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Importance: Pediatric patients with cancer commonly experience severely bothersome symptoms. The effectiveness of routine symptom screening with symptom feedback and symptom management care pathways is unknown., Objective: To determine whether thrice-weekly symptom screening with symptom feedback and management care pathways, compared with usual care, improves overall self-reported symptom scores measured by the Symptom Screening in Pediatrics Tool (SSPedi) in pediatric patients with cancer., Design, Setting, and Participants: This cluster randomized trial enrolled participants between July 2021 and August 2023 from 20 pediatric cancer centers in the US. Patients newly diagnosed with cancer aged 8 to 18 years receiving any cancer treatment were included. Twenty sites were randomized to provide symptom screening (n = 10) vs usual care (n = 10); 221 participants were enrolled at intervention sites and 224 participants at control sites. The date of final follow-up was October 18, 2023., Intervention: Symptom screening included providing thrice-weekly symptom screening prompts to participants, email alerts to the health care team, and locally adapted symptom management care pathway implementation., Main Outcomes and Measures: The primary outcome was self-reported total SSPedi score at week 8 (range, 0-60; higher scores indicate more bothersome). Secondary outcomes were Patient-Reported Outcomes Measurement Information System Fatigue score (mean [SD] score, 50 [10]; higher scores indicate more fatigue), Pediatric Quality of Life 3.0 Acute Cancer Module scores (range, 0-100; higher scores indicate better health), symptom documentation and interventions at week 8, and unplanned health care encounters., Results: A total of 445 participants (median [range] age, 14.8 [8.1-18.9] years; 58.9% males) were enrolled. The mean (SD) 8-week SSPedi score was 7.9 (7.2) in the symptom screening group vs 11.4 (8.7) in the usual care group. Symptom screening was associated with significantly better 8-week total SSPedi scores (adjusted mean difference, -3.8 [95% CI, -6.4 to -1.2]) and less bothersome individual symptoms, with 12 of 15 symptoms being statistically significantly reduced. There was no difference in fatigue or quality of life. The mean (SD) number of emergency department visits was 0.77 (1.12) in the symptom screening group and 0.45 (0.81) in the usual care group. There were significantly more emergency department visits in the symptom screening group (rate ratio, 1.72 [95% CI, 1.03-2.87])., Conclusions: Symptom screening with symptom feedback and symptom management care pathways was associated with improved symptom scores and increased symptom-specific interventions. Future work should integrate symptom screening into routine clinical care., Trial Registration: ClinicalTrials.gov Identifier: NCT04614662.

Background: This study aimed to determine the proportion of patients receiving clinical practice guideline (CPG)-inconsistent care related to chemotherapy-induced vomiting (CIV) prophylaxis, and to describe the association between CPG-inconsistent care and site size. The association between delivery of CPG-inconsistent care and patient outcomes (CIV control, admission prolongation, and unplanned healthcare visits) was also described., Methods: This was a retrospective study conducted at Children's Oncology Group (COG) National Cancer Institute Community Oncology Research Program (NCORP) sites. Eligible patients received highly (HEC) or moderately emetogenic chemotherapy (MEC) as inpatients from January 2014 through December 2015, and were previously enrolled in a COG study. The COG generated a patient list from which patients were randomly selected for chart review by participating sites. A central panel adjudicated CIV prophylaxis received as CPG-consistent or -inconsistent., Results: Twenty-four sites participated. Over half of patients received CPG-inconsistent CIV prophylaxis (HEC: 59/112, 52.6%; MEC: 119/215, 55.3%). The most common reasons for CPG-inconsistency were shortened duration of antiemetic administration or omission of dexamethasone. Site size was not found to be associated with CPG-inconsistent care delivery (HEC: adjusted odds ratio [OR]: 0.96, 95% confidence interval [CI]: 0.76-1.23; MEC: adjusted OR: 1.07; 95% CI: 0.92-1.24). Additionally, there was no statistically significant association between receipt of CPG-inconsistent care and patient outcomes., Conclusions: Patients receiving MEC or HEC often received CPG-inconsistent CIV prophylaxis. Site size was not associated with receipt of CPG-inconsistent care. Future studies should evaluate strategies to improve CIV control among pediatric oncology patients including those aimed at improving CPG adherence., (© 2024 The Author(s). Pediatric Blood & Cancer published by Wiley Periodicals LLC.)

