Cardiac Fibrosis In Heart Failure Research Articles

Usefulness of Galectin-3 as a Biochemical Marker to Detect Ventricular and Supraventricular Arrhythmias in Children.

Galectin-3 (Gal-3) has been demonstrated to play a pivotal role in the pathogenesis of several fibrotic disorders. A number of studies have examined the relationship between galectin-3 levels and cardiac fibrosis in heart failure. Nevertheless, the role of galectin-3 in the etiology of supraventricular (SVa) and ventricular (Va) arrhythmias remains largely unexamined. The objective of this prospective study was to investigate the potential correlation between galectin concentration and the occurrence of idiopathic cardiac arrhythmias in pediatric patients. Biochemistry analysis was performed on 30 children (11-18 years; 14 boys and 16 girls). The control group consisted of 20 children. Cardiac arrhythmia was confirmed by a 24 h Holter ECG recording. Serum galectin-3 levels were measured via enzyme-linked immunosorbent assay (ELISA). Statistical analysis of the data showed significant associations between creatinine kinase (CK) and Gal-3 in patients with SVa (SVT-supraventricular tachycardia) arrhythmias, suggesting a potential effect of CK on Gal-3 levels. However, no correlation was identified between Gal-3 concentration and the occurrence of cardiac arrhythmias under investigation. We concluded that galectin-3 does not have the potential to be a biomarker in the diagnosis of idiopathic arrhythmias in pediatric patients.

Protein kinase N promotes cardiac fibrosis in heart failure by fibroblast-to-myofibroblast conversion

Chronic fibrotic tissue disrupts various organ functions. Despite significant advances in therapies, mortality and morbidity due to heart failure remain high, resulting in poor quality of life. Beyond the cardiomyocyte-centric view of heart failure, it is now accepted that alterations in the interstitial extracellular matrix (ECM) also play a major role in the development of heart failure. Here, we show that protein kinase N (PKN) is expressed in cardiac fibroblasts. Furthermore, PKN mediates the conversion of fibroblasts into myofibroblasts, which plays a central role in secreting large amounts of ECM proteins via p38 phosphorylation signaling. Fibroblast-specific deletion of PKN led to a reduction of myocardial fibrotic changes and cardiac dysfunction in mice models of ischemia-reperfusion or heart failure with preserved ejection fraction. Our results indicate that PKN is a therapeutic target for cardiac fibrosis in heart failure.

Axitinib targets cardiac fibrosis in pressure overload-induced heart failure through VEGFA-KDR pathway.

There are no specific clinical medications that target cardiac fibrosis in heart failure (HF). Recent studies have shown that tyrosine kinase inhibitors (TKIs) may benefit fibrosis in various organs. However, there is limited research on their application in cardiac fibrosis. Axitinib, an FDA-approved tyrosine kinase inhibitor, was used to evaluate its effects on cardiac fibrosis and function in pressure overload-induced heart failure. To build a pharmacological network, the pharmacological targets of axitinib were first retrieved from databases and coupled with key heart failure gene molecules for analysis and prediction. To validate the results outlined above, 8-week-old male C57BL/6 J mice were orally administrated of axitinib (30 mg/kg) daily for 8 weeks after Transverse Aortic Constriction (TAC) surgery. Mouse cardiomyocytes and cardiac fibroblasts were used as cell lines to test the function and mechanism of axitinib. We found that the pharmacological targets of axitinib could form a pharmacological network with key genes involved in heart failure. The VEGFA-KDR pathway was found to be closely related to the differential gene expression of human heart-derived primary cardiomyocyte cell lines treated with axitinib, based on analysis of the publicly available dataset. The outcomes of animal experiments demonstrated that axitinib therapy greatly reduced cardiac fibrosis and improved TAC-induced cardiac dysfunction. Further research has shown that the expression of transforming growth factor-β(TGF-β) and other fibrosis genes was significantly reduced in vivo and in vitro. Our study provides evidence for the repurposing of axitinib to combat cardiac fibrosis, and offers new insights into the treatment of patients with HF.

