Cardiac Fibrosis Markers Research Articles

Conditional knockout of activin like kinase-1 (ALK-1) leads to heart failure without maladaptive remodeling.

Activin like kinase-1 (AlK-1) mediates signaling via the transforming growth factor beta (TGFβ) family of ligands. AlK-1 activity promotes endothelial proliferation and migration. Reduced AlK-1 activity is associated with arteriovenous malformations. No studies have examined the effect of global AlK-1 deletion on indices of cardiac remodeling. We hypothesized that reduced levels of AlK-1 promote maladaptive cardiac remodeling. To test this hypothesis, we employed AlK-1 conditional knockout mice (cKO) harboring the ROSA26-CreER knock-in allele, whereby a single dose of intraperitoneal tamoxifen triggered ubiquitous Cre recombinase-mediated excision of floxed AlK-1 alleles. Tamoxifen treated wild-type (WT-TAM; n=5) and vehicle treated AlK-1-cKO mice (cKO-CON; n=5) served as controls for tamoxifen treated AlK-1-cKO mice (cKO-TAM; n=15). AlK-1 cKO-TAM mice demonstrated reduced 14-day survival compared to cKO-CON controls (13 vs 100%, respectively, p<0.01). Seven days after treatment, cKO-TAM mice exhibited reduced left ventricular (LV) fractional shortening, progressive LV dilation, and gastrointestinal bleeding. After 14days total body mass was reduced, but LV and lung mass increased in cKO-TAM not cKO-CON mice. Peak LV systolic pressure, contractility, and arterial elastance were reduced, but LV end-diastolic pressure and stroke volume were increased in cKO-TAM, not cKO-CON mice. LV AlK-1 mRNA levels were reduced in cKO-TAM, not cKO-CON mice. LV levels of other TGFβ-family ligands and receptors (AlK5, TBRII, BMPRII, Endoglin, BMP7, BMP9, and TGFβ1) were unchanged between groups. Cardiomyocyte area and LV levels of BNP were increased in cKO-TAM mice, but LV levels of β-MHC and SERCA were unchanged. No increase in markers of cardiac fibrosis, Type I collagen, CTGF, or PAI-1, were observed between groups. No differences were observed for any variable studied between cKO-CON and WT-TAM mice. Global deletion of AlK-1 is associated with the development of high output heart failure without maladaptive remodeling. Future studies exploring the functional role of AlK-1 in cardiac remodeling independent of systemic AVMs are required.

Heart function and structure during the first year of haemodialysis treatment: Cardiac Uraemic Fibrosis Detection in Dialysis Patients, an observational prospective study

BackgroundPatients with end-stage renal disease undergoing haemodialysis are at high risk of sudden cardiac death. Advanced renal disease is associated with cardiac hypertrophy, which increases risk of life-threatening arrhythmias. Using a new MRI technique, T1 mapping, and the blood biomarker brain natriuretic peptide (BNP), we aimed to measure changes in function and structure of the heart muscle during the first year of haemodialysis treatment in the Cardiac Uraemic Fibrosis Detection in Dialysis Patients study. Methods28 adults with kidney disease on haemodialysis within Glasgow, UK, participated in this single-centre observational study. To be eligible for the study patients had to have been on haemodialysis for less than 1 year. Patients with MRI contraindications or who were expected to be on dialysis for less than 6 months were excluded. Primary outcome was volume of myocardial fibrosis (septal T1 time) on MRI at baseline and at 6 months' follow-up. Secondary outcome was change in serum BNP. Patients gave written consent, and ethics approval was given (13/WS/0301). This study is registered with the ISCTRN registry, number ISCTRN99591655. Findings22 patients completed both baseline and follow-up visits. There was no significant change in septal T1 time after 6 months of haemodialysis (septal T1 time baseline 1276·727 ms, follow-up 1271·837 ms). Left ventricular mass index (LVMI) was reduced at follow-up (baseline mean LVMI 78·3 g/m2 [SD 18·2], follow-up 67·9 [19·0]; p<0·0001). Differences in septal T1 times correlated with difference in LVMI (r=0·545, p=0·009). Median BNP did not significantly change between baseline and follow-up (2908 pg/mL [IQR 1639–5215] vs 2274·5 [1550–7055]). Change in LVMI was correlated with baseline BNP (r=0·492, p=0·02). Correlation between change in BNP and difference in septal T1 time was not significant. Interpretation6 months of haemodialysis therapy was associated with significant improvement in left ventricular hypertrophy but no significant change in T1 mapping markers of cardiac fibrosis or BNP. This small study suggests that starting haemodialysis might be associated with changes in myocardial structure. These changes could potentially be linked to control of uraemia, blood pressure, or both, which commonly occur on starting renal replacement therapy. FundingKidney Research UK (Innovation Grant IN02/2013).

