The Biplane Modified Simpson's Method Accurately Estimates Pericardial Effusion Volume: A Comparison with Pericardiocentesis.

BACKGROUND: Pericardial effusion (PE) volume is often assessed semiqualitatively by echocardiography and categorized into minimal, small, moderate, or large. Several methods of echocardiographic quantification have been proposed, but their application is limited either by complexity or inaccuracy. We evaluated the accuracy of PE volume quantification by two-dimensional transthoracic echocardiogram (2DTTE) and commercially available volume quantification software in patients undergoing pericardiocentesis. METHODS: In a retrospective case series, immediate preprocedure echocardiograms of 33 patients for pericardiocentesis were analyzed. 2DTTE using the Simpson's method was adopted for volume measurement in the apical two- and four-chamber views. Pericardial fluid volume was calculated by taking the difference between volumes obtained by tracing the epicardial border of the heart and the pericardium. Postprocedure echocardiograms were performed to verify adequate pericardiocentesis. RESULTS: The mean pericardiocentesis fluid volume was 725.1 ± 299.5 mL (range, 250-1420 mL). The average volume estimated echocardiographically by the Simpson's method was 657.5 ± 276.9 mL (range, 205.7-1193.2 mL). There was strong direct linear correlation between echocardiographic and pericardiocentesis-derived volumes (P < 0.001, r = +0.823). Echocardiography underestimated PE volume by a mean of 9.3%. CONCLUSION: Two-dimensional transthoracic echocardiography using biplane Simpson's method of disks can simply and accurately estimate PE volume.

Tbx6 is a determinant of cardiac and neural cell fate decisions in multipotent P19CL6 cells.

Multipotent P19CL6 cells differentiate into cardiac myocytes or neural lineages when stimulated with dimethyl sulfoxide (DMSO) or retinoic acid (RA), respectively. Expression of the transcription factor Tbx6 was found to increase during cardiac myocyte differentiation and to decrease during neural differentiation. Overexpression of Tbx6 was not sufficient to drive P19CL6 cells to a cardiac myocyte fate or to accelerate DMSO-induced differentiation. In contrast, knockdown of Tbx6 dramatically inhibited DMSO-induced differentiation of P19CL6 cells to cardiac myocytes, as evidenced by the loss of striated muscle-specific markers and spontaneous beating. Tbx6 knockdown was also accompanied by almost complete loss of Nkx2.5, a transcription factor involved in the specification of the cardiac myocyte lineage, indicating that Nkx2.5 is downstream of Tbx6. In distinction to its positive role in cardiac myocyte differentiation, Tbx6 knockdown augmented RA-induced differentiation of P19CL6 cells to both neurons and glia, and accelerated the rate of neurite formation. Conversely, Tbx6 overexpression attenuated differentiation to neural lineages. Thus, in the P19CL6 model, Tbx6 is required for cardiac myocyte differentiation and represses neural differentiation. We propose a model in which Tbx6 is a part of a molecular switch that modulates divergent differentiation programs within a single progenitor cell.

A critical role for the protein apoptosis repressor with caspase recruitment domain in hypoxia-induced pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a lethal syndrome associated with the pathogenic remodeling of the pulmonary vasculature and the emergence of apoptosis-resistant cells. Apoptosis repressor with caspase recruitment domain (ARC) is an inhibitor of multiple forms of cell death known to be abundantly expressed in striated muscle. We show for the first time that ARC is expressed in arterial smooth muscle cells of the pulmonary vasculature and is markedly upregulated in several experimental models of PH. In this study, we test the hypothesis that ARC expression is essential for the development of chronic hypoxia-induced PH. METHODS AND RESULTS: Experiments in which cells or mice were rendered ARC-deficient revealed that ARC not only protected pulmonary arterial smooth muscle cells from hypoxia-induced death, but also facilitated growth factor-induced proliferation and hypertrophy and hypoxia-induced downregulation of selective voltage-gated potassium channels, the latter a hallmark of the syndrome in humans. Moreover, ARC-deficient mice exhibited diminished vascular remodeling, increased apoptosis, and decreased proliferation in response to chronic hypoxia, resulting in marked protection from PH in vivo. Patients with PH have significantly increased ARC expression not only in remodeled vessels but also in the lumen-occluding lesions associated with severe disease. CONCLUSIONS: These data show that ARC, previously unlinked to pulmonary hypertension, is a critical determinant of vascular remodeling in this syndrome.

Regulation of p53 tetramerization and nuclear export by ARC.

