Publications by authors named "Carmen C Sucharov"

58 Publications

Circulating cyclic adenosine monophosphate concentrations in milrinone treated paediatric patients after congenital heart surgery.

Cardiol Young 2021 Feb 3:1-8. Epub 2021 Feb 3.

Department of Pediatrics, Division of Pediatric Cardiology, University of Colorado Denver, Anschutz Medical Campus, Children's Hospital Colorado, Aurora, CO, USA.

Background: Milrinone is a phosphodiesterase type 3 inhibitor that results in a positive inotropic effect in the heart through an increase in cyclic adenosine monophosphate. The purpose of this study was to evaluate circulating cyclic adenosine monophosphate and milrinone concentrations in milrinone treated paediatric patients undergoing congenital heart surgery.

Methods: Single-centre prospective observational pilot study from January 2015 to December 2017 including children aged birth to 18 years. Milrinone and circulating cyclic adenosine monophosphate concentrations were measured at four time points through the first post-operative day and compared between patients with and without low cardiac output syndrome, defined using clinical and laboratory criteria.

Results: Fifty patients were included. Nine (18%) developed low cardiac output syndrome. For all patients, 22% had single ventricle heart disease. The density and distribution of cyclic adenosine monophosphate concentrations varied between those with and without low cardiac output syndrome but were not significantly different. Milrinone concentrations increased in all patients. Paired t-tests demonstrated an increase in circulating cyclic adenosine monophosphate concentrations during the post-operative period among patients without low cardiac output syndrome.

Conclusions: In this prospective observational study, circulating cyclic adenosine monophosphate concentrations increased in those without low cardiac output syndrome during the first 24 post-operative hours and milrinone concentrations increased in all patients. Further study of the utility of cyclic adenosine monophosphate concentrations in milrinone treated patients is necessary.
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http://dx.doi.org/10.1017/S1047951121000251DOI Listing
February 2021

Circulating microRNAs differentiate Kawasaki Disease from infectious febrile illnesses in childhood.

J Mol Cell Cardiol 2020 09 4;146:12-18. Epub 2020 Jul 4.

Pediatric Cardiology, Children's Hospital Colorado, University of Colorado School of Medicine, Aurora, CO, USA.

Background: Kawasaki Disease (KD) is an acute vasculitis of unknown etiology in children that can lead to coronary artery lesions (CAL) in 25% of untreated patients. There is currently no diagnostic test for KD, and the clinical presentation is often difficult to differentiate from other febrile childhood illnesses. Circulating microRNAs (miRNAs) are small noncoding RNA molecules that control gene expression by inducing transcript degradation or by blocking translation. We hypothesize that the expression of circulating miRNAs will differentiate KD from non-KD febrile illnesses in children.

Methods: Circulating miRNA profiles from 84 KD patients and 29 non-KD febrile controls (7 viral and 22 bacterial infections) were evaluated. 3 ul of serum from each subject was submitted to 3 freeze/heat cycles to ensure miRNA release from microvesicles or interaction with serum proteins. miRNAs were reverse transcribed using a pool of primers specific for each miRNA. Real-time PCR reactions were performed in a 384 well plate containing sequence-specific primers and TaqMan probes in the ABI7900. '.

Results: KD patients (3.6 ± 2.2 yrs., 58% male) were found to have a unique circulating miRNA profile, including upregulation of miRNA-210-3p, -184, and -19a-3p (p < .0001), compared to non-KD febrile controls (8.5 ± 6.1 yrs., 72% male).

Conclusions: Circulating miRNAs can differentiate KD from infectious febrile childhood diseases, supporting their potential as a diagnostic biomarker for KD.
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http://dx.doi.org/10.1016/j.yjmcc.2020.06.011DOI Listing
September 2020

MicroRNA regulation postbleomycin due to the R213G extracellular superoxide dismutase variant is predicted to suppress inflammatory and immune pathways.

Physiol Genomics 2020 06 18;52(6):245-254. Epub 2020 May 18.

Developmental Lung Biology and Cardiovascular Pulmonary Research Laboratories, Departments of Pediatrics and Medicine, University of Colorado, Anschutz Medical Campus, Aurora, C Colorado.

Oxidative stress is a key contributor to the development of dysregulated inflammation in acute lung injury (ALI). A naturally occurring single nucleotide polymorphism in the key extracellular antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), results in an arginine to glycine substitution (R213G) that promotes resolution of inflammation and protection against bleomycin-induced ALI. Previously we found that mice harboring the R213G mutation in EC-SOD exhibit a transcriptomic profile consistent with a striking suppression of inflammatory and immune pathways 7 days postbleomycin. However, the alterations in noncoding regulatory RNAs in wild-type (WT) and R213G EC-SOD lungs have not been examined. Therefore, we used next-generation microRNA (miR) Sequencing of lung tissue to identify dysregulated miRs 7 days after bleomycin in WT and R213G mice. Differential expression analysis identified 92 WT and 235 R213G miRs uniquely dysregulated in their respective genotypes. Subsequent pathway analysis identified that these miRs were predicted to regulate approximately half of the differentially expressed genes previously identified. The gene targets of these altered miRs indicate suppression of immune and inflammatory pathways in the R213G mice versus activation of these pathways in WT mice. Triggering receptor expressed on myeloid cells 1 (TREM1) signaling was identified as the inflammatory pathway with the most striking difference between WT and R213G lungs. miR-486b-3p was identified as the most dysregulated miR predicted to regulate the TREM1 pathway. We validated the increase in TREM1 signaling using miR-486b-3p antagomir transfection. These findings indicate that differential miR regulation is predicted to regulate the inflammatory gene profile, contributing to the protection against ALI in R213G mice.
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http://dx.doi.org/10.1152/physiolgenomics.00116.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311677PMC
June 2020

Dysregulated micro-RNAs and long noncoding RNAs in cardiac development and pediatric heart failure.

Am J Physiol Heart Circ Physiol 2020 05 27;318(5):H1308-H1315. Epub 2020 Mar 27.

Division of Cardiology, Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado.

Noncoding RNAs (ncRNAs) are broadly described as RNA molecules that are not translated into protein. The investigation of dysregulated ncRNAs in human diseases such as cancer, neurological, and cardiovascular diseases has been under way for well over a decade. Micro-RNAs and long noncoding RNAs (lncRNAs) are the best characterized ncRNAs. These ncRNAs can have profound effects on the regulation of gene expression during cardiac development and disease. Importantly, ncRNAs are significant regulators of gene expression in several congenital heart diseases and can positively or negatively impact cardiovascular development. In this review, we focus on literature involving micro-RNAs and lncRNAs in the context of pediatric cardiovascular diseases, preclinical models of heart failure, and cardiac development.
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http://dx.doi.org/10.1152/ajpheart.00511.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346540PMC
May 2020

Paracrine Factors in Uremic Cardiomyopathy.

