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Objective: To investigate how permanent inhibition of guanylyl cyclase A receptor (CC-A) affects cardiac function. Methods: Hearts of GC-A--/-- and corresponding wild type mice (GC-A+/+) were characterised by histological, western [...]

Myomesin is a structural component of the M-band that is expressed in all types of striated muscle. Its primary function may be the maintenance of the thick filament lattice and its anchoring to the elastic filament system composed of titin. Different myomesin isoforms have been described in chicken and mice, but no particular function has been assigned to them. Here we investigate the spatio-temporal expression pattern of myomesin isoforms by means of reverse transcriptase-polymerase chain reaction and isoform-specific antibodies. We find that two alternative splicing events give rise to four myomesin isoforms in chicken contrary to only one splicing event with two possible isoforms in mice. A splicing event at the C terminus results in two splice variants termed H-myomesin and S-myomesin, which represent the major myomesin species in heart and skeletal muscle of avian species, respectively. In contrast, in mammalian heart and skeletal muscle only S-myomesin is expressed. In embryonic heart of birds and mammals, alternative splicing in the central part of the molecule gives rise to the isoform that we termed EH-myomesin. It represents the major myomesin isoform at early embryonic stages of heart but is rapidly down-regulated around birth. Thus, the strict developmental regulation of the EH-myomesin makes it an ideally suited marker for embryonic heart.

Myofibrillogenesis in situ was investigated by confocal microscopy of immunofluorescently labelled whole mount preparations of early embryonic chicken heart rudiments. The time-course of incorporation of several components into myofibrils was compared in triple-stained specimens, taken around the time when beating starts. All sarcomeric proteins investigated so far were already expressed before the first contractions and myofibril assembly happened within a few hours. No typical stress fibre-like structures or premyofibrils, structures observed in cultured cardiomyocytes, could be detected during myofibrillogenesis in the heart. Sarcomeric proteins like (α)-actinin, titin and actin were found in a defined localisation pattern even in cardiomyocytes that did not yet contain myofibrils, making up dense body-like structures. As soon as the heart started to beat, all myofibrillar proteins were already located at their exact position in the sarcomere. The maturation of the sarcomeres was characterised by a short delay in the establishment of the pattern for M-line epitopes of titin with respect to Z-disk epitopes and the incorporation of the M-line component myomesin, which preceded that of myosin binding protein-C. Thus dense body-like structures, made up of titin, (α)-actinin and actin filaments serve as the first organised complexes also during myofibrillogenesis in situ and titin functions as a ruler for sarcomere assembly as soon as its C termini have become localised. We suggest that assembly of thin and thick filament occurs independently during myofibrillogenesis in situ and that myomesin might be important for integrating thick filaments with the M-line end of titin.

Regulation of epithelial and endothelial permeability is essential for proper function of compartmentalized organisms, and tyrosine phosphorylation plays an important role in this process. We analyzed the impact of protein tyrosine phosphatase (PTP) inhibition on the structure of endothelial junctional proteins. In human umbilical vein endothelial cells (HUVECs) the PTP inhibitors phenylarsine oxide (PAO) and pervanadate induced proteolysis of the tight junction protein occludin. Occludin proteolysis was inhibited by the metalloproteinase inhibitor 1,10-phenanthroline (PHEN), but not by inhibitors against other types of proteases. The junctional proteins ZO-1, cadherin and beta-catenin were not cleaved. Under conditions of occludin proteolysis, PAO treatment elevated permeability for FITC-dextran. Simultaneous incubation of HUVECs with PAO and PHEN inhibited the rise in permeability by more than 60%. PAO treatment lead to progressive disappearance of occludin from the cell periphery. In contrast, ZO-1, cadherin and beta-catenin retained their positions at the sites of intercellular contact. Simultaneous administration of PAO and PHEN greatly prevented the redistribution of occludin. These results demonstrate a selective cleavage of occludin by a metalloproteinase and suggest that this process can contribute to the control of paracellular permeability in endothelial cells.

