Emergence of muscle and neural hematopoiesis in humans.
(17/1087)
During human development, hematopoiesis is thought to be compartmentalized to the fetal circulation, liver, and bone marrow. Here, we show that combinations of cytokines together with bone morphogenetic protein-4 and erythropoietin could induce multiple blood lineages from human skeletal muscle or neural tissue. Under defined serum-free conditions, the growth factors requirements, proliferation, and differentiation capacity of muscle and neural hematopoiesis were distinct to that derived from committed hematopoietic sites and were uniquely restricted to CD45(-)CD34(-) cells expressing the prominin AC133. Our study defines epigenetic factors required for the emergence of hematopoiesis from unexpected tissue origins and illustrates that embyronically specified microenvironments do not limit cell fate in humans. (+info)
Cyclic stretch induces vascular smooth muscle cell alignment via NO signaling.
(18/1087)
We investigated the effects of cyclic stretch on vascular smooth muscle cell (VSMC) alignment and potential overlap of signaling modalities with stretch-induced proliferation. VSMC were subjected to graded stretch (1 Hz at 100-124% of resting length) for 48 h. Graded stretch resulted in graded VSMC alignment from a minimum of completely random orientation to a maximum of ~80-85 degrees to the stretch vector. Alignment was reversible within 48 h of stretch cessation and independent of signaling modalities mediating stretch-induced proliferation: modulation of IGF-1, MAPK, phosphatidylinositol 3-kinase, tyrosine kinase, and stretch-activated calcium channels did not affect alignment. Nitric oxide (NO) synthase (NOS) blockade uncoupled alignment. Neither the NO donor, cytokine-induced NOS activity, nor L-citrulline affected alignment, but inhibited VSMC proliferation. Therefore, stretch-induced proliferation and alignment are differentially regulated, with NO a common signaling molecule for both. Targeting NOS in states such as restenosis and hypertension may prove to be beneficial. (+info)
Reperfusion, not simulated ischemia, initiates intrinsic apoptosis injury in chick cardiomyocytes.
(19/1087)
Although ischemia-reperfusion (I/R) can initiate apoptosis, the timing and contribution of the mitochondrial/cytochrome c apoptosis death pathway to I/R injury is unclear. We studied the timing of cytochrome c release during I/R and whether subsequent caspase activation contributes to reperfusion injury in confluent chick cardiomyocytes. One-hour simulated ischemia followed by 3-h reperfusion resulted in significant cell death, with most cell death evident during the reperfusion phase and demonstrating mitochondrial cytochrome c release within 5 min after reperfusion. By contrast, cells exposed to prolonged ischemia for 4 h had only marginally increased cell death and no detectable cytochrome c release into the cytosol. Caspase activation could not be detected after ischemia only, but it significantly increased after reperfusion. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, Ac-Asp-Gln-Thr-Asp-H, or benzyloxycarbonyl-Leu-Glu (Ome)-His-Asp-(Ome)-fluoromethyl ketone given only at reperfusion significantly attenuated cell death and resulted in return of contraction. Antixoxidants decreased cytochrome c release, nuclear condensation, and cell death. These results suggest that reperfusion oxidants initiate cytochrome c release within minutes, and apoptosis within hours, significant enough to increase cell death and contractile dysfunction. (+info)
Regulation of the S100B gene by alpha 1-adrenergic stimulation in cardiac myocytes.