JACC Basic Transl Sci 2020 Feb 24;5(2):167-168. Epub 2020 Feb 24.

Division of Cardiology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

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http://dx.doi.org/10.1016/j.jacbts.2020.01.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046544PMC
February 2020

Alteration of cardiolipin biosynthesis and remodeling in single right ventricle congenital heart disease.

Am J Physiol Heart Circ Physiol 2020 04 14;318(4):H787-H800. Epub 2020 Feb 14.

Division of Cardiology, Department of Pediatrics, University of Colorado School of Medicine, Children's Hospital Colorado, Aurora, Colorado.

Despite advances in both medical and surgical therapies, individuals with single ventricle heart disease (SV) remain at high risk for the development of heart failure (HF). However, the molecular mechanisms underlying remodeling and eventual HF in patients with SV are poorly characterized. Cardiolipin (CL), an inner mitochondrial membrane phospholipid, is critical for proper mitochondrial function, and abnormalities in CL content and composition are known in various cardiovascular disease etiologies. The purpose of this study was to investigate myocardial CL content and composition in failing and nonfailing single right ventricle (RV) samples compared with normal control RV samples, to assess mRNA expression of CL biosynthetic and remodeling enzymes, and to quantitate relative mitochondrial copy number. A cross-sectional analysis of RV myocardial tissue from 22 failing SV (SVHF), 9 nonfailing SV (SVNF), and 10 biventricular control samples (BVNF) was performed. Expression of enzymes involved in CL biosynthesis and remodeling were analyzed using RT-qPCR and relative mitochondrial DNA copy number determined by qPCR. Normal phase high-pressure liquid chromatography coupled to electrospray ionization mass spectrometry was used to quantitate total and specific CL species. While mitochondrial copy number was not significantly different between groups, total CL content was significantly lower in SVHF myocardium compared with BVNF controls. Despite having lower total CL content however, the relative percentage of the major tetralinoleoyl CL species is preserved in SVHF samples relative to BVNF controls. Correspondingly, expression of enzymes involved in CL biosynthesis and remodeling were upregulated in SVHF samples when compared with both SVNF samples and BVNF controls. The mechanisms underlying heart failure in the single ventricle (SV) congenital heart disease population are largely unknown. In this study we identify alterations in cardiac cardiolipin metabolism, composition, and content in children with SV heart disease. These findings suggest that cardiolipin could be a novel therapeutic target in this unique population of patients.
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http://dx.doi.org/10.1152/ajpheart.00494.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7191493PMC
April 2020

Increased myocyte calcium sensitivity in end-stage pediatric dilated cardiomyopathy.

Am J Physiol Heart Circ Physiol 2019 12 18;317(6):H1221-H1230. Epub 2019 Oct 18.

Division of Cardiology, Department of Medicine, University of Colorado Denver, Aurora, Colorado.

Dilated cardiomyopathy (DCM) is the most common cause of heart failure (HF) in children, resulting in high mortality and need for heart transplantation. The pathophysiology underlying pediatric DCM is largely unclear; however, there is emerging evidence that molecular adaptations and response to conventional HF medications differ between children and adults. To gain insight into alterations leading to systolic dysfunction in pediatric DCM, we measured cardiomyocyte contractile properties and sarcomeric protein phosphorylation in explanted pediatric DCM myocardium ( = 8 subjects) compared with nonfailing (NF) pediatric hearts ( = 8 subjects). Force-pCa curves were generated from skinned cardiomyocytes in the presence and absence of protein kinase A. Sarcomeric protein phosphorylation was quantified with Pro-Q Diamond staining after gel electrophoresis. Pediatric DCM cardiomyocytes demonstrate increased calcium sensitivity (pCa =5.70 ± 0.0291), with an associated decrease in troponin (Tn)I phosphorylation compared with NF pediatric cardiomyocytes (pCa =5.59 ± 0.0271, = 0.0073). Myosin binding protein C and TnT phosphorylation are also lower in pediatric DCM, whereas desmin phosphorylation is increased. Pediatric DCM cardiomyocytes generate peak tension comparable to that of NF pediatric cardiomyocytes [DCM 29.7 mN/mm, interquartile range (IQR) 21.5-49.2 vs. NF 32.8 mN/mm, IQR 21.5-49.2 mN/mm; = 0.6125]. In addition, cooperativity is decreased in pediatric DCM compared with pediatric NF (Hill coefficient: DCM 1.56, IQR 1.31-1.94 vs. NF 1.94, IQR 1.36-2.86; = 0.0425). Alterations in sarcomeric phosphorylation and cardiomyocyte contractile properties may represent an impaired compensatory response, contributing to the detrimental DCM phenotype in children. Our study is the first to demonstrate that cardiomyocytes from infants and young children with dilated cardiomyopathy (DCM) exhibit increased calcium sensitivity (likely mediated by decreased troponin I phosphorylation) compared with nonfailing pediatric cardiomyocytes. Compared with published values in adult cardiomyocytes, pediatric cardiomyocytes have notably decreased cooperativity, with a further reduction in the setting of DCM. Distinct adaptations in cardiomyocyte contractile properties may contribute to a differential response to pharmacological therapies in the pediatric DCM population.
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http://dx.doi.org/10.1152/ajpheart.00409.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960778PMC
December 2019

Redistribution of EC-SOD resolves bleomycin-induced inflammation increased apoptosis of recruited alveolar macrophages.

FASEB J 2019 12 27;33(12):13465-13475. Epub 2019 Sep 27.

Cardiovascular Pulmonary Research Laboratories, Departments of Pediatrics and Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

A human single nucleotide polymorphism (SNP) in the matrix-binding domain of extracellular superoxide dismutase (EC-SOD), with arginine to glycine substitution at position 213 (R213G), redistributes EC-SOD from the matrix into extracellular fluids. We reported that, following bleomycin (bleo), knockin mice harboring the human R213G SNP (R213G mice) exhibit enhanced resolution of inflammation and protection against fibrosis, compared with wild-type (WT) littermates. In this study, we tested the hypothesis that the EC-SOD R213G SNP promotes resolution accelerated apoptosis of recruited alveolar macrophage (AM). RNA sequencing and Ingenuity Pathway Analysis 7 d postbleo in recruited AM implicated increased apoptosis and blunted inflammatory responses in the R213G strain exhibiting accelerated resolution. We validated that the percentage of apoptosis was significantly elevated in R213G recruited AM WT at 3 and 7 d postbleo . Recruited AM numbers were also significantly decreased in R213G mice WT at 3 and 7 d postbleo. ChaC glutathione-specific γ-glutamylcyclotransferase 1 (Chac1), a proapoptotic γ-glutamyl cyclotransferase that depletes glutathione, was increased in the R213G recruited AM. Overexpression of Chac1 induced apoptosis of macrophages and was blocked by administration of cell-permeable glutathione. In summary, we provide new evidence that redistributed EC-SOD accelerates the resolution of inflammation through redox-regulated mechanisms that increase recruited AM apoptosis.-Allawzi, A., McDermott, I., Delaney, C., Nguyen, K., Banimostafa, L., Trumpie, A., Hernandez-Lagunas, L., Riemondy, K., Gillen, A., Hesselberth, J., El Kasmi, K., Sucharov, C. C., Janssen, W. J., Stenmark, K., Bowler, R., Nozik-Grayck, E. Redistribution of EC-SOD resolves bleomycin-induced inflammation increased apoptosis of recruited alveolar macrophages.
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http://dx.doi.org/10.1096/fj.201901038RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894081PMC
December 2019