Neonatal and adult rat cardiomyocytes display differences when isolated and culturedin vitro. Whereas cells obtained from juvenile hearts adapt quite rapidly as judged by their beating, cells from adult animals undergo a complex degeneration – regeneration process of their myofibrillar apparatus. These differences are also reflected by a distinct sensitivity to drugs that affect the non-sarcomeric cytoskeleton. After long-term treatment with nocodazole, which disassembles microtubules, neonatal rat cardiomyocytes (NRC) remain relatively unaffected, whereas adult rat cardiomyocytes (ARC) are unable to spread on the substrate or to undergo the remodelling process of their myofibrils. If microfilaments are destroyed by cytochalasin D, neither NRC nor ARC spread, and they lose the capacity to assemble new myofibrils. The effects of drug treatment with both cytochalasin and nocodazole, respectively, were reversible, since normal myofibrillogenesis took place after the cells had been washed and cultivated in standard medium for 4 days. This study demonstrates that microfilaments are essential for assembly of new sarcomeresin vitro, and underlines intrinsic differences between NRC and ARC in their requirement for intact microtubules. Adult cardiomyocytes have lost a certain degree of flexibility due to their longer adaptation to the specific situation in the heart, whereas cardiomyocytes isolated from neonatal animals can maintain and assemble myofibrilsin vitroeven after their microtubules were destroyed.

Vertebrate tropomyosins (TMs) are expressed from four genes, and at least 18 distinct isoforms are generated via a complex pattern of alternative RNA splicing and alternative promoters. The functional significance of this isoform diversity is largely unknown and it remains to be determined whether specific isoforms are required for assembly and integration into distinct actin-containing structures. The ability of nonmuscle (TM-1, -2, -3, -4, -5(NM1), -5a or -5b) and striated muscle (skeletal muscle (α)-TM) isoforms to incorporate into actin filaments of neonatal rat cardiomyocytes (NRCs) was studied using expression plasmids containing TM-fusions with GFP (green fluorescent protein) as well as with VSV- or HA-epitope tags. All isoforms, except of fibroblast TM-4, were able to incorporate into the I-band of NRCs. When TM-4 was co-transfected with other low molecular weight (LMW) isoforms of TM (TM-5, TM-5a and TM-5b), it was able to incorporate into sarcomeres of NRCs. This result was not obtained when TM-4 was co-transfected with high molecular weight (HMW) TMs (TM-1, TM-2 or skeletal muscle (α)-TM). These data demonstrate that the ability of TM-4 to bind to actin filaments can be specifically influenced by its interaction with other LMW TM isoforms. In addition, cells that incorporated the muscle or nonmuscle GFP-TMs into their sarcomeres continued to beat and exhibited sarcomeric contraction. These studies provide the first in vivo demonstration of synergistic effects between TM isoforms for binding to actin filaments. These results have important implications in understanding actin filament dynamics in nonmuscle cell systems, especially during development and in transformed cells, where alterations in the ratio of different LMW isoforms might lead to changes in their interactions with actin filaments. Furthermore, these studies demonstrate that GFP-TM can be used to study thin-filament dynamics in muscle cells and actin filament dynamics in nonmuscle cells.

Zusammenfassung An 3 Serien von Gipsmodellen von Kindern im Alter von 4 Monaten bis 12 Jahren wurden die Formen der Zahnbögen und die der Alveolarfortsatzbögen des Ober- und Unterkiefers untersucht. In jedem Altersstadium sind die Alveolarfortsatzbögen der Form einer Parabel angenähert. Das gleiche gilt für die Formen der Zahnbögen, sobald die ersten Milchzähne durchgebrochen sind. In der überwiegenden Zahl der Fälle handelt es sich um kubische Parabeln. Damit decken sich die von uns bei Erwachsenen erhobenen Befunde. Die Meinung, daß die Form des oberen Zahnbogens beim Menschen einer halben Ellipse entspreche, ist daher zu revidieren.