Background: The primary objective was to measure adherence to clinical practice guideline (CPG) recommendations for fertility preservation (FP) in pediatric cancer patients treated in National Cancer Institute Community Oncology Research Program (NCORP) sites. Secondary objectives were to describe factors such as site size associated with CPG-inconsistent care delivery and cryopreservation completion. Methods: This retrospective, multicenter study included patients 15 to 21 years old with a first cancer diagnosis from January 2014 through December 2015 who were previously enrolled to a Children's Oncology Group (COG) study and received care at a participating NCORP site. Patients were randomly selected from a list generated by the COG for chart review by participating sites. Primary outcome was care delivery that was inconsistent with a strong CPG recommendation on FP, namely discussion and offering of FP options before cancer treatment initiation, as adjudicated centrally by a panel. Results: A total of 129 patients from 25 sites were included. Among these, 48% (62/129) received CPG-inconsistent care. Most CPG-inconsistent care was due to lack of FP discussion documentation (93.5%, 58/62). Small site size, treatment at a pediatric (vs mixed adult/pediatric) site, and female sex were associated with higher odds of CPG-inconsistent care delivery. Conclusions: Newly diagnosed pediatric cancer patients often received CPG-inconsistent care for FP, with disproportionate gaps noted for females, and those treated at smaller or pediatric NCORP sites. The primary reason for CPG-inconsistent care is lack of FP discussion from clinicians. Opportunities to improve FP CPG implementation are highlighted.

Purpose: Individuals diagnosed with cancer between 15 and 39 years (adolescent and young adult [AYA]) face unique vulnerability. Detail is lacking about care delivery for these patients, especially those with ALL. We address these knowledge gaps by describing AYA ALL care delivery details at National Cancer Institute Community Oncology Research Program (NCORP) (sub)affiliates by model of care., Methods: Participating institutions treated at least one AYA with ALL from 2012 to 2016. Study-specific criteria were used to determine the number of unique clinical facilities (CFs) per NCORP and their model of care (adult/internal medicine [IM], pediatric, mixed [both]). Surveys completed by NCORPs for each CF by model of care captured size, resources, services, and communication., Results: Among 84 participating CFs (adult/IM, n=47; pediatric, n=15; mixed, n=24), 34% treated 5-10 AYAs with ALL annually; adult/IM CFs more often treated <5 (adult/IM, 60%; pediatric, 40%; mixed, 29%). Referral decisions were commonly driven by an age/diagnosis combination (58%), with frequent ALL-specific age minimums (87%) or maximums (80%). Medical, navigational, and social work services were similar across models while psychology was available at more pediatric CFs (pediatric, 80%; adult/IM, 40%; mixed, 46%-54%). More pediatric or mixed CFs reported oncologists interacting with pediatric/adult counterparts via tumor boards (pediatric, 93%; adult/IM, 26%; mixed, 96%) or initiating contact (pediatric, 100%; adult/IM, 77%; mixed 96%); more pediatric CFs reported an affiliated counterpart (pediatric, 53%; adult, 19%). Most CFs reported no AYA-specific resources (79%) or meetings (83%-98%)., Conclusion: System-level aspects of AYA ALL care delivery have not been examined previously. At NCORPs, these characteristics differ by models of care. Additional work is ongoing to investigate the impact of these facility-level factors on guideline-concordant care in this population. Together, these findings can inform a system-level intervention for diverse practice settings.

Background: The primary objective was to measure the proportion of episodes where care delivery was inconsistent with selected recommendations of a clinical practice guideline (CPG) on fever and neutropenia (FN) management. The influence of site size on CPG-inconsistent care delivery, and association between patient outcomes and CPG-inconsistent care were described., Methods: This retrospective, multicenter study included patients less than 21 years old with cancer who were at high risk of poor FN outcomes and were previously enrolled to a Children's Oncology Group (COG) study at participating National Cancer Institute Community Oncology Research Program (NCORP) institutions from January 2014 through December 2015. Patients were randomly selected for chart review by participating sites from a COG-generated list. Care delivered in each episode was adjudicated (CPG-consistent or CPG-inconsistent) against each of five selected recommendations., Results: A total of 107 patients from 22 sites, representing 157 FN episodes, were included. The most common CPG-inconsistent care delivered was omission of pulmonary computerized tomography in patients with persistent FN (60.3%). Of 74 episodes where assessment of four (episodes without persistent FN) or five (episodes with persistent FN) recommendations was possible, CPG-inconsistent care was delivered with respect to at least one recommendation in 63 (85%) episodes. Site size was not associated with CPG-inconsistent care delivery. No statistically significant association between CPG-inconsistent care and fever recurrence was observed., Conclusions: In this cohort of pediatric patients at high risk of poor FN outcomes, CPG-inconsistent care was common. Opportunities to optimize resource stewardship by boosting supportive care CPG implementation are highlighted., (© 2024 The Authors. Pediatric Blood & Cancer published by Wiley Periodicals LLC.)