Transcatheter aortic valve replacements alter circulating serum factors to mediate myofibroblast deactivation.

Sci Transl Med 2019 09;11(509)

Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO 80303, USA.

The transcatheter aortic valve replacement (TAVR) procedure has emerged as a minimally invasive treatment for patients with aortic valve stenosis (AVS). However, alterations in serum factor composition and biological activity after TAVR remain unknown. Here, we quantified the systemic inflammatory effects of the TAVR procedure and hypothesized that alterations in serum factor composition would modulate valve and cardiac fibrosis. Serum samples were obtained from patients with AVS immediately before their TAVR procedure (pre-TAVR) and about 1 month afterward (post-TAVR). Aptamer-based proteomic profiling revealed alterations in post-TAVR serum composition, and ontological analysis identified inflammatory macrophage factors implicated in myofibroblast activation and deactivation. Hydrogel biomaterials used as valve matrix mimics demonstrated that post-TAVR serum reduced myofibroblast activation of valvular interstitial cells relative to pre-TAVR serum from the same patient. Transcriptomics and curated network analysis revealed a shift in myofibroblast phenotype from pre-TAVR to post-TAVR and identified p38 MAPK signaling as one pathway involved in pre-TAVR-mediated myofibroblast activation. Post-TAVR serum deactivated valve and cardiac myofibroblasts initially exposed to pre-TAVR serum to a quiescent fibroblast phenotype. Our in vitro deactivation data correlated with patient disease severity measured via echocardiography and multimorbidity scores, and correlations were dependent on hydrogel stiffness. Sex differences in cellular responses to male and female sera were also observed and may corroborate clinical observations regarding sex-specific TAVR outcomes. Together, alterations in serum composition after TAVR may lead to an antifibrotic fibroblast phenotype, which suggests earlier interventions may be beneficial for patients with advanced AVS to prevent further disease progression.
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http://dx.doi.org/10.1126/scitranslmed.aav3233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754739PMC
September 2019

Elamipretide Improves Mitochondrial Function in the Failing Human Heart.

JACC Basic Transl Sci 2019 Apr 29;4(2):147-157. Epub 2019 Apr 29.

Department of Medicine/Division of Cardiology, University of Colorado School of Medicine, Aurora, Colorado.

Negative alterations of mitochondria are known to occur in heart failure (HF). This study investigated the novel mitochondrial-targeted therapeutic agent elamipretide on mitochondrial and supercomplex function in failing human hearts ex vivo. Freshly explanted failing and nonfailing ventricular tissue from children and adults was treated with elamipretide. Mitochondrial oxygen flux, complex (C) I and CIV activities, and in-gel activity of supercomplex assembly were measured. Mitochondrial function was impaired in the failing human heart, and mitochondrial oxygen flux, CI and CIV activities, and supercomplex-associated CIV activity significantly improved in response to elamipretide treatment. Elamipretide significantly improved failing human mitochondrial function.
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http://dx.doi.org/10.1016/j.jacbts.2018.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488757PMC
April 2019

A PDE3A Promoter Polymorphism Regulates cAMP-Induced Transcriptional Activity in Failing Human Myocardium.

J Am Coll Cardiol 2019 03;73(10):1173-1184

Division of Cardiology and Cardiovascular Institute, University of Colorado Denver, Aurora, Colorado; ARCA Biopharma, Westminster, Colorado.

Background: The phosphodiesterase 3A (PDE3A) gene encodes a PDE that regulates cardiac myocyte cyclic adenosine monophosphate (cAMP) levels and myocardial contractile function. PDE3 inhibitors (PDE3i) are used for short-term treatment of refractory heart failure (HF), but do not produce uniform long-term benefit.

Objectives: The authors tested the hypothesis that drug target genetic variation could explain clinical response heterogeneity to PDE3i in HF.

Methods: PDE3A promoter studies were performed in a cloned luciferase construct. In human left ventricular (LV) preparations, mRNA expression was measured by reverse transcription polymerase chain reaction, and PDE3 enzyme activity by cAMP-hydrolysis.

Results: The authors identified a 29-nucleotide (nt) insertion (INS)/deletion (DEL) polymorphism in the human PDE3A gene promoter beginning 2,214 nt upstream from the PDE3A1 translation start site. Transcription factor ATF3 binds to the INS and represses cAMP-dependent promoter activity. In explanted failing LVs that were homozygous for PDE3A DEL and had been treated with PDE3i pre-cardiac transplantation, PDE3A1 mRNA abundance and microsomal PDE3 enzyme activity were increased by 1.7-fold to 1.8-fold (p < 0.05) compared with DEL homozygotes not receiving PDE3i. The basis for the selective up-regulation in PDE3A gene expression in DEL homozygotes treated with PDE3i was a cAMP response element enhancer 61 nt downstream from the INS, which was repressed by INS. The DEL homozygous genotype frequency was also enriched in patients with HF.

Conclusions: A 29-nt INS/DEL polymorphism in the PDE3A promoter regulates cAMP-induced PDE3A gene expression in patients treated with PDE3i. This molecular mechanism may explain response heterogeneity to this drug class, and may inform a pharmacogenetic strategy for a more effective use of PDE3i in HF.
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http://dx.doi.org/10.1016/j.jacc.2018.12.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499384PMC
March 2019

Targeted delivery of YSA-functionalized and non-functionalized polymeric nanoparticles to injured pulmonary vasculature.

Artif Cells Nanomed Biotechnol 2018 19;46(sup3):S1059-S1066. Epub 2018 Nov 19.

a Department of Pharmaceutical Sciences , University of Colorado Anschutz Medical Campus , Aurora , CO , USA.