Human epididymis protein 4 indicates progression of cardiac fibrosis in Heart Failure with preserved Ejection Fraction

Abstract Background Cardiac fibrosis is one of the main causes of diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF). Human epididymis protein 4 (HE4) is a novel pro-fibrotic protein expressed especially in activated fibroblast, so-call myofibroblast [1]. Although our previous study showed that HE4 functioned as a secretory factor promoting cardiac fibrosis in dilated cardiomyopathy [2], little is known about the role of HE4 in HFpEF. Purpose The aim of this study is to evaluate the pathophysiological role of HE4 in HFpEF and the association between serum HE4 levels and clinical course of HFpEF. Methods In basic research, we administered high-fat diet and NOS inhibitor (L-NAME) to mice for 5 weeks to induce HFpEF. We analyzed echocardiographic findings and mRNA expressions of fibrosis-related genes including HE4 by real time PCR. In clinical research, we measured serum HE4 levels of 79 patients with HFpEF and divided these patients into low and high HE4 group using median values of HE4 (102.0 pmol/L). Then, we evaluated their mortality and cardiovascular events retrospectively. Results In vivo, the administration of high fat diet and L-NAME induced significant increase in left ventricular mass on echocardiography compared with control group (p=0.006). The mRNA expressions of aSMA and collagen3a1 in heart tissue was upregulated in HFpEF model mice (p=0.008 and p=0.003, respectively). Furthermore, the mRNA expression of HE4 was also upregulated in HFpEF model mice (n=6) than in control mice (n=5), although these differences did not reach statistical significance (p=0.238). In vitro, recombinant HE4 upregulated mRNA expression of pro-fibrotic genes, such as periostin (p=0.001), collagen3a1 (p=0.037), aSMA (p=0.095), and PAI-1 (p=0.17), indicating HE4 facilitate the activation of fibroblast and cardiac fibrosis although some did not reach statistical significance. In clinical research, Kaplan-Meier curve revealed that the rate of all cause death and rehospitalization for worsening heart failure were significantly higher in the high than the low HE4 group, suggesting HE4 can predicts prognosis in patients with HFpEF. Conclusion HE4 induces the activation of cardiac fibroblasts and is upregulated in the heart of HFpEF model mice. Furthermore, it is a useful biomarker for predicting cardiovascular events in patients with HFpEF. Funding Acknowledgement Type of funding sources: None.

Cardiac Mesenchymal Stem Cells Promote Fibrosis and Remodeling in Heart Failure: Role of PDGF Signaling

Heart failure (HF) is characterized by progressive fibrosis. Both fibroblasts and mesenchymal stem cells (MSCs) can differentiate into pro-fibrotic myofibroblasts. MSCs secrete and express platelet-derived growth factor (PDGF) and its receptors. We hypothesized that PDGF signaling in cardiac MSCs (cMSCs) promotes their myofibroblast differentiation and aggravates post-myocardial infarction left ventricular remodeling and fibrosis. We show that cMSCs from failing hearts post-myocardial infarction exhibit an altered phenotype. Inhibition of PDGF signaling invitro inhibited cMSC-myofibroblast differentiation, whereas invivo inhibition during established ischemic HF alleviated left ventricular remodeling and function, and decreased myocardial fibrosis, hypertrophy, and inflammation. Modulating cMSC PDGF receptor expression may thus represent a novel approach to limit pathologic cardiac fibrosis in HF.

Fibroblast growth factor 12 attenuated cardiac remodeling via suppressing oxidative stress

Fibroblast growth factors (FGFs) mediate key cardiac functions from development to homeostasis and disease. The current research was to explore the effects of FGF12 in the fibrosis of cardiac function and heart failure, and whether FGF12 alleviated cardiac fibrosis via inhibition of oxidative stress. Ligation of left coronary artery in mice induced heart failure and myocardial infarction (MI). Angiotensin II (Ang II) was administered to cardiac fibroblasts (CFs). FGF12 upregulation or FGF12 transgenic (Tg) mice could improve cardiac dysfunction of MI mice, and attenuated cardiac fibrosis of heart failure induced by MI in mice. FGF12 overexpression suppressed the increases of collagen I, collagen III and fibronectin which was induced by Ang II in CFs. The oxidative stress was enhanced in the heart of MI mice and CFs treated with Ang II, and these enhances were attenuated via FGF12 overexpression. Superoxide dismutase (SOD) overexpression inhibited the enhancements of collagen I, collagen III and fibronectin in the heart of MI mice, and in the CFs treated with Ang II. Overexpression of nicotinamide adenine dinucleotide phosphate oxidases (Nox1) reversed the attenuating influences of FGF12 on the enhancements of collagen I, collagen III and fibronectin in the CFs induced by Ang II. These outcomes showed that FGF12 upregulation can improve cardiac dysfunction and heart fibrosis of heart failure. FGF12 attenuates oxidative stress to suppress the cardiac fibrosis.

