Late-onset Cardiomyopathy Research Articles

Neonatal lupus with left bundle branch block and cardiomyopathy: a case report

BackgroundCardiac manifestations of neonatal lupus include an array of structural and conduction abnormalities due to placental transference of maternal anti-SSA/Ro and anti-SSB/La autoantibodies. Late-onset neonatal lupus cardiomyopathies, occurring outside the neonatal period, is an infrequently reported manifestation with unknown pathophysiology and poorly defined treatment regimens. Due to the rarity of this condition, additional studies and case reports are required to better understand and manage late-onset neonatal lupus cardiomyopathies.Case presentationA 4-week-old female, born to a mother with known anti-SSA/Ro and anti-SSB/La autoantibodies, presents with classic cutaneous manifestations for neonatal lupus and is found to have left bundle branch block, severely dilated cardiomyopathy with an ejection fraction of 25%, and a thin echogenic dyskinetic ventricular septum. Weekly second trimester and 30-week fetal echocardiograms showed no signs of structural or conduction abnormalities. There were no histologic signs of inflammation on cardiac tissue biopsy. After a complicated hospital course, she was successfully treated with biventricular pacemaker, intravenous immunoglobulin, and plasmapheresis.ConclusionsWe present a case of late-onset neonatal lupus with severe dilated cardiomyopathy, a dyskinetic ventricular septum, and left bundle branch block. To our knowledge, the dyskinetic ventricular septum has never been reported and left bundle branch block is rarely reported in NL. This case further validates the need for long term cardiac follow up for patients born with NL, even if lacking cardiac manifestations in the peripartum period. We characterize a unique presentation of a rare clinical entity, highlighting the diagnostic challenges, and describe a successful treatment course.

CRISPR-Cas9 generated Pompe knock-in murine model exhibits early-onset hypertrophic cardiomyopathy and skeletal muscle weakness

Infantile-onset Pompe Disease (IOPD), caused by mutations in lysosomal acid alpha-glucosidase (Gaa), manifests rapidly progressive fatal cardiac and skeletal myopathy incompletely attenuated by synthetic GAA intravenous infusions. The currently available murine model does not fully simulate human IOPD, displaying skeletal myopathy with late-onset hypertrophic cardiomyopathy. Bearing a Cre-LoxP induced exonic disruption of the murine Gaa gene, this model is also not amenable to genome-editing based therapeutic approaches. We report the early onset of severe hypertrophic cardiomyopathy in a novel murine IOPD model generated utilizing CRISPR-Cas9 homology-directed recombination to harbor the orthologous Gaa mutation c.1826dupA (p.Y609*), which causes human IOPD. We demonstrate the dual sgRNA approach with a single-stranded oligonucleotide donor is highly specific for the Gaac.1826 locus without genomic off-target effects or rearrangements. Cardiac and skeletal muscle were deficient in Gaa mRNA and enzymatic activity and accumulated high levels of glycogen. The mice demonstrated skeletal muscle weakness but did not experience early mortality. Altogether, these results demonstrate that the CRISPR-Cas9 generated Gaac.1826dupA murine model recapitulates hypertrophic cardiomyopathy and skeletal muscle weakness of human IOPD, indicating its utility for evaluation of novel therapeutics.

Deep learning for improved prediction of late-onset cardiomyopathy among childhood cancer survivors: A report from the St. Jude Lifetime Cohort (SJLIFE).