(20/1087)
We previously reported that S100B, a 20-kDa Ca(2+)-binding homodimer, inhibited the postinfarct myocardial hypertrophic response mediated by alpha(1)-adrenergic stimulation through the protein kinase C (PKC) signaling pathway. In the present study, we examined whether the same pathway induced the S100B gene, supporting the hypothesis that S100B is a feedback negative regulator of this pathway. We transfected cultured neonatal rat cardiac myocytes with a luciferase reporter gene driven by the maximal human S100B promoter and progressively shorter segments of this promoter sequentially deleted from the 5' end. We identified a basic promoter essential for transcription spanning 162 bp upstream of the transcription initiation site and positive (at -782/-162 and -6,689/-4,463) and negative (at -4,463/-782) myocyte-selective regulatory elements. We showed that the basic and maximal S100B promoters were activated specifically by alpha(1)-adrenergic agonists through the alpha(1A)-adrenergic receptor, but not by any other trophic hormonal stimuli. The activation of the S100B promoter was mediated through the PKC signaling pathway. Transcription enhancer factor-1 (TEF-1) and related to TEF-1 (RTEF-1) influenced transcription from the maximal, but not the basic, promoter implicating active MCAT elements upstream from the basic promoter. Acting in opposing fashions, TEF-1 transrepressed the S100B promoter and RTEF-1 transactivated the promoter. Our results suggest that alpha(1)-adrenergic stimulation induces the S100B gene after myocardial infarction through the PKC signaling pathway and that this induction is modulated by TEF-1 and RTEF-1. (+info)
Exercise training alters length dependence of contractile properties in rat myocardium.
(21/1087)
Myocardial function is enhanced by endurance exercise training, but the cellular mechanisms underlying this improved function remain unclear. Exercise training increases the sensitivity of rat cardiac myocytes to activation by Ca(2+), and this Ca(2+) sensitivity has been shown to be highly dependent on sarcomere length. We tested the hypothesis that exercise training increases this length dependence in cardiac myocytes. Female Sprague-Dawley rats were divided into sedentary control (C) and exercise-trained (T) groups. The T rats underwent 11 wk of progressive treadmill exercise. Heart weight increased by 14% in T compared with C rats, and plantaris muscle citrate synthase activity showed a 39% increase with training. Steady-state tension was determined in permeabilized myocytes by using solutions of various Ca(2+) concentration (pCa), and tension-pCa curves were generated at two different sarcomere lengths for each myocyte (1.9 and 2.3 microm). We found an increased sarcomere length dependence of both maximal tension and pCa(50) (the Ca(2+) concentration giving 50% of maximal tension) in T compared with C myocytes. The DeltapCa(50) between the long and short sarcomere length was 0.084 +/- 0.023 (mean +/- SD) in myocytes from C hearts compared with 0.132 +/- 0.014 in myocytes from T hearts (n = 50 myocytes per group). The Deltamaximal tension was 5.11 +/- 1.42 kN/m(2) in C myocytes and 9.01 +/- 1.28 in T myocytes. We conclude that exercise training increases the length dependence of maximal and submaximal tension in cardiac myocytes, and this change may underlie, at least in part, training-induced enhancement of myocardial function. (+info)
Assessment of O2 uptake dynamics in isolated single skeletal myocytes.
(22/1087)
The purpose of this research was to develop a technique for rapid measurement of O(2) uptake (Vo(2)) kinetics in single isolated skeletal muscle cells. Previous attempts to measure single cell Vo(2) have utilized polarographic-style electrodes, thereby mandating large fluid volumes and relatively poor sensitivity. Thus our laboratory has developed an approximately 100-microl, well-stirred chamber for the measurement of Vo(2) in isolated Xenopus laevis myocytes using a phosphorescence quenching technique [Ringer solution with 0.05 mM Pd-meso-tetra(4-carboxyphenyl)porphine] to monitor the fall in extracellular Po(2) (which is proportional to cellular Vo(2) within the sealed chamber). Vo(2) in single living myocytes dissected from Xenopus lumbrical muscles was measured from rest across a bout of repetitive tetanic contractions (0.33 Hz) and in response to a ramp protocol utilizing an increasing contraction frequency. In response to the square-wave contraction bout, the increase in Vo(2) to steady state (SS) was 16.7 +/- 1.3 ml x 100 g(-1) x min(-1) (range 13.0-21.9 ml x 100 g(-1) x min(-1); n = 6). The rise in Vo(2) at contractions onset (n = 6) was fit with a time delay (2.1 +/- 1.2 s, range 0.0-7.7 s) plus monoexponential rise to SS (time constant = 9.4 +/- 1.5 s, range 5.2-14.9 s). Furthermore, in two additional myocytes, Vo(2) increased progressively as contraction frequency increased (ramp protocol). This technique for measuring Vo(2) in isolated, single skeletal myocytes represents a novel and powerful investigative tool for gaining mechanistic insight into mitochondrial function and Vo(2) dynamics without potential complications of the circulation and other myocytes. (+info)
High glucose and insulin promote O-GlcNAc modification of proteins, including alpha-tubulin.