Subcutaneous patient-derived xenografts (PDXs) are an important tool for childhood cancer research. Here, we describe a resource of 68 early passage PDXs established from 65 pediatric solid tumor patients. Through genomic profiling of paired PDXs and patient tumors (PTs), we observe low mutational similarity in about 30% of the PT/PDX pairs. Clonal analysis in these pairs show an aggressive PT minor subclone seeds the major clone in the PDX. We show evidence that this subclone is more immunogenic and is likely suppressed by immune responses in the PT. These results suggest interplay between intratumoral heterogeneity and antitumor immunity may underlie the genetic disparity between PTs and PDXs. We further show that PDXs generally recapitulate PTs in copy number and transcriptomic profiles. Finally, we report a gene fusion LRPAP1-PDGFRA. In summary, we report a childhood cancer PDX resource and our study highlights the role of immune constraints on tumor evolution., (© 2023. The Author(s).)

Patient-derived xenografts (PDX) remain valuable models for understanding the biology and for developing novel therapeutics. To expand current PDX models of childhood leukemia, we have developed new PDX models from Hispanic patients, a subgroup with a poorer overall outcome. Of 117 primary leukemia samples obtained, successful engraftment and serial passage in mice were achieved in 82 samples (70%). Hispanic patient samples engrafted at a rate (51/73, 70%) that was similar to non-Hispanic patient samples (31/45, 70%). With a new algorithm to remove mouse contamination in multi-omics datasets including methylation data, we found PDX models faithfully reflected somatic mutations, copy-number alterations, RNA expression, gene fusions, whole-genome methylation patterns, and immunophenotypes found in primary tumor (PT) samples in the first 50 reported here. This cohort of characterized PDX childhood leukemias represents a valuable resource in that germline DNA sequencing has allowed the unambiguous determination of somatic mutations in both PT and PDX., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors.)

Over the past few decades, 5-year cancer survival has steadily improved for all adolescents and young adults (AYA, 15-39 years at diagnosis) combined. While encouraging, this progress simultaneously highlights a compelling need for improving survival in higher risk AYA subsets and for addressing health outcomes and health-related quality of life (HRQoL) among long-term survivors. The Children's Oncology Group (COG), in collaboration with the National Cancer Institute (NCI) and the adult network groups within the NCI National Clinical Trials Network (NCTN), has developed a large and growing portfolio of therapeutic AYA cancer clinical trials to identify optimal treatment approaches for common AYA cancers. Additional initiatives, led by the COG AYA Oncology Discipline Committee for increasing collaboration between the COG and the adult network groups, optimizing AYA clinical trial enrollment, and standardizing the assessment of HRQoL, have been highly successful to date. Further, NCTN-wide collaborations are currently underway focused on improving survival for AYA malignancies with poor prognosis and, through development of supportive care and care delivery trials, reducing the short- and long-term toxicity caused by cancer treatment. Leveraging the research infrastructure within the NCTN and the NCI Community Oncology Research Program, the COG will continue to champion meaningful advancements in health and survival for AYAs with cancer., (© 2023 Wiley Periodicals LLC.)

Objective: To develop a summary format of clinical practice guideline (CPG) recommendations to improve understandability among health care professionals., Methods: We developed a summary format based on current research and used the "Think Aloud" technique in one-on-one cognitive interviews to iteratively improve it. Interviews of health care professionals from Children's Oncology Group-member, National Cancer Institute Community Oncology Research Program sites were conducted. After every five interviews (a round), responses were reviewed, and changes made to the format until it was well understood and no new, substantive suggestions for revision were raised. We took a directed (deductive) approach to content analysis of the interview notes to identify concerns related to recommendation summary usability, understandability, validity, applicability and visual appeal., Results: During seven rounds of interviews with 33 health care professionals, we identified important factors that influenced understandability. Participants found understanding weak recommendations more challenging than strong recommendations. Understanding was improved when the term 'conditional' recommendation was used instead of 'weak' recommendation. Participants found a Rationale section to be very helpful but desired more information when a recommendation entailed a practice change. In the final format, the recommendation strength is clearly indicated in the title, highlighted, and defined within a text box. The rationale for the recommendation is in a column on the left, with supporting evidence on the right. In a bulleted list, the Rationale section describes the benefits and harms and additional factors, such as implementation, that were considered by the CPG developers. Each bullet under the supporting evidence section indicates the level of evidence with an explanation and the supporting studies with hyperlinks when applicable., Conclusions: A summary format to present strong and conditional recommendations was created through an iterative interview process. The format is straightforward, making it easy for organizations and CPG developers to use it to communicate recommendations clearly to intended users., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Santesso et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Clinical practice guideline (CPG)-consistent care improves patient outcomes, but CPG implementation is poor. Little is known about CPG implementation in pediatric oncology. This study aimed to understand supportive care CPG implementation facilitators and barriers at pediatric oncology National Cancer Institute (NCI) Community Oncology Research Program (NCORP) institutions., Methods: Healthcare professionals at 26 pediatric, Children's Oncology Group-member, NCORP institutions were invited to participate in face-to-face focus groups. Serial focus groups were held until saturation of ideas was reached. Supportive care CPG implementation facilitators and barriers were solicited using nominal group technique (NGT), and implementation of specific supportive care CPG recommendations was discussed. Notes from each focus group were analyzed using a directed content analysis. The top five themes arising from an analysis of NGT items were identified, first from each focus group and then across all focus groups., Results: Saturation of ideas was reached after seven focus groups involving 35 participants from 18 institutions. The top five facilitators of CPG implementation identified across all focus groups were organizational factors including charging teams with CPG implementation, individual factors including willingness to standardize care, user needs and values including mentorship, system factors including implementation structure, and implementation strategies including a basis in science. The top five barriers of CPG implementation identified were organizational factors including tolerance for inconsistencies, individual factors including lack of trust, system factors including administrative hurdles, user needs and values including lack of inclusivity, and professional including knowledge gaps., Conclusions: Healthcare professionals at pediatric NCORP institutions believe that organizational factors are the most important determinants of supportive care CPG implementation. They believe that CPG-consistent supportive care is most likely to be delivered in organizations that prioritize evidence-based care, provide structure and resources to implement CPGs, and eliminate implementation barriers., Trial Registration: ClinicalTrials.gov Identifier: NCT02847130. Date of registration: July 28, 2016., (© 2021. The Author(s).)

