Cardiomyocytes suppress contraction and O 2 consumption during hypoxia. Cytochrome oxidase undergoes a decrease in V max during hypoxia, which could alter mitochondrial redox and increase generation of reactive oxygen species (ROS). We therefore tested whether ROS generated by mitochondria act as second messengers in the signaling pathway linking the detection of O 2 with the functional response. Contracting cardiomyocytes were superfused under controlled O 2 conditions while fluorescence imaging of 2,7-dichlorofluorescein (DCF) was used to assess ROS generation. Compared with normoxia (PO 2 ϳ 107 torr, 15% O 2 ), graded increases in DCF fluorescence were seen during hypoxia, with responses at PO 2 ‫؍‬ 7 torr > 20 torr > 35 torr. The antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline attenuated these increases and abolished the inhibition of contraction. Superfusion of normoxic cells with H 2 O 2 (25 M) for >60 min mimicked the effects of hypoxia by eliciting decreases in contraction that were reversible after washout of H 2 O 2 . To test the role of cytochrome oxidase, sodium azide (0.75-2 M) was added during normoxia to reduce the V max of the enzyme. Azide produced graded increases in ROS signaling, accompanied by graded decreases in contraction that were reversible. These results demonstrate that mitochondria respond to graded hypoxia by increasing the generation of ROS and suggest that cytochrome oxidase may contribute to this O 2 sensing.Alterations in oxygen tension (PO 2 ) elicit a variety of functional responses in different cell types, including gene expression, altered metabolic function, altered ion channel activation, and release of neurotransmitters (1). In spontaneously contracting embryonic cardiomyocytes, we previously found significant decreases in contractile activity during prolonged moderate hypoxia (PO 2 ϭ 20 torr for Ͼ2 h) (2). This inhibition was not associated with a depletion of ATP or phosphocreatine stores, and was reversible when normoxic conditions were restored. Similar findings of decreased contractile function during hypoxia (48 h at 1% O 2 ) have also been seen in rat cardiac myocytes (3), which suggests that this response is not unique to embryonic cells. An ability to respond to changes in oxygen tension within the physiological range implies the existence of a cellular O 2 sensor linked to a signal transduction pathway. When activated by hypoxia, the sensor presumably would initiate a signaling cascade which ultimately leads to the functional response (e.g. diminished contractile activity). However, the O 2 sensing mechanism and the subsequent signal transduction pathways involved in the cardiomyocyte responses to hypoxia are not known.A number of different potential mechanisms of cellular O 2 sensing have been identified (1). Mitochondria are responsible for most of the O 2 consumption by the cell and would seem to be well suited because their local PO 2 responds to changes in the ratio of O 2 supply to demand. However, the low apparent K m of cytochrome oxidas...

The p38 mitogen-activated protein kinase (MAPK) is phosphorylated in response to oxidative stress. Mitochondria in cardiomyocytes increase their generation of reactive oxygen species (ROS) during hypoxia (1-5% O2). These ROS participate in signal transduction pathways involved in adaptive responses, including ischemic preconditioning and gene transcription. The present study therefore tested the hypothesis that hypoxia induces p38 MAPK phosphorylation by augmenting mitochondrial ROS generation. In cardiomyocytes, phosphorylation of p38 was observed in a PO2-dependent manner during hypoxia. This response was inhibited by rotenone, thenoyltrifluoroacetone, and myxothiazol, inhibitors of mitochondrial complexes I, II, and III, respectively. A similar inhibition was observed in the cells pretreated with anion channel inhibitor DIDS, which may block ROS release from mitochondria. During normoxia, increases in mitochondrial ROS elicited by azide (1-2 mM) or by the mitochondrial inhibitor antimycin A caused increased phosphorylation of p38. Brief treatment with exogenous H2O2 during normoxia also induced phosphorylation of p38 as hypoxia, but this effect was not abolished by myxothiazol or DIDS. The antioxidant N-acetyl-cysteine abolished the p38 response to hypoxia, presumably by scavenging H2O2, but the mitogen extracellular receptor kinase inhibitor PD-98059 did not inhibit p38 phosphorylation during hypoxia. Thus physiological hypoxia leads to p38 phosphorylation through a mechanism that requires electron flux in the proximal region of the mitochondrial electron transport chain, which suggests that either H2O2 or superoxide participates in activating that process. hydrogen peroxide; superoxide; respiration; protein kinases; oxidant stress MITOGEN-ACTIVATED PROTEIN KINASES (MAPK) comprise a superfamily of serine/threonine protein kinases that are critical for various cellular functions in different cell types. MAPK themselves are activated by phosphorylation on threonine and tyrosine residues in a Thr-X-Tyr motif residing in the activation loop proximal to the ATP-and substrate-binding sites of the protein (20). This phosphorylation is carried out by a dual-specificity MAPK kinase, which itself is activated by a phosphorylation event in response to an either intracellular or extracellular stimulus (13, 24). MAPK can be categorized further into three subfamilies based on the size of the activation loop and the identity of the amino acid present between the Thr and Tyr in the activation motif. One of these is p38 MAPK, which contains a Gly in the motif (12,16,25). Activation of p38 MAPK occurs in response to ultraviolet light, increased extracellular osmolarity, proinflammatory cytokines, and chemical stress (10,17,23).Oxidative signaling has been implicated in a variety of experimental interventions that lead to the initiation of gene transcription or other adaptive responses (19,26,28). Several groups have shown that p38 MAPK is activated by reactive oxygen species (ROS) generated intracellularly, as well as by hydr...

