The estimation of oxidative stress markers and apoptosis in right atrium auricles cardiomyocytes of patients undergoing surgical heart revascularisation with the use of warm blood cardioplegia.

Oxidative stress markers and apoptosis were estimated during elective surgical heart revascularization. Eight patients with good ejection fraction underwent coronary artery bypass grafting (CABG) with the use of warm blood cardioplegia. Two right atrium auricle biopsy specimens were collected before and after the operation. Specimens underwent immunocytochemical analysis of mitochondrial manganese superoxide dismutase (MnSOD) expression and apoptosis estimation by the TUNEL method. Ultrastructure analysis under electron microscope was made. Satisfactory results of the operation were obtained. After CABG the MnSOD expression increase in sections of auricles was observed through the increase of stain intensity and the percentage of cells with positive stain (from 30 to 80%). The apoptotic cells percentage remained at approximately the same level. Under the electron microscope insignificant pathological changes were observed. On this basis one may assume that in the case of cardiosurgical procedures with short aorta cross-clamping time and low operation risk level the application of cardioplegia sufficiently prevents reactive oxygen forms (ROF) cytotoxic activity although it does not inhibit the expression of oxidative stress (OS) markers. In our opinion the method of examining right atrium sections is safe and provides results comparable with other publications. It may also be a voice in the discussion on new methods of heart protection during cardiac surgery procedures

Shaping leg muscles in Drosophila: role of ladybird, a conserved regulator of appendicular myogenesis

Legs are locomotor appendages used by a variety of evolutionarily distant vertebrates and invertebrates. The primary biological leg function, locomotion, requires the formation of a specialised appendicular musculature. Here we report evidence that ladybird, an orthologue of the Lbx1 gene recognised as a hallmark of appendicular myogenesis in vertebrates, is expressed in leg myoblasts, and regulates the shape, ultrastructure and functional properties of leg muscles in Drosophila. ladybird expression is progressively activated in myoblasts associated with the imaginal leg disc and precedes that of the founder cell marker dumbfounded. The RNAi-mediated attenuation of ladybird expression alters properties of developing myotubes, impairing their ability to grow and interact with the internal tendons and epithelial attachment sites. It also affects sarcomeric ultrastructure, resulting in reduced leg muscle performance and impaired mobility in surviving flies. The over-expression of ladybird also results in an abnormal pattern of dorsally located leg muscles, indicating different requirements for ladybird in dorsal versus ventral muscles. This differential effect is consistent with the higher level of Ladybird in ventrally located myoblasts and with positive ladybird regulation by extrinsic Wingless signalling from the ventral epithelium. In addition, ladybird expression correlates with that of FGF receptor Heartless and the read-out of FGF signalling downstream of FGF. FGF signals regulate the number of leg disc associated myoblasts and are able to accelerate myogenic differentiation by activating ladybird, leading to ectopic muscle fibre formation. A key role for ladybird in leg myogenesis is further supported by its capacity to repress vestigial and to down-regulate the vestigial-governed flight muscle developmental programme. Thus in Drosophila like in vertebrates, appendicular muscles develop from a specialised pool of myoblasts expressing ladybird/Lbx1. The ladybird/Lbx1 gene family appears as a part of an ancient genetic circuitry determining leg-specific properties of myoblasts and making an appendage adapted for locomotion

<i>lb</i> is required for proper leg muscle performance and walking behaviour

<p>A The ball test see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000122#pone.0000122.s012" target="_blank">Videos S10</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000122#pone.0000122.s014" target="_blank">S12</a>. The abilities of flies to catch, maintain and rotate a polystyrene ball were tested. The number of individuals tested males only is indicated in upper case after the genotype. Each male performed each of the tests three times. The number of asterisks max. 5 illustrates the average performance. Notice that the RNAi-based attenuation of <i>lb</i> leads to a reduced ability to catch about 20% of failures and especially to maintain the ball about 60% of failures with slower and irregular rotations. Defects in catching, maintaining and rotating the ball were comparatively stronger in flies overexpressing <i>lb</i>. About 60% of flies were unable to catch the ball and more than 80% lost it in less than 30 s. B The ‘leg-print’ test for walking pattern. Two-day old flies were allowed to walk on a carbon-soot coated glass slide and their tracks were examined. The direction of movement is towards the top of each panel. The imprints made by the first 1 second 2 and third leg 3 of the left hemisegment are marked in each panel. Wild type flies B′ show a stereotypic pattern of prints, a consequence of a ‘tripod’ gait. In male flies where UAS-lbRNAi expression is under the control of the 1151GAL4 driver B″ the legs are held closer to the body and the leg-print is the consequence of a shuffling gait. In male flies where UAS-lbe expression is under the control of the 1151GAL4 driver the pattern of prints B′″ illustrates a bias towards one side, a consequence of the legs being abnormally positioned with respect to the body.</p