Association between reverse electrical remodeling and cardiac fibrosis markers in patients with cardiac resynchronization therapy.

Cardiac resynchronization therapy (CRT) induces structural and electrical reverse remodeling of the failing heart. However, the association between native QRS narrowing and cardiac fibrosis markers has not been investigated in patients with an implanted CRT device. A total of 41 symptomatic patients diagnosed with systolic heart failure who underwent CRT implantation were included in this study. Electrocardiogram findings and cardiac fibrosis marker levels [galectin-3, growth-differentiation factor-15 (GDF-15) and procollagen III N-terminal propeptide (P3TD)] were collected before and 12 months after initiation of biventricular pacing. Reverse electrical remodeling was defined as a decrease in 12-month intrinsic QRS (iQRS) duration by ≥20 milliseconds after CRT implantation. The median QRS duration decreased from 155 milliseconds (interquartile range [IQR]: 142-178 milliseconds) before CRT to 142 milliseconds (IQR: 130-161 milliseconds) (p=0.001) after 12 months of CRT. According to the predefined criteria, electrical remodeling was detected in 16 (39.0%) patients. The median galectin-3, GDF-15, and P3TD levels were significantly decreased after CRT implantation in patients with electrical remodeling [27.65 ng/mL (IQR: 24.4-35.2 ng/mL) vs 23.00 ng/mL (IQR: 16.0-36.7 ng/mL), p=0.017; 3104 pg/mL (IQR: 2923-4825 pg/mL) vs 2276 pg/mL (IQR: 1294-3209 pg/mL), p=0.002; 0.43 ng/mL (IQR: 0.23-0.64) vs 0.15 ng/mL (IQR: 0.04-0.29 ng/mL), p=0.034, respectively]. The galectin-3, GDF-15, and P3TD levels were not significantly changed in patients without electrical remodeling [26.80 ng/mL (IQR: 23.9-31.5 ng/mL) vs 28.80 ng/mL (IQR: 23.0-34.8 ng/mL), p=0.211; 4221 pg/mL (IQR: 2709-4995 pg/mL) vs 3035 pg/mL (IQR: 2038-4872 pg/mL), p=0.143; and 0.34 ng/mL (IQR: 0.11-0.68 ng/mL) vs 0.21 ng/mL (IQR: 0.09-0.37 ng/mL), p=0.112, respectively]. The results from the small sample used in this study indicated that electrical reverse remodeling after CRT was associated with a decrease in cardiac fibrosis.

Baicalin Attenuates Cardiac Dysfunction and Myocardial Remodeling in a Chronic Pressure-Overload Mice Model

Background/Aims: Baicalin has been shown to be effective for various animal models of cardiovascular diseases, such as pulmonary hypertension, atherosclerosis and myocardial ischaemic injury. However, whether baicalin plays a role in cardiac hypertrophy remains unknown. Here we investigated the protective effects of baicalin on cardiac hypertrophy induced by pressure overload and explored the potential mechanisms involved. Methods: C57BL/6J-mice were treated with baicalin or vehicle following transverse aortic constriction or Sham surgery for up to 8 weeks, and at different time points, cardiac function and heart size measurement and histological and biochemical examination were performed. Results: Mice under pressure overload exhibited cardiac dysfunction, high mortality, myocardial hypertrophy, increased apoptosis and fibrosis markers, and suppressed cardiac expression of PPARα and PPARβ/δ. However, oral administration of baicalin improved cardiac dysfunction, decreased mortality, and attenuated histological and biochemical changes described above. These protective effects of baicalin were associated with reduced heart and cardiomyocyte size, lower fetal genes expression, attenuated cardiac fibrosis, lower expression of profibrotic markers, and decreased apoptosis signals in heart tissue. Moreover, we found that baicalin induced PPARα and PPARβ/δ expression in vivo and in vitro. Subsequent experiments demonstrated that long-term baicalin treatment presented no obvious cardiac lipotoxicity. Conclusions: The present results demonstrated that baicalin attenuates pressure overload induced cardiac dysfunction and ventricular remodeling, which would be due to suppressed cardiac hypertrophy, fibrosis, apoptosis and metabolic abnormality.

Autoimmune Fibrotic Adverse Reactions in One-Year Treatment with Cabergoline for Women with Prolactinoma.