Purpose: Treatment-related pancreatitis (TRP) is a serious complication occurring in children with acute lymphoblastic leukemia (ALL). Those affected are at high risk for severe organ toxicity and treatment delays that can impact outcomes. TRP is associated with asparaginase, a standard therapeutic agent in childhood ALL. Native American ancestry, older age, high-risk leukemia, and increased use of asparaginase are linked to pancreatitis risk. However, dedicated genetic studies evaluating pancreatitis in childhood ALL include few Hispanics. Thus, the genetic basis for higher risk of pancreatitis among Hispanic children with ALL remains unknown., Methods: Cases of children with ALL treated in from 1994 through 2013 were reviewed and identified 14, all Hispanic, who developed pancreatitis related to asparaginase therapy. Forty-six controls consisting of Hispanic children treated on the same regimens without pancreatitis were selected for comparison. Total DNA isolated from whole blood was used for targeted DNA sequencing of 23 selected genes, including genes associated with pancreatitis without ALL and genes involved in asparagine metabolism., Results: Non-synonymous polymorphisms and frameshift deletions were detected in 15 genes. Most children with TRP had variants in ABAT, ASNS, and CFTR. Notably, children with TRP harbored many more CFTR variants (71.4%) compared with controls (39.1%). Among these, V470M (rs213950) was most frequent (OR 4.27, p = 0.025)., Conclusions: This is the first study of genetic factors in treatment-related pancreatitis in Hispanic children with ALL. Identifying correlative variants in ethnically vulnerable populations may improve screening to identify which patients with ALL are at greatest risk for pancreatitis.

Rationale: With a prevalence of 1 in 200 individuals, hypertrophic cardiomyopathy (HCM) is thought to be the most common genetic cardiac disease, with potential outcomes that include severe hypertrophy, heart failure, and sudden cardiac death (SCD). Though much research has furthered our understanding of how HCM-causing mutations in genes such as cardiac myosin-binding protein C ( MYBPC3 ) impair contractile function, it remains unclear how such dysfunction leads to hypertrophy and/or arrhythmias, which comprise the HCM phenotype. Identification of early response mediators could provide rational therapeutic targets to reduce disease severity. Our goal was to differentiate physiologic and pathophysiologic hypertrophic growth responses and identify early genetic mediators in the development of cardiomegaly in the cardiac myosin-binding protein C-null (cMyBP-C -/- ) mouse model of HCM. Methods and Results: We performed microarray analysis on left ventricles of wild-type (WT) and cMyBPC -/- mice ( n = 7 each) at postnatal day (PND) 1 and PND 9, before and after the appearance of an overt HCM phenotype. Applying the criteria of ≥2-fold change, we identified genes whose change was exclusive to pathophysiologic growth ( n = 61), physiologic growth ( n = 30), and genes whose expression changed ≥2-fold in both WT and cMyBP-C -/- hearts ( n = 130). Furthermore, we identified genes that were dysregulated in PND1 cMyBP-C -/- hearts prior to hypertrophy, including genes in mechanosensing pathways and potassium channels linked to arrhythmias. One gene of interest, Xirp2 , and its protein product, are regulated during growth but also show early, robust prehypertrophic upregulation in cMyBP-C -/- hearts. Additionally, the transcription factor Zbtb16 also shows prehypertrophic upregulation at both gene and protein levels. Conclusion: Our transcriptome analysis generated a comprehensive data set comparing physiologic vs. hypertrophic growth in mice lacking cMyBP-C. It highlights the importance of extracellular matrix pathways in hypertrophic growth and early dysregulation of potassium channels. Prehypertrophic upregulation of Xirp2 in cMyBP-C -/- hearts supports a growing body of evidence suggesting Xirp2 has the capacity to elicit both hypertrophy and arrhythmias in HCM. Dysregulation of Xirp2 , as well as Zbtb16 , along with other genes associated with mechanosensing regions of the cardiomyocyte implicate stress-sensing in these regions as a potentially important early response in HCM.

