Pediatric Dilated Cardiomyopathy Patients Research Articles

Clinical Characteristics and Outcomes of Dilated Cardiomyopathy in Chinese Children: A Single-Center Retrospective Study.

The reported outcomes of pediatric dilated cardiomyopathy (DCM) have varied across studies. There are few outcome data concerning DCM in Chinese children. Therefore, we conducted a retrospective study to describe clinical features and determine risk factors for poor outcomes in children with DCM. We enrolled 121 children with DCM in our hospital from 2003 to 2021. General information and laboratory and echocardiographic data were collected and analyzed. Cox regression analysis was performed to determine risk factors for poor outcomes. This study included 121 patients (69 males and 52 females). The median age at diagnosis was 10.8 years, and the follow-up time was 10.0 months. Eighty-two patients (67.8%) exhibited cardiac function classes III-IV at the time of diagnosis. Tachypnea was the most common symptom (78.5%). In echocardiography, the mean left ventricular end-diastolic dimension z score was 7.36 ± 2.73, and the left ventricular ejection fraction z score was -6.58 ± 2.17. The 1-, 2-, and 5-year survival rates were 51.2%, 43.8%, and 32.2%, respectively. Cox analysis revealed that cardiac function classes III-IV (hazard ratio [HR] = 1.801, 95% confidence interval [95% CI] = 1.030-3.149, p = 0.039) and calcium levels (HR = 0.219, 95% CI = 0.084-0.576, p = 0.002) were predictors of poor outcomes in children with DCM. Children with DCM are at high risk of death. Cardiac function class III-IV and calcium levels were related to the prognosis of pediatric DCM patients.

Circulating and Cardiac Tissue miRNAs in Children with Dilated Cardiomyopathy.

microRNAs (miRs) are small non-coding single-stranded RNAs that regulate gene expression. We previously evaluated expression of miRs in the cardiac tissue of children with dilated cardiomyopathy (DCM) using miRNA-seq. However, a comparative analysis of serum and cardiac miRs has not been performed in this population. The current study aimed to evaluate miR levels in the serum of pediatric DCM patients compared to healthy non-failing (NF) donor controls and investigate the association between miR levels in tissue and sera from the same pediatric DCM patients. Defining the relationship between serum and tissue miRs may allow the use of circulating miRs as surrogate markers of cardiac miRs. miR levels were investigated through miR-array in sera [n = 10 NF, n = 12 DCM] and miR-seq in tissue (n = 10 NF, n = 12 DCM). Pathway analysis was investigated using the miR enrichment analysis and annotation tool (miEAA) for the five miRs commonly dysregulated in the sera and tissue of pediatric DCM patients. Functional analysis of miRs commonly dysregulated in the sera and tissue of pediatric DCM patients suggests altered pathways related to cell growth, differentiation and proliferation, inflammation, mitochondrial function, and metabolism. These findings suggest that circulating miRs could reflect altered levels of cardiac tissue miRs.

The microtubule signature in cardiac disease: etiology, disease stage, and age dependency

Employing animal models to study heart failure (HF) has become indispensable to discover and test novel therapies, but their translatability remains challenging. Although cytoskeletal alterations are linked to HF, the tubulin signature of common experimental models has been incompletely defined. Here, we assessed the tubulin signature in a large set of human cardiac samples and myocardium of animal models with cardiac remodeling caused by pressure overload, myocardial infarction or a gene defect. We studied levels of total, acetylated, and detyrosinated α-tubulin and desmin in cardiac tissue from hypertrophic (HCM) and dilated cardiomyopathy (DCM) patients with an idiopathic (n = 7), ischemic (n = 7) or genetic origin (n = 59), and in a pressure-overload concentric hypertrophic pig model (n = 32), pigs with a myocardial infarction (n = 28), mature pigs (n = 6), and mice (n = 15) carrying the HCM-associated MYBPC32373insG mutation. In the human samples, detyrosinated α-tubulin was increased 4-fold in end-stage HCM and 14-fold in pediatric DCM patients. Acetylated α-tubulin was increased twofold in ischemic patients. Across different animal models, the tubulin signature remained mostly unaltered. Only mature pigs were characterized by a 0.5-fold decrease in levels of total, acetylated, and detyrosinated α-tubulin. Moreover, we showed increased desmin levels in biopsies from NYHA class II HCM patients (2.5-fold) and the pressure-overload pig model (0.2–0.3-fold). Together, our data suggest that desmin levels increase early on in concentric hypertrophy and that animal models only partially recapitulate the proliferated and modified tubulin signature observed clinically. Our data warrant careful consideration when studying maladaptive responses to changes in the tubulin content in animal models.Graphical

