Biology subjects

Kanisicak, O.

Publications and source records attributed to Kanisicak, O..

9 recordsLinked to original sources

bioRxiv2026

WISP1 drives a mechanically active immune modulatory and proliferative cardiac myofibroblast state

Pathological cardiac remodeling is driven by the proliferation and differentiation of resident fibroblasts into active myofibroblasts and results in excessive extracellular matrix (ECM) deposition and tissue stiffening. Expression of the matricellular protein WISP1 has previously been shown to be increased with cardiac fibrosis and promote myofibroblast activity, but the mechanisms by which this occurs remain unknown. Primary cardiac fibroblasts were isolated from adult mouse hearts and treated with recombinant WISP1 or TGF{beta}1 both alone and in combination to determine the functional role of the matricellular protein WISP1 in driving cardiac myofibroblast activity. WISP1 significantly increased alpha-smooth muscle actin and collagen type I expression, total collagen secretion, collagen gel contractility, and wound healing equally in fibroblasts from both male and female mice. However, WISP1 alone failed to induce expression of periostin, a hallmark myofibroblast marker, suggesting the resulting WISP1-dependent cell phenotype is unique and/or acting through non-canonical pathways. Indeed, inhibition of P38 MAPK completely ablated the WISP1-dependent increase in SMA and collagen expression, while having little to no impact on TGF{beta}1-dependent expression of myofibroblast marker genes. We next employed a multi-omics approach to define the functional impact of WISP1 on fibroblast cell-state within the transcriptome, cytosolic, and secreted ECM proteome. RNA-seq results show that WISP1 broadly promotes the expression of proliferative and immune modulatory genes at the transcriptomic level, while having very little impact on traditional myofibroblast and ECM modifying gene expression programs. At the proteome level, WISP1 was again a much weaker mediator of traditional myofibroblast and ECM proteins. However, in agreement with RNA-seq data, we observed a strong WISP1-dependent enrichment for proliferation-associated proteins in the cytosolic proteome and inflammation-associated proteins in the ECM proteome. Interestingly, WISP1 also showed a context-dependent response with TGF{beta}1, suggesting a more complex and yet to be elucidated signaling interaction between these independent mediators of myofibroblast activity. In conclusion, our data suggests that WISP1 promotes a unique proliferative and immune-modulatory myofibroblast phenotype. HighlightsO_LIWISP1 is sufficient to drive myofibroblast SMA and collagen expression and ECM deposition C_LIO_LIWISP1 promotes canonical myofibroblast contractility and wound healing activity C_LIO_LIWISP1 mediates myofibroblast activity via a non-canonical, P38 MAPK-dependent signaling pathway C_LIO_LIMulti-omics analysis of WISP1-dependent RNA and protein expression show that WISP promotes a proliferative and immune modulatory myofibroblast phenotype C_LI

cell biology↗

bioRxiv2024

Pulmonary fibroblast activation during Aspergillus fumigatus infection enhances lung defense via immunomodulation and tissue remodeling

Aspergillus fumigatus is the etiologic agent of invasive aspergillosis, a life- threatening fungal pneumonia that is initiated by the inhalation of conidia (spores) into the lung. If the conidia are not cleared, they secrete large quantities of hydrolytic enzymes and toxins as they grow, resulting in extensive damage to pulmonary tissue. Stromal fibroblasts are central responders to tissue damage in many organs, but their functional response to pulmonary injury caused by A. fumigatus has not been explored. In this study, we employed cell lineage tracing, targeted cell ablation, and single-cell RNA sequencing to monitor the dynamics of fibroblast behavior upon exposure to A. fumigatus in both immunocompetent and immunosuppressed hosts. The results demonstrate that a subset of pulmonary fibroblasts becomes activated in an immunocompetent host in response to a challenge with A. fumigatus conidia, acquiring a gene expression program reflecting the acquisition of new immunomodulatory properties as well as enhanced extracellular matrix (ECM)-secreting ability. Remarkably, through targeted ablation of fibroblasts that express the profibrotic activation marker periostin, we demonstrate that the progression of an invasive A. fumigatus infection in an immunosuppressed host is accelerated by the absence of periostin lineage cells and is accompanied by severe alveolar hemorrhage and angioinvasion. These findings uncover a novel protective role for fibroblasts in limiting the severity of A. fumigatus-induced pulmonary injury and emphasize the importance of the pulmonary stroma in host defense against this invasive fungal infection.

cell biology↗

bioRxiv2024

MCM2 mediates post-MI cardioprotection by promoting the pro-angiogenic cardiosome signaling