(23/1087)
Increased flux through the hexosamine biosynthesis pathway has been implicated in the development of glucose-induced insulin resistance and may promote the modification of certain proteins with O-linked N-acetylglucosamine (O-GlcNAc). L6 myotubes (a model of skeletal muscle) were incubated for 18 h in 5 or 25 mM glucose with or without 10 nM insulin. As assessed by immunoblotting with an O-GlcNAc-specific antibody, high glucose and/or insulin enhanced O-GlcNAcylation of numerous proteins, including the transcription factor Sp1, a known substrate for this modification. To identify novel proteins that may be O-GlcNAc modified in a glucose concentration/insulin-responsive manner, total cell membranes were separated by one- or two-dimensional gel electrophoresis. Selected O-GlcNAcylated proteins were identified by mass spectrometry (MS) analysis. MS sequencing of tryptic peptides identified member(s) of the heat shock protein 70 (HSP70) family and rat alpha-tubulin. Immunoprecipitation/immunoblot studies demonstrated several HSP70 isoforms and/or posttranslational modifications, some with selectively enhanced O-GlcNAcylation following exposure to high glucose plus insulin. In conclusion, in L6 myotubes, Sp1, membrane-associated HSP70, and alpha-tubulin are O-GlcNAcylated; the modification is markedly enhanced by sustained increased glucose flux. (+info)
Bupivacaine attenuates contractility by decreasing sensitivity of myofilaments to Ca2+ in rat ventricular muscle.
(24/1087)
BACKGROUND: Bupivacaine exhibits a cardiodepressant effect, the molecular mechanism(s) of which have yet to be fully understood. Bupivacaine may directly act on contractile proteins and thereby decrease myofibrillar Ca2+ sensitivity. METHODS: Rat ventricular muscle was used. First, the effect of bupivacaine was examined on tetanic contractions in isolated intact myocytes. Next, Triton X-100-treated ventricular trabeculae were used to investigate the effect of bupivacaine on the pCa (= -log [Ca2+ ])-tension relation as well as on maximal Ca2+ -activated tension. Furthermore, to test whether bupivacaine inhibits the pathway downstream from Ca2+ binding to troponin C, tension was elicited in the skinned preparations by lowering the Mg-adenosine triphosphate (MgATP) concentration in the absence of Ca2+. The effect of bupivacaine on the pMgATP (= -log [MgATP])-tension relation was examined. RESULTS: In myocytes, 3 microm bupivacaine significantly (P < 0.01) increased intracellular Ca2+ concentration required for 5% cell shortening from the resting cell length. In skinned preparations, bupivacaine shifted the pCa-tension relation to the lower pCa side; the midpoint of the pCa curve (pCa50) was significantly (P < 0.05) changed by 10 and 100 microm bupivacaine. A highly correlated linear relation (R = 0.81; P< 0.0005) was present between pCa50 and maximal Ca2+ -activated tension. Bupivacaine (10 and 100 microm) significantly (P < 0.05) shifted the midpoint of the pMgATP-tension relation to the higher pMgATP side. CONCLUSIONS: Bupivacaine decreases myofibrillar Ca2+ sensitivity in ventricular muscle, and this is coupled with the compound's inhibitory effect on the pathway beyond Ca2+ binding to troponin C, possibly on the actomyosin interaction. The current results may partly explain the overall cardiodepressant effect of bupivacaine in vivo. (+info)