Long noncoding RNA NEAT1 promotes cardiac fibrosis in heart failure through increased recruitment of EZH2 to the Smad7 promoter region

Cardiac fibrosis, a well-known major pathological process that ultimately leads to heart failure, has attracted increasing attention and focus in recent years. A large amount of research indicates that long noncoding RNAs (lncRNAs) play an important role in cardiac fibrosis, but little is known about the specific function and mechanism of the lncRNA NEAT1 in the progression of cardiac fibrosis to heart failure. In the present study, we have demonstrated that the lncRNA NEAT1 is upregulated in patients with heart failure. Similarly, the expression of Neat1 was also increased in the left ventricular tissue of transverse aortic constriction (TAC) surgery mice and cardiac fibroblasts treated with TGF-β1. Further, gain-of-function and loss-of-function experiments showed that silencing of Neat1 attenuated cardiac fibrosis, while overexpression of Neat1 with adenovirus significantly aggravated the in vitro progression of fibrosis. With regard to the underlying mechanism, our experiments showed that Neat1 recruited EZH2 to the promoter region of Smad7 through physical binding of EZH2 to the promoter region, as a result of which Smad7 expression was inhibited and the progression of cardiac fibrosis was ultimately exacerbated. We found that the introduction of shNeat1 carried by adeno-associated virus-9 significantly ameliorated cardiac fibrosis and dysfunction caused by TAC surgery in mice. Overall, our study findings demonstrate that the lncRNA Neat1 accelerates the progression of cardiac fibrosis and dysfunction by recruiting EZH2 to suppress Smad7 expression. Thus, NEAT1 may serve as a target for the treatment of cardiac fibrosis.

Abstract 14103: Loss of Bone Morphogenetic Protein (BMP) 9 Activity Reduces Survival and Increases Collagen Turnover via Enhanced Matrix Metalloproteinase Activity After Acute Myocardial Infarction

Introduction: Acute myocardial infarction (AMI) is a major cause of heart failure and mortality. The extracellular matrix plays a central role in post-infarct remodeling. We recently reported that BMP9 negatively regulates cardiac fibrosis in heart failure. A functional role for BMP9 in post-infarct remodeling remains poorly defined. We hypothesize that BMP9 signaling via activin like kinase 1 (ALK1) promotes survival after AMI by negatively regulating fibrosis and limiting matrix metalloproteinase (MMP) activity. Methods: AMI was induced by left coronary artery ligation in wild type (WT), BMP9-/- and ALK-/+ mice. After 14 days, LV function was assessed using conductance catheter and myocardial BMP9 signaling pathways were studied. In vitro siRNA mediated protein knockdown in human atrial fibroblasts (AtFB) further tested this signaling pathway. Results: BMP9-/- and ALK-/+ mice had significantly reduced survival after AMI compared to WT (p=0.04 and 0.008 respectively) and was associated with an increased incidence of cardiac rupture. LV ejection fraction decreased significantly among BMP9-/- and ALK1+/- survivors compared to sham (p<0.01 for both). pSMAD3 was increased and pSMAD1 decreased in both BMP9-/- and ALK1-/+ mice after AMI. Reduced pSMAD1 expression was directly associated with increased type 1 collagen levels in BMP9-/- (R 2 =0.48, p=0.02) and ALK1-/+ (R 2 =0.42, p=0.01) mice compared to WT. LV protein levels of endoglin increased significantly in both BMP9-/- and ALK+/- compared to WT sham (p<0.01 for both) after AMI. LV levels and activity of MMP9 were increased in BMP9-/-, but not in ALK1+/-, compared to WT after AMI (p=0.02 and 0.8 respectively). Circulating biomarkers of collagen degradation:synthesis (ICTP:PINP ratio) increased significantly (p=0.02) in BMP9-/- compared to WT after AMI. BMP9 but not ALK1 knockdown in AtFBs increased expression and activity of MMP9 (p=0.01 and 0.3 respectively). Conclusions: These findings identify a novel functional role for BMP9 and ALK1 as determinants of post-infarct survival and cardiac remodeling and further suggest that loss of BMP9-ALK1 signaling increases collagen degradation and turnover. BMP9/ALK1 signaling may be a novel target of therapy for post-infarct remodeling and survival.