Components of FGF signalling pathway are expressed in all Lbe myoblasts

<p>A–L Confocal images of leg imaginal discs triple-stained with anti-Lbe A, E, I and green in merge D, H, L, anti-B-galactosidase against Htl lacZ B and red in merge D, anti-B-galactosidase against Duf –lacZ F, J and red in merge H, L, and anti-Dof antibodies C, G, K and blue in merge D, H, L. A–D A third instar leg disc showing coexpression of FGF receptor Heartless and the downstream target of Htl signalling, Dof in Lbe-positive myoblasts see outlined area and myoblasts indicated by arrows. E–L Progressive activation of Duf expression in Lbe myoblasts with active FGF signalling. In third instar leg discs E–H Duf is co-expressed with Lbe and Dof in dorsal femur myoblasts outlined area but not present in the ventral myoblasts arrows. At 3 hr-APF I–L in addition to dorsal myoblasts outlined area Duf is progressively activated in ventral Lbe- and Dof-positive leg myoblasts within the femur, tibia arrows in J and coxa yellow arrow in J segments. All leg disc myoblasts showing Lbe expression have active FGF signalling, shown by expression of Dof D, H, L. Arrowhead in L points to Lbe expressing long tendon.</p

Wg signals are required for Lbe expression and play a key role in leg myogenesis

<p>A–I Confocal images of third instar leg imaginal discs. A–C Wild type leg disc stained for Lbe and Vestigial Vg. Lbe positive myoblasts are seen in leg disc proper B and green in merge C, while the myoblasts associated with the dorsal proximal region corresponding to the ventral thorax express Vg A and red in merge C. Vg myoblasts are devoid of Lbe asterisk in B. A Yellow arrowhead shows myoblasts expressing high levels of Vg and white arrowhead those expressing Vg at lower levels. D–F Leg disc, in which a dominant-negative TCF has been expressed in the myoblasts. Myoblasts are present as shown by anti-Twist antibody staining D and blue in merge F, but Lbe expression in myoblasts is lost asterisks in E and green in F. G–I Leg disc, in which activated Armadillo Arm transgene has been expressed in the myoblasts. Lbe is ectopically expressed in dorsal proximal myoblasts arrow in H and green in merge I, normally devoid of Lbe. Myoblasts are visualised with anti-Twi antibody G and blue in I. J–J′ show adult muscle phenotypes in the femur region induced by 1151-Gal4-driven forced expression of a dominant-negative TCF. Ventral muscles tidm are partially lost asterisks in J or completely lost asterisks in J′ and dorsal muscles tilm are severely affected. Muscles are visualised using MHC-tauGFP. Anterior views from the 3D reconstructions of confocal scans. K A schematic showing position of Lbe-expressing dorsal tilm and ventral tidm precursors of femur muscles green areas with respect to epithelial Wg expression domain violet triangle. The dorsal tilm myoblasts, located comparatively far from the Wg domain, receive a lower level of Wg morphogen long thin arrow than ventrally located tidm myoblasts short thick arrow.</p

Htl-transduced FGF signals regulate number of leg myoblasts and resulting muscle fibres and when overexpressed promote expression of Lbe and Duf.