Ephrin type-A receptor 2 (EphA2) is a transmembrane receptor which is upregulated in injured lungs, including those treated with bleomycin. YSA peptide (YSAYPDSVPMMS), a mimic of ephrin ligands, binds to EphA2 receptors on cell surface with high affinity. In this study, we assessed the ability of YSA-functionalized and non-functionalized poly (dl-lactide-co-glycolide) (PLGA) nanoparticles to enhance delivery to bleomycin treated cultured vascular endothelial cells and, in a bleomycin induced lung injury mouse model. Nanoparticles were loaded with a lipophilic fluorescent dye. Human umbilical vein endothelial cells (HUVEC) with or without 2-day bleomycin pretreatment (25 µg/ml) and adult mice with or without intratracheal instillation of bleomycin (0.1 U) were dosed with nanoparticles. Mice received nanoparticles via tail vein injection 4 days after bleomycin treatment. Three days after nanoparticle injection, tissues (lung, heart, kidney, spleen, liver, brain, eyes and whole blood) were harvested and quantified for fluorescence using IVIS imaging. Mean particle uptake increased with time and concentration for both types of particles in HUVEC, with the uptake being higher for YSA-functionalized nanoparticles. Bleomycin treatment increased the 3-h uptake of both types of nanoparticles in HUVEC by about two-fold, with the YSA-functionalized nanoparticle uptake being 1.66-fold compared to non-functionalized nanoparticles (p < .05). In mice, bleomycin injury resulted in 2.3- and 4.7-fold increase in the lung levels of non-functionalized and YSA-functionalized nanoparticles (p < .05), respectively, although the differences between the two particle types were not significant. In conclusion, PLGA nanoparticle delivery to cultured vascular endothelial cells and mouse lungs in vivo is higher following bleomycin treatment, with the delivery tending to be higher for YSA functionalized nanoparticles.
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http://dx.doi.org/10.1080/21691401.2018.1528984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443406PMC
June 2019

Phosphodiesterase-5 Is Elevated in Failing Single Ventricle Myocardium and Affects Cardiomyocyte Remodeling In Vitro.

Circ Heart Fail 2018 09;11(9):e004571

Division of Cardiology, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Children's Hospital Colorado, Aurora (A.M.G., S.J.N., S.D.M.).

Background Single ventricle (SV) congenital heart disease is fatal without intervention, and eventual heart failure is a major cause of morbidity and mortality. Although there are no proven medical therapies for the treatment or prevention of heart failure in the SV heart disease population, phosphodiesterase-5 inhibitors (PDE5i), such as sildenafil, are increasingly used. Although the pulmonary vasculature is the primary target of PDE5i therapy in patients with SV heart disease, the effects of PDE5i on the SV heart disease myocardium remain largely unknown. We sought to determine PDE5 expression and activity in the single right ventricle of SV heart disease patients relative to nonfailing controls and to determine whether PDE5 impacts cardiomyocyte remodeling using a novel serum-based in vitro model. Methods and Results PDE5 expression (n=9 nonfailing; n=7 SV heart disease), activity (n=8 nonfailing; n=9 SV heart disease), and localization (n=3 SV heart disease) were determined in explanted human right ventricle myocardium. PDE5 is expressed in SV heart disease cardiomyocytes, and PDE5 protein expression and activity are increased in SV heart disease right ventricle compared with nonfailing right ventricle. Isolated neonatal rat ventricular myocytes were treated for 72 hours with nonfailing or SV heart disease patient serum±sildenafil. Reverse transcription quantitative polymerase chain reaction (n=5 nonfailing; n=12 SV heart disease) and RNA sequencing (n=3 nonfailing; n=3 SV heart disease) were performed on serum-treated neonatal rat ventricular myocytes and demonstrated that treatment with SV heart disease sera results in pathological gene expression changes that are attenuated with PDE5i. Conclusions PDE5 is increased in failing SV heart disease myocardium, and pathological gene expression changes in SV heart disease serum-treated neonatal rat ventricular myocytes are abrogated by PDE5i. These results suggest that PDE5 represents an intriguing myocardial therapeutic target in this population.
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http://dx.doi.org/10.1161/CIRCHEARTFAILURE.117.004571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6206883PMC
September 2018

CELF1 regulates gap junction integrity contributing to dilated cardiomyopathy.

Noncoding RNA Investig 2018 Mar 7;2. Epub 2018 Mar 7.

Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

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http://dx.doi.org/10.21037/ncri.2018.02.03DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128295PMC
March 2018

A novel genetic marker of decreased inflammation and improved survival after acute myocardial infarction.

Basic Res Cardiol 2018 08 10;113(5):38. Epub 2018 Aug 10.

Cardiovascular Division, Department of Medicine, Washington University School of Medicine, 660 S. Euclid Ave., Campus, Box 8086, Saint Louis, MO, 63110, USA.

The CHRNA5 gene encodes a neurotransmitter receptor subunit involved in multiple processes, including cholinergic autonomic nerve activity and inflammation. Common variants in CHRNA5 have been linked with atherosclerotic cardiovascular disease. Association of variation in CHRNA5 and specific haplotypes with cardiovascular outcomes has not been described. The aim of this study was to examine the association of CHRNA5 haplotypes with gene expression and mortality among patients with acute myocardial infarction (AMI) and explore potential mechanisms of this association. Patients (N = 2054) hospitalized with AMI were genotyped for two common variants in CHRNA5. Proportional hazard models were used to estimate independent association of CHRNA5 haplotype with 1-year mortality. Both individual variants were associated with mortality (p = 0.0096 and 0.0004, respectively) and were in tight LD (D' = 0.99). One haplotype, HAP3, was associated with decreased mortality one year after AMI (adjusted HR = 0.42, 95% CI 0.26, 0.68; p = 0.0004). This association was validated in an independent cohort (N = 637) of post-MI patients (adjusted HR = 0.23, 95% CI 0.07, 0.79; p = 0.019). Differences in CHRNA5 expression by haplotype were investigated in human heart samples (n = 28). Compared with non-carriers, HAP3 carriers had threefold lower cardiac CHRNA5 mRNA expression (p = 0.023). Circulating levels of the inflammatory marker hsCRP were significantly lower in HAP3 carriers versus non-carriers (3.43 ± 4.2 versus 3.91 ± 5.1; p = 0.0379). Activation of the inflammasome, an important inflammatory complex involved in cardiovascular disease that is necessary for release of the pro-inflammatory cytokine IL-1 β, was assessed in bone marrow-derived macrophages (BMDM) from CHRNA5 knockout mice and wild-type controls. In BMDM from CHRNA5 knockout mice, IL-1β secretion was reduced by 50% compared to wild-type controls (p = 0.004). Therefore, a common haplotype of CHRNA5 that results in reduced cardiac expression of CHRNA5 and attenuated macrophage inflammasome activation is associated with lower mortality after AMI. These results implicate CHRNA5 and the cholinergic anti-inflammatory pathway in survival following AMI.
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http://dx.doi.org/10.1007/s00395-018-0697-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292447PMC
August 2018

Acute isoproterenol leads to age-dependent arrhythmogenesis in guinea pigs.

