Uhl's anomaly: perspective of fetal echocardiography and histopathological correlation.

We report a case of Uhl's anomaly imaged at 19 weeks of gestation by fetal echocardiography with pathological confirmation by anatomical gross heart specimen and tissue histology. Uhl's anomaly of the right ventricle is a rare cardiac disorder with isolated right ventricular enlargement with almost complete absence of the right ventricular myocardium.

Knockout of Lmod2 results in shorter thin filaments followed by dilated cardiomyopathy and juvenile lethality.

Leiomodin 2 (Lmod2) is an actin-binding protein that has been implicated in the regulation of striated muscle thin filament assembly; its physiological function has yet to be studied. We found that knockout of Lmod2 in mice results in abnormally short thin filaments in the heart. We also discovered that Lmod2 functions to elongate thin filaments by promoting actin assembly and dynamics at thin filament pointed ends. Lmod2-KO mice die as juveniles with hearts displaying contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy. Lmod2-null cardiomyocytes produce less contractile force than wild type when plated on micropillar arrays. Introduction of GFP-Lmod2 via adeno-associated viral transduction elongates thin filaments and rescues structural and functional defects observed in Lmod2-KO mice, extending their lifespan to adulthood. Thus, to our knowledge, Lmod2 is the first identified mammalian protein that functions to elongate actin filaments in the heart; it is essential for cardiac thin filaments to reach a mature length and is required for efficient contractile force and proper heart function during development.

Tbx20 transcription factor is a downstream mediator for bone morphogenetic protein-10 in regulating cardiac ventricular wall development and function.

Bone morphogenetic protein 10 (BMP10) belongs to the TGFß-superfamily. Previously, we had demonstrated that BMP10 is a key regulator for ventricular chamber formation, growth, and maturation. Ablation of BMP10 leads to hypoplastic ventricular wall formation, and elevated levels of BMP10 are associated with abnormal ventricular trabeculation/compaction and wall maturation. However, the molecular mechanism(s) by which BMP10 regulates ventricle wall growth and maturation is still largely unknown. In this study, we sought to identify the specific transcriptional network that is potentially mediated by BMP10. We analyzed and compared the gene expression profiles between α-myosin heavy chain (αMHC)-BMP10 transgenic hearts and nontransgenic littermate controls using Affymetrix mouse exon arrays. T-box 20 (Tbx20), a cardiac transcription factor, was significantly up-regulated in αMHC-BMP10 transgenic hearts, which was validated by quantitative RT-PCR and in situ hybridization. Ablation of BMP10 reduced Tbx20 expression specifically in the BMP10-expressing region of the developing ventricle. In vitro promoter analysis demonstrated that BMP10 was able to induce Tbx20 promoter activity through a conserved Smad binding site in the Tbx20 promoter proximal region. Furthermore, overexpression of Tbx20 in myocardium led to dilated cardiomyopathy that exhibited ventricular hypertrabeculation and an abnormal muscular septum, which phenocopied genetically modified mice with elevated BMP10 levels. Taken together, our findings demonstrate that the BMP10-Tbx20 signaling cascade is important for ventricular wall development and maturation.

Nebulette mutations are associated with dilated cardiomyopathy and endocardial fibroelastosis.

OBJECTIVES: Four variants (K60N, Q128R, G202R, and A592E) in the nebulette gene were identified in patients with dilated cardiomyopathy (DCM) and endocardial fibroelastosis. We sought to determine if these mutations are cardiomyopathy causing. BACKGROUND: Nebulette aligns thin filaments and connects them with the myocardial Z-disk, playing a role in mechanosensation. METHODS: We generated transgenic mice with cardiac-restricted overexpression of human wild-type or mutant nebulette. Chimera and transgenic mice were examined at 4, 6, and 12 months of age by echocardiography and cardiac magnetic resonance imaging. The hearts from embryos and adult mice were assessed by histopathologic, immunohistochemical, ultrastructural, and protein analyses. Rat H9C2 cardiomyoblasts with transient expression of nebulette underwent cyclic mechanical strain. RESULTS: We identified lethal cardiac structural abnormalities in mutant embryonic hearts (K60N and Q128R). Founders of the mutant mouse lines developed DCM with severe heart failure. An irregular localization pattern for nebulette and impaired desmin expression were noted in the proband and chimeric Q128R mice. Mutant G202R and A592E mice exhibited left ventricular dilation and impaired function with specific changes in I-band and Z-disk proteins by 6 months of age. The mutations modulated distribution of nebulette in the sarcomere and Z-disk during stretch of H9C2 cells. CONCLUSIONS: Nebulette is a new susceptibility gene for endocardial fibroelastosis and DCM. Different mutations in nebulette trigger specific mechanisms, converging to a common pathological cascade leading to endocardial fibroelastosis and DCM.