Rationale: Hypertrophic cardiomyopathy (HCM) occurs in ~0.5% of the population and is a leading cause of sudden cardiac death (SCD) in young adults. Cardiomyocyte hypertrophy has been the accepted mechanism for cardiac enlargement in HCM, but the early signaling responsible for initiating hypertrophy is poorly understood. Mutations in cardiac myosin binding protein C ( MYBPC3 ) are among the most common HCM-causing mutations. Ablation of Mybpc3 in an HCM mouse model (cMyBP-C -/- ) rapidly leads to cardiomegaly by postnatal day (PND) 9, though hearts are indistinguishable from wild-type (WT) at birth. This model provides a unique opportunity to explore early processes involved in the dramatic postnatal transition to hypertrophy. Methods and Results: We performed microarray analysis, echocardiography, qPCR, immunohistochemistry (IHC), and isolated cardiomyocyte measurements to characterize the perinatal cMyBP-C -/- phenotype before and after overt hypertrophy. cMyBP-C -/- hearts showed elevated cell cycling at PND1 that transitioned to hypertrophy by PND9. An expanded time course revealed that increased cardiomyocyte cycling was associated with elevated heart weight to body weight ratios prior to cellular hypertrophy, suggesting that cell cycling resulted in cardiomyocyte proliferation. Animals heterozygous for the cMyBP-C deletion trended in the direction of the homozygous null, yet did not show increased heart size by PND9. Conclusions: Results indicate that altered regulation of the cell cycling pathway and elevated proliferation precedes hypertrophy in the cMyBP-C -/- HCM model, and suggests that increased cardiomyocyte number contributes to increased heart size in cMyBP-C -/- mice. This pre-hypertrophic period may reflect a unique time during which the commitment to HCM is determined and disease severity is influenced.

We previously reported that the cardiomyocyte-specific leucine-rich repeat containing protein (LRRC)10 has critical functions in the mammalian heart. In the present study, we tested the role of LRRC10 in the response of the heart to biomechanical stress by performing transverse aortic constriction on Lrrc10-null (Lrrc10(-/-)) mice. Mild pressure overload induced severe cardiac dysfunction and ventricular dilation in Lrrc10(-/-) mice compared with control mice. In addition to dilation and cardiomyopathy, Lrrc10(-/-) mice showed a pronounced increase in heart weight with pressure overload stimulation and a more dramatic loss of cardiac ventricular performance, collectively suggesting that the absence of LRRC10 renders the heart more disease prone with greater hypertrophy and structural remodeling, although rates of cardiac fibrosis and myocyte dropout were not different from control mice. Lrrc10(-/-) cardiomyocytes also exhibited reduced contractility in response to β-adrenergic stimulation, consistent with loss of cardiac ventricular performance after pressure overload. We have previously shown that LRRC10 interacts with actin in the heart. Here, we show that His(150) of LRRC10 was required for an interaction with actin, and this interaction was reduced after pressure overload, suggesting an integral role for LRRC10 in the response of the heart to mechanical stress. Importantly, these experiments demonstrated that LRRC10 is required to maintain cardiac performance in response to pressure overload and suggest that dysregulated expression or mutation of LRRC10 may greatly sensitize human patients to more severe cardiac disease in conditions such as chronic hypertension or aortic stenosis., (Copyright © 2016 the American Physiological Society.)

It has been reported that in chondrichthyans the cardiac outflow tract is composed of the myocardial conus arteriosus, while in most teleosteans it consists of the nonmyocardial bulbus arteriosus. Classical studies already indicated that a conus and a bulbus coexist in several ancient actinopterygian and teleost groups. Recent work has shown that a cardiac outflow tract consisting of a conus and a bulbus is common to both cartilaginous and bony fishes. Nonetheless and despite their position at the base of the actinopterygian phylogenetic lineage, the anatomical arrangement of the cardiac outflow tract of the Polypteriformes remained uncertain. The present study of hearts from gray bichirs was intended to fill this gap. The cardiac outflow tract of the bichir consists of two main components, namely a very long conus arteriosus, furnished with valves, and a short, intrapericardial, arterial-like bulbus arteriosus, which differs from the ventral aorta because it is covered by epicardium, shows a slightly different spatial arrangement of the histological elements and is crossed by coronary arteries. Histomorphologically, the outflow tract consists of three longitudinal regions, distal, middle and proximal, an arrangement which has been suggested to be common to all vertebrates. The distal region corresponds to the bulbus, while the conus comprises the middle and proximal regions. The present findings reinforce the notion that the bulbus arteriosus of fish has played an essential role in vertebrate heart evolution as it is the precursor of the intrapericardial trunks of the aorta and pulmonary artery of birds and mammals., (Copyright © 2014. Published by Elsevier GmbH.)

