Requirement of a novel gene, Xin, in cardiac morphogenesis.
A novel gene, Xin, from chick (cXin) and mouse (mXin) embryonic hearts, may be required for cardiac morphogenesis and looping. Both cloned cDNAs have a single open reading frame, encoding proteins with 2,562 and 1,677 amino acids for cXin and mXin, respectively. The derived amino acid sequences share 46% similarity. The overall domain structures of the predicted cXin and mXin proteins, including proline-rich regions, 16 amino acid repeats, DNA-binding domains, SH3-binding motifs and nuclear localization signals, are highly conserved. Northern blot analyses detect a single message of 8.9 and 5.8 kilo base (kb) from both cardiac and skeletal muscle of chick and mouse, respectively. In situ hybridization reveals that the cXin gene is specifically expressed in cardiac progenitor cells of chick embryos as early as stage 8, prior to heart tube formation. cXin continues to be expressed in the myocardium of developing hearts. By stage 15, cXin expression is also detected in the myotomes of developing somites. Immunofluorescence microscopy reveals that the mXin protein is colocalized with N-cadherin and connexin-43 in the intercalated discs of adult mouse hearts. Incubation of stage 6 chick embryos with cXin antisense oligonucleotides results in abnormal cardiac morphogenesis and an alteration of cardiac looping. The myocardium of the affected hearts becomes thickened and tends to form multiple invaginations into the heart cavity. This abnormal cellular process may account in part for the abnormal looping. cXin expression can be induced by bone morphogenetic protein (BMP) in explants of anterior medial mesoendoderm from stage 6 chick embryos, a tissue that is normally non-cardiogenic. This induction occurs following the BMP-mediated induction of two cardiac-restricted transcription factors, Nkx2.5 and MEF2C. Furthermore, either MEF2C or Nkx2.5 can transactivate a luciferase reporter driven by the mXin promoter in mouse fibroblasts. These results suggest that Xin may participate in a BMP-Nkx2.5-MEF2C pathway to control cardiac morphogenesis and looping. (+info)
Coupling of the cell cycle and myogenesis through the cyclin D1-dependent interaction of MyoD with cdk4.
Proliferating myoblasts express the muscle determination factor, MyoD, throughout the cell cycle in the absence of differentiation. Here we show that a mitogen-sensitive mechanism, involving the direct interaction between MyoD and cdk4, restricts myoblast differentiation to cells that have entered into the G0 phase of the cell cycle under mitogen withdrawal. Interaction between MyoD and cdk4 disrupts MyoD DNA-binding, muscle-specific gene activation and myogenic conversion of 10T1/2 cells independently of cyclin D1 and the CAK activation of cdk4. Forced induction of cyclin D1 in myotubes results in the cytoplasmic to nuclear translocation of cdk4. The specific MyoD-cdk4 interaction in dividing myoblasts, coupled with the cyclin D1-dependent nuclear targeting of cdk4, suggests a mitogen-sensitive mechanism whereby cyclin D1 can regulate MyoD function and the onset of myogenesis by controlling the cellular location of cdk4 rather than the phosphorylation status of MyoD. (+info)
Cloning and characterization of the promoters of the maxiK channel alpha and beta subunits.
Large conductance, calcium-activated potassium (maxiK) channels are expressed in nerve, muscle, and other cell types and are important determinants of smooth muscle tone. To determine the mechanisms involved in the transcriptional regulation of maxiK channels, we characterized the promoter regions of the pore forming (alpha) and regulatory (beta) subunits of the human channel complex. Maximum promoter activity (up to 12.3-fold over control) occurred between nucleotides -567 and -220 for the alpha subunit (hSlo) gene. The minimal promoter is GC-rich with 5 Sp-1 binding sites and several TCC repeats. Other transcription factor-binding motifs, including c/EBP, NF-kB, PU.1, PEA-3, Myo-D, and E2A, were observed in the 5'-flanking sequence. Additionally, a CCTCCC sequence, which increases the transcriptional activity of the SM1/2 gene in smooth muscle, is located 27 bp upstream of the TATA-like sequence, a location identical to that found in the SM1/2 5'-flanking region. However, the promoter directed equivalent expression when transfected into smooth muscle and other cell types. Analysis of the hSlo beta subunit 5'-flanking region revealed a TATA box at position -77 and maximum promoter activity (up to 11.0-fold) in a 200 bp region upstream from the cap site. Binding sites for GATA-1, Myo-D, c-myb, Ets-1/Elk-1, Ap-1, and Ik-2 were identified within this sequence. Two CCTCCC elements are present in the hSlo beta subunit promoter, but tissue-specific transcriptional activity was not observed. The lack of tissue-specific promoter activity, particularly the finding of promoter activity in cells from tissues in which the maxiK gene is not expressed, suggests a complex channel regulatory mechanism for hSlo genes. Moreover, the lack of similarity of the promoters of the two genes suggests that regulation of coordinate expression of the subunits does not occur through equivalent cis-acting sequences. (+info)
Perioperative growth hormone treatment and functional outcome after major abdominal surgery: a randomized, double-blind, controlled study.
