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Vascular smooth muscle cell (VSMC) phenotype switching from a contractile state to a synthetic phenotype has been implicated in intimal remodeling during vascular injury. While multiple studies have focused on dedifferentiation of VSMCs, prevention of VSMC-mediated excessive repair remains poorly understood. In this issue of the JCI, Zeng et al. identified a mechanism by which platelet-derived microRNA-223 (miRNA-223) reverses VSMC dedifferentiation. The authors show that suppression of proliferation occurs after platelet internalization by VSMCs. Moreover, they demonstrate that miRNA-223 inhibits dedifferentiation and intimal hyperplasia in diabetic mice by decreasing PDGFR[beta] expression in VSMCs. Together, these results identify platelet-derived miRNA-223 as a potential therapeutic target in vascular injury., Platelets and vascular injury It is well established that following vascular injury, platelet surface receptors bind to the exposed subendothelial extracellular matrix, which leads to platelet activation and the release [...]

Members of the formin family are important for actin filament nucleation and elongation. We have identified a novel striated muscle--specific splice variant of the formin FHOD3 that introduces a casein kinase 2 (CK2) phosphorylation site. The specific targeting of muscle FHOD3 to the myofibrils in cardiomyocytes is abolished in phosphomutants or by the inhibition of CK2. Phosphorylation of muscle FHOD3 also prevents its interaction with p62/sequestosome 1 and its recruitment to autophagosomes. Furthermore, we show that muscle FHOD3 efficiently promotes the polymerization of actin filaments in cardiomyocytes and that the down-regulation of its expression severely affects myofibril integrity. In murine and human cardiomyopathy, we observe reduced FHOD3 expression with a concomitant isoform switch and change of subcellular targeting. Collectively, our data suggest that a muscle-specific isoform of FHOD3 is required for the maintenance of the contractile structures in heart muscle and that its function is regulated by posttranslational modification. doi/ 10.1083/jcb.201005060

Regulator of G protein signaling 4 (RGS4) regulates the strength and duration of G protein signaling and plays an important role in smooth muscle contraction, cardiac development, and psychiatric disorders. Little is known about the posttranscriptional regulation of RGS4 expression. We cloned the full-length cDNA of rabbit RGS4, which contains a long 3'-untranslated region (UTR) with several AU-rich elements (AREs). We determined whether RGS4 mRNA stability is mediated by the RNA-binding protein human antigen R (HuR) and contributes to IL-1[beta]-induced upregulation of RGS4 expression. We show that IL-1[beta] treatment in colonic smooth muscle cells doubled the half-life of RGS4 mRNA. Addition of RGS4 3'-UTR to the downstream of Renilla luciferase reporter induced dramatic reduction in the enzyme activity and mRNA expression of luciferase, which was attenuated by the site-directed mutation of the two 3'-most ARE sites. IL-1[beta] increased luciferase mRNA stability in a UTR-dependent manner. Knockdown of HuR significantly aggravated UTR-mediated instability of luciferase and inhibited IL-1[beta]-induced upregulation of RGS4 mRNA. In addition, IL-1[beta] increased cytosolic translocation and RGS4 mRNA binding of HuR. These findings suggest that 3'-most ARE sites within RGS4 3'-UTR are essential for the instability of RGS4 mRNA and IL-1[beta] promotes the stability of RGS4 mRNA through HuR. regulators of G protein signaling; Interleukin; AU-rich element doi: 10.1152/ajpcell.00093.2010.

Recent advances have identified an important role of bone morphogenetic protein 4 (BMP4) in pulmonary vascular remodeling, yet the underlying mechanisms remain largely unexplored. We have previously found that [Ca.sup.2+] influx through store-operated calcium channels (SOCC), which are mainly thought to be composed of canonical transient receptor potential (TRPC) proteins, likely contribute to the pathogenic development of chronic hypoxic pulmonary hypertension. In this study, we investigated the effect of BMP4 on expression of TRPC and store-operated [Ca.sup.2+] entry (SOCE) in pulmonary arterial smooth muscle cells (PASMCs). Real-time quantitative PCR and Western blotting revealed that treatment with BMP4 (50 ng/ml, 60 h) increased TRPC1, TRPC4, and TRPC6 mRNA and protein expression in growth-arrested rat distal PASMCs. Moreover, in comparison to vehicle control, cells treated with BMP4 also exhibited enhanced SOCE, and elevated basal intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) as determined by fluorescent microscopy using the [Ca.sup.2+] indicator Fura-2 AM. Perfusing cells with [Ca.sup.2+]-free Krebs-Ringer bicarbonate solution (KRBS) or KRBS containing SOCC antagonists SKF-96365 or Ni[Cl.sub.2] attenuated the increases in basal [[[Ca.sup.2+]].sub.i] caused by BMP4. Specific knockdown of BMP4 by small interference RNA significantly decreased the mRNA and protein expression of TRPC1, TRPC4, and TRPC6 and reduced SOCE and basal [[[Ca.sup.2+]].sub.i] in serum-stimulated PASMCs. We conclude that BMP4 regulates calcium signaling in PASMCs likely via upregulation of TRPC expression, leading to enhanced SOCE and basal [[[Ca.sup.2+]].sub.i] in PASMCs, and by this mechanism contributes to pulmonary vascular remodeling during pulmonary arterial hypertension. calcium signaling; intracellular calcium concentration doi: 10.1152/ajpcell.00040.2010.

Recent studies in Drosophila have implicated actin cytoskeletal remodeling in myoblast fusion, but the cellular mechanisms underlying this process remain poorly understood. Here we show that actin polymerization occurs in an asymmetric and cell type--specific manner between a muscle founder cell and a fusion-competent myoblast (FCM). In the FCM, a dense F-actin--enriched focus forms at the site of fusion, whereas a thin sheath of F-actin is induced along the apposing founder cell membrane. The FCM-specific actin focus invades the apposing founder cell with multiple finger-like protrusions, leading to the formation of a single-channel macro fusion pore between the two muscle cells. Two actin nucleation-promoting factors of the Arp2/3 complex, WASP and Scar, are required for the formation of the F-actin foci, whereas WASP but not Scar promotes efficient foci invasion. Our studies uncover a novel invasive podosome-like structure (PLS) in a developing tissue and reveal a previously unrecognized function of PLSs in facilitating cell membrane juxtaposition and fusion. doi/ 10.1083/jcb.201006006

Recent animal studies have reported that exercise pressor reflex (EPR)-mediated increases in blood pressure are exaggerated in hypertensive (HTN) rodents. Whether these findings can be extended to human hypertension remains unclear. Mean arterial pressure (MAP), muscle sympathetic nerve activity (MSNA), and venous metabolites were measured in normotensive (NTN; n = 23; 60 [+ or -] 1 yr) and HTN 01 = 15; 63 [+ or -] 1 yr) subjects at baseline, and during static handgrip at 30 and 40% maximal voluntary contraction (MVC) followed by a period of post-exercise ischemia (PEI) to isolate the metabolic component of the EPR. Changes in MAP from baseline were augmented in HTN subjects during both 30 and 40% MVC handgrip (P < 0.05 for both), and these group differences were maintained during PEI (30% PEI trial: [DELTA]15 [+ or -] 2 NTN vs. [DELTA]19 [+ or -] 2 HTN mmHg; 40% PEI trial: [DELTA]16 [+ or -] 1 NTN vs. [DELTA]23 [+ or -] 2 HTN mmHg; P < 0.05 for both). Similarly, in HTN subjects, MSNA burst frequency was greater during 30 and 40% MVC handgrip (P < 0.05 for both), and these differences were maintained during PEI [30% PEI trial: 35 [+ or -] 2 (NTN) vs. 44 [+ or -] 2 (HTN) bursts/min; 40% PEI trial: 36 [+ or -] 2 (NTN) vs. 48 [+ or -] 2 (HTN) bursts/min; P < 0.05 for both]. No group differences in metabolites were observed. MAP and MSNA responses to a cold pressor test were not different between groups, suggesting no group differences in generalized sympathetic responsiveness. In summary, compared with NTN subjects, HTN adults exhibit exaggerated sympathetic and pressor responses to handgrip exercise that are maintained during PEI, indicating that activation of the metabolic component of the EPR is augmented in older HTN humans. blood pressure regulation; exercise pressor reflex; muscle sympathetic nerve activity; static exercise; hypertension doi: 10.1152/aioheart.00556.2010.