AbstractO_ST_ABSBackgroundC_ST_ABSIn the past decade, induced cardiac rejuvenation has emerged as a leading approach to repair cardiac injury. Recent studies demonstrate that promoting cell cycle reentry in adult cardiomyocytes (CM) enhances cardiac rejuvenation by influencing paracrine signaling. We previously demonstrated that the inhibition of two cell cycle inhibitors, Retinoblastoma 1 (Rb1) and Meis homeobox 2 (Meis2), in the adult CM enhances angiogenesis and cardiac function following ischemic injury, but the underlying mechanisms have yet to be elucidated. The goal of this study is to determine the mechanisms by which inhibition of Rb1 and Meis2 promotes cardiac rejuvenation in a mouse model of myocardial infarction. MethodsMyocardial infarction was induced in adult C57/Bl6 mice via permanent LAD occlusion followed by direct injection of either control or Rb1+Meis2 siRNA cocktail to the ischemic myocardium. MCM2 overexpression done via direct myocardial injection of an MCM2-plasmid DNA expression cassette. Cardiac function and LV wall motion was assessed via echocardiography, and fibrosis and CM hypertrophy were assessed via histology. RNA-sequencing was performed on isolated adult murine CMs with siRNA-mediated Rb1+Meis2 knockdown to delineate the downstream mechanisms. Further identification of Rb1, Meis2, and MCM2-dependent mechanisms were done using in vitro techniques in isolated CMs and HUVEC cells. ResultsWe show that siRNA-mediated knockdown of Rb1 and Meis2 in vivo reduces pathological LV remodeling and preserves cardiac structure and function in adult mouse hearts post-MI. RNA-seq analyses revealed MCM2 as a potential downstream target of Rb1/Meis2 to enhance protective paracrine signaling in primary adult CMs. Indeed, re-expression of MCM2, which is developmentally lost from neonatal to adult CM in the heart, improves cardiac function and LV wall motion while reducing myocyte hypertrophy and fibrotic scar size post-MI. Mechanistically, re-expression of MCM2 promotes the secretion of pro- angiogenic factors from adult CM, and transfer of conditioned media from MCM2 expressing CM induced vasculogenesis in HUVEC cells. Proteomic analysis of the MCM2 interactome confirmed a significant enrichment of angiogenic mediators and suggests an MCM2-dependent protein packaging of pro- angiogenic factors in CM-derived small extracellular vesicles (cardiosomes). ConclusionRe-expression of MCM2 in adult CM promotes the secretion of pro-angiogenic cardiosomes that induce paracrine revascularization of endothelial cells and mitigates cardiac injury post-MI. SUMMARY DIAGRAM O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/628232v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@17eaeforg.highwire.dtl.DTLVardef@40f0f5org.highwire.dtl.DTLVardef@18b7664org.highwire.dtl.DTLVardef@109cb1c_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Fast skeletal myosin binding protein-C expression exacerbates dysfunction in heart failure

During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We previously observed that fast skeletal myosin binding protein-C (fMyBP-C) is upregulated in diseased mouse hearts. While fMyBP-C shares significant homology with its cardiac paralog, cardiac myosin binding protein-C (cMyBP-C), there are key differences that may affect cardiac function. However, it is unknown if the expression of fMyBP-C expression in the heart is a pathological or compensatory response. We aim to elucidate the cardiac consequence of either increased or knockout of fMyBP-C expression. To determine the sufficiency of fMyBP-C to cause cardiac dysfunction, we generated cardiac-specific fMyBP-C over-expression mice. These mice were further crossed into a cMyBP-C null model to assess the effect of fMyBP-C in the heart in the complete absence of cMyBP-C. Finally, fMyBP-C null mice underwent transverse aortic constriction (TAC) to define the requirement of fMyBP-C during heart failure development. We confirmed the upregulation of fMyBP-C in several models of cardiac disease, including the use of lineage tracing. Low levels of fMyBP-C caused mild cardiac remodeling and sarcomere dysfunction. Exclusive expression of fMyBP-C in a heart failure model further exacerbated cardiac pathology. Following 8 weeks of TAC, fMyBP-C null mice demonstrated greater protection against heart failure development. Mechanistically, this may be due to the differential regulation of the myosin super-relaxed state. These findings suggest that the elevated expression of fMyBP-C in diseased hearts is a pathological response. Targeted therapies to prevent upregulation of fMyBP-C may prove beneficial in the treatment of heart failure. Significance StatementRecently, the sarcomere - the machinery that controls heart and muscle contraction - has emerged as a central target for development of cardiac therapeutics. However, there remains much to understand about how the sarcomere is modified in response to disease. We recently discovered that a protein normally expressed in skeletal muscle, is present in the heart in certain settings of heart disease. How this skeletal muscle protein affects the function of the heart remained unknown. Using genetically engineered mouse models to modulate expression of this skeletal muscle protein, we determined that expression of this skeletal muscle protein in the heart negatively affects cardiac performance. Importantly, deletion of this protein from the heart could improve heart function suggesting a possible therapeutic avenue.