10545 Background: Early identification of survivors at high risk for treatment-induced cardiomyopathy may allow for prevention and/or early intervention. We utilized deep learning methods using COG guideline-recommended baseline electrocardiography (ECG) to improve prediction of future cardiomyopathy. Methods: SJLIFE is a cohort of 5-year clinically assessed childhood cancer survivors including baseline ECG measurements. Development of cardiomyopathy was identified from clinical and echocardiographic measurement using CTCAE criteria (grade 3-4). We applied deep learning approaches to ECG, treatment exposure and demographic data obtained at baseline SJLIFE assessment. We trained a cascaded model combining a 12-layer 1D convolutional neural network to extract features from waveform ECG signals with a 2-layer dense neural network to embed features from other phenotypic data in tabular format to determine if use of deep learning with ECG data could improve prediction of cardiomyopathy. Results: Among 1,218 subjects (median age 31.7 years, range 18.4-66.4) without cardiomyopathy at baseline evaluation, 616 (51%) were male, 1,041 (85%) white, 157 (13%) African American and 792 (65%) were survivors of lymphoma/leukemia. Follow-up averaged 5 (0.5 to 9) years from baseline examination. Mean chest radiation dose was 1350 cGy (range 0 to 6,200 cGy) and mean cumulative anthracycline dose was 191 mg/m2 (range o to 734 mg/m2). A total of 114 (9.4%) survivors developed cardiomyopathy after baseline. A cascaded deep learning model built on a training set (N = 974 participants) classified cardiomyopathy in the test set (N = 244 participants) using both clinical and ECG data with a sensitivity of 70%, specificity of 73%, and AUC of 0.74 (95% CI 0.63-0.85), compared to a model using clinical data alone (sensitivity 61%, specificity 62%, and AUC 0.67, 95% CI 0.56-0.79). In subgroup analyses, models predicting cardiomyopathy within 0-4 years following baseline had a sensitivity, specificity, and AUC of 77%, 78%, and 0.78 (0.65-0.91), respectively. When predicting cardiomyopathy 5-9 years following baseline, model performance dropped to a sensitivity, specificity, and AUC of 70%, 70%, and 0.68 (0.50-0.87), respectively. Conclusions: Deep learning using ECG at baseline evaluation significantly improved prediction of cardiomyopathy in childhood cancer survivors at high risk for cardiomyopathy. Future directions will incorporate deep learning approaches to echocardiography to further improve prediction.

Abstract P1-01-03: Association of cardiac energetics and plasma biomarkers with anthracycline-based chemotherapy in breast cancer patients

Abstract BACKGROUND Anthracyclines are widely used in the treatment of breast cancer and are well recognised to carry increased risk of cardiotoxicity. This can occur as an early, acute manifestation or many years after treatment as late onset cardiomyopathy. It is increasingly apparent that there may be a chronic subclinical phase associated with low grade cardiac injury with no apparent clinical impact on the contractile function of the heart. This period may remain latent for many years with the patient remaining asymptomatic and with apparently normal cardiac function. Identification of early and subtle cardiac dysfunction during this period could provide a window of opportunity for therapeutic intervention if those at risk or those experiencing this low-grade decline could be accurately identified. This study explores the potential utility of phosphorus magnetic resonance spectroscopy (31P-MRS) to identify very early anthracycline-induced cardiac toxicity and baseline predisposition to cardiac injury. This technique measures the phosphocreatine/adenosine triphosphate concentration ratio (PCr/ATP), which reflects cardiac cellular energetics. The normal range of PCr/ATP is approximately 1.8 - 2.2, whereas decreased PCr/ATP is associated with a drop in available energy reserve in the heart, associated with many forms of heart failure. Conventional cardiac investigations of electrocardiogram, multiple gated acquisition radionuclide scans (MUGA) and ultra-high sensitivity plasma cardiac troponin-I (cTn-I) have also been applied. METHODS 20 newly diagnosed breast cancer patients, receiving anthracycline-based chemotherapy, had magnetic resonance imaging to assess left ventricular ejection fraction and 31P magnetic resonance spectroscopy (31P MRS) to assess phosphocreatine to adenosine triphosphate concentration ratio (“PCr/ATP”). Measurements were performed pre-chemo, mid-chemo and post-chemo. Plasma high sensitivity cardiac troponin-I (cTn-I) and electrocardiograms were performed. RESULTS There was a significant negative correlation between change in PCr/ATP pre- to mid- chemo and change mid- to post-chemo (r=-0.68, p=0.04). Mean ejection fraction reduced significantly (5.1%) pre- to post-chemo (p=0.02). Change in PCr/ATP ratios pre- to post-chemo correlated inversely with changes in LVEF over the same period (r=-0.65, p=0.006) and mid- to post-chemotherapy (r=-0.77, p=0.002). Plasma cTn-I increased pre- to mid-chemo (1.35±0.81 to 4.40±2.64 ng/L; p=0.0001) and mid to post-chemo (4.40±2.64 to 18.33±13.23 ng/L; p=0.0001). CONCLUSIONS This study has demonstrated the use of 31P MRS in exploring cardiac energetics of breast cancer patients undergoing chemotherapy. Pre-chemotherapy PCr/ATP ratios were in line with healthy control values obtained using our system. This technique detected a pattern of recovery of PCr/ATP ratios following an initial 3 cycles of anthracyclines. Changes in PCr/ATP ratios were found to be significantly negatively correlated with changes in LVEF from pre to post chemotherapy. The significant decrease in LVEF from pre-post chemo may be due to the delayed response of LVEF to the initial 3 cycles of anthracyclines. Small but significant increases in plasma levels of cardiac troponin-I were observed early after initiation of anthracyclines. PCr/ATP ratios were not found to be related to cTn-I values but LVEF and cTn-I were found to be positively correlated. Future studies should take account of background factors that could influence cardiac PCr/ATP ratio such as age, physical fitness and regular medication whilst the improved sensitivity of 7T MRI scanners may also help in detecting earlier changes in cardiac energetics in this patient group. Citation Format: Olga Oikonomidou, Gillian Macnaught Macnaught, Christopher T Rodgers, William Clarke, Annette Cooper, Heather McVicars, Arran K Turnbull, Saeed Mirsadraee, Scott Semple, Martin Denvir. Association of cardiac energetics and plasma biomarkers with anthracycline-based chemotherapy in breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-01-03.

