Development Of Cardiac Fibrosis Research Articles

Vaccination targeting senescence-associated transmembrane protein alleviated cardiovascular pathology in the mice

Abstract Introduction Accumulation of senescent cells is observed in various organs including vasculature, heart and white adipose tissue with age, and known to become a substrate of pathophysiology of various cardiovascular-metabolic diseases. We have previously developed a vaccine to eliminate senescent cells, so-called "senolytic vaccine" by targeting Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) as a cellular-senescence-associated cell surface protein. We have demonstrated that GPNMB senolytic vaccine could remove senescent cells and alleviate the pathology cardiovascular-metabolic diseases. Senescence-associated adhesion molecule 1 (SAAM-1) is also known as a cell-surface membrane to increase its expression during cellular senescence in endothelial cells as well as the development of cardiovascular diseases. Here, we identified SAAM-1 as another senescence antigen and created a vaccine to eliminate senescent cells by targeting SAAM-1. Purpose This study was purposed to evaluate the efficacy of SAAM-1 vaccine for the treatment of cardiovascular diseases in mice models. Methods As a pressure overload-induced heart failure (HF) model, C57BL/6N wild type (WT) mice were underwent transverse aortic constriction (TAC) or sham opretaion at 13 weeks of age. SAAM-1 or control vaccination were given at 8 and 10 weeks of age before operation. Echocardiography examination was conducted two weeks after TAC operation, followed by tissue sampling 3 weeks after the examination. As an atherosclerosis model, ApoE KO mice were subjected to a high-fat diet (HFD) starting at 4 weeks of age. Vaccination of ApoE KO mice was administered at 8 weeks of age with SAAM-1-vaccine or control-vaccine, followed by sacrifice 4 weeks later for further analysis. ApoE-KO mice were also crossed with p16-mediated Luciferase expressing mice (p16-Luc mice) and subjected to generation of atherosclerosis model. Results SAAM-1 expression was significantly increased in left ventricle (LV) in TAC mice but its increase was suppressed by SAAM-1 vaccination. Interestingly, both worsening systolic function and dilatation of LV by pressure overload were significantly alleviated by SAAM-1 vaccination. Furthermore, expression level of senescent markers including p16 and p21, as well as several inflammation markers were increased in failing heart along with development of cardiac fibrosis, but SAAM-1 vaccine could significantly attenuate this pathological change. In ApoE KO mice, SAAM-1 vaccine decreased expression level of several inflammation markers in the mice, along with a decreasing p16 signal in the aorta. Conclusions Targeting SAAM-1 for vaccination would be a novel and promising approach to alleviating age-related cardiovascular disease.

Inorganic Polyphosphate Is in the Surface of Trypanosoma cruzi but Is Not Significantly Secreted.

Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that can lead to the development of cardiac fibrosis, which is characterized by the deposition of extracellular matrix (ECM) components in the interstitial region of the myocardium. The parasite itself can induce myofibroblast differentiation of cardiac fibroblast in vitro, leading to increased expression of ECM. Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that can also induce myofibroblast differentiation and deposition of ECM components and is highly abundant in T. cruzi. PolyP can modify proteins post-translationally by non-enzymatic polyphosphorylation of lysine residues of poly-acidic, serine-(S) and lysine (K)-rich (PASK) motifs. In this work, we used a bioinformatics screen and identified the presence of PASK domains in several surface proteins of T. cruzi. We also detected polyP in the external surface of its different life cycle stages and confirmed the stimulation of host cell fibrosis by trypomastigote infection. However, we were not able to detect significant secretion of the polymer or activation of transforming growth factor beta (TGF-β), an important factor for the generation of fibrosis by inorganic polyP- or trypomastigote-conditioned medium.

Abstract P355: Multiomic Analyses Reveal Sex-specific Mechanisms Governing Cardiac Fibrosis.