physiology↗

bioRxiv2023

Myocardial IGF2R is a critical mediator of inflammation and fibrosis after ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury is a common occurrence in various surgical procedures used to treat heart diseases. However, the role of insulin-like growth factor 2 receptor (IGF2R) during the process of myocardial I/R remains unclear. Therefore, this study aims to investigate the expression, distribution, and functionality of IGF2R in various I/R-associated models (such as reoxygenation, revascularization, and heart transplant). Loss-of-function studies (including myocardial conditional knockout and CRISPR interference) were performed to clarify the role of IGF2R in I/R injuries. Following hypoxia, IGF2R expression increased, but this effect was reversed upon restoration of oxygen levels. Loss of myocardial IGF2R was found to enhance the cardiac contractile functions, and reduced cell infiltration or cardiac fibrosis of I/R mouse models compared to the genotype control. CRISPR-inhibition of IGF2R decreased cell apoptotic death under hypoxia. RNA sequencing analysis indicated that myocardial IGF2R played a critical role in regulating the inflammatory response, innate immune response, and apoptotic process following I/R. Integrated analysis of the mRNA profiling, pulldown assays, and mass spectrometry identified granulocyte-specific factors as potential targets of myocardial IGF2R in the injured heart. In conclusion, myocardial IGF2R emerges as a promising therapeutic target to ameliorate inflammation or fibrosis following I/R injuries.

cell biology↗

bioRxiv2023

HuR inhibition reduces post-ischemic cardiac remodeling by dampening acute inflammatory gene expression and the innate immune response

Myocardial ischemia/reperfusion (I/R) injury and the resulting cardiac remodeling is a common cause of heart failure. The RNA binding protein Human Antigen R (HuR) has been previously shown to reduce cardiac remodeling following both I/R and cardiac pressure overload, but the full extent of the HuR-dependent mechanisms within cells of the myocardium have yet to be elucidated. In this study, we applied a novel small molecule inhibitor of HuR to define the functional role of HuR in the acute response to I/R injury and gain a better understanding of the HuR-dependent mechanisms during post-ischemic myocardial remodeling. Our results show an early (two hours post-I/R) increase in HuR activity that is necessary for early inflammatory gene expression by cardiomyocytes in response to I/R. Surprisingly, despite the reductions in early inflammatory gene expression at two hours post-I/R, HuR inhibition has no effect on initial infarct size at 24-hours post-I/R. However, in agreement with previously published work, we do see a reduction in pathological remodeling and preserved cardiac function at two weeks post-I/R upon HuR inhibition. RNA-sequencing analysis of neonatal rat ventricular myocytes (NRVMs) at two hours post-LPS treatment to model damage associated molecular pattern (DAMP)-mediated activation of toll like receptors (TLRs) demonstrates a broad HuR-dependent regulation of pro-inflammatory chemokine and cytokine gene expression in cardiomyocytes. We show that conditioned media from NRVMs pre-treated with HuR inhibitor loses the ability to induce inflammatory gene expression in bone marrow derived macrophages (BMDMs) compared to NRVMs treated with LPS alone. Functionally, HuR inhibition in NRVMs also reduces their ability to induce endocrine migration of peripheral blood monocytes in vitro and reduces post-ischemic macrophage infiltration to the heart in vivo. In summary, these results suggest a HuR-dependent expression of pro-inflammatory gene expression by cardiomyocytes that leads to subsequent monocyte recruitment and macrophage activation in the post-ischemic myocardium.

molecular biology↗

bioRxiv2022

HuR-dependent expression of Wisp1 is necessary for TGF????-induced cardiac myofibroblast activity

Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGF{beta}-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGF{beta}-induced myofibroblast activation upon HuR inhibition. To delineate HuR-dependent mechanisms, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify eleven mRNA transcripts that showed enriched HuR binding following TGF{beta} treatment as well as significant co-expression correlation with HuR, SMA, and periostin using single-cell RNA-sequencing from ischemic-zone isolated fibroblasts. Of these, Wnt1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 is able to partially rescue myofibroblast contractile function following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGF{beta}. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGF{beta}, in part through post-transcriptional regulation of Wisp1. HIGHLIGHTSO_LIThe RNA binding protein HuR is highly expressed in cardiac fibroblasts and its expression strongly correlates with markers of active myofibroblasts C_LIO_LIHuR inhibition reduces migration, contraction, and ECM production activity of cardiac fibroblasts C_LIO_LIExpression of the secreted matricellular protein, Wisp1, is increased in a HuR-dependent manner following TGF{beta} treatment C_LIO_LIRecombinant Wisp1 rescues myofibroblast contractile function following HuR inhibition C_LIO_LIHuR-dependent expression of Wisp1 is necessary for myofibroblast activation C_LI

molecular biology↗

bioRxiv2021

S-Palmitoylation-Dependent Regulation of Cardiomyocyte Rac1 Signaling Activity and Cardiac Hypertrophy