Skeletal muscle satellite cells are adult stem cells responsible for postnatal skeletal muscle growth and regeneration. Paired-box transcription factor Pax7 plays a central role in satellite cell survival, self-renewal, and proliferation. However, how Pax7 is regulated during the transition from proliferating satellite cells to differentiating myogenic progenitor cells is largely unknown. In this study, we find that miR-1 and miR-206 are sharply upregulated during satellite cell differentiation and downregulated after muscle injury. We show that miR-1 and miR-206 facilitate satellite cell differentiation by restricting their proliferative potential. We identify Pax7 as one of the direct regulatory targets of miR-1 and miR-206. Inhibition of miR-1 and miR-206 substantially enhances satellite cell proliferation and increases Pax7 protein level in vivo. Conversely, sustained Pax7 expression as a result of the loss of miR-1 and miR-206 repression elements at its 3' untranslated region significantly inhibits myoblast differentiation. Therefore, our experiments suggest that microRNAs participate in a regulatory circuit that allows rapid gene program transitions from proliferation to differentiation. doi/ 10.1083/jcb.200911036

Stem ceils that naturally reside in adult tissues, such as muscle stem ceils (MuSCs), exhibit robust regenerative capacity in vivo that is rapidly lost in culture. Using a bioengineered substrate to recapitulate key biophysical and biochemical niche features in conjunction with a highly automated single-cell tracking algorithm, we show that substrate elasticity is a potent regulator of MuSC fate in culture. Unlike MuSCs on rigid plastic dishes ([~10.sup.6] kilopascals), MuSCs cultured on soft hydrogel substrates that mimic the elasticity of muscle (12 kilopascals) self-renew in vitro and contribute extensively to muscle regeneration when subsequently transplanted into mice and assayed histologically and quantitatively by noninvasive bioluminescence imaging. Our studies provide novel evidence that by recapitulating physiological tissue rigidity, propagation of adult muscle stem cells is possible, enabling future cell-based therapies for muscle-wasting diseases. 10.1126/science.1191035

Endothelin 1 (ET-1) is an endogenous peptide that promotes vasoconstriction, endothelial and smooth muscle cell (SMC) proliferation, and fibrosis. ET-1 receptor antagonists are an important treatment strategy for pulmonary arterial hypertension, but less effective in systemic vascular disease. This observation suggests a special role for ET-1 in the pulmonary circulation. We hypothesized that ET-1 contributes to the pathogenesis of pulmonary arterial hypertension, in part, by promoting pulmonary vascular SMC migration. ET-1 treatment promoted migration in 3 distinct types of cultured pulmonary SMC. Pulmonary SMC migration was blocked by an [ET.sub.A] receptor selective agonist and a combined [ET.sub.A]-[ET.sub.B] antagonist, but not by a selective [ET.sub.B] antagonist. In contrast to the effect on pulmonary SMCs, ET-1 had no effect on migration of aortic SMCs. Flow cytometry showed that the ETA receptor was expressed at comparable levels on pulmonary and aortic SMCs, excluding receptor density as an explanation for the divergent effect. ET-1-induced pulmonary SMC migration was blocked by the structurally distinct MEK inhibitors PD98059 and U0126, consistent with a role for ERK1/2 MAP kinase. By Western blot in cultured cells and immunohistochemistry in ex vivo vessels, ET-1 stimulated phosphorylation of ERK1/2 as efficaciously as platelet-derived growth factor in pulmonary, but not aortic, SMCs. In conclusion, ET-1 induces SMC migration, with the [ET.sub.A] receptor tightly coupled to ERK1/2 phosphorylation only in the pulmonary circulation. This finding may help explain the striking difference in the efficacy of endothelin receptor blockers for pulmonary hypertension as compared to that for systemic cardiovascular disease. Key words: endothelin, migration, vascular smooth muscle cell, MAP kinase. L'endotheline (ET-1) est un peptide endogene qui favorise la vasoconstriction, la proliferation des cellules musculaires lisses et endotheliales, et la fibrose. L'emploi d'antagonistes des recepteurs de l'ET-1 constitue une strategie importante en matiere de traitement de l'hypertension arteerielle pulmonaire, mais d'avere moins efficace dans le traitement des affections vasculaires systemiques. Cette observation autorise a penser que l'ET-1 joue un role particulier dans la circulation pulmonaire. Nous avons emis l'hypothese que l'ET-1 contribue a la pathogenese que l'hypertension arterielle pulmonaire en favorisant la migration des cellules musculaires lisses (CML) vasculaires pulmonaires. Un traitement au moyen de l'ET-1 a facilite; la migration dans trois types distincts de CML pulmonaires en culture. La migration des CML pulmonaires a ete bloqueie par un agoniste selectif du recepteur [ET.sub.A] et un antagoniste des recepteurs [ET.sub.A]-[ET.sub.B] combines, mais pas par un antagoniste selectif du recepteur [ET.sub.B]. En revanche, l'ET- 1 n'a pas eu d'effet sur la migration des CML aortiques. La cytome;trie de flux a montre que le taux d'expression du recepteur ETA aeete comparable sur les CML pulmonaires et aortiques, ce qui exclut la densite des recepteurs comme explication de l'effet divergent. La migration des CML pulmonaires induite par l'ET-1 a etee bloquee par les inhibiteurs structurellement distincts de MEK, PD98059 et U0126, ce qui est compatible avec un role pour la ERK1/2 MAP kinase. Le transfert de western dans les cellules cultiveees et l'immunohistochimie dans les vaisseaux ex vivo ont indique que l'ET-1 a stimule la phosphorylation d'ERK1/2 aussi efficacement que facteur de croissance derive des plaquettes dans les CML pulmonaires, mais pas dans les CML aortiques. En conclusion, la migration des CML induite par l'ET-1 et le recepteur [ET.sub.A] est eetroitement coupleea la phosphorylation de ERK1/2 uniquement dans la circulation pulmonaire. Ce resultat pourrait expliquer la difference marqueee dans l'efficacite des bloqueurs des recepteurs de l'endotheline en ce qui a trait au traitement de l'hypertension pulmonaire et des affections cardiovasculaires systemiques. Mots-cles: endotheeline, migration, muscle lisse vasculaire, MAP kinase. [Traduit par la Redaction], Introduction The endothelin signaling system consists of 4 peptides (endothelin-1 to -4) and 2 G-protein coupled receptors ([ET.sub.A] and [ET.sub.B]). The most prominent of the endothelins in the cardiovascular system [...]

Skeletal muscle atrophy and weakness are major contributors to frailty and impact significantly on quality of life of older people. Muscle aging is characterized by a loss of maximum tetanic force ([P.sub.o]) generation, primarily due to muscle atrophy, to which mitochondrial dysfunction is hypothesized to contribute. We hypothesized that lifelong overexpression of the mitochondrial heat shock protein (HSP) HSP10 in muscle of mice would protect against development of these deficits. [P.sub.o] generation by extensor digitorum longus muscles of adult and old wild-type and HSP10-overexpressing mice was determined in situ. Muscles were subjected to damaging lengthening contractions, and force generation was remeasured at 3 h or 28 days to examine susceptibility to, and recovery from, damage, respectively. Muscles of old wild-type mice had a 23% deficit in [P.sub.o] generation and a 10% deficit in muscle cross-sectional area compared with muscles of adult wild-type mice. Overexpression of HSP10 prevented this age-related fall in [P.sub.o] generation and reduction in cross-sectional area observed in muscles of old wild-type mice. Additionally, overexpression of HSP10 protected against contraction-induced damage independent of age but did not improve recovery if damage occurred. Preservation of muscle force generation and CSA by HSP10 overexpression was associated with protection against the age-related accumulation of protein carbonyls. Data demonstrate that development of age-related muscle weakness may not be inevitable and show, for the first time, that lifelong overexpression of an HSP prevents the age-related loss of [P.sub.o] generation. These findings support the hypothesis that mitochondrial dysfunction is involved in the development of age-related muscle deficits. aging; heat shock protein; Cpnl0; Hspel; atrophy doi: 10.1152/ajpregu.00334.2009.