Cardiac fibrosis—the deposition of excess extracellular matrix in the heart—occurs during pathological cardiac remodelling and precedes cardiac dysfunction and heart failure. Experimental and clinical data show that both cardiac fibrosis and heart failure exhibit sex-specific differences, particularly in the absence of ischemic injury. However, no study to date has systematically examined this, or the hormonal mechanisms driving these differences, using high-resolution single-cell and spatial omics. To address this gap, we applied single-cell and spatial omics to map the quality and distribution of non-ischemic cardiac fibrosis in human and mouse hearts. Further, to query the impact of sex hormones on cardiac fibrosis development, we analysed hearts of castrated (CAST) and ovariectomized (OVX) mice, which were ectopically infused with angiotensin-II (AngII). Using single cell transcriptomic data of hypertensive mouse hearts, we identified a fibroblast population driving cardiac fibrosis with a unique gene signature. We confirmed the presence of these cells in multiple mouse models and aged donor human hearts. Spatial mapping of fibrosis showed a sex-specific distribution of fibrosis, specifically in the development of perivascular fibrosis in the heart. Hypertensive male mice exhibited extensive perivascular fibrosis, and coronary artery adventitial hypertrophy, which was absent in females. However, OVX resulted in the development of perivascular fibrosis in hypertensive mice suggesting a cardioprotective role of estrogen. Confirming these observations, spatial transcriptomic analyses showed distinct fibrotic signatures driving perivascular and interstitial fibrosis, and transcriptomic differences between females and males. Our findings provide fundamental new insights towards the importance of biological sex in driving cardiac fibrosis, pointing to new mechanisms that may be manipulated to ameliorate development of heart failure.

Macrophage MKL1 contributes to cardiac fibrosis in a mouse model of myocardial infarction

AimsCardiac fibrosis is characterized by aberrant collagen deposition in the heart. Macrophage polarization or infiltration is the main reason to accelerate the collagen deposition. We attempted to investigate the involvement of MKL1 in macrophages during the development of cardiac fibrosis. Materials and methodsCardiac fibrosis is induced by myocardial infarction (MI). The MKL1f/f mice were crossed to the Lyz2-cre mice to generate macrophage conditional MKL1 knockout mice (MKL1ΔMφ). In addition, macrophage conditional MKL1 overexpression mice (MKL1Mϕ-OE) were constructed by crossing Lyz2-cre mice to MKL1ΔN200-Rosa26 mice. Key findingsMKL1 expression was significantly increased in macrophages of both ischemic cardiomyopathy (ICM) patients and mice induced to develop myocardial infarction. Deletion of MKL1 in macrophages improved the heart function after MI-induced cardiac fibrosis. Consistently, MKL1Mϕ-OE mice displayed more severe cardiac fibrosis and worsened heart function than the control mice after MI. Moreover, administration of a small-molecule MKL1 inhibitor CCG-1423 also decreased the collagen deposition after MI. SignificanceOur data demonstrate that MKL1 in macrophages contributes to cardiac fibrosis pathogenesis and reinforce the notion that targeting MKL1 may yield effective antifibrotic therapeutics in the heart.

Novel Impact of Colchicine on Interleukin-10 Expression in Acute Myocardial Infarction: An Integrative Approach.

Background: Inflammation plays a critical role in myocardial infarction as a critical process in the development of heart failure, involving the development of cardiac fibrosis. Colchicine is a well-established anti-inflammatory drug, but its scientific application in controlling post-acute myocardial infarction (AMI) inflammatory processes has not been established. IL-10 is a key cytokine in modulating inflammatory responses, underscoring its potential as a crucial therapeutic target of colchicine. The objective was to explore the protective role of IL-10 modulated by colchicine in myocardial healing and repair following AMI, particularly cardiac fibrosis. Methods: The predicted protein of colchicine was assessed using WAY2DRUG PASS as probability active value. Proteins associated with colchicine, cardiac fibrosis, and acute myocardial infarction were analyzed with DisGeNET and Open Target databases. Analysis and visualization of protein-protein interactions were conducted using STRING and Cytoscape. A 3T3 cell line treated with CoCl2 was used to mimic hypoxic. HIF-1α and IL-10 expression were measured by flow cytometry and analyzed using a one-way ANOVA test. This observational clinical trial examined acute myocardial infarction patients undergoing immediate and delayed primary percutaneous coronary interventions. Subjects were randomized into control groups receiving placebo and intervention groups treated with colchicine. Assessments occurred at 24 h and five days after the intervention. IL-10 expression in the clinical trial was measured by ELISA and analyzed using a T-test. Results: Colchicine demonstrates promising bioactivity in treating acute myocardial infarction, with notably activity values highlighting its probable role as a tubulin antagonist (0.744), beta-tubulin antagonist (0.673), and NOS2 inhibitor (0.529). Its primary action targets IL-10, with the protein-protein interactions analysis indicating interactions between IL-10 and key inflammatory mediators-IL-1β, IFN-γ, CCL2, TNF, and TGF-β1-during acute myocardial infarction and cardiac fibrosis. Hypoxic conditions in the CoCl2-induced 3T3 cell model show significantly elevated HIF-1α compared to controls (p < 0.0001). Colchicine use significantly increased IL-10 expression in CoCl2-treated cells (p < 0.0001) and in AMI patients within five days (p < 0.05). Conclusions: Colchicine may bolster the anti-inflammatory response post-myocardial infarction by activating IL-10 pathways in fibroblasts and in clinical settings, potentially reducing inflammation after AMI. Further investigation into broader aspects of this pathway, particularly in cardiac fibroblasts, is required.