Mutations in cardiac myosin binding protein C (cMyBP-C) are prevalent causes of hypertrophic cardiomyopathy (HCM). Although HCM-causing truncation mutations in cMyBP-C are well studied, the growing number of disease-related cMyBP-C missense mutations remain poorly understood. Our objective was to define the primary contractile effect and molecular disease mechanisms of the prevalent cMyBP-C E258K HCM-causing mutation in nonremodeled murine engineered cardiac tissue (mECT). Wild-type and human E258K cMyBP-C were expressed in mECT lacking endogenous mouse cMyBP-C through adenoviral-mediated gene transfer. Expression of E258K cMyBP-C did not affect cardiac cell survival and was appropriately incorporated into the cardiac sarcomere. Functionally, expression of E258K cMyBP-C caused accelerated contractile kinetics and severely compromised twitch force amplitude in mECT. Yeast two-hybrid analysis revealed that E258K cMyBP-C abolished interaction between the N terminal of cMyBP-C and myosin heavy chain sub-fragment 2 (S2). Furthermore, this mutation increased the affinity between the N terminal of cMyBP-C and actin. Assessment of phosphorylation of three serine residues in cMyBP-C showed that aberrant phosphorylation of cMyBP-C is unlikely to be responsible for altering these interactions. We show that the E258K mutation in cMyBP-C abolishes interaction between N-terminal cMyBP-C and myosin S2 by directly disrupting the cMyBP-C-S2 interface, independent of cMyBP-C phosphorylation. Similar to cMyBP-C ablation or phosphorylation, abolition of this inhibitory interaction accelerates contractile kinetics. Additionally, the E258K mutation impaired force production of mECT, which suggests that in addition to the loss of physiological function, this mutation disrupts contractility possibly by tethering the thick and thin filament or acting as an internal load.

Hypertrophic cardiomyopathy (HCM) caused by mutations in cardiac myosin-binding protein-C (cMyBP-C) is a heterogenous disease in which the phenotypic presentation is influenced by genetic, environmental, and developmental factors. Though mouse models have been used extensively to study the contractile effects of cMyBP-C ablation, early postnatal hypertrophic and dilatory remodeling may overshadow primary contractile defects. The use of a murine engineered cardiac tissue (mECT) model of cMyBP-C ablation in the present study permits delineation of the primary contractile kinetic abnormalities in an intact tissue model under mechanical loading conditions in the absence of confounding remodeling events. We generated mechanically integrated mECT using isolated postnatal day 1 mouse cardiac cells from both wild-type (WT) and cMyBP-C-null hearts. After culturing for 1 wk to establish coordinated spontaneous contraction, we measured twitch force and Ca(2+) transients at 37°C during pacing at 6 and 9 Hz, with and without dobutamine. Compared with WT, the cMyBP-C-null mECT demonstrated faster late contraction kinetics and significantly faster early relaxation kinetics with no difference in Ca(2+) transient kinetics. Strikingly, the ability of cMyBP-C-null mECT to increase contractile kinetics in response to adrenergic stimulation and increased pacing frequency were severely impaired. We conclude that cMyBP-C ablation results in constitutively accelerated contractile kinetics with preserved peak force with minimal contractile kinetic reserve. These functional abnormalities precede the development of the hypertrophic phenotype and do not result from alterations in Ca(2+) transient kinetics, suggesting that alterations in contractile velocity may serve as the primary functional trigger for the development of hypertrophy in this model of HCM. Our findings strongly support a mechanism in which cMyBP-C functions as a physiological brake on contraction by positioning myosin heads away from the thin filament, a constraint which is removed upon adrenergic stimulation or cMyBP-C ablation.