We have sought evidence that arachidonic acid (AA) induces mitochondrial depolarization in isolated myocytes by a lipoxygenase (LOX)-dependent mechanism and that such depolarization might contribute to arrhythmogenesis following ischemia-reperfusion injury. A method was developed for measuring mitochondrial depolarization in isolated adult rat myocytes in suspension, using tetramethylrhodamine ethyl ester. The addition of AA to myocytes resulted in mitochondrial depolarization that was inhibited by the LOX inhibitor baicalein, by the reactive oxygen species (ROS) scavenger mercaptoproprionylglycine, and by the anion channel inhibitor diisothiocyanatostilbenedisulfonic acid (DIDS). AA induced mitochondrial uncoupling and mitochondrial ATPase activity in myocytes, but both were insensitive to baicalein. We conclude that the metabolic effect of AA in myocytes puts mitochondria into an energetically compromised state where membrane potential is easily changed by the DIDS-sensitive LOX/ ROS-mediated opening of an inner membrane anion channel. In an in vivo anesthetized rat model of coronary artery occlusion, baicalein was found to strongly inhibit arrhythmias induced by ischemia-reperfusion injury. Arrhythmias following ischemia-reperfusion injury have been previously associated with DIDS-sensitive ROS-mediated mitochondrial depolarization, and free fatty acids including AA were previously found to accumulate during such injury. We therefore conclude that arrhythmias following ischemia-reperfusion injury might originate from mitochondrial depolarization mediated by LOX and AA. mitochondria; ischemia; reperfusion; reactive oxygen species; anion channels doi: 10.1152/ajpheart.00906.2009.

Muscle contraction stimulates glucose uptake acutely to increase energy supply, but suitable cellular models that faithfully reproduce this complex phenomenon are lacking. To this end, we have developed a cellular model of contracting [C.sub.2][C.sub.12] myotubes overexpressing GLUT4 with an exofacial mycepitope tag (GLUT4myc) and explored stimulation of GLUT4 traffic by physiologically relevant agents. Carbachol (an acetylcholine receptor agonist) induced a gain in cell surface GLUT4myc that was mediated by nicotinic acetylcholine receptors. Carbachol also activated AMPK, and this response was sensitive to the contractile myosin ATPase inhibitor N-benzyl-p-toluenesulfonamide. The gain in surface GLUT4myc elicited by carbachol or by the AMPK activator 5-amino-4-carboxamide-1 [beta]-ribose was sensitive to chemical inhibition of AMPK activity by compound C and partially reduced by siRNA-mediated knockdown of AMPK catalytic subunits or LKB1. In addition, the carbachol-induced gain in cell surface GLUT4myc was partially sensitive to chelation of intracellular calcium with BAPTA-AM. However, the carbachol-induced gain in cell surface GLUT4myc was not sensitive to the CaMKK inhibitor STO-609 despite expression of both isoforms of this enzyme and a rise in cytosolic calcium by carbachol. Therefore, separate AMPK- and calcium-dependent signals contribute to mobilizing GLUT4 in response to carbachol, providing an in vitro cell model that recapitulates the two major signals whereby acute contraction regulates glucose uptake in skeletal muscle. This system will be ideal to further analyze the underlying molecular events of contraction-regulated GLUT4 traffic. carbachol; glucose transporter 4; adenosine 5'-monophosphate-activated protein kinase; acetylcholine doi:10.1152/ajpendo.00773.2009.

One proposed explanation for the V[O.sub.2] slow component is that lower-threshold motor units may fatigue and develop little or no tension but continue to use [O.sub.2], thereby resulting in a dissociation of cellular respiration from force generation. The present study used intact isolated single myocytes with differing fatigue resistance profiles to investigate the relationship between fatigue, tension development, and aerobic metabolism. Single Xenopus skeletal muscle myofibers were allocated to a fast-fatiguing (FF) or a slow-fatiguing (SF) group, based on the contraction frequency required to elicit a fall in tension to 60% of peak. Phosphorescence quenching of a porphyrin compound was used to determine [DELTA] intracellular [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]; a proxy for V[O.sub.2]), and developed isometric tension was monitored to allow calculation of the time-integrated tension (TxT). Although peak [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]: was not different between groups (P = 0.36), peak tension was lower (P < 0.05) in SF vs. FF (1.97 [+ or -] 0. 17 V vs. 2.73 [+ or -] 0.30 V, respectively) and time to 60% of peak tension was significantly longer in SF vs. FF (242 [+ or -] 10 s vs. 203 [+ or -] l0 s, respectively). Before fatigue, both[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and TxT rose proportionally with contraction frequency in SF and FF, resulting in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]/TxT being identical between groups. At fatigue, TxT fell dramatically in both groups, but [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] decreased proportionately only in the FF group, resulting in an increase in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]/TxT in the SF group relative to the prefatigue condition. These data show that more fatigue-resistant fibers maintain aerobic metabolism as they fatigue, resulting in an increased [O.sub.2] cost of contractions that could contribute to the Vo2 slow component seen in whole body exercise. oxidative phosphorylation; skeletal muscle; fiber type; oxygen uptake doi: 10.1152/ajpregu.00715.2009.

Nitric oxide signaling has an important role in regulating pulmonary development and function. Expression of soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase I (PKGI), both critical mediators of nitric oxide (NO) signaling, is diminished in the injured newborn lung through unknown mechanisms. Recent studies suggest that excessive transforming growth factor-[beta] (TGF-[beta]) activity inhibits injured newborn lung development. To explore mechanisms that regulate pulmonary NO signaling, we tested whether TGF-[beta] decreases sGC and PKGI expression in the injured developing lung and pulmonary vascular smooth muscle cells (SMC). We found that chronic oxygen-induced lung injury decreased pulmonary sGC[[alpha].sub.1] and PKGI immunoreactivity in mouse pups and that exposure to a TGF-[beta]-neutralizing antibody prevented this reduction of sGC and PKGI protein expression. In addition, TGF-[[beta].sub.1] decreased expression of NO signaling enzymes in freshly isolated pulmonary microvascular SMC/ myofibroblasts, suggesting that TGF-[beta] has a direct role in modulating NO signaling in the pup lung. Moreover, TGF-[[beta].sub.1] decreased sGC and PKGI expression in pulmonary artery and aortic SMC from adult rats and mice, suggesting a general role for TGF-[beta] in modulating NO signaling in vascular SMC. Although other cytokines decrease sGC mRNA stability, TGF-[beta] did not modulate sGC[[alpha].sub.1] or PKGI[beta] mRNA turnover in vascular SMC. These studies indicate for the first time that TGF-[beta] decreases NO signaling enzyme expression in the injured developing lung and pulmonary vascular SMC. Moreover, they suggest that TGF-[beta]-neutralizing molecules might counteract the effects of injury on NO signaling in the newborn lung. soluble guanylate cyclase; cGMP-dependent protein kinase I; bronchopulmonary dysplasia; hyperoxia doi:10.1152/ajplung.00181.2009.

Vasomotion consists of cyclic arterial diameter variations induced by synchronous contractions and relaxations of smooth muscle cells. However, the arteries do not contract simultaneously on macroscopic distances, and a propagation of the contraction can be observed. In the present study, our aim was to investigate this propagation. We stimulated endothelium-denuded rat mesenteric arterial strips with phenylephrine (PE) to obtain vasomotion and observed that the contraction waves are linked to intercellular calcium waves. A velocity of -100 [micro]/s was measured for the two kinds of waves. To investigate the calcium wave propagation mechanisms, we used a method allowing a PE stimulation of a small area of the strip. No calcium propagation could be induced by this local stimulation when the strip was in its resting state. However, if a low PE concentration was added on the whole strip, local PE stimulations induced calcium waves, spreading over finite distances. The calcium wave velocity induced by local stimulation was identical to the velocity observed during vasomotion. This suggests that the propagation mechanisms are similar in the two cases. Using inhibitors of gap junctions and of voltage-operated calcium channels, we showed that the locally induced calcium propagation likely depends on the propagation of the smooth muscle cell depolarization. Finally, we proposed a model of the propagation mechanisms underlying these intercellular calcium waves. conducted vasomotor response; smooth muscle cell; rat mesenteric artery doi: 10.1152/ajpheart.00281.2009

The mechanisms by which mitochondria regulate the sustained phase of agonist-induced responses in cytosolic [Ca.sup.2+] concentration as an independent organelle in whole is not clear. By exposing to ethidium bromide and supplying pyruvate and uridine, we established mitochondrial DNA (mtDNA)-depleted rat airway smooth muscle cells (RASMCs) with maintained cellular energy. Upon an exposure to 2 [mu]M histamine, [[[Ca.sup.2+].sub.i] in control RASMCs increased to a peak followed by a plateau above baseline, whereas [[[Ca.sup.2+]].sub.i] in mtDNA-depleted RASMCs jumped to a peak and then declined to baseline without any plateau, mtDNA depletion apparently attenuated intracellular reactive oxygen species generation induced by histamine. By coexposure to 2 [mu]M histamine and 0.1 [mu]M exogenous [H.sub.2][O.sub.2], which did not affect [[[Ca.sup.2+]].sub.i] by itself, the above difference in [[[Ca.sup.2+]].sub.i] kinetics in mtDNA-depleted RASMCs was reversed. Intracellular [H.sub.2][O.sub.2] decomposition abolishes histamine-induced sustained elevation in [[[Ca.sup.2+]].sub.i] in RASMCs. Thus, mitochondria regulate agonist-induced sustained [[[Ca.sup.2+]].sub.i] elevation by a [H.sub.2][O.sub.2]-dependent mechanism. histamine; calcium; reactive oxygen species doi: 10.1152/ajplung.00134.2009

