ML, Radke MH, Liss M, Govindarajan T, Maatz H, Schulz H, Li S, Parrish AM, Dauksaite V. RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing. Nature medicine. 2012 May;18(5):766-73
  180. 180. Ma G, Samad I, Motz K, et al. Metabolic variations in normal and fibrotic human laryngotracheal-derived fibroblasts: a Warburg-like effect. Laryngoscope 2017;127:E107-13
  181. 181. Weeland CJ, van den Hoogenhof MM, Beqqali A, Creemers EE. Insights into alternative splicing of sarcomeric genes in the heart. Journal of molecular and cellular cardiology. 2015 Apr 1;81:107-13
  182. 182. Rexiati M, Sun M, Guo W. Muscle-specific Mis-splicing and heart disease exemplified by RBM20. Genes. 2018 Jan;9(1):18
  183. 183. Brauch KM, Karst ML, Herron KJ, De Andrade M, Pellikka PA, Rodeheffer RJ, Michels VV, Olson TM. Mutations in ribonucleic acid binding protein gene cause familial dilated cardiomyopathy. Journal of the American College of Cardiology. 2009 Sep 1;54(10):930-41
  184. 184. Dauksaite V, Gotthardt M. Molecular basis of titin exon exclusion by RBM20 and the novel titin splice regulator PTB4. Nucleic acids research. 2018 Jun 1;46(10):5227-38
  185. 185. Watanabe T, Kimura A, Kuroyanagi H. Alternative splicing regulator RBM20 and cardiomyopathy. Frontiers in molecular biosciences. 2018 Nov 28;5:105
  186. 186. Morinaga A, Ito J, Niimi T, Maturana AD. RBM20 regulates CaV1. 2 surface expression by promoting exon 9* Inclusion of CACNA1C in neonatal rat cardiomyocytes. International journal of molecular sciences. 2019 Jan;20(22):5591
  187. 187. Cai H, Zhu C, Chen Z, Maimaiti R, Sun M, McCormick RJ, Lan X, Chen H, Guo W. Angiotensin ii influences pre-mRNA splicing regulation by enhancing RBM20 transcription through activation of the MAPK/ELK1 signaling pathway. International Journal of Molecular Sciences. 2019 Jan;20(20):5059
  188. 188. Briganti F, Sun H, Wei W, Wu J, Zhu C, Liss M, Karakikes I, Rego S, Cipriano A, Snyder M, Meder B. iPSC modeling of RBM20-deficient DCM identifies upregulation of RBM20 as a therapeutic strategy. Cell Reports. 2020 Sep 8;32(10):108117
  189. 189. Upadhyay SK, Mackereth CD. Structural basis of UCUU RNA motif recognition by splicing factor RBM20. Nucleic acids research. 2020 May 7;48(8):4538-50
  190. 190. Lennermann D, Backs J, van den Hoogenhof MM. New Insights in RBM20 Cardiomyopathy. Current Heart Failure Reports. 2020 Aug 13:1-3
  191. 191. Filippello A, Lorenzi P, Bergamo E, Romanelli MG. Identification of nuclear retention domains in the RBM20 protein. FEBS letters. 2013 Sep 17;587(18):2989-95
  192. 192. Murayama R, Kimura-Asami M, Togo-Ohno M, Yamasaki-Kato Y, Naruse TK, Yamamoto T, Hayashi T, Ai T, Spoonamore KG, Kovacs RJ, Vatta M. Phosphorylation of the RSRSP stretch is critical for splicing regulation by RNA-Binding Motif Protein 20 (RBM20) through nuclear localization. Scientific reports. 2018 Jun 12;8(1):1-4
  193. 193. Refaat MM, Lubitz SA, Makino S, Islam Z, Frangiskakis JM, Mehdi H, Gutmann R, Zhang ML, Bloom HL, MacRae CA, Dudley SC. Genetic variation in the alternative splicing regulator RBM20 is associated with dilated cardiomyopathy. Heart Rhythm. 2012 Mar 1;9(3):390-6