Mesenchymal stem cells may alleviate angiotensin II-induced myocardial fibrosis and hypertrophy by upregulating SFRS3 expression

Introduction and objectivesThe development of cardiac fibrosis (CF) and hypertrophy (CH) can lead to heart failure. Mesenchymal stem cells (MSCs) have shown promise in treating cardiac diseases. However, the relationship between MSCs and splicing factor arginine/serine rich-3 (SFRS3) remains unclear. In this study, our objectives are to investigate the effect of MSCs on SFRS3 expression, and their impact on CF and CH. Additionally, we aim to explore the function of the overexpression of SFRS3 in angiotensin II (Ang II)-treated cardiac fibroblasts (CFBs) and cardiac myocytes (CMCs). MethodsRat cardiac fibroblasts (rCFBs) or rat cardiac myocytes (rCMCs) were co-cultured with rat MSCs (rMSCs). The function of SFRS3 in Ang II-induced rCFBs and rCMCs was studied by overexpressing SFRS3 in these cells, both with and without the presence of rMSCs. We assessed the expression of SFRS3 and evaluated the cell cycle, proliferation and apoptosis of rCFBs and rCMCs. We also measured the levels of interleukin (IL)-β, IL-6 and tumor necrosis factor (TNF)-α and assessed the degree of fibrosis in rCFBs and hypertrophy in rCMCs. ResultsrMSCs induced SFRS3 expression and promoted cell cycle, proliferation, while reducing apoptosis of Ang II-treated rCFBs and rCMCs. Co-culture of rMSCs with these cells also repressed cytokine production and mitigated the fibrosis of rCFBs, as well as hypertrophy of rCMCs triggered by Ang II. Overexpression of SFRS3 in the rCFBs and rCMCs yielded identical effects to rMSC co-culture. ConclusionMSCs may alleviate Ang II-induced cardiac fibrosis and cardiomyocyte hypertrophy by increasing SFRS3 expression in vitro.

MTOR Inhibition Prolongs Survival and Has Beneficial Effects on Heart Function After Onset of Lamin A/C Gene Mutation Cardiomyopathy in Mice.

Mutations in LMNA encoding nuclear envelope proteins lamin A/C cause dilated cardiomyopathy. Activation of the AKT/mTOR (RAC-α serine/threonine-protein kinase/mammalian target of rapamycin) pathway is implicated as a potential pathophysiologic mechanism. The aim of this study was to assess whether pharmacological inhibition of mTOR signaling has beneficial effects on heart function and prolongs survival in a mouse model of the disease, after onset of heart failure. We treated male LmnaH222P/H222P mice, after the onset of heart failure, with placebo or either of 2 orally bioavailable mTOR inhibitors: everolimus or NV-20494, a rapamycin analog highly selective against mTORC1. We examined left ventricular remodeling, and the cell biological, biochemical, and histopathologic features of cardiomyopathy, potential drug toxicity, and survival. Everolimus treatment (n=17) significantly reduced left ventricular dilatation and increased contractility on echocardiography, with a 7% (P=0.018) reduction in left ventricular end-diastolic diameter and a 39% (P=0.0159) increase fractional shortening compared with placebo (n=17) after 6 weeks of treatment. NV-20494 treatment (n=15) yielded similar but more modest and nonsignificant changes. Neither drug prevented the development of cardiac fibrosis. Drug treatment reactivated suppressed autophagy and inhibited mTORC1 signaling in the heart, although everolimus was more potent. With regards to drug toxicity, everolimus alone led to a modest degree of glucose intolerance during glucose challenge. Everolimus (n=20) and NV-20494 (n=20) significantly prolonged median survival in LmnaH222P/H222P mice, by 9% (P=0.0348) and 11% (P=0.0206), respectively, compared with placebo (n=20). These results suggest that mTOR inhibitors may be beneficial in patients with cardiomyopathy caused by LMNA mutations and that further study is warranted.