Exploring a Functional Role for BMP10 in Heart Failure

<h3>Purpose</h3> Cardiac fibrosis leads to adverse clinical outcomes in heart failure. Published studies have identified that bone morphogenetic protein-10 (BMP-10), a member of the transforming growth factor-b (TGB-b) superfamily, may form a heterodimer with BMP-9, which binds to the TGF-b co-receptor endoglin with high affinity and has been shown to limit cardiac fibrosis in heart failure. We hypothesized that a model of pressure overload-induced heart failure increases BMP-10 expression in the left ventricle, which limits cardiac fibrosis by increasing signaling via Smad1. <h3>Methods</h3> Adult, male 10-week-old mice were subjected to transverse aortic constriction (TAC) using a 27G needle for all studies. C57 Bl/6 wild-type mice were subjected to TAC for 2 (acute) or 8 (chronic) weeks to quantify BMP-9 and BMP-10 expression. LV pressure-volume loop and echocardiographic data were obtained. <h3>Results</h3> A total of 6 mice underwent sham operations, 8 underwent 2-week TAC, and 8 underwent 8-week TAC. All mice survived until harvest. Compared to sham, there was a reduced ejection fraction and a dilated LV internal diastolic dimension in the chronic TAC cohort (Sham: 71±4 % v 8-week: 30±14%, p=0.001; Sham: 2.2±0.3 mm v 8-week: 3.8±0.6 mm, p=0.02). Additionally, compared to sham, both LV end-diastolic and end-systolic volumes were increased in the chronic TAC group (Sham: 57±11 uL v 8-week: 91±419 uL, p=0.02; Sham: 17±5 uL v 8-week: 64±23 uL, p=0.007). Compared to sham, minimum LV pressure was elevated in the chronic TAC cohort (Sham: -4.5±1.3 mmHg v 8-week: 8.8±10.3 mmHg, p=0.04). BMP-10 mRNA expression was elevated in the right atrium, but reduced in the ventricles, at 8 weeks of TAC, while BMP-10 protein expression was increased at 8 weeks of TAC (Figure 1A-B). BMP-9 expression was reduced in the chronic TAC cohort in the left ventricle while endoglin levels were unchanged (Figure 1C). <h3>Conclusion</h3> BMP-10 mRNA levels are downregulated, while protein levels are upregulated, in the LV at 8 weeks of TAC. Further studies are needed to fully explore the functional role of BMP-10 in the heart.

Trophoblast Stem-Cell-Derived Exosomes Improve Doxorubicin-Induced Dilated Cardiomyopathy by Modulating the let-7i/YAP Pathway

Trophoblast stem cells (TSCs) have been confirmed to play a cardioprotective role in heart failure. However, whether TSC-derived exosomes (TSC-exos) can protect against cardiac injury remains unclear. In the present study, TSC-exos were isolated from the supernatant of TSCs using the ultracentrifugation method and characterized by transmission electron microscopy and western blotting. Utilizing the public Gene Expression Omnibus (GEO) database, we found that let-7i and Yes-associated protein 1 (YAP) could participate in the development of heart failure. In vitro, AC16 cardiomyocytes subjected to doxorubicin (DOX) were treated with TSC-exos or let-7i mimic. Flow cytometry showed that TSC-exos and let-7i both decreased cardiomyocyte apoptosis. In vivo, mice that were intraperitoneally injected into DOX received either PBS, TSC-exos, or AAV9-let7iup for let-7i overexpression. Mice receiving TSC-exos and AAV9-let7iup showed improved cardiac function and decreased inflammatory responses, accompanied by downregulated YAP signaling. Mechanistically, TSC-exos could transfer let-7i to cardiomyocytes and silence the YAP signaling pathway. In conclusion, TSC-exos could alleviate DOX-induced cardiac injury via the let-7i/YAP pathway, which sheds new light on the application of TSC-exos as a potential therapeutic tool for heart failure.

Targeting cardiac fibrosis in heart failure with preserved ejection fraction: mirage or miracle?