OBJECTIVE: To evaluate short- and long-term effects of perioperative human growth hormone (hGH) treatment on physical performance and fatigue in younger patients undergoing a major abdominal operation in a normal postoperative regimen with oral nutrition. SUMMARY BACKGROUND DATA: Muscle wasting and functional impairment follow major abdominal surgery. METHODS: Twenty-four patients with ulcerative colitis undergoing ileoanal J-pouch surgery were randomized to hGH (12 IU/day) or placebo treatment from 2 days before to 7 days after surgery. Measurements were performed 2 days before and 10, 30, and 90 days after surgery. RESULTS: The total muscle strength of four limb muscle groups was reduced by 7.6% in the hGH group and by 17.1% in the placebo group at postoperative day 10 compared with baseline values. There was also a significant difference between treatment groups in total muscle strength at day 30, and at the 90-day follow-up total muscle strength was equal to baseline values in the hGH group, but still significantly 5.9% below in the placebo group. The work capacity decreased by approximately 20% at day 10 after surgery, with no significant difference between treatment groups. Both groups were equally fatigued at day 10 after surgery, but at day 30 and 90 the hGH patients were less fatigued than the placebo patients. During the treatment period, patients receiving hGH had reduced loss of limb lean tissue mass, and 3 months after surgery the hGH patients had regained more lean tissue mass than placebo patients. CONCLUSIONS: Perioperative hGH treatment of younger patients undergoing major abdominal surgery preserved limb lean tissue mass, increased postoperative muscular strength, and reduced long-term postoperative fatigue. (+info)
Spinal cord-evoked potentials and muscle responses evoked by transcranial magnetic stimulation in 10 awake human subjects.
Transcranial magnetic stimulation (TCMS) causes leg muscle contractions, but the neural structures in the brain that are activated by TCMS and their relationship to these leg muscle responses are not clearly understood. To elucidate this, we concomitantly recorded leg muscle responses and thoracic spinal cord-evoked potentials (SCEPs) after TCMS for the first time in 10 awake, neurologically intact human subjects. In this report we provide evidence of direct and indirect activation of corticospinal neurons after TCMS. In three subjects, SCEP threshold (T) stimulus intensities recruited both the D wave (direct activation of corticospinal neurons) and the first I wave (I1, indirect activation of corticospinal neurons). In one subject, the D, I1, and I2 waves were recruited simultaneously, and in another subject, the I1 and I2 waves were recruited simultaneously. In the remaining five subjects, only the I1 wave was recruited first. More waves were recruited as the stimulus intensity increased. The presence of D and I waves in all subjects at low stimulus intensities verified that TCMS directly and indirectly activated corticospinal neurons supplying the lower extremities. Leg muscle responses were usually contingent on the SCEP containing at least four waves (D, I1, I2, and I3). (+info)
Wasting of the small hand muscles in upper and mid-cervical cord lesions.
Four patients are described with destructive rheumatoid arthritis of the cervical spine and neurogenic wasting of forearm and hand muscles. The pathological connection is not immediately obvious, but a relationship between these two observations is described here with clinical, radiological, electrophysiological and necropsy findings. Compression of the anterior spinal artery at upper and mid-cervical levels is demonstrated to be the likely cause of changes lower in the spinal cord. These are shown to be due to the resulting ischaemia of the anterior part of the lower cervical spinal cord, with degeneration of the neurones innervating the forearm and hand muscles. These findings favour external compression of the anterior spinal artery leading to ischaemia in a watershed area as the likeliest explanation for this otherwise inappropriate and bizarre phenomenon. (+info)
Fas and Fas ligand interaction induces apoptosis in inflammatory myopathies: CD4+ T cells cause muscle cell injury directly in polymyositis.
OBJECTIVE: To investigate the involvement of the Fas/Fas ligand (Fas/FasL) system in the inflammatory myopathies. METHODS: Frozen muscle sections obtained from 7 patients with polymyositis (PM), 4 patients with dermatomyositis (DM), and 3 controls were studied by immunochemistry. Apoptosis was detected by DNA electrophoresis and in situ labeling using the TUNEL method. RESULTS: Fas was detected on muscle fibers and infiltrating mononuclear cells (MNC) in 6 PM patients and 2 DM patients. FasL was expressed mainly on CD4+ T cells and some CD8+ T cells, and on macrophages surrounding Fas-positive muscles in 4 PM patients and 1 DM patient. In 3 of the 5 patients with FasL-positive MNC, the TUNEL method showed that both invaded myonuclei and MNC underwent apoptosis. Chromosomal DNA from the muscle tissue of these patients showed ladder formation. CONCLUSION: Fas/FasL is involved in muscle cell apoptosis in at least 2 of the inflammatory myopathies, PM and DM. Although CD8+-mediated cytotoxicity is thought to be the main mechanism of muscle injury in PM, our data suggest that CD4+ T cells also directly cause muscle cell damage. (+info)
A novel interaction mechanism accounting for different acylphosphatase effects on cardiac and fast twitch skeletal muscle sarcoplasmic reticulum calcium pumps.
In cardiac and skeletal muscle Ca2+ translocation from cytoplasm into sarcoplasmic reticulum (SR) is accomplished by different Ca2+-ATPases whose functioning involves the formation and decomposition of an acylphosphorylated phosphoenzyme intermediate (EP). In this study we found that acylphosphatase, an enzyme well represented in muscular tissues and which actively hydrolyzes EP, had different effects on heart (SERCA2a) and fast twitch skeletal muscle SR Ca2+-ATPase (SERCA1). With physiological acylphosphatase concentrations SERCA2a exhibited a parallel increase in the rates of both ATP hydrolysis and Ca2+ transport; in contrast, SERCA1 appeared to be uncoupled since the stimulation of ATP hydrolysis matched an inhibition of Ca2+ pump. These different effects probably depend on phospholamban, which is associated with SERCA2a but not SERCA1. Consistent with this view, the present study suggests that acylphosphatase-induced stimulation of SERCA2a, in addition to an enhanced EP hydrolysis, may be due to a displacement of phospholamban, thus to a removal of its inhibitory effect. (+info)