ALKBH5-mediated m6A modification of IL-11 drives macrophage-to-myofibroblast transition and pathological cardiac fibrosis in mice

Cardiac macrophage contributes to the development of cardiac fibrosis, but factors that regulate cardiac macrophages transition and activation during this process remains elusive. Here we show, by single-cell transcriptomics, lineage tracing and parabiosis, that cardiac macrophages from circulating monocytes preferentially commit to macrophage-to-myofibroblast transition (MMT) under angiotensin II (Ang II)-induced hypertension, with accompanying increased expression of the RNA N6-methyladenosine demethylases, ALKBH5. Meanwhile, macrophage-specific knockout of ALKBH5 inhibits Ang II-induced MMT, and subsequently ameliorates cardiac fibrosis and dysfunction. Mechanistically, RNA immunoprecipitation sequencing identifies interlukin-11 (IL-11) mRNA as a target for ALKBH5-mediated m6A demethylation, leading to increased IL-11 mRNA stability and protein levels. By contrast, overexpression of IL11 in circulating macrophages reverses the phenotype in ALKBH5-deficient mice and macrophage. Lastly, targeted delivery of ALKBH5 or IL-11 receptor α (IL11RA1) siRNA to monocytes/macrophages attenuates MMT and cardiac fibrosis under hypertensive stress. Our results thus suggest that the ALKBH5/IL-11/IL11RA1/MMT axis alters cardiac macrophage and contributes to hypertensive cardiac fibrosis and dysfunction in mice, and thereby identify potential targets for cardiac fibrosis therapy in patients.

JAK/STAT3 signaling in cardiac fibrosis: a promising therapeutic target.

Cardiac fibrosis is a serious health problem because it is a common pathological change in almost all forms of cardiovascular diseases. Cardiac fibrosis is characterized by the transdifferentiation of cardiac fibroblasts (CFs) into cardiac myofibroblasts and the excessive deposition of extracellular matrix (ECM) components produced by activated myofibroblasts, which leads to fibrotic scar formation and subsequent cardiac dysfunction. However, there are currently few effective therapeutic strategies protecting against fibrogenesis. This lack is largely because the molecular mechanisms of cardiac fibrosis remain unclear despite extensive research. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling cascade is an extensively present intracellular signal transduction pathway and can regulate a wide range of biological processes, including cell proliferation, migration, differentiation, apoptosis, and immune response. Various upstream mediators such as cytokines, growth factors and hormones can initiate signal transmission via this pathway and play corresponding regulatory roles. STAT3 is a crucial player of the JAK/STAT pathway and its activation is related to inflammation, malignant tumors and autoimmune illnesses. Recently, the JAK/STAT3 signaling has been in the spotlight for its role in the occurrence and development of cardiac fibrosis and its activation can promote the proliferation and activation of CFs and the production of ECM proteins, thus leading to cardiac fibrosis. In this manuscript, we discuss the structure, transactivation and regulation of the JAK/STAT3 signaling pathway and review recent progress on the role of this pathway in cardiac fibrosis. Moreover, we summarize the current challenges and opportunities of targeting the JAK/STAT3 signaling for the treatment of fibrosis. In summary, the information presented in this article is critical for comprehending the role of the JAK/STAT3 pathway in cardiac fibrosis, and will also contribute to future research aimed at the development of effective anti-fibrotic therapeutic strategies targeting the JAK/STAT3 signaling.

Potential therapeutic effects of baicalin and baicalein.

