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Muscle-specific RING finger protein 1 (MuRF1) is a sarcomere- associated protein that is restricted to cardiac and skeletal muscle. In skeletal muscle, MuRF1 is up-regulated by conditions that provoke atrophy, but its function in the heart is not known. The presence of a RING finger in MuRF1 raises the possibility that it is a component of the ubiquitin-proteasome system of protein degradation. We performed a yeast two-hybrid screen to search for interaction partners of MuRF1 in the heart that might be targets of its putative ubiquitin ligase activity. This screen identified troponin 1 as a MuRF1 partner protein. MuRF1 and troponin 1 were found to associate both in vitro and in vivo in cultured cardiomyocytes. MuRF1 reduced steady-state troponin I levels when coexpressed in COS-7 cells and increased degradation of endogenous troponin I protein in cardiomyocytes. The degradation of troponin I in cardiomyocytes was associated with the accumulation of ubiquitylated intermediates of troponin I and was proteasome-dependent. In vitro, MuRF1 functioned as a ubiquitin ligase to catalyze ubiquitylation of troponin I through a RING finger-dependent mechanism. In isolated cardiomyocytes, MuRF1 reduced indices of contractility. In cardiomyocytes, these processes may determine the balance between hypertrophic and antihypertrophic signals and the regulation of myocyte contractile responses in the setting of heart failure. [ABSTRACT FROM AUTHOR]

Pyrrolidine dithiocarbamate (PDTC), a metal chelating compound, is known to induce cell death in vascular smooth muscle cells (VSMC). However, the molecular mechanism for PDTC-induced VSMC death is not well understood. Addition of PDTC reduced cell growth and DNA synthesis on VSMC in low density conditions. However, in serum depleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. Several metal chelators prevented the cell death induced by PDTC. In a serum-deprived condition, addition of exogenous metals, copper, iron, and zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as copper, iron, and zinc in serum may mediate the cytotoxic effect of PDTC. At low VSMC density in 10% FBS, treatment of PDTC, which induced a cell-cycle block in G1-phase, induced down-regulation of cyclins and CDKs and up-regulation of the CDK inhibitor p21 expression, whereas up-regulation of p27 or p53 by PDTC was not observed. Finally, we determined PDTC-mediated signaling pathway involved in VSMC death. Among relevant pathways, PDTC induced marked activation of p38MAPK and JNK. Expression of dominant negative p38MAPK and SB203580, a p38MAPK specific inhibitor, blocked PDTC-dependent p38MAPK, growth inhibition, and p21 expression. These data demonstrate that the p38MAPK pathway participates in p21 induction, which consequently leads to decrease of cyclin D1/cdk4 and cyclin E/cdk2 complexes and PDTC-dependent VSMC growth inhibition. In conclusion, an understanding of the molecular mechanisms of PDTC in VSMC provides a theoretical basis for clinical approaches using antioxidant therapies in atherosclerosis. J. Cell. Physiol. 198: 310–323, 2004© 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

: Aims Homeobox (Hox) proteins are transcriptional regulators in embryonic patterning, cell differentiation, proliferation, and migration in vertebrates and invertebrates. A growing body of evidence suggests that Hox proteins are involved in endothelial cell regulation. We have shown earlier that HoxB5 upregulates vascular endothelial growth factor receptor-2 and thereby contributes to enhanced endothelial precursor cell differentiation. Here we aim to elucidate the role of HoxB5 in angiogenesis. : Methods and results Endothelial cell sprouting was investigated in the human umbilical vein endothelial cell spheroid assay. We investigated in vivo angiogenesis in the chick (Gallus gallus) chorioallantoic membrane assay. Expression profiling of proangiogenic factors was done by quantitative PCR. The angiopoietin-2 (Ang2) promoter and deletion fragments thereof were cloned into the pGL3 reporter system for analysis of transcriptional activity. We observed that HoxB5 enhances endothelial cell sprouting and modulates the expression of adhesion molecules in vitro. Accordingly, we observed a modification of vascular growth by HoxB5 in vivo. The HoxB5 effect is reminiscent of the effects of angiopoietins. We demonstrate that Ang2 is upregulated upon HoxB5 overexpression and that the HoxB5 effect is abolished by the angiopoietin antagonist soluble Tie-2. : Conclusion HoxB5 has an activating effect on Ang2 that is essential for endothelial cell sprouting and coordinated vascular growth. [ABSTRACT FROM AUTHOR]

The assembly and maintenance of the cardiac sarcomere, which contains the basic contractile components of actin and myosin, are essential for cardiac function. While often described as a static structure, the sarcomere is actually dynamic and undergoes constant turnover, allowing it to adapt to physiological changes while still maintaining function. A host of new factors have been identified that play a role in the regulation of protein quality control in the sarcomere, including chaperones that mediate the assembly of sarcomere components and ubiquitin ligases that control their specific degradation. There is clear evidence of sarcomere disorganization in animal models lacking muscle-specific chaperone proteins, illustrating the importance of these molecules in sarcomere structure and function. Although ubiquitin ligases have been found within the sarcomere structure itself, the role of the ubiquitin proteasome system in cardiac sarcomere regulation, and the factors that control its activity, are only just now being elucidated. The number of ubiquitin ligases identified with specificity for sarcomere proteins, each with distinct target substrates, is growing, allowing for tight regulation of this system. In this review, we highlight the dynamic interplay between sarcomere-specific chaperones and ubiquitin-dependent degradation of sarcomere proteins that is necessary in order to maintain structure and function of the cardiac sarcomere. [ABSTRACT FROM AUTHOR]