Abstract 14006: Circulating microRNAs Predict Recovery in Pediatric Dilated Cardiomyopathy Patients

Introduction: microRNAs (miRs) are small single stranded RNAs capable of targeting expression of several genes. Circulating miRs are stable and can be important biomarkers of disease progression and diagnosis. Hypothesis: We hypothesized circulating miRs could be a reliable prognostic biomarker of recovery for pediatric dilated cardiomyopathy (DCM). Methods: TaqMan Open Array miR panel (ThermoFisher Scientific) was used to investigate expression of 381 circulating miRs. Samples originated from the Pediatric Cardiomyopathy Registry (PCMR) and were collected within 3 months of a new diagnosis of DCM. Inclusion criteria included <18 years of age at diagnosis of idiopathic or familial DCM (EF<50% or left ventricular end-diastolic dimension [LVEDd] z-score ≥+2). Patients with DCM secondary to musculoskeletal diseases or anthracycline toxicity were excluded. Primary outcomes, occurring within 1 year of enrollment, were defined as: [1] Death, transplant (tx) listing or initiation of mechanical circulatory support; [2] Recovered- normalization of ventricular size and function (EF≥50% and LVEDd z-score <+2) or [3] Stable- persistent DCM with EF<50% or LVEDd z-score ≥+2. Statistical analysis included random forest, hierarchical clustering and area under the receiver operator curve (AUC). Results: N=10 subjects were included in Group 1, of which 70% were female, 6 were white, 2 Hispanic and 3 of other/unknown races, median age 9.5y. N=5 samples were included in Group 2, of which 60% were female, 3 were white, 1 black and 1 native American, median age 1.9y. N=20 samples were included in Group 3, of which 40% were female, 10 were white, 5 black, 1 Pacific islander, 2 Hispanic and 4 of other/unknown races, median age 2.1y. The top 3 miRs differentiating Group 2 (Recovered) from Group 1 (Death/Tx) (AUC of 0.92) were hsa-miR-655, -410 and -636. The top 3 miRs differentiating Group 2 (Recovered) from Group 3 (Stable) were hsa-miR-17, -410 and -345 (AUC of 0.76). Conclusions: These results indicate circulating miRs can predict recovery in the pediatric DCM population. A unique biomarker signature of miRs specific to patients who have the potential to recover would improve risk stratification of this population.

Serum from pediatric dilated cardiomyopathy patients promotes dysregulation of cardiolipin biosynthesis and mitochondrial dysfunction in primary cardiomyocytes

Pediatric dilated cardiomyopathy (DCM) is a devastating and poorly understood disease with most clinical treatment paradigms extrapolated from the adult population. Our studies showed that aspects of metabolism and mitochondrial function are dysregulated in pediatric hearts. Cardiolipin (CL), a unique phospholipid in the inner mitochondrial membrane, is essential for optimal mitochondrial function and was shown to be dysregulated in failing adult and pediatric human heart. Our objective is to investigate if serum circulating factors from pediatric DCM patients can remodel CL resulting in mitochondrial dysfunction in vitro, similar to what is observed in the failing pediatric heart. Using a novel in vitro model that consists of treating neonatal rat ventricular myocytes (NRVMs) with serum from pediatric DCM patients or from non-failing (NF) healthy controls, mitochondrial respiration was assessed using the Agilent Seahorse, and reactive oxygen species (ROS) was assessed using Electron Paramagnetic Resonance Spectroscopy. Relative mtDNA copy number was determined by qPCR and expression of enzymes involved in CL biosynthesis were analyzed using RT-qPCR. Mass-spectrometry was used to quantitate total and specific CL species and to investigate the metabolite composition of NRVMs treated with NF or DCM serum. While mitochondrial ROS and mtDNA copy number were not significantly altered, we show that DCM serum decreases mitochondrial function, which is associated with alterations in CL content and composition and the downregulation of enzymes implicated in CL biosynthesis. Analysis of metabolite content showed an alteration of pathways involved in fatty acid metabolism, mitochondrial biogenesis and regulation of β-oxidation by PPARα. In conclusion, pediatric DCM serum circulating factors can promote CL remodeling resulting in mitochondrial dysfunction in primary cardiomyocytes. These findings suggest that CL could be a novel therapeutic target for this particular population.