Cabergoline is related to an elevated risk of fibrotic adverse reactions including cardiac valvular and pleuropulmonary fibrosis. We investigated pulmonary and cardiac valve fibrosis and immunological markers before and after 3 and 12 months of treatment with cabergoline in women with prolactinoma. The study included thirty-two women with newly diagnosed prolactinoma and 28 healthy women. CAB cumulative dose was 7.8±5.5 mg after 3-month therapy, and 31±22 mg after 12-month follow-up. The risk of autoimmune adverse fibrotic reactions related to CAB treatment including cardiac valvulopathy and pulmonary fibrosis were assessed by a transthoracic echocardiography and pulmonary function tests, respectively. Immunological markers including Antistreptolysin O, Rheumatoid factor, Immunglobuline E, Antinuchlear antibody were also evaluated. Before the start of CAB therapy, the total prevalence of trace grade of mitral, aortic, pulmonic, and tricuspid valve regurgitations were found as 34%, 3%, 6.3%, and 39 % respectively in women with prolactinoma. After improving of prolactin levels with CAB treatment, no change was found in the prevalence of the all valve regurgitations. There was no deterioration in pulmonary function tests. Rheumatoid factor was found higher in newly diagnosed women with prolactinoma than in healthy women (p=0.01), and this was improved by CAB therapy (p=0.005). The prospective study indicated that sufficient cabergoline doses for a period of one year treatment of prolactinoma were not found to be related to fibrotic adverse reactions including cardiac valvular and pulmonary fibrosis or increased levels of immunological marker, apart from rheumatoid factor. For the first time Rf was found higher in newly diagnosed women with prolactinoma and was improved after cabergoline therapy.

Long-Term Overexpression of Hsp70 Does Not Protect against Cardiac Dysfunction and Adverse Remodeling in a MURC Transgenic Mouse Model with Chronic Heart Failure and Atrial Fibrillation.

Previous animal studies had shown that increasing heat shock protein 70 (Hsp70) using a transgenic, gene therapy or pharmacological approach provided cardiac protection in models of acute cardiac stress. Furthermore, clinical studies had reported associations between Hsp70 levels and protection against atrial fibrillation (AF). AF is the most common cardiac arrhythmia presenting in cardiology clinics and is associated with increased rates of heart failure and stroke. Improved therapies for AF and heart failure are urgently required. Despite promising observations in animal studies which targeted Hsp70, we recently reported that increasing Hsp70 was unable to attenuate cardiac dysfunction and pathology in a mouse model which develops heart failure and intermittent AF. Given our somewhat unexpected finding and the extensive literature suggesting Hsp70 provides cardiac protection, it was considered important to assess whether Hsp70 could provide protection in another mouse model of heart failure and AF. The aim of the current study was to determine whether increasing Hsp70 could attenuate adverse cardiac remodeling, cardiac dysfunction and episodes of arrhythmia in a mouse model of heart failure and AF due to overexpression of Muscle-Restricted Coiled-Coil (MURC). Cardiac function and pathology were assessed in mice at approximately 12 months of age. We report here, that chronic overexpression of Hsp70 was unable to provide protection against cardiac dysfunction, conduction abnormalities, fibrosis or characteristic molecular markers of the failing heart. In summary, elevated Hsp70 may provide protection in acute cardiac stress settings, but appears insufficient to protect the heart under chronic cardiac disease conditions.

Lactobacillus reuteri GMNL-263 reduces hyperlipidaemia and the heart failure process in high-calorie diet-fed induced heart dysfunction in rats

Probiotics are reported to provide protective health benefits against various disorders. In this study, we established a high-calorie diet animal model with a diet prepared by fortifying normal diet with soybean oil and condensed milk and the animals were supplemented with different dose of probiotic Lactobacillus reuteri GMNL-263(Lr263) for 8 weeks to investigate the protective function. We measured the lipid levels in plasma, liver and faecal; heart weight, function and collagen accumulation and biochemical markers of cardiac disease. Lr263 reversed high-calorie diet-induced cardiac weight gain, systolic malfunction and prevented heart inflammation by decreasing inflammation proteins NF-κB and COX-2. Moreover, Lr263 reduced cardiac eccentric hypertrophy and the levels of cardiac fibrosis markers CTGF and SP1 in high-calorie diet-fed rat hearts. Thus, Lr263 is considered as a functional food that provides anti-inflammatory, anti-hypertrophic, anti-fibrotic functions and identified specific biologic effects in reducing the characteristics of the high-calorie diet-induced progression to heart failure.

Resveratrol is equipotent to perindopril in attenuating post-infarct cardiac remodeling and contractile dysfunction in rats