Anatomical and functional studies of the autonomic innervation in the conus arteriosus of the garfishes are lacking. This study reveals that the conus arteriosus of the longnose gar is primarily myocardial in nature, but additionally, large numbers of smooth muscle cells are present in the subendocardium. A well-developed system of adrenergic, cholinergic, substance P (SP) and neuronal nitric oxide synthase (nNOS) positive nerve terminals are found in the wall of the conus arteriosus. Coronary blood vessels running in the adventitia receive a rich supply of nNOS positive nerve fibers, thus suggesting their importance in the nitrergic control of blood flow in the conus arteriosus. The present data show that the patterns of autonomic innervation of the garfish conus arteriosus are more complex than previously appreciated., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Rationale: Cardiomyocytes cultured in a mechanically active 3-dimensional configuration can be used for studies that correlate contractile performance to cellular physiology. Current engineered cardiac tissue (ECT) models use cells derived from either rat or chick hearts. Development of a murine ECT would provide access to many existing models of cardiac disease and open the possibility of performing targeted genetic manipulation with the ability to directly assess contractile and molecular variables., Objective: To generate, characterize, and validate mouse ECT with a physiologically relevant model of hypertrophic cardiomyopathy., Methods and Results: We generated mechanically integrated ECT using isolated neonatal mouse cardiac cells derived from both wild-type and myosin-binding protein C (cMyBP-C)-null mouse hearts. The murine ECTs produced consistent contractile forces that followed the Frank-Starling law and accepted physiological pacing. cMyBP-C-null ECTs showed characteristic acceleration of contraction kinetics. Adenovirus-mediated expression of human cMyBP-C in murine cMyBP-C-null ECT restored contractile properties to levels indistinguishable from those of wild-type ECT. Importantly, the cardiomyocytes used to construct the cMyBP-C(-/-) ECT had yet to undergo the significant hypertrophic remodeling that occurs in vivo. Thus, this murine ECT model reveals a contractile phenotype that is specific to the genetic mutation rather than to secondary remodeling events., Conclusions: Data presented here show mouse ECT to be an efficient and cost-effective platform to study the primary effects of genetic manipulation on cardiac contractile function. This model provides a previously unavailable tool to study specific sarcomeric protein mutations in an intact mammalian muscle system.

The secondary heart field is a conserved developmental domain in avian and mammalian embryos that contributes myocardium and smooth muscle to the definitive cardiac arterial pole. This field is part of the overall heart field and its myocardial component has been fate mapped from the epiblast to the heart in both mammals and birds. In this study we show that the population that gives rise to the arterial pole of the zebrafish can be traced from the epiblast, is a discrete part of the mesodermal heart field, and contributes myocardium after initial heart tube formation, giving rise to both smooth muscle and myocardium. We also show that Isl1, a transcription factor associated with undifferentiated cells in the secondary heart field in other species, is active in this field. Furthermore, Bmp signaling promotes myocardial differentiation from the arterial pole progenitor population, whereas inhibiting Smad1/5/8 phosphorylation leads to reduced myocardial differentiation with subsequent increased smooth muscle differentiation. Molecular pathways required for secondary heart field development are conserved in teleosts, as we demonstrate that the transcription factor Tbx1 and the Sonic hedgehog pathway are necessary for normal development of the zebrafish arterial pole.

In chick and mouse embryogenesis, a population of cells described as the secondary heart field (SHF) adds both myocardium and smooth muscle to the developing cardiac outflow tract (OFT). Following this addition, at approximately HH stage 22 in chick embryos, for example, the SHF can be identified architecturally by an overlapping seam at the arterial pole, where beating myocardium forms a junction with the smooth muscle of the arterial system. Previously, using either immunohistochemistry or nitric oxide indicators such as diaminofluorescein 2-diacetate, we have shown that a similar overlapping architecture also exists in the arterial pole of zebrafish and some shark species. However, although recent work suggests that development of the zebrafish OFT may also proceed by addition of a SHF-like population of cells, the presence of a true SHF in zebrafish and in many other developmental biological models remains an open question. We performed a comprehensive morphological study of the OFT of a wide range of vertebrates. Our data suggest that all vertebrates possess three fundamental OFT components: a proximal myocardial component, a distal smooth muscle component, and a middle component that contains overlapping myocardium and smooth muscle surrounding and supporting the outflow valves. Because the middle OFT component of avians and mammals is derived from the SHF, our observations suggest that a SHF may be an evolutionarily conserved theme in vertebrate embryogenesis., (© 2010 Wiley Periodicals, Inc.)

A large number of congenital heart defects associated with mortality in humans are those that affect the cardiac outflow tract, and this provides a strong imperative to understand its development during embryogenesis. While there is wide phylogenetic variation in adult vertebrate heart morphology, recent work has demonstrated evolutionary conservation in the early processes of cardiogenesis, including that of the outflow tract. This, along with the utility and high reproductive potential of fish species such as Danio rerio, Oryzias latipes etc., suggests that fishes may provide ideal comparative biological models to facilitate a better understanding of this poorly understood region of the heart. In this review, the authors present the current understanding of both phylogeny and ontogeny of the cardiac outflow tract in fishes and examine how new molecular studies are informing the phylogenetic relationships and evolutionary trajectories that have been proposed. The authors also attempt to address some of the issues of nomenclature that confuse this area of research.

