Heather L. Bartlett

Early Predictors of Survival to and After Heart Transplantation in Children With Dilated Cardiomyopathy

Background— The importance of clinical presentation and pretransplantation course on outcome in children with dilated cardiomyopathy listed for heart transplantation is not well defined. Methods and Results— The impact of age, duration of illness, sex, race, ventricular geometry, and diagnosis of myocarditis on outcome in 261 children with dilated cardiomyopathy enrolled in the Pediatric Cardiomyopathy Registry and Pediatric Heart Transplant Study was studied. End points included listing as United Network for Organ Sharing status 1, death while waiting, and death after transplantation. The median age at the time of diagnosis was 3.4 years, and the mean time from diagnosis to listing was 0.62±1.3 years. Risk factors associated with death while waiting were ventilator use and older age at listing in patients not mechanically ventilated (P=0.0006 and P=0.03, respectively). Shorter duration of illness (P=0.04) was associated with listing as United Network for Organ Sharing status 1. Death after transplantation was associated with myocarditis at presentation (P=0.009), nonwhite race (P<0.0001), and a lower left ventricular end-diastolic dimension z score at presentation (P=0.04). In the myocarditis group, 17% (4 of 23) died of acute rejection after transplantation. Conclusions— Mechanical ventilator use and older age at listing predicted death while waiting, whereas nonwhite race, smaller left ventricular dimension, and myocarditis were associated with death after transplantation. Although 97% of children with clinically or biopsy-diagnosed myocarditis at presentation survived to transplantation, they had significantly higher posttransplantation mortality compared with children without myocarditis, raising the possibility that preexisting viral infection or inflammation adversely affects graft survival.

Examining the cardiac NK-2 genes in early heart development.

The cardiac NK-2 transcription factors are the vertebrate relatives of the Drosophila tinman gene. Without the Drosophila tinman gene, fruit flies fail to form their heart (“dorsal vessel”), and mutations or altered expression of cardiac NK-2 genes may lead to abnormal heart formation in vertebrates. Although the cardiac NK-2 gene NKX2-5 is recognized as an important factor in cases of human congenital heart disease and heart development in vertebrates, the roles of the other cardiac NK-2 genes are less clear. This report reviews what is known about the cardiac NK-2 genes in cardiac development, comparing studies in several different model systems.

Early Outcomes of Tricuspid Valve Replacement in Young Children

Background— Early outcomes after tricuspid valve replacement in young children are ill defined. The experience of the Pediatric Cardiac Care Consortium (45 centers, 1984 to 2002) was reviewed to evaluate the results of tricuspid valve replacement in children <6 years of age. Methods and Results— Ninety-seven patients who underwent initial tricuspid valve replacement are included in the present analysis. The most frequent cardiac diagnoses were Ebstein’s anomaly (40%), pulmonary atresia (11%), and tetralogy of Fallot (8%). Age at tricuspid valve replacement was 2.9±1.7 years (mean±SD). Mean patient weight was 12.7±6.1 kg. The major outcome was survival to discharge. Associations among age, diagnosis, valve type/size, and outcome were evaluated through the use of &khgr;2 analysis and logistic regression model fitting approaches. Hospital mortality was 26% and was very high (64%) in patients <1 year of age. A large size-to-weight ratio was the strongest predictor of mortality based on multivariable analysis (P<0.001). Mortality was 54% for patients with a size-to-weight ratio >2.5. Other complications included heart block requiring a pacemaker (13%) and thrombosis (5%). Pacemaker implantation was associated with the use of a mechanical valve (23% versus 6% bioprosthetic valve; P=0.01) Conclusions— Tricuspid valve replacement in young children is associated with high mortality, especially in infants <1 year of age. Surgical options other than tricuspid valve replacement such as transplantation may need to be considered in infants.

Thoracic Aortic Aneurysm (TAAD)-causing Mutation in Actin Affects Formin Regulation of Polymerization

Background: The biochemical mechanisms underlying α-smooth muscle actin-mediated vascular disease are unknown. Results: The R256H mutation in actin alters polymerization kinetics and causes misregulation by the nucleation factor, formin. Conclusion: Mutation-based changes in conformation affect filament stability and regulation of polymerization. Significance: The Arg-256 residue stabilizes the actin helix and maintains filament conformation required for formin regulation. More than 30 mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause autosomal dominant thoracic aortic aneurysm and dissection. The mutation R256H is of particular interest because it also causes patent ductus arteriosus and moyamoya disease. R256H is one of the more prevalent mutations and, based on its molecular location near the strand-strand interface in the actin filament, may affect F-actin stability. To understand the molecular ramifications of the R256H mutation, we generated Saccharomyces cerevisiae yeast cells expressing only R256H yeast actin as a model system. These cells displayed abnormal cytoskeletal morphology and increased sensitivity to latrunculin A. After cable disassembly induced by transient exposure to latrunculin A, mutant cells were delayed in reestablishing the actin cytoskeleton. In vitro, mutant actin exhibited a higher than normal critical concentration and a delayed nucleation. Consequently, we investigated regulation of mutant actin by formin, a potent facilitator of nucleation and a protein needed for normal vascular smooth muscle cell development. Mutant actin polymerization was inhibited by the FH1-FH2 fragment of the yeast formin, Bni1. This fragment strongly capped the filament rather than facilitating polymerization. Interestingly, phalloidin or the presence of wild type actin reversed the strong capping behavior of Bni1. Together, the data suggest that the R256H actin mutation alters filament conformation resulting in filament instability and misregulation by formin. These biochemical effects may contribute to abnormal histology identified in diseased arterial samples from affected patients.