Cardiac fibrosis is central to the pathology of heart failure, particularly heart failure with preserved ejection fraction (HFpEF). Irrespective of the underlying profibrotic condition (e.g. ageing, diabetes, hypertension), maladaptive cardiac fibrosis is defined by the transformation of resident fibroblasts to matrix‐secreting myofibroblasts. Numerous profibrotic factors have been identified at the molecular level (e.g. TGFβ, IL11, AngII), which activate gene expression programs for myofibroblast activation. A number of existing HF therapies indirectly target fibrotic pathways; however, despite multiple clinical trials in HFpEF, a specific clinically effective antifibrotic therapy remains elusive. Therapeutic inhibition of TGFβ, the master‐regulator of fibrosis, has unfortunately proven toxic and ineffective in clinical trials to date, and new approaches are needed. In this review, we discuss the pathophysiology and clinical implications of interstitial fibrosis in HFpEF. We provide an overview of trials targeting fibrosis in HFpEF to date and discuss the promise of potential new therapeutic approaches and targets in the context of underlying molecular mechanisms.

MicroRNA-132 attenuated cardiac fibrosis in myocardial infarction-induced heart failure rats.

The aim of the present study was to determine the effect of microRNA (miR)-132 on cardiac fibrosis in myocardial infarction (MI)-induced heart failure and angiotensin (Ang) II-treated cardiac fibroblasts (CFs). Experiments were carried out in Sprague-Dawley rat treatment with ligation of left coronary artery to induce heart failure, and in CFs administration of Ang II to induce fibrosis. The level of miR-132 was increased in the heart of rats with MI-induced heart failure and the Ang II-treated CFs. In MI rats, left ventricle (LV) ejection fraction, fractional shortening, the maximum of the first differentiation of LV pressure (LV +dp/dtmax) and decline (LV -dp/dtmax) and LV systolic pressure (LVSP) were reduced, and LV end-systolic diameter (LVESD), LV end-diastolic diameter (LVEDD), LV volumes in systole (LVVS) and LV volumes in diastole (LVVD) were increased, which were reversed by miR-132 agomiR but deteriorated by miR-132 antagomiR. The expression levels of collagen I, collagen III, transforming growth factor-β (TGF-β), and α-smooth muscle actin (α-SMA) were increased in the heart of rat with MI-induced heart failure and CFs administration of Ang II. These increases were inhibited by miR-132 agomiR but enhanced by miR-132 antagomiR treatment. MiR-132 inhibited PTEN expression, and attenuated PI3K/Akt signal pathway in CFs. These results indicated that the up-regulation of miR-132 improved the cardiac dysfunction, attenuated cardiac fibrosis in heart failure via inhibiting PTEN expression, and attenuating PI3K/Akt signal pathway. Up-regulation of miR-132 may be a strategy for the treatment of heart failure and cardiac fibrosis.

Ablation of RAGE Attenuated Cardiac Fibrosis by Inhibition of Autophagy medicated Endothelial‐to‐mesenchymal transition

Endothelial‐to‐mesenchymal transition (EndMT) has been found involved in the progression of heart failure (HF), which maybe one of the source of myofibroblasts. Emerging evidence support the notion that EndMT was regulated by autophagy. Our previous studies demonstrated deletion of receptor for advanced glycation end products (RAGE) inhibited autophagy and attenuated cardiac fibrosis in HF. Thus, here we investigated whether reduction of cardiac fibrosis by ablation of RAGE was through inhibition autophagy medicated EndMT. Using transverse aortic constriction (TAC) in mice as a model of pressure overload induced HF, we demonstrated that EndMT and autophagy were induced simultaneously at 8 weeks after TAC. Co‐localization of CD31 and α‐smooth muscle actin (α‐SMA) was observed in myocardium. Knockout of RAGE displayed reduced cardiac fibrosis and significantly improved cardiac function in TAC mice. Importantly, knockout of RAGE deceased the expression of autophagy related proteins (LC3BII/I and Beclin1), which was accompanied with the decrease of co‐expression of CD31 and α‐SMA in TAC mouse hearts. Moreover, 3‐methyladenine (3‐MA) and chloroquine (CQ), inhibitors of autophagy, attenuated EndMT and cardiac fibrosis in TAC mice. Taken together autophagy inducing EndMT may contribute to the development of cardiac fibrosis. Inhibition of this process by RAGE deletion lead to cardiac function improvement and reduction of cardiac fibrosis. Thus, we concluded that inhibition of the RAGE‐autophagy‐EndMT axis could be a novel strategy for treatment of heart failure.Support or Funding InformationThe National Natural Science Foundation of China (81373570, 81673920, 81473621, 81673796 and 81973776). The Natural Science Foundation of Guangdong Province(2016A030311030).Reduction of cardiac fibrosis especially cardiovascular fibrosis after TAC by inhibition of RAGE and autophagy (Scale bar= 1,000 μm. n = 4. Values are shown as means ± SEM, *P &lt; 0.05 vs Sham group, #P &lt; 0.05 vs TAC group.)Figure 1Deceased expression of autophagy and EndMT after TAC by inhibition of RAGE and autophagy. (n = 4. Values are shown as means ± SEM, *P &lt; 0.05 vs Sham group, #P &lt; 0.05 vs TAC group.)Figure 2