Am J Physiol Heart Circ Physiol 2018 10 20;315(4):H1051-H1062. Epub 2018 Jul 20.

Division of Cardiology, Department of Medicine, University of Colorado Denver School of Medicine , Aurora, Colorado.

Sudden cardiac death from ventricular arrhythmias is more common in adult patients with with heart failure compared with pediatric patients with heart failure. We identified age-specific differences in arrhythmogenesis using a guinea pig model of acute β-adrenergic stimulation. Young and adult guinea pigs were exposed to the β-adrenergic agonist isoproterenol (ISO; 0.7 mg/kg) for 30 min in the absence or presence of flecainide (20 mg/kg), an antiarrhythmic that blocks Na and ryanodine channels. Implanted cardiac monitors (Reveal LINQ, Medtronic) were used to monitor heart rhythm. Alterations in phosphorylation and oxidation of ryanodine receptor 2 (RyR2) were measured in left ventricular tissue. There were age-specific differences in arrhythmogenesis and sudden death associated with acute β-adrenergic stimulation in guinea pigs. Young and adult guinea pigs developed arrhythmias in response to ISO; however, adult animals developed significantly more premature ventricular contractions and experienced higher arrhythmia-related mortality than young guinea pigs treated with ISO. Although there were no significant differences in the phosphorylation of left ventricular RyR2 between young and adult guinea pigs, adult guinea pigs exposed to acute ISO had significantly more oxidation of RyR2. Flecainide treatment significantly improved survival and decreased the number of premature ventricular contractions in young and adult animals in association with lower RyR2 oxidation. Adult guinea pigs had a greater propensity to develop arrhythmias and suffer sudden death than young guinea pigs when acutely exposed to ISO. This was associated with higher oxidation of RyR2. The incidence of sudden death can be rescued with flecainide treatment, which decreases RyR2 oxidation. NEW & NOTEWORTHY Clinically, adult patients with heart failure are more likely to develop arrhythmias and sudden death than pediatric patients with heart failure. In the present study, older guinea pigs also showed a greater propensity to arrhythmias and sudden death than young guinea pigs when acutely exposed to isoproterenol. Although there are well-described age-related cardiac structural changes that predispose patients to arrhythmogenesis, the present data suggest contributions from dynamic changes in cellular signaling also play an important role in arrhythmogenesis.
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http://dx.doi.org/10.1152/ajpheart.00061.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230911PMC
October 2018

MicroRNA dysregulation in lung injury: the role of the miR-26a/EphA2 axis in regulation of endothelial permeability.

Am J Physiol Lung Cell Mol Physiol 2018 10 19;315(4):L584-L594. Epub 2018 Jul 19.

Cardiovascular Pulmonary Research Laboratories, University of Colorado Denver Anschutz Medical Center , Aurora, Colorado.

MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression in many diseases, although the contribution of miRNAs to the pathophysiology of lung injury remains obscure. We hypothesized that dysregulation of miRNA expression drives the changes in key genes implicated in the development of lung injury. To test our hypothesis, we utilized a model of lung injury induced early after administration of intratracheal bleomycin (0.1 U). Wild-type mice were treated with bleomycin or PBS, and lungs were collected at 4 or 7 days. A profile of lung miRNA was determined by miRNA array and confirmed by quantitative PCR and flow cytometry. Lung miR-26a was significantly decreased 7 days after bleomycin injury, and, on the basis of enrichment of predicted gene targets, it was identified as a putative regulator of cell adhesion, including the gene targets EphA2, KDR, and ROCK1, important in altered barrier function. Lung EphA2 mRNA, and protein increased in the bleomycin-injured lung. We further explored the miR-26a/EphA2 axis in vitro using human lung microvascular endothelial cells (HMVEC-L). Cells were transfected with miR-26a mimic and inhibitor, and expression of gene targets and permeability was measured. miR-26a regulated expression of EphA2 but not KDR or ROCK1. Additionally, miR-26a inhibition increased HMVEC-L permeability, and the disrupted barrier integrity due to miR-26a was blocked by EphA2 knockdown, shown by VE-cadherin staining. Our data suggest that miR-26a is an important epigenetic regulator of EphA2 expression in the pulmonary endothelium. As such, miR-26a may represent a novel therapeutic target in lung injury by mitigating EphA2-mediated changes in permeability.
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http://dx.doi.org/10.1152/ajplung.00073.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230876PMC
October 2018

R213G polymorphism in SOD3 protects against bleomycin-induced inflammation and attenuates induction of proinflammatory pathways.

Physiol Genomics 2018 09 13;50(9):807-816. Epub 2018 Jul 13.

Developmental Lung Biology and Cardiovascular Pulmonary Research Laboratories, Departments of Pediatrics and Medicine, University of Colorado, Anschutz Medical Campus, Aurora, Colorado.

Extracellular superoxide dismutase (EC-SOD), one of three mammalian SOD isoforms, is the sole extracellular enzymatic defense against superoxide. A known human single nucleotide polymorphism (SNP) in the matrix-binding domain of EC-SOD characterized by an arginine-to-glycine substitution at position 213 (R213G) redistributes EC-SOD from the matrix into extracellular fluids. We previously reported that knock-in mice harboring the human R213G SNP (R213G mice) exhibited enhanced resolution of inflammation with subsequent protection against fibrosis following bleomycin treatment compared with wild-type (WT) littermates. Herein we set out to determine the underlying pathways with RNA-Seq analysis of WT and R213G lungs 7 days post-PBS and bleomycin. RNA-Seq analysis uncovered significant differential gene expression changes induced in WT and R213G strains in response to bleomycin. Ingenuity Pathways Analysis was used to predict differentially regulated up- and downstream processes based on transcriptional changes. Most prominent was the induction of inflammatory and immune responses in WT mice, which were suppressed in the R213G mice. Specifically, PKC signaling in T lymphocytes, IL-6, and NFΚB signaling were opposed in WT mice when compared with R213G. Several upstream regulators such as IFNγ, IRF3, and IKBKG were implicated in the divergent responses between WT and R213G mice. Our data suggest that the redistributed EC-SOD due to the R213G SNP attenuates the dysregulated inflammatory responses observed in WT mice. We speculate that redistributed EC-SOD protects against dysregulated alveolar inflammation via reprogramming of recruited immune cells toward a proresolving state.
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http://dx.doi.org/10.1152/physiolgenomics.00053.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172610PMC
September 2018

Optimization of phenol-chloroform RNA extraction.