Machine-learning–based exploration to identify remodeling patterns associated with death or heart-transplant in pediatric-dilated cardiomyopathy

We investigated left ventricular (LV) remodeling, mechanics, systolic and diastolic function, combined with clinical characteristics and heart-failure treatment in association to death or heart-transplant (DoT) in pediatric idiopathic, genetic or familial dilated cardiomyopathy (DCM), using interpretable machine-learning. Echocardiographic and clinical data from pediatric DCM and healthy controls were retrospectively analyzed. Machine-learning included whole cardiac-cycle regional longitudinal strain, aortic, mitral and pulmonary vein Doppler velocity traces, age and body surface area. We used unsupervised multiple kernel learning for data dimensionality reduction, positioning patients based on complex conglomerate information similarity. Subsequently, k-means identified groups with similar phenotypes. The proportion experiencing DoT was evaluated. Pheno-grouping identified 5 clinically distinct groups that were associated with differing proportions of DoT. All healthy controls clustered in groups 1 to 2, while all, but one, DCM subjects, clustered in groups 3 to 5; internally validating the algorithm. Cluster-5 comprised the oldest, most medicated patients, with combined systolic and diastolic heart-failure and highest proportion of DoT. Cluster-4 included the youngest patients characterized by severe LV remodeling and systolic dysfunction, but mild diastolic dysfunction and the second-highest proportion of DoT. Cluster-3 comprised young patients with moderate remodeling and systolic dysfunction, preserved apical strain, pronounced diastolic dysfunction and lowest proportion of DoT. Interpretable machine-learning, using full cardiac-cycle systolic and diastolic data, mechanics and clinical parameters, can potentially identify pediatric DCM patients at high-risk for DoT, and delineate mechanisms associated with risk. This may facilitate more precise prognostication and treatment of pediatric DCM.

Abstract P372: Serum From Pediatric Dilated Cardiomyopathy Patients Promotes Dysregulation Of Cardiolipin Biosynthesis And Mitochondrial Dysfunction In Primary Cardiomyocytes

Pediatric dilated cardiomyopathy (DCM) is a devastating and poorly understood disease with most clinical treatment paradigms extrapolated from the adult population. Our studies have demonstrated that aspects of metabolism and mitochondrial function are dysregulated in pediatric DCM hearts. Cardiolipin (CL), a unique phospholipid in the inner mitochondrial membrane, is essential for optimal mitochondrial function and was shown to be dysregulated in both the failing adult and pediatric human heart. The objective of this study is to investigate if serum circulating factors from pediatric DCM patients can remodel CL resulting in mitochondrial dysfunction in vitro , similar to what is observed in the failing pediatric heart. Using a novel in vitro model that consists of treating neonatal rat ventricular myocytes (NRVMs) with serum from pediatric DCM patients or from non-failing (NF) healthy controls, mitochondrial respiration was assessed using the Agilent Seahorse, and reactive oxygen species (ROS) was assessed using Electron Paramagnetic Resonance Spectroscopy. Relative mitochondrial DNA (mtDNA) copy number was determined by qPCR and expression of enzymes involved in CL biosynthesis and remodeling were analyzed using RT-qPCR. Mass-spectrometry was used to quantitate total and specific CL species and to investigate the metabolite composition of NRVMs treated with NF or DCM serum. While mitochondrial ROS and mtDNA copy number were not significantly altered, we show that DCM serum decreases mitochondrial function, which is associated with alterations in CL content and composition and the downregulation of enzymes implicated in CL biosynthesis and remodeling. Analysis of metabolite content showed an alteration of pathways involved in fatty acid metabolism, mitochondrial biogenesis and regulation of β-oxidation by the transcription factor PPARα. In conclusion, pediatric DCM serum circulating factors can promote CL remodeling resulting mitochondrial dysfunction in primary cardiomyocytes. These findings suggest that CL could be a novel therapeutic target for this particular population.