Cardiomyopathy associated with the Ala143Thr variant of the α-galactosidase A gene

ObjectiveTo investigate whether the Ala143Thr variant of the α-galactosidase A gene (A143T/GLA), with conflicting interpretations of pathogenicity, is associated with Fabry cardiomyopathy.MethodsThe index patient, a woman in her 60s with...

Splicing mutation in LAMP2 gene leading to exon skipping and cardiomyopathy development

Mutations in LAMP2 (Xq24) encoding a lysosomal-associated membrane protein 2 crucial to the maintenance and adhesion of the lysosome cause a Danon disease. It is an X-linked glycogen storage disease that includes cardiomyopathy (hypertrophic or dilated), skeletal myopathy and mental retardation. Here, we report a woman, carrying the mutation c.864 + 1G > A (IVS6 + 1G > A, rs727503119) in heterozygosity and affected with late-onset dilated cardiomyopathy. We demonstrated the effects of the mutation on the splicing LAMP2 in cardiac muscle. The variant c.864 + 1G > A is located in the canonical splice donor site in intron 6. Functional analysis reveals that mutation c.864 + 1G > A leads to the skipping of exon 6 in cardiac muscle. The protein loses 41 amino acids in the second luminal domain of LAMP2 without frameshift and premature termination of protein synthesis. Using qRT-PCR we revealed a significantly low level of wild-type LAMP2 mRNA in patient that can be explained by X-inactivation.

A cardiac α-actin (ACTC1) p. Gly247Asp mutation inhibits SRF-signaling in vitro in neonatal rat cardiomyocytes

We recently identified a novel, heterozygous, and non-synonymous ACTC1 mutation (p.Gly247Asp or G247D) in a large, multi-generational family, causing atrial-septal defect followed by late-onset dilated cardiomyopathy (DCM). Molecular dynamics studies revealed possible actin polymerization defects as G247D mutation resides at the juncture of side-chain interaction, which was indeed confirmed by in vitro actin polymerization assays. Since polymerization/de-polymerization is important for the activation of Rho-GTPase-mediated serum response factor (SRF)-signaling, we studied the effect of G247D mutation using luciferase assay. Overexpression of native human ACTC1 in neonatal rat cardiomyocytes (NRVCMs) strongly activated SRF-signaling both in C2C12 cells and NRVCMs, whereas, G247D mutation abolished this activation. Mechanistically, we found reduced GTP-bound Rho-GTPase and increased nuclear localization of globular actin in NRVCMs overexpressing mutant ACTC1 possibly causing inhibition of SRF-signaling activation. In conclusion, our data suggests that human G247D ACTC1 mutation negatively regulates SRF-signaling likely contributing to the late-onset DCM observed in mutation carrier patients.

Cardiac α-Actin (ACTC1) Gene Mutation Causes Atrial-Septal Defects Associated With Late-Onset Dilated Cardiomyopathy.