Cardiomyopathy in offspring of diabetic rats is associated with activation of the MAPK and apoptotic pathways.

BACKGROUND: Maternal diabetes affects the developing fetal cardiovascular system. Newborn offspring of diabetic mothers can have a transient cardiomyopathy. We hypothesized that cardiomyopathic remodeling is associated with activation of the mitogen activated protein kinase (MAPK) signaling and apoptotic pathways. METHODS: To evaluate the effects of moderate and severe maternal hyperglycemia, pregnant rats were made diabetic with an injection of 50 mg/kg of streptozotocin. Moderately well controlled maternal diabetes was achieved with twice daily glucose checks and insulin injections. No insulin was given to severely diabetic dams. Offspring of moderate and severe diabetic mothers (OMDM and MSDM, respectively) were studied on postnatal days 1 (NB1) and 21 (NB21). Echocardiograms were performed to evaluate left ventricular (LV) dimensions and function. Myocardial MAPK and apoptotic protein levels were measured by Western blot. RESULTS: OMDM had increased cardiac mass at NB1 compared to controls that normalized at NB21. OSDM demonstrated microsomia with relative sparing of cardiac mass and a dilated cardiomyopathy at NB1. In both models, there was a persistent increase in the HW:BW and significant activation of MAPK and apoptotic pathways at NB21. CONCLUSION: The degree of maternal hyperglycemia determines the type of cardiomyopathy seen in the offspring, while resolution of both the hypertrophic and dilated cardiomyopathies is associated with activation of MAPK signaling and apoptotic pathways.

Muscle-specific loss of apoptosis-inducing factor leads to mitochondrial dysfunction, skeletal muscle atrophy, and dilated cardiomyopathy.

Cardiac and skeletal muscle critically depend on mitochondrial energy metabolism for their normal function. Recently, we showed that apoptosis-inducing factor (AIF), a mitochondrial protein implicated in programmed cell death, plays a role in mitochondrial respiration. However, the in vivo consequences of AIF-regulated mitochondrial respiration resulting from a loss-of-function mutation in Aif are not known. Here, we report tissue-specific deletion of Aif in the mouse. Mice in which Aif has been inactivated specifically in cardiac and skeletal muscle exhibit impaired activity and protein expression of respiratory chain complex I. Mutant animals develop severe dilated cardiomyopathy, heart failure, and skeletal muscle atrophy accompanied by lactic acidemia consistent with defects in the mitochondrial respiratory chain. Isolated hearts from mutant animals exhibit poor contractile performance in response to a respiratory chain-dependent energy substrate, but not in response to glucose, supporting the notion that impaired heart function in mutant animals results from defective mitochondrial energy metabolism. These data provide genetic proof that the previously defined cell death promoter AIF has a second essential function in mitochondrial respiration and aerobic energy metabolism required for normal heart function and skeletal muscle homeostasis.

Dilated cardiomyopathy presenting during fetal life.

OBJECTIVES: To describe the echocardiographic features, underlying causes, and outcome of fetuses with dilated cardiomyopathy. DESIGN: A retrospective observational study between 1983 and 2003 at a tertiary centre for fetal cardiology. PATIENTS: Affected fetuses were identified using a computerised database. We included fetuses with dilation and reduced systolic function of either the right ventricle, left ventricle, or both. We excluded fetuses with abnormal cardiac connections, arrhythmias, or stenosis of the aortic or pulmonary valves. In all, we identified 50 fetuses, born to 46 mothers. Of the fetuses, 24 had biventricular cardiomyopathy, 17 had isolated right ventricular cardiomyopathy, and 9 had isolated left ventricular cardiomyopathy. Two-thirds of the fetuses (32) were hydropic at some point during gestation. MAIN OUTCOMES: A cause of cardiomyopathy was identified in 37 cases (74 per cent). This was genetic or metabolic in 11 fetuses; infective in 11; fetal anaemia, without proven parvovirus infection, in 5; of cardiac origin in 5; and an association with renal disease in 5. In 10 cases (20 per cent), the pregnancy was terminated. Based on an intention to treat, the survival to delivery was 25 of 40 (62.5 per cent, 95 per cent confidence intervals from 46 to 77 per cent), at 28 days was 17 of 40 (42.5 per cent, 95 per cent confidence intervals from 27 to 59 per cent), and at 1 year was 15 of 40 (37.5 per cent, 95 per cent confidence intervals from 23 to 54 per cent). The overall survival of non-hydropic fetuses was 9 of 18 (50 per cent), compared to 6 of 32 (18 per cent) hydropic fetuses. CONCLUSIONS: Genetic, metabolic, infective, and cardiac diseases may present with dilated cardiomyopathy during fetal life. There is a high rate of spontaneous intra-uterine and early neonatal death. The prognosis is particularly poor for hydropic fetuses.