Cardiac Fibroblasts and Cardiac Fibrosis: Precise Role of Exosomes.

Exosomes are a group of extracellular microvesicles that deliver biologically active RNAs, proteins, lipids and other signaling molecules to recipient cells. Classically, exosomes act as a vehicle by which cells or organs communicate with each other to maintain cellular/tissue homeostasis and to respond to pathological stress. Most multicellular systems, including the cardiovascular system, use exosomes for intercellular communication. In heart, endogenous exosomes from cardiac cells or stem cells aid in regulation of cell survival, cell proliferation and cell death; and thus tightly regulate cardiac biology and repair processes. Pathological stimulus in heart alters secretion and molecular composition of exosomes, thus influencing the above processes. The past decade has yielded increasing interest in the role of exosomes in the cardiovascular system and significant contribution of cardiac fibroblast (CF) and mediated cardiac fibrosis in heart failure, in this review we had overviewed the relevant literatures about fibroblast exosomes, its effect in the cardiovascular biology and its impact on cardiovascular disease (CVD). This review briefly describes the communication between fibroblasts and other cardiac cells via exosomes, the influence of such on myocardial fibrosis and remodeling, and the possibilities to use exosomes as biomarkers for acute and chronic heart diseases.

The Roles of a Matricellular CCN Family Protein CCN5 in Cardiac Fibrosis of Heart Failure

Cardiac fibrosis is a major player in cardiovascular disease, both as a contributor to the development of disease and a post-injury response that drives progression to heart failure. Despite the identification of many mechanisms responsible for cardiovascular fibrosis, such as angiotensin II, TGF-[Formula: see text] and endothelin-1, to date no treatments have emerged that have effectively reduced the excess deposition of extracellular matrix associated with fibrotic conditions. Matricellular CCN proteins spatiotemporally regulated nonstructural components of the extracellular matrix (ECM) and participated in many essential biological functions, including wound healing and fibrotic diseases. CCN5 exhibited the opposing effects of CCN2 on the development of cardiac hypertrophy and fibrosis, and overexpression of matricellular protein CCN5 in the heart by adenoviral deliver significantly improved cardiac fibrosis in severe heart failure. Future time- and cell-specific study of CCN5 effect and its domain-specific function on fibrotic development and progression will advance our understanding of cardiac fibrosis, and meanwhile provide opportunities for therapeutic intervention of heart failure.

Bone Morphogenetic Protein 9 Reduces Cardiac Fibrosis and Improves Cardiac Function in Heart Failure.