Familial atrial septal defect (ASD) has previously been attributed primarily to mutations in cardiac transcription factors. Here, we report a large, multi-generational family (78 members) with ASD combined with a late-onset dilated cardiomyopathy and further characterize the consequences of mutant α-actin. We combined a genome-wide linkage analysis with cell biology, microscopy, and molecular biology tools to characterize a novel ACTC1 (cardiac α-actin) mutation identified in association with ASD and late-onset dilated cardiomyopathy in a large, multi-generational family. Using a genome-wide linkage analysis, the ASD disease locus was mapped to chromosome 15q14 harboring the ACTC1 gene. In 15 affected family members, a heterozygous, nonsynonymous, and fully penetrant mutation (p. Gly247Asp) was identified in exon 5 of ACTC1 that was absent in all healthy family members (n=63). In silico tools predicted deleterious consequences of this variant that was found absent in control databases. Ultrastructural analysis of myocardial tissue of one of the mutation carriers showed sarcomeric disarray, myofibrillar degeneration, and increased apoptosis, while cardiac proteomics revealed a significant increase in extracellular matrix proteins. Consistently, structural defects and increased apoptosis were also observed in neonatal rat ventricular cardiomyocytes overexpressing the mutant, but not native human ACTC1. Molecular dynamics studies and additional mechanistic analyses in cardiomyocytes confirmed actin polymerization/turnover defects, thereby affecting contractility. A combined phenotype of ASD and late-onset heart failure was caused by a heterozygous, nonsynonymous ACTC1 mutation. Mechanistically, we found a shared molecular mechanism of defective actin signaling and polymerization in both cardiac development and contractile function. Detection of ACTC1 mutations in patients with ASD may thus have further clinical implications with regard to monitoring for (late-onset) dilated cardiomyopathy.

Genetic susceptibility in pharmacodynamic and pharmacokinetic pathways underlying drug-induced arrhythmia and sudden unexplained deaths

Drug-induced arrhythmia is an adverse drug reaction that can be potentially fatal since it is mostly related to drug-induced QT prolongation, a known risk factor for Torsade de Pointes and sudden cardiac death (SCD). Several risk factors have been described in association to these drug-induced events, such as preexistent cardiac disease and genetic variation. Our objective was to study the genetic susceptibility in pharmacodynamic and pharmacokinetic pathways underlying suspected drug-induced arrhythmias and sudden unexplained deaths in 32 patients. The genetic component in the pharmacodynamic pathway was studied by analysing 96 genes associated with higher risk of SCD through massive parallel sequencing. Pharmacokinetic-mediated genetic susceptibility was investigated by studying the genes encoding cytochrome P450 enzymes using medium-throughput genotyping. Pharmacodynamic analysis showed three probably pathogenic variants and 45 variants of uncertain significance in 28 patients, several of them previously described in relation to mild or late onset cardiomyopathies. These results suggest that genetic variants in cardiomyopathy genes, in addition to those related with channelopathies, could be relevant to drug-induced cardiotoxicity and contribute to the arrhythmogenic phenotype. Pharmacokinetic analysis showed three patients that could have an altered metabolism of the drugs they received involving CYP2C19 and/or CYP2D6, probably contributing to the arrhythmogenic phenotype. The study of genetic variants in both pharmacodynamic and pharmacokinetic pathways may be a useful strategy to understand the multifactorial mechanism of drug-induced events in both clinical practice and forensic field. However, it is necessary to comprehensively study and evaluate the contribution of the genetic susceptibility to drug-induced cardiotoxicity.

A CARDIAC α-ACTIN (ACTC1) GENE MUTATION CAUSES ATRIAL-SEPTAL DEFECTS ASSOCIATED WITH DILATED CARDIOMYOPATHY

Atrial septal defect (ASD) is a common congenital heart defect. Its pathogenesis is not well defined, but mutations in cardiac transcription factor genes have been described in familial forms. Here, we report a large, multi-generational family with ASD combined with late-onset dilated cardiomyopathy

LATE-ONSET HYPERTROPHIC CARDIOMYOPATHY

Hypertrophic cardiomyopathy (HCM) is often considered a disease of the youth; however, it is also diagnosed in the elderly. The presentation of late-onset hypertrophic cardiomyopathy is not ubiquitous and precise diagnosis requires a multimodality approach. A 71-year-old man presented without