Baicalin and baicalein are natural flavonoids reported for the first time from Scutellaria baicalensis Georgi. Recently, attention has been paid to these valuable flavonoids due to their promising effects. This paper aims to have a comprehensive review of their pharmacological effects. An extensive search through scientific databases including Scopus, PubMed, and ISI Web of Science was established. According to literature, these compounds have been mainly effective in the treatment of neurological and neurodegenerative diseases, hepatic and cardiovascular disorders, metabolic syndrome, and cancers through anti-inflammatory and antioxidant pathways. Induction of apoptosis and autophagy, and inhibition of migration and metastasis are the main mechanisms for their cytotoxic and antitumor activities. Decreasing inflammation, reducing oxidative stress, regulating the metabolism of lipids, and decreasing fibrosis, apoptosis, and steatosis are their main hepatoprotective mechanisms. Inhibiting the development of cardiac fibrosis and reducing inflammation, oxidative stress, and apoptosis are also the mechanisms suggested for cardioprotective activities. Decreasing the accumulation of inflammatory mediators and improving cognitive function and depressive-like behaviours are the main mechanisms for neurological and neurodegenerative activities. The findings suggest the therapeutic potential of baicalin and baicalein. However, complementary research in different in vitro and in vivo models to investigate their mechanisms of action as well as clinical trials to evaluate their efficacy and safety are suggested.

Atrazine exposure promotes cardiomyocyte pyroptosis to exacerbate cardiotoxicity by activating NF-κB pathway

Atrazine is a ubiquitous herbicide with persistent environmental presence and accumulation in the food chain, posing potential health hazards to organisms. Increasing evidence suggests that atrazine may have detrimental effects on various organ systems, including the nervous, digestive, and immune systems. However, the specific toxicity and underlying mechanism of atrazine-induced cardiac injury remain obscure. In this study, 4-week-old male C57BL/6 mice were administered atrazine via intragastric administration at doses of 50 and 200 mg/kg for 4 and 8 weeks, respectively. Our findings showed that atrazine exposure led to cardiac fibrosis, as evidenced by elevated heart index and histopathological scores, extensive myofiber damage, and interstitial collagen deposition. Moreover, atrazine induced cardiomyocyte apoptosis, macrophage infiltration, and excessive production of inflammatory factors. Importantly, atrazine upregulated the expressions of crucial pyroptosis proteins, including NLRP3, ASC, CASPASE1, and GSDMD, via the activation of NF-κB pathway, thus promoting cardiomyocyte pyroptosis. Collectively, our findings provide novel evidence demonstrating that atrazine may exacerbate myocardial fibrosis by inducing cardiomyocyte pyroptosis, highlighting its potential role in the development of cardiac fibrosis.

Regulation of cardiac fibroblast cell death by unfolded protein response signaling.

The endoplasmic reticulum (ER) is a tightly regulated organelle that requires specific environmental properties to efficiently carry out its function as a major site of protein synthesis and folding. Embedded in the ER membrane, ER stress sensors inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) serve as a sensitive quality control system collectively known as the unfolded protein response (UPR). In response to an accumulation of misfolded proteins, the UPR signals for protective mechanisms to cope with the cellular stress. Under prolonged unstable conditions and an inability to regain homeostasis, the UPR can shift from its original adaptive response to mechanisms leading to UPR-induced apoptosis. These UPR signaling pathways have been implicated as an important feature in the development of cardiac fibrosis, but identifying effective treatments has been difficult. Therefore, the apoptotic mechanisms of UPR signaling in cardiac fibroblasts (CFs) are important to our understanding of chronic fibrosis in the heart. Here, we summarize the maladaptive side of the UPR, activated downstream pathways associated with cell death, and agents that have been used to modify UPR-induced apoptosis in CFs.

Inhibitory effect of microRNA-21 on pathways and mechanisms involved in cardiac fibrosis development.

Cardiac fibrosis is a pivotal cardiovascular disease (CVD) process and represents a notable health concern worldwide. While the complex mechanisms underlying CVD have been widely investigated, recent research has highlighted microRNA-21's (miR-21) role in cardiac fibrosis pathogenesis. In this narrative review, we explore the molecular interactions, focusing on the role of miR-21 in contributing to cardiac fibrosis. Various signaling pathways, such as the RAAS, TGF-β, IL-6, IL-1, ERK, PI3K-Akt, and PTEN pathways, besides dysregulation in fibroblast activity, matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs cause cardiac fibrosis. Besides, miR-21 in growth factor secretion, apoptosis, and endothelial-to-mesenchymal transition play crucial roles. miR-21 capacity regulatory function presents promising insights for cardiac fibrosis. Moreover, this review discusses numerous approaches to control miR-21 expression, including antisense oligonucleotides, anti-miR-21 compounds, and Notch signaling modulation, all novel methods of cardiac fibrosis inhibition. In summary, this narrative review aims to assess the molecular mechanisms of cardiac fibrosis and its essential miR-21 function.