Pediatric and adult dilated cardiomyopathy are distinguished by distinct biomarker profiles.

Emerging evidence suggests that pediatric and adult dilated cardiomyopathy (DCM) represent distinct diseases. Few diagnostic tools exist for pediatric cardiologists to assess clinical status and prognosis. We hypothesized that pediatric DCM would have a unique biomarker profile compared to adult DCM and controls. We utilized a DNA aptamer array (SOMAScan) to compare biomarker profiles between pediatric and adult DCM. We simultaneously measured 1310 plasma proteins and peptides from 39 healthy children (mean age 3 years, interquartile range (IQR) 1-14), 39 ambulatory subjects with pediatric DCM (mean age 2.7 years, IQR 1-13), and 40 ambulatory adults with DCM (mean age 53 years, IQR 46-63). Pediatric and adult DCM patients displayed distinct biomarker profiles, despite similar clinical characteristics. We identified 20 plasma peptides and proteins that were increased in pediatric DCM compared to age- and sex-matched controls. Unbiased multidimensionality reduction analysis suggested previously unrecognized heterogeneity among pediatric DCM subjects. Biomarker profile analysis identified four subgroups of pediatric DCM with distinguishing clinical characteristics. These findings support the emerging concept that pediatric and adult DCM are distinct disease entities, signify the need to develop pediatric-specific biomarkers for disease prognostication, and challenge the paradigm that pediatric DCM should be viewed as a single disease. Pediatric and adult DCM patients displayed distinct biomarker profiles, despite similar clinical characteristics and outcomes. Our findings suggest that pediatric DCM may be a heterogeneous disease with various sub-phenotypes, including differing biomarker profiles and clinical findings. These data provide prerequisite information for future prospective studies that validate the identified pediatric DCM biomarkers, address their diagnostic accuracy and prognostic significance, and explore the full extent of heterogeneity amongst pediatric DCM patients.

Higher Serum Lysophosphatidic Acids Predict Left Ventricular Reverse Remodeling in Pediatric Dilated Cardiomyopathy.

Background: The prognosis of pediatric dilated cardiomyopathy (PDCM) is highly variable, ranging from death to cardiac function recovery. Left ventricular reverse remodeling (LVRR) represents a favorable prognosis in PDCM. Disturbance of lipid metabolism is associated with the change of cardiac function, but no studies have examined lipidomics data and LVRR.Methods: Discovery analyses were based on 540 targeted lipids in an observational, prospective China—AOCC (An Integrative-Omics Study of Cardiomyopathy Patients for Diagnosis and Prognosis in China) study. The OPLS-DA and random forest (RF) analysis were used to screen the candidate lipids. Associations of the candidate lipids were examined in Cox proportional hazards regression models. Furthermore, we developed a risk score comprising the significant lipids, with each attributed a score of 1 when the concentration was above the median. All significant findings were replicated in a validation set of the China-AOCC study.Results: There were 59 patients in the discovery set and 24 patients in the validation set. LVRR was observed in 27 patients (32.5%). After adjusting for age, left ventricular ejection fraction (LVEF), and left ventricular end-diastolic dimension (LVEDD) z-score, lysophosphatidic acids (LysoPA) 16:0, LysoPA 18:2, LysoPA 18:1, and LysoPA 18:0 were significantly associated with LVRR in the discovery set, and hazard ratios (HRs) were 2.793 (95% CI, 1.545–5.048), 2.812 (95% CI, 1.542–5.128), 2.831 (95% CI, 1.555–5.154), and 2.782 (95% CI, 1.548-5.002), respectively. We developed a LysoPA score comprising the four LysoPA. When the LysoPA score reached 4, LVRR was more likely to be observed in both sets. The AUC increased with the addition of the LysoPA score to the LVEDD z-score (from 0.693 to 0.875 in the discovery set, from 0.708 to 0.854 in the validation set) for prediction of LVRR.Conclusions: Serum LysoPA can predict LVRR in PDCM patients. When the LysoPA score was combined with the LVEDD z-score, it may help in ascertaining the prognosis and monitoring effects of anti-heart failure pharmacotherapy.

