BackgroundThe transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1.Methodology/Principal FindingsIn the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death.Conclusions/SignificanceAppropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death.

Background-Cardiac resynchronization therapy (CRT) improves chamber mechanoenergetics and morbidity and mortality of patients manifesting heart failure with ventricular dyssynchrony; however, little is known about the molecular changes underlying CRT benefits. We hypothesized that mitochondria may play an important role because of their involvement in energy production. Methods and Results-Mitochondria isolated from the left ventricle in a canine model of dyssynchronous or resynchronized (CRT) heart failure were analyzed by a classical, gel-based, proteomic approach. Two-dimensional gel electrophoresis revealed that 31 mitochondrial proteins where changed when controlling the false discovery rate at 30%. Key enzymes in anaplerotic pathways, such as pyruvate carboxylation and branched-chain amino acid oxidation, were increased. These concerted changes, along with others, suggested that CRT may increase the pool of Krebs cycle intermediates and fuel oxidative phosphorylation. Nearly 50% of observed changes pertained to subunits of the respiratory chain. ATP synthase-␤ subunit of complex V was less degraded, and its phosphorylation modulated by CRT was associated with increased formation (2-fold, Pϭ0.004) and specific activity (ϩ20%, Pϭ0.05) of the mature complex. The importance of these modifications was supported by coordinated changes in mitochondrial chaperones and proteases. CRT increased the mitochondrial respiratory control index with tightened coupling when isolated mitochondria were reexposed to substrates for both complex I (glutamate and malate) and complex II (succinate), an effect likely related to ATP synthase subunit modifications and complex quantity and activity. Conclusions-CRT potently affects both the mitochondrial proteome and the performance associated with improved cardiac function. (Circ Cardiovasc Genet. 2010;3:78-87.)Key Words: cardiac resynchronization therapy Ⅲ mitochondria Ⅲ proteomics Ⅲ ATP synthase H eart failure (HF) is a leading cause of morbidity and mortality in older adults, affecting 5 million individuals in the US alone. 1 A subset of patients with HF also develops regional conduction delay, resulting in discoordinate contraction that worsens symptoms and ultimate prognosis. Since the turn of the millennium, cardiac resynchronization therapy (CRT), also referred to as biventricular pacing, has become a clinical treatment for such patients, improving heart function, clinical symptoms, and survival. 2,3 Some recent studies 4 -6 have revealed changes in gene expression and molecular remodeling of stress response kinases and cell survival signaling associated with CRT. Among the earliest findings in clinical CRT studies was the demonstration that CRT improves chamber energetic efficiency. This effect is rapid, resulting from the retiming of contraction to synchronously occur in both sides of the myocardium, reducing wasted chamber work much like tuning a car engine. Given the importance of abnormal mechanoenergetics in HF and centrality of ATP cycling, 7 we hypothesized that CR...