MethodsX 2018 30;5:599-608. Epub 2018 May 30.

Department of Medicine, Division of Cardiology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.

Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse transcription, that can skew data collection and interpretation. To overcome the issue of RNA contamination in the conventional phenol-chloroform based RNA extraction method, we have optimized the protocol by adding one chloroform extraction step, and several RNA washing steps. Importantly, RNA quality and purity and accuracy in the quantification of RNA concentration were significantly improved with the modified protocol, resulting in reliable data collection and interpretation in downstream gene expression analysis. •Our protocol is customized by the addition of a second chloroform extraction step. Chloroform is carefully pipetted so as to not disturb the interphase layer. Any contaminants accidentally removed from interphase will be present in subsequent steps and can result in RNA contaminated with protein or phenol. The additional chloroform step increases RNA purity.•Additionally, the addition of 2 additional ethanol washes, initially intended to remove any residual salts from the isopropanol RNA precipitation step, also removed residual phenol contamination, enhancing RNA purity.•In summary, these modifications serve to enhance not only the purity of the RNA but, also increase the accuracy and reliability of RNA quantification.
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http://dx.doi.org/10.1016/j.mex.2018.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031757PMC
May 2018

Differential Response to Heart Failure Medications in Children.

Prog Pediatr Cardiol 2018 Jun;49:27-30

Division of Cardiology, Department of Medicine, University of Colorado Denver School of Medicine, 12700 E 19 Ave, Aurora, CO USA 80045.

There have been many advances in the treatment of heart failure over the past several years. While these advancements have resulted in improved outcomes in adults with heart failure, these same treatments do not seem to be as efficacious in children with heart failure. Investigations of the failing pediatric heart suggest that there are unique phenotypic, pathologic and molecular differences that could influence how children with heart failure response to adult-based therapies. In this review, several recent studies and the potential implications of their findings on informing the future of the management of pediatric heart failure are discussed.
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http://dx.doi.org/10.1016/j.ppedcard.2018.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023416PMC
June 2018

Improved Detection of Circulating miRNAs in Serum and Plasma Following Rapid Heat/Freeze Cycling.

Microrna 2018 ;7(2):138-147

Department of Medicine, Division of Cardiology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, United States.

Background: The measurement of circulating miRNAs has proven to be a powerful biomarker tool for several disease processes. Current protocols for the detection of miRNAs usually involve an RNA extraction step, requiring a substantial volume of patient serum or plasma to obtain sufficient input material.

Objective: Here, we describe a novel methodology that allows detection of a large number of miRNAs from a small volume of serum or plasma without the need for RNA extraction.

Methods: Three μl of serum or plasma was subjected to three cycles of high and low temperatures (heat/freeze cycles) followed by miRNA arrays.

Results: Our results indicate that miRNA detection following this process is highly reproducible when comparing multiple samples from the same subject. Moreover, this protocol increases the reproducibility of miRNA detection in samples that were previously subjected to multiple freeze-thaw cycles. Importantly, the detection of miRNAs from serum vs. plasma following heat/freeze cycling are highly comparable, indicating that this heat/freeze process effectively eliminates differences in detection between serum and plasma samples that have been reported using other sample preparation methodologies.

Conclusion: We propose that this method is a potent alternative to current RNA extraction protocols, substantially reducing the amount of sample necessary for miRNA detection while simultaneously improving miRNA detection and reproducibility.
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http://dx.doi.org/10.2174/2211536607666180416152112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198569PMC
November 2018

Fibrosis-Related Gene Expression in Single Ventricle Heart Disease.

J Pediatr 2017 12 16;191:82-90.e2. Epub 2017 Oct 16.

Department of Pediatrics, Division of Cardiology, University of Colorado Anschutz Medical Campus, Children's Hospital Colorado, Aurora, CO. Electronic address:

Objective: To evaluate fibrosis and fibrosis-related gene expression in the myocardium of pediatric subjects with single ventricle with right ventricular failure.

Study Design: Real-time quantitative polymerase chain reaction was performed on explanted right ventricular myocardium of pediatric subjects with single ventricle disease and controls with nonfailing heart disease. Subjects were divided into 3 groups: single ventricle failing (right ventricular failure before or after stage I palliation), single ventricle nonfailing (infants listed for primary transplantation with normal right ventricular function), and stage III (Fontan or right ventricular failure after stage III). To evaluate subjects of similar age and right ventricular volume loading, single ventricle disease with failure was compared with single ventricle without failure and stage III was compared with nonfailing right ventricular disease. Histologic fibrosis was assessed in all hearts. Mann-Whitney tests were performed to identify differences in gene expression.

Results: Collagen (Col1α, Col3) expression is decreased in single ventricle congenital heart disease with failure compared with nonfailing single ventricle congenital heart disease (P = .019 and P = .035, respectively), and is equivalent in stage III compared with nonfailing right ventricular heart disease. Tissue inhibitors of metalloproteinase (TIMP-1, TIMP-3, and TIMP-4) are downregulated in stage III compared with nonfailing right ventricular heart disease (P = .0047, P = .013 and P = .013, respectively). Matrix metalloproteinases (MMP-2, MMP-9) are similar between nonfailing single ventricular heart disease and failing single ventricular heart disease, and between stage III heart disease and nonfailing right ventricular heart disease. There is no difference in the prevalence of right ventricular fibrosis by histology in subjects with single ventricular failure heart disease with right ventricular failure (18%) compared with those with normal right ventricular function (38%).

Conclusions: Fibrosis is not a primary contributor to right ventricular failure in infants and young children with single ventricular heart disease. Additional studies are required to understand whether antifibrotic therapies are beneficial in this population.
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http://dx.doi.org/10.1016/j.jpeds.2017.08.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705574PMC
December 2017

Midkine's Role in Cardiac Pathology.

J Cardiovasc Dev Dis 2017 Sep 24;4(3). Epub 2017 Aug 24.

Department of Medicine, University of Colorado Denver, Denver, CO 80045, USA.

Midkine (MDK) is a heparin-binding growth factor that is normally expressed in mid-gestational development mediating mesenchymal and epithelial interactions. As organisms age, expression of MDK diminishes; however, in adults, MDK expression is associated with acute and chronic pathologic conditions such as myocardial infarction and heart failure (HF). The role of MDK is not clear in cardiovascular disease and currently there is no consensus if it plays a beneficial or detrimental role in HF. The lack of clarity in the literature is exacerbated by differing roles that circulating and myocardial MDK play in signaling pathways in cardiomyocytes (some of which have yet to be elucidated). Of particular interest, serum MDK is elevated in adults with chronic heart failure and higher circulating MDK is associated with worse cardiac function. In addition, pediatric HF patients have higher levels of myocardial MDK. This review focuses on what is known about the effect of exogenous versus myocardial MDK in various cardiac disease models in an effort to better clarify the role of midkine in HF.
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http://dx.doi.org/10.3390/jcdd4030013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599136PMC
September 2017

Circulating miRNAs in Pediatric Pulmonary Hypertension Show Promise as Biomarkers of Vascular Function.