Heart failure is a growing cause of morbidity and mortality worldwide. Transforming growth factor beta (TGF-β1) promotes cardiac fibrosis, but also activates counterregulatory pathways that serve to regulate TGF-β1 activity in heart failure. Bone morphogenetic protein 9 (BMP9) is a member of the TGFβ family of cytokines and signals via the downstream effector protein Smad1. Endoglin is a TGFβ coreceptor that promotes TGF-β1 signaling via Smad3 and binds BMP9 with high affinity. We hypothesized that BMP9 limits cardiac fibrosis by activating Smad1 and attenuating Smad3, and, furthermore, that neutralizing endoglin activity promotes BMP9 activity. We examined BMP9 expression and signaling in human cardiac fibroblasts and human subjects with heart failure. We used the transverse aortic constriction-induced model of heart failure to evaluate the functional effect of BMP9 signaling on cardiac remodeling. BMP9 expression is increased in the circulation and left ventricle (LV) of human subjects with heart failure and is expressed by cardiac fibroblasts. Next, we observed that BMP9 attenuates type I collagen synthesis in human cardiac fibroblasts using recombinant human BMP9 and a small interfering RNA approach. In BMP9-/- mice subjected to transverse aortic constriction, loss of BMP9 activity promotes cardiac fibrosis, impairs LV function, and increases LV levels of phosphorylated Smad3 (pSmad3), not pSmad1. In contrast, treatment of wild-type mice subjected to transverse aortic constriction with recombinant BMP9 limits progression of cardiac fibrosis, improves LV function, enhances myocardial capillary density, and increases LV levels of pSmad1, not pSmad3 in comparison with vehicle-treated controls. Because endoglin binds BMP9 with high affinity, we explored the effect of reduced endoglin activity on BMP9 activity. Neutralizing endoglin activity in human cardiac fibroblasts or in wild-type mice subjected to transverse aortic constriction-induced heart failure limits collagen production, increases BMP9 protein levels, and increases levels of pSmad1, not pSmad3. Our results identify a novel functional role for BMP9 as an endogenous inhibitor of cardiac fibrosis attributable to LV pressure overload and further show that treatment with either recombinant BMP9 or disruption of endoglin activity promotes BMP9 activity and limits cardiac fibrosis in heart failure, thereby providing potentially novel therapeutic approaches for patients with heart failure.

Th1 effector T cells selectively orchestrate cardiac fibrosis in nonischemic heart failure.

Despite emerging data indicating a role for T cells in profibrotic cardiac repair and healing after ischemia, little is known about whether T cells directly impact cardiac fibroblasts (CFBs) to promote cardiac fibrosis (CF) in nonischemic heart failure (HF). Recently, we reported increased T cell infiltration in the fibrotic myocardium of nonischemic HF patients, as well as the protection from CF and HF in TCR-α-/- mice. Here, we report that T cells activated in such a context are mainly IFN-γ+, adhere to CFB, and induce their transition into myofibroblasts. Th1 effector cells selectively drive CF both in vitro and in vivo, whereas adoptive transfer of Th1 cells, opposite to activated IFN-γ-/- Th cells, partially reconstituted CF and HF in TCR-α-/- recipient mice. Mechanistically, Th1 cells use integrin α4 to adhere to and induce TGF-β in CFB in an IFN-γ-dependent manner. Our findings identify a previously unrecognized role for Th1 cells as integrators of perivascular CF and cardiac dysfunction in nonischemic HF.

C53: A Novel CNP-Based Therapeutic Strategy Targeting Cardiac Fibrosis and Heart Failure

Background: Cardiac fibrosis is a characteristic feature of the failing heart for which there is an unmet need of fibro-inhibiting therapies targeting heart failure (HF). C-type natriuretic peptide (CNP), a human endothelial derived 22 amino acid (AA) peptide, is a guanylyl cyclase B (GC-B) activator that mediates potent anti-fibrotic actions via its second messenger, cGMP. However, the endogenous anti-fibrotic actions of CNP are limited, in part, due to its rapid metabolic clearance by the clearance receptor (NPRC), which has been reported to be up regulated in HF patients. Considering the fibro-suppressing properties of GC-B activation, we engineered a novel 53 AA peptide (C53) based upon the CNP and proCNP sequence with the goal of advancing C53‘s therapeutic potential including reduced NPRC binding. Hypothesis: We hypothesized C53 would have enhanced cGMP generating actions in vivo due to reduced NPRC binding compared to CNP and C53 would activate cGMP in human cardiac fibroblasts (hCFs). Methods: Therapeutic dose of CNP, equimolar dose of C53 and vehicle were IV infused into normal anesthetized rats (n = 12) and in vivo CNP and cGMP levels were assessed. NPRC binding with C53 and CNP were assessed using a modified AlphaScreen luminescent proximity assay (ALPA). In vitro cGMP activating property of C53 (10−8M and 10−6M) compared to no treatment in hCFs was assessed. Data are mean ± SE and significance was achieved with P < .05. Results: In vivo, C53 significantly increased plasma and urinary cGMP compared to equimolar CNP and vehicle (P < .05), which correlated with higher plasma CNP levels with C53 IV infusion (P < .05). Binding studies using the ALPA demonstrated that C53 had reduced binding to NPRC compared to CNP. In hCFs, C53 generated cGMP at 10−8M and markedly activated cGMP at 10−6M compared to no treatment (no treatment: 0.00 ± 0.00; 10−8M: 0.17 ± 0.03; 10−6M: 1.79 ± 0.02, pmol/well, P < .05). Conclusion: We report a newly engineered CNP-based peptide, C53, has cGMP activating actions in hCFs and has superior in vivo cGMP generation than CNP which may, in part, be related to reduced binding to the metabolic clearance receptor NPRC. These studies suggest that C53 may be a novel therapeutic strategy for the prevention and treatment of cardiac fibrosis in HF and further investigations are warranted.