Next-Generation Sequencing Reveals Novel Genetic Variants for Dilated Cardiomyopathy in Pediatric Chinese Patients.

Dilated cardiomyopathy (DCM) is a myocardial disease characterized by bilateral or left ventricular cardiac dilation and systolic dysfunction that can lead to heart failure and sudden cardiac death in children. Many studies have focused on genetic variation in DCM-related genes in adult populations; however, the mutational landscape in pediatric DCM patients remains undetermined, especially in the Chinese population. We applied next-generation sequencing (NGS) technology to genetically analyze 46 pediatric DCM patients to reveal genotype-phenotype correlations. Our results indicated DCM-associated pathogenic mutations in 10 genes related to the structure or function of the sarcomere, desmosome, and cytoskeleton. We also identified 6 pathogenic mutations (5 novel) in the Titin (TTN) gene that resulted in truncated TTN variants in 6 (13%) out of 46 patients. Correlations between TTN mutations and clinical outcomes were assessed. Our data indicate that one-third of pediatric DCM cases are caused by genetic mutations. The role of TTN variants should not be underestimated in pediatric DCM and age-dependent pathogenic penetrance of these mutations should be considered for familial DCM cases. We argue that genetic testing of DCM cases is valuable for predicting disease severity, prognosis, and recurrence risk, and for screening first-degree relatives.

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

BackgroundmicroRNAs (miRNAs) are short single stranded nucleotides that can regulate gene expression. Although we previously evaluated expression of miRNAs in pediatric dilated cardiomyopathy (DCM) by miRNA array, pathway prediction based on changes in mRNA expression has not been previous analyzed in this population. The current study aimed to determine regulation of miRNA expression by miRNA-Seq and, through mRNA-Seq, analyze their putative target genes and altered pathways in pediatric DCM hearts.MethodsmiRNA 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.ResultsExpression 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 up-regulated pathways related regulation of stem cell differentiation and cardiac muscle contraction, and down-regulated pathways related to regulation of protein phosphorylation, signal transduction and cell communication.ConclusionOur 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.

Serum from pediatric dilated cardiomyopathy patients promotes dysregulation of cardiolipin biosynthesis and mitochondrial dysfunction

Pediatric dilated cardiomyopathy (DCM) is a devastating and poorly understood disease with most clinical treatment paradigms extrapolated from the adult population. Our studies showed that aspects of metabolism and mitochondria function are dysregulated in pediatric hearts. Cardiolipin (CL), a unique phospholipid in the inner mitochondrial membrane, is essential for optimal mitochondrial function and was shown to be dysregulated in failing adult hearts. We investigate if serum circulating factors from pediatric DCM patients can remodel CL resulting in mitochondrial dysfunction similar to what is observed in the failing pediatric heart. Using a novel in vitro model that consists of treating neonatal rat ventricular myocytes (NRVMs) with serum from pediatric DCM patients or from non-failing (NF) healthy controls, mitochondrial respiration was assessed using Seahorse assay, and reactive oxygen species (ROS) was assessed using Electron Paramagnetic Resonance Spectroscopy. Expression of enzymes involved in CL biosynthesis and remodeling were analyzed using RT-qPCR and relative mtDNA copy number determined by qPCR. Mass spectroscopy was used to quantitate total and specific CL species and to investigate the metabolite composition of NRVMs treated with NF or DCM serum. While mitochondrial ROS and copy number were not significantly altered, we showed that DCM serum decreases mitochondrial function, which is associated with alterations in CL content and composition and gene expression of enzymes implicated in CL biosynthesis and remodeling. Analysis of metabolite content showed an alteration of pathways involved in fatty acid metabolism, mitochondrial biogenesis and regulation of β-oxidation by PPARα. Pediatric DCM serum circulating factors can promote CL remodeling resulting in mitochondrial dysfunction in cardiomyocytes. These findings suggest that CL could be a therapeutic target for this particular population.

