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Aims The major cardiac cell types composing the adult heart arise from common multipotent precursor cells. Cardiac lineage decisions are guided by extrinsic and cell-autonomous factors, including recently discovered long noncoding RNAs (lncRNAs). The human lncRNA CARMEN , which is known to dictate specification toward the cardiomyocyte (CM) and the smooth muscle cell (SMC) fates, generates a diversity of alternatively spliced isoforms. Methods and results The CARMEN locus can be manipulated to direct human primary cardiac precursor cells (CPCs) into specific cardiovascular fates. Investigating CARMEN isoform usage in differentiating CPCs represents therefore a unique opportunity to uncover isoform-specific functions in lncRNAs. Here, we identify one CARMEN isoform, CARMEN-201, to be crucial for SMC commitment. CARMEN-201 activity is encoded within an alternatively spliced exon containing a MIRc short interspersed nuclear element. This element binds the transcriptional repressor REST (RE1 Silencing Transcription Factor), targets it to cardiogenic loci, including ISL1 , IRX1 , IRX5 , and SFRP1 , and thereby blocks the CM gene program. In turn, genes regulating SMC differentiation are induced. Conclusions These data show how a critical physiological switch is wired by alternative splicing and functional transposable elements in a long noncoding RNA. They further demonstrated the crucial importance of the lncRNA isoform CARMEN-201 in SMC specification during heart development. [ABSTRACT FROM AUTHOR]

Recent evidence supports that TNF-[alpha], long considered a catabolic factor, may also have a physiological function in skeletal muscle. The catabolic view, mainly based on correlative studies in human and in vivo animal models, was challenged by experiments with myoblasts, in which TNF-[alpha] induced differentiation. The biological effects of TNF-[alpha] in differentiated muscle, however, remain poorly understood. In the present study, we tested whether TNF-[alpha] has growth-promoting effects in myotubes, and we characterized the mechanisms leading to these effects. Treatment of [C.sub.2][C.sub.12] myotubes with TNF-[alpha] for 24 h increased protein synthesis (PS) and enhanced cellular dehydrogenase activity by 22 and 26%, respectively, without changing cell numbers. These effects were confirmed in myotubes differentiated from primary rat myoblasts. TNF-[alpha] activated two signaling cascades: 1) ERK1/2 and its target eIF4E and 2) Akt and its downstream effectors GSK-3, [p70.sup.S6K], and 4E-BP1. TNF-[alpha]-induced phosphorylation of Akt, and ERK1/2 was inhibited by an antibody against TNF-[alpha] receptor 1 (TNF-R1). PD-98059 pretreatment abolished TNF-[alpha]-induced phosphorylation of ERK1/2 and eIF4E, whereas PS was only partially inhibited. LY-294002 completely abolished TNF-[alpha]-induced stimulation of PS as well as phosphorylation of Akt and its downstream targets GSK-3, [p70.sup.S6K], and 4E-BP1. Rapamycin inhibited TNF-[alpha]-induced phosphorylation of the roTOR C1 target [p70.sup.S6K] without altering TNF-[alpha]-induced PS and 4E-BP1 phosphorylation. In conclusion, our results provide evidence that TNF-[alpha] enhances PS in myotubes and that this is based on enhanced protein translation mediated by the TNF-R1 and PI3K-Akt and MEK-ERK signaling cascades. tumor necrosis factor-[alpha]; tumor necrosis factor-[alpha] receptor 1; phosphatidylinositol 3-kinase; mitogen-activated protein kinase/extracellular signal-related kinase

Endogenous protein phosphatase 1 runs down gap junctional communication of rat ventricular myocytes. Am J Physiol Cell Physiol 281: C1648-C1656, 2001.--Gap junctional channels are essential for normal cardiac impulse propagation. In ventricular myocytes of new-born rats, channel opening requires the presence of ATP to allow protein kinase activities; otherwise, channels are rapidly deactivated by the action of endogenous protein phosphatases (PPs). The lack of influence of [Mg.sup.2+] and of selective PP2B inhibition is not in favor of the involvements of [Mg.sup.2+]-dependent PP2C and PP2B, respectively, in the loss of channel activity. Okadaic acid (1 [micro]M) and calyculin A (100 nM), both inhibitors of PP1 and PP2A activities, significantly retarded the loss of channel activity. However, a better preservation was obtained in the presence of selective PP1 inhibitors heparin (100 [micro]g/ml) or protein phosphatase inhibitor 2 (I2; 100 nM). Conversely, the stimulation of endogenous PP1 activity by p-nitrophenyl phosphate, in the presence of ATP, led to a progressive fading of junctional currents unless I2 was simultaneously added. Together, these results suggest that a basal phosphorylation-dephosphorylation turnover regulates gap junctional communication which is rapidly deactivated by PP1 activity when the phosphorylation pathway is hindered. connexin; patch clamp; protein phosphorylation