Am J Physiol Gastrointest Liver Physiol 298: G10-G13, 2010. First published November 5, 2009; doi: 10.1152/ajpgi.00426.2009.--How nerves transmit their signals to regulate activity of smooth muscle is of fundamental importance to autonomic and enteric physiology, clinical medicine, and therapeutics. A traditional view of neurotransmission to smooth muscles has been that motor nerve varicosities release neurotransmitters that act on receptors on smooth muscles to cause their contraction or relaxation via electromechanical and pharmacomechanical signaling pathways in the smooth muscle. In recent years, an old hypothesis that certain interstitial cells of Cajal (ICC) may transduce neural signals to smooth muscle cells has been resurrected. This later hypothesis is based on indirect evidence of closer proximity and presence of synapses between the nerve varicosities and ICC, gap junctions between ICC and smooth muscles, and presence of receptors and signaling pathways for the neurotransmitters and ICC. This indirect evidence is at best circumstantial. The direct evidence is based on the reports of loss of neurotransmission in mutant animals lacking ICC due to c-Kit receptor deficiency. However, a critical analysis of the recent data show that animals lacking ICC have normal cholinergic and purinergic neurotransmission and tachykinergic neurotransmission is actually increased. The status of nitrergic neurotransmission in c-Kit deficient animals has been controversial. However, reports suggest that 1) nitrergic neurotransmission in the internal anal sphincter does not require ICC and 2) the in vivo phenotype of ICC deficiency does not resemble nNOS deficiency. 3) The most recent report, in this issue of the Journal, concludes that impaired nitrergic neurotransmission may be due to smooth muscle defects associated with c-Kit receptor deficiency. bile gastritis; gastroparesis; intestinal phenotype of Ws/Ws; nitrergic neurotransmission

Am J Physiol Gastrointest Liver Physiol 298: G14--G24, 2010. First published October 22, 2009; doi: 10.1152/ajpgi.00266.2009.--To address the controversy surrounding the role of interstitial cells of Cajal (ICC) in nitrergic neurotransmission to gastrointestinal smooth muscle, circular smooth muscle from the lower esophageal sphincter (LES) of W/[W.sup.v] wild-type and mutant (ICC-deficient) mice were studied by using intracellular and tension recordings in vitro. Resting membrane potential was more negative, and the spontaneous unitary potentials diminished in mutant mice. In wild-type mice, nerve stimulation induced a biphasic inhibitory junction potential (IJP) consisting of a fast initial IJP followed by a long-lasting slow IJP (LSIJP). The IJP was markedly impaired in a significant proportion of mutant mice, whereas in others it was normal. Pharmacological studies in the mice with markedly impaired LIPs revealed that cholinergic and purinergic components of the nerve-mediated responses appeared intact. In wild-type mice, caffeine hyperpolarized smooth muscle cells, inhibited the initial fast IJP, and completely abolished the LSIJP. In mutant mice, caffeine depolarized smooth muscle cells and abolished the impaired LSIJP but did not affect the initial fast IJP. Immunohistochemical staining for c-Kit confirmed deficiency of ICC in mutant mice with a normal nitrergic IJP. Rings of LES circular smooth muscle from W/[W.sup.v] mutant mice generated significantly less spontaneous tone than controls. When tone was restored with carbachol, normal nitrergic LES relaxation was recorded. These data suggest that 1) there is significant variability in the generation of nitrergic neurotransmission in the LES of W/[W.sup.v] mutant mice, whereas purinergic and cholinergic neurotransmission are intact; 2) the altered nitrergic responses appear to be associated with abnormal [Ca.sup.2+]-dependent signaling initiated by spontaneous [Ca.sup.2+] release from sarcoplasmic reticulum in smooth muscle cells; and 3) c-Kit-positive ICC are not essential for nitrergic neurotransmission in mouse LES smooth muscle. smooth muscle; [Ca.sup.2+]-activated [Cl.sup.-] channels; sarcoplasmic reticulum; interstitial cells of Cajal

Kato K, Yamanouchi D, Esbona K, Kamiya K, Zhang F, Kent KC, Liu B. Caspase-mediated protein kinase C-5 cleavage is necessary for apoptosis of vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 297: H2253-H2261, 2009. First published October 16, 2009; doi: 10.1152/ajpheart.00274.2009.--Apoptotic death of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling and various vascular diseases. We have previously shown that protein kinase C-8 (PKC-[delta]) plays a critical role in SMC apoptosis. In this study, we tested the importance of PKC-[delta] proteolytic cleavage and tyrosine phosphorylation within the apoptosis pathway. Using hydrogen peroxide as a paradigm for oxidative stress, we showed that proteolytic cleavage of PKC-[delta] occurred in SMCs that underwent apoptosis, while tyrosine phosphorylation was detected only in necrotic cells. Furthermore, using a peptide (z-DIPD-fmk) that mimics the caspase-3 binding motif within the linker region of PKC-[delta], we were able to prevent the cleavage of PKC-[delta], as well as apoptosis. Inhibition of PKC-[delta] with rottlerin or small-interfering RNA diminished caspase-3 cleavage, caspase-3 activity, cleavage of poly (AD-Pribose) polymerase, cleavage of PKC-[delta], and DNA fragmentation, confirming the previously reported role of PKC-[delta] in initiation of apoptosis. In contrast, z-DIPD-fmk markedly diminished caspase-3 activity, cleavage of PKC-[delta], and DNA fragmentation without affecting cleavage of caspase-3 and poly (ADP-ribose) polymerase. Taken together, our data suggest that caspase-3-mediated PKC-[delta] cleavage underlies SMC apoptosis induced by oxidative stress, and that PKC-[delta] acts both upstream and downstream of caspase-3. oxidative stress; remodeling; phosphorylation; necrosis doi: 10.1152/ajpheart.00274.2009.

Frid MG, Li M, Gnanasekharan M, Burke DL, Fragoso M, Strassheim D, Sylman JL, Stenmark KR. Sustained hypoxia leads to the emergence of cells with enhanced growth, migratory, and promitogenic potentials within the distal pulmonary artery wall. Am J Physiol Lung Cell Mol Physiol 297: L1059-L1072, 2009. First published September 18, 2009; doi: 10.1152/ajplung.90611.2008.--All forms of chronic pulmonary hypertension (PH) are characterized by structural remodeling of the pulmonary artery (PA) media, a process previously attributed solely to changes in the phenotype of resident smooth muscle cells (SMC). However, recent experimental evidence in both systemic and pulmonary circulations suggests that other cell types, including circulating and local progenitors, contribute significantly to this process. The goal of this study was to determine if hypoxia-induced remodeling of distal PA (dPA) media involves the emergence of cells with phenotypic and functional characteristics distinct from those of resident dPA SMC and fibroblasts. In vivo, in contrast to the phenotypically uniform SMC composition of dPA media in control calves, the remodeled dPA media of neonatal calves with severe hypoxia-induced PH comprised cells exhibiting a distinct phenotype, including the expression of hematopoetic (CD45), leukocytic/monocytic (CD11b, CD14), progenitor (cKit), and motility-associated (S100A4) cell markers. Consistent with these in vivo observations, primary cell cultures isolated from dPA media of hypertensive calves yielded not only differentiated SMC, but also smaller, morphologically rhomboidal (thus termed here 'R') cells that transiently expressed CD11b, constitutively expressed the mesenchymal cell marker type I procollagen, expressed high mRNA levels of progenitor cell markers cKit, CD34, CD73, as well as for inflammatory mediators, IL-6 and MCP-1, and, with time in culture, gained expression of a myofibroblast marker, [alpha]-SM-actin. R cells exhibited highly augmented proliferative, migratory, invasive, and potent promitogenic capabilities, which were due, at least in part, to the production of PDGFs, SDF-1/CXCL12, and S100A4. These data suggest that the cellular mechanisms of dPA remodeling include the emergence of cells with phenotypic and functional characteristics markedly distinct from those of resident dPA cells. pulmonary hypertension; vascular remodeling; progenitor cells; inflammation; S100A4 doi: 10.1152/ajplung.90611.2008