Doxorubicin-induced cardiomyopathy associated with inhibition of autophagic degradation process and defects in mitochondrial respiration

Doxorubicin (Dox) is a highly effective anticancer drug but cause acute ventricular dysfunction, and also induce late-onset cardiomyopathy and heart failure. Despite extensive studies, the pathogenic sequelae leading to the progression of Dox-associated cardiomyopathy remains unknown. We assessed temporal changes in autophagy, mitochondrial dynamics, and bioenergetics in mouse models of acute and chronic Dox-cardiomyopathy. Time course study of acute Dox-treatment showed accumulation of LC3B II in heart lysates. Autophagy flux assays confirmed that the Dox-induced accumulation of autophagosomes occurs due to blockage of the lysosomal degradation process. Dox-induced autophagosomes and autolysosome accumulation were confirmed in vivo by using GFP-LC3 and mRFP-GFP-LC3 transgenic (Tg) mice. Mitochondria isolated from acute Dox-treated hearts showed significant suppression of oxygen consumption rate (OCR). Chronic Dox-cardiotoxicity also exhibited time-dependent accumulation of LC3B II levels and increased accumulation of green puncta in GFP-LC3 Tg hearts. Mitochondria isolated from chronic Dox-treated hearts also showed significant suppression of mitochondrial OCR. The in vivo impairment of autophagic degradation process and mitochondrial dysfunction data were confirmed in vitro using cultured neonatal cardiomyocytes. Both acute and chronic Dox-associated cardiomyopathy involves a multifocal disease process resulting from autophagosomes and autolysosomes accumulation, altered expression of mitochondrial dynamics and oxidative phosphorylation regulatory proteins, and mitochondrial respiratory dysfunction.

Derivation of Anthracycline and Anthraquinone Equivalence Ratios to Doxorubicin for Late-Onset Cardiotoxicity

Anthracyclines are part of many effective pediatric cancer treatment protocols. Most pediatric oncology treatment groups assume that the hematologic toxicity of anthracycline agents is equivalent to their cardiotoxicity; for example, Children's Oncology Group substitution rules consider daunorubicin and epirubicin isoequivalent to doxorubicin, whereas mitoxantrone and idarubicin are considered 4 to 5 times as toxic as doxorubicin. To determine optimal dose equivalence ratios for late-onset cardiomyopathy between doxorubicin and other anthracyclines or the anthraquinone mitoxantrone. This multicenter cohort study of childhood cancer survivors who survived 5 or more years analyzed data pooled from 20 367 participants in the Childhood Cancer Survivor Study treated from 1970 to 1999, 5741 participants in the Dutch Childhood Oncology Group LATER study diagnosed between 1963 and 2001, and 2315 participants in the St Jude Lifetime study treated from 1962 to 2005. Cumulative doses of each agent (the anthracyclines doxorubicin, daunorubicin, epirubicin, and idarubicin; and the anthraquinone mitoxantrone) along with chest radiotherapy exposure were abstracted from medical records. Cardiomyopathy (severe, life-threatening, or fatal) by 40 years of age. Agent-specific Cox proportional hazards models evaluated cardiomyopathy risk, adjusting for chest radiotherapy, age at cancer diagnosis, sex, and exposure to anthracyclines or to an anthraquinone. An agent-specific cardiomyopathy equivalence ratio (relative to doxorubicin) was estimated for each dose category as a ratio of the hazard ratios, and then a weighted mean determined the overall agent-specific equivalence ratio across all dose categories. Of 28 423 survivors (46.4% female; median age at cancer diagnosis 6.1 years [range, 0.0-22.7 years]), 9330 patients received doxorubicin, 4433 received daunorubicin, 342 received epirubicin, 241 received idarubicin, and 265 received mitoxantrone. After a median follow-up of 20.0 years (range, 5.0-40.0 years) following receipt of a cancer diagnosis, 399 cardiomyopathy cases were observed. Relative to doxorubicin, the equivalence ratios were 0.6 (95% CI, 0.4-1.0) for daunorubicin, 0.8 (95% CI, 0.5-2.8) for epirubicin, and 10.5 (95% CI, 6.2-19.1) for mitoxantrone. Outcomes were too rare to generate idarubicin-specific estimates. Ratios based on a continuous linear dose-response relationship were similar for daunorubicin (0.5 [95% CI, 0.4-0.7]) and epirubicin (0.8 [95% CI, 0.3-1.4]). The relationship between mitoxantrone and doxorubicin appeared better characterized by a linear exponential model. In a large data set assembled to examine long-term cardiomyopathy risk in childhood cancer survivors, daunorubicin was associated with decreased cardiomyopathy risk vs doxorubicin, whereas epirubicin was approximately isoequivalent. By contrast, the current hematologic-based doxorubicin dose equivalency of mitoxantrone (4:1) appeared to significantly underestimate the association of mitoxantrone with long-term cardiomyopathy risk.