Recent evidence supports that TNF-α, long considered a catabolic factor, may also have a physiological function in skeletal muscle. The catabolic view, mainly based on correlative studies in human and in vivo animal models, was challenged by experiments with myoblasts, in which TNF-α induced differentiation. The biological effects of TNF-α in differentiated muscle, however, remain poorly understood. In the present study, we tested whether TNF-α has growth-promoting effects in myotubes, and w& characterized the mechanisms leading to these effects. Treatment of C2C12 myotubes with TNF-α for 24 h increased protein synthesis (PS) and enhanced cellular dehydrogenase activity by 22 and 26%, respectively, without changing cell numbers. These effects were confirmed in myotubes differentiated from primary rat myoblasts. TNF-α activated two signaling cascades: 1) ERK1/2 and its target eIF4E and 2) Akt and its downstream effectors GSK-3, p70S6K and 4E-BP1. TNF-α-induced phosphorylation of Akt, and ERK1/2 was inhibited by an antibody against TNF-α receptor 1 (TNF-R1). PD-98059 pretreatment abolished TNF-α-induced phosphorylation of ERK1/2 and eIF4E, whereas PS was only partially inhibited. LY-294002 completely abolished TNF-α-induced stimulation of PS as well as phosphorylation of Akt and its downstream targets GSK-3, p70S6K, and 4E-BP1. Rapamycin inhibited TNF-α-induced phosphorylation of the mTOR C1 target p70S6K without altering TNF-α-induced PS and 4E-BP1 phosphorylation. In conclusion, our results provide evidence that TNF-α enhances PS in myotubes and that this is based on enhanced protein translation mediated by the TNF-R1 and PI3K-Akt and MEK-ERK signaling cascades. [ABSTRACT FROM AUTHOR]

In cardiac myocytes of new-born rats, the degree of intercellular communication through gap junctional channels closely depends on the metabolic state of the cells. In contrast, in stably transfected HeLa cells expressing rat cardiac connexin43 (Cx43, the main channel-forming protein present in ventricular myocytes), a major part of junctional communication persisted in ATP-depleted conditions, in the presence of a metabolic inhibitor (KCN) or of a broad spectrum inhibitor of protein kinases (H7). However, another metabolic inhibitor, antimycin A, which like cyanide inhibits electron transfer in the respiratory chain, totally interrupted cell-to-cell communication between Cx43-HeLa cells, even in whole-cell conditions, when ATP (5 mM) was present. Antimycin A caused a modest increase in cytosolic calcium concentration; however, junctional uncoupling still occurred when this rise was prevented. Conditions of ischemic insult (e.g. ischemia or chemical hypoxia) frequently cause the activation of protein kinases, particularly of Src and MAP kinases, and such activations are known to markedly disrupt gap junctional communication. Antimycin-induced junctional uncoupling occurred even in the presence of inhibitors of these kinases. Antimycin A appears able to cause junctional uncoupling either through the ATP depletion it induces as a metabolic poison or via a direct action on gap junction constituents. [ABSTRACT FROM AUTHOR]

Abstract: β1-Integrin mediates cardiomyocyte growth and survival and its proper regulation is essential for the structural and functional integrity of the heart. β1-Integrin expression is enhanced in hypertrophy, but the mechanism and significance of its up-regulation are unknown. Because reactive oxygen species (ROS) are important mediators of myocardial remodeling we examined their role in regulated β1-integrin expression. Hypertrophy was induced in neonatal cardiomyocytes by endothelin-1 (ET-1), which activated the regulatory NADPH oxidase subunit Rac1, evoked ROS, and enhanced fetal gene expression and cardiomyocyte size. ET-1 also enhanced cell adhesion and FAK phosphorylation and inhibited oxidative stress-induced cardiomyocyte apoptosis. Further, ET-1 increased β1-integrin mRNA and protein expression via Rac1–ROS-dependent MEK/ERK and EGF receptor–PI3K/Akt activation as shown by adenoviral dominant-negative Rac1 or overexpression of copper/zinc-superoxide dismutase. The relevance of regulated β1-integrin expression was examined in cardiomyocytes, in which targeting siRNA impeded the ET-1-induced β1-integrin up-regulation. In these cells, ET-1-induced cell adhesion, FAK phosphorylation, and hypertrophic response were significantly blunted, whereas its antiapoptotic effect was predominantly unchanged, suggesting at least partial dissociation of prohypertrophic and prosurvival signaling elicited by ET-1. In conclusion, β1-integrin up-regulation in response to ET-1 is mediated via Rac1–ROS-dependent activation of prohypertrophic pathways and is mandatory for ET-1-induced FAK activation, cell adhesion, and hypertrophic response. [Copyright &y& Elsevier]