Myofibrillar protein and gene expression in acute quadriplegic myopathy.

The dramatic muscle wasting, preferential loss of myosin and impaired muscle function in intensive care unit (ICU) patients with acute quadriplegic myopathy (AQM) have traditionally been suggested to be the result of proteolysis via specific proteolytic pathways. In this study we aim to investigate the mechanisms underlying the preferential loss of thick vs. thin filament proteins and the reassembly of the sarcomere during the recovery process in muscle samples from ICU patients with AQM. Quantitative and qualitative analyses of myofibrillar protein and mRNA expression were analyzed using SDS-PAGE, confocal microscopy, histochemistry and real-time PCR. The present results demonstrate that the transcriptional regulation of myofibrillar protein synthesis plays an important role in the loss of contractile proteins, as well as the recovery of protein levels during clinical improvement, myosin in particular, presumably in concert with proteolytic pathways, but the mechanisms are specific to the different thick and thin filament proteins studied.

Transcription factors in muscle atrophy caused by blocked neuromuscular transmission and muscle unloading in rats.

The muscle wasting associated with long-term intensive care unit (ICU) treatment has a negative effect on muscle function resulting in prolonged periods of rehabilitation and a decreased quality of life. To identify mechanisms behind this form of muscle wasting, we have used a rat model designed to mimic the conditions in an ICU. Rats were pharmacologically paralyzed with a postsynaptic blocker of neuromuscular transmission, and mechanically ventilated for one to two weeks, thereby unloading the limb muscles. Transcription factors were analyzed for cellular localization and nuclear concentration in the fast-twitch muscle extensor digitorum longus (EDL) and in the slow-twitch soleus. Significant muscle wasting and upregulation of mRNA for the ubiquitin ligases MAFbx and MuRF1 followed the treatment. The IkappaB family-member Bcl-3 displayed a concomitant decrease in concentration, suggesting altered kappaB controlled gene expression, although NFkappaB p65 was not significantly affected. The nuclear levels of the glucocorticoid receptor (GR) and the thyroid receptor alpha1 (TRalpha1) were altered and also suggested as potential mediators of the MAFbx- and MuRF1-induction in the absence of induced Foxo1. We believe that this model, and the strategy of quantifying nuclear proteins, will provide a valuable tool for further, more detailed, analyses of the muscle wasting occurring in patients kept on a mechanical ventilator.

Overexpression of semicarbazide-sensitive amine oxidase in smooth muscle cells leads to an abnormal structure of the aortic elastic laminas.

Elevated semicarbazide-sensitive amine oxidase (SSAO) activity has been observed in several human conditions, eg, diabetes, and it has been speculated that SSAO contributes to the development of vasculopathies associated with this disease. To investigate in vivo consequences of elevated expression of SSAO in vascular tissues, we have developed a transgenic model for overexpression of human SSAO in mice. A smooth muscle-specific promoter, smooth muscle alpha-actin promoter 8 (SMP8) was used. Transgenic expression of human SSAO in tissues with a high content of smooth muscle cells was confirmed by Northern blot analysis. Enzymatic analysis of homogenates from transgenic tissues showed elevated levels of SSAO activity compared to non-transgenic littermates. Furthermore, when plasma SSAO activity was analyzed, much higher activity was detected compared to plasma from control mice, indicating that plasma SSAO may originate from smooth muscle cells. Histopathological evaluation of aorta and renal artery from transgenic mice revealed an abnormal structure of the elastin tissue. Instead of the regularly folded elastic laminae normally found in tunica media of sacrificed mice, the elastic laminae were straight and unfolded with irregularly arranged elastic fibers, forming tangled webs, between the intercalating elastic laminae. These alterations of the elastin structures suggest that overexpression of SSAO has led to a reduced elasticity of the arteries. Moreover, the mean femoral arterial pressure of the SMP8 SSAO transgenic mice was significantly lower in comparison to non-transgenic littermates. This suggests that the transgenic mice have a defect in their ability to regulate blood pressure.

Semicarbazide-sensitive amine oxidase (SSAO) gene expression in alloxan-induced diabetes in mice.

BACKGROUND: Plasma activity of semicarbazide-sensitive amine oxidase (SSAO) has been reported to be significantly higher in diabetic patients compared to healthy controls. Due to the production of highly angiotoxic substances in SSAO-catalyzed reactions, it has been speculated that this could be a cause for the vascular complications frequently associated with diabetes. Little is known about how the enzyme activity is regulated, and why it is high in these patients. In the present study, we assessed the possibility of transcriptional regulation by analyzing SSAO activity and SSAO-mRNA levels in mice with alloxan-induced diabetes. MATERIALS AND METHODS: Diabetes was induced in NMRI mice by a single intravenous injection of alloxan. The enzyme activity was analyzed by a radiometric assay using (14) C-benzylamine as a substrate, and the mRNA-levels were analyzed by real-time PCR. RESULTS: We found that the enzyme activity was increased in lung and adipose tissue 1 day after induction, as the glucose levels start to rise. Seven days after the injection of alloxan, the activity in serum was increased, and this activity was positively correlated with blood glucose levels in the alloxan-treated animals. Although the enzyme activity was increased in adipose tissue as a result of the treatment, SSAO-mRNA levels in this tissue were decreased, possibly suggesting a negative feedback on the gene expression. CONCLUSIONS: The main conclusion from this study is that the increased enzyme activity observed in diabetes is not a result of increased SSAO gene transcription. We speculate that the enzyme activity is controlled by posttranslational modifications of the protein, and that the catalytic activity controls the gene expression.