Oxid Med Cell Longev 2017 27;2017:4957147. Epub 2017 Jul 27.

Children's Hospital Colorado, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Background/objectives: The objective of this study was to evaluate the utility of circulating miRNAs as biomarkers of vascular function in pediatric pulmonary hypertension.

Method: Fourteen pediatric pulmonary arterial hypertension patients underwent simultaneous right heart catheterization (RHC) and blood biochemical analysis. Univariate and stepwise multivariate linear regression was used to identify and correlate measures of reactive and resistive afterload with circulating miRNA levels. Furthermore, circulating miRNA candidates that classified patients according to a 20% decrease in resistive afterload in response to oxygen (O) or inhaled nitric oxide (iNO) were identified using receiver-operating curves.

Results: Thirty-two circulating miRNAs correlated with the pulmonary vascular resistance index (PVRi), pulmonary arterial distensibility, and PVRi decrease in response to O and/or iNO. Multivariate models, combining the predictive capability of multiple promising miRNA candidates, revealed a good correlation with resistive ( = 0.97, < 0.0001) and reactive ( = 0.86, < 0.005) afterloads. Bland-Altman plots showed that 95% of the differences between multivariate models and RHC would fall within 0.13 (mmHg-min/L)m and 0.0085/mmHg for resistive and reactive afterloads, respectively. Circulating miR-663 proved to be a good classifier for vascular responsiveness to acute O and iNO challenges.

Conclusion: This study suggests that circulating miRNAs may be biomarkers to phenotype vascular function in pediatric PAH.
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http://dx.doi.org/10.1155/2017/4957147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551515PMC
April 2018

Pediatric dilated cardiomyopathy hearts display a unique gene expression profile.

JCI Insight 2017 Jul 20;2(14). Epub 2017 Jul 20.

Division of Cardiology, University of Colorado School of Medicine, Aurora, Colorado, USA.

Our previous work showed myocellular differences in pediatric and adult dilated cardiomyopathy (DCM). However, a thorough characterization of the molecular pathways involved in pediatric DCM does not exist, limiting the development of age-specific therapies. To characterize this patient population, we investigated the transcriptome profile of pediatric patients. RNA-Seq from 7 DCM and 7 nonfailing (NF) explanted age-matched pediatric left ventricles (LV) was performed. Changes in gene expression were confirmed by real-time PCR (RT-PCR) in 36 DCM and 21 NF pediatric hearts and in 20 DCM and 10 NF adult hearts. The degree of myocyte hypertrophy was investigated in 4 DCM and 7 NF pediatric hearts and in 4 DCM and 9 NF adult hearts. Changes in gene expression in response to pluripotency-inducing factors were investigated in neonatal rat ventricular myocytes (NRVMs). Transcriptome analysis identified a gene expression profile in children compared with adults with DCM. Additionally, myocyte hypertrophy was not observed in pediatric hearts but was present in adult hearts. Furthermore, treatment of NRVMs with pluripotency-inducing factors recapitulated changes in gene expression observed in the pediatric DCM heart. Pediatric DCM is characterized by unique changes in gene expression that suggest maintenance of an undifferentiated state.
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http://dx.doi.org/10.1172/jci.insight.94249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518568PMC
July 2017

Myocardial microRNAs associated with reverse remodeling in human heart failure.

JCI Insight 2017 01 26;2(2):e89169. Epub 2017 Jan 26.

Division of Cardiology, Department of Medicine.

Background: In dilated cardiomyopathies (DCMs) changes in expression of protein-coding genes are associated with reverse remodeling, and these changes can be regulated by microRNAs (miRs). We tested the general hypothesis that dynamic changes in myocardial miR expression are predictive of β-blocker-associated reverse remodeling.

Methods: Forty-three idiopathic DCM patients (mean left ventricular ejection fraction 0.24 ± 0.09) were treated with β-blockers. Serial ventriculography and endomyocardial biopsies were performed at baseline, and after 3 and 12 months of treatment. Changes in RT-PCR (candidate miRs) or array-measured miRs were compared based on the presence (R) or absence (NR) of a reverse-remodeling response, and a miR-mRNA-function pathway analysis (PA) was performed.

Results: At 3 months, 2 candidate miRs were selectively changed in Rs, decreases in miR-208a-3p and miR-591. PA revealed changes in miR-mRNA interactions predictive of decreased apoptosis and myocardial cell death. At 12 months, 5 miRs exhibited selective changes in Rs (decreases in miR-208a-3p, -208b-3p, 21-5p, and 199a-5p; increase in miR-1-3p). PA predicted decreases in apoptosis, cardiac myocyte cell death, hypertrophy, and heart failure, with increases in contractile and overall cardiac functions.

Conclusions: In DCMs, myocardial miRs predict the time-dependent reverse-remodeling response to β-blocker treatment, and likely regulate the expression of remodeling-associated miRs.

Trial Registration: ClinicalTrials.gov NCT01798992.

Funding: NIH 2R01 HL48013, 1R01 HL71118 (Bristow, PI); sponsored research agreements from Glaxo-SmithKline and AstraZeneca (Bristow, PI); NIH P20 HL101435 (Lowes, Port multi-PD/PI); sponsored research agreement from Miragen Therapeutics (Port, PI).
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http://dx.doi.org/10.1172/jci.insight.89169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5256135PMC
January 2017

Exosomes from pediatric dilated cardiomyopathy patients modulate a pathological response in cardiomyocytes.

Am J Physiol Heart Circ Physiol 2017 Apr 27;312(4):H818-H826. Epub 2017 Jan 27.