BackgroundAngiotensin-converting enzyme (ACE) inhibitors improve prognosis in patients with post-myocardial infarction (MI) related cardiac dysfunction. Resveratrol is a polyphenol that has been reported to be beneficial in hypertension, ischemic heart disease, and cardiotoxicity in preclinical studies. Accordingly, we investigated the comparative and combinatorial efficacy of resveratrol and perindopril (ACE inhibitor) treatment on MI-related cardiac remodeling and contractile dysfunction. MethodsLeft anterior descending artery-ligated and sham-operated male Sprague–Dawley rats were gavaged with vehicle, resveratrol, perindopril, and combination of resveratrol+perindopril (2.5 mg/kg bodyweight/day) for 8 weeks (starting immediately after acute MI). Echocardiography was performed to assess cardiac structure and function at baseline and 8 weeks. ResultsAt 8 weeks, vehicle-MI rats had a significantly lower left ventricular ejection fraction (LVEF) and increased LV dilatation compared to vehicle-sham rats. MI rats treated with resveratrol, perindopril and a combination of both had significantly improved LVEF and reduced LV dilatation. Vehicle-treated MI rats also had increased level of lipid peroxidation product- malondialdehyde (MDA), proinflammatory protein- tumor necrosis factor-alpha (TNF-α) and cardiac fibrosis marker- collagen and decreased enzymatic activity of superoxide dismutase and catalase compared to vehicle-sham rats. Resveratrol, perindopril and combination of both significantly prevented the /ed to determine systolic functional parameter increase in MDA, TNF-α and collagen and improved the activity of superoxide dismutase and catalase in MI rats compared to vehicle-MI rats. ConclusionTreatment with resveratrol or perindopril was equivalent in significantly improving remodeling and attenuation of contractile dysfunction in MI rats. Combination treatment also attenuated the cardiac abnormalities. The improvement in cardiac abnormalities may partly be through reducing oxidative stress by preventing the decrease in the activity of superoxide dismutase and catalase, and decreasing cardiac inflammation and fibrosis.

Effect of Mineralocorticoid Receptor Antagonists on Cardiac Structure and Function in Patients With Diastolic Dysfunction and Heart Failure With Preserved Ejection Fraction: A Meta-Analysis and Systematic Review.

BackgroundThere has been an increasing interest in use of mineralocorticoid receptor antagonists (MRAs) in patients with heart failure with preserved ejection fraction (HFPEF). However, a comprehensive evaluation of MRA effects on left ventricular (LV) structure and function in these patients is lacking. In this meta‐analysis, we evaluated the effects of MRAs on LV structure and function among patients with diastolic dysfunction or HFPEF.Methods & ResultsRandomized, controlled clinical trials evaluating the efficacy of MRAs in patients with diastolic dysfunction or HFPEF were included. The primary outcome was change in E/e’, a specific measure of diastolic function. Secondary outcomes included changes in other measures of diastolic function, LV structure, surrogate markers for myocardial fibrosis (carboxy‐terminal peptide of procollagen type I [PICP] and amino‐terminal peptide of pro‐collagen type‐II [PIIINP]), blood pressure, and exercise tolerance. In the pooled analysis, MRA use was associated with significant reduction in E/e’ (weighted mean difference [WMD] [95% confidence interval {CI}]: −1.68 [−2.03 to −1.33]; P<0.0001) and deceleration time (WMD [95% CI]: −12.0 ms [−23.3 to −0.7]; P=0.04) as compared with control, suggesting and improvement in diastolic function. Furthermore, blood pressure and levels of PIIINP and PICP were also significantly reduced with MRA therapy with no significant change in LV mass or dimensions.Conclusion MRA therapy in patients with asymptomatic diastolic dysfunction or HFPEF is associated with significant improvement in diastolic function and markers of cardiac fibrosis without a significant change in LV mass or dimensions.

Abstract P070: Exercise Prevents Cardiac Diastolic Dysfunction in a Female Model of Over-Nutrition Induced Obesity by Reducing Oxidant Stress and Fibrosis without Lowering Bodyweight

Obesity is being classified as a global epidemic by both the WHO and the CDC. Sedentary lifestyle and consumption of a high-fat/high-fructose Western diet (WD) are implicated in this epidemic. Women are particularly vulnerable to obesity related cardiovascular disease. Obese women suffer higher rates of hypertension, insulin resistance and heart failure, especially diastolic heart failure early in their lives. There are no evidence based treatments for diastolic heart failure. We hypothesized that voluntary daily exercise would prevent WD induced diastolic dysfunction by reducing oxidant stress, fibrosis and inflammation. To test this hypothesis, we developed a diastolic heart failure model by subjecting C57BL6/J female mice to a solely WD fed regimen for 16 weeks. We treated a parallel cohort with daily exercise, via voluntary wheel running, for the entire 16 weeks of WD feeding alongside control diet (CD) groups (n=7 for each group). We monitored food consumption, running activity and body weight. After 16 weeks, we assessed diastolic function by cardiac MRI and echocardiography. Detailed myocardial staining and Western blotting were done for cardiac oxidant stress, fibrosis and inflammatory markers. Both imaging modalities revealed diastolic dysfunction with WD feeding that was normalized by voluntary exercise. Mice ran similarly high intensities on both CD (6.5 km/d) and WD (7.1km/d). While WD feeding increased bodyweight, there was no reduction in weight with exercise. Body composition analysis showed WD fed mice treated with voluntary exercise had increases in visceral fat weight similar to sedentary WD fed mice. There was a notable increase in lean body mass with exercise. WD feeding resulted in insulin resistance that was prevented by exercise. Finally, while WD feeding markedly increased oxidant stress and fibrosis in sedentary mice, exercise prevented myocardial oxidant stress and fibrosis. Our work provides seminal evidence that diastolic dysfunction of over-nutrition induced obesity can be prevented by exercise. Surprisingly, our study suggests that the mechanisms behind the amelioration of diastolic dysfunction are predominantly through reductions in oxidant stress and fibrosis without reductions in body weight or visceral fat.