Late-onset and fast progressive neuropathy and cardiomyopathy in Val32Ala transthyretin gene mutation.

More than 100 mutations of the transthyretin gene have been reported in autosomal dominant familial amyloid polyneuropathy. This rare disease causes severe motor and sensory disability, dysautonomia, and in some patients also cardiomyopathy. The diagnosis can be challenging mainly in sporadic adult patients showing clinical, laboratory, and neurophysiological findings overlapping other forms of chronic neuropathy. We describe the clinical features and course of a patient harboring the rare p.V32A (c.155T>C) variant that was previously described in only two patients and whose pathogenicity was unclear.

Inadequate ubiquitination-proteasome coupling contributes to myocardial ischemia-reperfusion injury.

The ubiquitin-proteasome system (UPS) degrades a protein molecule via 2 main steps: ubiquitination and proteasomal degradation. Extraproteasomal ubiquitin receptors are thought to couple the 2 steps, but this proposition has not been tested in vivo with vertebrates. More importantly, impaired UPS performance plays a major role in cardiac pathogenesis, including myocardial ischemia-reperfusion injury (IRI), but the molecular basis of UPS impairment remains poorly understood. Ubiquilin1 is a bona fide extraproteasomal ubiquitin receptor. Here, we report that mice with a cardiomyocyte-restricted knockout of Ubiquilin1 (Ubqln1-CKO mice) accumulated a surrogate UPS substrate (GFPdgn) and increased myocardial ubiquitinated proteins without altering proteasome activities, resulting in late-onset cardiomyopathy and a markedly shortened life span. When subject to regional myocardial ischemia-reperfusion, young Ubqln1-CKO mice showed substantially exacerbated cardiac malfunction and enlarged infarct size, and conversely, mice with transgenic Ubqln1 overexpression displayed attenuated IRI. Furthermore, Ubqln1 overexpression facilitated proteasomal degradation of oxidized proteins and the degradation of a UPS surrogate substrate in cultured cardiomyocytes without increasing autophagic flux. These findings demonstrate that Ubiquilin1 is essential to cardiac ubiquitination-proteasome coupling and that an inadequacy in the coupling represents a major pathogenic factor for myocardial IRI; therefore, strategies to strengthen coupling have the potential to reduce IRI.

Incidence, risk factors, and mortality of neonatal and late-onset dilated cardiomyopathy associated with cardiac neonatal lupus

BackgroundDilated cardiomyopathy (DCM), a well-known complication of cardiac neonatal lupus, is associated with high mortality rate. Its risk factors remain unclear. MethodsWe analyzed occurrence of postnatal DCM among children with high-degree congenital heart block (CHB) and mothers with anti-SSA and/or anti-SSB antibodies. ResultsAmong 187 neonates with CHB, 35 (18.8%, one missing data) had DCM and 22 (11.8%) died during a median follow-up of 7years [range: birth–36years]. On multivariate analysis, factors associated with postnatal DCM were in utero DCM (P=0.0199; HR=3.13 [95% CI: 1.20–8.16]), non-European origin (P=0.0052; HR=4.10 [95% CI: 1.81–9.28]) and pacemaker implantation (P=0.0013; HR=5.48 [95% CI: 1.94–15.47]).Postnatal DCM could be categorized in two subgroups: neonatal DCM (n=13, diagnosed at a median age of 0day [birth–4days]) and late-onset DCM (n=22, diagnosed at a median age of 15.2months [3.6months–22.8years]). Factors associated with neonatal DCM were in utero DCM, hydrops, endocardial fibroelastosis and pericardial effusion, whereas those associated with late-onset DCM were non-European origin, in utero mitral valve insufficiency, and pacemaker implantation. Fluorinated steroids showed no protective effect against late-onset DCM (P=0.27; HR=1.65 [95% CI: 0.63–4.25]). Probability of survival at 10years was 23.1% for newborns diagnosed neonatally with DCM, 53.9% for those who developed late-onset DCM, and 98.6% for those without DCM. ConclusionNeonatal and late-onset DCM appear to be two different entities. None of the known risk factors associated with neonatal DCM predicted late-onset DCM. Long-term follow-up of cardiac function is warranted in all children with CHB.