S-palmitoylation is a reversible lipid modification that regulates trafficking, localization, activity, and/or stability of protein substrates by serving as a fatty acid anchor to cell membranes. However, S-palmitoylation-dependent control of signal transduction in cardiomyocytes and its effects on cardiac physiology are not well understood. We performed an in vivo gain-of-function screen of zinc finger Asp-His-His-Cys (zDHHC) family S-acyl transferases that catalyze S-palmitoylation and identified the Golgi-localized enzyme zDHHC3 as a critical regulator of cardiac maladaptation. The closely-related enzyme, zDHHC7, also induced severe cardiomyopathy but this effect was not observed with overexpression of plasma membrane enzyme zDHHC5, endoplasmic reticulum enzyme zDHHC6, or Golgi enzyme zDHHC13. To identify effectors that may underlie zDHHC3-induced cardiomyopathy we performed quantitative site-specific S-acyl proteomics in zDHHC3-overexpressing cells that revealed the small GTPase Rac1 as a novel substrate. We generated cardiomyocyte-specific transgenic mice overexpressing zDHHC3, which develop severe cardiac disease. Cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice are preceded by enhanced S-palmitoylation of Rac1 and induction of additional Rho family small GTPases including RhoA, Cdc42, and the Rho family-specific chaperone RhoGDI. In contrast, transgenic mice overexpressing an enzymatically-dead mutant of zDHHC3 do not exhibit this profound induction of RhoGTPase signaling or develop cardiac disease. Rac1 S-palmitoylation, plasma membrane localization, activity, and downstream hypertrophic signaling were substantially increased in zDHHC3 overexpressing hearts. Taken together, these data suggest inhibition of zDHHC3/7 S-acyl transferase activity at the cardiomyocyte Golgi or disruption of Rac1 S-palmitoylation as novel therapeutic strategies to treat cardiac disease or other diseases associated with enhanced RhoGTPase signaling.

molecular biology↗

bioRxiv2021

Adipocyte deletion of the RNA binding protein HuR induces cardiac hypertrophy and fibrosis

Adipose tissue continues to gain appreciation for its broad role as an endocrine organ, and disruptions in adipose tissue homeostasis plays a central role in cardiovascular physiology. We have previously shown that expression of the RNA binding protein HuR in adipose tissue mediates energy expenditure, but the potential cardiovascular impacts of this finding have not been explored. We show here that adipose tissue-specific deletion of HuR (Adipo-HuR-/-) is sufficient to induce the spontaneous development of cardiac hypertrophy and fibrosis. Hearts from Adipo-HuR-/- mice have increased left ventricular (LV) ejection fraction, rate of pressure generation, and LV posterior wall thickness that is accompanied by an increase in LV/body weight ratio and hypertrophic gene expression. Furthermore, Adipo-HuR-/- hearts display increased fibrosis by picrosirius red staining and periostin expression. To identify underlying mechanisms, we applied both RNA-seq and weighted gene co-expression network analysis (WGCNA) to define HuR-dependent changes in gene expression as well as significant relationships between adipose tissue gene expression and LV mass. RNA-seq results demonstrate a significant increase in pro-inflammatory gene expression in the subcutaneous white adipose tissue (scWAT) from Adipo-HuR-/- mice that is accompanied by an increase in serum levels of both TNF- and IL-6. WGCNA identified a significant enrichment in inflammation, apoptosis/cell death, and vesicle-mediated transport genes among those whose expression most significantly associated with CVD in Adipo-HuR-/-. In conclusion, we demonstrate that the loss of HuR expression in adipose tissue promotes the development of cardiac hypertrophy and fibrosis, potentially through modulation of inflammation and vesicle-mediated transport in scWAT. NEW AND NOTEWORTHYThis work demonstrates the spontaneous development of cardiac hypertrophy and fibrosis upon adipose tissue-specific deletion of the RNA binding protein HuR that appears to be mechanistically driven by HuR-dependent changes in inflammatory and extracellular vesicle transport mediating genes in the subcutaneous white adipose tissue. These results suggest that loss of HuR expression in adipose tissue in obesity, as demonstrated in mouse and humans by our group and others, may contribute to obesity-mediated CVD.

physiology↗