Oleanolic acid alleviated pressure overload-induced cardiac remodeling.

Previous study has demonstrated that oleanolic acid (OA) possessing the anti-inflammatory and anti-oxidant properties blunted high-glucose-induced diabetic cardiomyopathy and ameliorated experimental autoimmune myocarditis in mice. However, little is known about its effects on pressure overload-induced cardiac remodeling. Herein, we investigated the effect of OA on cardiac remodeling and underlying mechanism. Mice, subjected to aortic banding (AB), were randomly assigned into control group and experimental group. OA premixed in diets was administered to mice after 3days of AB. Echocardiography and catheter-based measurements of hemodynamic parameters were performed after 8weeks' treatment of OA. Histologic examination and molecular analyses were used to assess cardiac hypertrophy and tissue fibrosis. In addition, the inhibitory effects of OA on H9c2 cardiomyocytes and cardiac primary fibroblast responded to the stimulation of AngII were also investigated. OA ameliorated the systolic and diastolic dysfunction induced by pressure overload evidenced by echocardiography and catheter-based measurements. OA also decreased the mRNA expression of cardiac hypertrophy and fibrosis markers evidenced by RT-PCR. It has been shown in our study that pressure overload activated the phosphorylations of Akt, mTOR, p70s6k, S6, GSK3β, and FoxO3a, and treatment of OA attenuated the phosphorylation of these proteins. In addition, hypertrophy of cardiomyocytes and fibrosis markers induced by AngII was inhibited by OA in vitro. Our findings uncover that OA suppressed AB-induced cardiac hypertrophy, partly by inhibiting the activity of Akt/mTOR pathway, and suggest that treatment of OA may have a benefit on retarding the progress of cardiac remodeling under long terms of pressure overload.

Galectin-3 levels in patients with hypertrophic cardiomyopathy and its relationship with left ventricular mass index and function.

Objective:Cardiac fibrosis is an important contributor to adverse left ventricular (LV) remodeling and arrhythmias in patients with hypertrophic cardiomyopathy (HCM). Galectin-3 (Gal-3) is a novel marker of cardiac fibrosis and inflammation. In this study, we investigated Gal-3 levels in patients with HCM and controls and assessed the relationship between Gal-3 level and echocardiographic indices using strain echocardiography in patients with HCM.Methods:Forty patients with HCM in sinus rhythm and 35 healthy controls were prospectively enrolled in this case-control study. The HCM diagnosis was based on two-dimensional echocardiographic demonstration of a hypertrophied and non-dilated left ventricle (LV) with a wall thickness ≥15 mm in one or more LV myocardial segments in the absence of any cardiac or systemic disease capable of inducing LV hypertrophy. Patients with one of the followings were excluded: coronary artery disease, atrial fibrillation episodes on 24-h Holter electrocardiogram (ECG) monitoring, history of an invasive intervention to alleviate an LV outflow (LVOT) obstruction, inadequate image quality, renal disease, diabetes mellitus, hyperlipidemia, liver cirrhosis, and pulmonary fibrosis. Global LV longitudinal, circumferential strain and strain rates, peak torsion, and LV mass index (LVMI) of all subjects were assessed by echocardiography. Gal-3 levels were measured in all subjects.Results:Left ventricular global longitudinal strain (-13.37±4.6% vs. -18.93±2.5%, p<0.001) and strain rate (0.66±0.22 s-1 vs. 1.08±0.14 s-1, respectively; p<0.001) values were lower in patients with HCM than in controls. Gal-3 levels were significantly higher in patients with HCM than in controls (16.9±6.64 ng/mL vs. 13.21±3.42 ng/mL, p=0.005). Gal-3 levels were associated with the thickness of the interventricular septum (r=0.444, p=0.004) and LVMI (r=0.365, p=0.021); however, they were not associated with LV global longitudinal strain (p=0.42) or strain rate (p=0.28).Conclusion:Gal-3 levels increased and were correlated with the degree of LV hypertrophy in patients with HCM. Gal-3 is not a good marker of decreased myocardial LV diastolic and systolic functions in these patients.

Increased levels of galectin-3 were associated with prediabetes and diabetes: new risk factor?

