Nonmuscle myosin II-B is required for normal development of the mouse heart

We used targeted gene disruption in mice to ablate nonmuscle myosin heavy chain B (NMHC-B), one of the two isoforms of nonmuscle myosin II present in all vertebrate cells. Approximately 65% of the NMHC-B−/− embryos died prior to birth, and those that were born suffered from congestive heart failure and died during the first day. No abnormalities were detected in NMHC-B+/− mice. The absence of NMHC-B resulted in a significant increase in the transverse diameters of the cardiac myocytes from 7.8 ± 1.8 μm (right ventricle) and 7.8 ± 1.3 μm (left ventricle) in NMHC-B+/+ and B+/− mice to 14.7 ± 1.1 μm and 13.8 ± 2.3 μm, respectively, in NMHC-B−/− mice (in both cases, P < 0.001). The increase in size of the cardiac myocytes was seen as early as embryonic day 12.5 (4.5 ± 0.2 μm for NMHC-B+/+ and B+/− vs. 7.2 ± 0.6 μm for NMHC-B−/− mice (P < 0.01)). Six of seven NMHC-B−/− newborn mice analyzed by serial sectioning also showed structural cardiac defects, including a ventricular septal defect, an aortic root that either straddled the defect or originated from the right ventricle, and muscular obstruction to right ventricular outflow. Some of the hearts of NMHC-B−/− mice showed evidence for up-regulation of NMHC-A protein. These studies suggest that nonmuscle myosin II-B is required for normal cardiac myocyte development and that its absence results in structural defects resembling, in part, two common human congenital heart diseases, tetralogy of Fallot and double outlet right ventricle.

Synpolydactyly phenotypes correlate with size of expansions in HOXD13 polyalanine tract

Synpolydactyly (SPD) is a dominantly inherited congenital limb malformation. Typical cases have 3/4 finger and 4/5 toe syndactyly, with a duplicated digit in the syndactylous web, but incomplete penetrance and variable expressivity are common. The condition has recently been shown to be caused by expansions of an imperfect trinucleotide repeat sequence encoding a 15-residue polyalanine tract in HOXD13. We have studied 16 new and 4 previously published SPD families, with between 7 and 14 extra residues in the tract, to analyze the molecular basis for the observed variation in phenotype. Although there is no evidence of change in expansion size within families, even over six generations, there is a highly significant increase in the penetrance and severity of phenotype with increasing expansion size, affecting both hands (P = 0.012) and feet (P < 0.00005). Affected individuals from a family with a 14-alanine expansion, the largest so far reported, all have a strikingly similar and unusually severe limb phenotype, involving the first digits and distal carpals. Affected males from this family also have hypospadias, not previously described in SPD, but consistent with HOXD13 expression in the developing genital tubercle. The remarkable correlation between phenotype and expansion size suggests that expansion of the tract leads to a specific gain of function in the mutant HOXD13 protein, and has interesting implications for the role of polyalanine tracts in the control of transcription.

Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.

Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.

Homocysteine induces congenital defects of the heart and neural tube: Effect of folic acid

The biological basis or mechanism whereby folate supplementation protects against heart and neural tube defects is unknown. It has been hypothesized that the amino acid homocysteine may be the teratogenic agent, since serum homocysteine increases in folate depletion; however, this hypothesis has not been tested. In this study, avian embryos were treated directly with d,l-homocysteine or with l-homocysteine thiolactone, and a dose response was established. Of embryos treated with 50 μl of the teratogenic dose (200 mM d,l-homocysteine or 100 mM l-homocysteine thiolactone) on incubation days 0, 1, and 2 and harvested at 53 h (stage 14), 27% showed neural tube defects. To determine the effect of the teratogenic dose on the process of heart septation, embryos were treated during incubation days 2, 3, and 4; then they were harvested at day 9 following the completion of septation. Of surviving embryos, 23% showed ventricular septal defects, and 11% showed neural tube defects. A high percentage of the day 9 embryos also showed a ventral closure defect. The teratogenic dose was shown to raise serum homocysteine to over 150 nmol/ml, compared with a normal level of about 10 nmol/ml. Folate supplementation kept the rise in serum homocysteine to ≈45 nmol/ml, and prevented the teratogenic effect. These results support the hypothesis that homocysteine per se causes dysmorphogenesis of the heart and neural tube, as well as of the ventral wall.

Characterization of human cardiac Na+ channel mutations in the congenital long QT syndrome

The congenital long QT syndrome (LQTS) is an inherited disorder characterized by a prolonged cardiac action potential. This delay in cellular repolarization can lead to potentially fatal arrhythmias. One form of LQTS (LQT3) has been linked to the human cardiac voltage-gated sodium channel gene (SCN5A). Three distinct mutations have been identified in the sodium channel gene. The biophysical and functional characteristics of each of these mutant channels were determined by heterologous expression of a recombinant human heart sodium channel in a mammalian cell line. Each mutation caused a sustained, non-inactivating sodium current amounting to a few percent of the peak inward sodium current, observable during long (>50 msec) depolarizations. The voltage dependence and rate of inactivation were altered, and the rate of recovery from inactivation was changed compared with wild-type channels. These mutations in diverse regions of the ion channel protein, all produced a common defect in channel gating that can cause the long QT phenotype. The sustained inward current caused by these mutations will prolong the action potential. Furthermore, they may create conditions that promote arrhythmias due to prolonged depolarization and the altered recovery from inactivation. These results provide insights for successful intervention in the disease.