Dilated cardiomyopathy: a disease of the intercalated disc?

The contractile tissue of the heart is composed of individual cells, making specific cell-cell contacts necessary to ensure mechanical and electrochemical coupling during beating. These contact sites, termed the intercalated discs, have gained increased attention recently due to their potential involvement in cardiac disease. This article discusses how the intercalated discs are assembled during heart development and how they are affected in cardiomyopathy, with particular emphasis on dilated cardiomyopathy. A model is proposed to relate the alterations that are seen at a molecular level with changes in function observed in that kind of cardiac disease.

Ventricular septal defect and cardiomyopathy in mice lacking the transcription factor CHF1/Hey2.

Ventricular septal defects are common in human infants, but the genetic programs that control ventricular septation are poorly understood. Here we report that mice with a targeted disruption of the cardiovascular basic helix-loop-helix factor (CHF)1Hey2 gene show isolated ventricular septal defects. These defects result primarily in failure to thrive. Mice often succumbed within the first 3 wk after birth and showed pulmonary and liver congestion. The penetrance of this phenotype varied, depending on genetic background, suggesting the presence of modifier genes. Expression patterns of other cardiac-specific genes were not affected. Of the few animals on a mixed genetic background that survived to adulthood, most developed a cardiomyopathy but did not have ventricular septal defects. Our results indicate that CHF1 plays an important role in regulation of ventricular septation in mammalian heart development and is important for normal myocardial contractility. These mice provide a useful model for the study of the ontogeny and natural history of ventricular septal defects and cardiomyopathy.

Cardiomyopathy in zebrafish due to mutation in an alternatively spliced exon of titin.

The zebrafish embryo is transparent and can tolerate absence of blood flow because its oxygen is delivered by diffusion rather than by the cardiovascular system. It is therefore possible to attribute cardiac failure directly to particular genes by ruling out the possibility that it is due to a secondary effect of hypoxia. We focus here on pickwickm171 (pikm171), a recessive lethal mutation discovered in a large-scale genetic screen. There are three other alleles in the pik complementation group with this phenotype (pikm242, pikm740, pikm186; ref. 3) and one allele (pikmVO62H) with additional skeletal paralysis. The pik heart develops normally but is poorly contractile from the first beat. Aside from the edema that inevitably accompanies cardiac dysfunction, development is normal during the first three days. We show by positional cloning that the 'causative' mutation is in an alternatively-spliced exon of the gene (ttn) encoding Titin. Titin is the biggest known protein and spans the half-sarcomere from Z-disc to M-line in heart and skeletal muscle. It has been proposed to provide a scaffold for the assembly of thick and thin filaments and to provide elastic recoil engendered by stretch during diastole. We found that nascent myofibrils form in pik mutants, but normal sarcomeres are absent. Mutant cells transplanted to wildtype hearts remain thin and bulge outwards as individual cell aneurysms without affecting nearby wildtype cardiomyocytes, indicating that the contractile deficiency is cell-autonomous. Absence of Titin function thus results in blockage of sarcomere assembly and causes a functional disorder resembling human dilated cardiomyopathies, one form of which is described in another paper in this issue.

Ventricular myosin light chain-2 gene expression in developing heart of chicken embryos.

Recent gene knock-out studies in mice have suggested that ventricular myosin light chain-2 (vMLC2) has a role in the regulation of cardiogenic development and that perturbation in expression of vMLC2 is linked to the onset of dilated cardiomyopathy. In an attempt to develop an avian model for such studies, we examined the expression pattern of vMLC2 in chicken embryos at various stages and analyzed the effect of antisense oligonucleotide-mediated interference of vMLC2 function in cultures of whole embryos. Our results showed vMLC2 to be a specific marker for ventricular chamber throughout chicken embryonic development and antisense vMLC2 treatment of primitive streak stage (stage 4) embryos to produce pronounced dilation of heart tube with severe deficiency in formation of striated myofibrils. Further studies with antisense mRNA techniques of whole embryo cultures should, therefore, be useful to evaluate the role of vMLC2 and other putative regulatory factors in cardiac myofibrillogenesis.