Galectin-3 (Gal-3) is a marker of cardiac fibrosis and predicts incident heart failure. Gal-3-deficient mice are resistant to multiple low-dose streptozotocin-induced diabetes. Recent experimental studies suggested an important role for Gal-3 in the regulation of adiposity, metaflammation and type 2 diabetes. This study aimed to examine the relationship between Gal-3 and newly diagnosed prediabetes and diabetes. Gal-3 concentrations were measured in 118 participants and 56 age- and sex-matched healthy controls. All subjects underwent a 75-g oral glucose tolerance test and were stratified into normal, prediabetic, and diabetes mellitus subgroups. DM was defined as a plasma glucose level ≥126 mg/dL in the fasting state or ≥200 mg/dL in the second hour after glucose loading. Impaired fasting glucose was defined as an FPG level of 100-125 mg/dL, and impaired glucose tolerance was defined as a 2-h plasma glucose level of 140-199 mg/dL. Sixty-one patients had prediabetes (Group 1), 57 had diabetes (Group 2), and 56 had neither diabetes nor prediabetes (Group 3). Gal-3 levels correlated with FPG (r = 0.787, P < 0.01), 2hPG (r = 0.833, P < 0.01), CRP (r = 0.501, P < 0.01), and HOMA-IR (r = 0.518, P < 0.01). Gal-3 levels were higher in Group 2 than in Groups 1 and 3 [1,053.9 (358.1) and 744.1 (119.3) vs. 481.7 (175.4) pg/mL; P < 0.001]. Gal-3 is an independent predictor of diabetes in multivariate logistic analysis. In ROC analysis, a Gal-3 cutoff value of 803.55 pg/mL diagnoses diabetes with a sensitivity of 80.7 % and a specificity of 85.5 % (AUC = 0.912). Gal-3 is a promising biomarker for detecting prediabetes and diabetes.

Ex vivo and in vivo administration of fluorescent CNA35 specifically marks cardiac fibrosis.

Cardiac fibrosis is a major hallmark of cardiac diseases. For evaluation of cardiac fibrosis, the development of highly specific and preferably noninvasive methods is desired. Our aim was to evaluate CNA35, a protein known to specifically bind to collagen, as a specific marker of cardiac fibrosis. Fluorescently labeled CNA35 was applied ex vivo on tissue sections of fibrotic rat, mouse, and canine myocardium. After quantification of CNA35, sections were examined with picrosirius red (PSR) and compared to CNA35. Furthermore, fluorescently labeled CNA35 was administered in vivo in mice. Hearts were isolated, and CNA35 labeling was examined in tissue sections. Serial sections were histologically examined with PSR. Ex vivo application of CNA35 showed specific binding to collagen and a high correlation with PSR (Pearson r = .86 for mice/rats and r = .98 for canine; both p < .001). After in vivo administration, CNA35 labeling was observed around individual cardiomyocytes, indicating its ability to penetrate cardiac endothelium. High correlation was observed between CNA35 and PSR (r = .91, p < .001). CNA35 specifically binds to cardiac collagen and can cross the endothelial barrier. Therefore, labeled CNA35 is useful to specifically detect collagen both ex vivo and in vivo and potentially can be converted to a noninvasive method to detect cardiac fibrosis.

Increased gene expression of the cardiac endothelin system in obese mice.

Obesity is a well-known risk factor of atherosclerosis and heart failure. In the human heart, a local endothelin system containing prepro-endothelin-1, endothelin-converting enzyme-1, and endothelin receptors A and B has been described. The endothelin system is activated in heart failure; however, the impact of obesity on the cardiac endothelin system is unknown. In this study, 18-week-old male C57BL/6 mice fed either a control diet or a high-fat diet for 10 weeks were analyzed. High-fat diet significantly increased the body weight of the animals and augmented low-density lipoprotein, high-density lipoprotein, and cholesterol plasma levels, compared to control. The animal groups showed no significant differences in left ventricular size or function (heart rate, ejection fraction, fractional shortening, left ventricular posterior wall thickness, cardiac output) after control or high-fat diet. We did not observe signs of cardiac hypertrophy or changes in markers of cardiac fibrosis in these heart samples. The cardiac expression of prepro-endothelin-1 mRNA, endothelin-converting enzyme-1 mRNA, and protein and endothelin receptors A and B mRNA was increased in 18-week-old obese C57BL/6 mice compared to animals with normal weight (p<0.05 vs. control). Furthermore, endothelin-1 plasma levels showed an increasing trend. In conclusion, an increased expression of genes of the endothelin system was observed in the hearts of 18-week-old mice after high-fat diet, possibly contributing to later cardiovascular complications of obesity.