Recent work on the PDZ-LIM protein family has revealed that it has important activities at the cellular level, mediating signals between the nucleus and the cytoskeleton, with significant impact on organ development. We review and integrate current knowledge about the PDZ-LIM protein family and propose a new functional role, sequestering nuclear factors in the cytoplasm. Characterized by their PDZ and LIM domains, the PDZ-LIM family is comprised of evolutionarily conserved proteins found throughout the animal kingdom, from worms to humans. Combining two functional domains in one protein, PDZ-LIM proteins have wide-ranging and multi-compartmental cell functions during development and homeostasis. In contrast, misregulation can lead to cancer formation and progression. New emerging roles include interactions with integrins, T-box transcription factors, and receptor tyrosine kinases. Facilitating the assembly of protein complexes, PDZ-LIM proteins can act as signal modulators, influence actin dynamics, regulate cell architecture, and control gene transcription.Keywords: actin cytoskeleton; organogenesis; PDZ-LIM; scaffold; T-box Evolutionarily conserved building blocks for a heterogeneous protein familyThe PDZ-LIM family is comprised of proteins with multiple functional domains, and all share a PDZ domain combined with at least one LIM domain. As protein-protein interaction modules, PDZ and LIM domains act as scaffolds, binding to filamentous actin-associated proteins, a range of cytoplasmic signaling molecules, and nuclear proteins, allowing this family to carry out diverse functions during development and adulthood.( Figure 1 shows the architecture of the PDZ-LIM protein subfamilies and their phylogenetic relationship. While both the PDZ and the LIM domains are often found in combination with other peptide motifs or domains, the coevolution of the two invariant functional domains, PDZ and LIM, indicate high functional relevance and, thus, is of particular interest and the focus of this review. All family members associate with the actin cytoskeleton, and this property, organizing protein complexes at the cytoskeleton, may serve a range of unexpected, yet important biological roles. PDZ domainFound in bacteria, yeast, and throughout the animal kingdom, the PDZ domain is one of the most common protein-protein binding domains. (17,19) It is characterized by a highly conserved sequence of 80-90 amino acids consisting of

The limb- and heart-specific Tbx5 transcription factor coexpresses with and directly binds to the novel PDZ-LIM domain protein, LMP4. LMP4 is distributed in the cytoplasm associated with the actin cytoskeleton. In the presence of LMP4, Tbx5 shuttles dynamically between the nucleus and cytoplasm and, in a complex with LMP4, localizes to actin filaments. Nuclear and cytoplasmic Tbx5 distribution in developing chicken wings suggests the functional significance of the LMP4–Tbx5 interaction. In primary epicardial cells, we demonstrate that Tbx5 protein subcellular relocalization can be stimulated by external signals that induce cell differentiation. To test whether the relocalization from nuclear to cytoplasmic sites interferes with downstream gene expression, we used limb-specific Fgf10 and heart-specific Anf promoter-luciferase reporters and demonstrate that LMP4 acts as a repressor of Tbx5 activity. These studies reveal a previously unknown mechanism for Tbx transcription factor regulation in vertebrate limb and heart development and provide a better understanding of the molecular basis of hand/heart birth defects associated with Tbx5 mutations.

During cardiac development, the T-box transcription factor Tbx5 displays dynamic changes in localization from strictly nuclear to both nuclear and cytoplasmic to exclusively cytoplasmic along the actin cytoskeleton in cells coexpressing its binding protein LMP4. Although nuclear localization signals (NLSs) have been described, the mechanism by which Tbx5 exits the nucleus remained elusive. Here, we describe for Tbx5 a nuclear export signal (NES) that is recognized by the CRM1 export protein. Site-directed mutagenesis of a critical amino acid(s) within this sequence determined the functionality of this NES. Confocal localization studies and luciferase transcriptional reporter assays with NES mutant Tbx5 forms demonstrated retention in the nucleus, regardless of the presence of LMP4. Coimmunoprecipitation and pharmacological interference studies demonstrated a direct interaction between Tbx5 and CRM1, revealing that Tbx5 is using the CRM1 pathway for nuclear export. In addition to Tbx5, we identified NESs in all T-box proteins and demonstrated interaction of the family members Tbx3 and Brachyury with the CRM1 exporter, suggesting general significance. This first demonstration of evolutionarily conserved NESs in all T-box proteins in conjunction with NLSs indicates a primordial function of T-box proteins to dynamically shuttle between nuclear and cytoplasmic compartments of the cell.