Novel Roles for Obscurin Proteins in Cardiac Muscle

Obscurin is a giant myofibrillar protein important for sarcomere structure, signaling, and maintenance of sarcoplasmic reticulum (SR) organization. Surprisingly, obscurin knockout mice develop normally and only show signs of a mild skeletal muscle myopathy. However, little is known about cardiac functions of obscurin. We characterized cardiac roles of obscurin and found no changes to cardiac physiology. One explanation for the lack of a cardiac phenotype in obscurin knockout mice is that other obscurin protein family members possess a functionally redundant role. We hypothesized that obscurin-like 1 (Obsl-1) is responsible for this redundancy, in part due to its shared ability to bind the sarcomeric proteins titin and myomesin. We generated double knockout (dKO) mice by crossing the obscurin-KO mice with a cardiac specific Obsl1-KO line to investigate the roles for these proteins in cardiac development and function. Although a majority of the dKO-mice died after 12 months of age, microscopic and ultrastructural analysis failed to reveal overt changes to sarcomere organization. Dramatic changes were observed, however, in SR structure and function. Specifically we observed: 1.) loss of SR structural integrity and apparent loss of SR content, and 2.) alterations to Ca2+ transients and in the levels of Ca2+ handling proteins in KO animals. The majority of these changes were more severe in dKO-mice. Furthermore, we employed serial blockface TEM technology to precisely measure the volume of the SR and mitochondria and noticed significant differences between KO and control animals. Physiologically, these mice develop a late onset cardiomyopathy characterized by increased systolic left ventricular internal dimension and alterations in hemodynamic properties at ∼12 mo. of age. Our data suggests that obscurin and Obsl1 are not critical for sarcomerogenisis, however are important for jointly organizing SR organization, cellular Ca2+ handling, and long-term survival in mice.

330 - Doxorubicin-Mediated Toxic Effects Are Mediated Via NO/eNOS in a Novel 3D in Vitro Model of the Human Heart

Background Nitric oxide (NO●) and endothelial nitric oxide synthase (eNOS) play a dual role in the human heart by both preventing and mediating the toxic effects of chemotherapeutics such as doxorubicin (DOX). Treatment of cancer patients with DOX can result in severe late-onset dilated cardiomyopathy more than one year after completion of treatment. This cardiotoxicity has been linked to DOX-mediated upregulation of eNOS, peroxynitrous acid generation and caspase 3 activation. However, currently available models used to study this signalling pathway have limited potential to identify the cellular and molecular targets of DOX and the mechanism of toxicity in humans. Aims to use a novel in vitro 3D co-culture model, the “cardiac spheroid” (CS), generated from human endothelial cells (ECs), cardiac myocytes (CMs) and fibroblasts (CFs) to recapitulate biochemical and morphological features typical of the human heart and examine the mechanisms of DOX toxicity. Results DOX-mediated dose dependent toxic effects on CSs are shown to be dependent on NO● synthesis. Inhibition of NO● formation using L-NIO reduced DOX-mediated toxic effects in CSs at DOX concentrations detected in DOX-treated patients. Analysis at the cellular level showed that EC-derived NO● plays a protective role, whereas most of the toxic effects of DOX/NO was derived from CFs. Silencing of eNOS with lentiviral NOS3 shRNA particles in ECs confirmed a dual role of NO in mediating DOX-derived toxic effects in CSs., Genetic eNOS inhibition in CFs supported the toxic role played by these cells. Conclusions This study provides the first direct evidence for a dual role of NO. in mediating both toxic and protective DOX-mediated effects in the human heart, and cellular crosstalk responsible for this. Determining the molecular and cellular targets of novel drugs in CSs has the potential to prevent unexpected toxic effects.