Singh J, Mehendiratta V, Del Galdo F, Jimenez SA, Cohen S, DiMarino AJ, Rattan S. Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 297: G1206-G1213, 2009. First published September 24, 2009; doi: 10.1152/ajpgi.00286.2009.--Systemic sclerosis (SSc) IgGs affecting the [M.sub.3]-muscarinic receptor ([M.sub.3]-R) have been proposed to be responsible for the gastrointestinal (GI) dysmotility in this disease. However, the effect of SSc IgGs on smooth muscle cell (SMC) function has not been studied. We determined the effect of SSc IgGs on the muscarinic receptor activation by bethanechol (BeCh; methyl derivate of carbachol) in SMC and smooth muscle strips from rat internal anal sphincter. IgGs were purified from GI-symptomatic SSc patients and normal volunteers, with protein G-Sepharose columns. SMC lengths were determined via computerized digital micrometry. The presence of [M.sub.3]-R and IgG-[M.sub.3]-R complex was determined by Western blot. IgGs from SSc patients but not from normal volunteers caused significant and concentration-dependent inhibition of BeCh response (P < 0.05). The maximal shortening of 22.2 [+ or -] 1.2% caused by [10.sup.-4] M BeCh was significantly attenuated to 8.3 [+ or -] 1.2% by 1 mg/ml of SSc IgGs (P < 0.05). Experiments performed in smooth muscle strips revealed a similar effect of SSc IgG that was fully reversible. In contrast to the effect on BeCh, the SSc IgGs caused no significant effect (P > 0.05) on [K.sup.+] depolarization and [[alpha].sub.1]- adrenoceptor activation by phenylephrine. Western blot studies revealed the specific presence of SSc IgG-[M.sub.3]-R complex. SSc IgGs attenuated [M.sub.3]-R activation, which was reversible with antibody removal. These data suggest that SSc GI dysmotility may be caused by autoantibodies that inhibit the muscarinic neurotransmission. Future treatment of SSc patients may be directed at the removal or neutralization of these antibodies. systemic sclerosis; rectoanal function; muscarinic receptor; autoantibodies doi: 10.1152/ajpgi.00286.2009

Flynn RS, Mahavadi S, Murthy KS, Kellum JM, Kuemmerle JF. Insulin-like growth factor-binding protein-5 stimulates growth of human intestinal muscle cells by activation of G[alpha]i3. Am J Physiol Gastrointest Liver Physiol 297: G1232-G1238, 2009. First published October 1, 2009; doi: 10.1152/ajpgi.00323.2009.--In human intestinal smooth muscle cells, endogenous insulin-like growth factor-I (IGF-I) regulates growth and IGF-binding protein-5 (IGFBP-5) expression. The effects of IGF-I are facilitated by 1GFBP-5. We previously showed that IGFBP-5 acts independently of IGF-I in human intestinal muscle to stimulate proliferation and upregulate IGF-I production by activation of Erkl/2 and p38 MAPK. Thus a positive feedback loop exists between IGF-I and IGFBP-5, whereby both stimulate muscle growth and production of the other factor. In Crohn's disease, IGF-I and IGFBP-5 expression are increased and contribute to stricture formation through this effect on muscle growth. To determine the signaling pathways coupling IGFBP-5 to MAPK activation and growth, smooth muscle cells were isolated from muscularis propria of human intestine and placed into primary culture. Erkl/2 and p38 MAPK activation and type I collagen production were measured by immunoblot. Proliferation was measured by [[sup.3]H]thymidine incorporation. Activation of specific G proteins was measured by ELISA. AG1024, an IGF-I receptor tyrosine kinase inhibitor, was used to isolate the IGF-I-independent effects of IGFBP-5. IGFBP-5-induced phosphorylation of Erkl/2 and p38 MAPK and proliferation were abolished by pertussis toxin, implying the participation of Gi. IGFBP-5 specifically activated Gi3 but not other G proteins. Transfection of an inhibitory G[alpha]i minigene specifically inhibited MAPK activation, proliferation, and both collagen-I and IGF-I production. Our results indicate that endogenous IGFBP-5 activates Gi3 and regulates smooth muscle growth, IGF-I production, and collagen production via the [alpha]-subunit of Gi3, independently of IGF-I, in normal human intestinal muscle cells. smooth muscle cell; heterotrimeric G protein; p38 MAPK; Erkl/2 doi: 10.1152/ajpgi.00323.2009

Sundaram P, Pang Z, Miao M, Yu L, Wing SS. USP19-deubiquitinating enzyme regulates levels of major myofibrillar proteins in L6 muscle cells. Am J Physiol Endocrinol Metab 297: E1283-E1290, 2009. First published September 22, 2009; doi: 10.1152/ajpendo.00409.2009.--The ubiquitin-proteasome system plays an important role in the degradation of myofibrillar proteins that occurs in muscle wasting. Many studies have demonstrated the importance of enzymes mediating conjugation of ubiquitin. However, little is known about the role of deubiquitinating enzymes. We previously showed that the USP19- deubiquitinating enzyme is induced in atrophying skeletal muscle (Combaret L, Adegoke OA, Bedard N, Baracos V, Attaix D, Wing SS. Am J Physiol Endocrinol Metab 288: E693-E700, 2005). To further explore the role of USP 19, we used small interfering RNA (siRNA) in L6 muscle cells. Lowering USP19 by 70-90% in myotubes resulted in a 20% decrease in the rate of proteolysis and an 18% decrease in the rate of protein synthesis, with no net change in protein content. Despite the decrease in overall synthesis, there were ~1.5-fold increases in protein levels of myosin heavy chain (MHC), actin, and troponin T and a ~2.5-fold increase in tropomyosin. USP19 depletion also increased MHC and tropomyosin mRNA levels, suggesting that this effect is due to increased transcription. Consistent with this, USP19 depletion increased myogenin protein and mRNA levels approximately twofold. Lowering myogenin using siRNA prevented the increase in MHC and tropomyosin upon USP19 depletion, indicating that myogenin mediated the increase in myofibrillar proteins. Dexamethasone treatment lowered MHC and increased USP19. Depletion of USP19 reversed the dexamethasone suppression of MHC. These studies demonstrate that USP19 modulates transcription of major myofibrillar proteins and indicate that the ubiquitin system not only mediates the increased protein breakdown but is also involved in the decreased protein synthesis in atrophying skeletal muscle. ubiquitin-specific processing protease doi: 10.1152/ajpendo.00409.2009.

Akerud H, Ronquist G, Wiberg-Itzel E. Lactate distribution in culture medium of human myometrial biopsies incubated under different conditions. Am J Physiol Endocrinol Metab 297: E1414-E1419, 2009. First published October 13, 2009; doi: 10.1152/ajpendo.00458.2009.--It is generally believed that a relationship exists between muscle fatigue and intracellular accumulation of lactate. This reasoning is relevant to obstetrical issues. Myocytes in uterus work together during labor, and the contractions need to be strong and synchronized for a child to be delivered. At labor dystocia, the progress of labor becomes slow or arrested after a normal beginning. It has been described that, during labor dystocia, when the force of the contractions is low, the uterus is under hypoxia, and anaerobic conditions with high levels of lactate in amniotic fluid dominate. The purpose of this study was to examine whether myometrial cells are involved in the production of lactate in amniotic fluid and whether there are differences in production and distribution of lactate in cells incubated under aerobic and anaerobic conditions. We also wanted to elucidate the involvement of specific membrane-bound lactate carriers. Women undergoing elective caesarean section were included. Myometrial biopsies from uteri were collected and subjected to either immunohistochemistry to identify lactate carriers or in vitro experiments to analyze production of lactate. The presence of lactate carriers named monocarboxylate transporters 1 and 4 was verified. Myometrial cells produced lactate extracellularly, and the lactate carriers operated differently under anaerobic and aerobic conditions; while being mainly unidirectional under anaerobic conditions, they became bidirectional under aerobic conditions. Human myometrial cells produced and delivered lactate to the extracellular medium under both anaerobic and aerobic conditions. The delivery was mediated by lactate carriers. monocarboxylate transporter 1; monocarboxylate transporter 4; myometrial cells; uterus doi: 10.1152/ajpendo.00458.2009