Allele-specific Effects of Thoracic Aortic Aneurysm and Dissection α-Smooth Muscle Actin Mutations on Actin Function

Twenty-two missense mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause thoracic aortic aneurysm and dissection. Limited access to diseased tissue, the presence of multiple unresolvable actin isoforms in the cell, and lack of an animal model have prevented analysis of the biochemical mechanisms underlying this pathology. We have utilized actin from the yeast Saccharomyces cerevisiae, 86% identical to human α-smooth muscle actin, as a model. Two of the known human mutations, N115T and R116Q, were engineered into yeast actin, and their effect on actin function in vivo and in vitro was investigated. Both mutants exhibited reduced ability to grow under a variety of stress conditions, which hampered N115T cells more than R116Q cells. Both strains exhibited abnormal mitochondrial morphology indicative of a faulty actin cytoskeleton. In vitro, the mutant actins exhibited altered thermostability and nucleotide exchange rates, indicating effects of the mutations on monomer conformation, with R116Q the most severely affected. N115T demonstrated a biphasic elongation phase during polymerization, whereas R116Q demonstrated a markedly extended nucleation phase. Allele-specific effects were also seen on critical concentration, rate of depolymerization, and filament treadmilling. R116Q filaments were hypersensitive to severing by the actin-binding protein cofilin. In contrast, N115T filaments were hyposensitive to cofilin despite nearly normal binding affinities of actin for cofilin. The mutant-specific effects on actin behavior suggest that individual mechanisms may contribute to thoracic aortic aneurysm and dissection.

Transient Early Embryonic Expression of Nkx2-5 Mutations Linked to Congenital Heart Defects in Human Causes Heart Defects in Xenopus laevis

Nkx2‐5 is a homeobox containing transcription factor that is conserved and expressed in organisms that form hearts. Fruit flies lacking the gene (tinman) fail to form a dorsal vessel, mice that are homozygous null for Nkx2‐5 form small, deformed hearts, and several human cardiac defects have been linked to dominant mutations in the Nkx2‐5 gene. The Xenopus homologs (XNkx2‐5) of two truncated forms of Nkx2‐5 that have been identified in humans with congenital heart defects were used in the studies reported here. mRNAs encoding these mutations were injected into single cell Xenopus embryos, and heart development was monitored. Our results indicate that the introduction of truncated XNkx2‐5 variants leads to three principle developmental defects. The atrial septum and the valve of the atrioventricular canal were both abnormal. In addition, video microscopic timing of heart contraction indicated that embryos injected with either mutant form of XNkx2‐5 have conduction defects. Developmental Dynamics 236:2475–2484, 2007.

Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of fluorescently tagged proteins using total internal fluorescence (TIRF) microscopy is used to visualize and quantify changes in cytoskeletal dynamics. TIRF microscopy and the use of fluorescent tags also allows for quantification of the changes in cytoskeletal dynamics caused by mutations in actin. Using this technique, quantification of cytoskeletal function in live cells valuably complements in vitro studies of protein function. As an example, missense mutations affecting the actin residue R256 have been identified in three human actin isoforms suggesting this amino acid plays an important role in regulatory interactions. The effects of the actin mutation R256H on cytoskeletal movements were studied using the yeast model. The protein, Aip1, which is known to assist cofilin in actin depolymerization, was tagged with green fluorescent protein (GFP) at the N-terminus and tracked in vivo using TIRF microscopy. The rate of Aip1p movement in both wild type and mutant strains was quantified. In cells expressing R256H mutant actin, Aip1p motion is restricted and the rate of movement is nearly half the speed measured in wild type cells (0.88 ± 0.30 μm/sec in R256H cells compared to 1.60 ± 0.42 μm/sec in wild type cells, p < 0.005).

A 16-Week-Old Infant With Failure to Thrive and Hypotonia

A 16-week-old girl initially presented to neurology for evaluation of failure to thrive and hypotonia. Prenatal history was notable for a maternal history of 4 miscarriages. A murmur was auscultated shortly after birth and an echocardiogram was normal for age. Her parents described her as a sleepy infant who needed to be awakened for feeds. She had poor weight gain since birth gaining 8.2 g per day the first 2 months of life and only 6.7 g per day in her third month of life. Gross motor development was delayed; she was not able to raise her head until 15 weeks of age. Shortly after arrival to the clinic, she had an episode of cyanosis and pallor that resolved spontaneously with supplemental oxygen. She was admitted to the pediatric intensive care unit, and subsequent evaluation noted multiple cardiac concerns. Chest x-ray revealed cardiomegaly with normal pulmonary vascular markings. Electrocardiogram showed a short PR interval, ventricular preexcitation, and extreme voltages (Figure 1). Echocardiogram demonstrated biventricular hypertrophy without outflow tract obstruction. The indexed left ventricular mass was 795 g/m (Z score +14). The left ventricular posterior wall in diastole measured 1.7 cm (Z score +21) and the interventricular septum in diastole measured 2.0 cm (Z score +24; Figure 2).