Clinical and Genetic Determinants of Cardiomyopathy Risk among Hematopoietic Cell Transplantation Survivors

Cardiomyopathy has been recognized as a complication after hematopoietic cell transplantation (HCT). Using a nested case-cohort design, we examined the relationships between demographic, therapeutic, and selected cardiovascular disease risk factors among ≥1-year HCT survivors who developed cardiomyopathy before (n = 43) or after (n = 89) 1 year from HCT as compared to a randomly selected subcohort of survivors without cardiomyopathy (n = 444). Genomic data were available for 79 cases and 267 noncases. Clinical and genetic covariates were examined for association with the risk of early or late cardiomyopathy. Clinical risk factors associated with both early- and late-onset cardiomyopathy included anthracycline exposure ≥250 mg/m2 and pre-existing hypertension. Among late-onset cardiomyopathy cases, the development of diabetes and ischemic heart disease further increased risk. We replicated several previously reported genetic associations among early-onset cardiomyopathy cases, including rs1786814 in CELF4, rs2232228 in HAS3, and rs17863783 in UGT1A6. None of these markers were associated with risk of late-onset cardiomyopathy. A combination of demographic, treatment, and clinical covariates predicted early-onset cardiomyopathy with reasonable accuracy (area under the curve [AUC], .76; 95% confidence interval [CI], .68 to .83), but prediction of late cardiomyopathy was poor (AUC, .59; 95% CI .53 to .67). The addition of genetic polymorphisms with marginal associations (odds ratios ≥1.3) did not enhance prediction for either early- or late-onset cardiomyopathy. Conventional cardiovascular risk factors influence the risk of both early- and late-onset cardiomyopathy in HCT survivors. Although certain genetic markers may influence the risk of early-onset disease, further work is required to validate previously reported findings and to determine how genetic information should be incorporated into clinically useful risk prediction models.

Effect of myocardial dysfunction in cardiac morbidity and all cause mortality in childhood cancer subjects treated with anthracycline therapy.

BackgroundSubacute cardiotoxicity, consisting of acute myocyte damage and associated left ventricular dysfunction, occurs early during anthracycline therapy. We investigated the impact of myocardial dysfunction, defined herein by a shortening fraction (SF) < 29 % at any time during or after anthracycline therapy, on late onset cardiomyopathy and all-cause mortality, among childhood cancer survivors exposed to anthracyclines. In addition, we sought to identify subpopulations of subjects at highest risk for cardiomyopathy and death from all causes.MethodsFive hundred thirty-one childhood cancer survivors exposed to anthracyclines were enrolled and studied on average 10 (1.4–27.3) years following their initial exposure. The medical records were reviewed to identify known risk factors associated with cardiotoxicity, including cumulative anthracycline dose, length of post-therapy interval, administration of other cardiotoxic medications (vinca alkaloids), previous heart disease, radiation dose to the heart, history of bone marrow transplantation, age at treatment, gender, systolic dysfunction, and history of congestive heart failure during anthracycline therapy.ResultsNinety subjects (16.9 %) developed SF < 29 % and 71 patients (13.4 %) died on average 10 years after initial exposure (range 1.4–27.3 years). Total cumulative dose (OR 3.27, 95 % CI 1.94, 5.49, p < 0.001) and bone marrow transplantation (OR 2.57, 95 % CI 1.24, 5.30, p = 0.01) were found to be statistically significant risk factors for development of myocardial dysfunction. There was a 3-fold increase in the odds of having a SF < 29 % at any point during or following cancer therapy if a subject underwent bone marrow transplantation or had a total cumulative dose anthracycline therapy ≥ 240 mg/m2. The all-cause mortality ratio was almost seven-fold higher (95 % CI, 2.40-fold to 17.81-fold higher) if a subject developed systolic dysfunction, defined by a previous SF < 29 % anytime during or after anthracycline therapy. Nine deaths (12.7 %) were attributed to cardiovascular disease. The risk of dying as a result of cardiac disease also was significantly higher in individuals who had a SF < 29 % at any time during or after therapy.ConclusionsThis study demonstrates an almost seven-fold increase in all cause mortality in pediatric cancer survivors with a history of anthracycline induced myocardial dysfunction defined as SF < 29 %.