Division of Cardiology, Department of Medicine, University of Colorado Denver, Aurora, Colorado;

Stimulation of the renin-angiotensin-aldosterone system (RAAS) and β-adrenergic receptors plays an important role in adult heart failure (HF). Despite the demonstrated benefits of RAAS inhibition and β-adrenergic receptor blockade in adult HF patients, no substantial improvement in survival rate has been observed in children with HF. This suggests that the underlying disease mechanism is uniquely regulated in pediatric HF. Here, we show that treatment of human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) with serum from pediatric dilated cardiomyopathy (DCM) patients induces pathological changes in gene expression, which occur independently of the RAAS and adrenergic systems, suggesting that serum circulating factors play an important role in cardiac remodeling. Furthermore, exosomes purified from DCM serum induced pathological changes in gene expression in NRVMs and iPSC-CMs. Our results suggest that DCM serum exosomes mediate pathological responses in cardiomyocytes and may propagate the pediatric HF disease process, representing a potential novel therapeutic target specific to this population. The results of this work could alter the present paradigm of basing clinical pediatric heart failure (HF) treatment on outcomes of adult HF clinical trials. The use of serum-treated primary cardiomyocytes may define age-specific mechanisms in pediatric HF with the potential to identify unique age-appropriate and disease-specific therapy.
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http://dx.doi.org/10.1152/ajpheart.00673.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5407162PMC
April 2017

Molecular Changes in Children with Heart Failure Undergoing Left Ventricular Assist Device Therapy.

J Pediatr 2017 03 29;182:184-189.e1. Epub 2016 Nov 29.

Division of Cardiology, Department of Medicine, University of Colorado Denver, Aurora, CO; Division of Cardiology, Department of Medicine, Denver Health and Hospital Authority, Denver, CO.

Objective: To determine whether left ventricular assist device (LVAD) treatment in children with heart failure would result in the modification of molecular pathways involved in heart failure pathophysiology.

Study Design: Forty-seven explanted hearts from children were studied (16 nonfailing control, 20 failing, and 11 failing post-LVAD implantation [F-LVAD]). Protein expression and phosphorylation states were determined by receptor binding assays and Western blots. mRNA expression was measured with real-time quantitative polymerase chain reaction. To evaluate for interactions and identify correlations, 2-way ANOVA and regression analysis were performed.

Results: Treatment with LVAD resulted in recovery of total β-adrenergic receptor expression and β-adrenergic receptor (β-AR) in failing hearts to normal levels (β-adrenergic receptor expression : 67.2 ± 11.5 fmol/mg failing vs 99.5 ± 27.7 fmol/mg nonfailing, 104 ± 38.7 fmol/mg F-LVAD, P ≤ .01; β-AR: 52.2 ± 10.3 fmol/mg failing vs 83.0 ± 23 fmol/mg non-failing, 76.5 ± 32.1 fmol/mg F-LVAD P ≤ .03). The high levels of G protein-coupled receptor kinase-2 were returned to nonfailing levels after LVAD treatment (5.6 ± 9.0 failing vs 1.0 ± 0.493 nonfailing, 1.0 ± 1.3 F-LVAD). Interestingly, β-adrenergic receptor expression was significantly greater in F-LVAD (27.5 ± 12; P < .005) hearts compared with nonfailing (16.4 ± 6.1) and failing (15.1 ± 4.2) hearts. Phospholamban phosphorylation at serine 16 was significantly greater in F-LVAD (7.7 ± 11.7) hearts compared with nonfailing (1.0 ± 1.2, P = .02) and failing (0.8 ± 1.0, P = .01) hearts. Also, atrial natriuretic factor (0.6 ± 0.8) and brain natriuretic peptide (0.1 ± 0.1) expression in F-LVAD was significantly lower compared with failing hearts (2.8 ± 3.6, P = .01 and 0.6 ± 0.7, P = .02).

Conclusion: LVAD treatment in children with heart failure results in reversal of several pathologic myocellular processes, and G protein-coupled receptor kinase-2 may regulate β-AR but not β-adrenergic receptor expression in children with heart failure.
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http://dx.doi.org/10.1016/j.jpeds.2016.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328921PMC
March 2017

Cardiac Adenylyl Cyclase and Phosphodiesterase Expression Profiles Vary by Age, Disease, and Chronic Phosphodiesterase Inhibitor Treatment.

J Card Fail 2017 Jan 15;23(1):72-80. Epub 2016 Jul 15.

Division of Cardiology, Department of Medicine, University of Colorado Denver, Aurora, Colorado. Electronic address:

Background: Pediatric heart failure (HF) patients have a suboptimal response to traditional HF medications, although phosphodiesterase-3 inhibition (PDE3i) has been used with greater success than in the adult HF population. We hypothesized that molecular alterations specific to children with HF and HF etiology may affect response to treatment.

Methods And Results: Adenylyl cyclase (AC) and phosphodiesterase (PDE) isoforms were quantified by means of quantitative real-time polymerase chain reaction in explanted myocardium from adults with dilated cardiomyopathy (DCM), children with DCM, and children with single-ventricle congenital heart disease of right ventricular morphology (SRV). AC and PDE expression profiles were uniquely regulated in each subject group and demonstratde distinct changes in response to chronic PDE3i. There was unique up-regulation of AC5 in adult DCM with PDE3i (fold change 2.415; P = .043), AC2 in pediatric DCM (fold change 2.396; P = .0067), and PDE1C in pediatric SRV (fold change 1.836; P = .032). Remarkably, PDE5A expression was consistently increased across all age and disease groups.

Conclusions: Unique regulation of AC and PDE isoforms supports a differential molecular adaptation to HF in children compared with adults, and may help identify mechanisms specific to the pathogenesis of pediatric HF. Greater understanding of these differences will help optimize medical therapies based on age and disease process.
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http://dx.doi.org/10.1016/j.cardfail.2016.07.429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384997PMC
January 2017

Signal-Dependent Recruitment of BRD4 to Cardiomyocyte Super-Enhancers Is Suppressed by a MicroRNA.

Cell Rep 2016 08 14;16(5):1366-1378. Epub 2016 Jul 14.

Division of Cardiology, Department of Medicine, University of Colorado Denver, Aurora, CO 80045, USA; Medical Scientist Training Program, University of Colorado Denver, Aurora, CO 80045, USA. Electronic address:

BRD4 governs pathological cardiac gene expression by binding acetylated chromatin, resulting in enhanced RNA polymerase II (Pol II) phosphorylation and transcription elongation. Here, we describe a signal-dependent mechanism for the regulation of BRD4 in cardiomyocytes. BRD4 expression is suppressed by microRNA-9 (miR-9), which targets the 3' UTR of the Brd4 transcript. In response to stress stimuli, miR-9 is downregulated, leading to derepression of BRD4 and enrichment of BRD4 at long-range super-enhancers (SEs) associated with pathological cardiac genes. A miR-9 mimic represses stimulus-dependent targeting of BRD4 to SEs and blunts Pol II phosphorylation at proximal transcription start sites, without affecting BRD4 binding to SEs that control constitutively expressed cardiac genes. These findings suggest that dynamic enrichment of BRD4 at SEs genome-wide serves a crucial role in the control of stress-induced cardiac gene expression and define a miR-dependent signaling mechanism for the regulation of chromatin state and Pol II phosphorylation.
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http://dx.doi.org/10.1016/j.celrep.2016.06.074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972677PMC
August 2016