Differential Expression Profiles and Functional Prediction of Circular RNAs in Pediatric Dilated Cardiomyopathy

Circular RNAs (circRNAs) have emerged as essential regulators and biomarkers in various diseases. To assess the different expression levels of circRNAs in pediatric dilated cardiomyopathy (PDCM) and explore their biological and mechanistic significance, we used RNA microarrays to identify differentially expressed circRNAs between three children diagnosed with PDCM and three healthy age-matched volunteers. The biological function of circRNAs was assessed with a circRNA–microRNA (miRNA)–mRNA interaction network constructed from Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. Differentially expressed circRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR) in 25 children with PDCM and 25 healthy volunteers. We identified 257 up-regulated (fold change ≤ 0.5, P < 0.05) and 899 down-regulated (fold change ≥2, P < 0.05) circRNAs in PDCM patients when compared to healthy volunteers. The qRT-PCR experiments confirmed has_circ_0067735 down-regulation (0.45-fold, P < 0.001), has_circ_0070186 up-regulation (2.82-fold, P < 0.001), and has_circ_0069972 down-regulation (0.50-fold, P < 0.05). A functional analysis of these differentially expressed circRNAs suggests that they are associated with hypertrophy, remodeling, fibrosis, and autoimmunity. CircRNAs have been implicated in PDCM through largely unknown mechanisms. Here we report differentially expressed circRNAs in PDCM patients that may illuminate the mechanistic roles in the etiology of PDCM that could serve as non-invasive diagnostic biomarkers.

Single nuclei sequencing reveals novel insights into cardiac cell signatures in human pediatric dilated cardiopathy

Abstract The mechanism underlying dilated cardiomyopathy (DCM) in children without a known genetic disorder are unclear. In contrast to adult DCM patients, there is an unmet need for therapeutic options that improve survival in pediatric DCM. Therefore, we performed single nuclei RNA sequencing (snRNA-seq) from heart tissue obtained from children undergoing heart transplantation due to severe heart failure. We processed heart tissue from 6 children with DCM (EF: 18.67±2.11%) of an age of 0.5, 0.75, 5, 6, 12 and 13 years (y). After snRNA-seq, unsupervised clustering was performed identifying 8 major cell types, including cardiomyocytes (CM), fibroblasts (FB), endothelial cells, leukocytes, pericytes, smooth muscle cells, neuronal-like cells and an endothelial-fibroblast-like cluster. Relative numbers of FB clusters correlated with increasing age of the children which was clinically validated by measuring late enhancement (LE) with cardiac magnetic resonance imaging in 68 pediatric DCM patients. The mean age of patients with LE was 5.86±0.53y vs. 2.36±0.53y in patients without LE (p&amp;lt;0.05). Further analysis of unique highly expressed genes (DEGs) between the 3 age groups identified a profound alteration of gene expression in FB clusters. FBs of explants of &amp;lt;1y old patients showed high expression of anti-fibrotic, development and remodeling associated genes. In contrast, FBs of 12–13y old children highly expressed pro-fibrotic and FB activation associated genes as transforming growth factor beta binding protein (6.63 fold), cytochrome P450 1B1 (3.79 fold), and periostin (7.67 fold) (all p&amp;lt;0.05). Moreover, we observed a switch in collagen expression patterns and in thrombospondin isoforms (from THBS1 to THBS4). Furthermore, our analysis revealed most profound transcriptional changes in CMs. We identified a cluster of CMs with a pro-regenerative profile in &amp;lt;1y old patients, which could not be detected during adolescence. This CM cluster showed high expression of genes associated with proliferation (e.g. cyclin D2), glycolytic metabolism and anti-oxidant markers. Increased cyclin D2 was confirmed by immunostaining (1.43 fold higher in &amp;lt;1y vs. 12–13y). Since all of these gene expression patterns might be affected by the underlying disease of the pediatric heart recipients, we explored their expression in FBs and CMs of postnatal vs. adult mice. Importantly, we could recapitulate the vast majority of the findings from humans in the mice experiments. Together, these data demonstrate an age-dependent decrease in CM numbers concomitant with increased FBs in pediatric DCM. FBs of &amp;lt;1y old pediatric patients revealed a distinct collagen expression profile and showed lower levels of pro-fibrotic genes. CMs of &amp;lt;1y old donors where characterized with a regeneration enabling gene expression profile, which include pro-proliferative genes. The expression patterns of the CMs indicates, that regeneration might also occur in humans during the firs year of life. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): Dr. Rolf M. Schwiete Stiftung, DZHK