  194. 194. Li D, Morales A, Gonzalez-Quintana J, Norton N, Siegfried JD, Hofmeyer M, Hershberger RE. Identification of novel mutations in RBM20 in patients with dilated cardiomyopathy. Clinical and translational science. 2010 Jun;3(3):90-7
  195. 195. Wells QS, Becker JR, Su YR, Mosley JD, Weeke P, D’Aoust L, Ausborn NL, Ramirez AH, Pfotenhauer JP, Naftilan AJ, Markham L. Whole exome sequencing identifies a causal RBM20 mutation in a large pedigree with familial dilated cardiomyopathy. Circulation: Cardiovascular Genetics. 2013 Aug;6(4):317-26
  196. 196. Hey TM, Rasmussen TB, Madsen T, Aagaard MM, Harbo M, Mølgaard H, Møller JE, Eiskjær H, Mogensen J. Pathogenic RBM20-variants are associated with a severe disease expression in male patients with dilated cardiomyopathy. Circulation: Heart Failure. 2019 Mar;12(3):e005700
  197. 197. Robyns T, Willems R, Van Cleemput J, Jhangiani S, Muzny D, Gibbs R, Lupski JR, Breckpot J, Devriendt K, Corveleyn A. Whole exome sequencing in a large pedigree with DCM identifies a novel mutation in RBM20. Acta Cardiologica. 2019 Oct 3:1-6
  198. 198. Monaco I, Santacroce R, Casavecchia G, Correale M, Bottigliero D, Cordisco G, Leccese A, Di Biase M, Margaglione M, Brunetti ND. Double de novo mutations in dilated cardiomyopathy with cardiac arrest. Journal of Electrocardiology. 2019 Mar 1;53:40-3
  199. 199. Pantou MP, Gourzi P, Gkouziouta A, Tsiapras D, Zygouri C, Constantoulakis P, Adamopoulos S, Degiannis D. Phenotypic heterogeneity within members of a family carrying the same RBM20 mutation R634W. Cardiology. 2018;141(3):150-5
  200. 200. Sedaghat-Hamedani F, Haas J, Zhu F, Geier C, Kayvanpour E, Liss M, Lai A, Frese K, Pribe-Wolferts R, Amr A, Li DT. Clinical genetics and outcome of left ventricular non-compaction cardiomyopathy. European heart journal. 2017 Dec 7;38(46):3449-60
  201. 201. Long PA, Theis JL, Shih YH, Maleszewski JJ, Abell Aleff PC, Evans JM, Xu X, Olson TM. Recessive TAF1A mutations reveal ribosomopathy in siblings with end-stage pediatric dilated cardiomyopathy. Human molecular genetics. 2017 Aug 1;26(15):2874-81
  202. 202. Fedida J, Fressart V, Charron P, Surget E, Hery T, Richard P, Donal E, Keren B, Duthoit G, Hidden-Lucet F, Villard E. Contribution of exome sequencing for genetic diagnostic in arrhythmogenic right ventricular cardiomyopathy/dysplasia. PloS one. 2017 Aug 2;12(8):e0181840
  203. 203. Waldmüller S, Schroeder C, Sturm M, Scheffold T, Imbrich K, Junker S, Frische C, Hofbeck M, Bauer P, Bonin M, Gawaz M. Targeted 46-gene and clinical exome sequencing for mutations causing cardiomyopathies. Molecular and Cellular Probes. 2015 Oct 1;29(5):308-14
  204. 204. Liss M, Radke MH, Eckhard J, Neuenschwander M, Dauksaite V, von Kries JP, Gotthardt M. Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling. PloS one. 2018 Jun 11;13(6):e0198492
  205. 205. Maatz H, Jens M, Liss M, Schafer S, Heinig M, Kirchner M, Adami E, Rintisch C, Dauksaite V, Radke MH, Selbach M. RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing. The Journal of clinical investigation. 2014 Aug 1;124(8):3419-30
  206. 206. Lorenzi P, Sangalli A, Fochi S, Dal Molin A, Malerba G, Zipeto D, Romanelli MG. RNA-binding proteins RBM20 and PTBP1 regulate the alternative splicing of FHOD3. The international journal of biochemistry & cell biology. 2019 Jan 1;106:74-83