Abstract 403: MicroRNA-21 Ablation Exacerbates Aldosterone-mediated Cardiac Inflammation, Fibrosis, Hypertrophy and Dysfunction

Excess aldosterone (ALDO) causes hypertension and cardiac hypertrophy, inflammation and fibrosis that lead to cardiac dysfunction. We reported that ALDO/SALT treatment increases left ventricle miR-21 expression after 2 weeks of treatment, and the elevation persists for up to 8 weeks. We aim to study the role of genetic ablation of miR-21 in ALDO/SALT-mediated cardiac inflammation, fibrosis, hypertrophy and function. Eight-week old uninephrectomized male miR-21 knockout (miR21KO) or wild-type (WT) mice were assigned to 4 groups (N=12/grp): WT-Control, WT-ALDO/SALT, miR21KO-Control and miR21KO-ALDO/SALT. Animals were treated for 8 weeks. ALDO (0.15 μg/h) or vehicle (PEG 300) was administered by osmotic minipump. SALT (1.0% NaCl + 0.3% KCl) was administered in tap water. Cardiac function was determined by echocardiography. Tissues were weighed and standardized to body weight. mRNA levels were quantified by qPCR. Another set of animals (N=12/grp) was implanted with carotid artery catheters for conscious MAP determination. ALDO/SALT-mediated cardiac dysfunction was exacerbated in miR21KO mice (EF: 38.0 ± 2.0 vs. 43.0 ± 4.1 %, p &lt;0.05) compared with WT mice (EF: 50.0 ± 3.7 vs. 54.7 ± 3.1 %, NS). ALDO/SALT-mediated cardiac hypertrophy was exacerbated in miR21KO mice compared with WT mice (ALDO-SALT/control: 1.30 ± 0.04 vs . 1.18 ± 0.02, p &lt;0.05). Fibrosis marker connective tissue growth factor (Ctgf) mRNA expression remained elevated by ALDO/SALT only in miR21KO mice (7.22 ± 1.96 fold change vs . control, p &lt;0.05). Similar results were observed with collagen I and fibronectin. Inflammation marker Serpine1 (PAI-1) mRNA expression was upregulated by ALDO/SALT only in miR21KO mice (3.11 ± 0.54 fold change vs . control, p &lt;0.05). Despite the exacerbation in cardiac dysfunction, hypertrophy, and fibrosis and inflammation markers expression in miR21KO mice, ALDO/SALT raised MAP to similar levels in both strains (WT: 139 ± 9 vs . 106 ± 2 mm Hg, p &lt;0.05; miR21KO: 142 ± 8 vs . 114 ± 4, p &lt; 0.05). These results show that miR-21 ablation exacerbates ALDO/SALT-mediated cardiac inflammation, fibrosis, hypertrophy and dysfunction independently of blood pressure suggesting that therapies that increase cardiac levels of miR-21 may benefit patients with primary aldosteronism.

Inhibition of farnesyl pyrophosphate synthase prevents angiotensin II-induced cardiac fibrosis in vitro

Farnesyl pyrophosphate synthase (FPPS)-catalysed isoprenoid intermediates are important for the activation of Ras homologue gene family, member A (RhoA) in angiotensin (Ang) II-induced cardiac fibrosis. This study was designed to investigate the specific role of FPPS in the development of cardiac fibrosis. We demonstrated that FPPS expression was elevated in both in-vivo and in-vitro models of Ang II-mediated cardiac fibrosis. FPPS inhibition by zolendronate and FPPS knock-down by a silencing lentivirus decreased the expression of cardiac fibrosis marker genes, including collagen I, collagen III and transforming growth factor (TGF)-β1. FPPS inhibition was reversed by geranylgeraniol (GGOH) and mimicked by RhoA knock-down with siRhoA. The antagonistic effect of GGOH on the zolendronate-mediated modulation of RhoA activation in Ang II-stimulated cardiac fibroblasts was demonstrated by a pull-down assay. Furthermore, FPPS knock-down also prevented RhoA activation by Ang II in vitro. In conclusion, FPPS and RhoA may be part of a signalling pathway that plays an important role in Ang II-induced cardiac fibrosis in vitro.

Galectin-3 testing: Validity of a novel automated assay in heart failure patients with reduced ejection fraction

Circulating levels of galectin-3 (Gal-3), a marker of cardiac fibrosis and remodeling, contribute to the risk stratification of patients with heart failure (HF). The aim of our study was to determine the analytical validity and clinical validity of a novel automated Gal-3 assay in HF patients with reduced ejection fraction. We showed an excellent agreement between the VIDAS® Gal-3 automated assay and the ELISA reference method (r=0.90, p<0.001) and a mean difference of –1.3ng/mL was observed on the Bland and Altman plot. Gal-3 levels measured with the VIDAS® assay were significantly related to NYHA functional classes (p<0.001) and mean Gal-3 levels were 13.8ng/mL in NYHA II patients, 17.7ng/mL in NYHA III and 19.6ng/mL in NYHA IV. Furthermore, our results showed that Gal-3 levels measured with the VIDAS® assay were not only predictive of long-term cardiovascular death in patients with systolic HF but have also provided added value to natriuretic peptide testing in multimarker strategies. Therefore, our data are also supporting the clinical validity of the Gal-3 automated assay.