Wnt1 gene expression in the heart left ventricle as a response to the various durations of the intensive exercise: An experimental study.

Objective. Myocardial fibrosis is a devastating condition causing millions of deaths yearly. Several factors, such as aging, cause myocardial fibrosis. The Wnt/β-catenin pathway is one of the critical intracellular signaling for the development of cardiac fibrosis. Molecular and cellular mechanism of myocardial fibrosis induced by intensive exercise is not well-understood. The current study evaluates the effects of short- and long-term intensive exercise on the Wnt1 gene expression in a heart left ventricle in an animal model. Methods. Twenty-one male Wistar rats (mean weight 250±50 g) were divided into three groups (n=7): 1) control group (C); 2) short-term regular intensive exercise group (S-RIE, high-intensity exercise for one month six days weekly for 60 min with speed of 35 m/min), and 3) long-term regular intensive exercise group (L-RIE, high-intensity exercise for six months six days daily for 60 min with speed of 35 m/min). The heart left ventricle was isolated at the end of the experiment, and the relative gene expression of the Wnt1 gene was measured by the Real-Time PCR. Results. The L-RIE group showed a significant increase in the Wnt1 expression compared to the S-RIE and the control group. Although no difference was observed in the Wnt1 mRNA level in the S-RIE group compared to the control group, Wnt1 mRNA level increased in the L-RIE group compared to the S-RIE group. Conclusion. The exercise duration was of a great importance in the Wnt1 gene expression. Regular intensive exercise may be involved in the formation of the myocardial fibrosis by increasing the expression of the Wnt1 gene.

Simvastatin Attenuates Cardiac Fibrosis under Pathophysiological Conditions of Heart Failure with Preserved Left Ventricular Ejection Fraction by Inhibiting TGF-β Signaling

Introduction: There is still no effective treatment for heart failure with preserved left ventricular ejection fraction (HFpEF), and therapies to improve prognosis are urgently needed. Clinical studies in patients with HFpEF have shown that statins and HMG-CoA reductase inhibitors may reduce their mortality rate. However, the mechanisms underlying the effects of statins on HFpEF remain unknown. In the present study, we examined whether simvastatin administration inhibits the development of cardiac fibrosis in HFpEF model mice. We further examined the contribution of the Smad and mitogen-activated protein (MAP) kinase pathways to the transforming growth factor-β (TGF-β) signaling pathway in the development of HFpEF. Methods: HFpEF animals were prepared by feeding C57BL/6 N mice a high-fat diet and providing water containing N[w]-nitro-l-arginine methyl ester hydrochloride (l-NAME) for 15 weeks. Simvastatin (30 mg/kg/day) or vehicle was administered orally daily during the experimental period. Cardiac function was measured by echocardiography, and cardiac fibrosis was evaluated by Masson’s trichrome staining. Changes in the TGF-β signaling proteins in myocardial tissue were examined by Western blotting. Results: A high-fat diet and l-NAME solution load induced cardiac diastolic dysfunction with cardiac fibrosis. Simvastatin treatment markedly attenuated cardiac fibrosis and reduced cardiac diastolic dysfunction. In addition, simvastatin prevented the increase in phosphorylation levels of Smad (Smad2 and Smad3) and MAPK (c-Raf, Erk1/2) pathway proteins downstream of the TGF-β receptor in cardiac tissue. Conclusions: Our present study demonstrated that simvastatin attenuated diastolic dysfunction by reducing cardiac fibrosis in HFpEF hearts. Furthermore, our findings suggest that the mechanisms by which simvastatin attenuates HFpEF development involve, at least in part, inhibition of the TGF-β signaling pathway, which is activated in the HFpEF heart.