  207. 207. Bertero A, Fields PA, Ramani V, Bonora G, Yardimci GG, Reinecke H, Pabon L, Noble WS, Shendure J, Murry CE. Dynamics of genome reorganization during human cardiogenesis reveal an RBM20-dependent splicing factory. Nature communications. 2019 Apr 4;10(1):1-9
  208. 208. Morinaga A, Ito J, Niimi T, Maturana AD. RBM20 regulates CaV1. 2 surface expression by promoting exon 9* Inclusion of CACNA1C in neonatal rat cardiomyocytes. International journal of molecular sciences. 2019 Jan;20(22):5591
  209. 209. Tharp C, Mestroni L, Taylor M. Modifications of Titin Contribute to the Progression of Cardiomyopathy and Represent a Therapeutic Target for Treatment of Heart Failure. Journal of Clinical Medicine. 2020 Sep;9(9):2770
  210. 210. Vikhorev PG, Smoktunowicz N, Munster AB, Kostin S, Montgiraud C, Messer AE, Toliat MR, Li A, Dos Remedios CG, Lal S, Blair CA. Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes. Scientific reports. 2017 Nov 1;7(1):1-1
  211. 211. Hinson JT, Chopra A, Nafissi N, Polacheck WJ, Benson CC, Swist S, Gorham J, Yang L, Schafer S, Sheng CC, Haghighi A. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy. Science. 2015 Aug 28;349(6251):982-6
  212. 212. Chopra A, Kutys ML, Zhang K, Polacheck WJ, Sheng CC, Luu RJ, Eyckmans J, Hinson JT, Seidman JG, Seidman CE, Chen CS. Force generation via β-cardiac myosin, titin, and α-actinin drives cardiac sarcomere assembly from cell-matrix adhesions. Developmental cell. 2018 Jan 8;44(1):87-96
  213. 213. Zhou Q, Kesteven S, Wu J, Aidery P, Gawaz M, Gramlich M, Feneley MP, Harvey RP. Pressure overload by transverse aortic constriction induces maladaptive hypertrophy in a titin-truncated mouse model. BioMed research international. 2015 Oct 4;2015
  214. 214. Ware JS, Cook SA. Role of titin in cardiomyopathy: from DNA variants to patient stratification. Nature Reviews Cardiology. 2018 Apr;15(4):241
  215. 215. Tabish AM, Azzimato V, Alexiadis A, Buyandelger B, Knöll R. Genetic epidemiology of titin-truncating variants in the etiology of dilated cardiomyopathy. Biophysical Reviews. 2017 Jun 1;9(3):207-23
  216. 216. Shibayama J, Yuzyuk TN, Cox J, Makaju A, Miller M, Lichter J, Li H, Leavy JD, Franklin S, Zaitsev AV. Metabolic remodeling in moderate synchronous versus dyssynchronous pacing-induced heart failure: integrated metabolomics and proteomics study. PLoS One. 2015 Mar 19;10(3):e0118974
  217. 217. Schafer S, De Marvao A, Adami E, Fiedler LR, Ng B, Khin E, Rackham OJ, Van Heesch S, Pua CJ, Kui M, Walsh R. Titin-truncating variants affect heart function in disease cohorts and the general population. Nature genetics. 2017 Jan;49(1):46-53
  218. 218. Neishabouri SH, Hutson SM, Davoodi J. Chronic activation of mTOR complex 1 by branched chain amino acids and organ hypertrophy. Amino acids. 2015 Jun 1;47(6):1167-82
  219. 219. Methawasin M, Hutchinson KR, Lee EJ, Smith III JE, Saripalli C, Hidalgo CG, Ottenheijm CA, Granzier H. Experimentally increasing titin compliance in a novel mouse model attenuates the Frank-Starling mechanism but has a beneficial effect on diastole. Circulation. 2014 May 13;129(19):1924-36
  220. 220. Parikh VN, Caleshu C, Reuter C, Lazzeroni LC, Ingles J, Garcia J, McCaleb K, Adesiyun T, Sedaghat-Hamedani F, Kumar S, Graw S. Regional variation in RBM20 causes a highly penetrant arrhythmogenic cardiomyopathy. Circulation: Heart Failure. 2019 Mar;12(3):e005371