Abstract 555: Sex-Dependent Regulation of Cardiomyocyte Function by Midkine in Pediatric Dilated Cardiomyopathy

Background: Pediatric dilated cardiomyopathy (DCM) is a disease with a poor prognosis that affects 1 in 100,000 children. Girls with DCM have worse outcomes than boys, and the mechanisms that lead to these differences are not clear. We have identified a cytokine, midkine (MDK) that is significantly upregulated in the serum from pediatric DCM patients. Circulating MDK is significantly higher in girls with DCM requiring heart transplantation compared to girls with DCM who are stable. Objective: The objective of this study is to determine if MDK impacts cardiomyocyte function in a sex-specific manner in juvenile male and female cells. Methods: Cardiomyocytes isolated from 3 week old juvenile male and female rats (JRVMs) were treated with 1μg MDK for 48 hours. Cardiomyocyte function and calcium dynamics were assessed using an IonOptix system. Myofibril mechanics and calcium sensitivity were measured. In addition, RNA sequencing was completed to identify differentially regulated pathways in juvenile male and female cardiomyocytes in response to MDK. Results: Female JRVMs treated with MDK had higher peak calcium and slower calcium reuptake compared to vehicle-treated female JRVMs. In contrast, male JRVMs treated with MDK did not demonstrate a change in peak calcium and had faster calcium reuptake compared to vehicle-treated male JRVMs. Myofibril calcium sensitivity was decreased in female JRVMs in response to MDK whereas calcium sensitivity was unchanged in male MDK-treated JRVMs. Analysis of the genes that were differentially expressed in cardiomyocytes in response to MDK demonstrated a sex-dependent regulation. Specifically, pathways which regulate calcium handling were only altered in female JRVMs treated with MDK but not in MDK-treated male JRVMs. Conclusions: This study demonstrates sex-specific differences in cardiomyocyte function in response to MDK. Particularly, female cardiomyocytes respond to MDK by regulating genes involved in calcium handling pathways. This suggests that elevated circulating MDK in pediatric DCM patients may lead to different cardiac responses in male and female patients. Therefore, elucidating these sex-specific disease mechanisms is critical to define therapies focused on male and female pediatric DCM patients.

Detection of parvovirus B19 and human herpesvirus 6 in pediatric dilated cardiomyopathy: Impact after heart transplantation.

Objectives:The aim of this study is to evaluate HHV-6 and PVB19 infection using polymerase chain reaction (PCR) and immunofluorescent assay (IFA) in the myocardium of pediatric patients with dilated cardiomyopathy (DCM) and the impact of viral persistence in the cardiac allograft after heart transplantation (HT).Methods:Multiplex droplet digital PCR was used to analyze the prevalence of viral sequences in myocardial samples from 48 pediatric DCM patients and 10 control subjects. Of the 48 DCM patients, 44 underwent HT. After HT, consecutive endomyocardial biopsy (EMB) samples were analyzed for the presence of PVB19 and HHV-6 antigens using IFA and the patients were evaluated for rejections, coronary vasculopathy, and graft loss.Results:Of the 48 DCM patients, 14 had positive viral PCR results in explanted/autopsy hearts. Among them, PVB19 was found in 8/48, HHV6 in 4/48, both PVB19 and HHV6 in 1/48, and enterovirus in one, but no adenovirus was found. The EMB samples obtained after HT were positive for PVB19 and HHV-6 in 7/44 and 3/44 cases, respectively. Viral presence in both the explanted heart and the cardiac allograft was demonstrated in 4 patients, 3 of whom were positive for PVB19, and one of whom was positive for HHV-6 pretransplant. Coronary vasculopathy and graft loss were more common in patients with PVB19-positive myocardial tissues versus those who were PVB19-negative.Conclusions:There is an association between PVB19 and HHV-6 infection and DCM in children. The study suggests the persistence of PVB19 and HHV-6 in the host can lead to subsequent viral reactivation in the transplanted heart, even in those recipients who do not have active myocarditis. PVB19 in the cardiac allograft tended toward higher adverse post-HT events.