Reduced activin receptor-like kinase 1 activity promotes cardiac fibrosis in heart failure.

Activin receptor-like kinase 1 (ALK1) mediates signaling via the transforming growth factor beta-1 (TGFβ1), a pro-fibrogenic cytokine. No studies have defined a role for ALK1 in heart failure. We tested the hypothesis that reduced ALK1 expression promotes maladaptive cardiac remodeling in heart failure. In patients with advanced heart failure referred for left ventricular (LV) assist device implantation, LV Alk1 mRNA and protein levels were lower than control LV obtained from patients without heart failure. To investigate the role of ALK1 in heart failure, Alk1 haploinsufficient (Alk1+/-) and wild-type (WT) mice were studied 2 weeks after severe transverse aortic constriction (TAC). LV and lung weights were higher in Alk1+/- mice after TAC. Cardiomyocyte area and LV mRNA levels of brain natriuretic peptide and β-myosin heavy chain were increased similarly in Alk1+/- and WT mice after TAC. Alk-1 mice exhibited reduced Smad 1 phosphorylation and signaling compared to WT mice after TAC. Compared to WT, LV fibrosis and Type 1 collagen mRNA and protein levels were higher in Alk1+/- mice. LV fractional shortening was lower in Alk1+/- mice after TAC. Reduced expression of ALK1 promotes cardiac fibrosis and impaired LV function in a murine model of heart failure. Further studies examining the role of ALK1 and ALK1 inhibitors on cardiac remodeling are required.

Galectin-3 correlates with arrhythmogenic right ventricular cardiomyopathy and predicts the risk of ventricular ­arrhythmias in patients with implantable defibrillators

Background Arrhythmogenic right ventricular dysplasia (ARVD) is a heritable disorder characterized by fibro-fatty replacement of right ventricular myocytes, increased risk of ventricular arrhythmias, and sudden cardiac death. Galectin-3 (GAL3) is known to play an important role in a number of fibrotic conditions, including cardiac fibrosis. Many studies have focused on the association between GAL3 levels and cardiac fibrosis in heart failure. However, the role of GAL3 in the pathogenesis of ARVD and ventricular arrhythmias has not yet been evaluated thoroughly. The aim of this study was to explore GAL3 levels in patients with ARVD and its association with ventricular arrhythmias.Methods Twenty-nine patients with ARVD and 24 controls were included. All patients with ARVD had an implantable cardiac defibrillator (ICD) for primary or secondary prevention. Ventricular arrhythmia history was obtained from a chart review and ICD data interrogation. Galectin-3 levels were measured using an enzyme-linked immunosorbent assay.Results Patients with ARVD had higher plasma GAL3 levels (16.9 ± 2.6 ng/mL vs 11.3 ± 1.8 ng/mL, P < 0.001) than the control group. Ten patients had sustained or non-sustained ventricular arrhythmias during follow-up. In the multivariable analysis, left ventricular disease involvement (HR: 1.05; 95% CI: [1.01-1.12]; P = 0.03); functional capacity >2 (HR: 1.21; 95% CI: [1.13-1.31]; P < 0.005); and GAL3 levels (HR: 1.05; 95% CI: [1.00-1.11]; P = 0.01) independently predicted VT/VF.Conclusion We demonstrated that serum GAL3 was significantly elevated in patients with ARVD. Also, serum GAL 3 levels could be regarded as a candidate biomarker in the diagnosis of ARVD which needs to be tested in larger prospective studies. In addition, GAL3 levels were higher in patients with VT/VF as compared with those without VT/VF.