The effect of constitutively activated proto-oncogene H-ras (H-rasQ61L) on the regulation of myosin heavy chain (MHC) promoter activities was investigated in rabbit satellite cell-derived muscle cell culture during the proliferation stage and early and later stages of differentiation, respectively. During proliferation, overexpression of H-rasQ61L did not affect basal level of activity of the slow MHCI/[beta] or the fast MHCIId/x promoter luciferase reporter gene construct in transient transfection assays. By contrast, H-rasQ61L affected both MHC promoter activities during differentiation, and this effect changes from inactivation after 2 days to activation after 4 days of differentiation. The activating effect of H-rasQ61L on both MHC promoters after 4 days of differentiation was significantly reduced by LY-294002, a specific inhibitor of the phosphoinositol-3-kinase (PI3K), a downstream target of Ras. Furthermore, the protein kinase Akt (protein kinase B), a downstream target of PI3k, was activated 4 days after initiation of differentiation in myotubes overexpressing H-rasQ61L. By contrast, inhibition of another Ras downstream pathway, mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated protein kinase 1/2 (MKK1/ 2-ERK1/2-MAPK), increased activities of both MHC promoters, indicating a suppressive role of this pathway. Moreover, the Ras-PI3K-Akt signaling pathway is involved in the activation of MHCI/[beta] and IId/x promoters in a later stage of differentiation of muscle cells, presumably by a known inhibiting effect of activated Akt on the MKK1/2-ERK1/2-MAPK pathway. The experiments demonstrate that during differentiation of muscle cells activated H-ras is an important regulator of MHC isoform promoter function with opposite effects during early and later stages. extracellular signal regulated protein kinase 1/2; mitogen-activated protein kinase; phosphosinositol-3-kinase; Akt; mammalian target of rapamycin doi: 10.1152/ajpcell.00567.2008

Diastolic depolarization (DD) of atrial myocytes can lead to spontaneous action potentials (APs) and, potentially, atrial tachyarrhythmias. This study examined the hypotheses that 1) a slowly inactivating component of the [Na.sup.+] current (referred to as late [I.sub.Na]) may contribute to DD and initiate AP firing and that 2) blocking late [I.sub.Na] will reduce spontaneous and induced firing of APs by atrial myocytes. Guinea pig atrial myocytes without or with DD and spontaneous AP firing were studied using the whole cell patch-clamp technique. In experiments using cells with a stable resting membrane potential (no spontaneous DD or firing), hydrogen peroxide ([H.sub.2][O.sub.2], 50 [micro]mol/l) caused DD and AP firing. The [H.sub.2][O.sub.2]-induced activity was suppressed by the late [I.sub.Na] inhibitors tetrodotoxin (TTX, 1 [micro]mol/l) and ranolazine (5 [micro]mol/l). In cells with DD but no spontaneous APs, the late [I.sub.Na] enhancer anemone toxin II (ATX-II, 10 nmol/l) accelerated DD and induced APs. In cells with DD and spontaneous AP firing, TTX and ranolazine (both, 1 [micro]mol/l) significantly reduced the slope of DD by 81 [+ or -] 12% and 75 [+ or -] 11% and the frequency of spontaneous firing by 70 [+ or -] 15% and 74 [+ or -] 9%, respectively. Ramp voltage-clamp simulating DD elicited a slow inward current. TTX at 1, 3, and 10 [micro]mol/l inhibited this current by 41 [+ or -] 4%, 73 [+ or -] 2%, and 91 [+ or -] 1%, respectively, suggesting that a slowly inactivating IN~ underlies the DD. ATX-II and [H.sub.2][O.sub.2] increased the amplitude of this current, and the effects of ATX-II and [H.sub.2][O.sub.2] were attenuated by ranolazine or TTX. In conclusion, late [I.sub.Na] can contribute to the DD of atrial myocytes and the inhibition of this current suppresses atrial DD and spontaneous APs. late sodium current; action potential; spontaneous activity doi: 10.1152/ajpheart.00444.2009

We have produced mice in which expression of the rat cardiac [Na.sup.+]/[Ca.sup.2+] exchanger (NCX1) transgene was switched on when doxycycline was removed from the feed at 5 wk. At 8 to 10 wk, NCX1 expression in induced (Ind) mouse hearts was 2.5-fold higher but protein levels of sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase, [[alpha].sub.1]- and [[alpha].sub.2]-subunits of [Na.sup.+]-[K.sup.+]-ATPase, phospholamban, ryanodine receptor, calsequestrin, and unphosphorylated and phosphorylated phospholemman were unchanged compared with wild-type (WT) or noninduced (non-Ind) hearts. There was no cellular hypertrophy since WT, non-Ind, and Ind myocytes had similar whole cell membrane capacitance. In Ind myocytes, NCX1 current amplitude was ~42% higher, L-type [Ca.sup.2+] current amplitude was unchanged, and action potential duration was prolonged compared with WT or non-Ind myocytes. Contraction and intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]) transient amplitudes in Ind myocytes were lower at 0.6, not different at 1.8, and higher at 5.0 mM extracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.o]) compared with WT or non-Ind myocytes. Despite similar [Ca.sup.2+] current amplitude and sarcoplasmic reticulum (SR) [Ca.sup.2+] uptake, SR [Ca.sup.2+] content at 5.0 mM [[Ca.sup.2+].sub.o] was significantly higher in Ind compared with non-Ind myocytes, indicating that NCX1 directly contributed to SR [Ca.sup.2+] loading. Echocardiography demonstrated that heart rate, left ventricular mass, ejection fraction, stroke volume, and cardiac output were similar among the three groups of animals. In vivo close-chest catheterization demonstrated similar contractility and relaxation among the three groups of mice, both at baseline and after stimulation with isoproterenol. We conclude that induced expression of NCX1 transgene resulted in altered [[Ca.sup.2+].sub.i] homeostasis, myocyte contractility, and action potential morphology. In addition, heart failure did not occur 3 to 5 wk after NCX1 transgene was induced to be expressed at levels found in diseased hearts. tet-off; excitation-contraction; fura-2; isolated mouse myocytes; electrophysiology; sarcoplasmic reticulum; intracellular [Ca.sup.2+] concentration

Hypoxia-induced mitogenic factor (HIMF), also known as 'found in inflammatory zone 1' (FIZZI) or resistin-like molecule-[alpha] (RELM[alpha]), is a profound vasoconstrictor of the pulmonary circulation and a strong mitogenic factor in pulmonary vascular smooth muscle. To further understand the mechanism of these contractile and mitogenic responses, we examined the effect of HIMF on intracellular [Ca.sup.2+] in human pulmonary artery smooth muscle cells (SMC). [Ca.sup.2+] imaging in fluo 4-loaded human pulmonary artery SMC revealed that recombinant murine HIMF increased intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]) in a sustained and oscillatory manner. This increase occurred independent of extracellular [Ca.sup.2+] influx. Pretreatment of human pulmonary artery SMC with U-73122, a specific inhibitor of phosphatidylinositolphospholipase C (PLC) completely prevented the HIMF-induced [Ca.sup.2+] signal. The [[Ca.sup.2+]].sub.i] increase was also abolished by pretreatment with 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate ([IP.sub.3]) receptor antagonist. Ryanodine pretreatment did not affect initiation of [[Ca.sup.2+]].sub.i] activation or internal release but reduced [[Ca.sup.2+]].sub.i] at the plateau phase. Pretreatment with the G[[alpha].sub.i]-specific inhibitor pertussis toxin and the G[[alpha].sub.s]-specific inhibitor NF449 did not block the [Ca.sup.2+] signal. Knockdown of G[[alpha].sub.q/11] expression did not prevent [Ca.sup.2+] release, but the pattern of [Ca.sup.2+] release changed from the sustained oscillatory transients with prolonged plateau to a series of short [[Ca.sup.2+].sub.i] transients that return to baseline. However, pretreatment with the tyrosine kinase inhibitor genistein completely inhibited the internal [Ca.sup.2+] release. These results demonstrate that HIMF can stimulate intracellular [Ca.sup.2+] release in human pulmonary artery SMC through the PLC signaling pathway in an [IP.sub.3]- and tyrosine phosphorylation-dependent manner and that G[[alpha].sub.q/11] protein-coupled receptor and ryanodine receptor contribute to the increase of [[Ca.sup.2+].sub.i]. resistin-like molecule-[alpha]; inositol 1,4,5-trisphosphate receptor; phospholipase C; tyrosine phosphorylation; pulmonary vascular smooth muscle