We have used zebrafish and 3,3',4,4',5-pentachlorobiphenyl (PCB126) to investigate the developmental toxicity of polychlorinated biphenyls (PCBs) that exert their effects through the aryl hydrocarbon receptor (AHR). We found that cardiac and neural crest (NC)-derived jaw and branchial cartilages are specifically targeted early in development. The suite of malformations, which ultimately leads to circulatory failure, includes a severely dysmorphic heart with a reduced bulbus arteriosus and abnormal atrioventricular and outflow valve formation. Early NC migration and patterning of the jaw and branchial cartilages was normal. However, the jaw and branchial cartilages failed to grow to normal size. In the heart, the ventricular myocardium showed a reduction in cell number and size. The heart and jaw/branchial phenotype could be rescued by pifithrin-alpha, a blocker of p53. However, the function of pifithrin-alpha in this model may act as a competitive inhibitor of PCB at the AHR and is likely independent of p53. Morpholinos against p53 did not rescue the phenotype, nor were zebrafish with a mutant p53-null allele resistant to PCB126 toxicity. Morpholino knockdown of cardiac troponin T, which blocks the onset of cardiac function, prevented the PCB126-induced cardiac dysmorphogenesis but not the jaw/branchial phenotype. The cardiovascular characteristics appear to be similar to hypoplastic left heart syndrome (HLHS) and introduce the potential of zebrafish as a model to study this environmentally induced cardiovascular malformation. HLHS is a severe congenital cardiovascular malformation that has previously been linked to industrial releases of dioxins and PCBs.

It has been generally assumed that the outflow tract of the chondrichthyan heart consists of the conus arteriosus, characterized by cardiac muscle in its walls. However, classical observations, neglected for many years, indicated that the distal component of the cardiac outflow tract of several elasmobranch species was composed of tissue resembling that of the ventral aorta. The present study was outlined to test the hypothesis that this intrapericardial, non-myocardial component might be homologous to the actinopterygian bulbus arteriosus. The material consisted of Atlantic catshark adults and embryos, which were examined by means of histochemical and immunohistochemical techniques for light and fluorescence microscopy. In this species, the distal component of the outflow tract differs histomorphologically from both the ventral aorta and the conus arteriosus; it is devoid of myocardium, is covered by epicardium and is crossed by the coronary arterial trunks. In the embryonic hearts examined, this distal component showed positive reactivity for 4,5-diaminofluorescein 2-diacetate (DAF-2DA), a fluorescent nitric oxide indicator. These findings, together with other observations in holocephals and several elasmobranch species, confirm that chondrichthyans possess a bulbus arteriosus interposed between the conus arteriosus and the ventral aorta. Therefore, the primitive heart of gnathostomates consists of five intrapericardial components, sinus venosus, atrium, ventricle, conus arteriosus and bulbus arteriosus, indicating that the bulbus arteriosus can no longer be regarded as an actinopterygian apomorphy. The DAF-2DA-positive reactivity of the chondrichthyan embryonic bulbus suggests that this structure is homologous to the base of the great arterial trunks of birds and mammals, which derives from the embryonic secondary heart field.

White Spot Syndrome Virus (WSSV) is a highly pathogenic and prevalent virus affecting crustacea. A number of WSSV envelope proteins, including vp28, have been proposed to be involved in viral infectivity based on the ability of specific antibodies to attenuate WSSV-induced mortality in vivo. In the present study, a series of monoclonal and polyclonal antibodies targeting vp28 were tested for their ability to neutralize WSSV infectivity, with the purpose of identifying epitopes potentially involved in vp28-mediated infection of shrimp. Surprisingly, when used as protein A-purified immunoglobulin, none of the antibodies tested were capable of inhibiting WSSV infectivity. This included one polyclonal preparation that has been previously shown to inactivate WSSV, when used as whole rabbit serum. Moreover, strong inactivation of WSSV by some rabbit sera was observed, in a manner independent of anti-vp28 antibodies. These results underscore the problems associated with using heterogeneous reagents (e.g. whole rabbit antiserum) in viral neutralization experiments aimed at defining proteins involved in infection by WSSV. In light of this, the potential of anti-vp28 antibodies to specifically neutralize WSSV should be reconsidered.

It is a widely held belief that the arterial pole of the zebrafish heart is unusual among models of comparative cardiogenesis. This is based, in part, on the report that the bulbus arteriosus undergoes a striated-to-smooth muscle phenotypic transition during development. An implication of this is that the zebrafish, a model almost ubiquitously accepted in other fields of comparative biology, may be poorly suited to the study of conotruncal abnormalities in human disease. However, while the use of atrioventricular-specific molecular markers has allowed extensive characterization of the development of the atrium and ventricle, the lack of any bulbus-specific markers has meant that this region of the zebrafish heart is poorly characterized and quite possibly misunderstood. We have discovered that the fluorescent nitric oxide indicator 4,5-diaminofluorescein diacetate (DAF-2DA) specifically labels the bulbus arteriosus throughout development from approximately 48 h post-fertilization. Therefore, using DAF-2DA and an immunohistochemical approach, we attempted to further characterize the development of the bulbus. We have concluded that no such phenotypic transition occurs, that contrary to current thinking, aspects of zebrafish arterial pole development are evolutionarily conserved, and that the bulbus should not be considered a chamber, being more akin to the arterial trunk(s) of higher vertebrates.