Soluble ST2 (sST2) Is a Novel Valuable Prognostic Marker Among Patients With Immunoglobulin Light Chain (AL) Amyloidosis

The use of soluble cardiac biomarkers such as NT-proBNP and troponin has revolutionized prognostication for patients with AL amyloidosis and led to their use in the Mayo 2004 and 2012 staging systems. Novel soluble markers with different phenotypic targets may further improve this approach. Soluble ST2 (sST2), which is the circulating form of the IL-33 receptor, has been shown to be a marker of cardiac remodelling and fibrosis, is predictive of mortality in patients with congestive heart failure, and is predictive of risk of GVHD and GVHD mortality.Samples of blood of patients with AL amyloidosis collected and frozen at -20C under a biobank IRB protocol were retrieved and sST2 measured. Patients were eligible for present study if they had a research sample collected within 90 days of their AL amyloidosis diagnosis. We have validated that values do not change significantly with storage at -20C over a 90 day period. Concentrations of sST2 were determined using a novel high-sensitivity sandwich immunoassay (Presagew ST2; Critical Diagnostics, San Diego, CA, USA). The sST2 assay had within-run and total coefficients of variation of <7.5% and 13.0% across its measurement range. Samples from 273 consecutive patients meeting diagnostic criteria for AL amyloidosis seen at our institution from 1/9/2010 to 12/22/2011 were evaluated. Median age was 63. 58% were male. One hundred and thirty have died over 33.9 months. Correlation between sST2 and the other biomarkers was modest with correlation coefficients of 0.48 and 0.20 for NT-proBNP and Troponin T, respectively. Patients with sST2 above and below the median of 27.2 ng/mL (6.3-596) had significant differences in 2 year overall survival: 71.2% to 39% (figure). In multivariate modeling sST2's prognostic value was independent of the Mayo 2004 and the Mayo 2012 cardiac biomarker staging systems, the latter of which also includes prognostic information relating to plasma cell burden. In these multivariate models, sST2 median had a RR of 2.4, (95%CI 1.6, 3.6 p<0.001) with the Mayo 2004 Staging system (RR of 2.9, 95%CI 2.1, 4.1p<0.001); and sST2 median had a RR of 2.2 (95%CI 1.5, 3.4, p<0.001) with the Mayo 2012 staging system (RR 1.9, 95%CI 1.6, 2.3, p<0.001). [Display omitted] A larger cohort including patients with longer follow up will be presented at the meeting as will gender specific cut off values and a comparison of our values with those previously used to prognosticate in patients with heart failure. sST2 appears to be a novel powerful prognostic factor for patients with AL amyloidosis. Because sST2 functions as a decoy receptor, which neutralizes the benefits of IL-33, it may play a role in the deleterious phenotype seen in patients with AL, i.e. myocardial hypertrophy and reduction of contractility. Disclosures:Saenger:Critical Diagnostics : Research Funding; Roche: Research Funding. Jaffe:Critical Diagnostics: Consultancy, Research Funding; Roche: Consultancy.

Ex Vivo and In Vivo Administration of Fluorescent CNA35-Protein Specifically Marks Cardiac Fibrosis

Background Cardiac fibrosis has severe adverse effects on systolic and diastolic function and increases the risk of arrhythmias. To detect cardiac fibrosis, the development of highly specific and preferably noninvasive methods is desired. In this study, the potential of labeled CNA35, a protein that specifically binds to vascular collagen, is investigated as a specific marker of cardiac fibrosis. Picrosirius red (PSR) staining served as reference for cardiac fibrosis detection. Methods Fluorescently labeled CNA35 (2.5 µM) was applied to frozen tissue sections of dog myocardium (n = 36). After CNA35 quantification, sections were histologically examined with PSR and compared to CNA35. Furthermore, fluorescently labeled CNA35 (2 × 38 µM) was administered intravenously in mice (n = 8). Hearts were isolated, and CNA35 labeling was examined in frozen tissue sections (n = 34). Serial sections were examined with PSR. Results CNA35 application on frozen canine myocardium shows specific CNA35 binding to collagen. CNA35 labeling highly correlates with PSR (r = 0.98, P in vivo administration in mice, myocardial CNA35 labeling is clearly observed, suggesting that labeled CNA35 is able to penetrate the cardiac endothelium. Strong correlation is observed between CNA35 and PSR (r = 0.91, P Conclusions CNA35 specifically binds to cardiac collagen when applied to frozen sections and after in vivo application of CNA35. Strong correlation has been observed between CNA35 and PSR. These data indicate that CNA35 is useful for quantification and localization of myocardial fibrosis and warrants further research into its use for monitoring cardiac fibrosis in vivo .