We previously showed that an increase in the peroxisome proliferator-activated receptor-[delta] (PPAR[delta]), together with subsequent induction of inducible nitric oxide synthase (iNOS) by beraprost (BPS), inhibits aortic smooth muscle cell proliferation. Herein, we delineated the mechanisms of the antiproliferative effects of BPS through the induction of p21/p27. BPS concentration dependently induced the p21/p27 promoter- and consensus cAMP-responsive element (CRE)-driven luciferase activities, which were significantly suppressed by blocking PPAR[delta] activation. Surprisingly, other than altering the CRE-binding protein (CREB), BPS-mediated PPAR[delta] activation increased nuclear localization of the CREB-binding protein (CBP), a coactivator, which was further confirmed by chromatin immunoprecipitation. Furthermore, novel functional PPAR-responsive elements (PPREs) next to CREs in the rat p21/p27 promoter regions were identified, where PPAR[delta] interacted with CREB through CBP recruitment. BPS-mediated suppression of restenosis in mice with angioplasty was associated with p21/p27 induction. Herein, we demonstrate for the first time that BPS-mediated PPAR[delta] activation enhances transcriptional activation of p21/p27 by increasing CBP nuclear translocation, which contributes to the vasoprotective action of BPS. cAMP-responsive element; cAMP-responsive element-binding protein; cAMP-responsive element-binding protein-binding protein; antiproliferation

Cdc42GAP (GTPase-activating protein) has been implicated in the regulation of cell motility, adhesion, proliferation, and apoptosis. In this study, Cdc42GAP was cloned from smooth muscle tissues. Cdc42GAP, but not inactive R282A Cdc42GAP (alanine substitution at arginine-282), enhanced the GTP hydrolysis of Cdc42 in an in vitro assay. Furthermore, we developed an assay to evaluate the activity of Cdc42GAP in vivo. Stimulation of smooth muscle cells with 5-hydroxytryptamine (5-HT) resulted in the decrease in Cdc42GAP activity. The agonist-induced GAP suppression was reversed by reactive oxygen species inhibitors. Treatment with hydrogen peroxide also inhibited GAP activity in smooth muscle cells. Because the vimentin cytoskeleton undergoes dynamic changes in response to contractile activation, we evaluated the role of Cdc42GAP in regulating vimentin filaments. Smooth muscle cells were infected with retroviruses encoding wild-type Cdc42GAP or its R282A mutant. Expression of wild-type Cdc42GAP, but not mutant R282A GAP, inhibited the increase in the activation of Cdc42 upon agonist stimulation. Phosphorylation of p21-activated kinase (PAK) at Thr-423 (an indication of PAK activation), vimentin phosphorylation (Ser-56), partial disassembly and spatial remodeling, and contraction were also attenuated in smooth muscle cells expressing Cdc42GAP. Our results suggest that the activity of Cdc42GAP is regulated upon contractile activation, which is mediated by intracellular ROS. Cdc42GAP regulates the vimentin network through the Cdc42-PAK pathway in smooth muscle cells during 5-HT stimulation. p21-activated kinase; 5-hydroxytryptamine; smooth muscle cells; cytoskeleton

The regional manipulation of the microenvironment surrounding single adult cardiac myocytes in a microfluidic structure is described. The flow rates of laminar streams were adjusted such that the fluid interface between an injection flow and a perfusion flow was manipulated laterally to stimulate regions of the cell surface. Using this general principle, we were able to selectively expose defined regions of the cell to test solutions, with predefined pulse durations and frequencies. We demonstrate the transient depolarisation of the cardiomyocyte through the regional chemical stimulation of localized areas of the cell with elevated potassium concentrations (100 mM). The results show that chemical stimulation at frequencies [less than or equal to] 0.25 Hz evoked [Ca.sup.2+] transients and cell shortening, comparable to those induced by electrical (field) stimulation. At higher frequencies the membrane potential failed to recover sufficiently from the depolarisation with the high [K.sup.+] solution, possibly because of the slow clearance of the ion from the t-tubular system. To test this hypothesis, the clearance of fluorescently labeled 10 kDa dextran from the t-system was measured and found to be ~0.5 s delayed compared to that of the bulk extracellular space, indicating the slow diffusion inside the confined space of the tubular membrane invaginations.

How the nuclei in mammalian skeletal muscle fibers properly position themselves relative to the cell body is an interesting and important cell biology question. In the syncytial skeletal muscle cells, more than 100 nuclei are evenly distributed at the periphery of each cell, with 3-8 nuclei anchored beneath the neuromuscular junction (NMJ). Our previous studies revealed that the KASH domain--containing Syne-1/Nesprin-1 protein plays an essential role in anchoring both synaptic and nonsynaptic myonuclei in mice. SUN domain--containing proteins (SUN proteins) have been shown to interact with KASH domain--containing proteins (KASH proteins) at the nuclear envelope (NE), but their roles in nuclear positioning in mice are unknown. Here we show that the synaptic nuclear anchorage is partially perturbed in Sun1, but not in Sun2, knockout mice. Disruption of 3 or all 4 Sun1/2 wild-type alleles revealed a gene dosage effect on synaptic nuclear anchorage. The organization of nonsynaptic nuclei is disrupted in Sun1/2 double-knockout (DKO) mice as well. We further show that the localization of Syne-1 to the NE of muscle cells is disrupted in Sun1/2 DKO mice. These results clearly indicate that SUN1 and SUN2 function critically in skeletal muscle cells for Syne-1 localization at the NE, which is essential for proper myonuclear positioning. KASH domain | Nesprin | neuromuscular junction | nuclear envelope protein | Syne-1

The use of muscle progenitor cells (MPCs) for regenerative medicine has been severely compromised by their decreased proliferative and differentiative capacity after being cultured in vitro. We hypothesized the loss of pivotal niche factors to be the cause. Therefore, we investigated the proliferative and differentiative response of passage 0 murine MPCs to varying substrate elasticities and protein coatings and found that proliferation was influenced only by elasticity, whereas differentiation was influenced by both elasticity and protein coating. A stiffness of 21 kPa optimally increased the proliferation of MPCs. Regarding differentiation, we demonstrated that fusion of MPCs into myotubes takes place regardless of elasticity. However, ongoing maturation with cross-striations and contractions occurred only on elasticities higher than 3 kPa. Furthermore, maturation was fastest on poly-D-lysine and laminin coatings. muscle progenitor cells; substrate elasticity; matrix proteins; maturation

Muscle fibers that power swimming in the blue crab Callinectes sapidus are metabolism; mitochondria; nuclei; reaction-diffusion modeling; crustacean

Myofibrillar proteins must be removed from the myofibril before they can be turned over metabolically in functioning muscle cells. It is uncertain how this removal is accomplished without disruption of the contractile function of the myofibril. It has been proposed that the calpains could remove the outer layer of filaments from myofibrils as a first step in myofibrillar protein turnover. Several studies have found that myofilaments can be removed from myofibrils by trituration in the presence of ATP. These easily releasable myofilaments (ERMs) were proposed to be intermediates in myofibrillar protein turnover. It was unclear, however, whether the ERMs were an identifable entity in muscle or whether additional trituration would remove more myofilaments until the myofibril was gone and whether calpains could release ERMs from intact myofibrils. The present study shows that few ERMs could be obtained from the residue after the first removal of ERMs, and the yield of ERMs from well-washed myofibrils was reduced, probably because some ERMs had been removed by the washing process. Mild calpain treatment of myofibrils released filaments that had a polypeptide composition and were ultrastructurally similar to ERMs. The yield of calpain-released ERMs was two- to threefold greater than the normal yield. Hence, ERMs are an identifiable entity in myofibrils, and calpain releases filaments that are similar to ERMs. The role of ERMs in myofibrillar protein turnover is unclear, because only filaments on the surface of the myofibril would turn over, and changes in myofibrillar protein isoforms during development could not occur via the ERM mechanism. calpain; muscle; proteasome

The actin-myosin interaction of vascular smooth muscle cells (VSMCs) is regulated by myosin light chain kinase (MLCK), which is a fusion protein of the central catalytic domain with the N-terminal actin-binding and C-terminal myosin-binding domains. In addition to the regulatory role of kinase activity mediated by the catalytic domain, nonkinase activity that derives from both terminals is able to exert a regulatory role as reviewed by Nakamura et al. (32). We previously showed that nonkinase activity mediated the filopodia upon the stimulation by sphingosylphosphorylcholine (SPC) (25). To explore the regulatory role of nonkinase activity in chemotaxis, we constructed VSMCs where the expression of MLCK was totally abolished by using a lentivirus-mediated RNAi system. We hypothesized that the MLCK-downregulated VSMCs were unable to form filopodia and to migrate upon SPC stimulation and confirmed the hypothesis. We further constructed a kinase-inactive mutant from bovine cDNA coding wild-type (WT) MLCK by mutating the ATP-binding sites located in the catalytic domain, followed by confirming the presence (absence) of the kinase activity of WT (kinase-inactive mutant). We transfected WT and the mutant into MLCK-downregulated VSMCs. We expected that the transfected VSMCs will recover the ability to induce filopodia and chemotaxis toward SPC and found both constructs rescued the ability. Because they share the actin- and myosin-binding domains, we concluded nonkinase activity plays a major role for SPC-induced migration. chemotaxis; myosin light chain kinase