Inactivation of the transcription factor p53 is central to carcinogenesis. Yet only approximately one-half of cancers have p53 loss-of-function mutations. Here, we demonstrate a mechanism for p53 inactivation by apoptosis repressor with caspase recruitment domain (ARC), a protein induced in multiple cancer cells. The direct binding in the nucleus of ARC to the p53 tetramerization domain inhibits p53 tetramerization. This exposes a nuclear export signal in p53, triggering Crm1-dependent relocation of p53 to the cytoplasm. Knockdown of endogenous ARC in breast cancer cells results in spontaneous tetramerization of endogenous p53, accumulation of p53 in the nucleus, and activation of endogenous p53 target genes. In primary human breast cancers with nuclear ARC, p53 is almost always WT. Conversely, nearly all breast cancers with mutant p53 lack nuclear ARC. We conclude that nuclear ARC is induced in cancer cells and negatively regulates p53.

Microarray analysis of global changes in gene expression during cardiac myocyte differentiation.

Significant progress has been made in defining pathways that mediate the formation of the mammalian heart. Little is known, however, about the genetic program that directs the differentiation of cardiac myocytes from their precursor cells. A major hindrance to this kind of investigation has been the absence of an appropriate cell culture model of cardiac myocyte differentiation. Recently, a subline of P19 cells (P19CL6) was derived that, following dimethyl sulfoxide (DMSO) treatment, differentiate efficiently over 10 days into spontaneously beating cardiac myocytes. We demonstrate that these cells are indeed cardiac myocytes as they express cell type-specific markers and exhibit electrophysiological properties indicative of cardiac myocytes. The requirement for DMSO stimulation in this paradigm was shown to be limited to the first 4 days, suggesting that critical events in the differentiation process occur over this interval. To uncover relationships among known genes and identify novel genes that mediate cardiac myocyte differentiation, a detailed time course of changes in global gene expression was carried out using cDNA microarrays. In addition to the activation of genes encoding cardiac transcription factors and structural proteins, increases were noted in the expression of multiple known genes and expressed sequence tags (ESTs). Analysis of the former suggested the involvement of a variety of signaling pathways in cardiac myocyte differentiation. The 16 ESTs whose expression was increased during the early, stimulus-dependent phase of cardiac myocyte differentiation may be novel regulators of this process. Thus this first report of large-scale changes in gene expression during cardiac myocyte differentiation has delineated relationships among the expression patterns of known genes and identified a number of novel genes that merit further study.

The apoptosis inhibitor ARC undergoes ubiquitin-proteasomal-mediated degradation in response to death stimuli: identification of a degradation-resistant mutant.

Efficient induction of apoptosis requires not only the activation of death-promoting proteins but also the inactivation of inhibitors of cell death. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both central apoptosis pathways. Despite its potent inhibition of cell death, cells that express abundant ARC eventually succumb. A possible explanation is that ARC protein levels decrease dramatically in response to death stimuli. The mechanisms that mediate decreases in ARC protein levels during apoptosis and whether these decreases initiate the subsequent cell death are not known. Here we show that endogenous ARC protein levels decrease in response to death stimuli in a variety of cell contexts as well as in a model of myocardial ischemia-reperfusion in intact mice. Decreases in ARC protein levels are not explained by alterations in the abundance of ARC transcripts. Rather, pulse-chase experiments show that decreases in steady state ARC protein levels during apoptosis result from marked destabilization of ARC protein. ARC protein destabilization, in turn, is mediated by the ubiquitin-proteasomal pathway, as mutation of ARC ubiquitin acceptor residues stabilizes ARC protein and preserves its steady state levels during apoptosis. In addition, this degradation-resistant ARC mutant exhibits improved cytoprotection. We conclude that decreases in ARC protein levels in response to death stimuli are mediated by increased ARC protein degradation via the ubiquitin-proteasomal pathway. Moreover, these data demonstrate that decreases in ARC protein levels are a trigger, and not merely a consequence, of the ensuing cell death.

Inhibition of both the extrinsic and intrinsic death pathways through nonhomotypic death-fold interactions.

Death-fold domains constitute an evolutionarily conserved superfamily that mediates apoptotic signaling. These motifs, including CARD (caspase recruitment domain), DD (death domain), and DED (death effector domain), are believed to exert their effects solely through homotypic interactions. Herein we demonstrate that the CARD-containing protein ARC engages in nontraditional death-fold interactions to suppress both extrinsic and intrinsic death pathways. The extrinsic pathway is disrupted by heterotypic interactions between ARC's CARD and the DDs of Fas and FADD, which inhibit Fas-FADD binding and assembly of the death-inducing signaling complex (DISC). The intrinsic pathway is antagonized by ARC-Bax binding, involving ARC's CARD and the Bax C terminus. This inhibits Bax activation and translocation to the mitochondria. Knockdown of endogenous ARC facilitates DISC assembly and triggers spontaneous Bax activation and apoptosis. Conversely, physiological levels of ARC suppress these events. These studies establish a critical role for nonhomotypic death-fold interactions in the regulation of apoptosis.