Temporal expression and spatial distribution of the proteoglycan versican during cardiac fibrosis development

Cardiac fibrosis is a central pathological feature in several cardiac diseases, but the underlying molecular players are insufficiently understood. The extracellular matrix proteoglycan versican is elevated in heart failure and suggested to be a target for treatment. However, the temporal expression and spatial distribution of versican and the versican cleavage fragment containing the neoepitope DPEAAE in cardiac fibrosis remains to be elucidated. In this study, we have examined versican during cardiac fibrosis development in a murine pressure overload model and in patients with cardiomyopathies. We found that versican, mainly the V1 isoform, was expressed immediately after induction of pressure overload, preceding collagen accumulation, and versican protein levels extended from the perivascular region into the cardiac interstitium. In addition, we found increased production of versican by collagen expressing fibroblasts, and that it was deposited extensively in the fibrotic extracellular matrix during pressure overload. In cardiac cell cultures, the expression of versican was induced by the pro-fibrotic transforming growth factor beta and mechanical stretch. Furthermore, we observed that the proteolytic cleavage of versican (DPEAAE fragment) increased in the late phase of fibrosis development during pressure overload. In patients with hypertrophic and dilated cardiomyopathies, we found elevated levels of versican and a positive correlation between versican and collagen mRNA in the heart, as well as increased cleavage of full-length protein. Taken together, the temporal expression profile and the spatial distribution of both the full-length versican and the DPEAAE fragment observed in this study indicates a role for versican in development of cardiac fibrosis.

Cardiac-specific deletion of myosin phosphatase targeting subunit 2 alleviates cardiac fibrosis in mineralocorticoid receptor-related hypertension

Abstract Introduction Myosin phosphatase targeting subunit 2 (MYPT2) is a crucial subunit of cardiac myosin light chain (MLC) phosphatase. The phosphorylation of MLC in the ventricle plays a pivotal role in cardiac hypertrophy and function. Our previous study demonstrated that overexpression of MYPT2 activated MLC phosphatase leading to cardiac dysfunction. Mineralocorticoid receptor (MR)-related hypertension is associated with extensive cardiac fibrosis. It remains unknown whether MYPT2 may play a role in the development of cardiac fibrosis in MR-related hypertension. Purpose Our purpose is to investigate the effect of MYPT2 on cardiac fibrosis in MR-related hypertension. Methods After the knockdown of MYPT2 using MYPT2-siRNA, HL-1 cells (murine cardiomyocytes) were incubated with aldosterone (ALDO) for 24h. Cardiac-­specific MYPT2 knockout (c-MYPT2-/-) mice were generated using the Cre-lox system (αMHC-­Cre; MYPT2f/f). MR-related hypertension mice were induced in c-MYPT2-/- and wild-type (MYPT2+/+) mice with the subcutanouse infusion of ALDO and 8% NaCl food for 4 weeks after uninephrectomy. Systolic blood pressure (SBP) was measured by tail cuffs every week. WB, qPCR, cardiac echo, and histological examinations were performed. Results In HL-1 cells, ALDO increased the expression of connective tissue growth factor (CTGF), MYPT2, and the catalytic subunit of the type-1 phosphatase δ isoform (PP1cδ) in a concentration-dependent manner. MYPT2 protein levels significantly decreased by 80% in HL-1 cells transfected with MYPT2 siRNA compared with cells transfected with unspecific siRNA. Knockdown of MYPT2 significantly decreased the protein level of CTGF. In the mice model, the MYPT2 expression in the heart from MYPT2-/- mice was decreased to less than 30% compared with MYPT2+/+ mice. Heart rate, blood pressure, and cardiac systolic function were normal in c-MYPT2-/- mice. PP1cδ was decreased in c-MYPT2-/- mice compared with MYPT2+/+ mice. The expression of cardiac MLC kinase was not changed. The level of phosphorylation of MLC in c-MYPT2-/- mice was significantly higher than MYPT2+/+. Blood pressure elevation and left ventricular hypertrophy were observed in both MR-related hypertension mice. No significant difference in heart size and the nuclear localization of MR in cardiomyocytes. However, ejection fraction and fractional shortening in echocardiography were higher in c-MYPT2-/- than in MYPT2+/+ in MR-related hypertension mice. Histopathological examinations revealed the degree of fibrosis in c-MYPT2-/- mice was lower than in MYPT2+/+ mice with the decreased expression of CTGF. Conclusions Cardiac-specific deletion of MYPT2 resulted in a decrease of MLC phosphatase and an increase of phosphorylation in MLC in vivo. MYPT2 deletion can inhibit cardiac fibrosis in a MR-related hypertension model.