  221. 221. Wyles SP, Li X, Hrstka SC, Reyes S, Oommen S, Beraldi R, Edwards J, Terzic A, Olson TM, Nelson TJ. Modeling structural and functional deficiencies of RBM20 familial dilated cardiomyopathy using human induced pluripotent stem cells. Human molecular genetics. 2016 Jan 15;25(2):254-65
  222. 222. Streckfuss-Bömeke K, Tiburcy M, Fomin A, Luo X, Li W, Fischer C, Özcelik C, Perrot A, Sossalla S, Haas J, Vidal RO. Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes. Journal of molecular and cellular cardiology. 2017 Dec 1;113:9-21
  223. 223. Motz K, Samad I, Yin LX, Murphy MK, Duvvuri M, Ding D, Hillel AT. Interferon-γ Treatment of Human Laryngotracheal Stenosis–Derived Fibroblasts. JAMA Otolaryngology–Head & Neck Surgery. 2017 Nov 1;143(11):1134-40
  224. 224. Hillel AT, Samad I, Ma G, et al. Dysregulated Macrophages Are Present in Bleomycin-Induced Murine Laryngotracheal Stenosis. Otolaryngol Head Neck Surg 2015;153:244-50
  225. 225. Hillel AT, Ding D, Samad I, et al. T-Helper 2 Lymphocyte Immunophenotype Is Associated With Iatrogenic Laryngotracheal Stenosis. Laryngoscope 2019;129:177-86
  226. 226. Nesek-Adam V, Mršić V, Oberhofer D, Grizelj-Stojčić E, Košuta D, Rašić Ž. Post-intubation long-segment tracheal stenosis of the posterior wall: a case report and review of the literature. Journal of Anesthesia. 2010;24(4):621-625
  227. 227. Hirshoren N, Eliashar R. Wound-healing modulation in upper airway stenosis-Myths and facts. Head & Neck. 2009;31(1):111-126
  228. 228. Antón-Pacheco J, Usategui A, Martínez I, García-Herrero C, Gamez A, Grau M et al. TGF-β antagonist attenuates fibrosis but not luminal narrowing in experimental tracheal stenosis. The Laryngoscope. 2016;127(3):561-567
  229. 229. Loewen M, Walner D, Caldarelli D. Improved airway healing using transforming growth factor beta-3 In a rabbit model. Wound Repair and Regeneration. 2001;9(1):44-49
  230. 230. Yin LX, Motz KM, Samad I, et al. Fibroblasts in Hypoxic Conditions Mimic Laryngotracheal Stenosis. Otolaryngol Head Neck Surg 2017;156:886-892
  231. 231. Ghosh A, Malaisrie N, Leahy KP, et al. Cellular Adaptive Inflammation Mediates Airway Granulation in a Murine Model of Subglottic Stenosis. Otolaryngol Head Neck Surg 2011;144:927-33
  232. 232. Wynn TA. Fibrotic disease and the TH1/TH2 paradigm. Nat Rev Immunol 2004;4:583-94
  233. 233. Haft S, Lee JY, Ghosh A, et al. Inflammatory Protein Expression in Human Subglottic Stenosis Tissue Mirrors That in a Murine Model. Ann Otol Rhinol Laryngol 2014;123:65-70
  234. 234. Kendall RT, Feghali-Bostwick CA. Fibroblasts in fibrosis: novel roles and mediators. Front Pharmacol 2014;5:123
  235. 235. Singh T, Sandulache V, Otteson T, Barsic M, Klein E, Dohar J et al. Subglottic Stenosis Examined as a Fibrotic Response to Airway Injury Characterized by Altered Mucosal Fibroblast Activity. Archives of Otolaryngology–Head & Neck Surgery. 2010;136(2):163
  236. 236. Doolin E, Tsuno K, Strande L, Santos M. Pharmacologic Inhibition of Collagen in an Experimental Model of Subglottic Stenosis. Annals of Otology, Rhinology & Laryngology. 1998;107(4):275-279
