Mitochondrial respiration is lower in the intrauterine growth-restricted fetal sheep heart.

Fetuses affected by intrauterine growth restriction have an increased risk of developing heart disease and failure in adulthood. Compared with controls, late gestation intrauterine growth-restricted (IUGR) fetal sheep have fewer binucleated cardiomyocytes, reflecting a more immature heart, which may reduce mitochondrial capacity to oxidize substrates. We hypothesized that the late gestation IUGR fetal heart has a lower capacity for mitochondrial oxidative phosphorylation. Left (LV) and right (RV) ventricles from IUGR and control (CON) fetal sheep at 90% gestation were harvested. Mitochondrial respiration (states 1-3, LeakOmy, and maximal respiration) in response to carbohydrates and lipids, citrate synthase (CS) activity, protein expression levels of mitochondrial oxidative phosphorylation complexes (CI-CV), and mRNA expression levels of mitochondrial biosynthesis regulators were measured. The carbohydrate and lipid state 3 respiration rates were lower in IUGR than CON, and CS activity was lower in IUGR LV than CON LV. However, relative CII and CV protein levels were higher in IUGR than CON; CV expression level was higher in IUGR than CON. Genes involved in lipid metabolism had lower expression in IUGR than CON. In addition, the LV and RV demonstrated distinct differences in oxygen flux and gene expression levels, which were independent from CON and IUGR status. Low mitochondrial respiration and CS activity in the IUGR heart compared with CON are consistent with delayed cardiomyocyte maturation, and CII and CV protein expression levels may be upregulated to support ATP production. These insights will provide a better understanding of fetal heart development in an adverse in utero environment. KEY POINTS: Growth-restricted fetuses have a higher risk of developing and dying from cardiovascular diseases in adulthood. Mitochondria are the main supplier of energy for the heart. As the heart matures, the substrate preference of the mitochondria switches from carbohydrates to lipids. We used a sheep model of intrauterine growth restriction to study the capacity of the mitochondria in the heart to produce energy using either carbohydrate or lipid substrates by measuring how much oxygen was consumed. Our data show that the mitochondria respiration levels in the growth-restricted fetal heart were lower than in the normally growing fetuses, and the expression levels of genes involved in lipid metabolism were also lower. Differences between the right and left ventricles that are independent of the fetal growth restriction condition were identified. These results indicate an impaired metabolic maturation of the growth-restricted fetal heart associated with a decreased capacity to oxidize lipids postnatally.

Integrated analysis of miRNA-mRNA interaction in pediatric dilated cardiomyopathy.

BACKGROUND: MicroRNAs (miRNAs) are short single-stranded nucleotides that can regulate gene expression. Although we previously evaluated the expression of miRNAs in pediatric dilated cardiomyopathy (DCM) by miRNA array, pathway prediction based on changes in mRNA expression has not been previously analyzed in this population. The current study aimed to determine the regulation of miRNA expression by miRNA-sequencing (miRNA-seq) and, through miRNA-sequencing (mRNA-seq), analyze their putative target genes and altered pathways in pediatric DCM hearts. METHODS: miRNA expression was determined by miRNA-seq [n = 10 non-failing (NF), n = 20 DCM]. Expression of a subset of miRNAs was evaluated in adult DCM patients (n = 11 NF, n = 13 DCM). miRNA-mRNA prediction analysis was performed using mRNA-seq data (n = 7 NF, n = 7 DCM) from matched samples. RESULTS: Expression of 393 miRNAs was significantly different (p < 0.05) in pediatric DCM patients compared to NF controls. TargetScan-based miRNA-mRNA analysis revealed 808 significantly inversely expressed genes. Functional analysis suggests upregulated pathways related to the regulation of stem cell differentiation and cardiac muscle contraction, and downregulated pathways related to the regulation of protein phosphorylation, signal transduction, and cell communication. CONCLUSIONS: Our results demonstrated a unique age-dependent regulation of miRNAs and their putative target genes, which may contribute to distinctive phenotypic characteristics of DCM in children. IMPACT: This is the first study to compare miRNA expression in the heart of pediatric DCM patients to age-matched healthy controls by RNA sequencing. Expression of a subset of miRNAs is uniquely dysregulated in children. Using mRNA-seq and miRNA-seq from matched samples, target prediction was performed. This study underscores the importance of pediatric-focused studies.

Serum response factor deletion 5 regulates phospholamban phosphorylation and calcium uptake.

AIMS: Pediatric dilated cardiomyopathy (pDCM) is characterized by unique age-dependent molecular mechanisms that include myocellular responses to therapy. We previously showed that pDCM, but not adult DCM patients respond to phosphodiesterase 3 inhibitors (PDE3i) by increasing levels of the second messenger cAMP and consequent phosphorylation of phospholamban (PLN). However, the molecular mechanisms involved in the differential pediatric and adult response to PDE3i are not clear. METHODS AND RESULTS: Quantification of serum response factor (SRF) isoforms from the left ventricle of explanted hearts showed that PDE3i treatment affects expression of SRF isoforms in pDCM hearts. An SRF isoform lacking exon 5 (SRFdel5) was highly expressed in the hearts of pediatric, but not adult DCM patients treated with PDE3i. To determine the functional consequence of expression of SRFdel5, we overexpressed full length SRF or SRFdel5 in cultured cardiomyocytes with and without adrenergic stimulation. Compared to a control adenovirus, expression of SRFdel5 increased phosphorylation of PLN, negatively affected expression of the phosphatase that promotes dephosphorylation of PLN (PP2Cε), and promoted faster calcium reuptake, whereas expression of full length SRF attenuated calcium reuptake through blunted phosphorylation of PLN. CONCLUSIONS: Taken together, these data indicate that expression of SRFdel5 in pDCM hearts in response to PDE3i contributes to improved function through regulating PLN phosphorylation and thereby calcium reuptake.