Cardiac defects and altered ryanodine receptor function in mice lacking FKBP12.

FKBP12, a cis-trans prolyl isomerase that binds the immunosuppressants FK506 and rapamycin, is ubiquitously expressed and interacts with proteins in several intracellular signal transduction systems. Although FKBP12 interacts with the cytoplasmic domains of type I receptors of the transforming growth factor-beta (TGF-beta) superfamily in vitro, the function of FKBP12 in TGF-beta superfamily signalling is controversial. FKBP12 also physically interacts stoichiometrically with multiple intracellular calcium release channels including the tetrameric skeletal muscle ryanodine receptor (RyR1). In contrast, the cardiac ryanodine receptor, RyR2, appears to bind selectively the FKBP12 homologue, FKBP12.6. To define the functions of FKBP12 in vivo, we generated mutant mice deficient in FKBP12 using embryonic stem (ES) cell technology. FKBP12-deficient mice have normal skeletal muscle but have severe dilated cardiomyopathy and ventricular septal defects that mimic a human congenital heart disorder, noncompaction of left ventricular myocardium. About 9% of the mutants exhibit exencephaly secondary to a defect in neural tube closure. Physiological studies demonstrate that FKBP12 is dispensable for TGF-beta-mediated signalling, but modulates the calcium release activity of both skeletal and cardiac ryanodine receptors.

Energy deprivation and a deficiency in downstream metabolic target genes during the onset of embryonic heart failure in RXRalpha-/- embryos.

RXRalpha null mutant mice display ocular and cardiac malformations, liver developmental delay, and die from cardiac failure around embryonic day (E) 14.5 pc. To dissect the molecular basis of the RXRalpha-associated cardiomyopathy, we performed subtractive hybridization and systematically characterized putative downstream target genes that were selectively lacking in the mutant embryos, both at early (E10.5) and late (E13.5) stages of mouse embryonic development. Approximately 50% of the subtracted clones (61/115) encoded proteins involved in intermediary metabolism and electron transport, suggesting an energy deficiency in the RXRalpha-/- embryos. In particular, clone G1, which encodes subunit 14.5b of the NADH-ubiquinone dehydrogenase complex, displayed a dose-dependent expression in the wild-type, heterozygous and RXRalpha mutant mice. This gene was also downregulated in a retinoid-deficient rat embryo model. ATP content and medium Acyl-CoA dehydrogenase mRNA were lower in RXRalpha mutant hearts compared to wild-type mice. Ultrastructural studies showed that the density of mitochondria per myocyte was higher in the RXRalpha mutant compared to wild-type littermates. We propose a model whereby defects in intermediary metabolism may be a causative factor of the RXRalpha-/- phenotype and resembles an embryonic form of dilated cardiomyopathy.

Investigation of the selenium status of aborted calves with cardiac failure and myocardial necrosis.

Lesions of heart failure, specifically cardiac dilation or hypertrophy along with a nodular liver (chronic passive congestion) and ascites, have been found in 4-5% of aborted bovine fetuses. In this study, a group of 22 such fetuses was compared with groups of aborted fetuses without lesions of heart failure and with nonaborted fetuses obtained from a slaughterhouse. The fetuses were necropsied, tissues were taken for histopathology, and samples were collected for routine bacteriologic and virologic examinations. Liver and kidney tissue was saved for selenium analysis. Histopathologic examinations of myocardium of fetuses with cardiac failure revealed myocardial necrosis and mineralization in 7 fetuses, lymphocytic myocarditis in 5 fetuses, myocardial fibrosis in 5 fetuses, or no microscopic lesions in 5 fetuses. Mean liver selenium levels were 5.5 mumol/kg in the fetuses with heart lesions, 6.5 mumol/kg in the fetuses without heart lesions and 7.5 mumol/kg in fetuses from the slaughterhouse; these differences were statistically significant. The results suggest that selenium deficiency in bovine fetuses may cause myocardial necrosis and heart failure. This study also provides data on normal liver and kidney selenium levels in bovine fetuses from the analyses of 19 nonaborted fetuses.