Platelet-derived microRNAs play a pivotal role in cardiac remodeling after myocardial ischemia and reperfusion

Abstract Background Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of miRNAs, which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate, that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. Purpose The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. Methods and Results Combining an in vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (LSFM, PET&amp;MRI, speckle-tracking echocardiography), in vitro immune cell migration assays and Next-generation deep sequencing analysis of platelet miRNA expression pattern in mice with a MK/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating LV remodeling after myocardial I/R following transient LCA ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial ischemia and reperfusion. Worsened cardiac remodeling after myocardial infarction (MI) in mice with a platelet-specific Dicer deletion (Dicer Pf4Δ/PfΔ4) resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at d28 post-MI. Altogether, these observations culminated in an impaired LV function and hampered long-term cardiac recovery after experimental MI and reperfusion therapy. Treatment with the P2Y12 antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in Dicer Pf4Δ/Pf4Δ mice. Conclusions The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial ischemia and reperfusion.

Sleep-disordered breathing is associated with fibrosis and impaired diastolic function

Abstract Background Emerging evidence is linking sleep-disordered breathing (SDB) to diastolic dysfunction. SDB-induced hypoxia may stimulate the development of cardiac fibrosis which then causes diastolic dysfunction. We investigated the association between SDB and myocardial collagen and PDGF mRNA levels and how collagen mRNA levels are linked to parameters of diastolic dysfunction. Methods RA biopsies were collected from 31 patients undergoing elective coronary artery bypass grafting in the prospective observational study CONSIDER-AF. Tissue mRNA levels of collagen type I (Col1A), PDGFα, and PDGFβ were quantified using real-time qPCR on ViiA 7 real-time PCR system. NT-pro-BNP levels were measured in the serum via ELISA blots. The apnea-hypopnea-index (AHI) was registered in portable SDB-monitoring. Echocardiography was performed to assess diastolic function. Results First, we investigated an association of SDB and cardiac fibrosis. AHI was correlated with tissue mRNA levels of Col3a (p = 0.0239, Fig A), PDGFα (p = 0.0414, Fig B), and PDGFβ (p = 0.0024, Fig C). Then, we investigated an association between cardiac fibrosis and diastolic dysfunction. Tissue mRNA levels of Col3A were inversely correlated with lateral (p = 0.0196, Fig D) and septal E’ (p = 0.0006, Fig E) as a marker for diastolic relaxation. Higher ratios of E/E’mean were correlated with increased tissue mRNA levels of Col3A (p = 0.0368, Fig F). NT-pro-BNP which is a diagnostic criterion for heart failure with preserved ejection fraction was correlated with Col3 levels (p = 0.0247, Fig G). Discussion: Our findings indicate that SDB is linked to fibrosis which is associated with diastolic dysfunction.

Circular RNA circSMAD4 regulates cardiac fibrosis by targeting miR-671-5p and FGFR2 in cardiac fibroblasts

Heart failure is a leading cause of death and is often accompanied by activation of quiescent cardiac myofibroblasts, which results in cardiac fibrosis. In this study, we aimed to identify novel circular RNAs that regulate cardiac fibrosis. We applied transverse aortic constriction (TAC) for 1, 4, and 8weeks in mice. RNA sequencing datasets were obtained from cardiac fibroblasts isolated by use of a Langendorff apparatus and then further processed by use of selection criteria such as differential expression and conservation in species. CircSMAD4 was upregulated by TAC in mice or by transforming growth factor (TGF)-β1 in primarily cultured human cardiac fibroblasts. Delivery of si-circSMAD4 attenuated myofibroblast activation and cardiac fibrosis in mice treated with isoproterenol (ISP). si-circSmad4 significantly reduced cardiac fibrosis and remodeling at 8weeks. Mechanistically, circSMAD4 acted as a sponge against the microRNA miR-671-5p in a sequence-specific manner. miR-671-5p was downregulated during myofibroblast activation and its mimic form attenuated cardiac fibrosis. miR-671-5p mimic destabilized fibroblast growth factor receptor 2 (FGFR2) mRNA in a sequence-specific manner and interfered with the fibrotic action of FGFR2. The circSMAD4-miR-671-5p-FGFR2 pathway is involved in the differentiation of cardiac myofibroblasts and thereby the development of cardiac fibrosis.