  237. 237. Ma G, Samad I, Motz K, et al. Metabolic variations in normal and fibrotic human laryngotracheal-derived fibroblasts: a Warburg-like effect. Laryngoscope 2017;127:E107-13
  238. 238. Anis M, Zhao Z, Khurana J, Krynetskiy E, Soliman A. Translational genomics of acquired laryngotracheal stenosis. The Laryngoscope. 2014;124(5):E175-E179
  239. 239. Boehler A, Chamberlain D, Kesten S, Slutsky AS, Liu M, Keshavjee S. Lymphocytic airway infiltration as a precursor to fibrous obliteration in a rat model of bronchiolitis obliterans. Transplantation. 1997;64(2):311-7
  240. 240. Sandulache VC, Chafin B, Li-Korotky HS, Otteson TD, Dohar JE, Hebda PA. Elucidating the Role of Interleukin 1ß and Prostaglandin E2 in Upper Airway Mucosal Wound Healing. Acta Otolaryngol Head Neck Surg. 2007;133:365-74
  241. 241. Nicolli EA, Carey RM, Farquhar D, Haft S, Alfonso KP, Mirza N. Risk factors for adult acquired subglottic stenosis. J Laryngol Otol. 2017;131(3):264-7
  242. 242. Branski RC, Rosen CA, Verdolini K HP. Markers of wound healing in vocal fold secretions from patients with laryngeal pathology. Ann Otol Rhinol Laryngol 2004;113:23-9
  243. 243. Sharma M, Panda NK. Proteomic Profiling of Protease-Primed Virus-Permissive Caco-2 Cells Display Abortive-Interferon Pathway and Deregulated Thromboinflammatory SERPINS. Preprints. 2020 Jun 17
  244. 244. Zhao YG, Zhang H. Phase separation in membrane biology: the interplay between membrane-bound organelles and membraneless condensates. Developmental Cell. 2020 Jul 28
  245. 245. Dodson AE, Kennedy S. Phase separation in germ cells and development. Developmental Cell. 2020 Oct 1
  246. 246. Singh J. Phase Separation of RNA Helicase Triggers Stress-Responsive Translational Switch. Trends in Biochemical Sciences. 2020 Sep 1;45(9):726-8
  247. 247. Majumder S, Jain A. Osmotic Stress Triggers Phase Separation. Molecular Cell. 2020 Sep 17;79(6):876-7
  248. 248. Zbinden A, Pérez-Berlanga M, De Rossi P, Polymenidou M. Phase separation and neurodegenerative diseases: a disturbance in the Force. Developmental Cell. 2020 Oct 12;55(1):45-68
  249. 249. Jalihal AP, Pitchiaya S, Xiao L, Bawa P, Jiang X, Bedi K, Parolia A, Cieslik M, Ljungman M, Chinnaiyan AM, Walter NG. Multivalent proteins rapidly and reversibly phase-separate upon osmotic cell volume change. Molecular Cell. 2020 Sep 17;79(6):978-90
  250. 250. Szabo G, Momen-Heravi F. Extracellular Vesicles and Exosomes: Biology and Pathobiology. The Liver: Biology and Pathobiology. 2020 Feb 12:1022-7
  251. 251. Saha P, Sharma S, Mishra R, Guneshekaran M, Kaushal S, Davis ME, Vallabhajosyula P. Circulating Transplanted Progenitor Cell Specific Exosomes Predict Functional Recovery of Ischemic Myocardium. Circulation. 2018 Nov 6;138(Suppl_1):A16237-
  252. 252. Zhang ZC, Liu Y, Xiao LL, Li SF, Jiang JH, Zhao Y, Qian SW, Tang QQ, Li X. Upregulation of miR-125b by estrogen protects against non-alcoholic fatty liver in female mice. Journal of hepatology. 2015 Dec 1;63(6):1466-75

Written By

Maryada Sharma, Kavita Kaushal, Sanjay Singh Rawat, Manjul Muraleedharan, Seema Chhabra, Nipun Verma, Anupam Mittal, Ajay Bahl, Madhu Khullar, Anurag Ramavat and Naresh K. Panda

Submitted: 13 July 2020 Reviewed: 19 November 2020 Published: 17 February 2021