Amniotic fluid microRNA profiles in twin-twin transfusion syndrome with and without severe recipient cardiomyopathy.

BACKGROUND: Twin-twin transfusion syndrome presents many challenges for clinicians, and the optimal means of identifying pregnancies that will benefit most from intervention is controversial. There is currently no clinically available biomarker to detect twin-twin transfusion syndrome or to stratify cases based on the risk factors. microRNAs are small RNAs that regulate gene expression and are biomarkers for various disease processes, including adult and pediatric heart failure. To date, no studies have investigated amniotic fluid microRNAs as biomarkers for disease severity, specifically for severe recipient cardiomyopathy in twin-twin transfusion syndrome cases. OBJECTIVE: This study aimed to assess whether amniotic fluid microRNAs could be useful as biomarkers to identify pregnancies at greatest risk for severe recipient cardiomyopathy associated with twin-twin transfusion syndrome. STUDY DESIGN: Amniotic fluid was collected at the time of amnioreduction or selective fetoscopic laser photocoagulation from monochorionic diamniotic twin pregnancies with twin-twin transfusion syndrome at any stage. Fetal echocardiography was performed on all twins before the procedure, and severe cardiomyopathy was defined as a right ventricular myocardial performance index of the recipient fetus of >4 Z-scores. microRNA was extracted from the amniotic fluid samples and analyzed using an array panel assessing 379 microRNAs (TaqMan Open Array, ThermoFisher). Student t tests were performed to determine significant differences in microRNA expression between pregnancies with severe recipient cardiomyopathy and those with preserved cardiac function. A stringent q value of <.0025 was used to determine differential microRNA expression. Random forest plots identified the top 3 microRNAs that separated the 2 groups, and hierarchical cluster analysis was used to determine if these microRNAs properly segregated the samples according to their clinical groups. RESULTS: A total of 14 amniotic fluid samples from pregnancies with twin-twin transfusion syndrome with severe cardiomyopathy were compared with samples from 12 twin-twin transfusion syndrome control cases with preserved cardiac function. A total of 110 microRNAs were identified in the amniotic fluid samples. Twenty microRNAs were differentially expressed, and the top 3 differentiating microRNAs were hsa-miR-200c-3p, hsa-miR-17-5p, and hsa-miR-539-5p. Hierarchical cluster analysis based on these top 3 microRNAs showed a strong ability to differentiate severe cardiomyopathy cases from controls. The top 3 microRNAs were used to investigate the sensitivity and specificity of these microRNAs to differentiate between the 2 groups with a receiver operating characteristic curve demonstrating sensitivity and specificity of 80.8%. All 20 differentially expressed microRNAs were down-regulated in the group with severe cardiomyopathy. CONCLUSION: Amniotic fluid microRNAs demonstrated differential expression between twin-twin transfusion syndrome recipient fetuses with severe cardiomyopathy and those without and have the potential to be important biomarkers of disease severity in this population.

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

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.

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

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.

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

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.

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

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.

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

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.NEW & NOTEWORTHY 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.

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

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 via 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 vs. WT at 3 and 7 d postbleo in vivo. Recruited AM numbers were also significantly decreased in R213G mice vs. 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 in vitro 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 via increased apoptosis of recruited alveolar macrophages.

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

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 (N = 8 subjects) compared with nonfailing (NF) pediatric hearts (N = 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 (pCa50 =5.70 ± 0.0291), with an associated decrease in troponin (Tn)I phosphorylation compared with NF pediatric cardiomyocytes (pCa50 =5.59 ± 0.0271, P = 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/mm2, interquartile range (IQR) 21.5-49.2 vs. NF 32.8 mN/mm2, IQR 21.5-49.2 mN/mm2; P = 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; P = 0.0425). Alterations in sarcomeric phosphorylation and cardiomyocyte contractile properties may represent an impaired compensatory response, contributing to the detrimental DCM phenotype in children.NEW & NOTEWORTHY 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.

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

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.

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

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.

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

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 ß-adrenergic stimulation. Young and adult guinea pigs were exposed to the ß-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 ß-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.

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

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 ß, was assessed in bone marrow-derived macrophages (BMDM) from CHRNA5 knockout mice and wild-type controls. In BMDM from CHRNA5 knockout mice, IL-1ß 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.

Differential Response to Heart Failure Medications in Children.

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.

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

Stimulation of the renin-angiotensin-aldosterone system (RAAS) and ß-adrenergic receptors plays an important role in adult heart failure (HF). Despite the demonstrated benefits of RAAS inhibition and ß-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.NEW & NOTEWORTHY 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.

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

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 ß-adrenergic receptor expression and ß1-adrenergic receptor (ß1-AR) in failing hearts to normal levels (ß-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; ß1-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, ß2-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 ß1-AR but not ß2-adrenergic receptor expression in children with heart failure.

Fibrosis-Related Gene Expression in Single Ventricle Heart Disease.

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.