We have previously reported that in ovine fetal pulmonary venous smooth muscle cells (FPVSMC), decreased expression of cGMP-dependent protein kinase (PKG) by hypoxia could explain hypoxia-induced SMC phenotype modulation. In this study, we investigated the role of myocardin, a possible downstream effector of PKG, in SMC phenotype modulation induced by 1 and 24 h of hypoxia. Hypoxia for 1 h induced the phosphorylation of E-26-1ike protein 1 (Elk-1), indicating a quick activation of Elk-1 after hypoxia. Either hypoxia (1 h) or treatment with DT-3, a PKG inhibitor, increased associations of Elk-1 with myosin heavy chain (MHC) gene and serum response factor (SRF), which was paralleled by a decrease in association of myocardin with MHC gene and SRF. Exposure to hypoxia of FPVSMC for 24 h significantly decreased the promoter activity of multiple SMC marker genes, downregulated protein and mRNA expression of myocardin, and upregulated mRNA expression of Elk-1, but had no significant effects on the phosphorylation of Elk-1. Inhibition of myocardin by siRNA transfection downregulated the expression of SMC marker proteins, while overexpression of myocardin prevented the hypoxia-induced decrease in expression of SMC marker proteins. Inhibition of PKG by siRNA transfection downregulated the expression of myocardin, but upregulated that of Elk-1. Overexpression of PKG prevented hypoxia-induced effects on protein expression of myocardin and Elk-1. These data suggest that PKG induces displacement of myocardin from SRF and upregulates myocardin expression, thus activating the SMC genes transcription. The inhibitory effects of hypoxia on PKG may explain hypoxia-induced SMC phenotype modulation by decreasing the effects of PKG on myocardin. Elk-1

Mitochondria can sense signals linked to changes in energy demand to affect nuclear gene expression. This retrograde signaling pathway is presumed to be involved in the regulation of myoblast proliferation and differentiation. We have investigated the regulation of mitochondrial biogenesis and production of putative retrograde signaling agents [hydrogen peroxide ([H.sub.2][O.sub.2]) and [Ca.sup.2+]] during the cell cycle and the onset of differentiation in L6E9 muscle cells. The biosynthesis of cardiolipin and mitochondrial proteins was mainly achieved in S phase, whereas the expression of mitochondrial biogenesis factors [peroxisome proliferator-activated receptor (PPAR)-[alpha], PPAR-[delta], and neuronal nitric oxide synthase 1] was regularly increased from [G.sub.1] to [G.sub.2]M phase. In agreement with the increase in mitochondrial membrane potential, mitochondria in S and [G.sub.2]M phases have a significantly higher [H.sub.2][O.sub.2] level when compared with [G.sub.1] phase. By contrast, the onset of differentiation was characterized by a marked reduction in mitochondrial protein expression and mitochondrial [H.sub.2][O.sub.2] level. The capacity of mitochondria to release [Ca.sup.2+] in response to a metabolic challenge was significantly decreased at the onset of differentiation. Finally, an increase in calmodulin expression in S and [G.sub.2]M phases and a transitory increase in phosphorylated nuclear factor of activated T cells (NFAT) c3 in S phase was observed. NFATc3 phosphorylation was markedly decreased at the onset of differentiation. Our data point to functional links between the control of mitochondrial biogenesis and the regulation of the level of retrograde signaling agents during the cell cycle and the onset of differentiation in L6E9 muscle cells. mitochondria; myogenesis; reactive oxygen species; skeletal muscle

The effects of actin filaments (AFs) and microtubules (MTs) on quasi-in situ tensile properties and intracellular force balance were studied in cultured rat aortic smooth muscle cells (SMCs). A SMC cultured on substrates was held using a pair of micropipettes, gradually detached from the substrate while maintaining in situ cell shape and cytoskeletal integrity, and then stretched up to ~15% and unloaded three times at the rate of 1 [micro]m every 5 s. Cell stiffness was ~20 nN per percent strain in the untreated case and decreased by ~65% and ~30% following AF and MT disruption, respectively. MT augmentation did not affect cell stiffness significantly. The roles of AFs and MTs in resisting cell stretching and shortening were assessed using the area retraction of the cell upon noninvasive detachment from thermoresponsive gelatin-coated dishes. The retraction was ~40% in untreated cells, while in AF-disrupted cells it was cellular biomechanics; mechanical properties; hysteresis; cell retraction; cellular prestress

Nutrition availability is one of the major environmental signals influencing cell fate, such as proliferation, differentiation, and apoptosis, often functioning in concert with other humoral factors, including insulin. Herein, we show that low-serum-induced differentiation of [C.sub.2] [C.SUB.12] myocytes is significantly hampered under low glucose (LG; 5 raM) compared with high glucose (HG; 22.5 mM) conditions, concurrently with nuclear accumulation of SIRT1, an [NAD.sup.+]-dependent deacetylase, and FoxO3a, both of which are implicated in the negative regulation of myogenesis. Intriguingly, insulin appears to exert opposite actions, depending on glucose availability, with regard to the regulation of SIRT1 and FoxO3a abundance, which apparently contributes to modulating the potency of insulin's myogenic action. Namely, insulin exerts a potent myogenic effect in the presence of sufficient glucose, whereas insulin is unable to exert its myogenic action under LG conditions, since insulin evokes massive upregulation of both SIRT1 and FoxO3a in the absence of sufficient ambient glucose. In addition, the hampered differentiation state under LG is significantly restored by sirtinol, a SIRT1 inhibitor, whereas insulin abolished this sirtinoldependent restoration, indicating that insulin can function as a negative as well as a positive myogenic factor depending on glucose availability. Taken together, our data reveal the importance of ambient glucose levels in the regulation of myogenesis and also in the determination of insulin's myogenic potency, which is achieved, at least in part, through regulation of the cellular contents and localization of SIRT1 and FoxO3a in differentiating [C.sub.2][C.sub.12] myocytes. forkhead box O; differentiation

Previous studies indicate that 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), an endothelium-derived hyperpolarizing factor in the rabbit aorta, mediates a portion of the relaxation response to acetylcholine by sequential metabolism of arachidonic acid by 15-lipoxygenase, hydroperoxide isomerase, and epoxide hydrolase. To determine the stereochemical configuration of the endothelial 11,12,15-THETA, its activity and chromatographic migration were compared with activity and migration of eight chemically synthesized stereoisomers of 11, 12, 15(S)-THETA. Of the eight isomers, only 11(R), 12(S), 15(S)-trihydroxyeicosa-5(Z), 8(Z), 13(E)-trienoic acid comigrated with the biological 11, 12, 15-THETA on reverse- and normal-phase HPLC and gas chromatography. The same THETA isomer ([10.sup.-7] - [10.sup.-4] M) relaxed the rabbit aorta in a concentration-related manner (maximum relaxation = 69 [+ or -] 5%). These relaxations were blocked by apamin ([10.sup.-7] M), an inhibitor of small-conductance [Ca.sup.2+]-activated [K.sup.+] channels. In comparison, 11 (S), 12(R), 15(S), 5(Z), 8(Z), 13(E)-THETA ([10.sup.-4] M) relaxed the aorta by 22%. The other six stereoisomers were inactive in this assay. With use of the whole cell patch-clamp technique, it was shown that [10.sup.-4] M 11(R), 12(S), 15(S), 5(Z), 8(Z), 13(E)-THETA increased outward [K.sup.+] current in isolated aortic smooth muscle cells by 119 [+ or -] 36% at +60 mV, whereas [10.sup.-4] M 11(R), 12(R), 15(S), 5(Z), 8(Z), 13(E)-THETA increased outward [K.sup.+] current by only 20 [+ or -] 2%. The 11(R), 12(S), 15(S), 5(Z), 8(Z), 13(E)-THETA-stimulated increase in [K.sup.+] current was blocked by pretreatment with apamin. These studies suggest that 11(R), 12(S), 15(S)-trihydroxyeicosa-5(Z), 8(Z), 13(E)-trienoic acid is the active stereoisomer produced by the rabbit aorta. It relaxes smooth muscle by activating [K.sup.+] channels. The specific structural and stereochemical requirements for [K.sup.+] channel activation suggest that a specific binding site or receptor of 11, 12, 15-THETA is involved in these actions. endothelium-derived hyperpolarizing factor; smooth muscle cells; potassium channels; arachidonic acid