<p>A–L Confocal images of third instar leg discs stained for Twi, Lbe and Duf-lacZ. Leg disc A–D Control <i>duf</i> lacZ, and Leg disc E–H with attenuated Htl signalling <i>htl</i> RNAi, I–L with increased Htl signalling <i>htl</i>-ca, in the myoblasts. Attenuation of Htl expression leads to reduced number of myoblasts associated with the leg disc compare A and E. This is particularly obvious when comparing the number of Twi/Lbe/Duf expressing cells within the outlined area in the dorsal femur. In contrast, forced Htl expression leads to significantly increased number of leg myoblasts outlined area 1 in I–L. Moreover, Htl, when overexpressed, induces Lbe prematurely in all myoblasts in the leg disc proper and promotes differentiation of dorsally located myoblasts into Duf-positive cells K,L, outlined areas 2,3,4. Note that the only leg disc associated Duf-lacZ myoblasts that do not express Lbe are in area 4, corresponding to the proximal part of the leg disc that contributes to the ventral thorax. Surprisingly, Htlca also induces Lbe/Duf-lacZ expression in tarsal segments outlined area 3. These cells give rise to ectopic muscles in tarsal segments N,P otherwise devoid of muscles. M A wild type adult muscle pattern revealed in the leg expressing muscle-specific MHC-tauGFP green and tendon-specific 1151-DsRed red. Note that no muscles are detected in the tarsus. Arrowhead points to the dorsal tibia muscle, talm. N Gain of Htl signalling in leg myoblasts leads to the formation of supernumerary muscle fibres arrowhead in N. Ectopic muscles form in the tarsus arrow in N. O RNAi-mediated attenuation of Htl expression leads to the reduced number of muscle fibres arrowhead in O. P An enlarged view of tarsal segments shown in N. Ectopic muscle fibres align along the long tendon but most of them are not attached to the epithelium. Q An enlarged view of the distal tibia muscles in wild type R; in Htl gain-of-function and after RNAi-based Htl attenuation S The number of muscle fibres in the talm muscle indicated by asterisk is significantly increased in R and reduced in S.</p

Lbe is dynamically expressed in leg disc myoblasts.

<p>A–F Confocal images of leg imaginal discs stained with anti-Twi A, D and blue in merge C, F, and anti-Lbe B, E and green in merge C, F antibodies. A–C Third instar leg imaginal disc. B Lbe expression can be seen in subsets of Twi myoblasts, associated with regions of the leg disc that develop into different segments of the adult leg. Arrows in B, E show a group of myoblasts that give rise to dorsal femur muscle, tilm. The arrowheads in B, E point to precursors of ventral femur muscle, tidm. D–F″ 0 hr APF leg imaginal disc. Myoblasts are regionalised at this stage and are seen at future muscle-forming sites in adult tibia tadm, talm, femur tidm, tilm, trocanter Tr, coxa Co. Lbe is expressed in almost all Twist myoblast subsets at specific sites along the proximal-distal axis in the leg disc proper. Most proximal cells including the dorsal proximal myoblasts asterisks in C, F are devoid of Lbe expression. F′, F″ Two different views of a 3D reconstruction see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000122#pone.0000122.s003" target="_blank">Video 1S</a> of the disc presented in F showing spatial distribution of different groups of leg myoblasts. G The schematic of F showing positions of Lbe-positive myoblasts within the leg segments. Lbe is also expressed in leg disc epithelium in the ventral region black asterisks in B, E and the long tendon yellow arrow in E. Abbreviations: Ta, tarsus, Ti, tibia, Fe, femur, see also 22 for muscle nomenclature.</p

Effects of RNAi-based attenuation of Lb gene function.

<p>A–F Anterior views of tibia A–C and femur D–F musculature revealed in wild type A, D and lbRNAi B, C, E, F flies carrying MHC-tauGFP transgene. B, E show mild phenotypes whereas C, F show severe lbRNAi phenotypes. 3D reconstructions from confocal scans were used to generate the presented views see corresponding 3D videos in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000122#pone.0000122.s005" target="_blank">Video S3</a>-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000122#pone.0000122.s010" target="_blank">S8</a>. Muscle fibres from <i>lb</i>RNAi legs are smaller in both ventral arrows in A–C and dorsal arrowheads in A–C tibia. General muscle mass appears reduced in tibia C and femur asterisks in E and F. Loss of muscle mass and the abnormal attachment of muscle fibres to the leg epithelium lead to morphological defects most frequently manifested by bending of the femur segment arrows in E and F. Note also the altered shape of muscle fibers in tadm muscles B, C and tilm muscle E. G RNAi induced reduction in Lbe and Lbl protein levels revealed by Western blot using two different anti-Lb antibodies. Note that a low level of Lbe is still detected in embryos ubiquitously expressing <i>lb</i>RNAi constructs see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000122#s4" target="_blank">Materials and Methods</a> for details. H, J Wild type electron microscopy micrographs and I, K micrographs from lbRNAi femur muscles showing H, I sarcomeric ultrastructure and J, K muscle-tendon junction area. The Z line associated pairs of mitochondria dyads, arrows in H are absent in lbRNAi muscle asterisks in I and the few mitochondria still present arrow in I appear to have altered internal structures. Also, myofilaments from lbRNAi sarcomeres are highly disorganised and some of them are disrupted arrowheads in I. A lower intensity electron dense desmosomes are detected in muscle-tendon junctions from lbRNAi legs arrow in K when compared to the wild type arrow in J.</p