Sex-dependent regulation of autophagy by midkine in pediatric dilated cardiomyopathy

Background: Sex hormones are thought to influence sex-based clinical and molecular differences in adult patients with dilated cardiomyopathy (DCM). Interestingly, sex differences are also observed in pre-pubertal pediatric DCM patients, as girls with DCM have worse outcomes than boys. This suggests that sex-hormone-independent differences also play a role in cardiac pathology. One potential factor which may regulate sex differences in pediatric cardiac function is midkine (MDK), a protein that is highly elevated in the serum of pediatric DCM patients. While MDK’s role in cardiac dysfunction is unclear, in cancer models, MDK has been found to inhibit autophagy. The objective of this study was to determine if high exogenous MDK leads to sex differences in autophagy and cardiac function.

Remodeling of myocardial extracellular matrix and proteoglycans varies in pediatric versus adult patients with dilated cardiomyopathy

Background: Idiopathic dilated cardiomyopathy (DCM) is a common cause of heart failure in adult and pediatric patients, but the underlying mechanisms vary since pediatric DCM patients do not respond as well to treatments designed for the adult patient population.

Genetic Testing in Pediatric Cardiomyopathy.

Genetic testing is recommended in patients with dilated cardiomyopathy (DCM); however, limited studies demonstrate high yields of genetic testing in non-hypertrophic (HCM) patients. Furthermore, there is sparse genotype-phenotype data in pediatric DCM patients. We performed a retrospective review of 70 consecutive probands with cardiomyopathy (non-HCM) who underwent genetic evaluation. Mean age at presentation was 5.48years. Echocardiography revealed mean ejection fraction of 32.4%. The LVEDd z score ranged from - 5.7 to + 15.9. Cardiomyopathy was classified as dilated in 56, 10 with non-compaction, 2 with restrictive, and 2 with ARVC. TTN gene mutations were the most common gene involved. Genetic testing was negative in 16/70 (23%) giving a yield of 77% including VUS. 33% (23/70) of probands had a positive family history among whom the diagnostic yield was 57% (13/23) for pathogenic mutations. Yield for positive genetic testing in the DCM with positive family history group was 9/18 (50%). There were 6 deaths (9%) and 26/70 (37%) underwent transplantation. More frequent cardiac transplantations (48 vs. 34%) and deaths (17 vs. 2%) were seen in mutation-positive vs. mutation-negative subgroups. This study demonstrates an increasing yield of genetic testing in DCM although with a high rate of VUS detection. Use of genetic information for better management and prognostication will require big data analysis.

The role of Midkine in Children with Dilated Cardiomyopathy

Introduction: While current medical therapies benefit adult heart failure (HF) patients, these therapies do not confer the same improvement in survival in children with HF. This difference suggests that the underlying cellular mechanisms in failing myocytes are uniquely regulated. We have found that serum circulating factors are important in cardiomyocyte remodelling in pediatric patients with Dilated Cardiomyopathy (DCM). In our preliminary results, we identified a specific heparin-binding growth factor, midkine (MDK), which is highly upregulated in the serum of pediatric DCM patients. The objective of this study is to investigate the relation between serum MDK levels and clinical outcome, and to define the effect of MDK in modulating pathologic responses. Methods and results: Our current serum bank contains 5 non-failing (NF) and 44 DCM samples from individual patients. The level of serum (circulating) MDK was determined in the banked serum from pediatric DCM patients by ELISA, and correlated with the patient’s clinical outcome. Our results show that MDK levels in serum was elevated in pediatric DCM patients who died or were transplanted in comparison with pediatric DCM patients who are clinically stable. The contribution of serum MDK to primary cardiomyocytes was investigated by treating neonatal rat ventricular myocytes (NRVMs) with human recombinant MDK for 72 hours. Our results show an increase in the expression of genes associated with pathologic remodelling in MDK-treated NRVMs. In addition, there is an increase in cell area and ANF expression in MDK-treated NRVMs compared to NF serum-treated cells. In our unique mouse model of pediatric cardiac pathologic growth (isoproterenol infusion –30mg/kg/day), MDK treatment (3mg/kg/week) resulted in increased heart/body weight. Conclusions: Our results suggest that MDK is detrimental to cardiac function. This finding combined with increased circulating MDK in end-stage pediatric DCM patients, suggests an important regulatory role for MDK in pediatric HF.