Proliferation of embryonic cardiomyocytes in zebrafish requires the sodium channel scn5Lab.

In mice, homozygous deletion of the cardiac sodium channel Scn5a results in defects in cardiac morphology and embryonic death before robust sodium current can be detected. In zebrafish, morpholino knockdown of cardiac sodium channel orthologs scn5Laa and scn5Lab perturbs specification of precardiac mesoderm and inhibits growth of the embryonic heart. It is not known which developmental processes are perturbed by sodium channel knockdown and whether reduced cell number is from impaired migration of cardiac progenitors into the heart, impaired myocyte proliferation, or both. We found that embryos deficient in scn5Lab displayed defects in primary cardiogenesis specific to loss of nkx2.5, but not nkx2.7. We generated kaede reporter fish and demonstrated that embryos treated with anti-scn5Lab morpholino showed normal secondary differentiation of cardiomyocytes at the arterial pole between 30 and 48 h post-fertilization. However, while proliferating myocytes were readily detected at 48 hpf in wild type embryos, there were no BrdU-positive cardiomyocytes in embryos subjected to anti-scn5Lab treatment. Proliferating myocytes were present in embryos injected with anti-tnnt2 morpholino to phenocopy the silent heart mutation, and absent in embryos injected with anti-tnnt2 and anti-scn5Lab morpholinos, indicating cardiac contraction is not required for the loss of proliferation. These data demonstrate that the role of scn5Lab in later heart growth does not involve contribution of the secondary heart field, but rather proliferation of cardiomyocytes, and appears unrelated to the role of the channel in cardiac electrogenesis.

Characterization of a dominant inhibitory E47 protein that suppresses C2C12 myogenesis.

Skeletal muscle formation is controlled through the coordinated actions of the muscle regulatory factors (MRFs). The activities of these basic helix-loop-helix proteins is mediated in part through heterodimer formation with a family of ubiquitous bHLH proteins, referred to as E-proteins. The primary E-protein in skeletal muscle is the E2A splice variant, E47. To further address the role of E47 during skeletal myogenesis, we created a chimeric E47 repressor protein by replacing the transcriptional activation domain with the Drosophila Engrailed transcriptional repressor domain. The dominant inhibitory E-protein (EnDeltaE47) formed homodimers capable of binding DNA and abolished E47-directed gene transcription. Stable expression of EnDeltaE47 in mouse C2C12 myoblasts effectively blocked the cells' ability to differentiate into mature myofibers. Closer examination of the molecular basis for the inhibition of myogenesis revealed that EnDeltaE47 preferentially forms heterodimers with myogenin. Interestingly, the chimeric repressor did not form DNA-binding heterodimers with MyoD in C2C12 myocytes. The failure to detect MyoD:EnDeltaE47 heterodimers in myoblasts was not due to protein conformational defects as both wild-type E47 and EnDeltaE47 readily formed DNA binding complexes with MyoD in vitro. These results indicate that E47 plays a crucial role in C2C12 myogenesis by serving as the preferred heterodimer partner of the myogenin protein.

Reconstruction of the anterior and posterior cruciate ligaments after knee dislocation. Results using fresh-frozen nonirradiated allografts.

We reviewed the results in 13 patients who underwent simultaneous allograft reconstruction of both the anterior and posterior cruciate ligaments after a knee dislocation (nine acute and four chronic injuries). Seven patients sustained related medial collateral ligament injuries and six patients had posterolateral complex injuries. Ligament reconstructions were performed using fresh-frozen Achilles or patellar tendon allografts. At follow-up evaluation (mean of 38 months), only one patient described the reconstructed knee as normal. Six patients had returned to unrestricted sports activities and four had returned to modified sports. The average extension loss was 3 degrees (range, 0 degree to 10 degrees) and average flexion loss was 5 degrees (range, 0 degree to 15 degrees). The KT-1000 arthrometer measurements at 133 N anterior-posterior tibial load showed a mean side-to-side difference of 4.5 mm (range, 0 to 10) at 20 degrees and 5.0 mm (range, 0 to 9) at 70 degrees. The mean Lysholm score was 88 (range, 42 to 100). International Knee Documentation Committee ratings were six nearly normal, five abnormal, and one grossly abnormal. Two patients required manipulations for knee stiffness. This study demonstrates that reconstruction of both cruciate ligaments can restore stability sufficient to allow sports activity in most patients with knee dislocations, but "normal" results are difficult to achieve.